Chemical and Functional Properties of Food Components [4 ed.] 9781032199221

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Chemical and Functional Properties of Food Components Over three editions, this book described the contents of food raw materials and products, the chemistry/biochemistry of food components, as well as the changes occurring during post-harvest storage and processing affecting the quality of foods. The fourth edition of Chemical and Functional Properties of Food Components discusses the role of chemical compounds in the structure of raw materials and the formation of different attributes of food quality, including nutritional value, safety, and sensory properties. This new edition contains four new chapters: “Non-Protein Nitrogenous Compounds”; “Prooxidants and Antioxidants in Food”; “Non-Nutritive Bioactive Compounds in Food of Plant Origin”; and “Analytical Methods Used for Assessing the Quality of Food Products.” These chapters have been included because new research results have brought increasing knowledge on the effect of non-protein nitrogenous compounds, especially bioactive peptides, nucleic acids, and biogenic amines on the biological properties of foods; the role of natural and added prooxidants and antioxidants in the processing and biological impact of foods; numerous benefcial and harmful effects of bioactive components of plant foods; and new systems for control of food composition and the safety of foods. Features: • Stresses the effect of the chemical/biochemical reactions on the selection of optimum parameters of food processing without presenting details of the technological processes • Describes naturally occurring elements and compounds as well as those generated during food handling in view of health hazards they may bring to consumers • Discusses the risks and benefts of reactions occurring during food handling The knowledge of the chemistry and biochemistry of the components and their interactions presented in this book aids food scientists in making the right decisions for controlling the rate of benefcial and undesirable reactions, selecting optimal storage and processing parameters, as well as the best use of food raw materials.

Chemical and Functional Properties of Food Components Series SERIES EDITOR Zdzisław E. Sikorski

Chemical and Functional Properties of Food Components, Fourth Edition Edited by Hanna Staroszczyk and Zdzisław E. Sikorski Meat Quality: Genetic and Environmental Factors Edited by Wiesław Przybylski and David Hopkins Food Oxidants and Antioxidants: Chemical, Biological, and Functional Properties Edited by Grzegorz Bartosz Fermentation: Effects on Food Properties Edited by Bhavbhuti M. Mehta, Afaf Kamal-Eldin and Robert Z. Iwanski Methods of Analysis of Food Components and Additives, Second Edition Edited by Semih Otles Food Flavors: Chemical, Sensory and Technological Properties Edited By Henryk Jelen Environmental Effects on Seafood Availability, Safety, and Quality Edited by E. Grazyna Daczkowska-Kozon and Bonnie Sun Pan Chemical and Biological Properties of Food Allergens Edited By Lucjan Jedrychowski and Harry J. Wichers Chemical, Biological, and Functional Aspects of Food Lipids, Second Edition Edited by Zdzisław E. Sikorski and Anna Kołakowska Food Colorants: Chemical and Functional Properties Edited by Carmen Socaciu Mineral Components in Foods Edited by Piotr Szefer and Jerome O. Nriagu Chemical and Functional Properties of Food Components, Third Edition Edited by Zdzisław E. Sikorski Carcinogenic and Anticarcinogenic Food Components Edited by Wanda Baer-Dubowska, Agnieszka Bartoszek and Danuta Malejka-Giganti Toxins in Food Edited by Waldemar M. Dąbrowski and Zdzisław E. Sikorski Chemical and Functional Properties of Food Saccharides Edited by Piotr Tomasik Chemical and Functional Properties of Food Proteins Edited by Zdzisław E. Sikorski

Chemical and Functional Properties of Food Components

Fourth Edition Edited by

Hanna Staroszczyk and Zdzisław E. Sikorski

Fourth edition published 2023 by CRC Press 6000 Broken Sound Parkway NW, Suite 300, Boca Raton, FL 33487-2742 and by CRC Press 4 Park Square, Milton Park, Abingdon, Oxon, OX14 4RN CRC Press is an imprint of Taylor & Francis Group, LLC © 2023 selection and editorial matter, Hanna Staroszczyk and Zdzislaw E. Sikorski; individual chapters, the contributors First edition published by CRC Press 1997 Second edition published by CRC Press 2002 Third edition published by CRC Press 2007 Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microflming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, access www.copyright.com or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. For works that are not available on CCC please contact [email protected] Trademark notice: Product or corporate names may be trademarks or registered trademarks and are used only for identifcation and explanation without intent to infringe. Library of Congress Cataloging-in-Publication Data Names: Staroszczyk, Hanna, editor. | Sikorski, Zdzisław E., editor. Title: Chemical and functional properties of food components / edited by Hanna Staroszczyk, Zdzislaw Sikorski. Description: Fourth edition. | Boca Raton, FL : CRC Press, 2023. | Includes bibliographical references and index. | Contents: Food components and quality / Zdzisław E. Sikorski and Barbara Piotrowska, Merck -- Chemical composition and structure of foods / Jolanta Tomaszewska-Gras -- Water and food quality / Peter Edward Doe and Emilia Barbara Cybulska. Identifers: LCCN 2022049021 (print) | LCCN 2022049022 (ebook) | ISBN 9781032199221 (hbk) | ISBN 9781032209227 (pbk) | ISBN 9781003265955 (ebk) Subjects: LCSH: Food--Analysis. | Food--Composition. Classifcation: LCC TX545 .C44 2023 (print) | LCC TX545 (ebook) | DDC 664/.07--dc23/eng/20221214 LC record available at https://lccn.loc.gov/2022049021 LC ebook record available at https://lccn.loc.gov/2022049022 ISBN: 978-1-032-19922-1 (hbk) ISBN: 978-1-032-20922-7 (pbk) ISBN: 978-1-003-26595-5 (ebk) DOI: 10.1201/9781003265955 Typeset in Times by Deanta Global Publishing Services, Chennai, India

Contents Preface......................................................................................................................vii About the Editors ......................................................................................................ix Contributors ..............................................................................................................xi Chapter 1

Food Components and Quality ............................................................ 1 Zdzisław E. Sikorski and Barbara Piotrowska

Chapter 2

Chemical Composition and Structure of Foods ................................. 13 Jolanta Tomaszewska-Gras

Chapter 3

Water and Food Quality ..................................................................... 39 Peter Edward Doe and Barbara Emilia Cybulska

Chapter 4

The Role of Mineral Components...................................................... 73 Małgorzata Grembecka

Chapter 5

Saccharides....................................................................................... 105 Hanna Staroszczyk

Chapter 6

The Role of Proteins in Food ........................................................... 155 Zdzisław E. Sikorski and Izabela Sinkiewicz

Chapter 7

Non-Protein Nitrogenous Compounds ............................................. 203 Edyta Malinowska-Pańczyk

Chapter 8

Lipids and Food Quality .................................................................. 225 Izabela Sinkiewicz

Chapter 9

Factors Affecting the Rheological Properties of Foods................... 265 Robert Tylingo

Chapter 10 Food Colorants ................................................................................. 285 Anna Podsędek v

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Chapter 11 Prooxidants and Antioxidants in Food............................................. 303 Ronald B. Pegg and Ryszard Amarowicz Chapter 12 Food Allergens ................................................................................. 339 Barbara Wróblewska Chapter 13 Food Flavors..................................................................................... 363 Shwu-Pyng Joanna Chen and Bonnie Sun Pan Chapter 14 The Role of Food Additives ............................................................. 401 Joanna Le Thanh-Blicharz and Jacek Lewandowicz Chapter 15 Food Safety ...................................................................................... 419 Agata Witczak and Kamila Pokorska-Niewiada Chapter 16 Probiotics and Prebiotics in Food..................................................... 433 Edyta Malinowska-Pańczyk Chapter 17 Mood Food ....................................................................................... 457 Maria H. Borawska and Sylwia K. Naliwajko Chapter 18 Mutagenic and Carcinogenic Compounds in Food..........................469 Agnieszka Bartoszek and Serhii Holota Chapter 19 Non-Nutritive Bioactive Compounds in Food of Plant Origin......... 497 Barbara Kusznierewicz Chapter 20 Analytical Methods Used for Assessing the Quality of Food Products............................................................................................ 535 Widiastuti Setyaningsih

Preface Water, saccharides, proteins, lipids, mineral compounds, colorants, other constituents, and additives contribute to the nutritional value and sensory properties of food. During post-harvest storage and processing, these components change. The extent and nature of the alterations depend on the chemical properties of the compounds themselves. Knowledge of the chemistry and biochemistry of food components and their behavior in the face of various stressors aids in making the right decisions for controlling the rate of benefcial and undesirable reactions, selecting optimal storage and processing parameters, and defning the best use of food raw materials. The book draws from the personal research and teaching experience of scientists from universities and research institutions around the world. Beginning with an examination of food components both natural and added, this volume, like its predecessors, details the role of chemical compounds in the structure of raw materials and the formation of different attributes of food quality. All chapter authors are renowned specialists in their feld and the editors have extensive experience in editing food science books. This new edition contains the following new chapters: “Non-Protein Nitrogenous Compounds,” “Prooxidants and Antioxidants in Food,” “Non-Nutritive Bioactive Compounds in Food of Plant Origin,” and “Analytical Methods Used for Assessing the Quality of Food Products.” These chapters have been included because new research results have brought increasing knowledge on the effect of non-protein nitrogenous compounds, especially bioactive peptides, nucleic acids, and biogenic amines on the quality as well as biological properties of foods, the role of natural and added prooxidants and antioxidants in the processing and biological impact of foods, numerous benefcial and harmful effects of bioactive components of plant foods, and new systems for control of food composition and the safety of foods. It was possible to prepare the book only thanks to the dedication of all persons involved. As the editors we thank all of them for their contributions. These thanks are primarily addressed to those who followed strictly all our editorial suggestions and delivered their chapters well ahead of the deadline. We dedicate this volume to all scientists, who cooperated in writing the numerous books of the CRC series Chemical and Functional Properties of Food Components. Hanna Staroszczyk and Zdzisław E. Sikorski Gdańsk University of Technology

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About the Editors Hanna Staroszczyk earned her BS and MS at the Cracow University of Technology, her PhD at the Hugon Kołłątaj University of Agriculture in Cracow, and her DSc at the Gdańsk University of Technology (GUT), Poland. Currently, she is head of the Department of Chemistry, Technology, and Biotechnology of Food at GUT. She also worked as a researcher/postdoc/visiting professor at the Institute of Food Research, Norwich, UK, Academia Sinica, Taipei, Taiwan, and the University of Arkansas, Fayetteville, AR. Her research is focused mainly on biopolymer chemistry. She is an author/co-author of original papers on the modifcation of proteins and polysaccharides, as well as an editor of books on food chemistry. She is a member of the Polish Society of Food Technologists. Zdzisław E. Sikorski received his BS, MS, PhD, and DSc from the Gdańsk University of Technology (GUT), a doctorate honoris causa from the Agricultural University in Szczecin, and is a fellow of the International Academy of Food Science and Technology. He gained industrial experience in breweries, in fsh, meat, and vegetable processing plants, and on a deep-sea fshing trawler. He was the organizer, professor, and head of the Department of Food Chemistry and Technology at GUT, served as dean of the Faculty of Chemistry, was chairman of the Committee of Food Technology and Chemistry of the Polish Academy of Sciences, chaired the scientifc board of the Sea Fisheries Institute in Gdynia, and was an elected member of the Main Council of Science and Tertiary Education in Poland. He worked also as a researcher/professor at Ohio State University, OH; CSIRO in Hobart, Australia; DSIR in Auckland, New Zealand; and National Taiwan Ocean University in Keelung, Taiwan. Currently, he is an honorary professor emeritus in the Faculty of Chemistry at GUT. His research concentrated on the technology of fsh processing and changes in food proteins due to storage and handling. He published numerous papers containing the results of his investigations in the feld of food chemistry and technology and is the author/editor of a number of books in English, Polish, Russian, and Spanish.

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Contributors Ryszard Amarowicz Institute of Animal Reproduction and Food Research Polish Academy of Sciences Olsztyn, Poland Agnieszka Bartoszek Gdańsk University of Technology Department of Chemistry, Technology and Biotechnology of Food Gdańsk, Poland Maria H. Borawska Medical University of Białystok Department of Bromatology Białystok, Poland Shwu-Pyng Joanna Chen Chinese University of Hong Kong, Honorary Research Fellow State Key Laboratory of Agrobiotechnology Hong Kong, China Emilia Barbara Cybulska Gdańsk University of Technology formerly Department of Pharmaceutical Technology and Biochemistry Gdańsk, Poland Peter Edward Doe University of Tasmania School of Engineering Tasmania, Australia Małgorzata Grembecka Medical University of Gdańsk Department of Bromatology Gdańsk, Poland

Serhii Holota Danylo Halytsky Lviv National Medical University Department of Organic, Bioorganic and Pharmaceutical Chemistry Lviv, Ukraine Barbara Kusznierewicz Gdańsk University of Technology Department of Chemistry, Technology and Biotechnology of Food Gdańsk, Poland Jacek Lewandowicz Poznań University of Technology Institute of Logistics Poznań, Poland Edyta Malinowska-Pańczyk Gdańsk University of Technology Department of Chemistry, Technology and Biotechnology of Food Gdańsk, Poland Sylwia K. Naliwajko Medical University of Białystok Department of Bromatology Białystok, Poland Bonnie Sun Pan National Taiwan Ocean University Department of Food Science Keelung City, Taiwan Ronald B. Pegg University of Georgia Department of Food Science and Technology Athens, Georgia, USA

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Barbara Piotrowska Merck Life Science Poznań, Poland Anna Podsędek Lodz University of Technology Institute of Molecular and Industrial Biotechnology Łódź, Poland Kamila Pokorska-Niewiada West Pomeranian University of Technology Department of Toxicology, Dairy Technology and Food Storage Szczecin, Poland Widiastuti Setyaningsih Gadjah Mada University Department of Food and Agricultural Product Technology Yogyakarta, Indonesia Zdzisław E. Sikorski Gdańsk University of Technology Department of Chemistry, Technology and Biotechnology of Food Gdańsk, Poland Izabela Sinkiewicz Gdańsk University of Technology Department of Chemistry, Technology and Biotechnology of Food Gdańsk, Poland

Contributors

Hanna Staroszczyk Gdańsk University of Technology Department of Chemistry, Technology and Biotechnology of Food Gdańsk Poland Joanna Le Thanh-Blicharz Prof. Waclaw Dabrowski Institute of Agriculture and Food Biotechnology – State Research Institute Warszawa, Poland Jolanta Tomaszewska-Gras Poznań University of Life Sciences Department of Food Quality and Safety Management Poznań, Poland Robert Tylingo Gdańsk University of Technology Department of Chemistry, Technology and Biotechnology of Food Gdańsk, Poland Agata Witczak West Pomeranian University of Technology Department of Toxicology, Dairy Technology and Food Storage Szczecin, Poland Barbara Wróblewska Institute of Animal Reproduction and Food Research Polish Academy of Sciences Olsztyn, Poland

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Food Components and Quality Zdzisław E. Sikorski and Barbara Piotrowska

CONTENTS 1.1

1.5

Introduction ......................................................................................................1 1.1.1 Components of Food Raw Materials and Products ..............................1 1.1.2 Factors Affecting Food Composition ...................................................2 1.1.3 The Role of Food Components .............................................................3 Functional Properties........................................................................................4 Food Quality.....................................................................................................5 1.3.1 Attributes of Quality.............................................................................5 1.3.2 Safety and Nutritional Value ................................................................6 1.3.3 Sensory Quality .....................................................................7 Chemical Analysis in Ensuring Food Quality.................................................. 8 1.4.1 Introduction ..........................................................................................8 1.4.2 Requirements of the Producer ..............................................................8 1.4.3 Requirements of the Consumer .......................................................... 10 1.4.4 Limits of Determination ..................................................................... 10 Conclusion ...................................................................................................... 11

1.1

INTRODUCTION

1.2 1.3

1.4

1.1.1

COMPONENTS OF FOOD RAW MATERIALS AND PRODUCTS

Foods are derived from plants, carcasses of animals, and single-celled organisms. They are composed of water, saccharides, proteins, lipids, and minerals, as well as a host of other compounds present in minor quantities, albeit of signifcant impact on the quality of many products. Here belong especially the non-protein nitrogenous compounds, vitamins, colorants, favorings, functional additives, and numerous other items generated during the processing or storage of foods. The content of water in various foods ranges from a few percent in dried commodities, e.g. milk powder, through about 15% in grains, 16–18% in butter, 20% in honey, 35% in bread, 65% in manioc, and 75% in meat, to about 90% in many fresh fruits and vegetables. Saccharides are present in food raw materials in quantities ranging from about 1% in meats and fsh, through about 4.5% in milk, 18% in potatoes, and 15–21% in sugar beets, to about 70% in cereal grains.

DOI: 10.1201/9781003265955-1

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The protein content in foods regards mainly crude protein, i.e. N × 6.25. The nitrogen-to-protein conversion factor 6.25 has been recommended for most plant and animal foods under the assumption that the N content in their proteins is 16% and they do not contain non-protein N. The N content in the proteins, however, depends on the amino acid composition. Furthermore, the total N consists of protein N and N contained in numerous non-protein compounds, e.g. free peptides and amino acids, nucleic acids and their degradation products, amines, betaines, urea, vitamins, and alkaloids. In some foods, the non-protein N may constitute up to 30% of total N. In many of these compounds the C:N ratio is similar to the average in amino acids. However, the N content in urea, being 47%, is exceptionally high. The average conversion factor for estimation of true protein, based on the ratios of total amino acid residues to amino acid N, is in the range of 5.14–6.61 for different classes of foods. Crude protein makes up about 1% of the weight of fruits, 2% of potatoes, 3.2% of bovine milk, 12% of eggs, 12–22% of wheat grain, about 20% of meat, and 25–40% of different beans. Cereal grain and legume seeds deposit during their development large quantities of storage proteins in granules known also as protein bodies. In soybeans, these proteins constitute 60–70% of the total protein content. The granules are 80% made of proteins. The lipid content in foods is given in nutrition information labeling predominantly as “total fat,” which is often called also “crude fat.” This is a mixture of various classes of lipids, mainly different triacylglycerols. The lipids of numerous fshes, such as orange roughy, mullets, codfsh, and sharks, as well as some crustaceans and mollusks, also comprise wax esters. Some shark oils are very rich in hydrocarbons, particularly in squalene. Furthermore, the lipid fraction of food raw materials harbors different sterols, vitamins, and pigments that are crucial for the metabolism. Thus the composition of the extracted crude fat depends on the kind of food and the polarity of the solvent used for extraction. Lipids constitute about 1% of the weight of fruits, vegetables, and lean fsh muscle, 3.5% of milk, 6% of beef meat, 32% of egg yolk, and 85% of butter.

1.1.2

FACTORS AFFECTING FOOD COMPOSITION

The content of different components in food raw materials depends on the species and variety of the animal and plant crop, the conditions of cultivation and time of harvesting of the plants, the feeding, conditions of life, and age of the farm animals, or the season of catching the fsh and marine invertebrates. The post-harvest changes in the crop during storage are also important. The food industry, by establishing quality requirements for raw materials, can encourage the producers to control within limits the contents of the main components. This regards, e.g., starch in potatoes, fat in various meat cuts, pigments in fruits and vegetables and in the fesh of fsh, or protein in wheat and barley, as well as the fatty acid composition of lipids in oilseeds and meats. The contents of desirable minor components, e.g. of natural antioxidants, can also be effectively controlled to slow the oxidation of pigments and lipids in beef meat. Contamination of the raw material with organic and inorganic pollutants can be controlled, e.g. by observing recommended agricultural procedures

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in using fertilizers, herbicides, and insecticides, and by seasonally closing certain fshing areas to avoid marine toxins. The size of predatory fsh like swordfsh, tuna, or sharks that are fshed commercially can be limited to reduce the risk of too high a content of mercury and arsenic in the fesh. The composition of processed foods depends on the applied recipe as well as on changes taking place due to processing and storage. These are mainly brought about by added, endogenous, and microbial enzymes, active forms of oxygen, heating, chemical treatment, and low or high pH. Examples of such changes are: • leaching of soluble, desirable, and undesirable parts, e.g. vitamins, mineral components, and toxins during washing, blanching, or cooking, • dripping after thawing or due to cooking, • loss of moisture and volatiles by evaporation and sublimation, • absorption of valuable or harmful compounds, e.g. during salting, pickling, seasoning, frying, or smoking, • formation of desirable or unwanted compounds as a result of enzyme activity, e.g. development of typical favor in cheese or decarboxylation of amino acids in fsh marinades, • generation of welcome or objectionable products due to interactions of reactive groups induced by heating or chemical treatment, e.g. favors or carcinogenic compounds in roasted meats, or trans fatty acids in hydrogenated fats, • formation of different products of oxidation of components, mainly of lipids, pigments, and vitamins, • loss of nutrients and deterioration, e.g. of dried fsh because of attacks by fies, mites, and beetles. In recent decades, a novel type of product appeared known as designer food. It is manufactured using biotechnological/engineering methods and enriched with healthpromoting nutritive additives. Putting this type of product on the market should be approved by appropriate food legislation authorities.

1.1.3

THE ROLE OF FOOD COMPONENTS

The indigenous water is immobilized in plant and animal tissues by the structural elements and various solutes; it contributes to buttressing the conformation of the polymers, serves as a solvent for different constituents, and interacts in metabolic processes. Polysaccharides, proteins, and lipids serve as the building materials of different structures of plant and animal tissues. These structures are responsible for the form and tensile strength of the tissues and create the necessary conditions for metabolic processes to occur. The resulting compartmentation plays a crucial biological role in the organisms. Some of the main components, as well as other constituents, are either bound to different cell structures or are distributed in soluble form in the tissue fuids.

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Many saccharides, proteins, and lipids are stored in living organisms for reserve purposes. Polysaccharides are present in plants as starch in the form of granules and in muscles as glycogen. Other saccharides are dissolved in tissue fuids or perform various biological functions, e.g. in free nucleotides or as components of nucleic acids, or being bound to proteins and lipids. Proteins also play crucial metabolic roles in plants and animals as enzymes and enzyme inhibitors, participate in the transport and binding of oxygen and metal ions, and perform immunological functions. Some plant polysaccharides are only partly utilized for energy. However, as dietary fber, they affect various processes in the gastrointestinal tract in different ways. The distribution of lipids in food raw materials depends on their role in living animals and plants. In an animal body, the lipids occur primarily as an energyrich store of neutral fat in the subcutaneous adipose tissue, as well as kidney, leaf, and crotch fat, the intramuscular fat responsible for marbling, and intermuscular or seam fat. In fatty animals, most of the lipids are stored as depot fat in the form of triacylglycerols. In fsh of lean species, most of the fat occurs in the liver. The lipids contained in the food raw materials in low quantities serve mainly as components of protein-phospholipid membranes and have metabolic functions. The main food components supply the consumers with the necessary building material and source of energy, as well as elements and compounds indispensable for the metabolism. Some polysaccharides are utilized as dietary fber. Many of the minor components present originally in the raw materials are nutritionally essential, e.g. vitamins. Some, although not indispensable, can be utilized by the body, e.g. most free amino acids, or impart valuable sensory properties to the food products. Numerous groups, including tocopherols, ubiquinone, carotenoids, ascorbic acid, thiols, amines, and several other non-protein nitrogenous compounds serve as endogenous muscle antioxidants, playing an essential role in postmortem changes in meat. Other minor components are useless or even harmful if present in excessive amounts. Most food raw materials are infected with different microorganisms, putrefactive and often pathogenic, and some contain parasites and the products of microbial metabolism. A variety of compounds are added intentionally during processing to serve as preservatives, antioxidants, colorants, favorings, sweeteners, and emulsifying agents or to fulfll other technological purposes.

1.2 FUNCTIONAL PROPERTIES The term “functional properties” has evolved to have a broad range of meanings. The meaning corresponding to the term “technological properties” implies that the given component present in proper concentration, subjected to processing at optimum parameters, contributes to the expected desirable sensory characteristics of the product, usually by interacting with other food constituents. Hydrophobicity, hydrogen bonds, ionic forces, and covalent bonding are involved in the interactions. Thus the functional properties of food components are affected by the number of accessible reactive groups and by the exposure of hydrophobic areas in the material. Therefore in a system of known water activity, pH, and range of temperature, the functional properties can be to a large extent predicted from the structure of

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the respective saccharides, proteins, and lipids. They can also be improved by appropriate, intentional enzymatic or chemical modifcations of the molecules, mainly those that affect the size, charge density, or the hydrophilic/hydrophobic character of the compounds or change the environment. The functional properties of food components make it possible to manufacture products of desirable quality. Thus pectins contribute to the characteristic texture of ripe apples and make perfect jellies. Different other polysaccharides are effcient thickening and gelling agents at various ranges of acidity and concentration of ions. Alginates in the presence of Ca2+ form protective, unfrozen gels on the surface of frozen products. Some starches are resistant to retrogradation thereby retarding staling of bread. Fructose slows moisture loss from biscuits. Mono- and diacylglycerols, phospholipids, and proteins are used for emulsifying lipids and stabilizing food emulsions and foams. Antifreeze proteins inhibit ice formation in various products, and gluten plays a major role in producing the characteristic texture of wheat bread. Technologically required functional effects can also be achieved by intentionally employing food additives, i.e. colors, sweeteners, and a host of other compounds. These additives are not per se regarded as foodstuffs but are used to modify the rheological properties or acidity, increase the color stability or shelf life, and act as humectants or favor enhancers. In recent decades, the term “functional” has been predominantly given to a large group of products and components, also termed designer foods, pharmafoods, nutraceuticals, or foods for specifc health use, which are regarded as health-enhancing or potentiating the performance of the human organism. These foods, mainly drinks, meals, confectionery, ice cream, and salad dressings, contain various ingredients, e.g. oligosaccharides, sugar alcohols, or choline, which are claimed to have special physiological functions like neutralizing harmful compounds in the body and promoting recovery and general good health. Foods containing prebiotics, various oligosaccharides, and probiotics, mainly dairy products, have been treated in-depth in other chapters of this volume.

1.3 FOOD QUALITY 1.3.1

ATTRIBUTES OF QUALITY

The quality of a food product, i.e. the characteristic properties that determine the degree of excellence, is the sum of the attributes contributing to the satisfaction and good health of the consumer. The composition and the chemical nature of the food components affect all aspects of food quality. The total quality refects at least the following attributes: • compatibility with the local or international food law regulations and standards regarding mainly the proportions of main components, presence of compounds serving as identity indicators, contents of contaminants and additives, hygienic standards, packaging, and labeling, • nutritional aspects, i.e. the contents and availability of nutritionally desirable constituents, mainly proteins, essential amino acids, essential fatty

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•

• • •

•

acids, saccharides, vitamins, prebiotics, probiotics, fber, and mineral components, safety affected by the concentration of compounds that may constitute health hazards for the consumers and affect the digestibility and nutritional availability of the food, e.g. heavy metals, toxins of different origins, some enzymes and enzyme inhibitors, factors decreasing the availability of some metal components, pathogenic microorganisms, and parasites, sensory attributes, i.e. the color, size, form, favor, taste, and rheological properties affected by the chemical composition of the product, as well as by changes resulting from processing and culinary preparation, shelf life at specifc storage conditions, convenience aspects that are refected by the size and ease of opening/ reclosing the container, suitability of the product for immediate use or for different types of thermal treatment, ease of portioning or spreading, as well as transport and storage requirements, ecological aspects regarding suitability for recycling of the packaging material and pollution hazards.

For many foods, one of the most important quality criteria is freshness. This is especially so in the case of numerous species of vegetables, fruits, and seafood. Fish of valuable species at a state of prime freshness, suitable to be eaten raw, may have over ten times higher market price than the same fsh after several days of storage in ice, which is still very ft for human consumption.

1.3.2

SAFETY AND NUTRITIONAL VALUE

Food is regarded as safe if it does not contain harmful organisms or compounds in concentrations above the offcially accepted limits. The nutritional value of foods depends primarily on the contents of nutrients and nutritionally objectionable components in the products. Processing may increase the safety and biological value of food by inducing chemical changes increasing the digestibility of the components or by inactivating undesirable compounds, e.g. toxins or enzymes catalyzing the generation of toxic agents from harmless precursors. Freezing and short-term frozen storage of fsh inactivates the parasites Anisakis that could escape detection during visual inspection of herring fllets used as raw material for cold marinades produced at mild conditions. Thermal treatment inactivates myrosinase, the enzyme involved in the hydrolysis of glucosinolates. This arrests the reactions, which lead to the formation of goitrogenic products in oilseeds of Cruciferae. Heat pasteurization and sterilization reduce to the acceptable level the number of vegetative forms and spores, respectively, of pathogenic microorganisms. Several other examples of such improvements in the safety and biological quality of foods are given in the following chapters of this book. There are, however, also nutritionally undesirable side effects of processing, e.g. destruction of essential food components as a result of heating, chemical treatment, and oxidation. Generally known side effects are the partial thermal decomposition of

Food Components and Quality

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vitamins, especially thiamine, loss of available lysine and sulfur-containing amino acids, or generation of harmful compounds, e.g. carcinogenic heterocyclic aromatic amines, lysinoalanine, and lanthionine or position isomers of fatty acids, not present originally in foods. In recent decades new evidence of side effects has been accumulated with respect to the chemical processing of oils and fats. Commercial hydrogenation of oils leads not only to the intended saturation of selected double bonds in the fatty acids and thereby the required change in the rheological properties of the oil but also to the generation of trans-trans and cis-trans isomers which are absent in the unprocessed oils. 1.3.3 Sensory Quality Many of the desirable sensory attributes of foods stem from the properties of the raw material. The natural color of meat, fsh muscles and skin, vegetables, and fruits depends on the presence of a host of different pigments, which are water or lipid soluble. Chlorophylls impart a green color to vegetables, but also to olive oil. Some natural oils are yellow or red due to different carotenoids. Carotenes are present also in the fesh oil of redfsh (Sebastes marinus), while different carotenoproteins are responsible for the vivid colors of fsh skin. Many hydroxy carotenoids occur in plants in form of esters of long-chain fatty acids. The red, violet, or blue color of fruits and fowers is caused by anthocyanins. Betalains impart the color to red beets. The favor, taste, and texture of fresh fruits and vegetables, as well as the taste of nuts and milk, depend on the presence of natural compounds. These properties are in many cases carried through to the fnal products. In numerous other commodities, the characteristic sensory attributes are generated as a result of processing. The texture of bread develops due to interactions of proteins, lipids, and saccharides with each other and with various gases, while that of cooked meats appears as the result of thermal protein denaturation. The bouquet of wine is due to the presence of volatile components in the grape as well as the result of fermentation of saccharides and a number of other biochemical and chemical reactions. The delicious color, favor, texture, and taste of smoked salmon or sturgeon are generated by enzymatic changes in the tissues and the effect of salt and smoke. The favor of various processed meats develops due to the thermal degradation of predominantly nitrogenous compounds, the generation of volatile products of the Maillard reaction, interactions of lipid oxidation products, and the effect of added spices. Optimum foam performance of beer depends on the interactions of peptides, lipids, the surface-active components of hops, and gases. The favor, texture, and taste of cheese result from fermentation and ripening, while the appealing color and favor of different fried products are due to reactions of saccharides and amino acids. The sensory attributes of foods are related to the contents of many chemically labile components. These, however, just like most nutritionally essential compounds, are prone also to deteriorative changes in conditions of severe heat treatment, oxidation, or application of considerably high doses of chemical agents, e.g. acetic acid or salt, which are often required to ensure safety and suffciently long shelf life of the products. Thus loss in sensory quality takes place, e.g. in over-sterilized meat

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and fsh products due to degradation of sulfur-containing amino acids and development of off-favor; toughening of the texture of over-pasteurized ham or shellfsh due to excessive shrinkage of the tissues and drip; and deterioration of the texture and arresting of ripening in herring preserved at too high concentration of salt. Optimum parameters of storage and processing ensure the retention of the desirable properties of the raw material and lead to the development of the intended attributes of the product. In the selection of these parameters, the chemistry of food components and of the effect of processing must be studied. The eager food technology student can fnd all the necessary information in excellent textbooks on food chemistry and in numerous books on food lipids, proteins, and saccharides, as well as in the current international journals.

1.4 CHEMICAL ANALYSIS IN ENSURING FOOD QUALITY 1.4.1

INTRODUCTION

All aspects of food quality described earlier can be assured only by applying appropriate control in the manufacturing process and storage, based on sensory, physical, chemical, biochemical, and microbial techniques. According to the purpose of analysis, appropriate techniques and hardware are used, from the most simple procedures and gadgets to the very sophisticated analytical instruments known in analytical chemistry. A rational system of control is necessary for the producer of the raw material, the food processor, the retailer, and even the consumer organizations. The results of chemical and microbiological analyses are indispensable for selecting the most suitable parameters of processing and for their implementation, for designing and operating the hazard analysis and critical control points system of quality assurance in processing plants, and for securing the safety of the food products available on the market.

1.4.2

REQUIREMENTS OF THE PRODUCER

Thanks to the possibility of rapid and reliable determination of food composition and contaminants by applying appropriate techniques, the raw materials can be optimally used for manufacturing various products. Furthermore, loss in quality, as well as health hazards, can be avoided. In the relations between the primary producer and the food processor, usually the requirements regarding the contents and characteristics of the most important components, as well as freshness grades of the raw materials, are agreed upon. Depending on the commodity it may be, e.g.: • saccharose in sugar beets, • fat in milk or in mackerel as raw material for hot smoking, • color of vegetables and egg yolk depending on the concentration of carotenoid pigments,

Food Components and Quality

• • • • • • • •

9

proportion of lean tissue and marbling in pig or beef carcasses, connective tissue in meats used for least-cost formulations of sausages, contents and characteristics of gluten in wheat grains, starch and protein in barley used for malting, extract in tomatoes, oil in oil-bearing raw materials, free fatty acids and peroxide value in fat-containing commodities, trimethylamine, hypoxanthine, or other freshness indicators in marine fsh, elasticity of kamaboko, the Japanese-type fsh cake.

These components and characteristics are usually determined using standard chemical or physicochemical analyses or enzymatic sensors. For example, the texture of kamaboko is commonly determined by folding a 5 mm-thick slice of the product and observing the formed edge. The highest-quality kamaboko can be folded twice without any cracking; the lowest-quality product falls apart after the frst folding. Although this test is very simple, it may decide the price of a large consignment of surimi or kamaboko. Nowadays many companies supply the hardware, reagents, and analytical procedures for numerous applications in the food plant and for water feld analysis. Thanks to enormous progress in analytical methodology and instrumentation, the food chemist can use automated equipment for assaying, e.g., water, proteins, lipids, saccharides, fber, and mineral components. Online analyses provide for continuous control of processing parameters. Among the rapid tests for food and beverage analysis are those for the determination of ascorbic acid in vegetable products, calcium, chromium, nickel, and nitrate in drinking water, hydroxymethylfurfural in honey and tomato products, as well as saccharose in fruits and juices. To assist in routine analyses in dairy production many tests, photometric or refectometric techniques are offered, like refectometric detection of alkaline phosphatase for controlling milk pasteurization, photometric control of lactose fermentation and determination of urea, or photometric assay of ammonia in milk. The characteristic freshness attributes of different foods are usually evaluated by sensory methods and by the determination of specifc indices, predominantly by biochemical sensors. A typical example may be the examination of fsh freshness by a taste panel and by chemical tests or biochemical sensors suitable for assaying the volatile odorous compounds and products of nucleotide catabolism. The results of these kinds of analyses serve as the basis for technological decisions regarding the suitability of the raw materials for further storage or the given treatment, as well as for adjusting the processing parameters. They often decide also on the price of the commodity. The producer needs chemical analysis to ascertain that the raw material used in his plant does not contain any harmful components or contaminants in quantities higher than those accepted by national or international regulations, e.g. nitrates(V) and nitrates(III) in vegetables, pesticide residues in various crops, heavy metals in many plant and animal tissues including Hg in large predatory fsh, histamine in fsh meat, or mycotoxins in peanuts.

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1.4.3

Sikorski and Piotrowska

REQUIREMENTS OF THE CONSUMER

The results of routine analyses performed by the producer and by food inspection laboratories have to ascertain that most consumer expectations regarding nutritious and wholesome food of high sensory quality are fulflled. The consumer generally requires that foods offered on the market contain the components typical for the type of product and that their proportions are those as presented on the label. This regards e.g. the contents of protein and fat in meat products, milk fat in butter, vitamin C in fruit juices, the unique fatty acid composition of the product sold as extra virgin olive oil or as n-3 polyenoic fatty acids rich preparation, absence of pork in produce declared as made of other meats, or meat or fsh species other than that specifed in comminuted commodities. Food adulteration has been known as an age-old vice and chemical analysis helps to combat it. The nutrition-cautious person looks on the label for information regarding essential amino acids, polyunsaturated fatty acids, vitamins, mineral components, fber, and recently also functional additives or GMO products. Many consumers study carefully the labels on packaged foods, since their health or even life may depend on the information regarding the presence of different ingredients rich in allergens in the product, e.g. gluten or peanuts. However, small amounts of such compounds may originate from residues in processing machinery or stem from additives used by the processor. The safety of food products is safeguarded by determining e.g. heavy metals and their speciation, polycyclic aromatic hydrocarbons in oils, heavy smoked fsh and meat products, acrylamide in French fries, mycotoxins in a variety of commodities, and various additives. For determination of the very large number of hazardous components, additives, and impurities, many specialized chromatographic, spectroscopic, and physical techniques, as well as enzyme, microbial, and immunological sensors are used.

1.4.4

LIMITS OF DETERMINATION

By applying effcient procedures of enrichment and separation of analytes, combined with the use of highly selective and sensitive detectors, it is now possible to determine different additives and contaminants, as well as the products of various chemical and biochemical reactions in foods in extremely low concentrations. This is often necessary, since the national and international bodies responsible for the safety and authenticity of foods require that the producers conform to regulations allowing very low amounts of various characteristic components and natural toxic compounds and contaminants in their products. These requirements are especially rigorous with respect to foods destined for young children – e.g. the contents of nitrates in potatoes and other vegetables should not exceed 250 mg NO3/kg. The tolerance for various pesticide residues ranges in different foods from about 0.01 to 20 mg/kg. The content of benzo[a]pyrene (BaP), one of the recognized representatives of the carcinogenic PAHs, should be in smoked meat products no higher than 1 μg/kg; for meats treated with smoke preparations the upper limit of 0.03 μg/kg has been set by the EU. In Europe, the countries producing olive residual oil have established a maximum level of 2 μg/kg for each of the eight highly carcinogenic PAHs, but not above 5 μg/kg

Food Components and Quality

11

for the total amount of all eight compounds. In smoked meat and fshery products, in baby foods, and in food oils, according to the regulation of the EC, the maximum permissible level of BaP is 5 μg/kg, 1 μg/kg, and 2 μg/kg wet weight, respectively. The detection limit of acrylamide in foods is actually about 10 μg/kg wet weight. In selecting the most appropriate analytical procedure suitable for the detection or determination of a compound in a food sample, the properties of the matrix must be considered. This is especially important in the step of separation of the analyte from the food material, be it by digestion, membrane techniques, solvent extraction, supercritical fuid extraction, sorption, headspace technique, or steam distillation. By using procedures comprising extraction of hydrocarbons from the food matrix, clean-up, separation by GC or HPLC, followed by detection and quantifcation by mass spectrometry or in fuorescence detectors, it is possible to determine the individual carcinogenic PAHs at concentrations of the order of 0.1 or even 0.01 μg/kg wet weight. The accuracy of the results depends signifcantly on the quality of standards used for calibration. Certifed reference materials are now available containing up to 15 PAHs in food samples. For quantitative analysis, internal GC-MS calibration with stable isotopes added prior to extraction and an MS detector in selected ion mode may also be used. In studies and routine monitoring regarding nutritional requirements and food safety aspects, many toxic elements are determined in trace concentrations of 0.01–10 mg/kg or even in ultra-trace amounts of below 10 μg/kg by using mainly spectrometric techniques. The lowest dose inducing symptoms of allergy in highly sensitive persons is about 0.1 mg of peanut or egg protein. This means, that the applied chemical examination must guarantee the detection of a few μg of peanut material in one gram of food.

1.5 CONCLUSION The contemporary market offers consumers in various parts of the world a very large variety of foods, obtained, handled, and processed by different methods. The quality of the products depends upon the characteristics of the raw material and on the after-harvest treatment at home and in the industry. Traditional and science-based methods as well as reliable control systems of production make it possible to supply the population with safe food of high sensory quality. Food chemistry is of crucial importance for realizing this goal. The interested reader can fnd information on the best available books on food chemistry on the Internet.

2

Chemical Composition and Structure of Foods Jolanta Tomaszewska-Gras

CONTENTS 2.1

Meat ................................................................................................................ 14 2.1.1 Defnition of Meat............................................................................... 14 2.1.2 Structure of Meat................................................................................ 14 2.1.3 The Chemical Composition of Meat................................................... 17 2.2 Eggs ................................................................................................................ 19 2.2.1 Foreword ............................................................................................. 19 2.2.2 Egg Structure ...................................................................................... 19 2.2.3 Chemical Composition of a Hen’s Egg............................................... 21 2.3 Milk ................................................................................................................ 23 2.3.1 Defnition ............................................................................................ 23 2.3.2 Chemical Composition of Milk .......................................................... 23 2.4 Cereals ............................................................................................................25 2.4.1 Foreword .............................................................................................25 2.4.2 The Structure of the Grain .................................................................26 2.4.3 Chemical Composition of Cereals......................................................26 2.5 Legumes..........................................................................................................28 2.6. Fruits............................................................................................................... 30 2.6.1 Foreword ............................................................................................. 30 2.6.2 Structure of Fruits............................................................................... 30 2.6.3 The Chemical Composition of Fruits ................................................. 31 2.7 Vegetables ....................................................................................................... 32 2.7.1 Defnitions........................................................................................... 32 2.7.2 Chemical Composition of Vegetable .................................................. 33 2.7.3 Potato .................................................................................................. 33 2.8 Oil Seeds and Fruits ....................................................................................... 35 2.9 Honey.............................................................................................................. 36 References................................................................................................................ 36

DOI: 10.1201/9781003265955-2

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2.1 2.1.1

MEAT DEFINITION OF MEAT

Defnitions of the term “meat” can differ depending on the intention and purpose for which it is used. In the context of food legislation, for instance, the term “meat” is defned in Regulation (EC) No. 853/2004, as edible parts of domestic cattle (beef), pigs (pork), sheep (mutton), goats (goat), farmed birds (poultry), lagomorphs (rabbit), and wildlife such as deer, rabbits, and fsh. This defnition applies not only to the muscular system but also to other edible parts, e.g. internal organs (offal), as well as bones. However, from the food science point of view, meat most often refers to skeletal muscle with adjacent connective tissue and associated fat derived from slaughtered animals of various mammalian species (pigs, cattle, sheep, goats, etc.), poultry, and fsh, but also of seafood or insects. In the broadest sense, meat is the edible post-mortem component originating from domesticated live animals, as well as wildlife. In colloquial language, this term means skeletal muscle tissue containing more-or-less adhering fat and connective tissue. Skeletal muscle is skeletal striated tissue, which is one of three types of muscle tissue (cardiac muscle and smooth tissue).

2.1.2 STRUCTURE OF MEAT At the macroscopic level, meat consists of muscle tissue, which is the contractile part, and connective tissue, as an elastic part building the tendons and membranes. Tendons hold the muscle to the bone and the membranes keep the muscle fbers bundled together. From the outside, the muscle is covered by the thick and tough membrane called the epimysium, while inside the muscle there are bundles of muscle fbers (multiple fascicles) surrounded by the perimysium, with about 50 muscle fbers in each bundle (Figure 2.1). A single muscle fber is a polynuclear, cylindrical cell, surrounded on the outside by a connective tissue membrane called the endomysium, and on the inside by a cell membrane (sarcolemma). The muscle cell, 10–100 µm in diameter and 1–40 mm long, contains all cellular organelles, such as the nuclei, the Golgi apparatus, the mitochondria, and the sarcoplasmic reticulum. However, the most characteristic components of a muscle cell, necessary for motor activities, are myofbrils, about 1 µm in diameter, composed of actin and myosin flaments called myoflaments. These are repeated in units called sarcomeres, which are the basic functional, contractile units of the muscle fber necessary for muscle contraction. Each myofbril consists of: • thick myoflaments composed of myosin, • thin actin myoflaments, • protein structures of the M and Z lines and cytoskeleton (Figure 2.2). The segment of myofbrils bound on both sides by the Z line constitutes the sarcomere, the basic structural unit of the myofbrils. The specifc arrangement of structures such as myoflaments (along the fber) and the Z line (across the fber) make

Chemical Composition and Structure

FIGURE 2.1

15

Meat structural organization from whole muscle to subcellular myofbrils.

the cross-sectional image visible in the longitudinal section under the microscope, which is caused by differences in refraction. The darker bands of the A (anisotropic) zone, composed mainly of myosin myoflaments with overlapping actin myoflaments, refract light twice, while the lighter bands of zone I (isotropic), built only of actin myoflaments with the Z line visible in the center, refract light ones. Regulatory proteins tropomyosin and troponin are bound to the actin chain. The most abundant myofbrillar protein is myosin, which forms the thick flaments (Figure 2.1) and makes up about 50% of the total contractile proteins. The myosin with a molecular weight (MW) of about 500 kDa is composed of two heavy polypeptide chains and four light polypeptide chains. Contraction of the muscle fbers is possible due to the interaction of myosin with actin by the activity of ATPase, located in the myosin head. The second major myofbrillar protein is actin, a constituent of the thin flament, which can exist in two forms, G-actin, being a small globular molecule (monomer) of about 42 kDa MW, and F-actin, in which the G-actin beads are aggregated forming a double-stranded helix. In addition to myoflaments, the fber also has a system of structures called the cytoskeleton, which maintains cell integrity, connects the organelles of the cell, and binds them to the sarcolemma. Based on their

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Jolanta Tomaszewska-Gras

FIGURE 2.2 Structural arrangement of the muscle cell.

location, the cytoskeleton structures can be divided by whether they are internal or external to myofbrils. Titin and nebulin are among the cytoskeletal proteins forming the longitudinal skeleton inside the muscle cell, mainly for the contractile proteins of myosin and actin. As the largest known protein, titin has about 1 µm in length and a molecular weight of 2,800–3,000 kDa. Intermediate flaments, composed of desmin, synemin, and vimentin, among other proteins, are the transverse skeleton on the outside of the myofbrils that runs crosswise to the muscle fber to integrate and link adjacent myofbrils at the Z line level. Another group of cytoskeletal proteins found outside myofbrils is the submembrane proteins that make up structures called costameres. Among them are vinculin, dystrophin, ankyrin, talin, and spectrin. They connect the Z lines of the peripheral sarcomeres with the sarcolemma, i.e. they form a connection between the cell wall and the entire internal system of interconnected myofbrils, participating in the transmission of nerve impulses. In general, skeletal muscle can be divided into two types of muscle fbers: red, slow-twitch oxidative (SO) and white, fast-twitch glycolytic (FG). Fibers differ in composition, structure, metabolism, contractile protein isoforms (e.g. myosin heavy and light chain), regulatory proteins, muscle contraction rate, and the content of the sarcoplasmic reticulum, mitochondria, and oxidative phosphorylation enzymes and glycolytic enzymes and their substrates (Table 2.1). Muscle fbers can be further divided into three subtypes, depending on the activity of myosin ATPase, the level of metabolic enzymes, and myosin isoforms: I, IIA, and IIB. Type I (βR), also referred to as SO, are red fbers, the thinnest, slowcontracting muscles, with aerobic metabolism, and therefore containing the most myoglobin and mitochondria. The second, opposing type is IIB (αW), also referred to as FG, white fbers of the largest diameter, rapidly contracting, with dominant

17

Chemical Composition and Structure

TABLE 2.1 Comparison of the Properties of White and Red Muscle Fibers Feature of the muscle

White fbers IIB, αW (FG)

Red fbers I, βR (SO)

Fiber diameter

Higher

Lower

Glycogen content Myoglobin content Content of connective tissue and intramuscular fat Sarcoplasm content Number of myofbrils per unit Number of mitochondria Activity of oxidative enzymes Activity of glycolytic enzymes Activity of myosin ATPase enzymes The rate of contraction Ca2+ uptake by the sarcoplasmic reticulum Time of active contraction state Intensity of rigor mortis Duration of rigor mortis

Higher Lower Lower Lower Higher Lower Lower Higher Higher Higher Faster Longer Lower Lower

Lower Higher Higher Higher Higher Higher Lower Lower Lower Slower Shorter Higher Higher

Metabolism

Anaerobic

Aerobic

glycolytic metabolism. There is also an intermediate type IIA (αR), referred to as FO (fast-twitch oxidative), classifed as a fast-twitch red fber with oxidative-glycolytic metabolism. White fbers contain more glycogen, have a higher activity of ATPases, phosphorylases, and glycolytic enzymes, mainly with anaerobic metabolism, and contract faster and more vigorously. In turn, red fbers, mainly those involved in oxygen metabolism, contract more slowly, and their effciency depends on the rate of supply of oxygen and substrates for energy transformations. In addition, they show a high activity of oxidative enzymes and have a greater number of mitochondria. Most skeletal muscles are heterogeneous and contain all types of fbers in varying proportions, although the pectoral muscle of chickens is an example of a muscle that contains only white fbers of anaerobic metabolism, rapidly contracting.

2.1.3 THE CHEMICAL COMPOSITION OF MEAT Lean meat contains an average of 70–75% water, 19–21% protein, 1–5% fat, 0.8– 1.8% mineral components, and 0.4–1.2% saccharides. The content of saccharides decreases after slaughter as they undergo glycolytic changes. The chemical composition of meat is determined to the greatest extent by the genotype (species, utility type, breed, breeding line), age, body weight, feeding method, and the conditions of rearing, as well as the location of the muscles in the animals’ organism. The variation in meat composition is mainly related to the fat content, which can range between 0.7% for chicken breast and 29% for beef brisket, as shown in Table 2.2.

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TABLE 2.2 Fat and Cholesterol Content in the Meat of Animals of Different Species Type of meat Poultry chicken

Poultry goose Poultry duck Poultry turkey

Pork

Beef

Lamb

Part of carcass

Fat (g/100 g)

Cholesterol (mg/100 g)

Breast without skin

0.7

43.4

Breast with skin Leg without skin Leg with skin Breast without skin Breast with skin Breast without skin Breast with skin Breast Thigh Separable wing meat Tenderloin Belly Loin Ham Chuck steak Spare ribs Blade Striploin Chuck Brisket

6.2 6.45 15.1 5.9 28.2 6.7 16.1 1.5 2.4–3.8 0.9 3.3–7.4 10.3–35.1 1.1–7.1 1.6 11.9 5.1–17.6 2.9 11.3 6.8 14

61.4 84.0 84.6 80.7 76.8 87.3 81.7 53 37–62 46 45–91 70–120 31–62 51.3 62.2 46–102 56.7 46.7 55 52

Longissimus muscle

3.8–6.9

60–70

Source: Honikel and Arneth, 1996; Dinh et al., 2011.

Particularly in the case of fsh, there is a large variation in the amount of fat, depending on the species, which is the basis for their classifcation as fsh: • • • •

lean, containing less than 2% fat (pike, hake, pollock, halibut, cod), medium fat, 2–7% (carp, redfsh, catfsh, tuna, trout), fatty, 7–15% (salmon, sprat), full-fat, over 15% (herring, mackerel, sardine, eel).

On the other hand, within the same species, differences in the fat content can be observed between different breeds or genetic lines; for example, in pigs, the differences between breeds in the average fat content are approx. 2.5%, while in the case of ducks it is approx. 14%. In addition to the species of animals and the type of culinary element of the meat, gender has a signifcant impact on the chemical composition. In general, females have more fat than males, since males are more muscular. Likewise,

Chemical Composition and Structure

19

age and body weight have an infuence on fat content. Adipose tissue begins to grow more intensively after the animals reach their maximum muscle mass. Older animals tend to have higher fat and lower water content. The presence of fat is related to the occurrence of another component characteristic of animal tissue, which is cholesterol. Its content in the muscle tissue of various animal species may range from 43 to 87 mg in 100 g of tissue, as shown in Table 2.2. Cholesterol is present not only in the meat of mammals and birds, but also in the meat of fsh. For example, cod contains about 57 mg of it, herring 65 mg, and mackerel 72 mg per 100 g of muscle tissue. The cholesterol content of offal is even higher, for instance, in 100 g of poultry liver, it is 350–700 mg, and in 100 g of pig or bovine liver 200–400 mg. Meat is also a valuable source of mineral compounds and B vitamins. The heme pigments present in the muscle tissue, i.e. myoglobin and hemoglobin, contain iron ions with high bioavailability. Bovine meat and breast muscles of ducks and geese contain the most heme pigments, 4–4.5 mg per 1 g, while the breast muscles of chickens or turkeys contain 0.2–0.6 mg per 1 g. Among the B group vitamins, 100 g of meat contains approx. 0.7–0.85 mg of thiamine, 0.1–0.3 mg of ribofavin, 5–7 mg of niacin, and 0.7 mg of pantothenic acid.

2.2

EGGS

2.2.1 FOREWORD Eggs are edible products whose trade name refers to hen’s eggs, in accordance with Reg. EC No. 589/2008, although the eggs of other species are also sold. However, those of other birds (geese, ducks, plovers, seagulls, quail) are of lesser signifcance, thus the term “egg,” without a prefx, generally relates to hen’s eggs. The egg contains all the nutrients necessary for the embryo’s growth, without the need to access external sources of food. Due to the richness of highly digestible nutrients, it is a valuable food product for humans. The proteins present in eggs contain all the essential amino acids.

2.2.2 EGG STRUCTURE The whole egg is composed of an eggshell (approx. 10%), egg white (albumen) (57%), and egg yolk (33%). The weight of a hen egg varies considerably within the same species; it can range from 53 to 73 grams, which is the basis for qualitative classifcation (Reg. EC No. 589/2008). There is even greater variation between different bird species, from 12 grams for a quail egg to 1.5–2 kg for an ostrich egg. In the macroscopic structure of an egg (Figure 2.3), the following structures can be distinguished from the outside: • the shell with the membrane, • the egg white (albumen) with chalazae, • the yolk with embryonic disc (blastoderm). The eggshell, 0.2–0.4 mm thick, consisting of two layers, spongy (outer) and mammillary (inner), makes up 10–12% of the weight of the egg. It is composed mainly

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Jolanta Tomaszewska-Gras

FIGURE 2.3

Macrostructure of the egg.

of mineral substances (approx. 95%), mostly calcium carbonate. On the outside, it is covered with a thin flm, called the cuticle or bloom. The shell protects the contents of the egg from damage and contamination and allows gas exchange due to the pores it contains. Directly under the shell, there is the shell membrane, composed of two layers (48 and 22 μm, respectively), each of which is an interwoven network of proteins and polysaccharides fbers. As the egg is laid, an air cell is formed at the large end of the egg, approx. 5 mm in diameter and increases in size during storage, hence it can be used to determine the age of eggs. In the shell, funnel-shaped minute pores can be seen (7,000–17,000 per egg). They are partially sealed by the proteins of the cuticle but remain permeable to gases while restricting penetration by microorganisms. The diameter of the pore canals ranges from 10 to 30 µm. The egg albumen (egg white), constituting about 57% of the egg mass, protects the embryo against physical damage, but also against microbial infections. It contains approx. 88% water and consists of four layers: • • • •

external thin albumen, thick albumen, internal thin albumen, chalaziferous layer with chalazae.

These layers represent 23%, 57%, 17%, and 3% of the total protein weight, respectively. The viscosity of thick albumen, much higher than that of thin albumen, is caused by the four times higher content of ovomucin. In the long axis of the egg, the chalaziferous layer, mainly composed of mucin fbers, is twisted at both sides of the yolk, forming a thick rope-like structure named chalaza, which holds the yolk in the center. It is twisted clockwise at the small end of the egg and counterclockwise at the large end. The yolk, comprising 32% of the weight of the egg, in fact, is one of the biggest ova. It is surrounded by a vitelline membrane and contains genetic material in the form of the germinal disc and the nutrient material for the growing

21

Chemical Composition and Structure

embryo. Structurally, the inner content is composed of yellow yolk and white yolk, whose weight is less than 2% of the total egg yolk. The yellow yolk consists of alternate light layers and deep yellow layers. In the center of the egg yolk, the latebra is located, which is the basic nutritional material for the embryo in the frst days of life. It is connected by a tubule (neck of the latebra) to the embryonic disc in the nucleus of the pander on the surface of the yolk (2–3 mm in diameter).

2.2.3 CHEMICAL COMPOSITION OF A HEN’S EGG In terms of chemical composition, the egg contains the most water, approx. 73.5% (Table 2.3), while the content of saccharides and minerals is the lowest – approx. 1%. Proteins are present in both the albumen and yolk. Egg albumen contains ovalbumin, ovotransferrin, ovomucoid, ovomucin, lysozyme, and avidin, which mainly have an enzymatic and antibacterial function, while the yolk contains mainly complex proteins. Ovalbumin is the main albumen protein, consisting of 54% of total egg white proteins. Several albumen proteins have biological activity as enzymes (e.g. lysozyme), enzyme inhibitors (e.g., ovomucoid, ovoinhibitor) and complexforming agents for some coenzymes (e.g., favoprotein, avidin). Egg yolk is an oilin-water emulsion with about 50% dry weight. The main proteins of egg yolk are LDL-lipoproteins (68%), HDL-lipoproteins (16%), livetins (10%), and phosvitins (4%). Egg yolk can be separated by centrifugation into plasma (90% of yolk lipids and 50% of yolk proteins) and granules (7% of yolk lipids and 50% of yolk proteins). Plasma, which is 71–81% of the yolk dry weight, is composed of LDL (85%) and glycoproteins: α, β, and γ livetins (15%). The granules, constituting 19–23% of the yolk’s dry weight, are composed of the HDL: α- and β-lipovitellins (70%), phosvitin (16%), and LDL (12%). Lipids, on the other hand, are found only in the yolk and account for about 64% of the yolk’s dry weight. They include mainly triacylglycerols (TAGs), which constitute 65% of all lipids, phospholipids with a share of 31%, and sterols with 3%. TAGs contain the biggest amount of the following fatty acids: • oleic, about 42%, • palmitic, about 30%,

TABLE 2.3 Chemical Composition of Hen Egg (%) Whole egg (with shell)

Whole egg (without shell)

Egg yolk

Egg white (albumen)

Eggshell

Water

66

73

48

88

1.6

Proteins Lipids Saccharides

12 10 1.0

13 12 1.0

17 33 1.0

11 0.01 0.9

3.3 – –

Mineral compounds

11

1

1.0

0.6

95

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• linoleic, about 10%, • stearic and arachidonic, about 3%. In the total composition of all fatty acids, monounsaturated ones constitute approx. 42%, and polyunsaturated fatty acids approx. 12%. The ratio of n-6 to n-3 acids is 10:1. Among the phospholipids, lecithins (phosphatidylcholine) and cephalins (phosphatidylethanolamine) are the most abundant in the yolk (Table 2.4). The yolk also contains cholesterol, the content of which per 100 g of egg is approximately 370 mg, while a medium-sized hen’s egg contains approximately 200–215 mg of cholesterol. Carotenoids, the natural pigments of hen egg yolk, are mainly composed of carotene and xanthophylls (lutein, cryptoxanthin, zeaxanthin), which are present in the yolk lipids in an amount of less than 1%. During storage, a series of chemical and structural changes occurs in eggs, including a decrease in the acidity of the egg white and yolk. This is caused by the diffusion of CO2 through the pores of the shell, which starts soon after the egg is laid, especially in the egg white. The pH of the albumen varies from 7.6 to 9.7, while the pH of the yolk varies from 6.0 to 6.8. The chalazae are weakened and the density of the thick albumen drops, which causes the yolk to deviate from the central position. The air cell expands due to the evaporation of water through the shell pores. There is also diffusion of water from the egg white into the yolk, the volume of which increases. The yolk also becomes less resilient due to the weakening of the vitelline membrane, which is seen on the level surface after the egg is cracked as a fattening. This is measured as a yolk index for determining the age of an egg, expressed as the ratio of yolk height to diameter. Considering chemical changes in proteins of egg white, during storage the more heat-stable S-ovalbumin (temperature of denaturation 92.5° C) is formed from the native protein (temperature of denaturation 84.5° C), causing an increase in S-ovalbumin from 5% in fresh eggs to 81% in eggs cold-stored for six months. The whole egg weight loss during storage ranges from 3.0 to 6.5%.

TABLE 2.4 Phospholipids Composition in Egg Yolk as Percent of Phospholipid Fraction (%) Phospholipids

%

Phosphatidylcholine Phosphatidyl ethanolamine Lysophosphatidylcholine Sphingomyelin Lysophosphatidylethanolamine Plasmalogen

73 15.5 5.8 2.5 2.1 0.9

Phosphatidyl inositol

0.6

Source: Sugino, Nitoda, and Juneja, 1997

Chemical Composition and Structure

2.3

23

MILK

2.3.1 DEFINITION Long before recorded history, the frst food provided for humankind was from the woman’s mammary glands. In distant times, when it was not possible for the newborn child to suckle from the mother, and people found that milk from other animals was good, they began domesticating milk-producing animals. Milk is the secretion of the mammary gland of female mammals that appears during the lactation period. The defnition included in Regulation (EC) No. 853/2004 defnes raw milk as that obtained from the mammary glands of farmed animals which has not been heated to above 40° C or any other treatment having an equivalent effect. For humans, the most important milk is from cows, goats, sheep, and buffalo, but the milk of the donkey, reindeer, camel, buffalo, musk ox, and alpaca can also be used for consumption, although these milks are of lesser signifcance. Thus, the term “milk,” without a prefx, generally relates to cow’s milk. In terms of composition and structure, milk is an oil-in-water emulsion consisting of a three-phase system: • continuous, which is an aqueous solution of lactose, mineral salts, and water-soluble proteins, • casein micelles, • fat globules, which are stabilized by phospholipids and proteins.

2.3.2 CHEMICAL COMPOSITION OF MILK In the chemical composition of milk, as shown in Table 2.5, the biggest component is water (from 82 to 88%), then protein and fat in relatively similar proportions in cow’s and goat’s milk (from 3.5 to 4.0%), while in sheep’s milk the fat content is approx. 6.5% and the protein content approx. 6%. The composition of milk is determined to the greatest extent by the species of mammals, and to a lesser extent by the lactation period, feed composition, and the health condition of the udder. Cow’s milk proteins can be divided into three groups: caseins, whey proteins, and blood proteins (Table 2.6). Caseins, belonging to phosphoproteins, are the main proteins in milk (78–82% of the total amount of milk protein). They play a very important role in cheese-making due to their ability to form a curd. Proteins, belonging to the group of caseins: calcium-sensitive (α S1-casein, α S2-casein, and β-casein) and calcium-insensitive (κ-casein and γ-casein) exist in milk in a unique, highly hydrated spherical complex with calcium phosphate, named casein micelle. These micelles have a broad range of diameter from 30 to 600 nm, which are on average ten times smaller than fat globules. The next group of proteins, known as whey proteins, consists of β-lactoglobulin and α-lactalbumin, which are, in contrast, soluble in the nonmicellar, aqueous phase in monomeric and dimeric forms. These two groups of proteins are separated by agglomeration of casein micelles during the forming of the curd, for instance, by enzyme treatment in cheese production. In addition, there are also blood proteins (serum albumin and immunoglobulins [Igs]) in milk, which, in

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TABLE 2.5 Chemical Composition of Mammalian Milk of Various Species (%) Water

Protein

Lipid

Lactose

Ash

Alpaca

83.7

5.8

3.2

5.1

1.6

Buffalo Llama Moose Donkey Man Horse Cow Goat Donkey Sheep Reindeer Dromedary camel Bactrian camel

83.2 84.8 76.8 82.6 87.8 89.8 88,1 87.7 90.8 80.7 67.9 89.0 84.8

4.0 4.1 10.5 5.2 1.0 2.0 3.2 2.9 1.6 4.5 10.4 3.1 3.9

7.4 4.2 8.6 6.8 3.8 1.6 3.3 4.5 0.7 7.4 16.1 3.2 5.0

4.4 6.3 2.6 4.8 7.0 6.6 5.1 4.1 6.4 4.8 2.9 4.3 4.2

0.8 0.7 1.6 0.8 0.2 0.4 0.7 0.8 0.4 1.0 1.5 0.8 0.9

Musk ox

83.6

5.3

5.4

4.1

1.6

Source: Huppertz and Kelly, 2009; Medhammar et al., 2012.

TABLE 2.6 Composition of the Major Proteins of Cow’s Milk Proteins

Concentration (g/100 g)

Caseins

24.7–28.9

Isoionic point

Molecular weight (kDa)

αS1-casein αS2-casein β-casein κ-casein γ-casein Whey proteins β-lactoglobulin α-lactalbumin Proteose-peptones Blood proteins Serum albumin Immunoglobulins IgG1 IgG2 IgA IgM

12.4–15.5 3.1–4.1 9.3–11.3 3.1–4.1 1–2.1 5.2–7.2 2.1–4.1 1–1.5 0.6–1.9

4.92–5.35 5.20–5.85 5.77–6.07 5.8–6.0

23.6 25.2 24 19 12–21

5.35–5.41 4.2−4.5a 3.3–3.7

18.3 14.2 4–41

0.1–0.4 0.6–1.0

5.13

66.3

5.5–6.8 7.5–8.3

162 152 400b 950c

Notes: a: isoelectric point; b: dimer; c: pentamer. Source: Horne, 2017; Belitz, Grosch, and Schieberle, 2009.

25

Chemical Composition and Structure

TABLE 2.7 Lipid Composition of Cow’s Milk Cow’s milk lipid fractions

Percent of the total lipid (%)

Triacylglycerols Diacylglycerols Monoacylglycerols Keto acid glycerides Hydroxy acid glycerides Free fatty acids Phospholipids Sphingolipids Sterols (cholesterol, cholesterol esters)

95–96 1.3–1.6 0.02–0.04 0.9–1.3 0.6–0.8 0.1–0.4 0.8–1.0 0.06 0.2–0.4

Source: Belitz, Grosch, and Schieberle, 2009.

contrast to caseins and whey proteins, are not produced by the mammary glands, but are derived from the blood. The second signifcant component of milk is fat, which is used in the production of cream, butter, and anhydrous milk fat. Milk fat (milk lipids) consists of 98% TAG, concentrated in the form of globules of 2–6 µm in diameter. Minor components of fat globules are diacylglycerols, monoacylglycerols, free fatty acids, polar lipids, sterols, and fat-soluble vitamins (Table 2.7). The main fatty acids present in TAG are butyric, capric, lauric, myristic, palmitic, stearic, oleic, and linoleic. However, more than 400 different fatty acids have been detected in cow’s milk lipids. Milk fat is also a valuable source of conjugated linoleic – rumenic (CLA) and vaccenic acid (VA), which have a proven health-promoting effect. Lactose, with sweetness about one-ffth of that of sucrose, is the disaccharide characteristic only for milk. It is present in milk in α- and β-forms. The α-form can be responsible in dairy products like ice cream for a sandy mouthfeel. In the dairy manufacturing industry, β-galactosidase is commonly used to hydrolyze lactose into glucose and galactose to produce lactose-free dairy products.

2.4 CEREALS 2.4.1 FOREWORD Cereals and cereal products are amongst the most important basic foods for humans. The frst cereal grown systematically was probably barley, which was known as early as 5000 BC in Egypt and Babylon. Cereals are plants from the Poaceae family that are of great importance as raw materials for the production of four, groats, beer, and alcohol, as well as for animal feed, because of their high starch content. The raw materials for milling and groats processing are common wheat, also known as bread wheat (Triticum vulgare), durum wheat (Triticum durum), spelt (Triticum spelta L.),

26

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rye (Secale cereale), triticale, barley (Hordeum vulgare), and oats (Avena sativa L.), as well as maize (Zea mays L.), rice (Oryza sativa L.), buckwheat (Fagopyrum esculentum Moench), sorghum (Sorghum Moench), and millet (Panicum miliaceum). Botanically, buckwheat is not a cereal, but because of its widespread use in cereal products, it will be described in this chapter. Commercially, the most important cereals are wheat, maize, and rice. Durum semolina is generally the best type of wheat for the production of pasta.

2.4.2 THE STRUCTURE OF THE GRAIN Cereals form a relatively large fruit, termed a caryopsis, in which the pericarp and seed are adherents. The shape of grains varies from elongated (rye) to spherical (millet), but in structure they are similar. Cereal grain consists of the germ, endosperm, and seed coat, also known as bran, and consists of three major parts: • the germ (including the scutellum), which produces the new plant, • the endosperm, rich in starch, which serves as food for the germinating seed and is the raw material of four manufacture, • bran (husk, coat) with various covering layers protecting the grain (Figure 2.4). The average wheat grain composition is approx. 85% endosperm, 13% bran, and 2% germ. The bran is composed of fve layers, which protect the endosperm and embryo against damage and drying out. The germ is located at the base of the grain, and it contains the embryo, which is rich in lipids (28%), proteins (34%), and vitamins (6%). The membranous tissue called the scutellum separates the germ from the endosperm. The most important part from the technological point of view is the endosperm, which contains mainly starch and proteins. These serve as food during germination. The endosperm consists of two elements: the aleurone layer, which lies directly under the seed coat, and the endosperm cells with starch granules, the main part of the endosperm. The aleurone layer makes up about 7% of the grain weight and consists of proteins, fat, vitamins, and enzymes. During milling, this layer passes along with the coat to the bran. The main component of the endosperm is starch, which comprises 80% of the total compounds in this structure, and 13% of proteins. The polysaccharide molecules in starch granules are organized radially and vary in size and form between different cereals.

2.4.3 CHEMICAL COMPOSITION OF CEREALS The major saccharide storage form of cereals is starch, which occurs only in the endosperm cells (Table 2.8). Due to the presence of alternating amorphous (mainly amylose) and semicrystalline layers (amylopectin), differences in the refraction index can be observed. Cereal starches consist of about 25% amylose and 75% amylopectin, except waxy corn, which contains only amylopectin. Starch granules swell when heated in a water suspension and by gelatinizing, they lose their form.

27

Chemical Composition and Structure

FIGURE 2.4 Structure of a wheat grain caryopsis.

TABLE 2.8 Chemical Composition of Cereals (%) Moisture Lipids Saccharides Fiber Proteins (N × 6.25) Albumins Globulins Prolamins Glutelins

Wheat

Rye

Corn

Barley

Oats

Rice

Millet

13.2

13.7

12.5

11.7

13.0

13.1

12.1

2.2 1.7 3.8 2.1 7.1 59.6 60.7 64.2 63.3 55.7 13.3 13.2 9.7 9.8 9.7 11.7 9.5 9.2 10.6 12.6 Protein distribution (%) in Osborne fraction 14.7 44.4 4.0 12.1 20.2 7.0 10.2 2.8 8.4 11.9 32.6 20.9 47.9 25.0 14.0

2.4a 74.1 2.2 7.4

4.05 68.8 3.8 10.6

10.8 9.7 2.2

18.2 6.1 33.9

77.3

41.8

45.7

24.5

45.3

Note: a: polished rice: 0.8%. Sourc: Belitz, Grosch, and Schieberle, 2009.

54.5

53.9

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TABLE 2.9 Chemical Composition of Different Types of Wheat Flour 405

550

812

1,050

1,700

Starch

Type of wheat four

82

82

78

78

69

Proteins (N × 5.8) Lipids Dietary fber

12 1.0 5

12 1.2 5.0

13 1.5 6

13 2 6

13 2.3 13

Ash

0.4

0.6

0.8

1

2

The other grain saccharides are glucose, fructose, and sucrose, present mainly in the embryo, while in the coat, there are also cellulose and hemicellulose, which are the main components of the dietary fber in the bran (78%). There are also proteins in the grain, which, despite their low content (7.4–12.6%), play an important role in the process of making the dough. The main proteins of cereal grains are proteins, classifed by Thomas Burr Osborne according to different solubilities: • albumins can be removed with water and also remain in solution at the isoelectric point, • globulins can be extracted with saline solution, • prolamines can be extracted with 70% ethanol, • glutelins are not extractable; they remain in the residue. Osborne fractions derived from different cereals are often designated by special names. For instance, glutelins and prolamines of wheat are called glutenin and gliadin while in corn they are zeanin and zein, in rice they are oryzenin and orizin, and in barley they are hordenin and hordein. Wheat gluten proteins fractionated by the Osborne method provide glutenins and ω-, α-, γ-gliadins in a ratio of 1:1. Both fractions, in their hydrated form, have different effects on the rheological characteristics of dough: prolamins are responsible, preferentially, for viscosity, and glutelins for dough strength and elasticity. The basic product produced from the wheat grains in the milling process is four, for which different types are produced, depending on the composition (Table 2.9).

2.5 LEGUMES Legumes, also known as pulses, the common feature of which is the fruit in the form of pods, are annual plants from the family of Fabaceae Lindl. The ripe seeds of the pods are primarily an important source of proteins. The common pulses used as food are broad beans (Vicia faba L.), chickpeas (Cicer arietinum L.), beans (Phaseolus L.), peas (Pisum L.), lupins (Lupinus L.), peanuts (Arachis hypogaea L.), lentils (Lens culinaris L.), and soybeans (Glycine max). Beans are eaten not only in the form of seeds but also in the form of pods such as green beans (string beans). There

29

Chemical Composition and Structure

are many varieties of beans, such as the garden bean, black bean, red bean, borlotti bean, pinto bean, fageolet bean, mung bean, adzuki bean, rice bean, kidney bean, lima bean, and jack bean. Legume seeds are relatively high in protein and saccharides, and some also contain fat. Broad beans and soybeans are distinguished by their high protein content, 38% and 44%, respectively (Table 2.10). Due to its amino acid composition most similar to meat, soybean protein is used in the production of protein concentrates and isolates as the main component of vegetarian dishes. Moreover, soybeans are characterized by a high fat content, approx. 20%, which also makes them a raw material for the fat industry. Even more fat is found in peanuts, as much as 50%. Peanuts are also a good source of niacin, as they contain about fve times more (14.2 mg/100 g) than the seeds of other legumes. Peanut seeds ripen in the ground to a depth of about 5–8 cm, where the pod is pushed through the peduncle after the fower fades. Peanuts are the raw material for the production of peanut butter and peanut oil. Generally, in all legumes, three types of proteins are predominant: albumins, globulins, and glutelins. With regard to biological value, legume proteins are somewhat defcient in S-containing amino acids. Glycinin (350 kDa MW) and β-conglycinin (156 kDa MW) are two of the most important allergenic proteins in soybean with medium thermal stability. In peanuts, two glycoproteins are predominant allergens: Ara h1 (65 kDa MW, 7S globulin, vicilin) with high thermal stability and Ara h2 (17 kDa MW) with medium thermal stability. The major carbohydrate in legumes is starch, amounting to 75–80%. Oligosaccharides in legumes are present in higher concentrations than in cereals, with sucrose, stachyose, and verbascose being predominant. After legume consumption, oligosaccharides might cause fatulence, a symptom of gas accumulation in the stomach or intestines as a result of the growth of anaerobic microorganisms in the intestines, which hydrolyze the oligo- into monosaccharides and cause their further degradation to CO2, CH4, and H2. In terms of nutritional value, legume seeds contain ingredients with high nutritional value, such as proteins, fat, niacin, isofavones (phytoestrogens), as well as anti-nutritional components such as trypsin inhibitors, phenolic compounds, phytates, cyanogenic compounds, lectins, saponins, as well as allergenic proteins, most of which are inactivate after heat treatment and can be substantially reduced by milling, cooking, germination, and fermentation. TABLE 2.10 Chemical Composition of Various Legumes Type of legume

Protein

Lipid

Saccharide

Ash

Broad bean

38.0

1.7

52.0

3.7

White beans String bean Pea Peanut Lentils

21.0 22.0 30.0 30.0 30.0

1.6 2.0 1.4 50.0 3.0

62.0 72.0 67.0 14.0 62.0

1.9 1.7 1.5 3.0 2.5

Soy

44.0

19.6

33.0

3.4

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2.6. FRUITS 2.6.1

FOREWORD

In botanical terms, fruits are organs of angiosperms formed from the ovary of the pistil, or the fower bottom. However, in the culinary sense, this defnition is narrowed down to the edible parts of trees or shrubs, which no longer includes the fruits of herbaceous plants such as cucumbers or tomatoes. In turn, the exception to this defnition is strawberries, which are the fruit of perennials. Hence, all fruits can be divided into fruits of fruit trees from the temperate zone (pear, apricot, apple, cherry, plum, peach, hazelnut, walnut), fruits of fruit trees from the subtropical and tropical zones (lemon, fg, pomegranate, grapefruit, orange, mandarin, mango, kiwi, date, coconut, avocado), fruit bushes (gooseberry, chokeberry, blueberry, lingonberry, bilberry, blackberry, raspberry, blackcurrant, red currant, white currant, grape, cranberry), and fruits of perennials (wild strawberry, strawberry). Due to their structure, they can be divided into fruits as follows: • pome (apples, pears, quinces), which are formed from the ovary of the pistil and the bottom of the fower, • pitted (cherries, peaches, plums), which are formed exclusively from the ovary of the pistil, • berry (blueberries, gooseberries, chokeberries), characterized by a pericarp with numerous seeds. In terms of nutrition, fruit is one of the most valuable sources of natural antioxidants, such as ascorbic acid, carotenoids, and phenolic compounds.

2.6.2

STRUCTURE OF FRUITS

The anatomical structure of the fruit distinguishes seeds and the pericarp, which includes the exocarp, mesocarp, endocarp, and conductive bundles (Figure 2.5). The outer part of the pericarp is called the exocarp, which is the epidermis of the fruit, i.e. the skin, which is usually the cuticle, which protects the fruit against water loss,

FIGURE 2.5

Illustration of anatomical structures of different types of fruits.

31

Chemical Composition and Structure

and sometimes it is additionally a hair (e.g. peaches). The middle part of the pericarp is the mesocarp, which is the pith tissue of the fruit, and the inner part is the endocarp, which in the case of stone fruit hardens and becomes woody, forming a stone with the seed inside, unlike the berry, where this part is completely feshy.

2.6.3

THE CHEMICAL COMPOSITION OF FRUITS

Water is the most abundant in fruit (up to 96%) and saccharides are also key components of the fruit, with content ranging from 7.2% in strawberries to 23.5% in bananas (Table 2.11). Glucose and fructose are the most abundant saccharides, which are stored in the parenchyma tissue. The fruit peel contains cellulose, hemicellulose, and pectins, which are components of dietary fber. Fruits are also a rich source of other nutrients, such as vitamins (vitamin C, carotenoids), phenolic compounds, organic acids (citric, malic), pigments (chlorophylls, carotenoids, anthocyanins), and TABLE 2.11 Chemical Composition of Fruits per 100 g of Edible Tissue Type of fruit Acerola

Protein (g)

Lipid (g)

Saccharide (g)

Vitamin C (mg)

Fiber (g)

0.4

0.3

6.8

1300

0.4

Apple Apricot Avocado Banana Blackberry Blackcurrant Blueberry Cherry, sweet Cherry, sour Gooseberry Grape Grapefruit Hazelnuts Kiwi Lemon Orange Pear Pineapple Plum Pomegranate Raspberry Strawberry

0.2 1.0 2.1 1.2 1.2 1.7 0.7 1.3 0.9 0.8 0.6 0.5 14.4 0.9 1.1 0.9 0.7 0.3 0.8 0.5 1.5 0.7

0.6 0.2 16.4 0.2 0.9 0.1 0.5 0.3 0.4 0.2 0.3 0.1 63.0 0.5 0.3 0.2 0.4 0.2 0.2 0.3 1.4 0.5

14.1 12.8 6.3 29.0 12.9 13.1 15.3 17.4 10.9 9.7 17.3 10.6 14.9 13.9 8.2 12.2 15.3 13.7 19.7 16.4 15.7 8.4

7 10 14 15 21 200 14 10 12 30 4.0 30 3.0 71 53 50 4.0 17.0 4.0 4.0 18 59

2.0 0.6 1.6 0.4 4.1 2.4 1.5 0.4 1.0 1.9 0.5 0.2 8.9 2.1 0.4 0.5 1.4 0.4 0.4 0.2 5.1 1.3

Walnut

16.0

60.3

18.0

5.8

6.5

Source: Ensminger et al., 1995.

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Jolanta Tomaszewska-Gras

aromatic compounds giving them a characteristic aroma. Carotenoids are widespread in fruits; 26 different carotenoids have been detected and are the main factor determining color in some fruits, such as citrus fruits, peaches, and sweet melons. In fruits, several hundred polyphenols (classifed into six groups according to the number of phenol rings: hydroxybenzoic acids, hydroxycinnamic acids, favonoids, chalcones, stilbenes, and lignans) have also been identifed, which are antioxidatively active and contribute to the color and taste of many types of fruit. A special kind of fruit is nuts, consisting of a hard nutshell protecting a kernel, which is usually edible. A wide variety of dry seeds are called nuts in general usage or in a culinary sense, but in the botanical meaning, “nut” implies that the shell does not open to release the seed, like in the case of walnuts, hazelnuts, or chestnuts. The storage time of fruits varies from four to eight months for apples, two to six months for pears, two to three months for grapes, and a few days for strawberries, raspberries, and cherries. Commonly used conditions are a temperature of about 2–4° C at 80–90% relative humidity. Some fruits, like apples, kiwis, and pears, have high ethylene production (the volatile promoter of fruit ripening), in contrast to cherries, grapes, and pineapples, which can be used to ripen the fruit by enclosing both types of fruit in a paper bag. Weight losses occur during fruit storage due to moisture losses of 3–10%.

2.7 2.7.1

VEGETABLES DEFINITIONS

Vegetables are a group of annual, biennial, and perennial plants, which are a rich source of dietary fber, vitamins, phenolic compounds, and glucosinolates (cruciferous vegetables). The whole group of vegetables can be divided into: • alliums (onion, garlic, leek), • cucurbits (pumpkin, cucumber), • brassicas (caulifower, broccoli, kohlrabi, head cabbage, Chinese cabbage, savoy cabbage, Brussels sprout), • root (beetroot, carrot, parsley), • leafy (lettuce, spinach, lamb’s lettuce, arugula), • nightshades (aubergine, tomato, pepper, potato), • turnips (radish, turnip). The structure of root vegetables (carrots, beetroot) is characterized by vascular bundles in the form of wood (xylem) and phloem, through which water is delivered to other parts of the plant (Figure 2.6). In carrots, the vascular bundles are formed due to the activity of the creative pulp (cambium), which causes secondary root growth. The beetroot, on the other hand, is characterized by secondary growths, visible in the cross-section in the form of centrally located rings of the creative pulp, producing phloem and wood. The vascular bundles in the stalks of monocotyledons, e.g.

Chemical Composition and Structure

FIGURE 2.6

33

Illustration of anatomical structures of different types of vegetables.

asparagus, for example, are arranged differently, where they are dispersed throughout the crumb.

2.7.2 CHEMICAL COMPOSITION OF VEGETABLE Vegetables are more diverse in terms of chemical composition than fruit (Table 2.12). In vegetables, water is the most abundant component, from 60% in garlic to 98% in asparagus. Protein is relatively low, with the exception of Brussels sprouts, which are 4.2% protein. The lipid content of vegetables is also negligible (less than 1%). On the other hand, saccharides are present in greater amounts, from 3.2% in cucumbers to 21.1% in potatoes, which contain the most starch of all saccharides. Like fruit, vegetables are a valuable source of potassium (broccoli, Brussels sprout, leaf parsley, potato – over 400 mg/100 g), calcium (leaf parsley, kale – 150–190 mg/100 g), magnesium (leaf parsley, spinach – 50–70 mg/100 g), phosphorus (garlic up to 150 mg/100 g), iron (spinach, parsley – 3–5 mg/100 g), vitamins, e.g. vitamin C (celery parsley, red pepper, kale over 100 mg/100 g), provitamin A (pumpkin, kale, carrot, red pepper, leaf parsley, spinach over 300 µg of β-carotene/100 g). Among the most valuable components of vegetables are also polysaccharides, comprising dietary fber.

2.7.3 POTATO Since the potato is the fourth largest crop in the world after maize, rice, and wheat, it is described separately from other vegetables. Potato (Solanum tuberosum) is the name of the tuber of a plant belonging to the nightshade family, which is very

34

Jolanta Tomaszewska-Gras

TABLE 2.12 Chemical Composition of Vegetables per 100 g of Edible Tissue Type of vegetable

Protein (g)

Lipid (g)

Saccharide (g)

Vitamin C (mg)

Fiber (g)

Asparagus

2.2

0.2

3.6

26

0.7

Beets Broccoli Brussels sprouts Cabbage Carrot Caulifower Cucumber Onion Red pepper Potato Spinach

1.1 3.1 4.2 1.1 1.2 2.7 0.6 1.5 1.2 2.6 3.2

0.1 0.3 0.4 0.2 0.2 0.2 0.1 0.2 0.2 0.1 0.3

7.2 4.5 6.4 4.3 9.7 5.2 3.2 8.2 4.8 21.1 4.3

6.0 90 87 33 – 78 11 32 128 20 51

0.8 1.5 1.6 0.8 1.0 1.0 0.3 1.2 1.4 0.3 0.6

Tomato

1.0

0.2

4.7

23

0.5

Source: Ensminger et al., 1995.

FIGURE 2.7

Structure of a potato.

widespread in Europe. Potato tubers are used as food and animal feed. The main components of the potato tuber structure are the periderm, pith (inner medulla), perimedulla, cortex, and vascular ring (Figure 2.7). New potato tubers are covered with an epidermis, i.e. a thin skin that is gradually exfoliated, and in its place, secondary tissue is formed, which is called the periderm. This has a thickness of 80 to 200 μm, which protects against mechanical damage and water loss, with many spiracles allowing gas exchange. Below this layer is the primary cortex, the outer part of which is rich in proteins, lipids, and pigments, while the inner part is rich in starch. Under the cortex there is a vascular ring, supplying nutrients from the shoot

Chemical Composition and Structure

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to the tuber and to the plants germinating from it. The bundles form a vascular ring surrounding the perimedulla, the outer part of which is the main starch storage location in the tuber, while the interior of the inner medulla (pith) is more watery and less starchy. The potato tuber mostly contains water, about 75%, and then starch. Its starch content depends on the variety and ranges from 10 to 30% and is a store for the plant. The potato contains a relatively high amount of vitamin C (15–50 mg in 100 g), and there are also potassium 415 mg/100 g, magnesium 24 mg/100 g, and a small quantity of B vitamins. Green or sprouted potato tubers can cause gastrointestinal disturbances due to the formation of solanine, which is a toxic glycoalkaloid.

2.8

OIL SEEDS AND FRUITS

Oil plants, the seeds of which are used to obtain oil, are primarily rapeseed (Brassica napus), sunfower (Helianthus annuus), palm oil kernel (Elaeis guineensis), faxseed (Linum usitatissimum), sesame (Sesamum indicum), cottonseed (Gossypium), cocoa (Theobroma cocao), and coconut (Cocos nucifera). The source of the oil can also be the pulp of fruit, e.g. olives (Olea europaea sativa) or oil palm (Elaeis guineensis). Oil is also produced from legumes like soybean (Glycine max) or peanut (Arachis hypogaea) or from cereals, for instance, from corn (Zea mays). In Europe, olive oil is classifed into eight categories according to Commission Regulation (EEC) No. 2568/91, i.e. extra virgin olive oil, virgin olive oil, Lampante olive oil, refned olive oil, olive oil composed of refned olive oil and virgin olive oils, crude olive-pomace oil, refned olive-pomace oil, and olive-pomace oil. Rapeseed, sunfower, and soybean oils are mostly produced and sold as refned oils since olive oil or faxseed oil are mainly sold as cold-pressed oils. In the past, rapeseed oil was characterized by a high content of erucic acid (20:1), which was hazardous in human nutrition. Nowadays new cultivars have been developed, called canola, “double zero” cultivars, with low levels of this fatty acid. Rapeseed oil is distinguished from other oils by a very favorable n-6 to n-3 fatty acid ratio of 2.2:1. However, the best ratio was noted for faxseed oil, as it is 1:3.5. Due to the high content of linolenic acid (18:3, n-3), i.e. 58%, the oil is very susceptible to peroxidation, a process involving polymerization reactions, making the oil solidify (“fast drying oil”). Therefore, faxseed oil is also used as a base for oil paints, varnishes, and linoleum manufacturing. Recently, in order to extend the shelf life and to reduce peroxidation, new genotypes of soybean, rapeseed, sunfower, and faxseed with low linolenic and high oleic acid have been developed, named “high oleic.” Palm oil, cocoa fat, and coconut oil belong to fats that are solid or semi-solid at room temperature. The fruits from the oil palm, the utilization of which is constantly increasing, provide two different oils – the frst from the pulp (palm oil) and the second from the seeds (palm kernel oil). Crude palm oil has a high carotene content, hence the color of the oil is red. Oil palm is the largest source of natural carotenes – there are 500–700 mg/kg of carotenes in crude palm oil (CPO). The raw palm kernel oil lacks carotenoids and is not red. Palm oil and palm kernel oil also differ in saturated fat content: palm mesocarp oil is 49% saturated, while palm kernel oil is 82% saturated fats. However, crude red palm oil

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is mainly processed, i.e. refned, neutralized, bleached, and deodorized, which is called RBD (refned, bleached, and deodorized) palm oil and does not contain carotenoids. Many industrial food applications of palm oil use fractionated components of palm oil (stearin, olein). Cocoa butter, named confectionery fat, is the fat from cocoa beans, which is solid at room temperature. The specifc feature of this fat is the ability to crystallize in six different polymorphic forms, while the best form melts at body temperature, giving a pleasant, cooling sensation in the mouth.

2.9 HONEY Honey is produced by honeybees from the sugary secretions of plants (foral nectar) or from secretions of other insects (such as honeydew). Bees suck up nectar, store it in their honey sac, and enrich it with some enzymes. Honey is essentially an oversaturated aqueous solution of inverted sugar (glucose and fructose), very hygroscopic and sticky with a density of about 1.4 g/cm3. It also contains a very complex mixture of other carbohydrates, several enzymes (for instance, peroxidases), amino and organic acids, aroma substances, pigments, waxes, and pollen grains. Fructose (30–44%) and glucose (25–40%) are the predominant sugars in honey. Other monosaccharides have not been found. However, more than 20 di- and oligosaccharides have been identifed, with maltose predominating, followed by kojibiose. The composition of disaccharides depends largely on the plants, from which the honey was derived. The water content of honey should be less than 20%, otherwise, it can be readily fermented by osmophilic yeasts. The crystallization of honey is infuenced mainly by the ratio of two main sugars, glucose and fructose, which varies depending on the assortment of honey. Glucose, due to its low solubility in water, accelerates crystallization, while fructose slows it down, and it is 4.4 times more soluble in water. Honey with a high glucose/ fructose ratio crystallizes more rapidly (rapeseed and sunfower honey), while honey with a lower glucose/fructose ratio does so slowly (acacia, lime).

REFERENCES Belitz, H.-D., Grosch, W., Schieberle, P. Food Chemistry, Springer-Verlag, Berlin Heidelberg, 2009. Commission Regulation (EC) No 589/2008 of 23 June 2008, laying down detailed rules for implementing Council Regulation (EC) No 1234/2007 as regards marketing standards for eggs. Commission Regulation (EEC) No 2568/91 of 11 July 1991 on the characteristics of olive oil and olive-residue oil and on the relevant methods of analysis. Dinh, T.T.N., Thompson, L.D., Galyean, M.L., Brooks, J.C., Patterson, K.Y., Boylan, L.M. Cholesterol content and methods for cholesterol determination in meat and poultry. Comprehensive Reviews in Food Science and Food Safety, 10: 269–289, 2011. Ensminger, A.H, Ensminger, M.E., Konlande, J.E., Robson, J.R.K. The Concise Encyclopedia of Food and Nutrition, CRC Press, Boca Raton, London, Tokyo, 1995. Honikel, K.O., Arneth, W. Cholesteringehalt in Fleich und Eiern. Fleischwirtschaft, 1996. Horne, D.S Charactersitics of milk. In: Fennema’s Food Chemistry, Ed. S. Damodaran, K.L. Parkin, CRC Press, Taylor & Francis Group, Boca Raton, London, New York, 907–953, 2017.

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Huppertz, T., Kelly, A.L. Properties and constituents of cow’s milk. In: Milk Processing and Quality Management, Ed. A.Y. Tamine, Wiley-Blackwell, Oxford, 23-43, 2009. Medhammar, E., Wijesinha-Bettoni, R., Stadlmayr, B., Nilsson, E., Charrondiere, U.R., Burlingame, B. Composition of milk from minor dairy animals and buffalo breeds: A biodiversity perspective. Journal of the Science of Food and Agriculture, 92(3): 445– 474, 2012, nr 3. Regulation (EC) No 178/2002 of the European Parliament and of the Council of 28 January 2002 laying down the general principles and requirements of food law, establishing the European Food Safety Authority and laying down procedures in matters of food safety. Regulation (EC) No 853/2004 of the European Parliament and of the Council of 29 April 2004, laying down specifc hygiene rules for food of animal origin. Sugino, H., Nitoda, T., Juneja, L.R. General chemical composition of hen eggs. In: Hen Eggs: Their Basic and Applies Science, Ed. T. Yamamoto, L.R. Juneja, H. Hatta, M. Kim, CRC Press LLC, Boca Raton, Boston, London, New York, Washington, DC, 13–24, 1997.

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Water and Food Quality Peter Edward Doe and Barbara Emilia Cybulska

CONTENTS 3.1 3.2

Introduction .................................................................................................... 39 Structure and Properties of Water ..................................................................40 3.2.1 The Water Molecule ...........................................................................40 3.2.2 Hydrogen Bonds ................................................................................. 41 3.2.3 Properties of Bulk Water .................................................................... 43 3.2.4 Thermal Properties of Water .............................................................. 47 3.2.5 Water as a Solvent............................................................................... 47 3.2.6 Water in Biological Materials............................................................. 51 3.2.6.1 Properties ............................................................................. 51 3.2.6.2 Water Transport ................................................................... 54 3.3 Water in Food ................................................................................................. 55 3.3.1 Introduction ........................................................................................ 55 3.3.2 Sorption Isotherms and Water Activity .............................................. 56 3.3.2.1 Principle ............................................................................... 56 3.3.2.2 Measurement of Water Activity........................................... 58 3.3.2.3 Water Activity and Shelf Life of Foods ...............................60 3.3.3 Bottled Water...................................................................................... 61 3.3.3.1 Classifcation........................................................................ 61 3.3.3.2 Natural Mineral Water......................................................... 61 3.3.4 Bottled Water Other Than Natural Mineral Water............................. 63 3.3.4.1 Defnition ............................................................................. 63 3.3.4.2 Water Defned by Origin...................................................... 63 3.3.4.3 Hygiene, Labeling, and Health Benefts ..............................64 3.3.5 Water Supply, Quality, and Disposal .................................................. 65 3.3.5.1 Water Supply........................................................................ 65 3.3.5.2 Water Quality....................................................................... 65 3.3.6 Water Pollution ................................................................................... 67 3.3.7 Wastewater Treatment and Disposal................................................... 68 References................................................................................................................ 69

3.1

INTRODUCTION

Water is the most popular and most important chemical compound on our planet. It is a major chemical constituent of the Earth’s surface and it is the only substance that is abundant in solid, liquid, and vapor forms. Because it is ubiquitous, it seems to be a mild and inert substance. In fact, it is a very reactive compound characterized DOI: 10.1201/9781003265955-3

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by unique physical and chemical properties that make it very different from other popular liquids. The peculiar water properties determine the nature of the physical and biological world. Water is the major component of all living organisms. It constitutes 60% or more of the weight of most living things, and it pervades all portions of every cell. It existed on our planet long before the appearance of any form of life. The evolution of life was doubtlessly shaped by the physical and chemical properties of the aqueous environment. All aspects of living cells’ structure and function seem to be adapted to water’s unique properties. Water is the universal solvent and dispersing agent, as well as a very reactive chemical compound. Biologically active structures of macromolecules are spontaneously formed only in aqueous media. Intracellular water is not only a medium in which structural arrangement and all metabolic processes occur, but an active partner of molecular interactions, participating directly in many biochemical reactions as a substrate or a product. Its high heat capacity allows water to act as a heat buffer in all organisms. Regulation of water contents is important in the maintenance of homeostasis in all living systems. Only 0.003% of all freshwater reserve participates in its continuous circulation between the atmosphere and the hydrosphere. The remaining part is confned to the Antarctic ice. The geography of water availability has determined, to a large degree, the vegetation, food supply, and habitation in the various areas of the world. For example, Bangladesh has one of the world’s highest population densities, made possible through the regular fooding of the Ganges River and the rich silts it deposits in its wake. In Bangladesh, the staple food – rice – grows abundantly and is readily distributed. In other societies, the food must be transported long distances or kept over winter. Human well-being is closely linked to the availability of water and food. An expected increase in the world population by the year 2050 (65% or 3.7 billion) will create enormous pressure on freshwater resources and food production. Agriculture is by far the largest consumer of water and the key issue is to look for ways to improve water use effciency. The solution lies in producing more food from existing water and land resources (Wallace and Gregory, 2002). Stability, wholesomeness, and shelf life are signifcant features of foods that are, to a large degree, infuenced by the water content. Dried foods were originally developed to overcome the constraints of time and distance before consumption. Canned and frozen foods were developed next. The physical properties, quantity, and quality of water within food have a strong impact on food effectiveness, quality attributes, shelf life, textural properties, and processing.

3.2

STRUCTURE AND PROPERTIES OF WATER

3.2.1 THE WATER MOLECULE Water is a familiar material, but it has been described as the most anomalous of chemical compounds. Although its chemical composition, HOH or H2O, is universally

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known, the simplicity of its formula belies the complexity of its behavior. Its physical and chemical properties are very different from compounds of similar complexity, such as HF and H2S. To understand the reasons for water’s unusual properties, it is necessary to examine its molecular structure in some detail. Although a water molecule is electrically neutral as a whole, it has a dipolar character. The high polarity of water is caused by the direction of the H-O-H bond angle, which is 104.5°, and by an asymmetrical distribution of electrons within the molecule. In a single water molecule, each hydrogen atom shares an electron pair with the oxygen atom in a stable covalent bond. However, the sharing of electrons between H and O is unequal because the more electronegative oxygen atom tends to draw electrons away from the hydrogen nuclei. The electrons are more often in the vicinity of the oxygen atom than in the vicinity of the hydrogen atom. The result of this unequal electron sharing is the existence of two electric dipoles in the molecule, one along each of the H-O bonds. The oxygen atom bears a partial negative charge δ–, and each hydrogen bears a partial positive charge δ+. Because the molecule is not linear, H-O-H has a dipole moment (Figure 3.1). Because of this, water molecules can interact through electrostatic attraction between the oxygen atom of one water molecule and the hydrogen of another.

3.2.2 HYDROGEN BONDS Such interactions, which arise because the electrons on one molecule can be partially shared with the hydrogen on another, are known as hydrogen bonds. The H2O molecule, which contains two hydrogen atoms and one oxygen atom in a nonlinear arrangement, is ideally suited to engage in hydrogen bonding. It can act both as a donor and as an acceptor of hydrogen. The nearly tetrahedral arrangement of the orbital about the oxygen atom allows each water molecule to form hydrogen bonds with four of its neighbors (Figure 3.2). An individual, isolated hydrogen bond is very labile. It is longer and weaker than a covalent O-H bond (Figure 3.3). The hydrogen bond’s energy, that is, the energy required to break the bond, is about 20 kJ/mol. These bonds are intermediate between those of weak van der Waals interactions (about 1.2 kJ/mol) and those of covalent bonds (460 kJ/mol).

FIGURE 3.1 Water molecule as an electric dipole.

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FIGURE 3.2 Tetrahedral hydrogen bonding of fve water molecules.

FIGURE 3.3

Two water molecules connected by hydrogen bonds.

FIGURE 3.4 Directionality of the hydrogen bonds.

Hydrogen bonds are highly directional; they are stronger when the hydrogen and the two atoms that share it are in a straight line (Figure 3.4). Hydrogen bonds are not unique to water. They are formed between water and different chemical structures, as well as between other molecules (intermolecular) or

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FIGURE 3.5

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Some hydrogen bonds of biological importance.

even within a molecule (intramolecular). They are formed wherever an electronegative atom (oxygen or nitrogen) comes in close proximity to a hydrogen atom covalently bonded to another electronegative atom. Some representative hydrogen bonds of biological importance are shown in Figure 3.5. Intra- and intermolecular hydrogen bonding occurs extensively in biological macromolecules. A large number of hydrogen bonds and their directionality confer very precise three-dimensional structures upon proteins and nucleic acids.

3.2.3 PROPERTIES OF BULK WATER The key to understanding water structure in solid and liquid form lies in the concept and nature of the hydrogen bonds. In the crystal of ordinary hexagonal ice (Figure 3.6), each molecule forms four hydrogen bonds with its nearest neighbors. Each HOH acts as a hydrogen donor to two of the four water molecules, and as a hydrogen acceptor from the remaining two. These four hydrogen bonds are spatially arranged according to tetrahedral symmetry (Bjerrum, 1952). The crystal lattice of ice occupies more space than the same number of H2O molecules in liquid water. The density of solid water is thus less than that of liquid

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FIGURE 3.6

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Structure of ice.

water, whereas simple logic would have the more tightly bound solid structure be denser than its liquid. One explanation for ice being lighter than water at 0° C proposes a reforming of intermolecular bonds as the ice melts, so that on average, a water molecule is bound to more than four of its neighbors, thus increasing its density. But as the temperature of liquid water increases, the intermolecular distances also increase, giving a lower density. These two opposite effects explain the fact that liquid water has a maximum density at a temperature of 4° C. At any given instant in liquid water at room temperature, each water molecule forms hydrogen bonds with an average of 3.4 other water molecules (Nelson and Cox, 2021). The average translational and rotational kinetic energies of a water molecule are approximately 7 kJ/mol, the same order as that required to break hydrogen bonds; therefore, hydrogen bonds are in a continuous state of fux, breaking and reforming with high frequency on a picosecond time scale. A similar dynamic process occurs in aqueous media with substances that are capable of forming hydrogen bonds. At 100° C liquid water still contains a signifcant number of hydrogen bonds, and even in water vapor, there is a strong attraction between water molecules. The very large number of hydrogen bonds between molecules confers great internal cohesion in liquid water. This feature provides a logical explanation for many of its unusual properties. For example, its large values for heat capacity, melting point, boiling

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point, surface tension, and heat of various phase transitions are all related to the extra energy needed to break intermolecular hydrogen bonds. That liquid water has structure is an old and well-accepted idea; however, there is no consensus among physical chemists as to the molecular architecture of the hydrogen bond’s network in the liquid state. It seems that the majority of hydrogen bonds survive the melting process, but obviously, rearrangement of molecules occurs. The replacement of crystal rigidity by fuidity gives molecules more freedom to diffuse about and change their orientation. Any molecular theory for liquid water must take into account changes in the topology and geometry of the hydrogen bond network induced by the melting process. Many models have been proposed, but none has adequately explained all properties of liquid water. Historically, there are two competing theoretical approaches used to describe the molecular structure of liquid water: (1) the continuum (uniform) models, and (2) the mixture (cluster) models (Starzak and Mathlouthi, 2003; Dauchez et al., 2003). According to continuum models, liquid water is depicted as a continuous, threedimensional network in which water molecules are interconnected by somewhat distorted hydrogen bonds; hydrogen bonding is almost complete, the structural parameters (distances and angles) and bond energies have continuous distribution; all water molecules are qualitatively the same and a whole-water sample is considered a single entity with temperature-dependent local structure. The continuum models are incompatible with particular water properties such as the compressibility minimum and the density maximum. Most mixture models describe liquid water as an equilibrium mixture of a few classes of different structural species more or less defned. A paper by Röntgen (1892) in which water was described as a saturated solution of ice in a liquid composed of simpler molecules, started the mixture model history. Over the next century, a variety of structural arrangements, based on the equilibrium of small water aggregates were proposed and used to explain the properties of water and aqueous solutions. The most popular, the fickering clusters model (Figure 3.7), suggests that liquid water is highly organized on a local basis: the hydrogen bonds break and reform spontaneously, creating and destroying transient structural domains (Frank and Quist, 1961; Frank and Wen, 1957). However, because the half-life of any hydrogen bond is less than a nanosecond, the existence of these clusters has statistical validity only; even this has been questioned by some authors who consider water to be a continuous polymer. Experimental evidence obtained by x-rays and neutron diffractions strongly supports the persistence of a tetrahedral hydrogen bond order in the liquid water, but with substantial disorder present. Since the high-resolution Raman technique became available the spectra have been carefully analyzed in favor of the mixture models. The frst computer simulation was performed by Rahman and Stillinger (1971) with a model of 216 water molecules. The view that emerges from these studies is the following: liquid water consists of a macroscopically connected, random network of hydrogen bonds. This network has a local preference for tetrahedral geometry,

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FIGURE 3.7

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Flickering clusters of H2O molecules in bulk water.

but it contains a large proportion of strained and broken bonds, which are continually undergoing topological reformation. The properties of water arise from the competition between relatively bulky ways of connecting molecules into local patterns characterized by strong bonds and nearly tetrahedral angles and more compact arrangements characterized by more strain and bond breakage (Stillinger, 1980). With the advent of supercomputers, a food of quantitative studies on water structure based on quantum and statistical mechanics have been carried out. A number of new models have been proposed in which there are more and more complicated structural units as liquid water components have been suggested (Starzak and Mathlouthi, 2003). According to a model proposed by Wiggins (1990, 2002), two types of structure can be distinguished: high-density water and low-density water. In high-density water, the bent, relatively weak hydrogen bonds predominate over straight, stronger ones. Low-density water has many ice-like straight hydrogen bonds. Although hydrogen bonding is still continuous through the liquid, the weakness of the bonds allows the structure to be disrupted by thermal energy extremely rapidly. High-density water is extremely reactive and more liquid, whereas low-density water is inert and more viscous. A continuous spectrum of water structures between these two extremes can be imagined. The strength of water–water hydrogen bonding, which is the source of

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water density and reactivity, has great functional signifcance; this explains water’s solvent properties and its role in many biological events. A common feature of all theories is that a defnite structure of liquid water is due to the hydrogen bonding between molecules and that the structure is in a dynamic state as the hydrogen bonds break and reform with high frequency.

3.2.4

THERMAL PROPERTIES OF WATER

The unusually high melting point of ice, as well as the heat of water vaporization and specifc heat, are related to the ability of water molecules to form hydrogen bonds and the strength of these bonds. A large amount of energy, in the form of heat, is required to disrupt the hydrogenbonded lattice of ice. In the common form of ice, each water molecule participates in four hydrogen bonds. When ice melts, most of the hydrogen bonds are retained by liquid water, but the pattern of hydrogen bonding is irregular, due to the frequent fuctuation. The average energy required to break each hydrogen bond in ice has been estimated to be 23 kJ/mol, while the energy to break each hydrogen bond in water is less than 20 kJ/mol (Ruan and Chen, 1998). The heat of water vaporization is much higher than that of many other liquids. As is the case with melting ice, a large amount of thermal energy is required for breaking hydrogen bonds in liquid water, to permit water molecules to dissociate from one another and enter the gas phase. Perspiration is an effective mechanism of decreasing body temperature because the evaporation of water absorbs so much heat. A relatively large amount of heat is required to raise the temperature of 1 g of water by 1° C because multiple hydrogen bonds must be broken in order to increase the kinetic energy of the water molecules. Due to the high quantity of water in the cells of all organisms, temperature fuctuation within cells is minimized. This feature is of critical biological importance because most biochemical reactions and macromolecular structures are sensitive to temperature. The unusual thermal properties of water make it a suitable environment for living organisms, as well as an excellent medium for the chemical processes of life.

3.2.5 WATER AS A SOLVENT Many molecular parameters, such as ionization, molecular and electronic structure, size, and stereochemistry, will infuence the basic interaction between a solute and a solvent. The addition of any substance to water results in altered properties of that substance and of the water itself. Solutes cause a change in water properties because the hydrate envelopes that are formed around dissolved molecules are more organized and therefore more stable than the fickering clusters of free water. The properties of solutions that depend on a solute and its concentration are different from those of pure water. The differences can be seen in such phenomena as the freezing point depression, boiling point elevation, and increased osmotic pressure of solutions. The polar nature of the water molecule and the ability to form hydrogen bonds determine its properties as a solvent. Water is a good solvent for charged or polar

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compounds and a relatively poor solvent for hydrocarbons. Hydrophilic compounds interact strongly with water by an ion–dipole or dipole–dipole mechanism, causing changes in water structure and mobility and in the structure and reactivity of the solutes. The interaction of water with various solutes is referred to as hydration. The extent and tenacity of hydration depend on a number of factors, including the nature of the solute, salt composition of the medium, pH, and temperature. Water dissolves dissociable solutes readily because the polar water molecules orient themselves around ions and partially neutralize ionic charges. As a result, the positive and negative ions can exist as separate entities in a dilute aqueous solution without forming ion pairs. Sodium chloride is an example where the electrostatic attraction of Na+ and Cl– is overcome by the attraction of Na+ with the negative charge on the oxygen and Cl– with the positive charge on the hydrogen ions (Figure 3.8). The number of weak charge–charge interactions between water and the Na+ and Cl– ions is suffcient to separate the two charged ions from the crystal lattice. To acquire their stabilizing hydration shell, ions must compete with water molecules, which need to make as many hydrogen bonds with one another as possible. The normal structure of pure water is disrupted in a solution of dissociable solutes. The ability of a given ion to alter the net structure of water depends on the strength

FIGURE 3.8 Hydration shell around Na+ and Cl–.

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of its electric feld. Among ions of a given charge type (e.g., Na+ and K+ or Mg2+ and Ca2+), the smaller ions are more strongly hydrated than the larger ions, in which the charge is dispersed over a greater surface area. Most cations, except the largest ones, have a primary hydration sphere containing four to six molecules of water. Other water molecules, more distant from the ion, are held in a looser secondary sphere. Electrochemical transfer experiments indicate a total of 16 molecules of water around Na+ and about ten around K+. The bound water is less mobile and denser than HOH molecules in bulk water. At some distance, the bonding arrangements melt into a dynamic confguration of pure water. Water is especially effective in screening the electrostatic interaction between dissolved ions because, according to Coulomb’s law, the force (F) between two charges q+ and q– separated by a distance r is given as: F ˜ q° .

q˛

˝ r2

(3.1)

where ϵ is the dielectric constant of the medium. For a vacuum, ϵ = 1 Debye unit, whereas for bulk water, ϵ = 80; this implies that the energies associated with electrostatic interactions in aqueous media are approximately 100 times smaller than the energies of covalent association, but increase considerably in the interior of a protein molecule. In thermodynamic terms, the free energy change, ΔG, must have a negative value for a process to occur spontaneously. ∆G = ∆H – T∆S

(3.2)

where ΔG represents the driving force, ΔH (the enthalpy change) is the energy from making and breaking bonds, and ΔS (the entropy change) is the increase in randomness. Solubilization of salt occurs with a favorable change in free energy. As salt such as NaCl dissolves, the Na+ and Cl– ions leaving the crystal lattice acquire greater freedom of motion. The entropy (ΔS) of the system increases; where ΔH has a small positive value and TΔS is large and positive, ΔG is negative. Water in the multilayer environment of ions is believed to exist in a structurally disrupted state because of conficting structural infuences of the innermost vicinal water and the outermost bulk-phase water. In concentrated salt solutions, the bulkphase water would be eliminated, and the water structure common in the vicinity of ions would predominate. Small or multivalent ions, such as Li+, Na+, H3O+, Ca2+, Mg2+, F–, SO42–, and PO43–, which have strong electric felds, are classifed as water structure formers because solutions containing these ions are less fuid than pure water. Ions that are large and monovalent, most of the negatively charged ions and large positive ions, such as K+, Rb+, Cs+, NH4+, Cl–, Br–, I–, NO3–, ClO4 –, and CNS– disrupt the normal structure of water; they are structure breakers. Solutions containing these ions are more fuid than pure water (Fennema, 1996). Through their varying abilities to hydrate and to alter water structure and its dielectric constant, ions infuence all kinds of water–solute interactions. The

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conformation of macromolecules and the stability of colloids are greatly affected by the kinds and concentrations of ions present in the medium. Water is a good solvent for most biomolecules, which are generally charged or polar compounds. Solubilization of compounds with functional groups such as ionized carboxylic acids (COO –), protonated amines (NH3+), phosphate esters, or anhydrides is also a result of hydration and charge screening. Uncharged but polar compounds possessing hydrogen bonding capabilities are also readily dissolved in water, due to the formation of hydrogen bonds with water molecules. Every group that is capable of forming a hydrogen bond with another organic group is also able to form hydrogen bonds of similar strength with water. Hydrogen bonding of water occurs with neutral compounds containing hydroxyl, amino, carbonyl, amide, or imine groups. Saccharides dissolve readily in water, due to the formation of many hydrogen bonds between the hydroxyl groups or carbonyl oxygen of the saccharide and water molecules. Water–solute hydrogen bonds are weaker than ion–water interactions. Hydrogen bonding between water and polar solutes also causes some ordering of water molecules, but the effect is less signifcant than with ionic or nonpolar solutes. The introduction into water of hydrophobic substances such as hydrocarbons, rare gases, and the apolar groups of fatty acids, amino acids, or proteins is thermodynamically unfavorable because of the decrease in entropy. The decrease in entropy arises from the increase in water–water hydrogen bonding adjacent to apolar entities. Water molecules in the immediate vicinity of a nonpolar solute are constrained in their possible orientations, resulting in a shell of highly ordered water molecules around each nonpolar solute molecule (Figure 3.9a). The number of water molecules

FIGURE 3.9 Cagelike water structure around the hydrophobic alkyl chain (a) and hydrophobic interactions (b).

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in the highly ordered shell is proportional to the surface area of the hydrophobic solute. In the case of dissolved hydrocarbons, the enthalpy of formation of the new hydrogen bonds often almost exactly balances the enthalpy of creation in water, a cavity of the right size to accommodate the hydrophobic molecule. However, the restriction of water mobility results in a very large decrease in entropy. To minimize contact with water, hydrophobic groups tend to aggregate; this process is known as hydrophobic interaction (Figure 3.9b). The existence of hydrophobic substances barely soluble in water but readily soluble in many nonpolar solvents, and their tendency to segregate in aqueous media, have been known for a long time. However, the origin of this hydrophobic effect is still somewhat controversial. The plausible explanation is that hydrophobic molecules disturb the hydrogen-bonded state of water, without having any compensatory ordering effects. Apolar molecules are water structure formers; water molecules cannot use all four possible hydrogen bonds when in contact with hydrophobic, water-hating molecules. This restriction results in a loss of entropy, a gain in density, and an increased organization of bulk water. Amphipathic molecules, compounds that contain both polar or charged groups and apolar regions, disperse in water if the attraction of the polar group for water can overcome possible hydrophobic interactions of the apolar portions of the molecules. Many biomolecules are amphipathic: proteins, phospholipids, sterols, certain vitamins, and pigments have polar and nonpolar regions. When amphipathic compounds are in contact with water, the two regions of the solute molecule experience conficting tendencies: the polar or charged hydrophilic regions interact favorably with water and tend to dissolve, but the nonpolar hydrophobic regions tend to avoid contact with water. The nonpolar regions of the molecules cluster together to present the smallest hydrophobic area to the aqueous medium, and the polar regions are arranged to maximize their interactions with the aqueous solvent. In aqueous media, many amphipathic compounds are able to form stable structures, containing hundreds to thousands of molecules, called micelles. The forces that held the nonpolar regions of the molecules together are due to hydrophobic interactions. The hydrophobic effect is a driving force in the formation of clathrate hydrates and the self-assembly of lipid bilayers. Hydrophobic interactions between lipids and proteins are the most important determinants of biological membrane structure. The three-dimensional folding pattern of proteins is also determined by hydrophobic interactions between nonpolar side chains of amino acid residues.

3.2.6 WATER IN BIOLOGICAL MATERIALS 3.2.6.1 Properties Water behaves differently in different environments. The properties of water in heterogeneous systems, such as living cells or food, remain a feld of debate (Mathlouthi, 2001; Rückold et al., 2003). Water molecules may interact with macromolecular components and supramolecular structures of biological systems through hydrogen bonds and electrostatic interactions. Solvation of biomolecules such as lipids, proteins, nucleic acids, or saccharides resulting from these interactions determines their molecular structure and function.

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Various physical techniques, such as nuclear magnetic resonance (NMR), x-ray diffraction, and chemical probes (exchange of H by D), indicate that there is a layer of water bound to protein molecules, phospholipid bilayers, and nucleic acids, as well as at the surface of the cell membranes and other organelles. Water associated at the interfaces and with macromolecular components may have quite different properties from those in the bulk phase. Water can be expected to form locally ordered structures at the surface of water-soluble as well as water-insoluble macromolecules and at the boundaries of the cellular organelles. Biomacromolecules generally have many ionized and polar groups on their surfaces and tend to align near polar water molecules. This ordering effect exerted by the macromolecular surface extends quite far into the surrounding medium. According to the association–induction theory proposed by Ling (1962), fxed charges on macromolecules and their associated counterions constrain water molecules to form a matrix of polarized multilayers having restricted motion, compared with pure water. The monolayer of water molecules absorbed on the polar sorption site of the molecule is almost immobilized and thus behaves, in many respects, like part of the solid or like water in ice. It has different properties than the additional water layers, defned as multilayers, have. The association–induction theory has been shared by many researchers for many years. Unfortunately, elucidation of the nature of individual layers of water molecules has been less successful, due to the complexity of the system and lack of appropriate techniques. Measurements of the diffusion coeffcients of globular protein molecules in solution yield values for molecular size that are greater than the corresponding radii determined by x-ray crystallography. The apparent hydrodynamic radius can be calculated from the Stokes-Einstein relation: D ˜ kBT / 6°˛ aN

(3.3)

where D is the diffusion coeffcient, k B is the Boltzmann constant, T is the temperature, η is the solution viscosity, and aH is the molecule radius (Nossal and Lecar, 1991). Similarly, studies utilizing NMR techniques show that there is a species of associated water that has a different character than water in the bulk phase. By these and other methods, it was found that for a wide range of protein molecules, approximately 0.25 to 0.45 g of H2O are associated with each gram of protein. The hydration forces can stabilize macromolecular association or prevent macromolecular interactions with a strength that depends on the surface characteristic of the molecules and the ionic composition of the medium. The interaction between a solute and a solid phase is also infuenced by water. Hydration shells or icebergs associated with one or the other phase are destroyed or created in this interaction and often contribute to conformational changes in macromolecular structures – and ultimately to changes in biological and functional properties important in food processing. Biophysical processes involving membrane transport are also infuenced by hydration. The size of the hydration shell surrounding small ions and the presence

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of water in the cavities of ionic channels or in the defects between membrane lipids strongly affect the rates at which the ions cross a cell membrane. The idea that intracellular water exhibits properties different from those of bulk water has been around for a long time. The uniqueness of the cytoplasmic water was deduced from: The observation that cells may be cooled far below the freezing point of a salt solution iso-osmotic with that of the cytoplasm. Properties of the cytoplasm, which in the same conditions should bind water like a gel. Osmotic experiments in which it has often been observed that part of cell water is not available as a solvent. This water has been described as osmotically inactive water, bound water, or compartmentalized water. According to a recent view, three different kinds of intracellular water can be distinguished: a percentage of the total cell water appears in the form of usual liquid water. A relevant part is made up of water molecules that are bound to different sites of macromolecules in the form of hydration water, while a sizeable amount, although not fxed to any defnite molecular site, is strongly affected by macromolecular felds. This kind of water has been termed vicinal water. Most of the vicinal water surrounds the elements of the cell cytoskeleton. Vicinal water has been extensively investigated, and it has been found that some of its properties are different from those of normal water. It does not have a unique freezing temperature, but an interval ranging from –70 to –50° C; it is a very bad solvent for electrolytes, but nonelectrolytes have the same solubility properties as in usual water; its viscosity is enhanced, and its NMR response is anomalous (Giudice et al., 1986). The distribution of various types of water inside living cells is a question that cannot be answered yet, especially because in many cells marked changes have been noted in the state of intracellular water as a result of biological activity. The possibility that water in living cells may differ structurally from bulk water has prompted a search for parameters of cell water that deviate numerically from those of bulk water. The diffusion coeffcient for water in the cytoplasm of various cells has been determined with satisfactory precision. It has been found that the movement of water molecules inside living cells is not much different and is reduced by a factor of between 2 and 6, compared with the self-diffusion coeffcient for pure water. According to Mild and Løvtrup (1985), the most likely explanation of the observed values is that part of the cytoplasmic water, the vicinal water close to the various surface structures in the cytoplasm, is structurally changed to the extent that its rate of motion is signifcantly reduced compared with the bulk phase. In heterogeneous biological materials and foods, water exists in different states. It is thought that water molecules in different states function differently. Water associated with proteins and other macromolecules has traditionally been referred to as bound water. However, to designate such water as bound can be misleading because, for the most part, the water molecules are probably only transiently

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associated, and at least a portion of the associated water has to be constantly rearranged due to the thermal perturbations of weak hydrogen bonds. Water molecules are constantly in motion, even in ice. In fact, the translational and rotational mobility of water directly determines its availability. Water mobility can be measured by a number of physical methods, including NMR, dielectric relaxation, electron spin resonance (ESR), and thermal analysis (Chinachoti, 1993). The mobility of water molecules in biological systems may play an important role in a biochemical reaction’s equilibrium and kinetics, formation and preservation of chemical gradients and osmotic pressure, and macromolecular conformation. In food systems, the mobility of water may infuence the engineering processes, such as freezing, drying, and concentrating, chemical and microbial activities, and textural attributes (Ruan and Chen, 1998). Water determines the quality, stability, shelf life, and physical properties of food. It has an infuence on rheological, thermal, mass transfer, electrical, optical, and acoustic physical properties (Lewicki, 2004). 3.2.6.2 Water Transport Water transport is associated with various physiological processes in whole living organisms and single cells. When cells are exposed to hyper- or hypoosmotic solutions, they immediately lose or gain water, respectively. Even in an isotonic medium, a continuous exchange of water occurs between living cells and their surroundings. Most cells are so small and their membranes are so leaky that the exchange of water molecules measured with isotopic water reaches equilibrium in a few milliseconds. The degree of water permeability differs considerably between tissues and cell types. Mammalian red blood cells and renal proximal tubules are extremely permeable to water molecules. Transmembrane water movements are involved in diverse physiological secretion processes. How water passes through cells has begun to become clear only in the last few years. Water permeates living membranes through both the lipid bilayer and specifc water transport proteins. In both cases, water fow is passive and directed by osmosis. Water transport in living cells is therefore under the control of ATP (adenosine triphosphate) and ion pumps. The most general water transport mechanism is diffusion through lipid bilayers, with a permeability coeffcient of 2 to 5 × 104 cm/sec. The diffusion through lipid bilayers depends on lipid structure and the presence of sterol (Subczyński et al., 1994). It is suggested that the lateral diffusion of the lipid molecules and the water diffusion through the membrane is a single process (Haines, 1994). A small amount of water is transported through certain membrane transport proteins, such as a glucose transporter or the anion channel of erythrocytes (aquaporins). The major volume of water passes through water transport proteins. The frst isolated water-transporting protein was the channel-forming integral protein from red blood cells. The identifcation of this protein has led to the recognition of a family of related water-selective channels, the aquaporins, which are found in animals, plants, and microbial organisms. In addition to water, they permeate some other small molecules. The pore is formed by six membrane-spanning helices. In

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the membrane they aggregate into tetramers, but each monomer acts as a separate water channel. Water fow through the protein channel is controlled by the number of protein copies in the membrane. In red blood cells, there are 200,000 copies/cell that account for up to 90% of the water permeability of the membrane; in apical brush border cells of renal tubules, it constitutes 4% of the total protein (Engel et al., 1994). It is assumed that another important function of aquaporins is the detection of osmotic and turgor pressure gradients (Hill et al., 2004).

3.3 WATER IN FOOD 3.3.1

INTRODUCTION

Water, with a density of 1,000 kg m–3, is denser than the oil components of foods; oils and fats typically have densities in the range of 850–950 kg m–3. Glycerols and sugar solutions are denser than water. Unlike solid phases of most other liquids, ice is less dense than liquid water; ice has a lower thermal conductivity than water. These properties have an effect on the freezing of foods that are predominantly water-based; the formation of an ice layer on the surface of the liquids and the outside of solids has the effect of slowing down the freezing rate. Because a molecule of water vapor is lighter (molecular weight = 18) than that of dry air (molecular weight about 29), moist air is lighter than dry air at the same temperature. This is somewhat unexpected in that the popular conception is that humid air (which contains more water) is heavier than dry air. At room temperature, water has the highest specifc heat of any inorganic or organic compound with the sole exception of ammonia. It is interesting to speculate why the most commonly occurring substance on this planet should have one of the highest specifc heats. One of the consequences of this peculiarity in the food industry is that heating and cooling operations for essentially water-based foods are more energy-demanding. To heat a kilogram of water from 20 to 50° C requires about 125 kJ of energy, whereas heating the same mass of vegetable oil requires only 44 kJ. A sponge holds most of its water as liquid held in the intestacies of the sponge structure. Most of the water can be wrung out of the sponge, leaving a matrix of air and damp fbers. Some of the water may be loosely bound to the surfaces of the sponge – this is referred to as adsorbed water. Within the sponge fbers, the residual water is more strongly held – absorbed within the fber of the sponge. If the sponge is left to dry in the sun, this absorbed water will evaporate, leaving only a small proportion of water bound chemically to the salts and to the cellulose of the sponge fbers. As with the familiar example of water in a sponge, water is held in food by adsorption, absorption, and various other physical and chemical mechanisms (Table 3.1). It is a convenient oversimplifcation to distinguish between “free” and “bound” water. The defnition of bound water in such a classifcation poses problems. Fennema (1996) reports seven different defnitions of “bound” water. Some of these defnitions are based on the freezability of the “bound” component and others rely on

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TABLE 3.1 Classifcation of Water States in Foods

Class of water

Description

Proportion of typical 90% (wet basis) moisture content food < 0.03%

Constitutional

An integral part of non-aqueous constituent

Vicinal

“Bound” water that strongly acts with specifc hydrophilic sites of non-aqueous constituents to form a monolayer coverage; water–ion and water–dipole bonds “Bound” water that forms several additional layers around hydrophilic groups; water–water and water–solute hydrogen bonds Flow is unimpeded; properties close to dilute salt solutions; water–water bonds predominate

0.1–0.9%

Free water held within matrix or gel that impedes fow

5–96%

Multilayer

Free Entrapped

1–5%

5–96%

Source: Fennema (1985).

its availability as a solvent. He prefers a defnition in which “bound” water is: “that which exists in the vicinity of solutes and other non-aqueous constituents, exhibits reduced molecular activity and other signifcantly altered properties as compared with ‘bulk water’ in the same system, and does not freeze at –40° C.” Moisture content can be measured simply by weighing a sample and then oven drying it, usually at 105° C overnight, the difference in mass being the moisture in the original sample. However, much confusion is caused by reporting the moisture content simply as a percentage without specifying the basis of the calculation. It should be made clear whether the moisture content is calculated on a wet basis (moisture content divided by original mass) or on a dry basis (moisture content divided by the “bone dry” or “oven dry” mass). Even the term “bone dry” mass can cause confusion amongst non-English speakers; it was once misinterpreted as the “mass of the dry bones.” In foods containing signifcant quantities of fat or salt, for example, moisture content may be calculated as the mass of water in a sample divided by the dry solids that are not salt or fat, in which case the moisture content should be reported as “salt free, fat free, dry basis.”

3.3.2

SORPTION ISOTHERMS AND WATER ACTIVITY

3.3.2.1 Principle Since 1929 it has been recognized that the chemical and microbial stability, and hence the shelf life, of foods is not directly related to their moisture content, but to a property called water activity (Tomkins, 1929). Essentially water activity is the measure of the degree to which water is bound within the food and hence is unavailable for further chemical or microbial activity.

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Water activity is defned as the ratio of the partial pressure of water vapor in or around the food to that of pure water at the same temperature. Relative humidity of moist air is defned in the same way except that by convention, relative humidity is reported as a percentage whereas water activity is expressed as a fraction. Thus if a sample of meat sausage is sealed within an airtight container, the humidity of the air in the head-space will rise and eventually equilibrate to a relative humidity of say 83% which means that the water activity (aw) of the meat sausage is 0.83. The relationship between water activity and moisture content for most foods at a particular temperature is a sigmoidal-shaped curve called the sorption isotherm (Figure 3.10). The phrase equilibrium moisture content curve is also used. Sorption isotherms at different temperatures can be calculated using the Clausius-Clapeyron equation from classical thermodynamics, namely: d(ln aw ) °H ˜ d(1 / T ) R

(3.4)

where aw is the water activity, T is the absolute temperature, ∆H is the heat of sorption, and R is the gas constant. A complication arises from one of the methods of measuring sorption isotherms for a food. A food that has previously been dried and is then re-hydrated will have a different sorption isotherm (adsorption isotherm) from that which is in the process

FIGURE 3.10 A typical sorption isotherm for a food.

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of drying (desorption isotherm). This difference is due to a change in water binding capacity in foods that have been previously dried. Many mathematical descriptors for sorption isotherms have been proposed. One of the more famous is that of Brunauer, Emmett, and Teller (1938) (the BET isotherm) which is based on the concept of a measurable amount of mono-molecular layer (vicinal) water for a particular food. Wolf and Jung (1985) compiled 2,201 references to sorption isotherm data for foods. Iglesias et al. (1975) propose the following three-parameter equation to ft sorption isotherm data for a range of foods: aw = exp(-a’ θ r)

(3.5)

where a’ and r are the parameters as listed in Table 3.2, and θ = X/Xm. X is the equilibrium moisture content, and Xm, in units of g/100 g dry basis, is the BET monomolecular moisture content for the particular food as listed in Table 3.2. However, there are nearly as many equations for sorption isotherms as there are researchers in this feld. Basu, Shivhare, and Mujumdar (2006) present a comprehensive review of a dozen sorption isotherm models with a statistically rigorous review of their application to a range of foods. More recently Labuza and Altunakar (2020) published a review of water activity prediction methods noting there are commercially available computer programs that predict the water activity in foods based on their composition (Table 3.2). 3.3.2.2 Measurement of Water Activity Many methods of measuring water activity have been developed by researchers. These include direct vapor pressure measurement, equilibration with a stable hygroscopic substance that has a known sorption behavior, and various types of hygrometers (Doe, 1998). Water activity is most conveniently measured by the measurement of relative humidity in the head-space over a food sample in a sealed container. Commercially available instruments for water activity determination use different methods for measuring the relative humidity: resistive electrolytic hygrometers (REH), capacitance hygrometers, and dew point instruments. Hygrometer-based instruments are prone to drift and must be calibrated regularly against saturated solutions of various inorganic salts. Hygrometer-based instruments are also prone to hysteresis at high humidities. Dew point hygrometers detect water condensation on a chilled mirror (dewpoint temperature). These instruments are sensitive to less than 0.001 water activity units. Readings take fve minutes or less and are accurate to +0.003 water activity units. Care must be taken with any measurement of water activity to ensure that the sample is representative of the food under test. Dried fsh, for example, will have moisture and salt contents, and hence water activity, varying widely from thin, exposed, fesh to the relatively moist interior. If the “worst case” scenario for the growth of potentially toxic or spoilage organisms is of interest, the sample of fesh

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TABLE 3.2 Predicted Water Activity of Various Foods Fruits Apple juice Apples Apricots Bananas Bilberries Blackberries Blueberries Cherries Cherry juice Cranberries Currants Dates Dewberries Figs Gooseberries Grape juice Grapefruit Grapes Lemons Limes Mangoes Melons Nectarines Orange juice Oranges Papayas Pears Persimmons Plums Quinces Raspberries Raspberry juice Sour cherries Strawberries Strawberry juice Sweet cherries Tangerines Watermelons

aw 0.986 0.976–0.988 0.977–0.987 0.964–0.987 0.989 0.986–0.989 0.982 0.959–0.986 0.986 0.989 0.990 0.974 0.985 0.974 0.989 0.983 0.980–0.985 0.963–0.986 0.982–0.989 0.980 0.986 0.970–0.991 0.94 0.988 0.90 0.990 0.979–0.989 0.976 0.969–0.982 0.961–0.979 0.984–0.994 0.988 0.971–0.983 0.986–0.997 0.991 0.975 0.987 0.992

Source: Labuza and Altunakar (2020).

Vegetables Artichokes Asparagus Avocadoes Beans, green Beans, Inna Broccoli Brussels sprouts Cabbages Carrots Caulifower Celeriac Celery Celery leaves Com, sweet Cucumbers Eggplant Endive Green onions Leeks Lettuce Mushrooms Onions Parsnips Peas, green Peppers Potatoes Potatoes, sweet Pumpkins Radishes Rhubarb Rutabagas Radishes, small Spinach Squash Tomato pulp Tomatoes Turnips Other products Beef Lamb Pork Fish, cod Cream, 40%, fat Milk

aw 0.976–0.987 0.992–0.994 0.989 0.984–0.996 0.994 0.990 0.991 0.992 0.983–0.993 0.984–0.990 0.990 0.987–0.994 0.992–0.997 0.994 0.985–0.992 0.987–0.993 0.995 0.992–0.996 0.991–0.976 0.996 0.995–0.969 0.974–0.990 0.988 0.982–0.990 0.982–0.997 0.982–0.988 0.985 0.984–0.992 0.980–0.990 0.989 0.988 0.992–0.996 0.988–0.996 0.994–0.996 0.993 0.991–0.998 0.986 0.980–0.990 0.990 0.990 0.990–0.994 0.979 0.994–0.9

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for water activity determination should be excised from the thickest, most moist region of the fsh. 3.3.2.3 Water Activity and Shelf Life of Foods Many of the chemical and biological processes that cause the deterioration of foods, and ultimately spoilage, are water-dependent. Microbial growth is directly linked to water activity. No microbes can multiply at a water activity below 0.6. Dehydration is arguably the oldest form of food preservation; the sun drying of meat and fsh has been traced to the beginning of recorded history. Drying relies on removing water, thus making it unavailable for microbial growth. Salting or curing has a similar effect. A saturated solution of common salt has a water activity of close to 0.75. Thus by adding suffcient salt to food, the water activity can be lowered to a level where most pathogenic bacteria are inactivated, but the moisture content remains relatively high. Intermediate moisture content foods (IMF) such as pet food and continental sausages rely on fats and water-binding humectants such as glycerol to lower water activity. Fat, being essentially hydrophobic, does not bind water but acts as a fller for IMF to increase the volume of the product. The effect of several humectants is for each to sequester an amount of water independently of the other humectants that may be present in the food. Each thus lowers the water activity of the system according to the equation from Ross (1975): awn = aw0 . aw1 . aw2 . aw3 . etc

(3.6)

where awn is the water activity of the complex food system and aw0 etc. are the water activities associated with each component of the system. For example, the water activity of a food with a moisture content of 77% (wet basis) and a salt content of 3% (wet basis) can be calculated as follows:100 g of the food comprises 77 g of water, 20 g of “bone” dry matter, and 3 g of salt. The contribution to the water activity due to the salt and the molecular weights of water (18) and salt (58.5) can be calculated (according to Raoult’s law of dilute solutions) as: aw1 = (77 × 18) / (77 × 18 + 3 × 58.5) = 0.89

(3.7)

The water activity for the salt-free solid matter of the food is found from its sorption isotherm at that moisture content, aw0 = 0.90, say. Thus, the water activity of the salted food is: awn = aw0 . aw1 = 0.9 × 0.89 = 0.8

(3.8)

None of the dangerous pathogenic bacteria associated with food, such as Clostridium or Vibrio spp. which cause botulism and cholera, can multiply at water activity values below about 0.9. Thus drying, or providing suffcient water-binding humectants, are effective methods of preventing the growth of food-poisoning bacteria. Only osmophilic yeasts and some molds can grow at water activities in the range of 0.6 to 0.65. Thus, by reducing the water activity below this value, foods are microbially stable. That is, unless the packaging is such that the food becomes locally

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re-wet, in which case local spoilage can occur, for example, when condensation occurs within a hermetically sealed package subject to rapid cooling. There are various chemical reactions that proceed and may be accelerated at low values of water activity. Maillard reactions leading to lysine loss and brown color development peak at aw values around 0.5 to 0.8. Nonenzymatic lipid oxidation increases rapidly below aw = 0.4. Enzymic hydrolysis decreases with water activity down to aw = 0.3 and is then negligible.

3.3.3 BOTTLED WATER 3.3.3.1 Classifcation Water in glass or plastic bottles, or cartons, is becoming increasingly popular not only in areas where the tap water supply quality may be sub-standard but also where drinking bottled water is seen as culturally acceptable behavior. Health benefts are claimed for some products. Marketing of bottled water has been so successful that consumers spend from 240 to over 10,000 times more per liter for bottled water than for tap water. Tap water may contain hazardous chemicals and microorganisms; it may also contain additives such as fuoride to provide perceived health benefts to communities. Bottled water is required by law to meet standards of chemical and biological safety. In some aspects, standards for bottled water are more stringent than those for tap water because of the shelf-life requirement for bottled water. There are basically three types of bottled water: 1. Natural mineral water must come from an underground source. It receives no treatment other than fltration or carbonation and is bottled at the source. Natural mineral water must meet tight microbiological standards and be regularly tested. 2. Spring water may come from other sources. It must also be bottled at the source and meet microbiological and chemical standards. Permitted treatments are fltration and carbonation. However, spring water does not have to be stable in composition. 3. Table water is basically any water in a bottle. It could come from an underground source but might be tap water. Table water may, or may not, be treated. It could have additives to change its favor or chemical composition. 3.3.3.2 Natural Mineral Water The Codex Alimentarius Commission (CAC) has published a Standard for Natural Mineral Waters (CAC, 1997). This provides guidance to industry and regulating bodies that may choose to follow this standard, or make parts of it mandatory by law. The standard defnes natural mineral water as being clearly distinguishable from ordinary drinking water because it is: • Characterized by its content of certain mineral salts and their relative proportions and the presence of trace elements or of other constituents.

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• Obtained directly from natural or drilled sources from underground waterbearing strata for which all possible precautions should be taken within the protective perimeters to avoid any pollution of, or external infuence on, the chemical and physical qualities of natural mineral water. • Consistent in composition and stable in its discharge and its temperature, due account being taken of the cycles of minor natural infuences. • Collected under conditions that guarantee the original microbiological purity and chemical composition of essential components. • Packaged close to the point of emergence with particular hygiene precautions. • Not subjected to any treatment other than those permitted by this standard. The standard also defnes naturally carbonated natural mineral water, noncarbonated natural mineral water, decarbonated natural mineral water, natural mineral water fortifed with carbon dioxide from the source, and carbonated natural mineral water. The standard outlines permissible levels for a number of chemical substances as listed in Table 3.3. TABLE 3.3 Permissible Levels of Chemicals in Natural Mineral Water (mg/dm³) Chemical

Permissible level (mg/dm³ unless specifed)

Antimony

0.005

Arsenic Barium Borate Cadmium Chromium Copper Cyanide Fluoride Lead Manganese Mercury Nickel Nitrate Nitrite Selenium

0.01 calculated as total As 0.7 5 calculated as B 0.003 0.05 calculated as Cr 1 0.07 (See note 1) 0.01 0.5 0.001 0.02 50 calculated as nitrate 0.02 as nitrite (see note 2) 0.01

Notes: 1. If more than 1 mg/dm³ fuoride, the product must be labeled: contains fuoride. If more than 2 mg/dm³, the product must be labeled: The product is not suitable for infants and children under the age of seven years. 2. Set as a quality limit (except for infants). Source: Codex Standard 108-1981.

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The standard requires that natural mineral water shall not pose a microbiological risk to the consumer. Escherichia coli or thermotolerant coliforms must not be present in a 250-mL sample. If no more than two total coliform bacteria, fecal streptococci, Pseudomonas aeruginosa, or sulfde-reducing anaerobes are detected in a 250-mL sample, a second examination must be carried out; if more than two are detected the batch is rejected. Natural mineral water containers must be hermetically sealed and safe from possible adulteration or contamination. The label must identify the product as natural mineral water and must declare the chemical composition. No claims of medicinal effects may be shown, and any health beneft claims must be true and not misleading.

3.3.4

BOTTLED WATER OTHER THAN NATURAL MINERAL WATER

3.3.4.1 Defnition There is a Codex standard for packaged water for drinking purposes other than natural mineral water (CAC, 2001). Such water may contain minerals and carbon dioxide naturally occurring or intentionally added, but may not contain sugars, sweeteners, favorings, or other foodstuffs. No packaged water may contain substances that emit radioactivity in quantities that may be injurious to health. All packaged water shall comply with the Guidelines for Drinking Water Quality published by the World Health Organization (World Health Organisation, 2017). The addition of minerals must comply with the relevant Codex standards. The standard distinguishes between “waters defned by origin,” which originate from a specifc underground or surface resource and do not pass through a community water system, and “prepared waters,” which may originate from any supply. Prepared waters can be subjected to antimicrobial treatments, which modify the physical and chemical properties of the original water, provided that such treatment satisfes the WHO Guidelines for Drinking Water Quality. 3.3.4.2 Water Defned by Origin Water defned by origin is limited in treatment prior to packaging to: • Reduction and/or elimination of dissolved gases (and resulting possible changes in pH). • Addition of carbon dioxide (and resulting changes in pH) or re-incorporation of the original carbon dioxide present at emergence. • Reduction and/or elimination of unstable compounds such as iron, manganese, sulfur (as So or S--) compounds, and carbonates in excess, under normal conditions of temperature and pressure, of the calco-carbonate equilibrium. • Addition of air, oxygen, or ozone on condition that the concentration of byproducts resulting from ozone treatment is below the tolerance established under the health limits for chemical and radiological substances.

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• Decrease and/or increase in temperature. • Reduction and/or separation of elements originally present in excess of maximum concentrations or of maximum levels of radioactivity set according to the health limits for chemical and radiological substances. Anti-microbial treatments may be used to conserve the original microbial ftness for human consumption. 3.3.4.3 Hygiene, Labeling, and Health Benefts Prepared waters can be subjected to antimicrobial treatments that modify the physical and chemical properties of the original water provided that such treatment satisfes the WHO Guidelines for Drinking Water Quality (World Health Organization, 2017). The standard recommends that packaged waters comply with the Recommended International Code of Practice – General Principles of Food Hygiene (CAC/RCP 1-191) and also the Code of Hygienic Practice for Bottled/Packaged Drinking Waters (Other Than Natural Mineral Waters) (CAC/RCP 48-2001). There are requirements in the standard for approval and inspection of the source for waters defned by origin. Labeling must comply with the Codex General Standard for the Labelling of Prepackaged Foods (Codex Standard 1-1985, adopted 1985 and amended 1991, 1999, 2001, 2003, 2005, 2008, and 2010) together with any additional provisions in national legislation. Where appropriate, in the case of waters defned by origin, labeling may specify “naturally carbonated,” “naturally sparkling,” or “fortifed with carbon dioxide.” For all waters, “carbonated” or “sparkling” may be used if the carbon dioxide does not come from the same source. If there is no visible release of carbon dioxide, the words “non-carbonated,” “non-sparkling,” or “still” may be used. The total dissolved content of packaged waters may be shown, and in the case of waters defned by origin, the chemical composition that confers the characteristics of the product may be shown. Where required by local laws, the precise location of the source of water defned by origin must appear on the label. Water bottled from a tap water distribution system that has not undergone further treatment (e.g. the addition of carbon dioxide or fuoride) must bear the words “from a public or private distribution system.” The standard prohibits any claims for medicinal (preventive, alleviative, or curative) effects. Claims for other health benefts may only appear if true and not misleading. Any place name may not form part of the trade name unless it refers to water defned by origin collected from that place. The chemical and microbiological safeguards placed on bottled water make it a safer alternative to tap water in places where community water supplies may be substandard or compromised by drought or food. The mineral content of bottled water may be an important and necessary supplement to dietary mineral intake. For a very exhaustive presentation of the contents

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of mineral components in different food sources and their role in nutrition, see Chapter 4.

3.3.5

WATER SUPPLY, QUALITY, AND DISPOSAL

3.3.5.1 Water Supply Just as water is an integral part of any food, the supply, quality, and disposal of water are of prime consideration in the establishment and operation of all food processing. Potable (drinkable) water may be required for addition to the product and will certainly be required for clean-up. Non-potable water may be required for heat exchangers and cooling towers. Boiler feed water must be conditioned within close limits of pH and “hardness.” Brennan et al. (1990) in their book, Food Engineering Operations, list four types of water used in the food and beverage industries: (i) (ii) (iii) (iv)

General purpose water Process water Cooling water Boiler feed water

The siting and consequent viability of a food processing plant may well depend on a guaranteed, regular supply of suitable quality water and an environmentally acceptable method of disposal. Developed countries now have strict regulations for the emission of wastewater. Developing countries are becoming increasingly aware of the problems of wastewater disposal. In a recent symposium in Indonesia, a fsh drying processor was asked what his main technical problems were. He nominated water pollution, not for reasons of meeting environmental control regulations, but because the fsh farmers further down the river were complaining about his wastewater. 3.3.5.2 Water Quality 3.3.5.2.1 Standards and Treatment There are a number of international standards for potable (drinkable) water quality in existence. The World Health Organization (WHO) has a standard for potable water quality as part of the Codex Alimentarius. The standard detailed in Table 3.4 is from the United States Departmental Protection Agency. There is also a large EC Directive relating to the quality of water intended for human consumption (80/778/EEC) which is contained in the Joint Circular from the Department of the Environment Circular 20/82, 2 Marsham Street London SW1P 3EB, and the Welsh Offce Circular 33/82, Cathays Park Cardiff CF1 3NQ, issued on 19 August 1982. Jenkins (2010) assesses the political impact of facilitating the EU directives in England and Wales. A more recent study of water quality revealed differences in the allowable limits of constituent chemicals has been published by Koh and Ko (2018). In most cases, water will require some treatment to assure it is of the required standard to meet food hygiene requirements and not constitute a public health

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TABLE 3.4 Primary Maximum Contaminant Levels (mg/dm³ Unless Specifed) Contaminant

Level (mg/dm³ unless specifed)

Arsenic

0.01

Barium Cadmium Chromium Lead Mercury Nitrate (as N) Selenium Silver Fluoride Endrin Lindane Methoxychlor Toxaphene 2.4 D 2,4,5 TP Silvex Total trihalomethanes Trichloroethylene Carbon tetrachloride 1,2 dichloroethane Vinyl chloride Benzene Para-dichlorobenzene 1,1,dichloroethylene 1,1,1,trichloroethane Radium 226 and 228, combined Gross alpha particle activity Gross beta particle activity Turbidity

2 0.005 0.1 0.015 0.002 10 0.05 0.05 4.0 0.002 0.0002 0.04 0.003 0.1 0.05 0.08 0.005 0.005 0.005 0.002 0.005 0.075 0.007 0.2 5 pC/dm³ 15 pC/dm³ 4 millirem/year 1 tu up to 5 tu

Coliform bacteria

1 per 100 cm³, monthly average

Source: USDPA (1987)

hazard. Surface water from rain runoff into rivers or impoundments is likely to contain atmospheric solutes; minerals from the ground; organic matter from vegetation; microbial contamination from, birds, wild and domestic animals; and human waste. Water from underground aquifers will have much of the surface contamination fltered out but is likely to be high in dissolved mineral content. Water treatment to bring quality to within the required standard may involve screening, sedimentation, coagulation and focculation, fltration, and other physical

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or chemical treatments to remove microorganisms, organic matter, or dissolved minerals. Metal screens are used to remove particles larger than about 1 mm in size. Settling ponds remove smaller particles. Insoluble, suspended matter is usually removed by sand flters. Coagulating and focculating agents act to bind smaller particles into clumps which then settle, or can be screened or fltered. Microorganisms can be inactivated by heat, chemical disinfection, UV radiation, or ultrasonic treatment. Most town water supplies are chlorinated or have ozone added for chemical disinfection. Treatment to remove dissolved mineral matter is more complex. Dissolved bicarbonates of calcium, magnesium, sodium, and potassium cause alkalinity; soluble calcium and magnesium salts cause hardness. Alkalinity and hardness may need to be adjusted for some food procession operations. For example, the formation of a “head” on beer is critically dependent on water hardness. Excessively hard water may cause discoloration and toughening of certain foods. On the other hand, hardness may be required to prevent excessive foaming in clean-up operations. Iron and manganese salts may be present in water supplies forming organic slimes which tend to clog pipes. Aeration, fltering, and settling are effective for the removal of iron bicarbonates. Insoluble oxides of manganese are formed through chlorination. Excessive amounts of dissolved gases, carbon dioxide, oxygen, nitrogen, and hydrogen sulfde cause problems in boiler feed water, corrosion, and bacterial formation. Treatment is by boiling and venting off the non-condensable gases, or by chemical dosing. Small amounts of hydrocarbons such as kerosene and diesel cause tainting in foods. Separating fuels from processing areas, personal hygiene, cleaning stations around food procession operations, and good housekeeping can prevent this problem.

3.3.6

WATER POLLUTION

Polluted water is described as polluted if it poses a risk to the health of humans, fsh, or other animals. Microbiologically polluted water contains bacteria and other microorganisms which may be hazardous or toxic. Human and animal wastes from sewage and farmyard run-off are the principal sources of microbiological pollution. Polluted water can be an indirect hazard as fsh and shellfsh may become contaminated and eaten. Water can be polluted by organic and inorganic chemicals. Domestic and industrial pesticides are a major source of chemical pollution, as are detergents. Many of these substances are slow to degrade and may be concentrated in the food chain with disastrous consequences for fsh and bird life. Biodegradable substances tend to be oxygen-depleting, resulting in a reduction of aerobic bacteria and fsh. Food waste is high in biochemical oxygen demand (BOD). Food wastes contain large quantities of organic matter which break down naturally by oxidation; however, this oxygen demand is at the expense of other natural biochemical processes in waterways which become oxygen depleted and lifeless if

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the BOD is too high. BOD is defned as the quantity of oxygen (in units of mg/dm3) required for microorganisms to oxidize the waste at a particular temperature (20° C) in fve days. Food wastes can range in BOD from 500 to 4,000 mg/dm3 BOD which is higher than for domestic sewage (200–400 mg/dm3). An excess of nutrients, such as phosphorous and nitrogen, in polluted water will lead to excessive growth of plant matter and algal blooms in waterways. Besides clogging waterways and adding toxins, this extra plant material contributes to the BOD of the water. Suspended matter, even if chemically and biologically inert, can contribute to pollution. These particles will eventually settle out and cause silting and an anaerobic environment at the bottom of the waterway. Water pollution is most effectively prevented by removing the pollutants before they get into the waterways. This can be effected through good house-keeping practices, such as avoiding the discharge of fatty material and detergents into the domestic sewerage system; proper design of landfll areas; pre-treatment of industrial wastes; and separation of storm-water from sewage, so as not to overload treatment plants.

3.3.7 WASTEWATER TREATMENT AND DISPOSAL The ultimate aim of any food processing operation is to have an environmentally neutral impact. Re-use, recycling, and sustainability are today’s catchwords. For a food operation to be truly environmentally sustainable it should recycle all water not incorporated in the product or vented into the atmosphere. The reality is that it is currently considered uneconomic to recycle wastewater from food processing operations. Current practice is to treat wastewater to limit its effect on receiving waters. Treatment of wastewater mirrors the water treatment methods described earlier, that is, a combination of physical, chemical, and biological treatments. A physical process treats suspended rather than dissolved pollutants. The pollutants may be simply allowed to settle out or foat to the top naturally, or the process may be aided mechanically, which will cause smaller particles to stick together, forming larger particles that will settle or rise faster – a process known as focculation. Organic chemical focculants such as polyamine, or inorganic focculants such as aluminum sulfate (alum), are usually added to produce larger particles. Filtration through a medium such as sand as a fnal treatment stage can result in very clear water. Ultrafltration, nanofltration, and reverse osmosis are processes that force water through membranes and can remove colloidal material and even some dissolved matter (Hube et al., 2020). Absorption (adsorption, technically) on activated charcoal is a physical process that can remove dissolved chemicals. Air or steam stripping can be used to remove pollutants that are gasses or low-boiling liquids from water, and the vapors that are removed in this way are also often passed through beds of activated charcoal to prevent air pollution. These last processes are used mostly in industrial treatment plants, though activated charcoal is common in municipal plants for odor control.

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Wastewater from food processing operations usually contains signifcant solid matter that can be removed by physical processes. Fats and oils can be skimmed from the surface of settling tanks; heavier suspended matter can be removed as sludge which can then be dewatered, dried, and used as animal feed, fertilizer, or fuel. An alternative method for the removal of oils and fats is by aeration, in which air bubbles blown from the bottom of a settling tank carry fne solids and grease to the surface. Chemical treatments of wastewater are much the same as described earlier for process water treatment. BOD can be effectively reduced by biological treatment. Both aerobic and anaerobic fermentation of the organic material is used. Depending on the scale of the operation, bio-reactors range in size from 7.5 m in diameter and 2–3 m in depth to lagoons 1–2 m deep covering several hectares. However, for long-term sustainable operation, there must be provision for sludge removal. Where the area for treatment is not a limitation, and there is suffcient isolation for smell not to be a deterrent, wastewater is sprayed directly on the ground where it breaks down under the action of sunlight and in-ground bacteria. A typical municipal treatment plant would comprise a preliminary treatment plant in which large or hard solids are removed or crushed. The effuent then passes through a primary settling basin in which organic suspended matter will either settle out or foat to the surface to be skimmed off. The next part of the process is usually referred to as secondary treatment wherein the remaining dissolved or colloidal organic matter is removed by aerobic biodegradation. This promotes the formation of less offensive, oxidized products. The treatment unit should be suffciently large to remove enough of the pollutants to prevent signifcant oxygen demand in the receiving water after discharge. Sewage and wastewater can also be treated anaerobically. Closed reactors facilitate odor control, although anaerobic lagoons are also used. Such lagoons are deeper than aerobic types with grease allowed to accumulate on the surface to control odor emission. Methane produced as an end product of the biochemical pathway can be used for heating the reactors in cold weather. A problem with the anaerobic digestion process is its sensitivity to pH and temperature variation, and the susceptibility of the active microorganisms to chemical disinfectants.

REFERENCES Basu, S., Shivhare, U.S. and Mujumdar, A.S., 2006. Models for sorption isotherms for foods: A review. Drying Technology, 24(8), 917–930. https://doi.org/10.1080 /07373930600775979. Bjerrum, N., 1952. Structure and properties of ice. Science, 115(2989), 385–390. Brennan, J.G., Butters, J.R., Cowell, N.D. and Lilley, A.E.V., 1990. Food Engineering Operations. 3rd Edition. Elsevier Applied Science, London and New York, 523. Brunauer, S., Emmett, P.H. and Teller, E., 1938. Adsorption of gases in multimolecular layers. Journal of the American Chemical Society, 60(2), 309–319. https://doi.org/10.1021 /ja01269a023. CAC., 1997. Codex Alimentarius Commission, “Codex Standard for Natural Mineral Waters”, Codex Standard 108 – 1981, Rev.1 1997.

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CAC., 2001. Codex Alimentarius Commission, “Code of Hygienic Practice for Bottled/ Packaged Drinking Water (other than Natural Mineral Waters)”, Codex Standard 227 – 2001. CAC/RCP (Codex Alimentarius Commission/Recommended International Code of Practice) General Principles of Food Hygiene. FAO, Rome, 1-1969 Rev. 3 (1997) Amended 1999. CAC/RCP (Codex Alimentarius Commission/Recommended International Code of Practice) Code of Hygienic Practice for Bottled/Packaged Drinking Waters (other than Natural Mineral Waters). FAO, Rome, 48-2001. Chinachoti, P., 1993. Water mobility and its relation to functionality of sucrose-containing food systems. Food Technology (Chicago), 47(1), 134–140. Codex Standard. Codex General Standard for the Labelling of Packaged Foods. FAO, Rome, 1-1985 (Rev. 1-1991). Dauchez, M., Peticolas, W.L., Debelle, L. and Alix, A.J., 2003. Ab initio calculations of polyhedra liquid water. Food Chemistry, 82(1), 23–28. https://doi.org/10.1016/S0308 -8146(02)00589-7. Doe, P.E. ed., 1998. Fish Drying and Smoking Production and Quality. Technomic Publishing Co., Inc, Lancaster, 22. Engel, A., Walz, T. and Agre, P., 1994. The aquaporin family of membrane water channels. Current Opinion in Structural Biology, 4(4), 545–553. Fennema, O.R. 1996. Food chemistry. 3rd Edition. New York: Marcel Dekker Inc. Frank, H.S. and Quist, A.S., 1961. Pauling’s model and the thermodynamic properties of water. The Journal of Chemical Physics, 34(2), 604–611. https://doi.org/10.1063/1 .1700993. Frank, H.S. and Wen, W.Y., 1957. Ion-solvent interaction: Structural aspects of ion-solvent interaction in aqueous solutions: A suggested picture of water structure. Discussions of the Faraday Society, 24, 133–140. Giudice E., Doglia, S., Milani, M. and Vitiello, G., 1986. Water in biological systems. In Modern Bioelectrochemistry, F. Gutmann and H. Keyzer, eds. Plenum Press, New York and London, 282. Haines, T.H., 1994. Water transport across biological membranes. FEBS Letters, 346(1), 115–122. Hill, A.E., Shachar-Hill, B. and Shachar-Hill, Y., 2004. What are aquaporins for? The Journal of Membrane Biology, 197(1), 1–32. https://doi.org/10.1007/s00232-003-0639-6. Hube, S., Eskaf, M., Hrafnkelsdóttir, K.F., Bjarnadóttir, B., Bjarnadóttir, M.Á., Axelsdóttir, S. and Wu, B., 2020. Direct membrane fltration for wastewater treatment and resource recovery: A review. Science of the Total Environment, 710, 136375. https://doi.org/10 .1016/j.scitotenv.2019.136375. Iglesius, H.A., Chirife, J. and Lombardi, J.L., 1975. An equation for correlating equilibrium moisture content in foods. International Journal of Food Science & Technology, 10(3), 289–297. Jenkins, J.O., 2010. The impact of politics on the application of the Drinking Water Directive (80/778/EEC). Water and Environment Journal, 24(3), 228–236. Koh, D.C. and Ko, K.S., 2018. Recent trends of domestic and international management and research of natural mineral water used for bottled water. Journal of Soil and Groundwater Environment, 23(6), 9–27. https://doi.org/10.7857/JSGE.2018.23.6.009. Ling, G.N., 1962. A Physical Theory of the Living State. Ginn (Blaisdel), Boston, MA. Labuza, T.P. and Altunakar, B., 2020. Diffusion and sorption kinetics of water in foods. In Water Activity in Foods: Fundamentals and Applications. 2nd Edition. John Wiley & Sons, Inc., 287–309. Lewicki, P.P., 2004. Water as the determinant of food engineering properties. A review. Journal of Food Engineering, 61(4), 483–495. https://doi.org/10.1016/S0260-8774(03)00219-X.

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Mathlouthi, M., 2001. Water content, water activity, water structure and the stability of foodstuffs. Food Control, 12(7), 409–417. https://doi.org/10.1016/S0956 -7135(01)00032-9. Mild, K.H. and Løvtrup, S., 1985. Movement and structure of water in animal cells. Ideas and experiments. Biochimica et Biophysica Acta (BBA)-Reviews on Biomembranes, 822(2), 155–167. Nelson, A.L. and Cox, M.M., 2021. Lehninger, Principles of Biochemistry. 8th Edition. W. H. Freeman and Company, New York. Nossal, R. and H. Lecar, eds., 1991. Molecular and Cell Biophysics. Addison-Wesley Publishing Company, Reading, MA. Rahman, A. and Stillinger, F.H., 1971. Molecular dynamics study of liquid water. The Journal of Chemical Physics, 55(7), 3336–3359. https://doi.org/10.1063/1.1676585. Röntgen, W.C., 1892. Über die Constitution des füssigen Wassers. Annalen der Physik, 281(1), 91–97. Ross, K.D., 1975. Estimation of water activity in intermediate moisture foods. Food Technology, 29(3), 26–34. Ruan, R.R. and Chen, P.L., 1998. Water in Foods and Biological Materials. Technomic Publishing Co.Inc, Lancaster, PA. Rückold, S., Isengard, H.D., Hanss, J. and Grobecker, K.H., 2003. The energy of interaction between water and surfaces of biological reference materials. Food Chemistry, 82(1), 51–59. https://doi.org/10.1016/S0308-8146(02)00541-1. Starzak, M. and Mathlouthi, M., 2003. Cluster composition of liquid water derived from laser-Raman spectra and molecular simulation data. Food Chemistry, 82(1), 3–22. https://doi.org/10.1016/S0308-8146(02)00584-8. Stillinger, F.H., 1980. Water revisited. Science, 209(4455), 451–457. Subczynski, W.K., Wisniewska, A., Yin, J.-J., Hyde, J.S. and Kusumi, A., 1994. Hydrophobic barriers of lipid bilayer membranes formed by reduction of water penetration by alkyl chain unsaturation and cholesterol. Biochemistry, 33(24), 7670–7681. Tomkins, R.G., 1929. Studies of the growth of moulds.―I. Proceedings of the Royal Society of London. Series B, Containing Papers of a Biological Character, 105(738), 375–401. USDPA., 1987. The safe drinking water act. United States Departmental Protection Agency, Program Summary - October 1987. Wallace, J.S. and Gregory, P.J., 2002. Water resources and their use in food production systems. Aquatic Sciences, 64(4), 363–375. Wiggins, P.M., 1990. Role of water in some biological processes. Microbiological Reviews, 54(4), 432–449. Wiggins, P.M., 2002. Water in complex environments such as living systems. Physica A: Statistical Mechanics and its Applications, 314(1–4), 485–491. Wolf, W., Speiss, W.E.L. and Jung, G., 1985. Sorption Isotherms and Water Activity of Food Materials. Science and Technology Publishers, Hornchurch, Essex. World Health Organization, 2017. Guidelines for Drinking-Water Quality: First Addendum to the Fourth Edition. World Health Organization, Geneva. Licence: CC BY-NC-SA 3.0 IGO.

4

The Role of Mineral Components Małgorzata Grembecka

CONTENTS 4.1

The Origin and Contents of Mineral Components in Food Raw Materials and Products ................................................................................... 73 4.2 Factors Affecting the Appearance and Speciation of These Components ..... 75 4.2.1 Sources of Elements in Food .............................................................. 75 4.2.2 Speciation of Essential Elements........................................................ 78 4.2.3 Speciation of Toxic Elements ............................................................. 79 4.3 Changes in the Contents and Distribution of Mineral Components in Foods Due to Storage and Processing ............................................................80 4.3.1 Negative Effects of Food Processing..................................................80 4.3.2 Positive Effects of Food Processing ................................................... 81 4.3.3 Infuence of Thermal Processing........................................................ 81 4.3.4 Infuence of Processing on Cereals..................................................... 82 4.3.5 Infuence of Processing on Foods of Animal Origin.......................... 82 4.3.6 The Infuence of Packaging ................................................................ 82 4.4 The Effect of These Components on the Stability and Sensory Properties as Well as the Biological Value of Foods...................................... 83 4.4.1 Enzymatic Browning and Interactions between Metal Ions and Polysaccharides................................................................................... 83 4.4.2 Sodium Chloride and Alkali Metals Infuence................................... 83 4.4.3 Effect of Hard Water...........................................................................84 4.4.4 Effect on Oxidation ............................................................................ 85 4.5 Interactions of Mineral Elements with Other Food Components................... 86 4.6 Mineral Food Additives.................................................................................. 88 References.............................................................................................................. 102

4.1

THE ORIGIN AND CONTENTS OF MINERAL COMPONENTS IN FOOD RAW MATERIALS AND PRODUCTS

Chemical elements are ubiquitous in the natural environment. They may be of natural and/or anthropogenic origin. The natural sources include mainly rock weathering, as mineral components are natural inorganic constituents of the earth’s crust. However, anthropogenic sources (industry, transport) have a much greater infuence on the presence of chemical elements in the natural environment or agriculture. As DOI: 10.1201/9781003265955-4

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a consequence of human activity, chemical elements are released into the air, soil, and water and become an integral part of the food chain. The mineral components of food do not include carbon, oxygen, nitrogen, and hydrogen, which after burning do not leave ash. Mineral components are also constituents of animal, plant, and human tissues. Chemical elements found in plants and animals can be divided into macroelements and microelements. The frst group includes calcium (Ca), magnesium (Mg), phosphorus (P), sodium (Na), potassium (K), sulfur (S), and chlorine (Cl). These elements constitute almost 99.8% of plants and animals’ weight, and their content exceeds 50 mg/kg of dry tissue weight. They occur in proteins, lipids, sugars, nucleotides, the skeletal system, and the external skeleton of animals. In plants, their role is to catalyze biochemical reactions, change the state of cellular colloids, directly affect the cell metabolism, and change the protoplasm turgor and permeability. The latter group concerns iron (Fe), zinc (Zn), copper (Cu), manganese (Mn), cobalt (Co), nickel (Ni), chromium (Cr), molybdenum (Mo), vanadium (V), lithium (Li), rubidium (Rb), boron (B), selenium (Se), fuorine (F), iodine (I), silicon (Si), and others whose biological function have not yet been fully recognized. These elements regulate the activity of enzymes, hormones, vitamins, and other factors determining the course of processes in living organisms. Both groups of elements are essential for organisms; however, V, Cr, Ni, F, and I are necessary only for animals, while B is only for plants. An excess of elements usually has a detrimental effect. There are also elements derived directly or indirectly from a polluted environment, not fulflling a useful role in the physiological processes of organisms, harmful to humans, and at higher concentrations often toxic – cadmium (Cd), mercury (Hg), arsenic (As), lead (Pb), and aluminum (Al). Monovalent elements (Na, K, Cl) are present in foods as soluble salts, mostly in ionized form. On the other hand, polyvalent ions are usually present in the form of an equilibrium between ionic, dissolved, nonionic, and colloidal species. Moreover, metals can often be found in the form of chelates (Co in vitamin B12, Fe in hemoglobin) (deMan 2013). The human body contains approximately 60 elements, which accounts for 6% of a body mass of a young person and about 4% in older people. They are classifed as micronutrients required by living organisms for the maintenance of normal cellular metabolism and tissue function. However, they are not themselves metabolized, nor are they a source of energy. Bioelements are essential components that must be supplied with food, and they represent 0.2–0.3% of the total intake of all nutrients in the diet. According to the WHO Experts, mineral components can be classifed into three groups, based on their nutritional signifcance in humans, i.e., 1 – essential elements, 2 – possibly essential, and 3 – potentially toxic elements (WHO, 1996). They also can be divided into main elements (also called macroelements), trace elements (microelements), and ultra-trace elements (Belitz et al. 2009). Macroelements, which occur in the body in amounts greater than 0.01%, are needed in amounts greater than 100 milligrams per day and include Ca, P, K, S, Mg, Na, and Cl. Microelements such as Fe, Zn, Se, Cu, Mn I, F, Cr, and Co are required in much lower amounts than 100 milligrams per day. Their content in the human body does not exceed 0.01%.

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Bioelements play a fundamental role in the water and electrolyte balance (Na, K, Cl) and in the maintenance of the acid-base balance and neuromuscular excitability. Moreover, elements are necessary to maintain appropriate fuid balance, synthesize hormones, and protect against harmful free radicals in the body. They are also components of biologically active compounds such as hemoglobin, myoglobin (Fe), thyroxine (I), vitamin B12 (Co), ATP, ADP (P), and enzymes (Zn, Mn, Se) (Table 4.1). The source of mineral components for humans is primarily food products, including water and table salt. They can also be supplied by food to which bioelements have been intentionally added, e.g., Ca, Fe, as well as fortifed products and dietary supplements. A certain amount of elements present in food may come from additives as well as the apparatus used during its production. The content of individual mineral components in food depends on various factors such as cultivation and breeding conditions, weather and soil conditions, use of fertilizers, and the type and state of the plants’ and animals’ maturity. Elements’ concentration in meat varies to a lesser extent than in plant products. In addition, the use of various forms of food processing, i.e., peeling, grinding, cooking, and thawing, affects the loss of mineral components to a different extent. The amount of losses depends on the type of raw material, storage, and process conditions. Therefore, the varied diet should consist of both plant and animal products to supply a suffcient amount and the right proportions of the elements necessary for proper development and health. Elements occur in food in various chemical forms. However, they are absorbed as cations and anions in covalent and non-covalent linkages with organic compounds. Mineral components in food are not fully utilized and their absorption in the gastrointestinal tract depends on many factors that are related to both the food and the body. Many factors infuence the absorption of bioelements by the body: internal ones (sex, age, physiological state, nutritional status of the organism); the presence of compounds that reduce the bioavailability (oxalates, phytates, tannins, fber, toxic elements as well as micronutrients that may have a toxic effect as a result of accumulation in the body); and the source of the ingredient, i.e., the type of the consumed products, their composition, and interactions between nutrients.

4.2

FACTORS AFFECTING THE APPEARANCE AND SPECIATION OF THESE COMPONENTS

4.2.1 SOURCES OF ELEMENTS IN FOOD Food safety and quality depend on many, both biological and chemical, factors. One of these factors is the presence of elements in food products, both toxic and necessary for the proper functioning of the human body. Metals enter food mainly from air, soil, and water. The degree of plant uptake of elements from soil depends on soil temperature, pH, oxidation-reduction potential, the presence of complexing compounds, the concentration of these elements in soil, and the activity of microorganisms and fertilizers. In raw materials of animal origin, the content of mineral components varies to a lesser extent than in plant products and depends on the availability of the elements in the feed, as well as on the species, physiological condition,

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TABLE 4.1 Physiological Roles of the Selected Mineral Components and Their Content in Food Products Mineral element

Physiological role

Dietary sources (average content in mg/100 g ww or *μg/100 g ww)

Macroelements Ca

Mg

P

K

Na

Microelements Fe

Cu

Zn

The basic component of bones and teeth; muscle contraction, nerve stimulation; blood clotting; a cofactor of many enzymes; muscle contraction A component of bones and teeth, soft tissues; nerve stimulation; blood clotting; enzyme activator; muscle contraction The main component of bones and teeth; component of ATP and ADP, fats, proteins, carbohydrates, and DNA or RNA; acid-base balance Component of intracellular fuid, digestive juices; intracellular acid-base balance; water retention; osmotic pressure; nerve stimulation; muscle contraction; cell permeability A component of the extracellular fuid, digestive juices; acid-base balance; water balance; osmotic pressure; muscle contraction; nerve stimulation; cell permeability A component of hemoglobin, myoglobin, and many enzymes; necessary for transport and storage of oxygen Component of many enzymes; necessary for the proper iron utilization and the inactivation of free radicals; elastic tissue development Essential for over 200 enzymes; necessary for the synthesis of protein and nucleic acids; vitamin A utilization

Emmental cheese (1,029); cheddar cheese (752); camembert cheese, 30% fat content (600); soybean (201); parsley leaves (179); cow’s milk (120); cottage cheese (95) Wheat bran (490); cocoa (414); soybean, dry (220); almonds (170); white bean, dry (140); dark chocolate (100); buckwheat groats (48) Wheat bran (1,143); almonds (454); white bean, dry (426); camembert cheese, 30% fat content (385); roast beef (157); buckwheat groats (150); cow’s milk, UHT (92) Soybean, dry (1,799); white bean, dry (1337); tuna (363); roast beef (356); founder (288); rye bread (244); cow’s milk, UHT (157); Emmental cheese (95) Processed cheese, 45% fat content (1,260); ham, cooked (965); rye bread (523); founder smoked (481); Emmental cheese (275); white cabbage (12); soybean (4.7) Pig’s liver (18); soybean, dry (6.6); white bean, dry (6.2); almonds (4.1); spinach (3.8); baker’s yeast (3.5); ham, canned (2.5); roast beef (2); garlic (1.4); camembert cheese, 30% fat content (0.17) Cocoa powder (3.8); Brazil nut (1.3); pig’s liver (1.3); soybean seed, dry (1.2); mushroom (0.4); buckwheat groats (0.41); rye four type 1150 (0.318) Cocoa powder (8.2); pig’s liver (6.5); Emmental cheese (4.6); baker’s yeast (4.3); soybean seed, dry (4.2); roast beef (4.1); crispbread (3.1); white bean, dry (2.6) (Continued)

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TABLE 4.1 (CONTINUED) Physiological Roles of the Selected Mineral Components and Their Content in Food Products Mineral element Mn

I*

Se*

Physiological role Enzyme component; activator of many enzymes; important for the formation of connective tissue, bones, brain function, pancreas, and reproduction; role as an antioxidant (Mn-superoxide dismutase) Component of the thyroid hormone; prevents the formation of endemic goiter Component of the thyroid hormone; necessary for the proper functioning of enzymes; antioxidant role

Cr*

Trivalent chromium participates in the process of glucose metabolism; role in the metabolism of fats, carbohydrates, and proteins; component of enzymes and stimulation of their activity; hexavalent chromium is toxic

F

Component of bones and teeth; increases bone density

Dietary sources (average content in mg/100 g ww or *μg/100 g ww) Tea (73); soybean seed, dry (2.7); white bean, dry (1.6); peanut (1.6); pearl barley (1.3); rye bread (0.870); parsley leaf (0.756); broccoli (0.454); black currant (0.346) Halibut (52); Baltic sea herring (50); pea, dry seeds (14); soybean seed, dry (6.3); gouda cheese (3.6); cocoa powder (3.1) Coconut (810); Brazil nut (103); herring (43); soybean seed, dry (19); white bean, dry (14); Emmental cheese (11); chicken egg (10); mushroom (7) Cocoa powder (159); chocolate, milk free (40% cocoa) (30); hazelnut (12); rye bread (7.8); parsley leaf (7); soybean seed, dry (6.1); baker’s yeast (5); pig’s liver (3.8); wheat bran (3.3)

Tea (9.5); brown shrimp (0.160); peanut (0.130); oyster (0.120); chicken egg (0.110); coffee roast (0.90)

Source of content data: Souci, Fachmann, & Kraut, 2002. References: Grembecka & Szefer, 2011; Nabrzyski, 2007.

and age of the animal. An additional source of metals can be vessels, equipment, and apparatus used during production and processing as well as the packaging in which food is transported and stored. Particular food types differ signifcantly in the content of macro- and microelements (Table 4.1). To provide the human body with enough elements and in the right proportions, the diet should be composed of both plant and animal products. There are also other sources of these substances, such as sodium chloride, polyphosphates, preservatives, colorants, and drinking water. The toxicity and bioavailability of chemical elements depend not only on the dose or route of administration of a given element but also on its chemical form. Chemical elements can be divided into two groups based on their effect on the human body: toxic elements and elements necessary for life. In the case of the elements necessary for life, their role can be extremely diverse. Chemical elements can constitute a building material in the human body, take part in metabolic

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changes, regulate the balance of electrolytes, or fnally constitute an important element of enzymes and hormones (Table 4.1). However, even necessary microand macroelements in too high doses may cause toxic effects. Moreover, the physicochemical form of an element has a huge impact on its bioavailability and role in the human body.

4.2.2 SPECIATION OF ESSENTIAL ELEMENTS The toxicity of chromium (Cr) strongly depends on the oxidation state of this element. Cr(III) is recognized as a micronutrient necessary for the proper metabolism of lipids, glucose, and proteins in mammals (Table 4.1). Cr(VI), in turn, has a strong mutagenic and genotoxic effect and, therefore, is classifed by the International Agency for Research on Cancer (IARC) as Group 1 – carcinogen for humans. Chromium has not been confrmed to play any biological role in the physiology of plants. The excess of this element may provoke morphophysiological and biochemical processes in plants. Although this element is potentially toxic to plants, it can be collected from the soil through complexes formed with citric acid, aspartic acid, or oxalic acid. Weathering of the parent materials is the major source (natural source) of Cr in soils (Raj & Maiti 2020). Plants also tend to absorb toxic elements such as As, tellurium (Te), Cd, or Ni, along with the necessary microelements such as Se, Cu, and Zn. Selenium (Se) can be found in inorganic and organic compounds such as dimethyl selenide, selenomethionine, selenocysteine, and methyl selenocysteine. However, the bioavailability of Se is higher in the case of its organic compounds. The toxic properties of this metalloid strongly depend on its chemical speciation. Inorganic forms of Se are considered more toxic due to their reactivity with organic sulfur compounds, which leads to the formation of oxygen radicals. Food is the main source of Se supplied to the human body. The ability of plants to uptake and accumulate this element in tissues strongly depends both on the form in which it is present in soil and water and also on the plant species. Plants accumulate Se in the form of inorganic compounds, selenates(IV) or selenates(VI), which are then converted into organic forms, in particular selenomethionine and selenocysteine. In contrast, cabbage, beetroots, and garlic may contain up to 50% of the total Se as inorganic compounds. Animals can take this element from both water and the plants they eat. It is also possible to supplement farm animals with Se. Selenocysteine is the dominant form of Se in the tissues of terrestrial animals. Its combination with amino acids was also confrmed in fsh. It is assumed that the Se content in food increases with the increased protein content (Ullah et al. 2019). Selenium contained in dietary supplements can be present in organic form, for example, selenomethionine, or in the inorganic form of selenates(IV) and selenates(VI). In addition to yeast, an alternative form of supplementation of Se can be biomass of bacteria and plants enriched in this element. Also, fermenting lactic acid bacteria supports the excessive accumulation of Se (Kieliszek 2019). Zinc bioavailability for plants depends on the speciation form of this element, but also on environmental conditions such as soil pH, organic and inorganic matter content, and humidity. Plants take up this element from the soil mainly in the form

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of Zn(II) ion, which can be further metabolized to organic forms, i.e., Zn bound with proteins, peptides, amino acids, carotenoids, alcohols, phenolic compounds, favonols, glycosides, and stilbenoids (Ruzik & Kwiatkowski 2018). In aquatic organisms, the amount of Zn absorbed strongly depends on the speciation of the metal in the environment. Seafood contains generally protein-bound Zn, which does not undergo signifcant bioaccumulation (Cornelis et al. 2005).

4.2.3 SPECIATION OF TOXIC ELEMENTS Assessment of the total content of toxic elements such as As, antimony (Sb), tin (Sn), Hg, Cd, or Pb in food samples does not necessarily indicate the danger associated with the consumption of such products. The toxicity of these elements depends on their oxidation state or the presence of the element in the inorganic or organic form. The toxicity of organic As is low or practically negligible, while inorganic As causes signifcant acute and chronic toxicity. Arsenic in seafood occurs mainly in its non-toxic compounds, i.e., arsenobetaine and arsenocholine. Hence, there is no risk associated with consuming seafood extremely rich in arsenobetaine, although the health risks for As sugars still cannot be assessed. In the case of plants, it acts as an analog of P due to the chemical similarity, and inorganic forms of this element are usually taken up and accumulated in the roots, while organic forms can be absorbed through leaves or tree bark (Cornelis et al. 2005; Raj & Maiti 2020). Nevertheless, terrestrial foods contain much less As but the element is present mostly in inorganic form. Especially interesting is rice and its products which can contain very high concentrations of inorganic As, which might be of great danger to specifc groups of the population (Khouzam et al. 2012; Raj & Maiti 2020). The toxicity of Sn also depends on its form, and organotin compounds such as tributyltin (TBT), dibutyltin (DBT), and monobutyltin (MBT) are more toxic than inorganic compounds. Moreover, tributyltin tends to bioaccumulate in fsh and other edible marine organisms such as mollusks and crustaceans. The main source of butyltin in fsh and seafood is TBT-based antifoulants, widely used on ships (Capar & Szefer, 2005). In the case of Pb, it was observed that its organic species are generally more toxic than the inorganic ones (Timbrell 2008). Similarly to As, Pb can be accumulated by marine organisms such as clams, fsh, and algae to levels that exceed water concentrations (Ravipati et al. 2021). Due to the low mobility of Pb in plants, the greatest amounts of this element can be accumulated by root plants. Most of the Pb taken up by the plants remains in the roots, because of its binding to ion-exchangeable sites on the cell wall and extracellular precipitation deposited on the cell wall (mainly in the form of PbCO3) (Raj & Maiti 2020). Living organisms, to defend themselves against the toxic effects of Pb, bind this element with metallothioneins (Wong et al. 2017). Cadmium speciation form in which it occurs in food or the environment may affect the level of its absorption or migration path in the body. However, there is no connection between the degree of toxicity and the speciation form of Cd introduced into the organism. This element is accumulated mostly in the plant’s root, and the

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accumulation is even higher when Cd enters the plant system through foliar deposition (by atmospheric deposition). In plants, the chlorophyll content acts as the indicator of the upper critical limit of Cd, thus, the low chlorophyll concentration indicates high metal accumulation (Kabata-Pendias & Mukherjee 2007). Cadmium in living organisms is bound to metallothioneins but it also shows an affnity for phosphate groups and for other side chains of protein (Raj & Maiti 2020). Mercury toxicity is strongly dependent on the form in which it is absorbed, but the most toxic are methylated forms of Hg, not metallic Hg or its inorganic compounds. The greatest exposure to Hg is related to the intake of this element with food, in which the vast majority of Hg is in a highly toxic form – methylmercury (Cornelis et al. 2005). Plants can take up this element from the soil in both Hg(II) and methylated forms. Its transport from the root to the above-ground parts of plants is species dependent, nevertheless, it is assumed that most of it is accumulated in the root (Shahid et al. 2020). Mercuric cations have a strong affnity for –S groups and subsequently disturb almost any function where critical or non-protected proteins are involved. Mercury decreases the chlorophyll content, bioelements, and tissues’ water content. Food is considered the most important source of exposure to Hg poisoning. Mercury can be accumulated by aquatic organisms living in both fresh and salt water. This element accumulates primarily in the muscle tissue in the methylated form; much smaller amounts of Hg were determined in the liver and kidneys (Cornelis et al. 2005).

4.3

CHANGES IN THE CONTENTS AND DISTRIBUTION OF MINERAL COMPONENTS IN FOODS DUE TO STORAGE AND PROCESSING

4.3.1 NEGATIVE EFFECTS OF FOOD PROCESSING Food processing concerns industrial and homemade preparations, which have similar goals, i.e., elimination of undesirable impurities, microorganisms, and non-nutrients, enzyme inactivation, increasing the digestibility and absorption of nutrients, improving the structure and consistency, and giving the appropriate sensory characteristics to the food. However, negative effects such as loss of health-promoting components and exposure of the body to harmful substances generated during improper processing can occur. Mineral components during food processing behave differently, depending on the type of processed product and the operations and technological processes used during the preparation of the raw materials. Peeling, milling, cooking, thawing, blanching, or refning can lead to mineral losses, even up to 90%. Freezing, in general, has no effect on bioelements. However, preprocessing in the form of blanching or improperly conducted thawing can result in losses of watersoluble nutrients, including macro- and microelements. Losses of elements due to food processing can be compensated by the addition of mineral salts, yeast, or other organic products such as bran. Most losses of Mg, Ca, and Fe arise during the peeling of vegetables as their peels are richer in elements than the cortex. Moreover, frozen vegetables thawed at room

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temperature lose many mineral components and other nutrients in the thaw drip. Frozen fruits should be consumed just after thawing altogether with the juice.

4.3.2

POSITIVE EFFECTS OF FOOD PROCESSING

On the other hand, the positive effect of food preprocessing such as soaking, fermentation, and germination, is the improvement of mineral components’ bioavailability through phytase activation. The lower content of phytates results in the greater bioavailability of such macroelements as Ca or Mg. Also, blanching vegetables in water or steam allows the removal of excess nitrates that may be present in these products as a result of nitrogen fertilization of the soil (Synowiecki 2007).

4.3.3 INFLUENCE OF THERMAL PROCESSING During the traditional cooking or blanching of vegetables, the extraction of mineral components can be observed, but the food itself becomes more digestible. The related losses are in the range of 30–65% for K, 15–70% for Mg and Cu, and 20–40% for Zn (Szefer 2013). The effect of blanching on mineral components is a function of surface area per mass of product and depends on the degree of maturity of the product, blanching method, time, and method of cooling after blanching (Dandago 2009). In general, steam blanching results in smaller losses of nutrients as the leaching is minimized in this process. Blanching, cooking, and heating in a microwave preserve more nutrients, including bioelements, than in traditional cooking. During cooking some elements such as K, Ca, and Na can be lost by leaching into the cooking liquid (Rickman et al. 2007). Thermal processing of food products can lead to the loss of Se in the food because of the formation of its volatile compounds (Kieliszek 2019). To minimize the loss of mineral components during heat treatment of food, the processing time of fruits and vegetables should be shortened to the necessary minimum, and blanching or microwave heating should be chosen. The liquid or thaw drip should be consumed altogether with the meal. However, boiling products contaminated with Pb, Cd, As, and Al results in their transfer to the liquid. Therefore, the removal of water would be benefcial for the consumer (Lee et al. 2019). Although the heat treatment of canned food should not cause a loss of mineral components, it may occur due to the increased temperature and the extraction of bioelements to the liquid, rarely consumed. Canning can cause losses of Mn (87%), Co (71%), and Zn (40%) in spinach. Beans and tomatoes as a result of such processing might lose up to 60% and up to 83% of Zn, respectively. Cobalt can be extracted during canning from carrots (70%), beetroots (67%), and peas (89%) (Belitz et al. 2009). However, the concentration of some elements such as Na, Ca, or other elements might increase. Sodium comes from salt, which is usually added to canned products as a favor enhancement, whereas Ca, as well as other elements, are taken up from hard water used in processing. Calcium can be also added to some vegetables such as tomatoes to minimize softening during processing (Rickman et al. 2007). Mineral components’ concentrations usually do not change during the storage of canned products, except for Fe. This element’s content can increase in foods

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canned in tin-plated steel cans. On the other hand, the Cu amount can decrease due to a reaction with tin (Rickman et al. 2007).

4.3.4

INFLUENCE OF PROCESSING ON CEREALS

Cereals and legumes undergo pre-processing operations, including dehulling, milling, refning, and polishing, which change the nutritional composition of the product to varying degrees. The concentration of essential nutrients decreases with the degree of milling with minor alteration in energy density (Oghbaei & Prakash 2016). Milling removes the husk and sometimes the bran layers and results in an edible portion in the form of a powder with varying particle sizes that is free of impurities. This process decreases the contents of almost all nutrients in wheat four, including mineral components such as Mn (89%), Fe (76%), Co (68%), Cu (68%), Zn (78%), and Se (16%). Polishing of rice is also associated with bioelements’ content decrease, i.e., Cr (75%), Zn (75%), Cu (45%), and Mn (26%) (Belitz et al. 2009). In polished groats (barley, buckwheat) losses amount to 20–70%, and in semolina up to 85% in relation to the amount in grain. Therefore, four enrichment with mineral components after milling is necessary (Dandago 2009).

4.3.5 INFLUENCE OF PROCESSING ON FOODS OF ANIMAL ORIGIN Changes in mineral composition in dairy products may result from the precipitation of casein that binds Ca, Zn, Cu, and other metals. The losses may also be due to ultrafltration used in the production of whey (Synowiecki 2007). Meat, poultry, and fsh may lose signifcant amounts of mineral components as a result of leaching during washing, thermal processes, as well as during thawing. However, the losses of elements depend on their forms. Those present in the form of soluble dissociated salts (some Na and small amounts of Ca, P, and K) are leaching, while bound with proteins (almost all Fe) remain in the meat. Besides, a small addition of salt (approx. 1% in meat tissue) before freezing sometimes reduces thaw drip losses from frozen meat by almost 50%.

4.3.6 THE INFLUENCE OF PACKAGING The source of changes in the mineral components’ content in food may also be the packaging from which elements penetrate into the food during storage. Food product packaging is an integral part and has direct contact with the contents, so it plays an important role in consumer health protection. Materials for food packaging should not affect the quality of the products. The occurrence of leaks in the inner layer of food cans leads to the formation of corrosion products (Zalewska & Kowalik 2017). The age of the can and the storage time are important factors in determining Fe uptake (Reilly 2002). Damage to the inner protective coating of the can leads to Sn contamination of the food. Particularly high Sn concentration was found in canned fruit and vegetable preserves with an acidic pH. Lead can get into acidifed food products from cans or from pottery glaze. Also, wine stored for long periods

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in lead-based crystal carafes may become contaminated with Pb. Aluminum can also penetrate acidic foods and drinks from dishes, cans, and Al foils and poses a threat to human health. Reduced skeletal mineralization (osteopenia), neurotoxicity, dermatitis, and digestive disorders are the symptoms of Al toxicity (Grembecka & Szefer 2011). During the production of plastic packaging, low concentrations of heavy metals can be used as catalysts, migrating to food to a different degree. In addition to catalysts, various substances, such as antioxidants, deactivators, or stabilizers, added at certain production stages, may include Ni, Cu, Pb, Sb, Sn, Cd, and Zn.

4.4

4.4.1

THE EFFECT OF THESE COMPONENTS ON THE STABILITY AND SENSORY PROPERTIES AS WELL AS THE BIOLOGICAL VALUE OF FOODS ENZYMATIC BROWNING AND INTERACTIONS BETWEEN METAL IONS AND POLYSACCHARIDES

Mineral components cause changes in the quality and functional properties of food during its processing and storage. They infuence the gelling ability, solubility, denaturation and aggregation of proteins, meat maturation, and the rate of microbial growth, enzyme activity, and oxidation and reduction reactions. Their action can often be quite the opposite. While some of them can inhibit the growth of microorganisms, others in trace amounts are necessary for the proper course of fermentation. Copper and Fe ions as well as other transition metals accelerate non-enzymatic browning and cause deterioration of the taste and smell of food due to Maillard reactions. These ions are also catalysts for the thermal decomposition of trimethylamine to form dimethylamine, which has an impact on the formation of the smell of fsh meat. Pre-processed white vegetables (sliced, cut, chopped, or diced) are susceptible to enzymatic browning as the cells are broken down liberating enzymes from the tissues, in particular polyphenol oxidases. Enzymatic browning can be controlled by dipping the vegetables into a water solution containing ascorbic acid and citric acid at a maximum concentration of 1% for a couple of minutes (Commission Regulation [EU] 2020/1419). The rheological and functional properties of food products can be also affected by interactions between metal ions and polysaccharides. Neutral polysaccharides form weak complexes with cations in neutral or non-alkaline media, whereas in alkaline solution the affnity between cation and donor is great. Anionic polysaccharides (including alginate, xanthan gum, and carrageenan) have a strong affnity for metal counterions, even at low concentrations. The ability of alginates to form gels with Ca ions makes them suited to prepare fruits and meat analogs.

4.4.2

SODIUM CHLORIDE AND ALKALI METALS INFLUENCE

Sodium chloride has a preservative effect in the concentration range of 1% to 20%. The use of higher salt concentrations (e.g., in salted herring) allows for

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a long-lasting preservative effect, but the product requires desalination before consumption. Sodium chloride is an osmoactive substance that leads to shrinkage of the protoplasmic contents and loss of cell membrane semi-permeability, and even the separation of the cell wall from the cytoplasmic membrane, which leads to cell death. Salt also reduces the solubility of oxygen in liquid environments and weakens the activity of intracellular proteolytic enzymes. Moreover, its addition increases the water absorption and water-holding capacity of the meat. The highest water absorption is achieved by adding 4–5% of this compound. Alkali metal phosphates added in the process of salting or curing (most often mixtures of poly and sodium pyrophosphates) increase water absorption more than salt, and their effect is noticeable at concentrations lower than 1%. In the case of milk, the addition of various phosphates, especially polyphosphates, which are effective Ca complexing agents, can increase the caseinate stability of milk. The addition of Ca ions has the opposite effect and decreases the stability of milk. Salting or freezing food causes an increase in inorganic salts in liquids found in tissues, which affects the solubility and functional properties of proteins to a degree depending on the acidity of the environment. Low salt concentration increases the solubility or water absorption of proteins (Sikorski 1992). Sodium chloride also reduces the unwanted gelling of collagen during the sterilization of canned food. Magnesium, Ca, and Ba ions have the greatest ability to destroy water structures and stabilize the structure of macromolecules. In contrast, Na and K ions affect proteins to a lesser extent. Reducing the content of Ca and Mg in the tissues (by displacement with salt or dilute NaHCO3 solutions) facilitates the denaturation and digestion of proteins with proteolytic enzymes. Calcium cations, released after cell death, also make maturing meat more tender. The decomposition of Ca and Mg complexes with nucleotides is also the cause of a rapid decrease in water absorption in the primary stages of meat maturation. During maturation, proteins and glycogen, the components of the muscle tissue, are partially broken down and the desired technological and culinary properties (mainly tenderness, palatability, juiciness, color, ability to bind added water, emulsify fats, swell, and gel), as well as nutritional (e.g., increasing the digestibility of nutrients), are developed. The polysaccharideprotein complexes formed with Ca are less prone to coagulation at elevated temperatures or due to lowering pH than the original proteins. The formation of complex compounds of Ca with amino acids also partially inhibits the reactions of the non-enzymatic browning of food.

4.4.3 EFFECT OF HARD WATER The high degree of water hardness, which is mainly related to the content of Ca and Mg ions, has a signifcant effect on the tarnishing of vegetables and fruits subjected to heat treatment. In addition, vegetables and fruit turn gray and may seem much less appetizing. The high content of Ca and Mg ions in the water intended for cooking blocks pectins and the process of dissolving them, thus making it diffcult for the fruit to soften. However, the disadvantageous effect of hard water usage does not occur in the case of plant products with a high content of phytic or oxalic acid, which

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bind Ca and Mg in the form of hardly soluble salts. Calcium and Mg ions as well as other ingredients contained in hard water can have a signifcant impact on baking. A high degree of hardness can lead to a change in the structure of the gluten contained in the four and has a negative effect on yeast fermentation. As a result, the dough can become rubbery, sticky, and simply heavy. In addition, the proteins in the four fnd it more diffcult to absorb hard water.

4.4.4

EFFECT ON OXIDATION

Oxidation of lipids and pigments is considered one of the most important processes negatively affecting the quality (functional, sensory, texture, and nutritional value) of food products during storage. The rate of oxidation of lipid compounds is determined by several factors. First, the speed of the reaction increases signifcantly with increasing oxygen pressure as well as temperature. Light, the presence of heavy metals (Cu, Fe, and Mn), salt, water, and other non-lipid components also play an important role. Factors that slow down the oxidation reaction include reduced temperature, reduced oxygen pressure, an inert gas environment, and the presence of antioxidants. Among metals, the main prooxidants are Fe and Cu, which free transition ions (Fe[III] or Cu[II]), after being reduced, can react with hydrogen peroxides. As a result of this reaction, there are produced hydroxyl radicals that can initiate the lipid autooxidation process. Metal ions act catalytically both during the initiation stage of autooxidation as well as during propagation. High catalytic activity in animal products is characteristic especially of Fe, both heme (myoglobin, hemoglobin) and non-heme. Although Fe ions exhibit similar catalytic activity to Cu ions, they are present in greater amounts, i.e., in the form of mineral salts or as a component of heme, cytochromes, ferritin, transferrin, and many enzymes. Heme Fe can initiate lipid oxidation in both raw and heated meat, whereas non-heme Fe plays a greater role in accelerating the lipid oxidation processes in heated meat, especially in an acidic environment (pH value around 4.5) (Dai et al. 2014). Due to the greater content of Fe and myoglobin, beef is more susceptible to protein carbonylation than pork or poultry. Food products contain substances that accelerate auto-oxidation reactions and antioxidants that inhibit them. The effect of these substances depends on the raw material storage and processing conditions, such as temperature, oxygen concentration, pH, the amount of added sodium chloride, the storage period, as well as the concentration of metal ions, and the degree of cell damage during processing. The oxidation processes of food products can be effectively controlled and limited by the use of antioxidants, metal ion sequestrants, and oxidants, radical scavenging enzymes, or proteases that hydrolyze proteins that catalyze the production of radicals. These compounds may be used alone or in various mixtures, and may include a variety of additives, from synthetic phenolic antioxidants to natural plant-derived antioxidant ingredients. The most important antioxidants found in animal tissues include ascorbic acid, urate, tocopherols, glutathione, and other thiol compounds as well as enzymes: superoxide dismutase, catalase, and glutathione peroxidase which inactivate lipid radicals. These enzymes are activated by Cu, Mn, Zn, and Se ions, and even trace amounts of them have a strong antioxidant effect. Food acids and their

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salts as well as phosphates in all stages of condensation bind to Fe and Cu ions and, thus, prevent or limit catalytic reactions with oxygen. The addition of phosphates (pyrophosphates and hexametaphosphates) to meat delays the processes of lipid oxidation in heated meat by complexing metals, especially Fe. Similarly, tocopherols slow down lipid oxidation in muscle tissues. Fruit and vegetable products are more resistant to oxidation than meat products mainly due to the lack of heme Fe and the presence of tocopherols, β-carotene, and polyphenols. Flavonoids and their glycosides found in plants form chelate compounds with Fe and other transition metal ions and are inhibitors of Ca-activated lipoxygenase and other enzymes, which are involved in lipid oxidation. Metal ion sequestrants are citric, malic, tartaric, and ethylenediaminetetraacetic (EDTA) acids, as well as phosphates and lecithin. EDTA is a metal ion sequester, which is approved for use in the food industry as a stabilizer and antioxidant. It also acts as a bacteriostatic agent as it forms chelates with multivalent cations, thus, inhibiting the growth of Staphylococcus aureus cells. However, this effect is easily reversible by the addition of other cations for which EDTA has a higher affnity. Ascorbic acid can be both an antioxidant and an initiator of the oxidative reaction, depending on the concentration. When used in small amounts, it accelerates the oxidation reactions, while at higher concentrations it has an antioxidant effect. Reactions involving ascorbic acid reduce the nutritional value of the products due to excessive loss of vitamin C.

4.5

INTERACTIONS OF MINERAL ELEMENTS WITH OTHER FOOD COMPONENTS

Nutrient interaction means the impact of one nutrient on the other’s bioavailability. These interactions may affect bioavailability either in a positive or negative way. Mineral components can interact with all macronutrients, i.e., proteins, carbohydrates, and fats as well as micronutrients, i.e., vitamins and other bioelements. Also, non-nutrients and antinutritive compounds present in the diet, as well as drugs or supplements can have a large impact on elements’ bioavailability. Mineral components are particularly sensitive to certain substances that hinder their absorption. These substances include phytic acid, oxalic acid, fber, polyphosphates, tannins, and polyphenols. Proteins bind and carry certain mineral components such as Fe, Cu, and Ca, thus, their improper intake results in the impaired function of these elements. On the other hand, some large and poorly digestible proteins may bind bioelements tightly, thus, hindering their absorption. Increased intake of protein can sometimes help improve the body’s Mg status. Triacylglycerols and long-chain fatty acids may interact with Ca and Mg to form soaps and decrease their bioavailability. Some macromolecular carbohydrates such as pectins, cellulose, hemicellulose, and polymers produced in the Maillard reaction during cooking processing or storage may bind mineral components in the lumen (Nabrzyski 2007). Diet high in simple sugars increases the urinary excretion of Cr. On the other hand, lactose, along with vitamins C and D, increases Ca absorption.

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Calcium absorption is infuenced by many factors, including age, the amount needed, and foods consumed at the same time. Its absorption can be negatively affected by a diet high in dietary fber and phytates as well as excessive P intake (Fishbein, 2004). Moreover, oxalic acid, which can be found in spinach, beets, celery, and other food products, can form with this element insoluble complexes that are excreted in the feces. On the other hand, vitamin D accelerates the absorption of Ca from the gastrointestinal tract. A high intake of K reduces the urinary excretion of Ca, thus increasing the amount of K-containing foods in the diet may be helpful in maintaining the density and strength of bones. High consumption of Na, caffeine, or proteins increases the excretion of Ca. The bioavailability of mineral components is also infuenced by reactions between the individual components. Although Mg is required for Ca to maintain a balanced role in the body’s metabolism, these two macroelements compete with each other for intestinal absorption, thus, they should not be taken in the form of supplements at the same time. However, healthy diets almost always need to contain foods rich in both bioelements. Magnesium also has an important relationship with K and helps regulate its movement in and out of our cells. Calcium decreases the absorption of heme and nonheme Fe. However, both elements, Ca and Fe, are more easily absorbed in meat than in vegetables because the latter contains these mineral components mainly in the form of insoluble complexes with phytic acid, phosphates, oxalates, and carbonates. Therefore, less than 8% of Fe in plant food is bioavailable to humans. However, even 25 mg of vitamin C can double the absorption of non-heme Fe from the meal. Vitamin C can signifcantly interact with bioelements. Its food level amounts can signifcantly enhance Fe uptake and metabolism, whereas high doses can interfere with Cu metabolism. Other nutrients that support Fe metabolism are Cu and vitamin A. Ascorbic acid also enhances Se absorption, which is required to keep glutathione in its active form (Nabrzyski 2007). Besides glutathione, Se is indirectly responsible for keeping intact the body’s supply of vitamin C and vitamin E. Both Fe and Cu defciencies appear to increase the risk of Se defciency. Copper defciency increases the formation of free radicals and leads to a reduction of defense against oxidative stress. Copper reacts with many mineral components including Se, Fe, Zn, S, and Mo. Zinc in higher doses, taken on a daily basis over an extended period, may lower the availability of Cu. The presence of too much Fe in a feeding formula for infants can decrease the absorption of Cu from that product (Lönnerdal 2017). Zinc, even in doses available in food, can compromise the body’s supply of Cu unless foods rich in this element are also included in the diet. However, high intakes of Zn, usually obtained from supplements, can also decrease the absorption of Ca. Zinc and Cu or Mo and Cu affect the Zn/Cu and Cu/Mo ratios leading to Cu and Mo defciencies, respectively. Moreover, Zn reduces the bioavailability of Fe, which also diminishes the absorption of Zn. The bioavailability of Zn is higher in meat, eggs, and seafood due to the absence of compounds that inhibit its absorption and the presence of certain amino acids (cysteine and methionine) that improve Zn absorption. Zinc in whole grain products is less bioavailable due to the relatively high content of phytic acid and oxalates (Grembecka & Szefer 2011). Zinc absorption can also be

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infuenced by intestinal parasites, excessive sweating, drinking alcohol, and a diet rich in Ca (Nabrzyski, 2007). Although Zn is associated with potentially detrimental effects on Cu and Ca, it is also supportive of another nutrient, i.e., vitamin A. Without it, vitamin A cannot be effectively transported around the body and effciently used. Iodine is required in the production of thyroid hormones. The conversion of thyroxine (T4) to triiodothyronine (T3) requires the removal of the I molecule from T4. This reaction requires the mineral Se (Grembecka & Szefer 2011). In the case of selenium defciency, the conversion of T4 to T3 is slowed, and less I is available for the thyroid to produce new hormones. Also, As interferes with the uptake of I by the thyroid, leading to goiter. In addition, dietary defciency of vitamin A, vitamin E, Zn, and/or Fe can exaggerate the effects of I defciency. High doses of Mn may inhibit the absorption of Fe, Cu, and Zn. On the other hand, high intakes of Mg, Ca, P, Fe, Cu, and Zn may inhibit the absorption of Mn. Although teas are a rich source of Mn, the tannins present in tea can moderately reduce its absorption. Also, amino acids can interact with certain mineral components, whereas phytic acid, which can be found in whole grains, reduces the bioavailability of Fe, Ca, Zn, Mg, Mn, and Cr. However, diets rich in whole grains contain signifcant amounts of Cr, thus, the activity of phytic acid in grains is unlikely to increase our risk of Cr defciency. Moreover, vitamin C increases the absorption of Cr. The activity of bioelements can be also modifed by toxic elements such as Pb, Cd, and Hg. A diet rich in Ca and phytates limits Pb absorption. However, low Ca as well as P levels promote increased absorption and retention of Pb and Cd (Alonso López et al., 2004). The effects of P and Ca defciencies are additive (Nabrzyski, 2007). Absorption and retention of Pb and Cd may also result from Fe defciency. On the other hand, a low-fat diet has been found to reduce Pb absorption. Zinc malabsorption is observed in the presence of Hg and Cd (Alonso López et al., 2004; Nabrzyski, 2007). Certain medications, such as tetracycline, can also inhibit the absorption of mineral components, including Ca, Fe, Zn, and Mg. Patients treated with high doses of oral Al-based drugs (phosphate binders, antacids, anti-diarrheal drugs) may experience malabsorption of P, Ca, Mg, Fe, and Mn. Synergistic and antagonistic relationships between various elements present in food result in the ultimate biological activity of each of them.

4.6 MINERAL FOOD ADDITIVES Many mineral components are used in food production as additives supporting the course of technological processes, shaping the physical and sensory features of fnished products, inhibiting the growth of microorganisms, and extending product durability (Table 4.2). Food additives containing mineral compounds belong to different groups depending on their technological function and can be used when it is necessary and is not hazardous to health. Many mineral compounds are listed as preservatives, sequestrants, favor enhancers, and nutritive additives. Their characteristics can be found in Table 4.2. Moreover, calcium, and sodium stearoyl lactylates as well as sodium, potassium, calcium, and magnesium salts of fatty acids are used as emulsifers that are crumb

E263

E213; benzoic acid calcium salt

E227; calcium hydrogen sulphite; calcium hydrogensulfte

E282; propanoic acid calcium salt; ethylformic acid calcium salt; calcium propanoate

E203; calcium hexadienoate; sorbic acid calcium salt; hexadienic acid calcium salt; 2-propenylacrylic acid calcium salt

E226

Calcium benzoate

Calcium bisulfte

Calcium propionate

Calcium sorbate

Calcium sulfte

Synonyms

Calcium acetate

Preservatives

Name of compound

Bleaching agent; antimicrobial; preservative; dough modifer; vitamin C stabilizer; not for use in meats, sources of vitamin B1, raw fruits and vegetables or fresh potatoes

Baked goods; beverages; bread; cake batters; cake fllings, cake toppings; cheese, cottage cheese; fsh (smoked, salted); fruit juices (fresh); fruits (dried); margarine; pickled products; pie crusts; pie fllings; salad dressings; salads (fresh); sausage; seafood cocktails; syrups; wine

Baked products; cheese products

Bleaching agent; antimicrobial; preservative; dough modifer; vitamin C stabilizer; not for use in meats, sources of vitamin B1, raw fruits and vegetables, or fresh potatoes

Beverages; margarine; bakery products; fsh products; fruit juice; fruit pulp; jam; liquid egg; whole egg; egg yolk; mayonnaise; mustard; pickles; sauces; ketchup; sausage

Beer; ale; stout; porter; malt beverages

Food use

TABLE 4.2 The Selected Mineral Compounds Used as Food Additives

Preservative in cider and fruit juices; disinfectant in brewing vats; in sugar manufacture

Antimicrobial preservative; favoring; acidulant

Antimicrobial preservative; antimycotic; favoring agent; preservative additive; mold inhibitor

Antimicrobial preservative; antioxidant and bleach

Antimicrobial preservative

Antimold and antirope agent; stabilizer; buffer

Function in foods

(Continued)

UK and Europe – conditionally permitted USA, Canada, Australia – not permitted ADI 0–0.7 mg/kg bw

USA – permitted (GRAS) UK and Europe – conditionally permitted Canada, Australia, Japan – permitted in specifc foods ADI – 25 mg/kg bw

USA – permitted (GRAS) Canada, Australia – permitted in specifc foods UK and Europe – conditionally permitted

USA, Canada, Australia – not permitted UK and Europe – conditionally permitted ADI 0–0.7 mg/kg bw

UK and Europe – conditionally permitted Canada, Australia – permitted in selected foods ADI 0–5 mg/kg bw

USA, UK, and Europe – permitted (GRAS) Australia/NZ – permitted, restrictions of use Canada – not permitted

Legislation

The Role of Mineral Components 89

90

TABLE 4.2 (CONTINUED) The Selected Mineral Compounds Used as Food Additives Name of compound

Synonyms

Food use

Function in foods

Legislation

E 261(i)

Meat products; ready-to-eat meals

Preservative; acidity modulator

USA – permitted (GRAS) Canada – not permitted UK and Europe – permitted Australia – permitted in specifc foods

Potassium benzoate

E212; benzoic acid potassium salt

Beverages; margarine; bakery products; fsh products; fruit juice; fruit pulp; jam; liquid egg; whole egg; egg yolk; mayonnaise; mustard; pickles; sauces; ketchup; sausage

Antimicrobial preservative

USA – permitted Canada, Australia – permitted in specifc foods UK and Europe – conditionally permitted ADI 0–5 mg/kg bw

Potassium metabisulfte

E224; disulfurous acid dipotassium salt; potassium pyrosulfte; potassium disulfte; dipotassium disulfte; potassium pentaoxodisulfate

Food preservative; antioxidant; sterilizer; brewing; wine making; not for use in meats, sources of vitamin B1, raw fruits and vegetables, or fresh potatoes

Antimicrobial preservative; antioxidant; bleach

USA – permitted Canada, Australia - permitted in specifc foods UK and Europe – conditionally permitted ADI 0–0.7 mg/kg bw

Potassium nitrate

E252; saltpetre; nitre

Color retention agent; preservative

Formerly used as a curing agent, now superseded by nitrite

USA – permitted Canada, Australia - permitted in specifc foods UK and Europe – conditionally permitted ADI 0–3.7 mg/kg bw Expressed as nitrate ion; ADI does not apply to infants below the age of 3 months

Potassium nitrite

E249

Cured meats; meat products; fsh products

Antimicrobial preservative; color fxative; favor enhancer

USA – permitted Canada, Australia – permitted in specifc foods UK and Europe – conditionally permitted ADI 0–0.07 mg/kg bw Evaluated as nitrite and expressed as nitrite ion; ADI applies to all sources of intake but not to infants below the age of 3 months

Potassium propionate

E283

Baked products; cheese products

Antimicrobial preservative; antimycotic; favoring agent; preservative additive; mold inhibitor

USA, Canada – not permitted UK and Europe – conditionally permitted Australia/NZ – permitted in specifc foods

(Continued)

Małgorzata Grembecka

Potassium acetate

Synonyms

Food use

Potassium sorbate

Name of compound

E202; potassium hexadienoate; sorbic acid potassium salt; 2,4-hexadienoic acid potassium salt

Baked goods; beverages; bread; cake batters; cake fllings, cake toppings; cheese; fsh (smoked, salted); fruit juices (fresh); fruits (dried); margarine; pickled products; pie crusts and fllings; salad dressings; salads (fresh); sausage; seafood cocktails; syrups; wine

Antimicrobial preservative; favoring; acidulant

Function in foods

USA – permitted (GRAS) Canada, Australia – permitted in specifc foods UK and Europe – conditionally permitted ADI 0–25 mg/kg bw

Legislation

Sodium acetate

E262

Jams; jellies; preserves

Preservative; favoring

USA – permitted (GRAS) UK and Europe – permitted Canada – not permitted Australia/NZ – permitted in specifc foods

Sodium benzoate

E211; benzoic acid sodium salt

Beverages; margarine; bakery products; fsh products; fruit juice; fruit pulp; jam; liquid egg; whole egg; egg yolk; mayonnaise; mustard; pickles; sauces; ketchup; sausage

Preservative; antimicrobial agent; favoring agent; adjuvant; antimycotic migrating from food packaging

USA – permitted (GRAS) Canada, Australia, Japan – permitted in specifc foods UK and Europe – conditionally permitted ADI 0–5 mg/kg bw

Sodium bisulfte

E222; sodium acid sulfte; sulfurous acid monosodium salt; sodium sulfhydrate

Preservative; not for use in meats, sources of vitamin B1, raw fruits and vegetables, or fresh potatoes

Antimicrobial preservative; antioxidant; bleach

USA – permitted (GRAS) Canada, Australia, Japan – permitted in specifc foods UK and Europe – conditionally permitted

Sodium diacetate

262(ii); sodium hydrogen diacetate

Bread; meat, fsh, poultry, and their products

Antimold and antirope agent

USA – permitted (GRAS) Australia/NZ – permitted in specifc foods UK and Europe – permitted Canada – not permitted ADI 0–15 mg/kg bw

Sodium metabisulfte

E223; disulfurous acid disodium salt; sodium pyrosulfte; sodium bisulfte

Preservative; antioxidant; favoring in cherries and shrimp; boiler water additive for food contact; bleaching agent; not for use in meats, sources of vitamin B1, raw fruits and vegetables, or fresh potatoes

Antimicrobial preservative; antioxidant; bleach

USA – permitted (GRAS) Canada, Australia, Japan – permitted in specifc foods UK and Europe – conditionally permitted ADI 0–0.7 mg/kg bw

91

(Continued)

The Role of Mineral Components

TABLE 4.2 (CONTINUED) The Selected Mineral Compounds Used as Food Additives

92

TABLE 4.2 (CONTINUED) The Selected Mineral Compounds Used as Food Additives Name of compound

Synonyms

Food use

Function in foods

Legislation

E251; chile saltpetre; cubic or soda nitre

Antimicrobial preservative

Antimicrobial agent; preservative; source of nitrite; color fxative in cured meats, fsh, and poultry; boiler water additive; curing salt

USA, Canada, Australia/NZ – permitted in specifc foods UK and Europe – conditionally permitted ADI 0–3.7 mg/kg bw Expressed as nitrate ion; ADI does not apply to infants below the age of 3 months

Sodium nitrite

E250; nitrous acid sodium salt

Cured meats; meat products; fsh products

Antimicrobial agent; color fxative; favor enhancer

USA, Canada, Australia/NZ – permitted in specifc foods UK and Europe – conditionally permitted ADI 0–0.07 mg/kg bw Evaluated as nitrite and expressed as nitrite ion; ADI applies to all sources of intake but not to infants below the age of 3 months

Sodium propionate

E281; propanoic acid sodium salt; ethylformic acid sodium salt; sodium propanoate

Baked products; cheese products

Antimicrobial preservative; antimycotic; favoring agent; preservative additive; mold inhibitor

USA – permitted (GRAS) Canada, Australia/NZ – permitted in specifc foods UK and Europe – conditionally permitted

Sodium sorbate

E201; sodium hexadienoate; sorbic acid sodium salt; 2-propenylacrylic acid sodium salt

Baked goods; beverages; bread; cake batters; cake fllings, cake toppings; cheese, cottage cheese; fsh (smoked, salted); fruit juices (fresh); fruits (dried); margarine; pickled products; pie crusts; pie fllings; salad dressings; salads (fresh); sausage; seafood cocktails; syrups; wine

Antimicrobial preservative; favoring; acidulant

USA – permitted (GRAS) Canada, Australia, Japan – permitted in specifc foods UK and Europe – conditionally permitted ADI 0–25 mg/kg bw

Sodium sulfte

E221; sulfurous acid sodium salt (1:2); sulfurous acid disodium salt; sodium sulfte (2:1); disodium sulfte

Food preservative and antioxidant; boiler water additive; bleaching agent; not for use in meats, sources of vitamin B1, raw fruits and vegetables, or fresh potatoes

Antimicrobial preservative; antioxidant; bleach

USA – permitted (GRAS) Canada, Australia, Japan – permitted in specifc foods UK and Europe – conditionally permitted ADI 0–0.7 mg/kg bw

(Continued)

Małgorzata Grembecka

Sodium nitrate

Name of compound

Synonyms

Food use

Function in foods

Legislation

Sequestrants Calcium acetate

E263; lime acetate

Edible caseinates; instant puddings; sweet sauces; baked goods; gelatins; syrups; cake mixes; fllings; toppings

Used in the manufacture of acetic acid; source of calcium; emulsifer; frming agent; mold-control agent

USA – permitted (GRAS) UK, Europe, Canada, Japan – permitted

Calcium chloride

E509, calcium chloride anhydrous

Sliced apples; canned fruit; coffee and tea; apple pie mix; canned milk; processed fruit; jams and jellies; milk powders; fruit juice; canned tomatoes and vegetables; baked goods; cheese; beverages

Sequestrant; frming agent; anticaking agent; antimicrobial; curing agent; favor enhancer; humectant; nutrient supplement; pH control agent; pickling agent; processing aid; stabilizer; surfactant; texturizer; thickener in foods; solvent in preparation of hops for beer brewing

USA – approved for use at 3% maximum UK and Europe – approved, ADI not specifed Japan – approved for use at 1% maximum WHO limitation: 350–800 mg/kg in canned fruits/ vegetables; 200 mg/kg in preserves, processed cheese

Calcium citrate

E333; tricalcium citrate; tricalcium salt of 2-hydroxy-1,2,3 propanetricarboxylic acid; tricalcium salt of betahydroxytricarballylic acid

Confections; four; milk powders; jams and jellies; canned vegetables; processed cheese; saccharin; evaporated milk; cream; condensed milk; edible ices; frozen apples

Used in the production of citric acid; improves baking properties of fours; good stabilizer; soluble in fats

USA – permitted (GRAS) UK and Europe, Canada, Japan – permitted

Calcium disodium ethylenediaminetetraacetate

E385; calcium disodium EDTA; calcium disodium edetate

Salad dressing; fats and oils; canned legumes; sauces; nut meat and roasted nuts; prepeeled potatoes; spreads; fried and baked goods; soft drinks; cheese; fruit juices; canned seafood; vinegar and cider; milks; beer and wine; margarine; canned mushrooms; oleomargarine

Stabilizes fats and oils; preservative; prevents rancid odor in fried and baked products; retention of color/ favor in meat; stabilization of vitamins in aqueous systems; promotes texture retention in cucumbers; decreases poor favor in milk

USA, Canada – permitted in certain food products ADI 0–2.5 mg/kg bw

93

(Continued)

The Role of Mineral Components

TABLE 4.2 (CONTINUED) The Selected Mineral Compounds Used as Food Additives

94

TABLE 4.2 (CONTINUED) The Selected Mineral Compounds Used as Food Additives Name of compound

Synonyms

Food use

Function in foods

Legislation

E578; D-gluconic acid calcium salt; calcium di-gluconate

Pickled cucumbers; cured meats; sugar substitute; coffee powders; gelatin and puddings; dairy product analogs; canned fruit and vegetables; baked goods; jams and jellies

Firming agent in fruits and vegetables; acidity regulator; nutrient supplement; sequestrant

USA, Canada – permitted (GRAS) UK and Europe – permitted Japan – 1% as calcium in cured meat products

Calcium phosphate monobasic

E341; calcium phosphate monobasic anhydrous; monocalcium orthophosphate; phosphoric acid calcium salt (2:1)

Ice cream; ice milk mix; sherbet; dough conditioner; leavening agent; frming agent; supplement; yeast foods; acidity regulator; four treatment agent; sequestrant; texturizer; thickener

Slightly soluble in water; insoluble in alcohol; strong acidic taste

USA, Canada, Japan, Europe – permitted (GRAS) MTDI 70 mg/kg bw (as P)

Calcium phosphate tribasic

341(iii); calcium orthophosphate; calcium phosphate tertiary; phosphoric acid calcium salt (2:3)

Ice cream; ice milk mixes

Leavening agent; texturizer; yeast food; dough conditioner; mineral supplement; water retention agent in foods

USA, Canada, Japan; Europe – permitted (GRAS) MTDI 70 mg/kg bw (as P)

Calcium phytate

Hexacalcium phytate

Wine; vinegar; glazed fruit; calcium source for nutrition

Removes excess metals in wine and vinegar products; source of calcium

USA, Canada– permitted (GRAS)

Calcium sulfate

E516

Yeast food; brewing, sherry and wine; artifcially sweetened fruit; cottage cheese; bread (rolls and buns); jelly; canned tomatoes; cheese, cereal fours; canned potatoes and sweet peppers; soft ice cream; frozen apples; confections; puddings

Bleaching agent in four; alkali in cottage cheese; frming agent in canned vegetables; dough conditioner/strengthener, calcium source – with alginates forms dessert gels; nutritional supplement; four treatment agent; sequestrant; yeast food

USA, Canada, Japan, UK and Europe - permitted

(Continued)

Małgorzata Grembecka

Calcium gluconate

Name of compound

Synonyms

Food use

Function in foods

Legislation

Calcium disodium ethylenediaminetetraacetate (disodium EDTA)

E385; edetate disodium; EDTA disodium salt; calcium disodium ethylene diamine tetra-acetate; disodium EDTA; calcium disodium EDTA; calcium disodium edetate

Lard; canned legumes; creamed turkey; fats and oils; potatoes and French fries; dried banana; fruit juices; canned seafood; cereal; ham; bacon; frankfurters; milks; strawberry pie flling; canned corn; egg custard; mayonnaise; canned apples; cheese; frozen ground beef; processed vegetables and fruits

Stabilizes lard and vitamins in food systems; preservative; promotes color and favor retention in foods; decreases crystal formation; sequestrant

USA, Canada– permitted Europe – approved ADI 0–2.5 mg/kg bw

Disodium pyrophosphate

E450a; disodium dihydrogen diphosphate; disodium dihydrogen pyrophosphate

Evaporated milk; cheese; biscuits; pasta products; cured meats; four; cake mixes; doughnuts; poultry products; processed potatoes; canned fsh products

Increases water-holding capacity in meat; emulsifer in cheese; decreases cooked-out juices; buffering agent

USA, Japan; UK, Europe – permitted MTDI 70 mg/kg bw (as P)

Manganese citrate

–

Baked goods; fsh products; milk products; beverages; infant formula; poultry products; dairy products analogues; meat products

Sequestrant; nutritional supplement

USA – permitted (GRAS)

Potassium dihydrogen citrate

E332(i); monopotassium citrate; potassium citrate monobasic

Processed cheese; evaporated milk; margarine; edible ices and ice mix; condensed milk; jams and jellies; edible caseinates; milk powders; citrus marmalade

Chelates and deactivates metal contaminants; increases the effect of preservatives; good stabilizer

USA, Canada, Japan; UK, Europe – permitted

The Role of Mineral Components

TABLE 4.2 (CONTINUED) The Selected Mineral Compounds Used as Food Additives

(Continued)

95

96

TABLE 4.2 (CONTINUED) The Selected Mineral Compounds Used as Food Additives Name of compound

Synonyms

Food use

Function in foods

Legislation

Potassium phosphate dibasic

E340(ii); dipotassium hydrogen phosphate; dipotassium orthophosphate; dibasic potassium phosphate

Cured ham and chopped meat; milk and cream powders; luncheon meat; evaporated milk; processed cheese; edible ices and ice mixes; low sodium products; caramels; sparkling wines; yeast food; cream; cured pork shoulder; condensed milk

USA, Canada, Japan; UK, Europe – permitted MTDI 70 mg/kg bw (as P)

Potassium phosphate monobasic

E340(i); potassium dihydrogen orthophosphate; monopotassium monophosphate; potassium acid phosphate

Milk; whole eggs; caramels; meat products; yeast food; poultry food products; low sodium products; sparkling wine

potassium sodium L(+)–tartrate

E337; potassium sodium dextro-tartrate; rochelle salt; Seignette salt; potassium sodium dextro-tartrate E339(ii); sodium hydrophosphate; dibasic sodium phosphate; disodium hydrogen phosphate E450c; sodium hexametaphosphate; sodium tetraphosphate; sodium hexametaphosphate; sodium tetrapolyphosphate; Graham’s salt

Cheese products; citrus marmalade; margarine; minced meat; jams and jellies; edible ices and ice mixes

Yeast food in brewing industry; acidifying agent; forms soluble complexes with alkali and alkali earth metal ions; decreases amount of cooked-out juice; increases hydration of freeze-thaw products; stabilizes meat emulsions Preserves color in whole eggs; yeast food; acidifying agent; complexes metal ions; decreases amount of cooked-out juice; increases hydration of freeze-thaw products; stabilizes meat emulsions Sequestrant; pH control agent; buffering agent; prevents rancidity with antioxidants; stabilizer in minced meat Buffer; emulsifer; sequestrant; decreases amount of cooked-out juice; dietary supplement; increases and stabilizes pH Decreases amount of cooked-out juice; inactivates metallic ions; retards oxidation; decreases growth of microbes; stabilizer; decreases turbidity in wines/vinegars; sequestrant; emulsifer; thickener

Sodium monohydrogen phosphate 2:1:1

Sodium tartrate

E335(ii); disodium tartrate; sodium dextrotartrate

Processed cheese; cream powders and cream; sweetened condensed milk; luncheon meat and cured ham; cured pork; milk powders; frozen seafood; edible ices and ice mixes; dried eggs; evaporated milk; canned fruit and vegetables; carbonated vegetables; alcoholic beverages Meat products; cheeses

Stabilizer; used as a chemical reactant

USA, Canada, Japan; UK, Europe – permitted ADI 0–30 mg/kg bw

USA, Canada – approved Europe – permitted MTDI 70 mg/kg bw (as P)

USA, Canada, Japan; UK, Europe – permitted MTDI 70 mg/kg bw (as P)

USA, Canada, Japan; UK, Europe – permitted ADI 0–30 mg/kg bw

(Continued)

Małgorzata Grembecka

Sodium polyphosphate

Coffee whitener; meat food products; puddings; cream sauce; evaporated milk;

USA, Canada, Japan; UK, Europe – permitted MTDI 70 mg/kg bw (as P)

Name of compound Flavor enhancers Disodium 5'-guanylate

Disodium 5'-inosinate

Synonyms E627; disodium guanosine-5’monophosphate, disodium 5’-guanylate, guanosine monophosphate disodium salt, guanosine 5’-monophosphate disodium salt, guanylic acid sodium salt E631; Disodium inosinate, disodium inosine-5’phosphate, sodium 5’-inosinate

Food use

Function in foods

Legislation

Flavor enhancer in foods, favoring agent in pharmaceuticals, canned foods, poultry, sauces, snack

Flavor enhancer

USA, UK, Europe, Australia, Canada – approved

Flavor enhancer in foods

Flavor intensifer

USA, UK, Europe, Australia, Canada – approved

E518; magnesium sulfate (1:1); sulfuric acid magnesium salt (1:1); Epsom salts, bitter salts; magnesium sulfate

Various

Dietary supplement; fermentation aid; favor enhancer; nutrient; processing aid; tofu coagulant (Japan); yeast nutrient

USA – approved (GRAS) UK and Europe – approved Canada and Japan – approved for use in certain products

Magnesium sulfate heptahydrate

E518; bitter salts; Epsom salts; sulfuric acid magnesium salt (1:1) heptahydrate

Beer-making

Firming agent; favor enhancer; nutrient/dietary supplement; processing aid in food and beer-making

USA – approved (GRAS) UK and Europe – approved Canada and Japan – approved for use in certain products

Monosodium L-glutamate

E621; sodium hydrogen L-glutamate; glutamic acid monosodium salt

Meat; poultry, sauces; soups

Flavor enhancer; dietary supplement (Japan)

USA, Canada, UK, Europe, Australia – approved

Potassium chloride

E508; tripotassium trichloride, potassium monochloride; sylvine; sylvite

Jelly (artifcially sweetened); meat (raw cuts); poultry (raw cuts); preserves (artifcially sweetened); beer malting; infant formula

Direct food additive; dietary supplement; favor enhancer; favoring agent; gelling agent; nutrient; pH control agent; salt substitute; tissue-softening agent; yeast food; stabilizer; thickener; tissue softener

USA, Canada, UK, Europe, Australia – approved

97

Magnesium sulfate anhydrous

The Role of Mineral Components

TABLE 4.2 (CONTINUED) The Selected Mineral Compounds Used as Food Additives

(Continued)

98

TABLE 4.2 (CONTINUED) The Selected Mineral Compounds Used as Food Additives Name of compound

Synonyms

Food use

Function in foods

Legislation

E622; monopotassium glutaminate; potassium L-glutamate;

Meat

Flavor enhancer; salt substitute; nutrient; dietary supplement, replenisher

USA, Canada, UK, Europe, Japan – approved

Potassium lactate

E326; potassium 2-hydroxypropanoate

Confectionery; jams; jellies; marmalades; margarine

Direct food additive; favor enhancer; favoring agent; favoring adjuvant; humectant; pH control agent; antioxidant synergist; antimicrobial (meat and poultry products); adjuvant

USA, Canada, UK, Europe – approved

Sodium alginate

E401; algin; alginate sodium salt; alginic acid sodium salt

Candy (hard); condiments; confections; edible flms; frostings; fruit juices; fruits (processed); gelatins; puddings; relishes; sauces; toppings; ice-cream (as stabilizer); boiler water additive

boiler water additive; emulsifer; frming agent; favor enhancer; formulation aid; processing aid; stabilizer; surface-active agent; texturizer; thickener

USA, Canada, UK, Europe, Japan – approved

Sodium lactate

E325; lactic acid monosodium salt; lactic acid sodium salt

Biscuits; fruits; meat products; hog carcasses; trip; vegetable; nuts; sponge cake; Swiss roll; water (canned); water (bottled)

Cooked-out juice retention aid; emulsifer; favor enhancer; favoring agent/adjuvant; glycerol substitute; hog scald agent; humectant; pH control agent; washing agent; antioxidant synergist; bulking agent in foods; antimicrobial (meat and poultry); preservative; adjuvant

USA, Canada, UK, Europe, Japan – approved Neither D(–)-lactic acid nor DL-lactic acid should be used in infant foods

(Continued)

Małgorzata Grembecka

Potassium glutamate

Name of compound

Synonyms

Food use

Function in foods

Legislation

Nutritive additives Calcium glycerophosphate

β- glycerophosphate (calcium salt); calcium glycerinophosphate

Breads; cereals; cornmeal; dietary supplements; dietetic foods; four; formulated liquid diets; infant formulae; meal replacements; pasta rice; gelatins; puddings; fllings

Enrichment; fortifcation or restoration; stabilizer

USA, Canada, UK, Europe – approved Japan – restricted (1% max. as calcium)

Calcium lactate pentahydrate

2-hydroxypropanoic acid calcium salt

Beverages; breads; cereals; cornmeal; dietary supplements; dietetic foods; four; formulated liquid diets; juices; meal replacements; pasta; rice

Enrichment; fortifcation or restoration; preservative

USA, Canada, UK, Europe – approved

Calcium-Dpantothenate

Vitamin B5 (calcium salt); calcium pantothenate; calcium N-(2,4-dihydroxy-3,3-dime thyl-1-oxobutyl)-β-alanine

Cereals and cereal products; dairy products; soft drinks; sugars; sugar preserves and confectionery

Fortifcation of infant formulae; breakfast cereals; fruit drinks; sugar and cocoa confectionery; milk drinks; meal replacements

USA, Canada, UK, Europe – approved (GRAS) Japan – approved (1% max. as calcium)

Calcium phosphate dibasic

E341b; calcium hydrogen orthophosphate; calcium hydrogen phosphate anhydrous; calcium monohydrogen phosphate; dicalcium phosphate; phosphoric acid calcium salt (1:1)

Breads; cereals; cornmeal; dietary supplements; dietetic food; four; formulated liquid diets; infant formulae; meal replacements; pasta; rice; desserts gels

Enrichment; fortifcation or restoration; antioxidant synergist; dough conditioner; frming agent; stabilizer; yeast food for baked food

USA, Canada, UK, Europe – approved (GRAS) Japan – approved (1% max. as calcium) MTDI 70 mg/kg bw (as P)

Calcium phosphate tribasic

E341c; calcium hydroxide phosphate; calcium orthophosphate; calcium phosphate tertiary; tricalcium orthophosphate; tribasic calcium phosphate

Breads; cereals; cornmeal; dietary supplements; dietetic food; four; formulated liquid diets; infant formulae; meal replacements; pasta; rice

Enrichment; fortifcation or restoration; anti-caking agent; buffering agent; chewing gum base; clarifying agent; emulsifer (Japan); fat rendering aid; leavening agent; stabilizer; yeast nutrient

USA, Canada, UK, Europe – approved (GRAS) Japan – approved (1% max. as calcium), restricted intake MTDI 70 mg/kg bw (as P)

99

(Continued)

The Role of Mineral Components

TABLE 4.2 (CONTINUED) The Selected Mineral Compounds Used as Food Additives

100

TABLE 4.2 (CONTINUED) The Selected Mineral Compounds Used as Food Additives Name of compound

Synonyms

Food use

Function in foods

Legislation

Bis(D-gluconato) copper; copper(II) gluconate

Dietary supplements; dietetic foods; formulated liquid diets; infant formulae; meal replacements

Enrichment; fortifcation or restoration

USA, Canada, UK, Europe – approved (GRAS) Japan – approved (0.6 mg/L as copper in milk)

Ferric orthophosphate

Iron phosphate; iron(III) phosphate; ferric phosphate; ferriphosphate

Breads; cereals; cornmeal; dietary supplements; dietetic food; egg products; four; formulated liquid diets; fruit-favored drinks and bases; heat-and-serve dinners; infant cereals; infant formulae; meal replacements; rice; pasta; peanut spreads; simulated meat and poultry products

Enrichment; fortifcation or restoration

USA, Canada, UK, Europe – approved (GRAS)

Ferrous fumarate

Iron(III) fumarate

Breads; cereals; cornmeal; dietary supplements; dietetic food; egg products; four; formulated liquid diets; fruit-favored drinks and bases; heat-and-serve dinners; infant cereals; infant formulae; meal replacements; rice; pasta; peanut spreads; simulated meat and poultry products; waffes

Enrichment; fortifcation or restoration

USA, Canada, UK, Europe – approved (GRAS)

Ferrous sulphate, anhydrous

Iron(II) sulphate (1:1)

Breads; cereals; cornmeal; dietary supplements; dietetic food; egg products; four; formulated liquid diets; fruit-favored drinks and bases; heat-and-serve dinners; infant cereals; infant formulae; meal replacements; rice; pasta; peanut spreads; simulated meat and poultry products

Enrichment; fortifcation or restoration

USA, Canada, UK, Europe, Japan – approved (GRAS)

(Continued)

Małgorzata Grembecka

Cupric gluconate

Name of compound

Function in foods

Legislation

Synonyms

Food use

Magnesium carbonate hydroxide

E504(ii); magnesite; magnesium carbonate basic; magnesium hydroxide carbonate

Alimentary pastes; breakfast cereals; dietary supplements; dietetic foods; egg products; four; formulated liquid diets; infant formulae

Enrichment; fortifcation or restoration; anti-caking agent; four conditioner; lubricant; pH control agent; processing aid; release agent

USA, Canada, UK, Europe – approved (GRAS)

Magnesium oxide

E530; magnesia; calcinated magnesia

Alimentary pastes; breakfast cereals; dietary supplements; dietetic foods; egg products; four; formulated liquid diets; infant formulae

Enrichment; fortifcation or restoration; anti-caking agent; frming agent; lubricant; neutralizing agent; pH control agent; processing aid (Japan); release agent

USA, Canada, UK, Europe – approved (GRAS) Japan – restricted use

Reduced elemental iron

Carbonyl iron; reduced iron

Breads; cereals; cornmeal; dietary supplements; dietetic food; egg products; four; formulated liquid diets; fruit-favored drinks and bases; heat-and-serve dinners; infant cereals; infant formulae; meal replacements; rice; pasta; peanut spreads; simulated meat and poultry products

Enrichment; fortifcation or restoration; processing aid (Japan)

USA, Canada, UK, Europe – approved (GRAS) Japan – restricted use (processing aid)

Zinc sulphate monohydrate

Dried zinc sulfate; sulphuric acid zinc salt (1:1)

Beverages; breakfast cereals; dietary supplements; dietetic foods; egg products; formulated liquid diets; infant formulae; meal replacements; peanut spreads

Enrichment; fortifcation or restoration

USA, Canada, UK, Europe – approved (GRAS) Japan – restricted use

The Role of Mineral Components

TABLE 4.2 (CONTINUED) The Selected Mineral Compounds Used as Food Additives

Notes: GRAS – Generally Recognized as Safe; NZ – New Zealand; ADI – acceptable daily intake; MTDI – maximum tolerable daily intake; bw – body weight. References: Smith & Hong-Shum, 2011; World Health Organization, Evaluations of the Joint FAO/WHO Expert Committee on Food Additives (JECFA), https://apps .who.int/food-additives-contaminants-jecfa-database/.

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softeners in bread and dough strengtheners. They can be found in bread, baked goods, coffee whiteners, confectionery products, dairy products, margarine, and spreads. Their usage is approved worldwide with an acceptable daily intake (ADI) set only for calcium and sodium stearoyl lactylates amounting to 20 mg/kg body weight (bw). Calcium and potassium alginates fnd application as antifoaming and bulking agents, carriers, gelling and glazing agents, humectants, sequestrants, stabilizers, and thickeners. They are approved in the USA (Generally Recognized as Safe, GRAS), the United Kingdom, Europe, and Japan with an ADI value of 25 mg/kg. Mineral salts of ascorbic acid, i.e., sodium ascorbate (E301) and calcium ascorbate (E302) are used as antioxidants, sources of vitamin C, and meat color preservatives. The latter compound is also a nitrosamine inhibitor in cured meat. As there is no risk associated with the consumption of these compounds, they are permitted in the USA, the UK, Europe, and Japan, and ADI was not specifed. Mineral colorants include Cu complexes of chlorophylls and chlorophyllins, iron oxides and hydroxides, calcium carbonate, Al, silver (Ag), and gold (Au). All these compounds are permitted in Europe for use in specifed products but are unallowed in the USA. They can be used as dispersed powders or leaves to surface-color sugar or chocolate confectionery and can be also added to certain liqueurs. All of them are used only as colorants with one exception – calcium carbonate. This substance also fnds application as a pH control agent, dough conditioner, frming agent, modifer for chewing gum, yeast food, and nutritive additive, which is used to fortify products in Ca. Calcium carbonate is also listed as a four additive as well as calcium phosphate monobasic, potassium bromate, and sodium acid pyrophosphate. The latter substance prevents canned meats and potatoes from darkening and is used together with baking powder as a leavening agent to release carbon dioxide. A similar role is played by calcium phosphate monobasic, which is also a yeast nutrient, buffer, and acidulant in beverage powders. On the other hand, potassium bromate is a strong oxidizing agent of the gluten proteins in four and yeast-raised bread products. However, it was banned from use in food products in the European Union, Argentina, Brazil, Canada, and other countries but is approved in the USA (in four and bread).

REFERENCES Alonso López, M., Montaña, F.P., Miranda, M., Castillo, C., Hernández, J., Benedito, J.L. Interactions between toxic (As, Cd, Hg and Pb) and nutritional essential (Ca, Co, Cr, Cu, Fe, Mn, Mo, Ni, Se, Zn) elements in the tissues of cattle from NW Spain. BioMetals 2004, 17: 389–397. https://doi.org/10.1023/b:biom.0000029434.89679.a2. Belitz, H.-D., Grosch, W., Schieberle, P. Food Chemistry. 4th revised and extended ed. Springer-Verlag, 2009. https://doi.org/10.1007/978-3-540-69934-7. Capar, S.G., Szefer, P. Determination and speciation of trace elements in foods. In Otles, S. (Ed.), Methods of Analysis of Food Components and Additives. CRC Press LLC, 2005, 111–158. Commission Regulation (EU) 2020/1419 of 7 October 2020 amending Annex II to Regulation (EC) No 1333/2008 of the European Parliament and of the Council as regards the use

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of ascorbic acid (E 300) and citric acid (E 330) on white vegetables intended for further processing. Cornelis, R., Caruso, J., Crews, H., Heumann, K. Handbook of Elemental Speciation II: Species in the Environment, Food, Medicine and Occupational Health. John Wiley & Sons, Ltd, 2005. https://doi.org/10.1002/0470856009. Dai, Y., Lua, Y., Wua, W., Lu, X., Han, Z., Liua, Y., Li, X., Dai, R. Changes in oxidation, color and texture deteriorations during refrigerated storage of ohmically and water bath-cooked pork meat. Innov. Food Sci. Emerg. 2014, 26: 341–346. https://doi.org/10 .1016/j.ifset.2014.06.009 Dandago, M.A. Changes in nutrients during storage and processing of foods - a review. Techno. Sci. Africana J. 2009, 3(1): 24–27. deMan, J.M. Principles of Food Chemistry. Springer-Verlag, 2013. https://doi.org/10.1007 /978-3-319-63607-8. Fishbein, L. Multiple sources of dietary calcium – Some aspects of its essentiality. Regul. Toxicol. Pharmacol. 2004, 39: 67–80. https://doi.org/10.1016/j.yrtph.2003.11.002. Grembecka, M., Szefer, P. Metals and metalloids in foods – Essentiality, toxicity, applicability. In Medina, D.A., Laine, A.M. (Eds.), Food Quality: Control, Analysis and Consumer Concerns. Nova Science Publishers, New York, USA, 2011, 1–60. Kabata-Pendias, A., Mukherjee, A.B. Trace Elements from Soil to Human. Springer-Verlag, 2007. https://doi.org/10.1007/978-3-540-32714-1. Khouzam, R.B., Szpunar, J., Holeman, M., Lobinski, R. Trace element speciation in food: State of the art of analytical techniques and methods. Pure Appl. Chem. 2012, 84(2): 169–179. https://doi.org/10.1351/PAC-CON-11-08-14. Kieliszek, M. Selenium–fascinating microelement, properties and sources in food. Molecules 2019, 24: 1298–1312. https://doi.org/10.3390/molecules24071298 Lee, J.-G., Hwang, J.-Y., Lee, H.-E., Kim, T.-H., Choi, J.-D., Gang, G.-J. Effects of food processing methods on migration of heavy metals to food. Appl. Biol. Chem. 2019, 62: 64–74. https://doi.org/10.1186/s13765-019-0470-0. Lönnerdal, B. Excess iron intake as a factor in growth, infections, and development of infants and young children. Am. J. Clin. Nutr. 2017, 106: 1681S–1687S. https://doi.org/10.3945 /ajcn.117.156042. Nabrzyski, M. Functional role of some minerals in food. In Szefer, P., Nriagu, J.O. (Eds.), Mineral Components in Foods. CRC Press, Taylor & Francis Group, 2007. https://doi .org/10.1201/9781420003987. Oghbaei, M., Prakash, J. Effect of primary processing of cereals and legumes on its nutritional quality: A comprehensive review. Cogent Food Agric. 2016, 2(1): 1136015. https://doi .org/10.1080/23311932.2015.1136015. Raj, D., Maiti, S.K. Sources, bioaccumulation, health risks and remediation of potentially toxic metal(loid)s (As, Cd, Cr, Pb and Hg): an epitomised review. Environ. Monit. Assess. 2020, 192: 108–128. https://doi.org/10.1007/s10661-019-8060-5. Ravipati, E.S., Mahajan, N.N., Sharma, S., Hatware, K.V., Patil, K. The toxicological effects of lead and its analytical trends: an update from 2000 to 2018. Crit. Rev. Anal. Chem. 2021, 1–16. https://doi.org/10.1080/10408347.2019.1678381 Reilly, C. Metal Contamination of Food: Its Signifcance for Food Quality and Human Health. Blackwell Science, 2002. https://doi.org/10.1002/9780470995105. Rickman, J.C., Bruhn, C.M., Barrett, D.M. Nutritional comparison of fresh, frozen, and canned fruits and vegetables II. Vitamin A and carotenoids, vitamin E, minerals and fber. J. Sci. Food Agric. 2007, 87: 1185–1196. https://doi.org/10.1002/jsfa.2824. Ruzik, L., Kwiatkowski, P. Application of CE-ICP-MS and CE-ESI-MS/MS for identifcation of Zn-binding ligands in Goji berries extracts. Talanta 2018, 183: 102–107. https://doi .org/10.1016/j.talanta.2018.02.040.

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Shahid, N.M., Shahid, M., Khalid, S., Bibi, I., Bundschuh, J., Niazi, N.K., Dumat, C. A critical review of mercury speciation, bioavailability, toxicity and detoxifcation in soilplant environment: Ecotoxicology and health risk assessment. Sci. Total Environ. 2020, 711: 134749. https://doi.org/10.1016/j.scitotenv.2019.134749 Sikorski, Z.E. Morskie surowce żywnościowe. Dostępnoś ć, właściwości i przechowywanie chłodnicze. Wydawnictwo WNT, 1992. Smith, J., Hong-Shum, L. Food Additives Databook. Wiley-Blackwell, Chichester, West Sussex, UK, 2011. Souci, S.W., Fachmann, W., Kraut, H. Food Composition and Nutrition Tables. Scientifc Publishers, Stuttgart, 2002. Synowiecki, J. Składniki mineralne. In Sikorski, Z.E. (Ed.), Chemia Żywności. Składniki Żywności. Wydawnictwo Naukowo-Techniczne, Warszawa, 2007, 88–106. Szefer, P. Składniki mineralne - ich niezbędność fzjologiczna, zagrożenia toksykologiczne oraz rola w żywności. In Sikorski, Z.E., Staroszczyk, H. (Eds.), Chemia żywności. T. 1. Główne składniki żywności. Wydawnictwo WNT, Warszawa, 2013, 53–86. Timbrell, J.A. Principles of Biochemical Toxicology. 4th ed. Informa Health Care, New York, USA, 2008. Ullah, H., Liu, G., Yousaf, B., Ali, M.U., Irshad, S., Abbas, Q., Ahmad, R. A comprehensive review on environmental transformation of selenium: Recent advances and research perspectives. Environ. Geochem. Health 2019, 41: 1003–1035. https://doi.org/10.1007/ s10653-018-0195-8. WHO. Trace Elements in Human Nutrition and Health. WHO, Geneva, 1996. Wong, D.L., Merrifeld-MacRae, M.E., Stillman, M.J. Lead(II) Binding in Metallothioneins. In Sigel, A., Sigel, H., Sigel, R.K.O. (Eds.), Lead – Its Effects on Environment and Health. De Gruyter, Berlin, Boston, 2017. https://doi.org/10.1515/9783110434330. World Health Organization, Evaluations of the Joint FAO/WHO Expert Committee on Food Additives (JECFA). https://apps.who.int/food-additives-contaminants-jecfa-database. Zalewska, A., Kowalik, J. Powłoka cynowa jako zabezpieczenie opakowań metalowych przeznaczonych do żywności. Inż. Ap. Chem. 2017, 56(6): 224–225.

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Saccharides Hanna Staroszczyk

CONTENTS 5.1

Chemical Properties ..................................................................................... 106 5.1.1 Introduction ...................................................................................... 106 5.1.2 Chemical Structure........................................................................... 106 5.1.2.1 Monosaccharides ............................................................... 106 5.1.2.2 Alginates ............................................................................ 109 5.1.2.3 Carrageenans ..................................................................... 110 5.1.2.4 Cellulose ............................................................................ 111 5.1.2.5 Chitosan ............................................................................. 113 5.1.2.6 Cyclodextrins ..................................................................... 115 5.1.2.7 Pectin Polysaccharides....................................................... 115 5.1.2.8 Starch ................................................................................. 120 5.1.2.9 Bacterial Polysaccharides .................................................. 122 5.1.3 Chemical Reactivity ......................................................................... 126 5.1.3.1 Reduction ........................................................................... 126 5.1.3.2 Oxidation ........................................................................... 127 5.1.3.3 Metal Interactions .............................................................. 128 5.1.3.4 Esterifcation ...................................................................... 130 5.1.3.5 Etherifcation...................................................................... 131 5.1.3.6 Glycosylation ..................................................................... 131 5.2 Functional Properties.................................................................................... 131 5.2.1 Introduction ...................................................................................... 131 5.2.2 Color, Flavor, and Aroma ................................................................. 132 5.2.2.1 Non-Enzymatic Browning/Maillard Browning................. 132 5.2.2.2 Degradation of Ascorbic Acid ........................................... 137 5.2.2.3 Caramelization................................................................... 138 5.2.3 Taste.................................................................................................. 139 5.2.4 Texture .............................................................................................. 144 5.2.5 Nutritional Value .............................................................................. 150 References.............................................................................................................. 151

DOI: 10.1201/9781003265955-5

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5.1 CHEMICAL PROPERTIES 5.1.1

INTRODUCTION

Saccharides are widely distributed in nature as the major component of fruits and vegetables (primarily monosaccharides), milk and malt (mainly disaccharides), and grains (mostly polysaccharides). They are an important source of energy, structural material, as well as favor and taste factors. Molecules of this class of compounds contain mainly carbon, hydrogen, and oxygen atoms; however, most natural saccharides do not have a simple chemical composition but occur as oligomers or polymers, in pure, oxidized, or reduced form, as derivatives of amines, esters, and ethers, as well as protein- or lipid-bound molecules. The variety of saccharide structures, the size, shape, and chemical composition of their molecules, as well as the diversity of their physicochemical properties, affect the functional properties and nutritional value of foods.

5.1.2

CHEMICAL STRUCTURE

5.1.2.1 Monosaccharides Hydroxyaldehyde monosaccharides or their hemiacetals are aldoses. Their names end with the suffx “ose,” e.g., trioses, tetroses, pentoses, hexoses, etc. according to the number of C atoms they contain. Hydroxycetone monosaccharides or their hemiketals are ketoses, denoted by inserting “ul” into the name of the corresponding aldoses, e.g., triuloses, tetruloses, pentuloses, hexuloses, etc. (Tomasik, 2004). The cyclic structure with fve-membered rings – pentoses or pentuloses – are called furanoses (f), and those with six-membered rings – hexoses or hexuloses – are referred to as pyranoses (p), although some of them are also furanoses. Due to torsional and steric strains, ring structures are not fat but assume different conformations, most often boat or chair. The chair forms two possible conformations, e.g., 4C1 and 1C4. In both rings, the axial positions of the hydroxyl groups at C-2 in furanoses and at C-1 in pyranoses are more stable than the equatorial positions.

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In the solid state, saccharides exist in chain (acyclic) form, whereas in an aqueous solution, an equilibrium state is established between the chain and cyclic (hemiacetal or hemiketal) forms. The aqueous solution of D-glucose consists almost exclusively of two p, with 36% α- and 64% β-anomer, two f accounting for less than 1%, and the acyclic form only present in trace amounts. One cyclic form has a hydroxyl group on the anomeric C atom below the ring plane, the α anomer, and the other above this plane, the β anomer.

Saccharides can occur in the D or L forms, but most natural saccharides are in the D form. Monosaccharides with the L confguration are less common in nature, although L-arabinose and L-galactose are present as saccharide units in many polysaccharide chains. All monosaccharides (except dihydroxyacetone) contain one or more chiral C atoms and thus occur in optically active forms. In general, a molecule with n chiral centers can have 2n stereoisomers, e.g., hexoses with four chiral centers have 24 stereoisomers. Of the 16 isomers possible of aldohexoses, eight are in the D form and eight are in the L form. Due to the direction of rotation of polarized light, they are written as (+)D when the rotation is clockwise or (–)L when the rotation is counterclockwise. The most abundant monosaccharide found in nature is D-glucose. Most others do not occur in the free state in nature but appear as condensed products, i.e., disaccharides, oligosaccharides, polysaccharides, or glycosides, held together by a glycosidic linkage. These can be converted into monosaccharides by hydrolysis using acids or enzymes.

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5.1.2.2 Alginates Alginates are matrix cell wall polysaccharides of brown algae (Phaeophyceae). The main sources of alginates are Laminariales and Fucales, with the former including four subfamilies: Chordaceae, Laminariacace, Lessoniaceae, and Alariaceae, and the latter three, including subfamilies: Fucaceae, Cystoseiraceae, and Sargassaceae. Alginate can also be produced by heterotrophic bacteria from two families: the Pseudomonadaceae and the Azotobacteriaceae. Although Azotobacter vinelandii has been evaluated as a source for industrial production, most of the alginate used commercially is extracted from algal sources, generally from three genera: Macrocystis, Laminaria, and Ascophyllum (Draget et al., 2006). Alginate is a linear polysaccharide composed of β-D-ManpA (see 5.21) and α-LGulpA (see 5.19), joined by (1→4)-glycosidic bonds and arranged in a sequential fashion. Depending on the source, these two monomers can be present either as uniform blocks of β-D-ManpA or α-L-GulpA units only (M-block and G-block, respectively), as blocks of alternating β-D-ManpA and α-L-GulpA units (MG-block), or as blocks of alternating two β-D-ManpA and one α-L-GulpA units and two α-L-GulpA and one β-D-ManpA units (MMG-block and GGM-block, respectively). Microbial alginate differs from algae alginate in that a fraction of the acid β-D-ManpA residue is O-acetylated at the C-2 or C-3 position. In addition, unlike alginate derived from algae and Azotobacter, the one derived from Pseudomonas does not contain closely linked α-L-GulpA residues in its structure, as these bacteria are unable to produce polymers containing blocks of α-L-GulpA units only. The latter is formed by C-5 epimerization of β-D-ManpA chains induced by an epimerase located in several algae and A. Vinelandii (Figure 5.1). α-L-GulpA residues in G-blocks are in the 1C4 conformation, while β-D-ManpA residues in M-blocks have the 4C1 conformation. Hence, various blocks have different chain conformations: M-block regions are fat and ribbon-like, and the G-block regions have a pleated (corrugated) conformation. The conformation of guluronate

FIGURE 5.1 Alginate chain structure: M-β-D-mannuronic acid (ring conformation 4C1), G-α-L-guluronic acid (ring conformation 1C4).

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in alternating sequences is still not resolved, although it is well known that the stiffness of the chain blocks increased in the order MG-block < M-block < G-block. Alginate contains all four possible glycosidic linkages: diequatorial (M-block), diaxial (G-block), equatorial-axial (MG-block), and axial-equatorial (GM-block). As the 1C conformation of the α-L-GulpA residues makes the glycosidic linkage diaxial in 4 G-blocks, a hindered rotation around the glycosidic linkage results in the stiff and extended nature of the alginate chain. Alginates show ion-binding properties, and their affnity for multivalent cations depends on their composition. This affnity is a property exclusive to polyguluronate, while polymannuronate has almost no selectivity. The affnity of alginates to alkaline earth metals increases in the order Mg TΔS S→L), which means that the free energy of the system is small. The entropy factor increases as the temperature increases, and the system has the lowest energy above the melting point ΔH S→L < TΔS S→L. Cooling of the TAG in the liquid state below the melting point does not result in immediate crystallization – a supercooling state occurs and the fats still remain liquid even in the range of several degrees Celsius. The state of fat changes only after crossing the activation energy barrier necessary for nucleation, i.e., the formation of stable crystal nuclei – groups of molecules capable of growing into macrocrystals under favorable conditions. The temperature to which the system can be subcooled is affected by the type of fat, the presence of impurities, and the cooling rate. Nucleation is accompanied by changes in free energy – enthalpy and entropy inside the nuclei and surface tension at the nucleus/liquid phase. In a very pure oil cooled much below the freezing point, primary spontaneous (homogeneous) nucleation takes place, and in oil containing impurities, heterogeneous nucleation. The stable nuclei transform into TAG crystals by attaching molecules from the liquid phase. The phenomenon in which small fragments detach from the crystals and form new nuclei is called secondary nucleation. The crystal growth rate depends on the rate of the molecules’ migration to the interface, determined, among others, by the liquid phase viscosity, the possibility of their incorporation into the crystal structure, and the yield of crystallization heat dissipation. In the oil cooled to the temperature at which the rate of stable nuclei formation is faster than that of their growth, many fne crystals are formed. However, when the rate of new molecules attaching is greater than that of nucleation, the number of crystals is lower and their dimensions are larger. In general, rapid cooling well below the crystallization point leads to the formation of many small crystals, and at a low cooling rate to the temperature just below the melting point, fewer large crystals are formed. TAG can occur in the solid state in several polymorphic crystal forms which differ in their stability and affect the sensory properties of fats. The typical three

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polymorphic phases of TAG in various fats are, α, β’, and β, and the existence of subα, α, β’, and β has been shown in milk fat. In general, in β phase, there are unit cells of two acyls long, β (2L), but in TAG that contain various acyls, β phase is formed with cells three FA (3L) long (Figure 8.6). The melting point of crystals depends on the structure of the acyls included in the TAG, which determines the packing density of the molecules and the forces of intermolecular interactions. According to the stability and the melting point they can be arranged as α < β ’< β. The melting points of these three forms of tristearoyl glycerol are 54.7, 64.0, and 73.3, respectively (Drozdowski 2007). Although the β form is the thermodynamically most stable, many TAG crystallize frst in the α form because it has the lowest activation energy for crystal nucleation. Over time under appropriate conditions, the transformation occurs through the β’ to β form. The rate of α → β’→ β transformation depends on temperature, pressure, and the presence of impurities. In general, the rate of the frst reaction step is high, the second one is lower. The composition of the TAG has a signifcant effect. In fats with less varying TAG composition, the rate α → β’→ β of conversion is high, and in fats composed of various TAG, it is low. The formation of the various crystal forms depends on the distribution of the acyls in the TAG and their chemical properties, in particular the length of the hydrocarbon chain, the number of double bonds, and the cis/trans confguration. The symmetrical and asymmetrical TAG crystallize differently. The most stable form of 1,3-dipalmitoyl-2-oleoyl-sn-glycerol is the β crystals, while 1,2-dipalmitoyl-3-oleoyl-sn-glycerol forms the stable β’ forms. TAG containing three saturated acyls easily forms the most stable β phase. However, if in the mixture there are other TAG, the stable β’ crystals or the stable mixtures of β’ and β crystals are formed. Some desirable properties of fats can be obtained by interesterifcation (see section 8.5.3). In the mixtures of bovine tallow and rapeseed oil (canola), it reduces the content of saturated and tri-unsaturated TAG and increases the proportion of TAG containing one SFA and two residues of unsaturated acids.

FIGURE 8.6

The units in which TAG containing saturated acyls crystallize.

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Polymorphic occurrence and mixing phase behavior of TAG are largely dictated by the complex intermolecular interactions of hydrocarbon chains and the presence of functional groups at the interface, as well as the crystallization kinetics, in particular the cooling rate. In experiments with a mixture of 1,3-dilauroyl-2-stearoyl-sn-glycerol (LSL) and 1,2-dilauroyl-3-stearoyl-sn-glycerol (LLS), it was found that only β’ phase was formed during cooling at a rate of 0.1° C/min, and at 3° C/min α and β’ forms. The most stable phase is formed during the crystallization of symmetrical LSL. Depending on the proportion of LSL and LLS in the mixture, the balance between the stabilizing effect of symmetric acylglycerol and the kinetic effects is established. The asymmetric TAG species have a lower crystallization temperature than their symmetrical counterparts, regardless of the cooling rate (Bouzidi et al. 2010). In the milk fat cooled from 65 to 20° C at 10° C/min, the crystallization takes place in two steps and involves the α → β’ polymorphic transformation. Under these conditions, a higher number of small crystals of similar size are formed than during cooling at 0.1° C/min, when the larger crystals are formed in the one-step process (Wiking et al. 2009). In cocoa butter, depending on the heating/cooling conditions, as many as six polymorphic crystal forms are formed with a melting point from 17 to 36° C. In chocolate, the βV form is the most desired with a melting point of 34° C. These crystals give the chocolate the required hardness, characteristic texture, and strong gloss. To obtain mainly such crystals, the chocolate mass is tempered – frst heated to 45° C to melt all the crystalline forms, then cooled to 27° C with stirring to form a lot of small βIV and βV crystals, and in the last step heated again to 31° C to remove the βIV crystals. During the crystallization, mixing with a suffciently high shear force increases the number of nuclei and the rate of the desired polymorph formation. In the fnished product, over time the βV form transform into the most stable but less desirable βVI form. The gloss of the chocolate bars disappears and blooms appear during storage at too high or variable temperatures due to the migration of some fat fractions to the surface. The storage temperature fuctuations cause the melting of the crystals of the lowest melting point and recrystallization under uncontrolled conditions. In properly tempered chocolate the blooms contain only TAG, while in non-tempered chocolate there are mixtures of fat and sugar (James and Smith 2009). TAG crystallization can be used to isolate specifc components from fat. It is possible to separate, e.g., tallow, palm kernel oil, or milk fat into fractions with the desired properties using fractionated crystallization. The TAG are extracted by cooling below their freezing point, and then crystals are formed which are separated from the liquid phase by fltration or centrifugation. The products with the desired properties can be obtained by selecting the proportion of the different fat fractions, and they meet the requirements for various applications in the food industry. For example, milk fat fractions with high melting points are used as shortenings with the desired butter favor, the chocolate bloom inhibitors as the components of frozen desserts, and the low melting point fats are applied in baking and meal preparation. 8.1.3.3 Crystalline Network Formation In a crystallization process, a crystalline network is formed in the solidifed fat, which determines the rheological properties of TAG. During nucleation and crystal

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growth, aggregation under Van der Waal’s forces occurs, resulting in the growth of a three-dimensional network consisting of joined TAG crystals, their spherulites, and larger structures. The crystals start to join up already in the presence of a low proportion of the solid phase. As a result, after the solidifcation of the fat, there are in the network various structures with dimensions from about 0.5 to 200 μm. The composition and distribution of acyls in the TAG and the presence of other components greatly affect the crystallization rate and the crystalline network formation, as well as the properties of the structures. The addition of tripalmitin to the palm oil increases the crystallization rate and the β-crystals formation and increases the size of the TAG crystals. The introduction of saturated MAG leads to an increase in the number of crystallization seeds and accelerated palm oil crystallization, favoring the β-crystals formation and reducing crystal size (Baso et al. 2010). In turn, the addition of PL to milk fat affect signifcantly crystal morphology. At low concentrations, PL increase the size of the spherulites composed of the TAG crystals, while at a concentration of over 2%, they inhibit the formation of spherulites. The crystals are dispersed at low temperatures and the butter is harder and has less perceptible granular texture. Fedotova and Lencki (2010) found direct prevention of the TAG secondary nucleation on the crystal surface by the PL as one of the reasons for the inhibition of the spherulite formation. The type of crystalline forms generated in the cooled fats, the number of crystals and their aggregates, dimensions, shape, and interaction of all components of the three-dimensional colloidal network affect the properties of solid fats important in processing and in food products, including hardness and yield strength. Margarine with large spherulites and a low number of TAG crystals is less hard than the product containing more TAG crystals and smaller spherulites, although the solid fat content in the former is larger.

8.1.4

PHOSPHOLIPIDS

PL include glycerophospholipids and sphingolipids. The glycerophospholipids are derivatives of sn-glycerol-3-phosphate in which the hydroxyl groups form ester bonds with the LCFA (Figure 8.7a), or in the sn-1 position there is a 1-alkenyl ether residue and in the sn-2 position a long-chain acyl (Figure 8.7b). When the FA residues are cleaved off, the derivatives have the prefx “lyso” in their name. A mixture of the phosphatidyl esters found in vegetable oils is commonly called raw lecithin (Figure 8.8), and lecithin was formerly called phosphatidylcholine. Sphingolipids are derivatives of sphingosine in which the primary hydroxyl group is esterifed with phosphoric acid (Figure 8.9). Plant and animal tissues, as well as microbial biomass, contain 1–2% (dry weight) PL. They are mainly bound to proteins as components of cell membranes. Their proportion in the total mass of lipids in fatty raw materials is usually small – 0.1–0.5% in crude rapeseed oil and 1–3.5% in soybean oil. However, in lean cod meat, containing less than 1% fat, there are practically no other lipids than PL. Chicken egg yolk is very rich in PL. In egg yolk is about 70% (dry weight) of fat, containing 56–64% TAG and 21–31% PL. In the total amount of PL from various raw materials, phosphatidylcholine generally has the largest contribution (Table 8.3).

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Chemical structure of: A – phosphatidic acid, B – plasmenic acid.

FIGURE 8.8 Chemical structure of phosphatidyl esters.

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FIGURE 8.9 Chemical structure of sphingolipid and sphingomyelin.

TABLE 8.3 Distribution of Different Components (%) in PL Concentrates from Various Raw Materials Component

Sources of PL Egg yolk

Milk fat

Soybean

Rapeseed

Phosphatidylcholine

68–86

10–26

18–32

18–26

Phosphatidylethanolamine Phosphatidylserine

8–24 tr–2

25–45 tr–6

6–17 tr–2

14–31 1–2

tr

tr–4

17–24

6–14

Phosphatidylinositol

Note: tr – traces. Source: data from Pokorný and Schmidt 2011.

In glycerophospholipids, acyl in the sn-2 position is usually more unsaturated than in the sn-1 position. The number of double bonds in unsaturated FA residues in PL, especially in meat and fsh, is generally greater than in TAG in the same raw materials. However, they have the ability to complex metal ions and act synergistically with tocopherols and favonoids. Thus they can participate in the inhibition of lipid oxidation in food. PL molecules have amphipathic nature because they contain a hydrophobic fragment composed of hydrocarbon FA chains and a hydrophilic part with the phosphoric acid residue with esterically bound polar groups. Therefore, they self-assemble into various micellar structures in aqueous media. PL dissolve in fatty solvents but are insoluble in acetone. In foods, they act as emulsifers, increase the volume of bread and reduce the rate of its staling, prevent the crystallization of high melting TAG in chocolate, increase the rate of dissolving and dispersing instant foods, and act as an anti-spattering agent in margarine.

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WAXES

Natural waxes are mixtures of FA esters and long-chain alcohols. Depending on their origin, the waxes may contain admixtures of long-chain hydrocarbons and plant or animal sterols and their esters. In plants, the waxes are a protective covering on leaves, fruits, and seeds against water loss and microbes. In marine organisms, they serve as aids in buoyancy and as storage lipids. The alcohols found in fsh oils are mainly: C16H33OH, C18H37OH, and C18H35OH. Natural waxes have many nonfood uses, including in cosmetology. The edible fsh oils and other marine animals’ oils generally contain no more than 1% of waxes, but there are exceptions to this rule. Barracuda oil contains 75% waxes, and deep-water fsh caught off the coast of New Zealand contain about 90 to 98%. One of the species of these fsh is the orange roughy with a very tasty, white meat. Although the waxes are not desirable food components, these fsh are valued on the market because the lipids are mainly present in the subcutaneous layer of the skin, which is removed by deep skinning. In the head of the sperm whale, a wax known as the spermaceti is present.

8.2

BIOLOGICAL EFFECTS OF LIPIDS IN FOODS

8.2.1 NUTRITIONAL VALUE Lipids are essential in the human diet. They are a source of energy, essential fatty acids, and cholesterol. Lipids provide energy in the amount of 37.6 kJ/g, while cholesterol is not used as an energy source. In addition, fats spare the use of proteins as energy, increase the satiating properties of foods as they delay the digestion process for up to four hours, and enable the absorption of fat-soluble vitamins. Lipids are also a component of cell membranes, digestive secretions, and hormones, and form the adipose tissue of the human body which protects internal organs by isolating and controlling the temperature. According to the recommendations of WHO/FAO experts, the fat consumption by children and adolescents should be 40% of energy, and by adults in the range of 20–30% of energy. The demand for fat depends on the energy requirements of the body, which, in addition to age, are affected by gender, the type of work performed, and in women the physiological state, i.e., the period of pregnancy and lactation. There is no recommended daily intake for each group of FA. However, it is believed that SFA intake should be kept as low as possible. However, not all SFA have adverse effects on the body. It has been shown that butyric acid contributes to the prevention of cancer. Caprylic acid may have a positive effect in reducing the incidence of bacterial diseases and mycoses of the digestive system. Short-chain FA and MCFA are used in dietetics because they are absorbed directly into the portal vein. It is benefcial for the prevention of atherosclerosis and heart disease if unsaturated FA are included in the diet instead of SFA, which lower the concentration of low-density lipoprotein (LDL) cholesterol and TGA in the blood. Olive oil and vegetable oils are the richest sources of MUFA. The greatest amounts of PUFA are found in vegetable

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oils and fsh, especially marine ones, i.e., mackerel, herring, sardine, and also in farmed trout and salmon. Very important is the correct ratio of n-6 FA to n-3 FA, amounting to about 4:1. This reduces the risk of oxidative stress and obesity development, and plays an important role in proper development in infants and children. It is also necessary for the proper development of the nervous system and cognitive functions. Nerve functions depend on the type of lipids in cell membranes. The neuronal part of the brain has a high level of essential FA. Numerous PUFA of the n-3 and n-6 FA families belong to the essential FA, which are not synthesized in the body. The essential FA perform numerous functions, including maintaining the function and integrity of cell membranes, the fuidity of cell membranes, and enzymatic activities, and they also serve as carriers for membrane receptors. They are the precursors to hormone-like compounds called eicosanoids such as prostaglandins, prostacyclins, leukotrienes, and thromboxanes. Their defciency, especially n-3 FA, in the diet for a prolonged period can lead to hypercholesterolemia because they determine the metabolism of cholesterol. A daily intake of linoleic acid constituting 4% of energy and linolenic acid in the range of 0.2–1% of dietary energy lowers the concentration of LDL cholesterol and TGA and increases that of high-density lipoprotein (HDL) cholesterol. According to the European Food Safety Authority (EFSA), the recommended dose of EPA and DHA is 250 mg per day, which is enough to maintain the overall health of the cardiovascular system. Therefore, it is recommended to consume fsh at least twice a week. In addition, DHA is essential for brain development, while EPA appears to be more involved in behavior and mood. Both help to support neurological health. It is proven that a mixture of EPA and DHA has a positive effect on perception and various learning disorders. EPA and DHA defciency leads among others to malfunction of the heart, liver, kidneys, or endocrine glands. Cholesterol is produced in the animal body in the amount of 60–80%, while 20–40% comes from the diet. During the endogenic synthesis, such an amount of cholesterol is formed which covers the body’s needs and a man does not need to take it with food. So far, the maximum cholesterol intake recommended by European expert groups is 300 mg/day. The main sources of cholesterol are eggs, cured meats, and milk fat.

8.2.2 HARMFUL EFFECTS 8.2.2.1 Natural Lipids Some lipids in their native state or after adverse changes occurring during food processing and storage are harmful to human health. Exceeding fat consumption in relation to the amount of the recommended intake poses a health risk for people. According to the Food and Agricultural Organization of the United Nations (FAO), excess dietary fat can lead to overweight and obesity, and to the development of type 2 diabetes, atherosclerosis, hypertension, and most cancers. Obesity is promoted more by excess dietary fat than by excess saccharides. After eating high-fat foods, satiety signals may be suppressed, which promotes over-eating. Compliance with the

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principles of proper nutrition – regularity of meals, not eating between meals, and a diet with the recommended intake levels – protects against obesity and is important in the prevention of many civilization diseases. In some countries, there is a lack of regulation limiting TFA content in food. To ensure the health safety of edible fats, an electronic database of TFA content in food products with a search engine has been developed. Not only the amount but also the type of fats consumed has a great effect on the occurrence of diet-related diseases. A diet with a relatively high intake of animal fats (pork lard, beef and sheep tallow, goose lard, butter) with a high content of SFA and cholesterol increases the concentration of cholesterol in the blood (hypercholesterolemia). This promotes atherosclerosis and, as a result, contributes to ischemic heart disease. In total cholesterol, which is bound to proteins in the form of lipoproteins, there are among others the LDL fraction, the so-called bad cholesterol, and the HDL fraction, the so-called good cholesterol, with an antiatherosclerotic effect. Atherosclerosis is one of the cardiovascular diseases where changes occur within the inner walls of the arteries. First, deposits of macrophages, LDL cholesterol, macrophages loaded with oxidized LDL, and extracellular cholesterol clusters accumulate in the space between the endothelium and the muscle layer of the vessel. Fibrous elements of connective tissue bind to these deposits and surround and separate them from the rest of the vessel. The result is an advanced lesion that causes narrowing of the vessel resulting in restriction of blood fow. Long-term atherosclerotic lesions cause a permanent oxygen defcit, and when blood fow is completely interrupted, an ischemic area is formed. The complete occlusion of the vessels, most often of the heart, brain, and legs, leads to tissue death. One of the most important ways to prevent atherosclerosis is a diet rich in vegetables, fruits, and legumes. An excessive SFA intake increases the risk of blood clots. Coconut oil (over 80%) and palm oil (over 40%) have a high content of SFA. Total SFA intake has been shown to strongly correlate with the sum of the intake of lauric, myristic, palmitic, and stearic acids, which increase the concentration of LDL cholesterol and TAG, apart from stearic acid, which reduces LDL but also lowers HDL. It has also been proven in many studies that diets rich in SFA have an infuence on the development of certain cancers. Excessive dietary intake of cholesterol leads also to an increase in total cholesterol in the blood, including LDL and VLDL fractions, which increase the risk of atherosclerosis and ischemic heart disease. Cholesterol intake is especially not recommended for patients with type 2 diabetes. 8.2.2.2 Oxidized Lipids The oxidation of lipids in food causes the formation of compounds harmful to human health. Secondary oxidation products are formed: aldehydes, ketones, lactones, alcohols, short-chain KT, keto acids, hydroxy acids, epoxides, ethers, hydrocarbons, and other volatile compounds which adversely affect, among other things, the biological properties of food. Many of these products are toxic. Linseed, grape seed, sunfower, soybean, and corn oils, as well as fsh fats, which are high in linoleic and linolenic acid, are the most susceptible to oxidation. Olive

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oil and rapeseed oil, rich in oleic acid, are more stable. For this reason, rapeseed oil is commonly used in frying. Animal fats are the most stable due to the presence of active antioxidants, lower PUFA content, and high oleic acid content. Oxidized sterols (oxysterols), cholesterol oxidation products, were not detected in either UHT milk or milk powder, indicating their high oxidative stability. The resulting short-chain aldehydes and ketones, which are volatile compounds, cause sensory changes in food products, most often leading to food spoilage. Many of these aldehydes, including hexanal, pentanal, octanal, and nonenal, are toxic compounds with the lowest threshold for rancid odor. They are also responsible for the grassy, rubbery, metallic, and bitter taste. Due to their high reactivity, these aldehydes easily react with other food components, e.g., proteins during food storage resulting in reduced digestibility and nutritional value. These secondary products, especially malondialdehyde, participate in carbonyl-amine reactions, Schiff base formation, and protein cross-linking. As a result, long-stored foods lose some amino acids, most often lysine, and also color deterioration occurs, i.e., browning, yellowing, discoloration, and undesirable changes in texture. Malondialdehyde is mutagenic. It has the ability to form covalent bonds with DNA, proteins, and PL. This damages the cell membranes and disrupts the protein structures of cells, resulting in their earlier aging. Interaction of lipid oxidation products with DNA leads to genetic instability, followed by an increase in somatic mutations and cancer formation. Harmful lipid oxidation products also include short chain, cytotoxic 4-hydroxy-2-alkenals, e.g., 4-hydroxy-2-hexenal (HHE), 4-hydroxy2-nonenal (HNE), which can react with DNA, contributing to cell dysfunctions. Moreover, these compounds cause infammation in the upper small intestine. Their intake by children aged three months to one year should not exceed 20 μg/kg body weight. In open packages of infant formula enriched with n-3 and n-6 PUFA, after ten days their content increases even to about 800 μg/kg. Toxic products of lipid oxidation are also hydroxy fatty acids, epoxy compounds, and oxysterols. Due to non-enzymatic reactions during heat treatment, processing, and storage of animal food products, dietary cholesterol is easily oxidized to a great number of derivatives. Salami and parmesan have a high content of oxysterols. The addition of onion and garlic to pork meat inhibits the formation of the predominant oxysterols, i.e., 7-ketocholesterol and 7-hydroxycholesterol, by up to 88%. Meat stored under aerobic conditions contains two to six times more oxysterols compared to vacuumstored meat. Oxysterols have mutagenic, carcinogenic, DNA synthesis–inhibiting, and cholesterol biosynthesis–inhibiting properties, and cause cell membrane dysfunctions. Studies indicate their potential involvement in the development of atherosclerosis and certain cancers, including colon. Atherosclerotic lesions result from their cytotoxic effects on the endothelial and smooth muscle cells of arteries. Oxidized lipids pose a health risk because they lead to the formation of intracellular damage and degenerative changes, and as a result, serious diseases such as atherosclerosis. They contribute to a change in the lipid composition of the mitochondrial membrane, which reduces the rate of energy metabolism. They have been shown to act as markers of oxidative stress, which play a role in the accelerated aging of the body cells. Especially, dimalonic aldehyde, HHE, HNE, acrylic aldehyde (acrolein),

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and crotonaldehyde are mutagenic. The current literature reports on the presence of oxidized PUFA at all stages of human tumor formation. Not the amount of fat in the diet, but a high content of linoleic acid promotes the induction of tumors. In addition, the health safety of edible oils is not guaranteed during shelf life. The amount of lipid oxidation products formed in foods can be reduced by using vacuum packaging or a protective gas atmosphere, light-free low storage temperatures, enriching products with antioxidants, and introducing antioxidant substances during processing. 8.2.2.3 Cold- and Hot-Pressed Oils Heat treatments used in food technology, gastronomy, or during the preparation of meals cause not only desired sensory quality and nutritional value, but also the formation of unfavorable products of lipid changes. Producing land animal fats has little effect on changes in the nutritional value of the products, unlike the processing of vegetable oils, where the various steps take place at different temperatures. Cold-pressed oils contain much fewer secondary oxidation products than refned oils because pressing proceeds at low temperatures of 38–42° C. In order to increase the pressing effciency, the ground seeds are usually conditioned at 70–105° C, and during pressing, the temperature is raised even to 160° C. At elevated temperatures, the content of associated substances increases, which deteriorates the quality of the oil. Therefore, multi-step refning is required, which also occurs at high temperatures, especially deodorization at 200–300° C, favoring the generation of TFA, TAG polymers, dimers, and oxidized TAG. After refning, the oils contain fewer antioxidant substances, i.e., tocopherols, carotenoids, sterols, PL, and polyphenols. Furthermore, KT with two and three conjugated double bonds are formed, which increases the susceptibility of oils to oxidation. Moreover, there are impurities formed during refning, i.e., 2-mono-chloropropane-1,3-diol (2-MCPD), 3-mono-chloropropane-1,2-diol (3-MCPD), and glycidyl esters, which are classifed as carcinogenic and mutagenic compounds. 8.2.2.4 Trans Fatty Acids During partial hydrogenation of cooking oils, isomerization of some double bonds occurs, resulting in the formation of acids with displaced bonds, some of which adopt the trans confguration. Trans isomers are nutritionally disadvantageous. Hydrogenated fats rich in TFA and SFA are resistant to oxidation, making them longer used for repeated frying. The baked goods made with them have a larger volume, better porosity, fner texture, and longer shelf life. The availability and consumption of products with a high TFA content have increased in recent years. In Europe, it is from 1.2 to 6.7 g per day. According to Commission Regulation (EU) 2019/649, the content of TFA in the daily food ration should not exceed 2 g. From 2 April 2021, food producers will be required to inform on the TFA content in the product, other than trans isomers naturally occurring in animal fat, if the amount exceeds 2 g per 100 g of fat. Hardened vegetable oils, i.e., margarine and shortening, are the main sources of TFA. In products containing hydrogenated vegetable fats, TFA can be up to 40% of the total KT. Hard margarine contains TFA up to about 35% of all KT, and soft margarine up to about 20%. In the

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case of blends, so-called mixes, which consist of butter and hydrogenated vegetable fats, the TFA content ranges from 2.4% to 14.8%. High consumption of foods fried in hydrogenated fats and frequent consumption of fast food products contribute to the increase in TFA intake. The same regards the diet of children, who prefer chocolate spreads on bread. In addition, children like chocolate bars, microwave-heated popcorn, and cookies. The TFA content in some such products exceeds the recommended daily allowance for one item or package; 100 g of cookies can contain 4.4 g of TFA, and in one 36 g bar, 2.5 g of these compounds. Research in Canada has shown that even children aged 1.5–5 years old are exposed to a high intake of TFA, which in 2006 averaged 4.8 g/day. Bakery products, especially cookies, are commonly consumed by adolescents, resulting in exposure to a greater intake of TFA, in amounts high enough to have an adverse effect on the blood lipid profle. In 1993, the reported intake of TFA by American teenagers exceeded 30 g/day. In Poland, among many types of confectionery products produced for children, cookies have become one of the most important sources of dietary fat. TFA increase total cholesterol level and the concentration of LDL and TAG and decrease HDL cholesterol level to a much greater extent than SFA. These isomers inhibit the activity of transacetylase catalyzing the esterifcation of cholesterol, which stops its metabolism. In addition, they reduce the size of LDL molecules, which is correlated with an increased likelihood of developing coronary heart disease. Moreover, they contribute to an increase in the concentration of lipoprotein A and infammatory biomarkers and also vascular endothelial dysfunction in the blood. Experimental and clinical studies proved that a high TFA intake increases the risk of developing atherosclerosis and cardiovascular diseases, and some types of cancer. Apart from that, TFA interfere with the synthesis of n-3 and n-6 family PUFA, promote the formation of free radicals, and increase insulin resistance. They are thought to be a factor in enhancing the risk of aggression in humans. Excessive consumption of TFA causes a loss of the integrity of cell membranes. TFA are incorporated into TAG or PL of cell membranes, which reduces the permeability and fuidity of these membranes. The changes in the function of membrane receptors and transporters, and ion channels occur, resulting in dysfunction of the body cells. The content of TFA in cell membranes corresponds to the dietary intake of these acids. Regular intake of TFA in the amount of 1.6% of energy increases the likelihood of myocardial infarction and the risk of coronary heart disease, which is related to their content in the erythrocyte membranes. The probability of coronary heart disease associated with TFA consumption is much higher than with other FA. It was also found that a 1.5-fold enhancement in the risk of sudden cardiac arrest is affected by increased TFA content in erythrocyte membranes, especially the presence of 18:2 trans isomers, in contrast to 18:1 trans isomers, which have no such effect. Diet rich in TFA also increases body weight and abdominal obesity. The reason for this is the reduction of cell sensitivity to insulin by trans isomers due to the changes in the structure and function of cell membranes, which contributes to hyperinsulinemia. Lipid deposition occurs in the cells of abdominal adipose tissue, which

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becomes more susceptible to insulin than subcutaneous adipose tissue as a result of a greater number of adipocytes, which are better innervated and vascularized. TFA of natural origin present in dairy products and ruminant meat delivered to the body in large amounts, corresponding to 3.7% of energy, also worsen the blood lipid profle similar to industrially produced TFA. However, TFA from ruminants are not as abundant in a proper diet. A diet containing a moderate amount of TFA, equal to 1.5% energy, does not induce changes in blood total cholesterol, LDL, and HDL cholesterol concentrations. The amount of partially hydrogenated fat used during processing refects the industrially derived TFA content of food products. It is better to avoid foods with a high TFA content by eating foods not containing partially hardened fat. This information is provided on the product label.

8.3 THE EFFECTS OF LIPIDS ON THE SENSORY VALUE OF FOOD 8.3.1 INTRODUCTION Lipids generally have a desirable effect on the sensory properties of foods, in many cases only as long as they are not hydrolyzed, oxidized, or polymerized. The fats affect the attributes of foods, mainly the color, favor, and rheological properties. The sensory properties are affected by the content and chemical contribution of the lipids, the degree of grinding and emulsifcation, the composition of acylglycerols, and also the chemical and enzymatic transformations of the lipids in food storage and processing. The desirable contribution of fats and lipochromes can disappear during the deteriorative reactions, mainly hydrolysis and oxidation, and also in the reaction with other components. An interesting study of the role of lipids in food was published by Stołyhwo (2007).

8.3.2 THE ROLE OF LIPIDS IN FOOD COLOR The lipids contribute to color formation by being carriers of many lipophilic colorants and by participating in the reactions leading to the formation, release, or modifcation of colored components. The carbonyl compounds generated during fat oxidation react with the protein amino groups resulting in the browning of the longstored food. The color of the skin and fns of marine animals comes from the numerous complexes of carotenoids and proteins. These complexes can be: yellow, orange, red, purple, blue, or green. Their color depends on the type of carotenoid, mainly astaxanthin, canthaxanthin, lutein, and β-carotene, and also on the properties of the protein. The dissociation of the protein fragment, especially exposed to light, causes fading of the color of the fsh skin. During long-term frozen storage, the released carotenoids diffuse into the lipid layer resulting in yellow discoloration. The cooking of crustaceans turns the blue or blue-gray carapace into red because heat denaturation of the carotenoprotein complex known as crustacyanin occurs and red astaxanthin is released. Carotenoid pigments also affect the color of the fsh fesh, e.g., salmonids, and redfsh oil. The astaxanthin-lipoprotein complex is responsible for

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the green color of the lobster eggs. Due to the carotenoid pigments, fsh roe is generally yellow, orange, orange-red, or even orange-purple. Carotenoids, usually in concentrations of about 0.1%, are also found in vegetable oils. The carotenoid colorants make palm oil orange. Lipid oxidation products increase the rate of meat browning. The desirable, especially in beef, cherry-red or bright red color comes from the non-oxidized forms of myoglobin and hemoglobin. During storage, the color of the meat on the crosssection gradually turns brown as the oxidation of myoglobin to metmyoglobin progresses (Figure 8.10). Pre-packaged meat displayed for sale retains the desired color over a period of one to fve days depending on: the reducing capacity of the muscle tissue, the proportion and quality of fat, the properties of the packaging, the composition of the atmosphere above the product, and the temperature. The secondary lipid oxidation products, especially unsaturated aldehydes, increase the rate of browning by covalently binding MbFe(II)O2, changing the conformation of the pigment molecule, thus facilitating heme iron oxidation. The meat hemoproteins participate in oxidation reactions because they can increase the rate of the hydroperoxides decomposition and the formation of alkoxy and peroxide radicals:

The rapid oxidation and undesirable discoloration of red meat can be prevented by adding appropriate antioxidants to the cattle feed. α-tocopherol inhibits the release of cell membrane lipid oxidation products in the meat resulting in delaying oxymyoglobin oxidation.

8.3.3 LIPIDS AND FOOD FLAVOR The food aroma depends to some extent on the lipid content in the raw material or product, since the lipophilic aromatic compounds dissolve in fat, which affects

FIGURE 8.10 Reduced and oxidized meat hemoproteins.

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their release and their concentration in the atmosphere. The role of fat as a carrier of numerous aromatic compounds of spices is particularly important in the culinary preparation of dishes. Reducing the fat proportion in the recipe of a specifc meat product leads to a change in the typical aroma of the product because it violates the ratio of the vapor concentration of lipophilic and water-soluble aroma components. The binding and release of aromatic compounds by various matrices were presented by Guichard (2011). In many foods, hydrolysis of fats causes an undesirable odor known as rancid. It appears in long-stored lard, especially in stale butter, where short-chain FA accumulate, mainly butyric acid with an unpleasant odor. The odor detection threshold for butyric acid is 50 mg/kg of fat, and for caproic acid about twice as high, and it increases very strongly with the increase of the pH. On the other hand, the odor of LCFA in food is practically undetectable. In cheeses, the hydrolysis and lipid oxidation products formed by the action of milk enzymes and microfora contribute to the typical, desired aroma. Among the aroma compounds formed in cheeses directly from fat are: short-chain FA, ketones, alcohols, and lactones (Figure 8.11). Interreactions of lipid degradation products with other milk components also lead to the formation of many odorous compounds. Lipoxygenases and hydroperoxide lyases play an important role in the formation of the aroma compounds of many plant food raw materials. Short-chain aldehydes and alcohols, secondary products of enzymatic lipid oxidation, are involved in generating the desired “green aroma” of fresh melons, cucumbers, tomatoes, bananas, and mushrooms. In low concentrations, they are responsible for the mild, seaweedy aroma of very fresh fsh. PUFA of fsh lipids are transformed into aldehydes, ketones, and alcohols with six, eight, and nine carbon atoms in reactions catalyzed

FIGURE 8.11

Aroma compounds of cheeses formed directly from milk lipids.

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by endogenous lipoxygenases, hydroperoxide lyases, isomerases, and alcohol dehydrogenases. The gradual reduction in the intensity of fresh fsh aroma is in part due to the formation of alcohols from aldehydes, catalyzed by microfora enzymes. The characteristic aroma of cooked shrimp is also caused by lipid oxidation with the participation of lipoxygenase. Undesirable odors of secondary lipid oxidation products appear during the long-term storage of frozen fsh. There is a view in the literature that meat odor comes from the non-fat fraction of meat, while the characteristic aroma of a particular species is affected by lipids. Dishes from fatty mutton have a more intense typical odor than those from lean meat. The characteristic aroma of cooked, baked and fried meat is largely due to the numerous secondary oxidation products of TAG and PL. Their importance is great because they are perceptible in very low concentrations. The meat of animals fed with a feed rich in PUFA can have a slight fshy odor, which comes, among others, from the reaction products of unsaturated aldehydes with amino acids. The aroma is also caused by other volatile compounds formed in the Maillard reaction with the participation of secondary lipid oxidation products. Among the approximately 1,000 volatile compounds found in meat, several hundred are formed by enzymatic and thermal transformations of lipids. The secondary lipid oxidation products, in particular carbonyl compounds, are also responsible for the aroma of long-heated food (warm-over favor). A characteristic compound responsible for the warm-over favor is hexanal, the concentration of which is about ten times higher in such products than in freshly cooked beef (Belitz et al. 2001). A comprehensive treatment of the subject of the role of volatile compounds formed by lipid transformations in food odor formation was published by Jeleń and Wąsowicz (2011).

8.3.4

LIPIDS AND FOOD TEXTURE

The rheological properties of butter and lard, natural fats used for bread spreading, depend on the proportion of TAG present in the solid state and on the factors determining their microstructure, i.e., the number, size, and shape of crystals, and their aggregates involved in the formation of the fat network. Lipids affect also strongly the rheological properties of food emulsions. Detailed information on it is given in section 8.6. However, lipids also have a signifcant effect on the rheological properties of many other foods, including bread, cheese, meat, and meat and fsh products. It concerns especially the texture and complex sensory impressions known as “mouthfeel” in relation to marbled meat containing several percent of intramuscular fat, whole milk, or creamy ice cream. The desired texture of high-quality culinary meat is largely due to its marbling. Marbling increases proportionally to the total intramuscular fat content. In general, beef with an intramuscular fat content of 1.5–7% has the highest sensory quality. The rheological properties of meat are also affected by the FA composition of the lipids in the animals’ feed. The increase in proportion of PUFA in the feed increases especially the tenderness of pork. In sausages, the fat content and the optimal diameter

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and uniform distribution of its droplets in the product structure are very important. The texture of fatty fsh meat depends on seasonal fuctuations in fat content. Therefore, Baltic sprats caught in the summer season, when they contain below 6% fat, are not suitable as a raw material for the production of “canned smoked sprats in oil,” as their texture is hard and not juicy enough. Very high-quality smoked mackerel can be obtained only from the raw material containing about 30% fat. The tender texture of the lightly salted maatjes is also associated with the high lipid content in the tissues of the immature herring. During the long frozen storage, especially at insuffciently low temperatures, fsh meat hardens due to the denaturation of muscle proteins. One reason for this phenomenon is cross-linking of proteins due to the interactions with secondary lipid oxidation products. The characteristic, desirable sensory sensation of melting chocolate in the mouth is affected by the fact that the fat of cocoa butter has a melting point close to the temperature of the human body. In bread and bakery products, lipids are involved in the formation and maintenance of the desired texture through interactions with proteins and polysaccharides. Wheat four contains 1.5–2.5% fat, including TAG, PL, and glycolipids. In the dough, they are in the form of microemulsion stabilized by PL distributed in a network formed by proteins. The surface-active polar lipids of the four and the added shortenings, modify the structure of the gluten network in the dough, stabilizing the porous structure of the bread crumb and forming gels as amylose-lipid complexes, thus resulting in the texture of the bread.

8.4 CHEMICAL AND BIOCHEMICAL REACTIONS OF LIPIDS IN STORAGE AND PROCESSING 8.4.1 INTRODUCTION Biochemical and chemical reactions of lipids occur in food during storage and processing, causing benefcial or undesirable changes in the sensory quality and biological value of food raw materials and products. Moreover, they are increasingly used to modify the properties of lipids and their suitability for various applications, not only food. The susceptibility of lipids to enzymatic and chemical changes depends on their chemical structure, including especially: the number of double bonds in hydrocarbon chains, the distribution of acyls in the acylglycerol molecule, the presence of reactive functional groups, the proportion of hydrophobic and hydrophilic fragments, and the degree of dispersion in the system. During the storage and processing of food, the stability of lipids is mainly affected by temperature, pH, access to oxygen and its reactive forms, enzyme activity, the presence of radicals, metal ions, prooxidants, and antioxidants, the effect of light, and possibly ionizing radiation. Typical reactions in which lipids in food are involved are hydrolysis of the ester bonds, esterifcation of hydroxyl groups, acyl exchange, hydrogenation of double bonds, isomerization, oxidation, and polymerization. The involvement of non-polar FA fragments and lipid acyls in hydrophobic interactions with other components, and surface activity resulting from the amphipathic properties of some lipids, are important for the rheological properties of food products.

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HYDROLYSIS

Ester bonds in TAG and PL present in food raw materials are hydrolyzed by endogenous and microbial enzymes. This leads to the release of free FA and PL hydrolysis products (Figure 8.12) and, depending on the reaction conditions, and on the type of enzyme, DAG, MAG, and glycerol. The specifcity of lipases and phospholipases can be used to study the structure of TAG and PL (Drozdowski 2007). Enzymatic hydrolysis of lipids causes deterioration of the quality of many raw materials during their storage. In fsh meat, lipids, especially PL, are hydrolyzed not only during storage in ice, but also in a frozen state. In trout meat stored in ice, after 14 days the FA content in the total lipid content may reach 8%, and at –15° C in herring fllets, it may increase from 1% after 16 days to 10% after about eight months (Kołakowska 2011). The rate of lipid hydrolysis in fsh meat is essentially affected by the state of gonadal development and animal nutrition. In oilseeds, the progress of the reaction depends mostly on the storage conditions, including mainly seed moisture and temperature. The acid number of rapeseed oil should not exceed 1.5 mg KOH/g of oil. Refned vegetable oils usually contain below 0.05% free FA. In the non-enzymatic hydrolysis of fats catalyzed by mineral acids, with excess water, glycerol and FA are obtained from TAG, and in the presence of NaOH or KOH glycerol and soaps sodium or potassium salts of FA are obtained, respectively. PL are easily completely hydrolyzed in an acidic medium, while in an alkaline medium, FA residues are rapidly cleaved off, and phosphatidyl esters are hydrolyzed at a lower rate.

8.4.3 ESTERIFICATION Generally, in stored foods, FA esterifcation does not occur. It is used for analytical purposes and in the processing of fats. The FA are directly converted to methyl esters in preparation for gas-liquid chromatographic analysis. The reaction is carried out in

FIGURE 8.12 Bonds hydrolyzed by phospholipases A1, A2, B, C, and D in glycerophospholipids.

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the presence of methanol and catalysts – H2SO4, HCl, or BF3, or, nowadays, probably most commonly with diazomethane:

In the processing of oils and fats, alcoholysis is used to obtain esters and incomplete acylglycerols directly from TAG (Figure 8.13). Using acidolysis, the individual acyls in TAG can be substituted by others by reaction with the appropriate FA in the presence of catalysts. In order to obtain fats with the desired rheological and nutritional properties, the distribution and composition of the acyls in the TAG can be changed by appropriate transesterifcation in the presence of alkaline catalysts or enzymes. The positions of acyls can be replaced within a TAG molecule (intramolecular interesterifcation) or between different TAG (intermolecular interesterifcation). When equilibrium is reached, in the transesterifed fat, the acyls are statistically distributed in all TAG positions. Intermolecular interesterifcation of mixtures of various fats and oils leads to the formation of fats with the desired properties by changing the composition and structure of the TAG. The chemical transesterifcation of the mixture of canola oil and fully hydrogenated cottonseed oil results in fat with a reduced content of trisaturated and an increased proportion of monounsaturated and diunsaturated TAG. It leads to changes in the temperature and crystallization enthalpy and modifcation of the product’s functional properties. A shortening with very good functional properties and without TFA can be obtained by chemical esterifcation of a mixture of sunfower oil and fully hydrogenated canola, and soybean oils in a ratio of 70:17:13 (Ahmadi and Marangoni 2009). Similarly favorable results were obtained by chemical interesterifcation of a mixture of beef tallow and canola oil (Liu et al. 2009). Fully hydrogenated cottonseed oil, containing a lot of palmitic acid residues, is a very good component of the mixtures subjected to interesterifcation. In fats rich in this acid, β’-crystals are formed after cooling and are benefcial for the product’s plasticity.

FIGURE 8.13 Alcoholysis of oils and fats.

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Directed interesterifcation at the appropriate temperature can cause the resulting fully saturated TAG to crystallize. The formed liquid and solid phases have different compositions and properties. In enzymatic transesterifcation, lipases are used, mainly of microbial origin, among them regiospecifc or selective toward PUFA, immobilized on various carriers. By using such enzymes, fats with desired properties can be obtained under mild conditions. Lipases are also used to produce concentrates of biologically active PUFA. The very strongly curved hydrocarbon chains of EPA and DHA are a steric hindrance for many lipases near the ester bond. Therefore, these enzymes release mainly SFA and MUFA from the TAG, thus enriching the fsh oil with the essential FA.

8.4.4

HYDROGENATION AND ISOMERIZATION

Double bonds in the FA chains are hydrogenated to increase the melting point and the oxidation resistance of fats. Thus, plastic fats at room temperature less sensitive to autooxidation are obtained from oils. The reaction is carried out with hydrogen under a pressure of 105 Pa at 120–220° C in the presence of catalysts containing various metals – Ni, Pt, Pd, Cu, or Co. The reaction rate depends on the degree of unsaturation and distribution of acyls in TAG, catalyst type and concentration, oil contamination, temperature, pressure, and mass transfer effciency in the autoclave. The hydrogenation rate of PUFA is higher than that of dienoic acids and MUFA. Some catalysts do not catalyze the reaction with MUFA. The hydrogenation rate of the same acyl in the sn-1 and sn-3 positions in TAG is higher than in the sn-2 position. The reaction progress is followed by the determination of the iodine number or the refractive index. The hydrogenation mechanism involves the chemisorption of hydrogen and unsaturated acyl on the surface of the catalyst resulting in the activation of the reaction substrates. The activated hydrogen atom binds to one carbon atom in – C=C–, and the other C atom binds to the catalyst; the unstable semi-hydrogenated acyl is formed. In a reversible reaction in defciency of hydrogen near the catalyst surface, it can be decomposed to an unsaturated acyl by cleaving a hydrogen atom:

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Hydrogen can dissociate from the carbon adjacent to the left or right side of the C-atom bound to the catalyst, resulting in the –C=C– bond reconstruction in the original position, or be displaced. In the case of PUFA hydrogenation, the positional isomerism affects the formation of conjugated double bond systems or –C=C– bonds separated by several –CH2 groups. By attaching a second hydrogen molecule to the semi-hydrogenated compound, an acyl containing one double bond less than the starting substrate is formed. The possibility of free rotation around –C=C– bonds in the intermediate product enables the formation of TFA isomers. The progress of the cis/trans isomerization reaction can be signifcantly limited by reducing hydrogen defciencies at the reaction site, using new types of catalysts, and one-phase hydrogenation in the membrane reactor. Hydrogenation removes valuable biological PUFA from the oil. In addition, the partially hydrogenated fats, still containing unsaturated acyls, raise concerns among human nutritionists due to the presence of TFA. Therefore, in industry, to obtain fats with desirable rheological and nutritional properties, hydrogenation is now avoided, being replaced by interesterifcation (see section 8.4.3). The reader interested in problems of edible oil hydrogenation may fnd more information in the work of Drozdowski (2007).

8.4.5

OXIDATION

Lipid oxidation by atmospheric oxygen is very often the reason for the loss of the desired quality of many raw materials and food products, not only fatty ones. Unsaturated FA or acyls in acylglycerols or PL, sterols, and carotenoids are oxidized at room temperature or under freezing conditions. The initiation of the reaction is the formation of an alkyl radical (L•) by direct cleavage of the C–H bond with the energy of UV and visible light, attack of singlet oxygen 1O2, generated by the action of light in the presence of a photosensitizer on unsaturated FA (LH), or, according to the autooxidation mechanism, any initiator (In) present in the environment. A free perhydroxyl radical HO•2, hydroxyl radical •OH, peroxyl radical LOO•, alkoxyl LO•, or L• detaches a hydrogen atom from the carbon in the FA chain, and a lipid radical is formed:

In photosensitized oxidation, an alkyl radical is not formed, but a hydroperoxide in the trans confguration:

On the other hand, the decomposition of hydroperoxides results in radicals:

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which are involved in further reactions just like in the process initiated by the initiator. Moreover, lipid oxidation in food can be initiated by the action of lipoxygenases which catalyze the stereo- and regiospecifc oxidation of PUFA with molecular oxygen leading to the formation of the corresponding hydroperoxides. Lipoxygenases also have the ability to form high-energy complexes with substrates (radicals) resulting in oxidation initiation: lipids, carotenoids, chlorophylls, tocopherols, thiol compounds, and proteins. The amount of energy required to cleave a hydrogen atom in the unsaturated FA chain depends on the position of the methylene group. In MUFA the most labile are the methylene groups in the position α relative to the double bond, and in PUFA, the groups located between the double bonds. The reaction rate increases with the number of double bonds in the lipid molecule. The second step of lipid oxidation is propagation, in which the lipid radical reacts with oxygen. A peroxide radical is formed, which in reaction with another molecule of unsaturated lipid cleaves the hydrogen atom; a hydroperoxide and a new lipid radical are formed. This sequence is repeated many times with the available lipid molecules:

In the third step, called termination, the radicals accumulated in the environment react with each other and break the chain of reactions:

Not only lipid radicals but also protein radicals (P•) can participate in termination leading to the formation of lipid-protein complexes:

Oxidized lipids undergo further transformations – secondary products are formed affecting unfavorably the aroma, favor, rheological properties, and biological properties of food. The decomposition of unstable hydroperoxides leads to alkoxyl radicals which due to the breaking of the C–C bond produce numerous low-molecular-weight compounds (Figure 8.14). The kinetic curve of lipid oxidation shows that in the initiation period, the increase in the concentration of hydroperoxides is very small; it increases rapidly in the propagation period, and in the termination step, the peroxide number gradually decreases (Figure 8.15). The effect of the fat type (acyl composition) and the presence of an antioxidant on the rate of hydroperoxide formation can be seen. The course of the kinetic curve also indicates that the concentration of hydroperoxides,

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FIGURE 8.14 Formation of secondary oxidation products.

FIGURE 8.15 Kinetics of fat oxidation: 1 – cod liver oil, 2 – refned rapeseed oil, 3 – refned rapeseed oil with the addition of antioxidant, 4 – pork lard.

expressed by the peroxide number, in milliequivalents of active oxygen per kilogram of the studied substance, can correctly correspond to the correct progress of oxidation only in the frst two steps of the process. During the termination period, a better measurement, signifcantly correlated with the results of the sensory evaluation, is the content of malondialdehyde and other compounds reacting with 2-thiobarbituric acid (TBARS test – 2-thiobarbituric reactive substances) and the anisidine number – the result of the determination of conjugated dienals and alka-2-enals using the p-anisidine reagent, or the concentration of secondary oxidation products determined by instrumental methods (Figure 8.16).

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FIGURE 8.16 Test TBARS.

The lipid oxidation rate in food can be effectively reduced, among others by using suitable antioxidants (see Chapter 11). The effectiveness of various antioxidants depends on their polarity and on their antioxidant activity (AA): AA= Ia / Io

(8.2)

where Ia is the induction period on the oxidation kinetic curve of fat containing the antioxidant addition and Io is the induction period of fat without antioxidants. The action of specifc antioxidants (AH) is to capture radicals, especially LOO•:

The AH effectiveness is greater the more labile its hydrogen is, which is transferred to the LOO• and the lower the energy of the antioxidant radical (A•) formed in the reaction. A• radicals terminate a chain oxidation reaction by reacting with themselves or with other free radicals.

8.5 FRYING FATS 8.5.1

INTRODUCTION

Frying is used in the preparation of dishes at home and in restaurants, and in the food industry in the production of potato fries, donuts, canned and marinated fsh, and many other foods. For home frying, lard, butter, margarine, or oils are used depending on the knowledge and preferences of the household and the contents of the refrigerator. The industry uses solid fats or oils with desirable sensory properties, called frying medium, resistant to adverse changes occurring at the temperature of deep frying. These can be refned vegetable oils, partially hydrogenated or interesterifed oils, solid vegetable or animal fats, or appropriately selected mixtures of oils and solid fats at different properties.

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During deep-fat frying, usually within a few minutes at 160–200° C, generally 180° C, steam is released from the water present in the fried product and various volatile components are generated. Water evaporation accelerates the distillation of volatile compounds and the removal of oxygen from the frying medium. The loss of water from the surface of the product causes it to heat up to a temperature exceeding 100° C, while in the moist interior of the fried product is the desired temperature 70–80° C. This leads to the desired sensory properties of the fried products, among others due to the Maillard reactions. During frying, the physical and sensory properties of the fat change due to: • leakage of fat from the fatty foods into the frying medium, including PL with foaming properties, • losses of frying medium components getting into the fried products, • numerous products accumulating interactions of lipids with components of the fried products, including dimers and oligomers, emulsifers, foaming compounds, color compounds, and components affecting the favor and aroma, and also having antioxidant properties, • frying medium contamination with debris of fried food – in old-style fryers, the fne pieces of the product deposited on the heated bottom form a layer with a higher temperature than the rest of the oil, in which numerous thermal reactions take place. As a result, the effect on the sensory and health properties of fried foods changes over the time of using the frying medium. In the initial period, in very fresh frying fat, the desired color and texture surface are not achieved until the product has lost too much water. Subsequent changes in the oil, up to several hours, are benefcial as they increase the desired effect on the juiciness, color, favor, and aroma of the products. The required frying medium properties are retained over the period of approximately 50% of its total usable life, after which its quality deteriorates – the product turns brown on the surface before reaching the required degree of doneness inside and excessively absorbs fat. During further frying in the unchanged fat, the products burn easily. The total, permissible duration of the use of a frying medium and the proportions of these periods of usefulness depend on the composition of fat and the frying conditions.

8.5.2 CHEMICAL REACTIONS Long heating can cause TAG and PL degradation, oxidation, and the formation of numerous new compounds. The rate of these reactions depends on the properties of the fat and the process conditions – temperature, heating time, the presence of air and thermostable antioxidants, and interactions with other food components. These factors change during frying – for example, the frying medium temperature decreases locally for a short time due to loading a new batch of raw material into the fryer, oil contamination gradually increases, and the release of water vapor varies over time (Boskou 2011).

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High oil temperature, steam, and a long period of the frying medium used to fry the subsequent batches of the products favor the TAG hydrolysis. The accumulated products – glycerol, FA, DAG, and MAG – change the properties of the frying oil, decreasing the permissible time of use. At a very high temperature of frying, acrolein with an irritating odor is formed from glycerol, which easily evaporates from the frying medium. DAG and MAG cause foaming of frying oil. A measurement of the suitability of the frying medium for further use in the fryer is, among others, the content of dimers, oligomers, and polymers. They are formed mainly in reactions of unsaturated acylglycerols and FA, both without oxygen due to C–C bond formation and as a result of interactions of various lipid oxidation products. Homolytic cleavage of C–C bonds adjacent to the double bond occurs under anaerobic conditions (Figure 8.17). The resulting radicals react themselves resulting in the formation of various products: FA, dicarboxylic acids, FA and acylglycerol dimers, oligomers, cyclic compounds, and trans isomers. In the presence of oxygen, heating at the frying temperature causes the oxidation of not only unsaturated but also saturated lipids. The attack of oxygen results in the formation of various alkoxy radicals. Their thermal degradation and reactions with other components of the frying medium are the source of aldehydes, ketones, epoxy compounds, hydroxy acids, and hydrocarbons. Oxidation of unsaturated lipids at high frying temperatures follows the same mechanism as described in section 8.4.5, but the reaction rate is much higher. Therefore, during the evaluation of the oxidation degree of the frying medium, the determination of the peroxide value cannot be used because the thermal hydroperoxide decomposition rate can be more rapid than its formation. The oxidation and polymerization of unsaturated fats result in products with ether or peroxide bonds and additional oxygen functions (Figure 8.18) (Belitz et al. 2001). The content of various color, aroma, and favor compounds and also substances harmful to health increases in the frying medium with the time of its use. The polymers increase the viscosity of the frying oil which can affect the fat absorption by the fried product. During frying, approximately 400 compounds are formed in oil, including about 200 volatile substances, most of which are responsible for the aroma and favor of the frying medium and fried products. Among the compounds harmful to health are mainly polymers, acrolein, and acrylamide.

FIGURE 8.17 Homolytic breakdown of the C–C bond.

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FIGURE 8.18 Formation of polymers in heated frying oil in the presence of oxygen.

The requirements for quality frying oil are high in order to reduce the formation rate of undesirable products of the transformation of components of the fried products and frying medium, as well as to prolong its useful life. The percentage of PUFA in heated oils should be very low; in particular, the content of linolenic acid should not exceed 2%. Due to the risk of introducing TFA into the product, the use of partially hydrogenated oils is avoided. Instead, fats with high thermo-oxidative stability are recommended, especially those rich in oleic acid and added antioxidants that retain activity at high frying temperatures. A good frying medium is palm oil, especially palm olein. The undesirable changes during frying due to the reactions occurring in the frying oil and in the products can be partially limited by rationally selecting the heat treatment parameters to the type of the fried product and the fat composition (Boskou 2011).

8.6

LIPID EMULSIONS

8.6.1 STRUCTURE Lipids mixed with an immiscible liquid form dispersed systems called emulsions. If the oil droplets are dispersed in a continuous water phase it is an oil-in-water (o/w) emulsion, such as milk, mayonnaise, or sauces. In the water-in-oil (w/o) emulsion, the water droplets are dispersed in the fat phase such as in butter, margarine, and ice cream. To produce an emulsion, energy is required to reduce the diameter of the globules of the dispersed phase. In general, the diameter of fat droplets in food emulsions is in the range of 0.2–100 μm. Natural surfactants present in the system or added emulsifers form an interfacial layer, usually 1–40 nm thick, separating the two phases. These can be polypeptides or proteins or low molecular surfactants. Their hydrophobic chains are placed in the lipid droplets, and the hydrophilic fragments protrude into the water phase. As a result, they facilitate the formation of the emulsion and stabilize it. Milk proteins in a concentration of 0.3–2% effectively stabilize o/w emulsions containing 10% to 45% fat.

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There are many attempts to modify the structure of lipid emulsions in order to increase their physical stability and resistance to oxidation and to improve functional properties. Thus, the multilayer envelopes around the dispersed phase globules, usually thicker and tighter than a monolayer, are formed. In o/w1/w2 emulsions, at pH 7, the oil droplets (o) are contained in hydrated casein granules (w1) dispersed in a pectin-rich continuous water phase (w2). Acidifcation of such an emulsion to pH 5 causes the adsorption of pectin on the surface of the granules (w1), which leads to the formation of flled hydrogel particles with a diameter of 3–4 μm consisting of emulsifed lipid droplets (Matalanis et al. 2010). Similar o/w1/w2 emulsions can also be prepared using other proteins and ionic polysaccharides, for example, whey proteins and acacia gum. Low molecular weight surfactants incorporated into the emulsion accumulate in the interfacial layer but are also dissolved in the water phase. At a high concentration in this phase, exceeding the so-called critical micellar concentration, surfactants spontaneously form micelles. These are clusters of molecules held by hydrophobic interactions, in which non-polar chains are grouped inside, and hydrophilic fragments face the aquatic environment. In addition, the interfacial layer contains antioxidants and prooxidants, lipid hydrolysis products, and metal ions.

8.6.2

PHYSICAL STABILITY

Emulsions are inherently unstable – their physicochemical properties change under the effect of many factors, and after a long enough period of time, complete phase stratifcation may even occur. These changes occur due to Ostwald ripening, creaming, aggregation, coalescence, and partial coalescence (Frederick et al. 2010). Ostwald ripening involves the increase to the larger extent of the dispersed phase particles due to the deposition of material from droplets with smaller diameters on their surface (Figure 8.19). The pressure on the concave side of the interface is greater than on the convex side in proportion to the product of the surface tension

FIGURE 8.19 Mechanisms of destabilization of lipid emulsions: A – creaming, B – Ostwald ripening, C – aggregation, D – coalescence, E – partial coalescence.

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and curvature, resulting in higher solubility in the surrounding medium of dispersed particles from large droplets than that from small ones. When the concentration of these compounds in the aqueous phase increases above the saturated state, they settle on the large droplets. As a result, the diameter of the small dispersed globules decreases with time, and that of the large ones increases. In o/w emulsions, Ostwald ripening is practically irrelevant because TAG are only minimally soluble in water. Creaming is manifested by the accumulation of fat droplets, for example, in milk, in the top layer of the liquid jar. The rate of fat globules movement (V) is proportional to the difference in density of the components of the two phases (df – dl), the square of the diameter of the globules (r), and the gravitational acceleration (g), and inversely proportional to the viscosity of the dispersing phase (η) according to the Stokes equation: V = 2r2g (df – dl)/ 9η

(8.3)

In practice, under food processing conditions, the creaming rate is affected the greatest by the acceleration, which can be higher thousands of times than g during centrifugation. Coalescence occurs when the lipid droplets come closer together by aggregation or creaming so that the layer of the continuous phase preventing them from joining is very thin. When this layer is damaged, in particular at low oil–water interfacial tension, the droplets fuse together. Partial coalescence occurs when the dispersed lipids are globules composed of TAG crystals and liquid oil molecules. Such a phenomenon takes place in many food emulsions. The crystals form a solid network in the globules, and some protrude outside. The crystals protruding into the continuous phase may, upon collision with another globule, pierce the protective flm of this globule and form a lipid bridge between the two globules. The merging of both globules into one, larger lipid droplet is counteracted by the crystal networks formed inside them. The emulsion can lose stability due to the simultaneous occurrence of several mechanisms which, under appropriate conditions, may act synergistically with each other. The physical instability of food lipid emulsions is generally not desirable because it is the cause of product deterioration. On the other hand, some changes in emulsion state are used in the production of many food products. Milk cream is obtained by creaming, and partial coalescence is a condition for the appearance of the desired sensory properties of, among others, butter, ice cream, and whipped creams.

8.6.3 SUSCEPTIBILITY TO OXIDATION Numerous foods, including mayonnaise, sauces, dressings, and baby food, are o/w emulsions. In order to meet the modern requirements of rational nutrition, these products contain oils comprising TAG, rich in PUFA, and therefore very easily oxidized. The susceptibility of fats in lipid emulsions to oxidation is especially high because the contact surface of the fnely divided fat globules with the water phase containing the water-soluble prooxidants is enormous. Even a very considerable

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reduction in the size of the fatty globules in the range of the values found in the emulsions does not cause a corresponding increase in the oxidation degree, because under these conditions the change in the size of the interfacial area does not largely affect the rate of the reaction. The factors affecting the oxidation rate of emulsifed lipids are related to the properties of both the emulsion phases and the interfacial phase (Table 8.4). Transition metals are among the most active prooxidants, especially Fe(II) as they increase the rate of hydroperoxides decomposition to radicals in the surface layer of fat droplets. Therefore, transferrin and lactoferrin, iron-binding milk proteins, and egg phosvitin inhibit oxidation very effectively (Waraho et al. 2011). Phosphorylated casein peptides are also good chelators. The oxidative stability of the o/w emulsion depends very strongly on how effectively the fat globules repel or attract metal ions, and therefore on the type and magnitude of the electric charge on the surface. Thus, the prooxidant activity of the transition metals can be affected by selecting the type of emulsifer that binds in the surface layer, and also the pH and ionic strength of the medium.

TABLE 8.4 Factors Affecting Lipid Oxidation in the o/w Emulsions Lipid phase Degree of acyl unsaturation Concentration and size of droplets Prooxidants: free FA, hydroperoxides Inherent antioxidants: free radical scavengers, chelators Added antioxidants Solubility, partition coeffcient, and diffusion rate of antioxidants and prooxidants Viscosity affecting diffusion rate of antioxidants and prooxidants Polarity affecting partition coeffcient Water phase pH and ionic strength Prooxidants: transition metals, photosensitizers, enzymes Inherent and added antioxidants Micelles affecting availability of antioxidants and prooxidants Reducing agents taking part in redox cycle transition metals Solubility, partition coeffcient, and diffusion rate of substrates and products Viscosity Polarity Interfacial phase Antioxidant and prooxidant activity Thickness and steric hindrance of interactions between both phase components Electrostatic attraction and repulsion of antioxidants and prooxidants Diffusion of antioxidants and prooxidants in both phases Source: Waraho et al. 2011.

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The oxidation rate of emulsifed lipids depends very signifcantly on the properties of the interfacial phase limiting the contact of the fat droplet with oxygen, radicals, and prooxidants present in the aqueous phase – thickness, cohesiveness, porosity, and viscosity, especially of the protein envelope. The proteins of this envelope can react with the radicals from the fat globule and with the aldehyde products of lipid oxidation, which affects their properties and the sensory effects of oxidation. Also, proteins present in the water phase can inhibit the oxidation of fat emulsions by acting as chelators and free radical scavengers in the environment. The effectiveness of the radical scavengers as antioxidants depends on their reactivity and location in the emulsion. In general, nonpolar antioxidants are more effective than polar antioxidants, because they are bound on the surface of the lipid droplets and react in places of the greatest risk. Surfactant micelles (see section 8.6.1) affect the kinetics of lipid oxidation because they can dissolve or bind low-molecular-weight components of the aqueous phase, including hydroperoxides, transition metal ions, prooxidants, and antioxidants.

REFERENCES Ahmadi, L., Marangoni, A.G. 2009. Functionality and physical properties of interesterifed high oleic shortening structured with stearic acid. Food Chem., 117(4), 668–674. https://doi.org/10.1016/j.foodchem.2009.04.072 Basso, R.C., Ribeiro, A.P.B., Masuchi, M.H, Gioielli, L.A, Gonçalves, L.A.G., dos Santos, A.O., Cardoso, L.P., Grimaldi, R. 2010. Tripalmitin and monoacylglycerols as modifers in the crystallisation of palm oil. Food Chem., 122(4), 1185–1192. https://doi.org /10.1016/j.foodchem.2010.03.113 Belitz, H.D, Grosch, W., Schieberle, P. 2001. Lehrbuch der Lebensmittelchemie. Berlin: Springer-Verlag. Boskou, D. 2011. Frying fats. In: Chemical, Biological, and Functional Properties of Food Lipids, Z.E. Sikorski and A. Kołakowska (eds.), pp. 429–454. Boca Raton: CRC Press. Bouzidi, L., Boodhoo, M.V., Kutek, T., Filip, V., Narine, S.S. 2010. The binary phase behavior of 1,3-dilauroyl-2-stearoyl-sn-glycerol and 1,2-dilauroyl-3-stearoyl-sn-glycerol. Chem. Phys. Lipids, 163(6), 607–629. https://doi.org/10.1016/j.chemphyslip.2010.05.002 Commission Regulation (EU) 2019/649 of 24 April 2019 amending Annex III to Regulation (EC) No 1925/2006 of the European Parliament and of the Council as regards trans fat, other than trans fat naturally occurring in fat of animal origin. Drozdowski, B. 2007. General characteristics of edible fats. In: Food Chemistry, Vol. 1, Fifth Edition, Z.E. Sikorski (ed.), pp. 145–166. Warsaw, Poland: Wydawnictwo NaukowoTechniczne (in Polish). Fedotova, Y., Lencki, R.W. 2010. The effect of phospholipids on butter physical and sensory properties. J. Amer. Oil Chem. Soc., 87(1), 75–82. https://doi.org/10.1007/s11746-009 -1468-2 Forycki, Z.F. 2011. Dietary lipids and coronary heart disease. In: Chemical, Biological, and Functional Aspects of Food Lipids, Z.E. Sikorski and A. Kołakowska (eds.), pp. 211– 226. Boca Raton: CRC Press. Frederick, E., Walstra, P., Dewettinck, K. 2010. Factors governing partial coalescence in oilin water emulsions. Adv. Coll. Inter. Sci., 153(1–2), 30–42. https://doi.org/10.1016/j.cis .2009.10.003 Guichard, E. 2011. Binding and release of favor compounds. In: Food Flavors: Chemical, Sensory and Technological Properties, H. Jeleń (ed.), pp. 137–154. Boca Raton: CRC Press.

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James, B.J., Smith, B.G. 2009. Surface structure and composition of fresh and bloomed chocolate analysed using X-ray photoelectron spectroscopy, cryo-scanning electron microscopy and environmental scanning electron microscopy. LWT – Food Sci. Technol., 42(5), 929–937. https://doi.org/10.1016/j.lwt.2008.12.003 Jeleń, H., Wąsowicz, E. 2011. Lipid derived favor compounds. In: Food Flavors. Chemical, Sensory and Technological Properties, H. Jeleń (ed.), pp. 65–93. Boca Raton: CRC Press. Kołakowska, A. 2011. Fish lipids. In: Chemical, Biological, and Functional Aspects of Food Lipids, Z.E. Sikorski and A. Kołakowska (eds.), pp. 273–312. Boca Raton: CRC Press. Liu, Y., Meng, Z., Shan, L., Jin, Q., Wang, X. 2009. Preparation of specialty fats from beef tallow and canola oil by chemical interesterifcation: Physico-chemical properties and bread applications of the products. Eur. Food Res. Technol., 230(3), 457–466. https:// doi.org/10.1007/s00217-009-1188-8 Matalanis, A., Lesmes, U., Decker, E.A., McClements, D.J. 2010. Fabrication and characterization of flled hydrogen particles based on sequential segregative and aggregative biopolymer phase separation. Food Hydrocoll., 24(8), 689–701. https://doi .org/10.1016/j.foodhyd.2010.04.009 Pokorný, J., Schmidt, Š. 2011. Phospholipids. In: Chemical, Biological, and Functional Aspects of Food Lipids, Z.E. Sikorski and A. Kołakowska (eds.), pp. 97–112. Boca Raton: CRC Press. Sikorski, Z.E., Kołakowska, A. (eds.) 2011. Chemical, Biological, and Functional Properties of Food Lipids, Second Edition. Boca Raton: CRC Press. Stołyhwo, A. 2007. Lipids and food quality. In: Chemical and Functional Properties of Food Components, Third Edition, Z.E. Sikorski (ed.), pp. 23–34. Boca Raton: CRC Press. Tunick, M.H. 2011. Milk lipids. In: Chemical, Biological, and Functional Aspects of Food Lipids, Z.E. Sikorski and A. Kołakowska (eds.), pp. 313–325. Boca Raton: CRC Press. Waraho, T., McClements, D.J., Decker, E.A. 2011. Mechanisms of lipid oxidation in food dispersions. Trends Food Sci. Technol., 22, 3–13. https://doi.org/10.1016/j.tifs.2010.11 .003 Wiking, L., De Graef, V., Rasmussenn, M., Dewettinck, K. 2009. Relations between crystallisation mechanisms and microstructure of milk fat. Int. Dairy J., 19(8), 424–430. https://doi.org/10.1016/j.idairyj.2009.03.003

9

Factors Affecting the Rheological Properties of Foods Robert Tylingo

CONTENTS 9.1 9.2

Introduction .................................................................................................. 265 Basic Dependencies and Research Methods of Rheologic Properties .........266 9.2.1 The Impact of Food Ingredients on Its Rheologic Properties ..........266 9.2.2 Methods of Testing Food Rheologic Properties – Defning Food Mechanical Properties ...................................................................... 269 9.2.3 The Research Methods of Food Rheologic Properties – Rheology of Liquids ......................................................................... 275 9.2.3.1 Laminar and Turbulent Flow ............................................. 275 9.2.3.2 Dynamic Viscosity............................................................. 275 9.2.3.3 Relative Viscosity .............................................................. 277 9.2.3.4 Kinematic Viscosity........................................................... 277 9.2.3.4 Shear Stress........................................................................ 277 9.2.3.5 Shear Speed γ .................................................................... 277 9.2.3.6 Viscosity Parameters.......................................................... 277 9.2.3.7 The Division of Liquids ..................................................... 279 9.2.3.8 Viscosity Measurements ....................................................280 References.............................................................................................................. 283

9.1 INTRODUCTION A pre-Socratic Greek philosopher, Heraclitus of Ephesus, in 500 BCE, while elaborating a change concept, stated that “panta rhei,” which means that everything fows, thus depicting, in two words, the essence of rheology as a science on matter fow and deformation. All the materials are characterized by rheologic properties and the assessment of these properties is signifcant in numerous scientifc felds. Food rheology is an interdisciplinary science concerning food technology, chemical engineering, physics, mathematics, and sensorics, which arises from its utility signifcance. It plays an important role in basic aspects connected with food and its production, enabling us to understand the infuence of food ingredient properties on the selection of operation and unit process parameters, e.g. during stirring, pumping,

DOI: 10.1201/9781003265955-9

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and gelatinizing. It also allows us to understand the infuence of food ingredients on the end product properties, e.g. hardness and elasticity, which are assessed by consumers. There has been long-term research conducted that determines dependencies between food ingredients and their rheologic properties, which results in plenty of literature data on the aspects of liquid and solid engineering (Joyner et al., 2017).

9.2

BASIC DEPENDENCIES AND RESEARCH METHODS OF RHEOLOGIC PROPERTIES

Rheology characterizes body deformations due to stresses in solids, liquids, and gases. It is divided into microrheology and macrorheology. Microrheology deals with determining dependencies between the body structure which is real at the molecular level and the rheologic properties of such a body. Macrorheology deals with the mathematical theory of continuum, fuid mechanics, and material strength. Perfect solids deform in an elastic manner, which means that it is possible to regain the total energy consumed for deformation after stress elimination. Perfect fuid continua (gases and liquids) deform in an irreversible manner. This phenomenon is called a fow and energy consumed for deformation is dispersed in the form of heat. In most cases, unprocessed food raw materials contain from 60% to 90% water; however, some products are solids and others are liquids. Differences in a physical state result from the microstructural impacts of food ingredients. This complex matrix in many cases consists of a mixture of solid and liquid ingredients, and it is mostly treated as a real body that is neither a perfect solid nor a perfect liquid continuum. Real solid bodies may also deform irreversibly under suffciently large forces. Thus, a creeping or fowing process occurs. Basic mathematical tests are limited by a sample shape, the size and direction of forces, and a deformation shape. It is really diffcult to transfer them to bodies that are a complex matrix and cannot satisfy these requirements. An advantage of mathematical tests is that it is possible to anticipate rheologic properties on the basis of known dependencies. If food is solid, it is possible to defne its mechanical properties, and if a liquid expressed with a parameter is viscosity, dependent on shear stresses generated during fow (Tabilo-Munizaga and Barbosa-Cánovas, 2005).

9.2.1 THE IMPACT OF FOOD INGREDIENTS ON ITS RHEOLOGIC PROPERTIES Food is a complex matrix and each ingredient plays its role in the formation of rheologic properties. An example here is the distribution of fatty acids in triacylglycerols, which determines their melting temperatures and crystalline properties (Staniewski et al., 2021). Interactions affecting the formation of ionic covalent bonds and inter-particle interactions between food ingredients determine such food properties as viscosity, crispness, or elasticity. Inter-particle interactions are also crucial in the generation and stabilization of emulsions in the following products: ice creams, creams, sauces, yogurts, and mayonnaises. The proper share of water, lipids, emulsifers, e.g. lecithin, and stabilizers, e.g. polysaccharides, determines the sensory

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acceptability of food products based on emulsions. Technological processes and operations, conducted during food processing, play separate roles in the formation of rheologic properties. These transformations result from ionic force changes, protein denaturation, fat hydrolysis, and water elimination, and they occur e.g. during thermal processing, the use of preservatives, and ripening of cold meat and cheese. Being familiar with the impact of respective food ingredients and interactions occurring between them allows technologists to anticipate and modify the end rheologic properties of food. Water is found in food in high quantities (considering the weight share in a product). If its molar ratio is demonstrated, it is present even in dried products in the number of particles comparable to solid substances. The presence of water in food and the interaction of water and food ingredients play a signifcant role in the formation of rheologic properties. Interactions between water and food ingredients determine such parameters as solution viscosity, gel hardness, or meat crispness. In rheologic terms, proteins fulfll many functions in food (Table 9.1). In addition, by means of modifying their properties, it is possible to obtain various end structures with different rheologic parameters. Muscle proteins and connective tissue proteins are responsible for the crispness and elasticity of meat undergoing thermal processing. The physical-chemical transformations of proteins during

TABLE 9.1 The Rheologic Functions of Selected Proteins Found in Food Protein source Meat

Protein type Actin, myosin

It plays an important role in the formation of a meat texture It forms stable gels in the wide range of temperatures They form stable emulsions They enable meat restructuring

Collagen

It plays an important role in the formation of a meat texture If cross-linked, it enables the formation of insoluble structures In the presence of Ca2+ they form durable gels During freezing, they form fbers Together with lipids, they form stable membranes During precipitation, they form fbers in an isoelectric point During extrusion, they form stable fbrous structures They give viscous-elastic properties to the kneaded dough It forms stable emulsions It forms a stable foam They form stable gels They form a stable foam They form stable emulsions

Legumes

Glycinin, conglycinin

Cereal grains Milk

Gliadin, glutenin, Casein Whey proteins

Eggs

Rheologic functions

Ovalbumin, ovotransferrin, ovomucoid

They form stable gels They form a stable foam They form stable emulsions

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technological processes related to food production play a signifcant role in the formation of the end product texture. Myosin, actomyosin, and collagen are mostly responsible for gelatinization and the elastic properties of the structure of readycold meat and fsh processed products. Owing to the specifc properties of actin and myosin found in white fsh meat, it is possible to generate a stable, gel structure of a kamaboko dish (popular in Japan) (Sankar and Ramachandran, 2002). The interactions of soya proteins with calcium ions are responsible for the formation of a tofu gel structure. Casein transformations during cheese ripening allow us to obtain specifc rheologic properties affecting the sensory quality of the product obtained (Lucey et al., 2003). Undenatured β-lactoglobulin found in whey has very good emulsifying, gelatinizing, and foaming properties (Moro et al., 2013). The diversity of proteins and their variable properties, depending on the physicalchemical conditions of food, enable obtaining a wide range of products with desirable rheologic properties. Fats fulfll an essential function in the rheologic properties of emulsions, obtaining a proper texture or viscosity of a food product. The chemical or enzymatic modifcations of lipids allow us to obtain products with specifc functionality. Some lipid forms play an important role in the formation of a food structure, and these are phospholipids. Lecithin (from soya or egg yolks) is frequently used as an emulsifer aiding the obtaining of water-oil mixtures. Sugars, in terms of structure, shape, and size, are a varied group of food ingredients. Polysaccharides with a high molecular weight determine rheologic properties, and monosaccharides and polysaccharides with a low molecular weight play a less signifcant role. Starch, cellulose, hemicellulose, pectins, and plant resins (rubbers) determine texture indicators, such as crispness, hardness, and sensory properties. Polysaccharides, in many cases, form gels, and as their molecular weight grows, they increase the viscosity of solutions. A monosaccharide ring is basically a rigid particle. After joining with another monosaccharide by means of a glycoside bond, it forms a secondary structure that is not so rigid. When a glycosidic bond is in the 1,6 position, it is possible to rotate the particle, which makes the entire structure more elastic. Tertiary and quaternary polysaccharide structures, for instance, in the form of joined helixes or spirals are often elastic. During polysaccharide hydration, water reaches amphoteric areas quickly and surrounds polymers. Then, polysaccharide chain segments may undergo complete solvation, creating a hydrocolloid. Polysaccharides with a function of food thickening are found in a solution in an unordered, coincidental conformation, which results in the increase of mixture viscosity, whereas polysaccharides forming gels have an ordered form. A gelatinization mechanism depends on the type of polysaccharide and it concerns mostly water confnement in the three-dimensional network of ordered polysaccharide chains. Most polysaccharides used in the food industry have a plant origin and their merchantability is connected mainly with the possibility of modifying the rheologic properties of end products. They are generally used as thickening additives, gelling additives, and synaeresis inhibitors. They are able to form hetero-dispersion systems which increase viscosity greatly even at low concentrations. In order to modify viscosity, the polysaccharide concentration

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used is mainly below 1% (Goff and Guo, 2019). Polysaccharides are also used as agents for stabilizing suspensions and membrane-generative substances, provided that capsules and microcapsules are received (Moro et al., 2013). Technological suitability depends on the presence of other food ingredients, time, temperature, and technological processing environment. Polysaccharides that are most abundant in nature include cellulose and starch. Cellulose is responsible for maintaining the structure of vegetables and fruit which contain about 80–90% of water. The main function of cellulose is forming a cell wall by joining hemicellulose, protein, and lignin, at the same time ensuring structural integrity. Starch is a polysaccharide which is a plant reserve substance. It is not soluble in cold water and after heating, in the presence of excessive water, it forms gels. Diluted starch solutions, after cooling down, may be subject to retrogradation. More concentrated dispersions form viscous-plastic gels (Lovegrove et al., 2017). Food, in practice, is a mixture of many ingredients, which determine its rheologic properties. In addition, interactions between proteins, lipids, and polysaccharides affect the sensory properties of food products. Some reactions taking place during technological processes, e.g. during meat or cheese ripening, will contribute to obtaining a product ft for consumption. Due to food complexity, the description of its rheologic properties requires conducting empirical tests; only then it will be possible to assess the impact of food ingredients on rheologic properties.

9.2.2 METHODS OF TESTING FOOD RHEOLOGIC PROPERTIES – DEFINING FOOD MECHANICAL PROPERTIES During food production, processing, and consumption, gels are subject to large deformations, which may cause irreversible changes in their structure and fractures. In order to assess such changes, a range of mechanical tests is carried out. Traditionally, tests determining gel hardness are conducted. These are point tests that do not demonstrate a full specifcation of the mechanical properties of gels because gels also possess the rheologic properties of liquids (small solid body parts are dispersed in a large quantity of liquid). The texture of food products, such as meat, bread, and many others, is also assessed by means of performing mechanical point tests. These defnitions enable the determination of the technological processing impact on the rheologic properties of end products. A basic phenomenon that describes mechanical properties is product destruction in the form of pressure. Gel fracture is a property sensed by people during food biting and chewing, and it determines the texture parameters of many food products, e.g. hot dog sausages. A fracture parameter is determined during sample deformation achieved by shearing, compression, or stretching. Compression changes the test sample shape, at the same time not changing its volume. Compression may lead to sample volume reduction. Stretching may lead to its volume increase. When determining a force needed for sample fracture, compression tests are used. Most universal testing machines are equipped with proper accessories allowing for conducting compression tests. An important element when testing food is a device measurement range which must be similar to stresses during

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food consumption. Breaking tests determine material behavior in the conditions of axial tensile loads. Data obtained in the tensile test are used for determining a proportionality limit, assumed plasticity limit, tensile strength, and other properties connected with stretching (Figure 9.1A). Tensile tests performed at higher temperatures provide data on creeping. A compression test consists of the axial compression of gel by means of a cylindrical head. As in puncture testing, the force needed for obtaining a specifc deformation is measured or the deformation obtained with the constant force applied to the sample is measured. Compression strength is the maximum stress which may be taken by a material in the conditions of a crushing load. This test enables obtaining data connected with compressive stress and deformation and then determining a “stress-strain” dependence which allows us to defne parameters similar to breaking test parameters (Figure 9.1B). The compressive strength of a material, which gets damaged due to fracture resulting in disintegration, may be determined in a relatively narrow range as an independent property. Nevertheless, compressive strength, in the event of materials that do not disintegrate under a load, must be defned as a stress value needed for material deformation to an arbitrarily defned degree. Compressive strength is calculated by dividing the maximum load by the primary cross-section feld of a sample under a compressive test. An advantage of compressive tests is the elimination of a problem connected with sample mounting (Aguilera and Stanley, 1999). An important parameter in food testing, especially for gels, is an elasticity modulus known as Young’s modulus (Equation 9.1). This modulus specifes a deformation change in the stress function during compressive or tensile tests. E˜

F tension = A extension °L L

(9.1)

FIGURE 9.1 Stress-strain dependence in breaking (a) and compressive tests (b). 1 – proportionality limit, 2 – assumed plasticity limit, 3 – maximum stress, compressive strength, or breaking strength (ε – deformation, σ – stress).

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Where: E – Young’s modulus; F – force perpendicular to the sample cross-section; A – sample section area; L – sample length or height; ΔL – length or height change resulting from F force impact. Young’s modulus is determined by defning the straight gradient of a part of the stress-strain graph. This modulus may be used only for elastic materials, and most food demonstrates viscous-elastic properties, is not uniform, and has an irregular shape. Some researchers claim that this modulus is applied incorrectly in food analysis and suggest calling it a “deformability modulus” in such cases. The exemplary values of Young’s modulus for selected food products are presented in Table 9.2. In order to analyze a food chewing moment, we need a shear elasticity modulus which is also called a G cross elasticity modulus. The shear elasticity modulus is sometimes referred to as a rigidity modulus but still a G symbol is used (Equation 9.2) (Aguilera and Stanley, 1999). G˜

F share tension = A share extension ° L

(9.2)

G – shear elastic modulus; F – force parallel to the surface determined as stress; γ – the largest distance of a given material dislocation during shearing; L – sample length or height. A protein that is intrinsically related to the gelatinizing phenomenon is gelatin. A gelatin is a mixture of polypeptides of varied lengths, which makes it a non-uniform solution in the form of a gel, sol, or liquid. With temperature decrease, sol transforms

TABLE 9.2 Deformation Modulus Values for Selected Food Products Material

E, apparent Young’s elasticity modulus (Pa)

Raw apple

60–140

Fresh banana Bread Raw carrot Gelatin gels Fresh peach Raw pear

8–30 0.1–0.3 200–400 2 20–200 120–300

Raw potatoes

60–140

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into gel; with temperature increase, gel transforms into sol. The reversible gelatin gelatinizing process is, in theory, unlimited and it is one of the most important physical-chemical parameters defning a gelatin quality (Djabourov et al., 1988). Other hydrocolloids, such as alginates, carrageenins, and pectins also form gels, yet this process reversibility is often limited signifcantly by additional chemical reactions which affect the gelatinization of such polysaccharides. Tests consisting of piercing a gel with a pin of a standardized size allow us to determine simply a dependence between stress and the deformation obtained. A pin is submerged in a gel at a specifed depth (Figure 9.2). The test results allow us to determine gel strength. This type of test is often used for solid and semi-solid food products. One of the variants of this type of test is the determination of gelatin gel hardness. A rheologic parameter of gelatin gels is the Bloom value (Netter et al., 2020). This name is taken from the surname of Oskar T. Bloom, who in 1925 developed a device used in gel hardness marking. A Bloom value is a pin mass in grams which is required for submerging a 0.5-inch pin at the depth of 4 mm in the gelatin gel with a concentration of 6.67% (w/v), regulated thermally for 17 hours at the temperature of 10° C (Figure 9.3). In the gel hardness marking technique, it is obligatory to adhere to the accuracy of operations connected with sample preparation; what is also crucial is the weight of the sample and solution volume, and the measurement vessel must have a proper shape (Figure 9.3). At the stage of gelatin dissolution, it is necessary to maintain the temperature below 60° C (optimally 50–55° C) for 20 minutes. The vessel must be closed in order to limit water evaporation and it is necessary to return

FIGURE 9.2 Piercing tests (A – exemplary pins used in piercing tests, B – piercing test). Photograph by the author.

The Rheological Properties of Foods

FIGURE 9.3

273

Determining gelatin hardness in Bloom test. Photograph by the author.

the condensate to the solution. It is required to avoid solution foaming. Gelatinization must take place slowly. Sudden solution cooling may lower gel hardness by 10% as compared to gels which during gelatinization underwent a slow cooling process of the gelatin solution. Furthermore, it is absolutely essential to maintain the temperature of the sample in the water bath at the value of 10 ± 0.1° C. The Bloom value of commercial gelatins falls within the range of 50–300. Gelatin with hardness from 200 to 300 Bloom value is defned as a high hardness gelatin, from 10 to 200 Bloom value medium hardness, and from 50 to 100 Bloom value low hardness. Gelatins with higher Bloom values have a higher melting temperature and gelatinizing temperature, shorter gelatinizing time, their color is lighter, and their odor and taste are neutral. Higher gel hardness enables the use of lower gelatin concentrations in order to achieve a desirable gel rigidity in ready-made products. A texture is a sensory and functional derivative of a structure; it comprises mechanical and surface food properties determined by means of the sense of eyesight, hearing, touch, and kinaesthesia (an experience connected with the movement of the object being touched). Texture, besides appearance, odor, taste, and nutritional value, is a signifcant discriminant of food quality (Szczesniak, 2002). For such products as meat, cornfakes, potato crisps, fruit, vegetables, and cheese, texture is a factor determining the consumer’s sensory acceptability. Furthermore, a texture may be characterized by non-destructive tests with the use of, for instance, acoustic waves, an own-resonance phenomenon, a method with the use of lasers, friction sensors, or non-destructive impacts (Miri, 2011). The use of destructive tests based mainly on strength methods allows us to imitate an eating process in which large product deformations and destructions take place. The applied mechanical tests include bending,

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stretching, and compressing. Compression is the most popular test because it is similar to the process of biting and chewing. Compressive tests are performed between parallel plates or piercing tests are applied. Food tests conducted in recent years demonstrate the presence of dependencies between texture profle analysis (TPA) tests, which enable the determination of a food’s mechanical properties and its structure (Peleg, 2019). The TPA test is conducted by the uniaxial compression of samples between two parallel plates in two cycles, each at a proper speed (e.g. 20 mm/min). A deformation level is selected experimentally in order to avoid destroying the sample. Most frequently, the deformation level is about 20% of the sample height. Based on the obtained TPA curves (Figure 9.4), it is possible to determine: hardness as the maximum force in the frst tests cycle (H1 [N]), elasticity expressed by a ratio of distance covered by the pin from the compression beginning to obtaining the maximum force in the second and frst cycle (L2/L1 non-dimensionless parameter), cohesiveness as a ratio of positive areas for the compression curve in the second and frst cycle (A2/A1 dimensionless parameter), and springiness expressed by the product of the hardness and cohesiveness value [N]. The area of a negative peak, A3, is defned as sample adhesiveness. This is work indispensable for separating the sample during the head lifting. This peak does not always appear during the TPA tests. It is connected with the surface impact of the sample at the point contacting the head. The parameters described enable the quantitative and qualitative assessment of a food texture and the determination of the conformity of the production process implemented with the high requirements to be met by food producers (Bourne, 2002). Additional information on the texture may be obtained by conducting shear and break tests. The shear strength of products, such as gels or meat, is determined by

FIGURE 9.4 The curve of the profle texture analysis – TPA (A1, A2, A3 – peak areas; H1, H2 – maximum peak heights; L1, L2 – distances from the peak beginning to obtaining its maximum) (Bourne, 2002).

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applying a shear load to the material in the torsion test. Shear strength is the maximum load needed for sample shearing so that a part after shearing is separated completely, expressed in psi pressure units after including the area of the edge sheared.

9.2.3 THE RESEARCH METHODS OF FOOD RHEOLOGIC PROPERTIES – RHEOLOGY OF LIQUIDS One of the frst scientists researching the phenomena occurring during liquid fowing was Isaac Newton (1642–1726), who stated that “resistances formed during liquid fowing are directly proportional to the fow rate.” Currently, this theory is connected directly with the rheologic properties of perfectly viscous fuids (Newtonian fuids). A French physicist and physician, Jean Louis Marie Poiseuille (1797–1869), while conducting tests on the human blood fow in thin capillary vessels, contributed to the development of contemporary viscometry. The word “rheology” is claimed to have been used for the frst time by E.C. Bingham (1876–1945), who was one of the pioneers of the mathematical interpretation of rheologic properties. Although works connected with the rheologic properties of liquids have been conducted for years, it is often diffcult to predict how a given liquid will behave in the technological process. The variability and complexity of a matrix which is food often force scientists to conduct the empirical defnition of rheologic properties. Some crucial defnitions related to viscometry are presented in the following. They are an introduction to the determination of liquid rheologic properties. 9.2.3.1 Laminar and Turbulent Flow Laminar, layered fow takes place in parallel liquid layers without any disturbances between them. After exceeding a certain fow rate value, laminar fow transforms into turbulent fow (Figure 9.5). A threshold transformation speed of one form into another is determined by Reynold’s number (Re). After exceeding the threshold value, laminar fow transforms into transient fow, partially turbulent, and then turbulent fow (Figure 9.5). 9.2.3.2 Dynamic Viscosity Dynamic viscosity, often called viscosity or absolute viscosity, is the property of liquids and plastic solid bodies which characterizes resistance occurring as a result of

FIGURE 9.5 The types of fow through a capillary vessel with a circular section surface. A – laminar fow, B – turbulent fow, C – transient fow (partially turbulent).

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friction during substance fow. This is one of the key properties of liquids and plastic bodies. The dynamic viscosity values of selected liquids are presented in Table 9.3. Dynamic viscosity is expressed by Equation 9.3:

˜˝

° ˛

(9.3)

Where: η– viscosity; σ – shear stress; γ – shear speed. A dynamic viscosity unit is Pa s (Pascal second) also expressed as m–1kg s–1. The following unit is also often used: mPa s (1,000 mPa s = 1 Pa s). A former unit was poise P (a name taken from the surname of Jean Louis Marie Poiseuille). 1P = 1 10 –1Pa s In practice, a unit 100 times smaller is often used: centipoise (cP). 1 cP = 1 mPa s A viscosity value is often conditioned by temperature (Table 9.2). One must note that water viscosity at the temperature of 20° C equals 1mPa s = 1cP. Sometimes, instead of viscosity, one may encounter the term liquidity. Liquidity ϕ is a conversion of viscosity and it is presented by Equation 9.4: ˜

° ˛

(9.4)

TABLE 9.3 The Dynamic Viscosity Values of Selected Liquids Liquid Air (0° C) Water (0° C) Water (20° C) Water (100° C) Ethanol (25° C) 20% saccharose solution (20° C) 80% saccharose solution (20° C) Diethyl ether (20° C) Glycerol (20° C) Honey Blood Source: Bourne, 2002.

Viscosity (mPa s) 1,708 · 10–4 1.7921 1,000 0.2838 1.07 1,967 40,000 0.23 1,759 2,000–10,000 ≈3

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9.2.3.3 Relative Viscosity Relative viscosity is also called a viscosity indexηrel. This is a ratio of dynamic viscosity η of a solution to the dynamic viscosity of pure solvent ηs and it is expressed by Equation 9.5:

˜rel °

˜ ˜s

(9.5)

9.2.3.4 Kinematic Viscosity Kinematic viscosity ν is a ratio of dynamic viscosity to liquid density (Equation 9.6):

˜˙

° ° ˙ ˛ ˛ ˆ ˝

(9.6)

Where: ν – kinematic viscosity; Η – dynamic viscosity; ρ – liquid density; Σ – shear stress; γ – shear speed. Kinematic viscosity in the SI system is expressed by m2·s–1. 9.2.3.4 Shear Stress Stress is expressed by the ratio of force to the area on which this force is applied. Shear stress occurs if the direction of the applied force is tangential to the area (shear force). In rheology, shear stress refers to liquid pressure (apparent shear stress is directly proportional to the pressure measured). Shear stress is expressed in Pa. 9.2.3.5 Shear Speed γ Shear speed is a ratio of the relative speed difference between adjacent liquid layers to the distance between them. Shear speed is connected directly with stresses occurring during liquid fow. In the SI system, a shear speed value is 1/s. For instance, shear speed during mouth wetting is about 50 s–1, and a household food processor works in the shear speed range of 100 to 1,000 s–1. The increase in shear speed depending on the liquid type causes viscosity to decrease or increase. In some liquids, time results in the generation of so-called “shear history.” It means that viscosity in such cases will depend on whether the sample, before starting the measurement, was at rest or whether it underwent continuous shearing (Macosko, 1994). 9.2.3.6 Viscosity Parameters Viscosity characterizing the properties of liquids, connected with resistance evoked by shearing, depends on seven separate parameters (Equation 9.7):  ˇ ˝ ˜ ˛ funkcja ˆ S, T, C, Mw, P, ° , t  ˙ ˘

(9.7)

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S – the physical-chemical properties of the substance measured; T – temperature; C, Mw, P – the concentration of the dissolved substance and its molecular mass and pressure. In practice, the dependence of viscosity on temperature is an inversely proportional value. In some cases, during viscosity measurements, a viscosity increase is observed during temperature growth. This is caused by physical-chemical transformations in the liquid tested, e.g. polymers cross-linking because the cross-linking reaction rate is directly proportional to the temperature growth. In such a case, viscosity should be determined in stable conditions after fnishing the transformations. The dependence of viscosity on temperature is a key technological parameter. Liquid temperature is often increased on purpose in order to reduce viscosity and facilitate processing. In practice, we can observe the non-linear increase of liquid viscosity along with the increase in dissolved substance concentration (Figure 9.6). In addition, substance concentration affects the fow properties. As concentration, the molecular weight increase results in viscosity growth. The molecular weight of some biopolymers (chitosans) is determined by viscosity measurements. The viscosity of most liquids is basically constant within the pressure range of 0–10 MPa, hence the

FIGURE 9.6 The dependence of viscosity on the concentration of saccharose solutions A, B, D, and E at selected temperatures and sodium chloride C (A – 0°, B – 20°, C – 20°, D – 60°, E – 100°) (Bourne, 2002).

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impact of pressure is often omitted in the measurements of food rheologic properties (Macosko, 1994). Liquid food products form two liquid systems. One group consists of juices and concentrates in which the insoluble ingredients of the cell wall are suspended in water containing soluble ingredients, such as sugars, organic acids, and mineral salts. The other group is constituted by the following products: mayonnaises and sauces, that is, emulsions. The increase of the insoluble fraction and the non-continuous phase content are crucial for the viscosity and fow type (Macosko, 1994). 9.2.3.7 The Division of Liquids Liquids are characterized by fow curves and viscosity curves. Depending on the curve course, Newtonian and non-Newtonian fuids are differentiated (Figure 9.7). A Newtonian fuid, known as a perfect liquid, is characterized by the Newtonian equation (9.8):

˜ ˝ ° ˙ ˛

(9.8)

Where: σ – shear stress; η– viscosity; γ – shear speed. The graphical interpretation of the Newtonian equation (Figure 9.7A, fow curve A) presents a directly proportional dependence of stress increase along the shear rate growth. In the analysis of the fow curve for a Newtonian fuid, it may be stated that the ratio of stress σ and rate γ shearing γ is constant, which means that viscosity η is not dependent on the shear rate (Figure 9.7, viscosity curve A). All the liquids for which said dependence is true are called Newtonian fuids (water, mineral oils).

FIGURE 9.7 Flow type (A – Newtonian fuid, B – pseudo-plastic liquid, C – dilatant liquid, D – pseudo-plastic liquid with plasticity limit, plastic liquid; B, C, D – Non-Newtonian fuids).

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All the remaining liquids whose fow is not perfect are referred to as non-Newtonian fuids. One of the varieties is pseudo-plastic liquids (Figure 9.7B), for which the shear rate increase results in the monotonous decrease of viscosity. Lots of liquids demonstrate a viscosity drop as the shear rate grows. Liquids become “thinner” when the fow rate increases. An example here is blood, ketchup, and whipped cream. These properties enable the fow of larger mass or the reduction of energy needed for maintaining the constant fow rate. These properties often result from the non-uniformity of liquids. The shape of substances contained in the liquid when increasing the shear rate may change and the substance layout changes toward the fow direction. Polymer chains extend and arrange in parallel to the force direction. The parallel arrangement of particles reduces friction, which facilitates their movement in relation to one another. For example, erythrocytes, which have the shape of a coin at rest, during fow are deformed into long tubes with a smaller diameter. This allows red blood cells to penetrate into small blood vessels and achieve higher fow rates. A similar phenomenon takes place in the fow of an emulsion, for example, oil in water. At rest, oil has the form of spheres; during fowing, these spheres are fattened. For most materials, the effect of thinning at shearing is reversible. The fow rate reduction causes a considerable increase in viscosity. A thinning phenomenon or pseudo-plastic fow is not constant in the entire shearing range. If the interrelations between particles are very strong in a liquid, it is necessary to exceed a certain limit of stress so that this liquid could fow. This type of liquid is plastic liquid with fow limits (Figure 9.7D). Furthermore, there are liquids whose viscosity increases along the shear rate growth. These are liquids with a dilatant fow (Figure 9.7C). This phenomenon occurs mainly in liquids in the presence of a plasticizer whose weight share in the volume decreases as the fow grows. It is a consequence of particles moving apart at higher fow intensity, which results in a volume increase. Such behavior of liquids is called thickening through shearing. This behavior is noticeable in sugar solutions in water (Schramm, 1994; Steffe, 1996). Except for the orientational effects of particles, in food, we can often observe interactions between particles in the dispersed system. This leads to the formation of a three-dimensional gel structure. At the time of the shearing process, weak bonds are broken and viscosity is reduced, which causes gel transformation into sol. Liquids that, after the shear process, at rest, become gels are thixotropic liquids. Flow curves in thixotropic liquids are not convergent. This is connected with the additional amount of energy supplied to the sheared volume of the sample (Figure 9.8). A property opposite to thixotropy is rheopexy, which is a phenomenon consisting of the increase of liquid viscosity dependent on the stress duration. In this case, the course of fow curves is opposite to thixotropy. Rheopexy is an extremely rare phenomenon, unlike thixotropy (Rao, 1999; Steffe, 1996; Rao, 2005). 9.2.3.8 Viscosity Measurements One of the most known and oldest viscometers is a capillary viscometer. Determining viscosity on this type of viscometer consists of measuring the fow time of a specifc liquid amount through properly calibrated capillary tubes due to the pressure gradient. The measurement of viscosity in this type of viscometer is similar. In Ostwald’s viscometer (Figure 9.9), a wider part (U) should be flled with liquid of a specifc

The Rheological Properties of Foods

FIGURE 9.8

Flow and viscosity curve characterizing thixotropy (Steffe, 1996).

FIGURE 9.9

Ostwald’s viscometer.

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volume (the left side of the U tube), then the liquid must be sucked to the narrower part (the right side of the U tube) and the liquid dropping time should be determined between the calibrated scale. Kinematic viscosity is calculated by multiplying the time obtained by the capillary constant (Schramm, 1994). Another approach to liquid fow simulation is the application of solutions consisting of the dropping of a calibrated ball in a tube flled with water (Höppler’s viscometer). When determining the ball dropping time and multiplying it by the viscometer constant and the density difference of the material of the ball and liquid, dynamic viscosity is obtained (Schramm, 1994). Currently, rotary viscometers are used more commonly; they enable direct measurements. The instruments available on the worldwide market often differ in design; there are rotameters with concentric cylinders, a cone-plate design, and a design making use of the parallel arrangement of measuring plates. Rotameters with the sensor system with concentric cylinders are the most popular. A sample placed in the gap between rings may be subject to shear forces or shear stresses for a longer time. Shearing is obtained by the rotary motion of one of the cylinders, the internal

FIGURE 9.10 by the author.

Pins used for viscosity measurements in Brookfeld’s viscometer. Photograph

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one or the external one. Internal cylinders may have different shapes depending on the viscosity ranges in which measurements are carried out (Figure 9.10). The use of interchangeable cylinders (pins) extended considerably the possibilities of using rotary viscometers, which currently made them the most popular viscometers. In all the viscometers, viscosity is dependent on temperature; each measurement must be carried out at a strictly controlled temperature. For this purpose, most viscometers are equipped with effcient and accurate temperature maintenance systems for samples or devices (Schramm, 1994).

REFERENCES Aguilera J. M., Stanley D. W. 1999. Microstructural Principles of Food Processing and Engineering (2nd ed.). Aspen Publishers, Inc, Gaitherburg, MD. Bourne M. C. 2002. Food Texture and Viscosity: Concept and Measurement (2nd ed.). Academic Press, New York. Brummer R. 2006. Rheology Essentials of Cosmetic and Food Emulsions. Springer, Berlin Heidelberg. Djabourov M., Leblond J., Papon P. 1988. Gelation of aqueous gelatin solutions. II. Rheology of the sol-gel transition. Journal de Physique, 49(2), 333–343. doi:10.1051/ jphys:01988004902033300 Goff H. D., Guo Q. 2019. The Role of Hydrocolloids in the Development of Food Structure. Joyner H. S., Daubert C. R. 2017 Rheological principles for food analysis. Food Analysis, 5, 511–552. doi:10.1039/9781788016155-00001 Lovegrove A., Edwards C. H., De Noni I., Patel H., El S. N., Grassby T., … Shewry P. R. 2017. Role of polysaccharides in food, digestion, and health. Critical Reviews in Food Science and Nutrition, 57(2), 237–253. doi:10.1080/10408398.2014.939263 Lucey J. A., Johnson M. E., Horne D. S. 2003. Invited review: Perspectives on the basis of the rheology and texture properties of cheese. Journal of Dairy Science, 86(9), 2725–2743. doi:10.3168/jds.S0022-0302(03)73869-7. Macosko C. W. 1994. Rheology: Principles, Measurements and Applications. Wiley VCH, Poughkeepsie, NY. Miri T. 2011. Practical Food Rheology An Interpretive Approach. Blackwell Publishing Ltd. Moro A., Báez G. D., Ballerini G. A., Busti P. A., Delorenzi N. J. 2013. Emulsifying and foaming properties of β-lactoglobulin modifed by heat treatment. Food Research International, 51(1), 1–7. doi:10.1016/j.foodres.2012.11.011 Netter A. B., Goudoulas T. B., Germann N. 2020. Effects of Bloom number on phase transition of gelatin determined by means of rheological characterization. LWT, 132, 109813. doi:10.1016/j.lwt.2020.109813 Peleg M. 2019. The instrumental texture profle analysis revisited. Journal of Texture Studies, 50(5), 362–368. doi:10.1111/jtxs.12392 Rao M. A. 2005. Rheological Properties of Fluid Foods in Engineering Properties of Foods (3rd ed.). Taylor and Francis Group, London. Rao M. A. 1999. Rheology of Fluid and Semisolid Foods Principles and Applications. Aspen Publishers, Inc, Gaithersburg, MD. Sankar T. V., Ramachandran A. 2002. Rheological characteristics of suwari and kamaboko gels made of surimi from Indian major carps. Journal of the Science of Food and Agriculture, 82(9), 1021–1027. doi:10.1002/jsfa.1139 Schramm G. 1994. A Practical Approach to Rheology and Rheometry. Haake, Crawley, Sussex.

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Staniewski B., Ogrodowska D., Staniewska K., Kowalik J. 2021. The effect of triacylglycerol and fatty acid composition on the rheological properties of butter. International Dairy Journal, 114, 104913. doi: 10.1016/j.idairyj.2020.104913 Steffe J. F. 1996. Rheological Methods in Food Process Engineering. Freeman Press, East Lansing, MI. Szczesniak A. S. 2002. Texture is a sensory property. Food Quality and Preference, 13, 215– 225. doi:10.1016/S0950-3293(01)00039-8 Tabilo-Munizaga G., Barbosa-Cánovas G. V. 2005. Rheology for the food industry. Journal of Food Engineering, 67(1–2), 147–156. doi:10.1016/j.jfoodeng.2004.05.062

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CONTENTS 10.1 Anthocyanins................................................................................................ 285 10.1.1 Infuence of Chemical Structure on Color........................................ 285 10.1.2 Occurrence of Anthocyanins............................................................ 287 10.1.3 Anthocyanins Stability and Alterations during Processing and Storage .............................................................................................. 289 10.2 Betalains ....................................................................................................... 290 10.2.1 Infuence of Chemical Structure on Color........................................ 290 10.2.2 Occurrence of Betalains ...................................................................290 10.2.3 Betalains Stability and Alterations during Processing and Storage .......................................................................................... 291 10.3 Chlorophylls.................................................................................................. 293 10.3.1 Infuence of Chemical Structure on Color........................................ 293 10.3.2 Occurrence of Chlorophylls.............................................................. 294 10.3.3 Chlorophylls Stability and Alterations during Processing and Storage .............................................................................................. 294 10.4 Carotenoids ................................................................................................... 296 10.4.1 Infuence of Chemical Structure on Color........................................ 296 10.4.2 Occurrence of Carotenoids in Food.................................................. 296 10.4.3 Carotenoids Stability and Alterations during Processing and Storage .............................................................................................. 297 10.5 Plant Pigments as Food Coloring Additives ................................................. 298 References.............................................................................................................. 299

10.1 ANTHOCYANINS 10.1.1

INFLUENCE OF CHEMICAL STRUCTURE ON COLOR

Among many types of natural pigments of plant origin, anthocyanins represent a large group of water-soluble colorants. So far, more than 635 anthocyanins have been identifed. They are responsible for the vivid blue, purple, and red color of many fruits and vegetables, as well as legumes and grains. Anthocyanins are a group of favonoids belonging to phenolic compounds. The anthocyanidins (or aglycons) are polyhydroxy and polymetoxy derivatives of 2-phenyl-benzo-pyrylium or favylium salts called anthocyanidins (Figure 10.1). These polyphenolic substances consist of two aromatic rings (A and B) bonded to a heterocyclic ring (C) that contains oxygen. Depending on the number and position of the hydroxyl and methoxyl substituents, DOI: 10.1201/9781003265955-10

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FIGURE 10.1 Basic structure of anthocyanidins (A) and the structures of the most common anthocyanidins.

25 different anthocyanidins have been described. Nevertheless, approximately 95% of all anthocyanins commonly found in fruits, vegetables, legumes, and grains are derived from only six of them. Substitution of hydroxyl and methoxyl groups infuences the color of anthocyanidins. Increments in the number of hydroxyl groups tend to deepen the color to a more bluish shade. On the other hand, increments in the number of methoxyl groups increase redness. Moreover, hydroxylation reduces stability, while methylation increases stability of the pigments. In nature, anthocyanidins are linked to one or more glycosidic units, usually at –OH groups in C3 and/or C5 positions. Generally, anthocyanidin glycosides are 3-monoglycosides and 3,5-diglycosides, having glucose and rhamnose as the most common saccharide attached. Nevertheless, xylose, galactose, arabinose, and rutinose can also occur. Saccharide moieties may be further linked to other through glycosidic bonds or acylated with organic aromatic or aliphatic acids (e.g., caffeic, p-coumaric, ferulic, sinapic, malonic, oxalic, and acetic) through ester or glycosidic bonds. Glycosylation increases water solubility while acylation reduces it and improves anthocyanin stability by forming an intramolecular H-bonding network within the anthocyanin molecule. In solution, anthocyanins are very sensitive to pH and change to a mixture of colored and colorless forms in equilibrium through acid–base, water addition– elimination, and isomerization reactions. At a pH below 2, anthocyanins exist predominantly in the red favylium cation form while other forms such as the colorless carbinol pseudobase, the colorless or pale-yellow chalcone, and the blue quinoidal base are formed at pH values above 3 (Figure 10.2). As a result of the transformation, frst, the red color brightens and disappears, and then a green-blue color appears depending on the anthocyanin structure. Acylated pigments retain more color at higher pH values than nonacylated forms. Notably, the favylium cation and quinoidal base are reversibly inter-convertible forms by pH changes while trans- and cis-chalcone can in principle be inter-converted by

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FIGURE 10.2 Forms of anthocyanins associated with a change in pH.

photo-excitation. A further pH increase (pH > 7) may induce the degradation of the pigments into aldehyde and phenolic acid. The variety of colors associated with anthocyanins is also the result of their interactions with other molecules. Phenolic compounds co-occur with anthocyanins and participate in the color as co-pigments. Anthocyanins also react with alkaloids, amino acids, organic acids, nucleotides, and metallic ions. Interactions between anthocyanin and co-pigment have been categorized as intermolecular and intramolecular co-pigmentation, self-association, and metal complexation. The basic role of co-pigments is to protect the colored favylium cation from the nucleophilic attack of the water molecule. The increases in absorbance intensity (hyperchromic effect) and in the maximum wavelength (bathochromic effect) are observed as results of co-pigmentation. These effects are evident under weakly acid conditions (pH 4–6) where anthocyanins exist in their colorless form.

10.1.2

OCCURRENCE OF ANTHOCYANINS

Fruits and vegetables, both in unprocessed and processed form, constitute the main source of anthocyanins in the human diet. These pigments are also present in certain

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grains (e.g., purple corn, black rice) and legumes (e.g., black soybeans, red beans) as well as herbs. Total anthocyanin content varies substantially across plant species and even cultivars (Table 10.1). Environmental factors such as light, temperature, and growing conditions also affect anthocyanin concentration considerably. Fruit juices and wine are also important sources of anthocyanins. Their content was 401 mg/dm3 of blackberry juice, 6–219 mg/dm3 of pomegranate juice, 100–670 mg/dm3 of chokeberry juice, and 370 mg/dm3 of black currant juice, while in wines anthocyanins varied from 151 to 250 mg/dm3 (Dobson et al. 2017; Sivilotti et al. 2016; Sosnowska et al. 2016; Ozgen et al. 2008; Wang and Xu 2007). The color of the food depends not only on the anthocyanin content but also on its qualitative composition. Some of the anthocyanin sources such as chokeberry, cherry, cranberry, red currant, raspberry, black and red bean, and eggplant contain only nonacylated anthocyanins, while blackberry, blueberry, black currant, gooseberry, strawberry, grape, lettuce, red cabbage, and red onion contain also acylated forms. Moreover, the color of plum, red cabbage, and lettuce is only derived from cyanidin derivatives, and strawberry and red radish are mainly from pelargonidin derivatives. For comparison, derivatives of as many as six anthocyanidins were identifed in cranberry and as many as fve in blueberry and red grape (except pelargonidin) and in black currant (except malvidin). The anthocyanin profle is sometimes very complex, as in purple Bordeaux radish and red cabbage there are 60 and 36 TABLE 10.1 Content (mg/100 g Fresh Weight) of Anthocyanins in Unprocessed Food Fruits

Anthocyanin content

Acai

282–504

Black carrot

Acerola Cranberry Blackberry Black chokeberry Black currant Blueberry Grape Pomegranate Red currant Red raspberry Strawberry Sweet cherry

6–8 41–207 70–326 357–1,480 130–587 25–495 27–750 6–30 12–67 23–92 13–315 2–463

Black bean Black rice Black soybean Eggplant Purple corn Purple sweet potato Red bean Red cabbage Red chicory Red leaf lettuce Red onion Red radish

Tart cherry

65–82

Vegetables/grains

Rhubarb

Anthocyanin content 22–126 24–44 10–493 20–1,420 6–86 1,642 42 7 23–322 39 2–5 23–48 100–154 4–200

Sources: Gonçalves et al. 2021; Bendokas et al. 2020; Vagiri and Jensen 2017; Lee at al. 2016; Sivilotti et al. 2016; Gavrilova et al. 2011; Zheng et al. 2012; Olsen at al. 2010; de Pascual-Teresa and Sanchez-Ballesta 2008; Podsędek et al. 2008; Silva et al. 2007; Wu et al. 2006.

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anthocyanins, respectively (Lin et al. 2011; Charron et al. 2007). Purple sweet potatoes contain 27 anthocyanins (Lee et al. 2013), and Concord grape and blueberry 16 and 14 anthocyanins, respectively (Wu and Prior 2005).

10.1.3

ANTHOCYANINS STABILITY AND ALTERATIONS PROCESSING AND STORAGE

DURING

In plant cells, anthocyanins are rather stable, but damage to the cell structure during technological processes affects their stability and, consequently, also the color of food. High temperature, pH, oxygen, increased sugar level, and ascorbic acid can affect the rate of their destruction. The degradation of anthocyanin is mainly done through three ways – cleavage, derivatization, and polymerization. In general, the colorless compounds are products of the anthocyanin cleavage reactions, whereas polymerization and derivatization cause browning and generation of colored molecules, respectively. During plant material crushing, anthocyanins are lost as a result of the action of oxygen and ortho-quinones (products of the action of polyphenol oxidases on other phenolic components). Also, native glycosidases can convert anthocyanins to less stable anthocyanidins. The inactivation of enzymes is ensured by blanching or the addition of inhibitors including sulftes and ascorbic acid. Ascorbic acid causes mutual and irreversible destruction of the pigments while sulfte bleaching is reversible and pH-dependent. Temperature is a particularly destructive factor in relation to anthocyanins. The kinetics of anthocyanins decomposition during heating of liquid products (e.g., juçara and “Italia” grapes, and blackberry juice and concentrate) followed the frstorder reactions (Peron et al. 2017; Wang and Xu 2007). Anthocyanins were readily degraded during the thermal treatment of chokeberries before pressing and during pasteurization (Wilkes et al. 2014) while blanching and vacuum frying were the most destructive steps in the production of purple yam chips (Fang et al. 2011). Anthocyanins of red raspberry juice degraded faster than those in the model system, indicating that the coexisting components in juice may have an acceleration effect on anthocyanin degradation (Chen et al. 2020). Jam processing reduced anthocyanin levels by 66–84% and by a further 36–62% within fve months of storage (Oancea and Calin 2016). During food storage, anthocyanins content decreased in time- and temperature-dependent manners and was higher in fruit concentrate than juice due to the higher content of soluble substances (Dobson et al. 2017; Wang and Xu 2007). The effect of the culinary processing on the stability of anthocyanins depends on temperature, time, pH, and amount of water. From the point of view of preserving pigments, steaming is recommended. Generally, the high temperature, short-time thermal processing, and low storage temperature were preferable for the retention of anthocyanins in a complex food matrix. The change in the color of processed food is related to, inter alia, the reactions of anthocyanins with other food ingredients, and with products of their transformation. Fortifying beverages with vitamin C improves their nutritional value but can accelerate anthocyanin degradation and color deterioration. The destruction of the pigments is likely due to the auto-oxidation of vitamins leading to the formation of

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free radicals that cleave the core structure of the favylium cation. Condensation of vitamin C with anthocyanins is also likely to produce a colorless product. In young red wines, anthocyanins extracted from grapes are the main coloring molecules that are partially transformed into anthocyanin-derived compounds. Anthocyanins can react directly with grape tannins or their binding is mediated by alcoholic fermentation products such as acetaldehyde. Red wine anthocyanins can also react with acetaldehyde, 4-vinylphenol, and pyruvic acid to form pyranoanthocyanins, They can then form linkages with phenolic acids or favonols. These derivatives are more stable than anthocyanins and tend to resist the effects of pH, oxygen, and SO2 bleaching. It has been estimated that about 25% of anthocyanins may have polymerized with catechins by the end of alcoholic fermentation, and about 40% after one year’s aging (Jackson 2014). Monomeric anthocyanins react with the sulftes, serving as antimicrobial agents in food processing, at the pH of most foods and beverages to form a colorless sulfonic acid addition adduct. Polymeric anthocyanins and pyranoanthocyanins do not undergo this reaction.

10.2 BETALAINS 10.2.1 INFLUENCE OF CHEMICAL STRUCTURE ON COLOR Betalains are water-soluble and nitrogen-containing pigments, divided into red-violet betacyanins and yellow-orange betaxanthins. The yellow pigments are composed of betalamic acid and biogenic amino acids or amines, and the betacyanins of betalamic acid and cyclo-DOPA. Betacyanins can be divided into four structural types: betanin, gomphrenin, amaranthin, and bougainvillein (Figure 10.3). They are widespread in the form of glycosides and acylated glycosides. Usually, glucose, apiose, and glucuronic acid attach to the aglycone by a glycosidic bond. In acylated forms, aliphatic (malic, 3-hydroxy-3-methylglutaric) or aromatic (coffee, p-coumaric, and ferulic) acids are linked by an ester bond with the hydroxyl group of the saccharide. Betacyanins are optically active compounds due to the presence of two chiral carbons. More than 90 different betalains (60 betacyanins and 33 betaxanthins) have been found in nature, especially in plants belonging to about 17 families in the order Caryophyllales. These pigments are produced in fowering petals, roots, stems, leaves, and fruits.

10.2.2 OCCURRENCE OF BETALAINS The most known edible sources of betalains are red beetroot, grainy or leafy amaranth, fruits of the cacti Opuntia sp., and the colored Swiss chard. Moreover, strawberry blite, Ulluco tubers, pigeonberry, and Celosia argenta are betalain-containing foods in some regions of the world. Red beetroot contains 18 betacyanins and 12 betaxanthins, among which betanin, isobetanin, and vulgaxanthin I are predominant. The concentrations of betacyanins and betaxanthins in red beetroot are 10–210 mg/100 g and 11–140 mg/100 g, respectively (Lee et al. 2014; Ninfali and Angelino 2013). For comparison, the prickly pear (Opuntia spp.) contains 14–70 mg betalains/100 g (Fernández-López et al. 2002). Prickly pear (Opuntia fcus-indica L.) tissues contain

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Examples of betacyanin and betaxanthin structures.

14 betalains, among which betanin and indicaxanthin are the most abundant (GarciaCayuela et al. 2019). However, there are differences in the betalains profle in the different varieties of the prickly pear. In cactus pears from O. stricta, only betanin and isobetanin were detected; from O. undulata and O. fcus-indica the predominant pigments were betanin and indicaxanthin, with very low levels of isobetanin (FernándezLópez et al. 2002). Contents of betacyanins and betaxanthins in red beet juice were 465–807 and 301–507 mg/dm3, respectively (Wruss et al. 2015). Betacyanin content in juices from Hylocereus polyrhizus, O. fcus-indica cv. Rossa, and O. fcusindica cv. Gialla was 525.3, 73.9, and 1.3 mg/dm3, respectively, whereas betaxanthins amounted to 48.3, 36.4, and 5.3 mg/dm3 in O. fcus-indica cv. Gialla, O. fcus-indica cv. Rossa, and H. polyrhizus, respectively (Stintzing et al. 2003). Betalains have never been found to co-occur with anthocyanins in the same plant.

10.2.3 BETALAINS STABILITY AND ALTERATIONS DURING PROCESSING AND STORAGE Water-soluble betalains are unstable, and their stability during processing and storage depends on the chemical structure, pigment concentration, food matrix, activity

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of redox enzymes, pH, temperature, presence of light and oxygen, and transition metal ions. Their degradation includes different reactions such as isomerization, deglycosylation, hydrolysis, decarboxylation, and dehydrogenation. Betacyanins show the greatest stability in the pH range of 4–6 and if stored at 4° C. At pH above 8, an increase in absorbance in the range of 400–460 nm is observed due to the release of betalamic acid. On the other hand, a pH below 3 causes isomerization and transformation of betanin and betanidin into iso-forms, and a change of color toward violet. Acylated betacyanins are more stable due to the proper arrangement of the molecule, which protects it against hydrolytic attack. Brightening or discoloration of betalain pigments also occurs with the participation of endogenous α-glucosidases, polyphenol oxidases, and peroxidases. Betacyanins turned out to be more susceptible to enzymatic degradation than betaxanthins. Betalains can be stabilized by ascorbic acid, isoascorbic acid, citric acids, as well as lactic, acetic, and betalamic acids. The effect of stabilizers depends not only on their dose but also on the pH, temperature, and composition of the matrix. Betaxanthin retention after heating (85° C, 1 h) of yellow-orange cactus pear juice was 63.9% and 37.5% at pH 6 and 4, respectively (Moßhammer et al. 2007). The stability of pigments in juice (pH 6) with 0.1% ascorbic acid decreased by 7.2%, while in the presence of isoascorbic acid did not change, and after adding citric acid increased by 6.1%. Contrary, the addition of ascorbic acid (1%) to the juice of violet-colored dragon fruit after six months of storage at 20° C in the presence and in the absence of light increased the stability of betacyanins two and eight times, respectively (Herbach et al. 2007). So, ascorbic acid acts both as a degradative and stabilizing agent for betacyanins. The adverse effect of ascorbic acid on pigments may be connected with the harmful effect of hydrogen peroxide resulting from the breakdown of vitamin C. On the other hand, as an antioxidant, ascorbic acid removes oxygen and prevents oxidation of pigments. Heavy metal ions accelerate the decomposition of betacyanins, probably as a result of higher oxygen uptake, which causes oxidation of betalains themselves and their hydrolysis products. Metal chelating agent EDTA stabilizes betalains by preventing metal-induced bleaching through the formation of an EDTA–metal complex. Citric acid can also reduce betalains oxidative damage by acidifying and chelating metal ions. Betalains are thermolabile food components, therefore their content decreases after cooking, and the changes depend on the temperature and time of heating, as well as on the red beet variety. In general, in the range of pH 3–7, the pigments polymerize during heating, which causes color changes toward lightening and browning. When cooked at temperatures above 80° C, betanin may undergo polymerization, decarboxylation, and dehydrogenation reactions to form unstable betalamic acid and colorless cyclo-DOPA glucoside. Yellow betaxanthins are rather less stable than betacyanins. Both betacyanins and betaxanthins are able to resynthesize from degradation products by spontaneous condensation. The process of boiling whole red beetroots and peeled roots for 60 min led to a reduction in the total betalains content by about 54% and 61%, respectively (Sawicki and Wiczkowski 2018). The seven-day spontaneous fermentation process caused a decline in betalains content in the beetroot by 61%, and by 88% when the process was extended up to 14 days. The quality of betalains changes during fermentation. Betanin and vulgaxanthin I were the

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predominant compounds in the fresh roots, while in the 14-day fermented roots and fermented juice, betanidin (the aglycone of betanin) and threonine-betaxanthin were the main compounds (Sawicki and Wiczkowski 2018). A six-month cold storage of fermented red beet at 5° C decreased red pigment content about three-fold while yellow pigment levels did not change (Czyżowska et al. 2020).

10.3

CHLOROPHYLLS

10.3.1 INFLUENCE OF CHEMICAL STRUCTURE ON COLOR Chlorophylls are green pigments containing four pyrrole rings (A–D) which are joined by methine bridges, and a magnesium atom in the center. The ffth ring (E) is made up of the carbon atoms themselves. They also include the aliphatic phytol tail which gives the whole molecule a hydrophobic character. The structural diversity of chlorophylls results from the type of substituents in the pyrrole rings, and the presence or absence of phytol (Figure 10.4). Chlorophyll a, greenish-yellow in solution, is the primary photosynthetic pigment in green plants for the transfer of light energy to a chemical acceptor. Chlorophyll

FIGURE 10.4

Structures of chlorophyll a and b, and chlorophyll a derivatives.

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a, alone, is found in blue-green and some red algae. Chloropyll b, blue-green in solution, is present in higher plants and green algae with chlorophyll a. Chlorophyll c and d are found with chlorophyll a in brown algae and in some red algae, respectively. Higher plants have two types of chlorophylls, chlorophyll a and chlorophyll b. Chlorophyll a is blue-green with absorption maxima at 433 and 663 nm, and chlorophyll b is green-yellow with maxima at 453 and 642 nm. Although chlorophyll a is less polar than chlorophyll b, both are insoluble in water but very soluble in ethanol and methanol. Chlorophyll c is water-soluble due to the lack of phytol. Various chlorophyll derivatives are also formed in plants as a result of pigment catabolism as well as in processed foods under the infuence of degrading factors. One is the formation of bright green chlorophyllide, due to the removal of the phytol chain from the chlorophyll structure catalyzed by the enzyme chlorophyllase. In cell free matrices, under the infuence of a weak acid, the magnesium atom is replaced by hydrogen ions to generate a hydrophobic olive-green pheophytin. At low pH, the magnesium ion and phytol are removed to form the olive-brown water-soluble pheophorbide. As a result of heating, green pyro-chlorophylls and hydroxy-chlorophylls are formed, which, due to the presence of phytol, are fat-soluble.

10.3.2 OCCURRENCE OF CHLOROPHYLLS The edible sources of chlorophylls are mainly several raw foods, including leafy, stem, and inforescence vegetables, some fruits, as well as herbs. The total content of chlorophylls varies substantially across plant species, and even cultivars (Table 10.2). The ratio of chlorophyll a/b in plants ranged from 1.5 to 4.7 and varies due to growth conditions and external factors, especially high-light intensity and sun exposure. The lower chlorophyll a/b ratio indicates the convenience of chlorophyll dissolving in an aqueous solvent. Chlorophyll a is less polar than chlorophyll b; thus a lower ratio of chlorophyll a/b resulted in higher solubility in a water-based solvent. Chlorophyll pigments with pheophytin as a dominant form are responsible for the greenish hues of virgin olive oil. Virgin olive oil contains 2.6–64.1 mg/kg total chlorophylls depending on olive fruit genetic factors, the stage of fruit ripeness, environmental conditions, the extraction process, and storage conditions (İnanç 2011).

10.3.3 CHLOROPHYLLS STABILITY AND ALTERATIONS DURING PROCESSING AND STORAGE When removed from their native environment, chlorophylls are highly sensitive molecules to light, low pH, high temperature, enzymes, and oxygen. Their degradation is the result of both enzymatic and non-enzymatic reactions. Enzymatic degradation of chlorophyll occurs due to the presence of endogenous plant enzymes such as peroxidase, Mg-dechelatase, pheophorbide a oxygenase, chlorophyll-reductase catabolite, and chlorophyllase. Enzymatic degradation takes place during storage while non-enzymatic changes usually develop during post-harvest processing. The formation of a gray-colored pheophytin a and a brown-colored pheophytin b becomes the frst stage of chlorophyll degradation during senescence, followed by the release

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TABLE 10.2 Chlorophyll Contents of Vegetables and Fruits (mg/100 g Fresh Weight) Vegetable/fruit Broccoli Brussels sprouts Celery Chicory Chinese cabbage Cucumber Fenugreek Green peas Green pepper Iceberg lettuce Kale Kiwifruit Leek Parsley Red leaf lettuce Romaine lettuce Savoy beet Spinach Apple fesh Apple peel Kiwifruit

Total content of chlorophylls

Chlorophyll a

Chlorophyll b

2.1–31

1.6–22

0.5–9.1

3.2–8.4 2.3 200–328 5.8–15.8 3.6 2.0 5.1–23.1 7.9–8.6 2.9 187–1,236 2.1–4.0 8.7–28.5 63.2 73 24.5–28.9 1.2 33–127 0.15–0.81 0.57–3.07

2.3–5.0 1.8 142–238 4.1–11.5 2.6 – 3.9–14.0 4.1–5.8 2.3 120–251 1.9–3.5 6.6–16.7 47.8 59 18.5–19.9 – 24–94.6 0.12–0.63 0.45–2.37

0.9–2.8 0.5 58–90 1.6–4.7 1.0 – 1.2–9.1 2.8–3.8 0.6 30–57 0.1–2.0 2.1–10.3 15.4 14 4.6–10.4 – 14.7–29 0.03–0.18 0.12–7.0

2.1–4.0

1.9–3.5

0.1–2.0

Sources: Zhong et al. 2021; Lee and Chandra 2018; Akdaş and Bakkalbaşı 2017; dos Reis et al. 2015; Delgado-Pelayo et al. 2014; Žnidarčič et al. 2011; Latocha et al. 2010; Kunieda et al. 2005; Khachik et al. 1986.

of phytol. The conformation of pheophytin from chlorophyll a occurs 2.5–10-fold faster than that of chlorophyll b. Non-enzymatic transformations of chlorophylls into pheophytins, pheophorbides, and pyro-chlorophylls take place under the infuence of acids (e.g., released from olives during extraction or during cooking of vegetables) and heating (Indrasti et al. 2018). Food storage conditions, including the type of packaging, have a signifcant impact on the preservation of green pigments. Generally, increased CO2 levels and lower O2 concentration may delay yellowing and chlorophyll and protein degradation, which improves the overall acceptance of leafy vegetables. The content of chlorophyll pigments in cooked vegetables depends on the food matrix and the heat treatment conditions. Retention of chlorophyll a in cooked vegetables ranged from 35% in steamed broccoli to 100% in microwaved peas while retention of chlorophyll b varied from 39% in boiled leek to 86% in boiled peas

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(Turkmen et al. 2006). In fve of six vegetables, chlorophyll a was found more heat resistant compared with chlorophyll b, except in peas. Pheophytins increased in all vegetables after cooking. Contrarily, some authors have found a higher sensitivity of chlorophyll a to thermal treatment, compared to chlorophyll b (Danowska‐Oziewicz et al. 2020; Guillén et al. 2017). Traditional boiling of broccoli resulted in an increase in chlorophyll content while steaming and microwaving had no effect (Danowska‐Oziewicz et al. 2020). The degradation rates in total chlorophylls after steaming, boiling, microwaving, and stir-frying of kale were 18.7%, 21.5%, 55.3%, and 71.0%, respectively (Akdaş and Bakkalbaşı 2017). Roasting of pistachio nuts resulted in an 85% reduction in pheophytin a and b levels as well as an increase (10–12-fold) in pyro-pheophytin a and b levels compared to raw pistachio (Pumilia et al. 2014). Microwave-convective drying had a signifcant impact on total chlorophylls in herbs; however, the level of the losses was different for each herb species. A greater rate of loss was observed in the case of chlorophyll a, amounting to 6–32%, whereas the degradation of chlorophyll b was at the level of 5–25% (Śledź and Witrowa-Rajchert 2012).

10.4 CAROTENOIDS 10.4.1

INFLUENCE OF CHEMICAL STRUCTURE ON COLOR

Carotenoids are yellow to red lipid-soluble pigments and usually consist of a 40-carbon atoms chain with conjugated double bonds, almost exclusively with transconfguration. Carotenoids are classifed by their chemical structure as: (1) carotenes that are constituted by carbon and hydrogen; (2) oxycarotenoids or xanthophylls that have carbon, hydrogen, and, additionally, oxygen functional groups such as hydroxyl, epoxy, and carbonyl. The basic linear and symmetrical skeleton of carotenoids is modifed in many ways, including cyclization, hydrogenation, dehydrogenation, introduction of oxygen-containing groups, chain shortening, or extension. Carotenoids may be acyclic (e.g., lycopene, ζ-carotene) or may have a six-carbon ring at one (e.g., γ-carotene, δ-carotene) or both ends (e.g., β-carotene, α-carotene) of the molecule, except capsanthin and capsorubin, which have a fve-carbon ring. Carotenoids can occur free or esterifed, and sometimes in acylated form. In plants, more than 700 carotenoids are reported. Carotenoids in which the carbon skeleton has been shortened are called apocarotenoids (e.g., bixin, crocetin).

10.4.2

OCCURRENCE OF CAROTENOIDS IN FOOD

Carotenoid pigments account for the natural yellow, orange, or red colors of many foods. Vegetables can be considered richer sources of carotenoids than fruits (Table 10.3). The content of pigments in fresh fruit and vegetables depends on many factors, including growing conditions, harvest time, and variety. Vegetables and fruits contain several to several dozen carotenoid pigments. The carotenoids most commonly encountered in foods are β-carotene, α-carotene, β-cryptoxanthin,

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TABLE 10.3 The Total Content (mg/100 g Fresh Weight) of Carotenoids and Their Predominant Form in Vegetables and Fruits Vegetable/fruit Brocolli Brussels sprouts Carrot Kale Lettuce Parsley Red pepper Spinach Tomato Grapefruit Nectarine Papaya Peach Sea buckthorn Tangerine Watermelon

Content of carotenoids 4–8 1–9 10–16 35–141 2–16 25 1–30 11–66 4–13 4 2 3–5 2–4 6–24 2 5

Dominant carotenoids Lutein, β-carotene Lutein, violaxanthin, β-carotene Β-carotene, α-carotene Lutein, β-carotene, violaxanthin, 9’-cis-neoxanthin Lutein, violaxanthin, β-carotene Lutein, β-carotene, violaxanthin Β-carotene, zeaxanthin Lutein, β-carotene, violaxanthin Lycopene Didehydrolycopene, β-carotene Lutein, β-carotene Didehydrolycopene, lycopene Β-carotene Β-carotene, lycopene, zeaxanthin β-cryptoxanthin, β-carotene, zeaxanthin Lycopene

Sources: Teleszko et al. 2015; Sánchez et al. 2014; Jaswir et al. 2011; Müller 1997.

lycopene, lutein, and zeaxanthin. The content of lutein in 18 varieties of cabbage ranged from 0.03 to 0.22 mg/100 g, and in 13 varieties of spinach from 6.49 to 12.98 mg/100 g (Walsh et al., 2015).

10.4.3 CAROTENOIDS STABILITY AND ALTERATIONS DURING PROCESSING AND STORAGE Carotenoids are very prone to degradation due to their high degree of unsaturation. The stability of carotenoids in food depends on their qualitative composition, the composition of the food matrix, the presence of oxygen, light, oxidizing enzymes, antioxidants, and pro-oxidants, as well as temperature. Oxidative processes are catalyzed by lipoxygenases, metal ions, and pro-oxidants, mainly products of lipid oxidation. They lead to the appearance of cis isomers, epoxycarotenoids, hydroxycarotenoids, apocarotenoids, and low molecular weight compounds. As a result of these changes, the color lightens, and sometimes it disappears and the characteristic, foreign smell appears. Enzymatic oxidation of carotenoids can occur to a greater extent than thermal decomposition in many foods. Blanching and cooking inactivate

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enzymes and loosen the structure of the raw material and, consequently, increase the extraction effciency and bioavailability of carotenoids. Blanching corn, broccoli, peppers, and chives resulted in an increase in the content of lutein and zeaxanthin by 20–118%, and of β-carotene by 1–84% (Walsh et al. 2015). Microwaves and conventional heating of kiwifruit puree led to marked changes in the carotenoid content (62–91% losses) (Benlloch-Tinoco et al. 2015). Carrots boiled in water contained 6% more carotenoids than steamed ones, and in the case of spinach, the opposite correlation was found – steamed vegetables contained 20% more (Mazzeo et al. 2011). Carotenoids can also be affected by drying temperature. Lutein and β-carotene levels for spinach and kale decreased by over 70% with the increase of drying temperature from –25 to 75° C (Lefsrud et al. 2008). The stability of carotenoids during storage depends on the type of product and storage conditions and is favored by food packaging in a modifed atmosphere and low storage temperature. The annual storage of frozen vegetables reduces the carotenoid content by 23%, and the fruit by 37% on average. Storage for six weeks of unpasteurized or pasteurized orange juice at 2° C resulted in a reduction in the concentration of carotenoids by 25 and 26%, respectively (Cortés et al. 2006).

10.5 PLANT PIGMENTS AS FOOD COLORING ADDITIVES Anthocyanins, carotenoids, betalains, and chlorophylls are used commercially and approved for use in the USA and the European Union. Chlorophylls and chlorophyllins are identifed as E140 and copper complexes of chlorophylls and chlorophyllins as E141. Carotenes and xanthophylls are grouped by E160 and E161 food additives, respectively. Anthocyanins are listed as E163 and betanin as E162. Anthocyanin colorants are produced from grape pomace, hibiscus fower, black carrot, and red cabbage while betanin is from red beetroot. The raw material for the production of carotenes is carrots, carrot oil, corn endosperm, and bell pepper as well as Bixa orellana L. (annatto) and marigold oleoresin (Calendula offcinalis) for lutein (xanthophyll) production. Chlorophylls are extracted from alfalfa (Medicago sativa), nettle (Urtica doica), and Festuca arundinacea grass (Luzardo-Ocampo et al. 2021; Rodrigues et al. 2019). Green chlorophylls are used in confectionery, icing, ice cream and frozen desserts, drink mixes and powders, yogurts, jellies, and puddings, among other food applications. Red-violet betanin is used, inter alia, in confections, ice cream, yogurt, ready-made frostings, cake mixes, beverages, sausages, and pates. Carotenoids can be applied to meat products, butter, fat emulsions, some cheeses, and confectionery, as well as in breakfast cereals. In contrast, anthocyanins are used for staining beverages, desserts, ice cream, and dairy products. Despite its safety and health benefts, the use of natural colors in food systems is still limited due to technological problems. Consequently, new sources of pigments need to be explored as well as new technologies to be developed to fnd more technologically stable color additives and provide good color stability for colored food products.

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Luzardo-Ocampo, I., Ramírez-Jiménez, A.K., Yañez, J., Mojica, L., Luna-Vital, D.A. (2021). Technological applications of natural colorants in food systems: A review. Foods, 10(3), 634, doi: 10.3390/foods10030634. Mazzeo, T., N’Dri, D., Chiavaro, E., Visconti, A., Fogliano, V., Pellegrini, N. (2011). Effect of two cooking procedures on phytochemical compounds, total antioxidant capacity and colour of selected frozen vegetables. Food Chem., 128, 627–633, doi: 10.1016/j. foodchem.2011.03.070. Moßhammer, M.R., Rohe, M., Stintzing, F.C., Carle, R. (2007). Stability of yellow-orange cactus pear (Opuntia fcus-indica [L.] Mill. cv.‘Gialla’) betalains as affected by the juice matrix and selected food additives. Eur. Food Res. Technol., 225, 21–32, doi: 10.1007/s00217-006-0378-x. Müller, H. (1997). Determination of the carotenoid content in selected vegetables and fruit by HPLC and photodiode array detection. Zeits. für Lebensm.- Unter. und – Forsch. A., 204, 88–94, doi: 10.1007/s002170050042. Ninfali, P., Angelino, D. (2013). Nutritional and functional potential of Beta vulgaris cicla and rubra. Fitoterapia, 89, 188–199, doi: 10.1016/j.ftote.2013.06.004. Oancea, S., Calin, F. (2016). Changes in total phenolics and anthocyanins during blackberry, raspberry and cherry jam processing and storage. Rom. Biotechnol. Lett., 21, 11232–11237. Olsen, H., Aaby, K., Borge, G.I.A. (2010). Characterization, quantifcation, and yearly variation of the naturally occurring polyphenols in a common red variety of curly kale (Brassica oleracea L. convar. acephala var. sabellica cv.‘Redbor’). J. Agric. Food Chem., 58, 11346–11354, doi: 10.1021/jf102131g. Ozgen, M., Durgaç, C., Serçe, S., Kaya, C. (2008). Chemical and antioxidant properties of pomegranate cultivars grown in the Mediterranean region of Turkey. Food Chem., 111, 703–706, doi: 10.1016/j.foodchem.2008.04.043. Peron, D.V., Fraga, S., Antelo, F. (2017). Thermal degradation kinetics of anthocyanins extracted from juçara (Euterpe edulis Martius) and “Italia” grapes (Vitis vinifera L.), and the effect of heating on the antioxidant capacity. Food Chem., 232, 836–840, doi: 10.1016/j.foodchem.2017.04.088. Podsędek, A., Sosnowska, D., Redzynia, M., Koziołkiewicz, M. (2008). Effect of domestic cooking on the red cabbage hydrophilic antioxidants. Int. Jo. Food Sci. Technol., 43, 1770–1777, doi: 10.1111/j.1365-2621.2007.01697.x. Pumilia, G., Cichon, M.J., Cooperstone, J.L., Giuffrida, D., Dugo, G., Schwartz, S.J. (2014). Changes in chlorophylls, chlorophyll degradation products and lutein in pistachio kernels (Pistacia vera L.) during roasting. Food Res. Int., 65, 193–198, doi: 10.1016/j. foodres.2014.05.047. Rodrigues, D.B., Mercadante, A.Z., Mariutti, L.R.B. (2019). Marigold carotenoids: Much more than lutein esters. Food Res. Int., 119, 653–664, doi: 10.1016/j. foodres.2018.10.043. Sánchez, C., Baranda, A.B., de Marañón, I.M. (2014). The effect of high pressure and high temperature processing on carotenoids and chlorophylls content in some vegetables. Food Chem., 163, 37–45, doi: 10.1016/j.foodchem.2014.04.041. Sawicki, T., Wiczkowski, W. (2018). The effects of boiling and fermentation on betalain profles and antioxidant capacities of red beetroot products. Food Chem., 259, 292–303, doi:10.1016/j.foodchem.2018.03.143. Silva, F.M., Bailon, M.T.E., Alonso, J.J.P., Gonzalo, J.C.R., Buelga, C.S. (2007). Anthocyanin pigment in strawberry. Lebensm. Wiss. Technol., 40, 374–382, doi: 10.1016/j. lwt.2005.09.018. Sivilotti, P., Herrera, J.C., Lisjak, K., Baša Česnik, H., Sabbatini, P., Peterlunger, E., Castellarin, S.D. (2016). Impact of leaf removal, applied before and after fowering, on

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anthocyanin, tannin, and methoxypyrazine concentrations in ‘Merlot’(Vitis vinifera L.) grapes and wines. J. Agric. Food Chem., 64, 4487–4496, doi: 10.1021/acs.jafc.6b01013. Śledź, M., Witrowa-Rajchert, D. (2012). Infuence of microwave-convective drying of chlorophyll content and colour of herbs. Acta Agrophys., 19, 865–876. Sosnowska, D., Podsędek, A., Kucharska, A.Z., Redzynia, M., Opęchowska, M., Koziołkiewicz, M. (2016). Comparison of in vitro anti-lipase and antioxidant activities, and composition of commercial chokeberry juices. Eur. Food Res. Technol., 242, 505– 515, doi: 10.1007/s00217-015-2561-4. Stintzing, F.C., Schieber, A., Carle, R. (2003). Evaluation of colour properties and chemical quality parameters of cactus juices. Eur. Food Res. Technol., 216, 303–311, doi: 10.1007/s00217-002-0657-0. Teleszko, M., Wojdyło, A., Rudzinska, M., Oszmianski, J., Golis, T. (2015). Analysis of lipophilic and hydrophilic bioactive compounds content in sea buckthorn (Hippophae rhamnoides L.) berries. J. Agric. Food Chem., 63, 4120–4129, doi: 10.1021/acs.jafc .5b00564. Turkmen, N., Poyrazoglu, E.S., Sari, F., Sedat Velioglu, Y. (2006). Effects of cooking methods on chlorophylls, pheophytins and colour of selected green vegetables. Int. J. Food Sci. Technol., 41, 281–288. Vagiri, M., Jensen, M. (2017). Infuence of juice processing factors on quality of black chokeberry pomace as a future resource for colour extraction. Food Chem., 217, 409– 417, doi: 10.1111/j.1365-2621.2005.01061.x. Walsh, R.P., Bartlett, H., Eperjesi, F. (2015). Variation in carotenoid content of kale and other vegetables: A review of pre-and post-harvest effects. J. Agric. Food Chem., 63, 9677– 9682, doi: 10.1021/acs.jafc.5b03691. Wang, W.D., Xu, S.Y. (2007). Degradation kinetics of anthocyanins in blackberry juice and concentrate. J. Food Eng., 82, 271–275, doi: 10.1016/j.jfoodeng.2007.01.018. Wilkes, K., Howard, L.R., Brownmiller, C., Prior, R.L. (2014). Changes in chokeberry (Aronia melanocarpa L.) polyphenols during juice processing and storage. J. Agric. Food Chem., 62, 4018–4025, doi: 10.1021/jf404281n. Wruss, J., Waldenberger, G., Huemer, S., Uygun, P., Lanzerstorfer, P., Müller, U., Weghuber, J. (2015). Compositional characteristics of commercial beetroot products and beetroot juice prepared from seven beetroot varieties grown in Upper Austria. J. Food Compos. Anal., 42, 46–55, doi: 10.1016/j.jfca.2015.03.005. Wu, X., Beecher, G.R., Holden, J.M., Haytowitz, D.B., Gebhardt, S.E., Prior, R.L. (2006). Concentrations of anthocyanins in common foods in the United States and estimation of normal consumption. J. Agric. Food Chem., 54, 4069–4075, doi: 10.1021/jf060300l. Wu, X., Prior, R.L. (2005). Systematic identifcation and characterization of anthocyanins by HPLC-ESI-MS/MS in common foods in the United States: Fruits and berries. J. Agric. Food Chem., 53, 2589–2599, doi: 10.1021/jf048068b. Zheng, J., Yang, B., Ruusunen, V., Laaksonen, O., Tahvonen, R., Hellsten, J., Kallio, H. (2012). Compositional differences of phenolic compounds between black currant (Ribes nigrum L.) cultivars and their response to latitude and weather conditions. J. Agric. Food Chem., 60, 6581–6593, doi: 10.1021/jf3012739. Zhong, S., Bird, A., Kopec, R.E. (2021). The metabolism and potential bioactivity of chlorophyll and metallo‐chlorophyll derivatives in the gastrointestinal tract. Mol. Nutr. Food Res., 65, 2000761, doi: 10.1002/mnfr.202000761. Žnidarčič, D., Ban, D., Šircelj, H. (2011). Carotenoid and chlorophyll composition of commonly consumed leafy vegetables in Mediterranean countries. Food Chem., 129, 1164–1168, doi: 10.1016/j.foodchem.2011.05.097.

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Prooxidants and Antioxidants in Food Ronald B. Pegg and Ryszard Amarowicz

CONTENTS 11.1 11.2 11.3 11.4 11.5

Introduction................................................................................................ 303 Oxidants in Foods and Measuring the Oxidation Potential ......................304 Mechanisms of Lipid Oxidation ................................................................306 Oxidation of Proteins in Foods ..................................................................307 The Effect of Oxidation on the Sensory and Biological Properties of Foods .....................................................................................................308 11.6 Benefcial Role of Added Antioxidants to Foods ......................................308 11.7 Sources of Natural Antioxidants in Foods.................................................309 11.8 Antioxidants Generated by Processing of Foods....................................... 314 11.9 Sources and Impact of Prooxidants in Foods ............................................ 315 11.9.1 Tocopherols .................................................................................. 315 11.9.2 Carotenoids................................................................................... 316 11.9.3 Vitamin C ..................................................................................... 318 11.9.4 Flavonoids .................................................................................... 319 11.9.5 Prooxidant Transition-Metal Ions ................................................ 320 11.9.6 Lipoxygenases .............................................................................. 321 11.9.7 Free Fatty Acids ........................................................................... 322 11.9.8 Salt................................................................................................ 323 11.10 Antioxidant Activity and Its Measurement................................................ 324 References.............................................................................................................. 326

11.1 INTRODUCTION Antioxidants are either naturally occurring enzymes or chemicals found in foods or synthetic additives added to foodstuffs. In foods, they provide defense against the deleterious effects of oxidation, both chemical and enzymatic in nature, as well as the free radicals generated in the product during its shelf-life. Many of the natural antioxidants found in or added to foods are of plant origin. These belong to different classes including polyphenolics, carotenoids, S-containing compounds like isothiocyanates, as well as antioxidant vitamins and minerals such as vitamins A, C, E, and selenium. Different antioxidants in food products can sometimes interact amongst themselves to provide synergistic activity; a good example is the vitamin antioxidants (C and E) in combination with phenolics and/or carotenoids. Much research

DOI: 10.1201/9781003265955-11

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remains, however, in terms of the multifaceted interactions among natural antioxidants in different food systems. When we consume foods, we ingest endogenous antioxidants. If suffcient types and quantities of antioxidants are consumed, it is believed that they play a role in helping to neutralize the adverse effects of oxidative stress; that is, the imbalance between oxidants and antioxidants within the body due to antioxidant defciency or increased reactive oxygen species (ROS), reactive nitrogen species (RNS), and reactive sulfur species (RSS) production, which leads to potential cellular damage. Free radicals and ROS are normal byproducts of metabolism that occur in our bodies; their generation, however, can be exacerbated by factors such one’s poor dietary choices, stress, and the environment (e.g., second-hand smoke and air pollution). If left unchecked, free radicals and ROS can damage cells in the body including a cell’s DNA; hence, the primary action attributed to antioxidants is their capability at quenching reactive free radicals before damage at the cellular level can be done. If the oxidative stress placed on the body is not alleviated via antioxidant mechanisms of action, an increase in the risk of developing chronic diseases is believed to exist. Much is unknown about the specifcs relating to the types and quantities of antioxidant constituents for optimal health, but the structural features of the antioxidant molecules in question as well as the food matrix, which may or may not have undergone some degree of processing, will dictate their action.

11.2 OXIDANTS IN FOODS AND MEASURING THE OXIDATION POTENTIAL Food, a term designated for what we ingest to provide macronutrients, micronutrients, and bioactives for life, is varied in its type/form, composition, and quality. For the most part, we look for nutritious, wholesome foods to be part of our daily diet. Yet, food is organic in nature and prone to decomposition by microbiological, enzymatic, and chemical means thereby resulting in a loss of its nutritional quality and potentially the production of harmful compounds. A key property in maintaining the wholesomeness of food is to limit the process of oxidation. Oxidation reactions of food constituents, including lipids, proteins, vitamins, pigments, and DNA, lead to loss of quality via the generation of new chemical species. In some cases, these new products do not have a deleterious effect on the sensory properties of the food, but others do. For instance, polyunsaturated fatty acid (PUFA) linoleic acid in, say, canola oil is an essential fatty acid for human nutrition. If not protected by either natural or synthetic antioxidants or a combination, in the presence of air it will oxidize generating cardboardy and painty volatile notes eventually leading to rancidity. Hexanal, a secondary lipid oxidation product of linoleic acid, is often a dominant volatile aldehyde measured by headspace–solidphase microextraction–gas chromatography (HS-SPME-GC) to track the loss in the lipid’s quality and the development of deleterious favor notes. Plants and animals that we consume have defense systems in the living host to protect against oxidation. Once a plant is harvested or an animal is slaughtered, an

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array of biochemical reactions take place before the product is available as food. Depending upon its state, that is, if it is raw, cooked, or has been processed in some manner, the food will have different degrees of protection against oxidation. In other words, the food will have an oxidation potential. Some foods will possess natural antioxidants or antioxidant enzymes. As an illustration, when muscle tissue is converted to meat via rigor mortis and glycolysis, antioxidant enzymes in the muscle tissue stay active afterward for a period. The enzymes involved include catalase, glutathione peroxidase, and superoxide dismutase. In the living animal, these enzymes help to counterbalance the generation of free radicals and ROS, which can lead to free radical–induced cell damage. In raw meat, the endogenous enzymes help to maintain the wholesomeness, including color and macro-/micronutrient preservation, by providing a redox potential to retard or quench lipid, protein, and pigment oxidation reactions. Typically, a well-treated piece of raw meat will spoil because of microbiological putrefaction before it does so by oxidation. The redox potential of food is infuenced by its chemical composition, the processing the food has undergone, its storage environment (i.e., air, vacuum, or modifed atmosphere), and the presence of different types of oxidizing (i.e., prooxidants) and reducing substances. Oxidation-reduction potential (ORP) electrodes can provide a numerical index of the intensity of oxidizing or reducing conditions within a system. Hence, the ORP value represents an indirect assay for the oxidation status by measuring the redox balance within a biological system. A measuring electrode containing a reference hydrogen electrode determines ORP values: positive values denote oxidizing conditions, while negative values indicate reducing ones. Foods contain endogenous reductants such as vitamin C, vitamin E, dithiothreitol, cysteine, thiol-containing biomolecules, and phenolics to name a few. During processing exogenous oxidizing (i.e., prooxidants) or reducing agents can be added and this will change the redox status of the food system. Furthermore, normal metabolism by microorganisms in foods will elicit the formation of oxidizing or reducing metabolites, which can also change the redox status of the foodstuff. Knowledge of how food has been handled, processed, and stored before being presented at a meal is key in helping to understand the oxidation status of the product. Constituents of food, such as lipids, saccharides, proteins, vitamins, pigments, and DNA, are vulnerable to oxidation reactions, which generate new chemical products (Kanner 1994). In certain situations, this is desirable; for example, the oxidative cross-linking of proteins to manipulate viscosity and gelation in dairy products. Typically, however, oxidative processes are associated with anti-nutritional and unfavorable sensory outcomes. In meat, oxidative cross-linking of proteins can result in toughness, reduced water-holding capacity, off-tastes and odors, and undesirable changes in color (Lund et al. 2011). In the context of food, the term “antioxidant” is widely employed, being associated with the chemical stabilization of oxidative processes throughout storage. Furthermore, antioxidant-rich foods are linked with a range of supposed health benefts for the consumer. Yet, the concept of oxidation potential in foods is neither well defned nor in common use. Here, oxidation potential will be defned as the extent or initial rate of oxidation of a suitable molecular marker present in a given food, which is sensitive to oxygen-mediated chemical

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change. This concept is distinct from that of oxidative stress in living organisms, which usually elicits an antioxidant biochemical response that may be supplemented by antioxidant-rich foods.

11.3

MECHANISMS OF LIPID OXIDATION

Food lipids can be oxidized by different mechanisms, of which the most common is autoxidation. This process involves a free-radical chain reaction involving initiation, propagation, and termination steps, as depicted in Figure 11.1. Although the hydroperoxides, the primary products of lipid oxidation, are odorless and colorless in nature and therefore do not impart negative sensory notes, they are prone to decomposition. The resulting secondary lipid oxidation products include an array of small molecular-weight volatiles, comprising aldehydes, ketones, alcohols, hydrocarbons, epoxides, and thiol compounds. Each has its own favor profle as well as imparting a decisive favor at the concentration at which it is present in the food system. As lipid oxidation is a dynamic process, the status of off-favor volatiles changes in the food system over time leading ultimately to rancidity. Not every fatty acid produces the same volatiles: it depends on a number of factors including the degree of unsaturation as well as the position of the double bonds in the fatty acid molecules. As aforementioned, hexanal levels can be determined by HS-SPME-GC and used as a marker compound to assess the extent of lipid oxidation in food systems. Sometimes oxygen atoms can be excited from the ground triplet to the singlet state by sunlight or other light energy sources, thereby forming singlet oxygen (1O2). The pigments in food systems, most notably chlorophyll and some vitamins, act as sensitizers. They can absorb a photon from visible or UV-light to form an excited singlet sensitizer. This excited species then relaxes and gives off its energy. During this process, highly electrophilic 1O2 can form. Then, the 1O2 can react directly with the double bond of PUFAs and cause oxidation, but by a different mechanism than that of autoxidation. In the case of oleic acid photooxidation, Kołakowska and Bartosz

FIGURE 11.1 The generated radical (1) can interact with molecular oxygen (3O2) (2) and undergo many subsequent propagation reactions (3) with endogenous or exogenous substrates resulting in a variety of ROS. It is important to note that R• (1) is relatively unreactive; however, once it propagates with 3O2 to form ROO•, it becomes highly reactive.

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(2014) reported it to be 30,000 times faster than autoxidation. Like autoxidation, photooxidation generates lipid hydroperoxides, which are unstable and can break down into secondary lipid oxidation products. Antioxidants in foods may offer a line of defense in the human body by providing an exogenous source to help defend against the detrimental effects of free-radical damage/oxidative stress. Antioxidants can be primary in nature; that is, they are chain-breaking and can react directly with free radicals. In this process, they quench ROS by transforming them into stable, non-radicals products. Furthermore, antioxidants have a secondary role by functioning indirectly to retard lipid oxidation. Here, they can act as sequestrants of transition-metal ions to limit the catalytic effects of these ions during the initiation of lipid autoxidation.

11.4

OXIDATION OF PROTEINS IN FOODS

Lipids are not the only macromolecule in food prone to oxidation. The various side chains of the different amino acids are subject to oxidation, most notably thiol groups. The oxidation of amino acids leads to the formation of reactive Strecker aldehydes resulting in the cross-linking of various protein residues in foods. These reactions can be initiated by reactive oxygen, nitrogen, or sulfur species. While protein oxidation does not lead to perceptible favor changes in food as lipid oxidation does, the oxidation of proteins can result in modifed textures of the foods in question. As aforementioned, the oxidative cross-linking of proteins in meat products can result in toughness, reduced water-holding capacity, off-tastes and odors, and undesirable changes in color (Lund et al. 2011). Food proteins do not react directly with triplet oxygen (3O2); rather, reactive intermediates must frst be created. One important mechanism of ROS formation in food systems is metal-catalyzed oxidation by free transition-metal ions such as iron and copper. Iron and copper ions can be present in food at parts-per-million concentrations, albeit, they are often bound in complexes with constituents such as hemoproteins or oxalic and phytic acid (Hellwig 2019). During oxidation of other targets (e.g., H2O2), transition-metal ions are reduced, but via redox cycling they can return to their oxidized state, thereby allowing the oxidation process to repeat. The generation of reactive hydroxyl radicals (•OH) by the classic Fenton reaction or the oxidation of 3O2 by Fe2+ yielding the superoxide anion radical (O2•‒), are examples of how transition-metal ions can generate the necessary ROS for protein oxidation. There is great structural diversity in terms of protein oxidation products: many are carbonylation derivatives; however, this is not necessarily so for amino acid oxidation products. Other reactions in food such as glycation, lipation, and proteinpolyphenol reactions can also generate protein-bound carbonyl compounds (Hellwig 2020). Protein oxidation is somewhat more of a random process leading to a plethora of reaction products, such that the choice of a reliable marker for quantitation is diffcult. The most widely used in vitro assay for protein oxidation assessment is the reaction of 2,4-dinitrophenylhydrazine (DNPH) to corresponding hydrazones followed by spectrophotometric quantitation. Due to many side reactions and the importance of protein oxidation products like methionine sulfoxide, cysteine, and

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3,4-dihydroxyphenylalanine that do not react in the DNPH assay, the measurement of hydrazones is fraught with inaccuracy (Hellwig 2020). The formation of disulfde bonds because of oxidative modifcation in proteins is another common in vitro assay. Free thiol groups are derivatized with Ellman’s reagent (5,5′-dithio-bis(2-nitrobenzoic acid) leading to the formation of a disulfde derivative and the release of 5-thio-2-nitrobenzoic acid, the latter which is measured by UV spectrophotometry. The content of disulfdes initially in the protein system can be quantifed as thiols after reduction by either sodium borohydride or 2-mercaptoethanol (Kehm et al. 2021). Assays involving the measurement of specifc individual oxidized protein species are more commonplace. Mass spectrometric techniques, under the umbrella of a proteomics approach, have been employed to identify and characterize individual oxidized protein species in food. Relative quantifcation of selected peptides and using tryptic marker peptides resulting from oxidation have been used to quantify the oxidation of individual proteins (Dyer et al. 2017).

11.5

THE EFFECT OF OXIDATION ON THE SENSORY AND BIOLOGICAL PROPERTIES OF FOODS

Protein and amino acid oxidation, particularly of sulfur-containing amino acids, can have a limited impact on favor. When food is subjected to irradiation or photooxidation, dimethyl mono-, di-, and trisulfde volatiles can form, giving rise to sulfurous and rotten egg-like off-favors (Brewer 2009). Yet, for the most part, the impact of protein oxidation does not have a marked effect on favor modifcation of food, as lipid oxidation does. Limited protein oxidation can be desirable in some food systems. For example, moderate oxidation of whey proteins can facilitate the stabilization of emulsions, but extensive oxidation has the opposite effect (Tan et al. 2016). The development of a stable gluten network via kneading of dough in bread making is critical for the formation of intermolecular disulfde bridges to provide the viscoelasticity for high-quality bread. In raw meat, the water-holding capacity decreases with increasing protein oxidation, which can ultimately affect the tenderness and juiciness of the cooked product. This has been attributed to a decrease in peptidase calpain activity via the oxidation and myosin crosslinking (Carvalho et al. 2017), with cysteine as the dominant crosslink in oxidized meat products (Bao et al. 2018).

11.6 BENEFICIAL ROLE OF ADDED ANTIOXIDANTS TO FOODS A number of foods contain natural antioxidants: a great example is the tocopherols in vegetable oils helping to protect the integrity of PUFAs against lipid oxidation and the development of rancidity. Oftentimes, foods are processed or refned, which strips away the natural protection of antioxidants afforded by Mother Nature. The resultant food products are often then enriched or fortifed with antioxidants, either synthetic or natural. The purpose of this is to prolong the shelf-life of the foodstuff and to protect various nutrients against degradation. Synthetic antioxidants, such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), tert-butylhydroquinone (TBHQ), and propyl gallate (PG),

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have been used to extend the shelf-life of foodstuffs, but their usage has fallen into disfavor. BHA and BHT have been found to be responsible for adverse effects on the liver and reported carcinogenesis in animal studies (Botterweck et al. 2000). Presently there is a trend for clean labels on products, which favors the addition of natural antioxidants from a number of sources to food systems. Natural antioxidants extracted from herbs and plants are the most common and can be divided into three main classes, namely phenolics, vitamins, and carotenoids. Caution in the choice of antioxidants and their usage levels is necessary, as a number are not approved by the agencies regulating the health and safety of a country’s food supply. Furthermore, many natural antioxidants have lower effcacy than their synthetic counterparts, and this necessitates greater addition levels to food systems. Unfortunately, this can lead to dosages that are harmful or cause favor defects in the food product, thereby requiring some form of favor masking. The phenolic compounds are diverse in terms of types and chemical structures. For example, there are simple phenolic acids like gallic and ferulic acids that can be added to food systems, as well as more complex polyphenolics like tannins. Phenolic compounds are not added as pure phytochemicals, rather they are added in the form of an extract such as those obtained from spices or herbs like rosemary, thyme, and sage. Within a rosemary extract, there are many phenolic constituents, but the extract is dominated by cyclic diterpene diphenols, carnosol, and carnosic acid (Nieto et al. 2018). Employment of herbal extracts is limited because of odor, color, and taste issues; hence, commercial means have been developed for the preparation of odorless and colorless products so that the natural antioxidants can be delivered to the food system without negatively affecting its sensory properties. In terms of antioxidant vitamins, the most important ones are E and C. Vitamin E is lipid soluble and comprises four tocopherols and four tocotrienols. The four isomers include α-, β-, γ- and δ-forms, but only α-tocopherol can be absorbed by the human body because of a receptor in the liver. In terms of foods, vitamin E is found mainly in oilseeds, legumes, and cereal grains. The water-soluble antioxidant vitamin is C and it is naturally present in many fruits and vegetables. At lipid interfaces, vitamin C works in tandem to regenerate vitamin E to help maintain the integrity of lipid bilayers. The last important class of natural antioxidants is carotenoids. Important examples of those with antioxidant activity are β-carotene, α-carotene, lycopene, and lutein. Besides the antioxidant capacity they afford to foods, these carotenoids can be used as food colorants.

11.7

SOURCES OF NATURAL ANTIOXIDANTS IN FOODS

There are many different classes of foods, such as vegetables, fruits, meats, cereals/ grains, legumes/seeds/nuts, etc. Within each class are foodstuffs rich in certain antioxidant constituents. Depending on the food in question, the antioxidants might be water-soluble, lipid-soluble, or possessing characteristics of both. Table 11.1 provides a list of specifc antioxidants and examples of foods that provide good sources of bioactives. When perusing the list, one can see that perhaps the rich source of antioxidants in our daily diet tends to come from fruits and vegetables.

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TABLE 11.1 Sources of Natural Antioxidants and Bioactives Found in Foods Specifc antioxidant/ bioactive constituent

Example food sources

Allium sulfur compounds

Onions, garlic, leeks, shallots

Anthocyanins

Vitamin E Zeaxanthin

Cherries, pomegranates, grapes, red cabbage, red onions, berries, black beans Butternut squash, pumpkin, mangoes, carrots, apricots, cantaloupe, spinach, parsley Green tea, red wine, broad beans, apples, raspberries, black grapes Seafood like oysters, lean meat, liver, milk, nuts Avocados, mangoes, oranges, papaya, peaches, tangerines, pumpkin, watermelon Pomegranates, strawberries, raspberries, blackberries, cherries, nuts like pecans and walnuts Teas, citrus fruits, apples, onions, grapes, red wine, berries Cruciferous vegetables such as cabbage, broccoli, brussel sprouts, turnips, bok choy Soybeans, tofu, lentils, peas, beans Flax seeds, sesame seeds, bran, whole grains like rye and oats Greens, leafy vegetables like spinach and kale, corn, bell peppers Tomatoes, apricots, watermelon, pink grapefruit, pink guavas Seafood, lean meats, milk, nuts Berries, herbs and spices, cocoa powder, nuts, coffee, tea Brazil nuts, seafood, offal, whole grains Liver, salmon, tuna, sweet potatoes, carrots, milk, egg yolks Oranges, grapefruit, black currants, kiwi fruit mangoes, broccoli, spinach, strawberries Vegetable oils, avocados, nuts like almonds, peanuts, seeds, whole grains Dark-green vegetables like kale, spinach, broccoli, kiwi fruit, grapes

Zinc

Shellfsh like oysters and crab, lean meat, legumes, milk, nuts like pecans

Β-carotene Catechin/epicatechin Copper Cryptoxanthins Ellagic acid Flavonoids Indoles Isofavonoids Lignans Lutein Lycopene Manganese Polyphenols Selenium Vitamin A Vitamin C

Several foods can be singled out for being excellent sources of antioxidants and are described in the following. Green tea is one: it is made from the unoxidized leaves of the Camellia sinensis bush. It is reported that daily consumption of green tea promotes mental alertness, relieves digestive systems and headaches, as well as promotes weight loss (Suzuki et al. 2012). Green tea contains ~30% polyphenols, including marked amounts of (+)-catechin/(–)-epicatechin (i.e., a favan-3-ol) and a derivative, epigallocatechin-3-O-gallate (EGCG). The polyphenols in green tea, notably EGCG, are believed to provide a protective effect against oxidative damage in the body, thereby reducing the risk of developing heart disease and cancer. Recently, the benefts of tea favonoid intake, as tea consumption has been suggested to be inversely associated with a decreased risk of cardiovascular disease (CVD),

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were reviewed by Chung et al. (2020). Databases were searched to include the fndings of randomized trials, prospective cohort studies, and nested case-control studies on tea consumption and the risk of cardiovascular events, stroke events, CVDspecifc mortality, or all-cause mortality. The strength of evidence was found to be low to moderate for CVD-specifc mortal outcomes and was rated as low for CVD events, stroke, and all-cause mortality outcomes. An umbrella review to describe and critically evaluate the totality of evidence that tea favonoid intake reduces the risk of CVD was conducted by Keller and Wallace (2021); they found a similar conclusion. Yet, they proposed that consuming two cups of unsweet tea per day offers the right level of favonoids to potentially decrease CVD risk and its progression. The possible associations between green tea consumption and the risk of cancer incidence and mortality as a primary outcome were reviewed by Filippini et al. (2020). Epidemiological studies, randomized control trials, and observational studies (142 in all) investigating the association of green tea consumption with cancer risk were considered. The researchers reported that the fndings yielded inconsistent results, thus providing limited evidence for the benefcial effect of green tea consumption on the overall risk of cancer. Noteworthy is that the National Cancer Institute (2022) does not recommend or oppose using green tea to reduce the risk of any type of cancer. Blueberries are a perennial fowering plant of the genus Vaccinium with blue or purple berries. They are a rich source of nutrients and bioactives in the diet, including vitamins (C and K), minerals (iron, phosphorous, calcium, magnesium, manganese, zinc), and phenolic compounds, most notably anthocyanins (Kalt et al. 2020). Anthocyanins are known for their powerful antioxidant and free-radical scavenging properties (Szymanowska et al. 2015). Zhou et al. (2020) identifed 14 anthocyanins from blueberries by liquid chromatography-diode array detector-electrospray ionization-tandem mass spectrometry (LC-DAD-ESI-MS2). Malvidin-3-O-glucoside was dominant, followed by malvidin-3-O-galactose and petunidin-3-O-glucoside. The researchers noted that these three anthocyanins comprised ~45% of the total anthocyanins. Huang et al. (2016) surveyed the antioxidant functional role of blueberry anthocyanins in endothelial cells. The results showed that anthocyanins, such as malvidin-3-O-glucoside, decreased the levels of ROS indicating the potential of blueberry at curbing oxidative stress. The antioxidant capacity of blueberry extracts for peroxyl-radical scavenging and inhibition of plasma lipid oxidation was assessed by Morita et al. (2017). The researchers found that the blueberry extracts exerted potent antioxidant effects in the plasma lipid oxidation model when induced by biologically relevant oxidants, including peroxyl radicals, peroxynitrite, hypochlorite, 15-lipoxygenase, and 1O2. Dark chocolate, which has not been subjected to Dutch processing, is known for high levels of favonoids, particularly (+)-catechin, (–)-epicatechin, and procyanidins: total concentrations range between 400 to 700 mg/kg (Żyżelewicz et al. 2016). Health benefts attributed to dark chocolate consumption include a reduction in the risk of coronary heart disease and positive neurocognitive effects (Yuan et al. 2017). The epicatechin content of cocoa is primarily responsible for dark chocolate’s favorable impact on vascular endothelium via its activation of endothelial NO synthase,

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leading to the generation of NO (Katz et al. 2011). According to these researchers, other cardiovascular effects attributed to dark chocolate are mediated through antiinfammatory effects of cocoa procyanidins and modulated through the activity of NF-κB. Furthermore, the antioxidant effects of cocoa may directly infuence insulin resistance and, thereby, reduce the risk for type-2 diabetes. In a more recent review, dark chocolate’s positive effects on helping to regulate blood pressure, insulin levels, vascular functions, oxidation processes, glucose homeostasis, and lipid metabolism were summarized by Montagna et al. (2019). Tree nuts are a rich source of phenolics belonging to several classes. Chlorogenic acid is typical for pistachios and almonds. The highest amounts of ellagic acid were found in Brazil nuts, pecans, and pine nuts (Amarowicz and Pegg, 2020). Pecans, walnuts, and hazelnuts are known as rich sources of condensed tannins (i.e., proanthocyanidins, PACs). The content of procyanidin dimers and trimers in hazelnuts was reported by Slatnar et al. (2014) and Ciemniewska-Żytkiewicz et al. (2015). Phenolic compounds present in tree nut and peanut extracts can protect lowdensity lipoprotein (LDL) against oxidation. Up to 48% inhibition of LDL oxidation by a peanut extract at a concentration of 100 ppm was observed by De Camargo et al. (2015). Extracts obtained from raw and roasted cashew kernels and pistachios hindered Cu2+-initiated oxidation of human LDL (Chandrasekara and Shahidi 2011; Shahidi et al. 2007). In recent years, pecans (Carya illinoinensis [Wangenh.] K. Koch) have gained increased attention for their purported health benefts. Robbins et al. (2015) determined the content of phenolics and antioxidant capacity of 18 US pecan cultivars. Tandem HPLC-ESI-MS was employed to identify the free, ester-linked, and glycoside-linked phenolics of pecans, which included gallic and ellagic acids, their derivatives, and PACs. Even though the in vitro assays performed by Robbins et al. (2015) revealed that pecans are rich in antioxidant constituents, their in vivo antioxidant activity needs verifcation. The fndings of a placebo-controlled, three-way crossover design clinical trial with a one-week washout period between pecan treatments revealed that γ-tocopherol and the PAC favan-3-ol monomers of pecans were absorbable and contributed to postprandial antioxidant defenses in the human body (Hudthagosol et al. 2011). More recently, Guarneiri et al. (2021) evaluated daily pecan consumption for eight weeks on fasting and postprandial lipid peroxidation, total antioxidant capacity, and tocopherol levels in adults suffering from hypercholesterolemia or elevated adiposity. These researchers found that daily pecan consumption (68 g) increased postprandial total antioxidant capacity and fasting γ-tocopherol, while reducing postprandial lipid peroxidation in adults at risk for CVD. The dominant natural antioxidants of legume seeds are phenolic acids, favonoids, and PACs (Amarowicz and Pegg 2019). With their colored coats, legumes are also rich in anthocyanins. The phenolic acids and favonoids are located mainly in the cotyledons of legume seeds and are rich in phenolic acids and favonoids, whereas PAC oligomers and polymers are mostly present in the seed coats. The strong antiradical-scavenging effect of PACs (i.e., tannin constituents) separated from acetonic extracts of red and green lentils, adzuki bean, red bean, fava bean, and broad bean was confrmed using the DPPH• and ABTS•+ assays by Amarowicz et

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al. (2008, 2009, 2010, 2017) and Amarowicz and Shahidi (2017, 2018). These tannin fractions also exhibited marked antioxidant activity when employed in emulsion systems. Germination and fermentation of legume seeds can increase the content of phenolic compounds and their antioxidant potential (Amarowicz and Pegg 2008). Aguilera et al. (2014) reported that the antioxidant potential of germinated lentil seeds was affected by the length of time for germination. Extracts obtained from defatted oilseeds can be used as a source of natural antioxidants. For instance, sinapine (i.e., the choline ester of sinapic acid), sinapic acid, its glucoside, and other derivatives from it are the main phenolics found in rapeseed and canola (Szydłowska-Czerniak 2013). According to Naczk et al. (2000), condensed tannins can be present in the hulls of some rapeseed cultivars. Canolol, as a product of sinapic acid decarboxylation catalyzed by sinapic acid decarboxylase, can be present in rapeseed/canola oil (Wijesundera et al. 2008). Soybean seeds are characterized by the presence of isofavones that include their aglycones (daidzein, genistein, glycitein), glucosides (daidzin, geinstin, glycitin), malonylglucosides (malonyldaidzin, malonylgeinstin, malonylglycitin), and acetylglucosides (acetyldaidzin, acetylgeinstin, acetylglycitin) (Luthria et al. 2007). In sunfower kernels, the predominant phenolic compounds are 5-O-caffeoylquinic acid (Aramendia et al. 2000), dicaffeoylquinic acids (1,3-di-O-caffeoylquinic and 1,4-di-O-caffeoylquinic acids), and isomers of coumaroylquinic acid such as 3-O-p-coumaroylquinic and 4-O-p-coumaroylquinic acids (Karamać et al. 2012). In faxseeds, lignans are present in the form of macromolecules composed of secoisolariciresinol diglucoside (SDG), hydroxymethylglutaric acid, 4-O-βglucopyranosyl-p-coumaric acid, 4-O-β-glucopyranosyl-ferulic acid, 4-O-βglucopyranosyl-caffeic acid, and 4-O-β-glucopyranosyl-caffeic acid (Struijs et al. 2008; Kosińska et al. 2011). The molecular weight of faxseed lignan macromolecules is reported in the range from 1,500 to 4,300 Da (Struijs et al. 2009). Cereals are also an important source of natural antioxidants in the human diet (Amarowicz and Pegg 2020). In wheat, ferulic acid is the main phenolic compound, while in rye, triticale, caffeic, p-coumaric, ferulic, and sinapic acids are dominant (Weidner et al. 1999). Alkylresorcinols are another class of phenolic compounds that can be found in wheat, rye, triticale, and barley (Verma et al. 2008). Alkylresorcinols comprise a single phenolic ring with a long alkyl side chain (Kozubek and Tyman 1999). Ferulic acid esterifed with triterpene alcohols and plant sterols, often referred to as γ-oryzanol, is a typical phenolic compound present in rice bran (Patel and Naik 2004). In the last decade, food scientists have focused on pseudocereals as sources of natural antioxidants. In the seeds of buckwheat, rutin, orientin, isoorientin, vitexin, isovitexin, derivatives of caffeic acid, and procyanidins have been identifed as the chief phenolic compounds, (Kiprovski et al. 2015); in canihua, its kaempferol glycosides (Peñarrieta et al. 2008); while in quinoa, its sinapic acid, ferulic acid, p-coumaric acid, rosmarinic acid, catechin, apigenin, apigenin-7-O-glucoside, quercetin, kaempferol, and chrysin (Al-Qabba et al. 2020). The presence of rosmarinic acid is typically found in plants belonging to the Lamiaceace family with examples including the herbs of sage, rosemary, thyme,

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oregano, and basil. On the list of the other natural antioxidants endogenous to these herbs are the following: apigenin, luteolin, hispidulin, and nepetin (in sage); epirosmanol, carnosol, carnosic acid, caffeic acid, and catechin (in rosemary); rosmarinic acid-glucoside, chlorogenic acid, lithospermic acid, gallic acid, 3,4-dihydroxyphenyl acetic acid, protocatechuic acid-hexoside, and protocatechuic acid (in thyme); hyperoside and isoquercitrin (in oregano); quercetin and rutin (in basil) (Lee et al. 2018; Shan et al. 2005; Sonmezdag et al. 2018; Oniga et al. 2018; Fratianni et al. 2017). Waller et al. (2017) determined 4-hydroxybenzoic acid, caffeic acid, chlorogenic acid, hesperetin, and rutin as the main phenolics in marjoram.

11.8

ANTIOXIDANTS GENERATED BY PROCESSING OF FOODS

When food is processed, there is a possibility that new chemical compounds possessing antioxidant activity may be generated. An excellent example to illustrate this point are Maillard reaction products (MRPs) formed during thermal processing, especially when there is a high temperature and low moisture levels. MRPs have been reported to exhibit marked antioxidant activity (Nooshkam et al. 2019) due to such mechanisms as scavenging free radicals, chelating prooxidant transitionmetal ions, and breaking down oxygen species (Nooshkam and Madadlou 2016). Michalska et al. (2008) reported that MRPs (mainly melanoidins) contributed to the total antioxidant potential of rye bread, as determined using the ABTS•+ assay and scavenging potential of the peroxyl radical. The antioxidant properties of MRPs extracted from cookies (Sun et al. 2008) and biscuits (Patrignani et al. 2016) have also been reported. Via model system studies, MRPs prepared under laboratory conditions were found to exhibit antioxidant activity in food models of ground beef patties (Fernández et al. 2016); ground chicken breast (Miranda et al. 2012); pork (Li et al. 2013); full-cream milk powder (McGookin and Augustin 1997); dairy beverages containing linseed oil (Giroux et al. 2010); and sardine lipids (Chiu et al. 1991). The water-soluble melanoidins, containing derivatives of chlorogenic acid, ferulic acid, caffeic acid, and p-coumaric acid, were determined to contribute greatly to the antioxidant activity of roasted coffee (Delgado-Andrade et al. 2005). Several studies have shown that after protein hydrolysis, the peptides generated possess antioxidant properties. For example, the inhibition of oxidation of meat and fsh lipids by whey, casein, soy, and egg yolk hydrolysates has been reported by Diaz and Decker (2005), Pena-Ramos and Xiong (2003), Sakanaka and Tachibana (2006), and Sakanaka et al. (2005). Strong antioxidant properties were exhibited by carnosine and anserine, as well as histidine-containing dipeptides (Chan et al. 1994). The antioxidant activities of peptides from fsh proteins have been confrmed using several chemical assays, as described by Je et al. (2007) and Kim et al. (2007). According to Saiga et al. (2003), a high content of hydrophobic amino acids in peptides prepared via hydrolysis could increase their solubility in lipids and thereby facilitate access to scavenging lipid-free radicals. In the study of Saito et al. (2003), a tripeptide containing Trp or Tyr residues at the C-terminus demonstrated strong radical-scavenging activity, but weak peroxynitrite-scavenging activity. A novel antioxidant peptide from a corn protein hydrolysate with the amino acid sequence

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of Gln-Gln-Pro-Gln-Pro-Trp was found to have marked antioxidant activity (Wang et al. 2014). An antioxidant peptide from chickpea protein hydrolysates was also identifed with the amino acid sequence of Asn-Arg-Tyr-His-Glu (Zhang et al. 2011).

11.9

SOURCES AND IMPACT OF PROOXIDANTS IN FOODS

Prooxidants are compounds that can initiate, facilitate, or accelerate lipid oxidation. Under specifc conditions, natural antioxidants found in foods like tocopherols, carotenoids, and vitamin C can act as prooxidants.

11.9.1

TOCOPHEROLS

The mechanism of prooxidant activity of tocopherols (i.e., vitamin E) is varied (Choe and Min 2006; Barouh et al. 2021). Tocopherols (TocOH) can act as a donor of hydrogen atoms to lipid peroxyl radicals (ROO•) resulting in the production of a hydroperoxide (ROOH) and a tocopheroxy radical (TocO•); the TocO• is of lower energy and therefore more stable than the original lipid peroxyl radical. ROO• + TocOH → ROOH + TocO• The tocopheroxy radical can also react with other hydroperoxides, thereby generating a lipid peroxyl radical. ROOH + TocO• → ROO• + TocOH Tocopherols, particularly α-tocopherol, act as prooxidants in oils when their concentration is high and when the concentration of lipid peroxyl radicals is very low. At such conditions, the tocopheroxy radical abstracts a hydrogen atom from the lipid yielding tocopherol and lipid alkyl radicals, which accelerates lipid autooxidation (Kamal-Eldin 2006). RH + TocO• → R• + TocOH This reaction is sometimes referred to as tocopherol-mediated peroxidation (Bowry and Stocker 1993). According to Yamamoto (2001), ascorbic acid can reduce tocopheroxy radicals and prevent tocopherol-mediated peroxidation from occurring. TocOH or TocO• can also reduce transition-metal ions from higher oxidation states to lower ones, and via the Fenton reaction generate reactive •OH. Tocopherol can also function as an electron donor to the O2•‒ leading to the production of H2O2, which is a precursor of •OH. Several experimental studies have confrmed the prooxidant properties of tocopherols. Based on regression analysis of peroxide values (PVs), 2-thiobarbituric acid reactive substances (TBARS), and tocopherol contents of edible vegetable oils (namely corn oil; sunfower oil; soybean oil; peanut oil; camellia oil; palm oil; blended oil with 55% soybean oil, 35% rapeseed oil, and 10% perilla oil; and blended

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oil with 90% soybean oil and 10% perilla oil), Cao et al. (2015) concluded that when α-tocopherol predominated over other homologs, it gave a prooxidant effect when present at a high concentration. However, the γ- or δ-tocopherol isoforms did not demonstrate this prooxidant action. In the study by Elisia et al. (2013), the naturally occurring α-tocopherol contents in 14 edibles were signifcantly correlated (r = 0.696) with the level of conjugated dienes during the primary stage of lipid oxidation, indicating a potential prooxidant effect of α-tocopherol. The prooxidant activity of α-tocopherol during linoleic acid autoxidation in an aqueous media (pH 6.9) was confrmed by an increase in the level of conjugated dienes after α-tocopherol addition (Cillard et al. 1980). The authors identifed α-tocopherylquinone and a dimer of α-tocopherol as two reaction products of α-tocopherol oxidation. During a long-term storage stability study of faxseed oil at 25, 40, and 60° C for 30 days, α-tocopherol was found to impart a prooxidant effect during almost every day at lower temperatures and displayed a prooxidant activity only on day 30 at 60° C (Mohanan et al. 2018). The results of Pinto et al. (2015) indicated that α-tocopherol at 0.5, 1.0, 1.5, and 2.0 g/kg acted as a prooxidant in two peanut oil biodiesels (i.e., blends of long-chain fatty acid alkyl esters); the prooxidant effects were measured using a Rancimat unit and by differential scanning calorimetry coupled to a pressure cell. The prooxidant effect of α-tocopherol at a concentration of 0.2% (w/w) in an experiment with salmon oil was reported by Belhaj et al. (2010). The addition of δ-tocopherols to corn chips enriched with linseed oil decreased the oxidative stability of α-linolenic acid during six months of storage, as measured by PVs and the loss of α-linolenic acid content. The greatest effects were observed at the highest addition level of δ-tocopherol (i.e., 800 mg/100 g chips; Rogalski and Szterk 2015). Under long-term storage conditions for encapsulated vegetable oils, α-tocopherol was found to induce a decrease in oil resistivity to oxidation (Le Priol et al. 2021).

11.9.2

CAROTENOIDS

Due to the extended conjugation of their π-bonding electrons, carotenoids can undergo oxidation and form relatively stable-ion radicals. The radical cation of β-carotene, with an electron reduction potential greater than that of a PUFA, can abstract a hydrogen atom from PUFAs and in doing so, produce a new fatty acid radical. Burton and Ingold (1984) showed that peroxide lipid radicals can be added to a molecule of β-carotene, thereby generating a new carotene peroxyl radical. After reaction with oxygen, this radical can propagate the chain reaction of lipid oxidation (Iannone et al. 1998). During quenching of 1O2 by β-carotene, relatively stable cyclic mono- and diendoperoxides are produced (Fiedor et al. 2005). The authors suggested that these endoperoxides are related to the prooxidant activity observed for β-carotene. Moreover, β-carotene and other carotenoids have been reported to display prooxidative activity at high oxygen tensions in foods and model systems. It was demonstrated that carotenoids could increase or decrease the total yield of free radicals depending on the oxidation potential of the carotenoids and the nature of the radicals

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in question. A reaction mechanism was proposed involving the reduction of Fe3+ to Fe2+ by carotenoids. The effectiveness of this “carotenoid-driven Fenton reaction” increases with a decrease in the scavenging rates for free radicals and oxidation potentials of carotenoids (Lee and Min 1998; Polyakov et al. 2001). β-carotene, at addition levels ranging from 50 to 300 μg/g, acted as a prooxidant in highly unsaturated sunfower oil, when oxidized using a Rancimat system (Zeb 2011). The prooxidant activity of β-carotene was observed after the third hour of thermal degradation of a high-oleic acid model of acylglycerols (Zeb and Murkovic 2010). The acylglycerols of refned olive oil were found to oxidize in a Rancimat system (at 110° C for 1–14 h) much faster when in the presence of β-carotene (300 mg/kg). The longer the exposure time, both β-carotene and astaxanthin (300 mg/kg) were found to signifcantly increase the PVs of olive oil (Zeb and Murkovic 2011). The researchers also reported that during thermal oxidation of edible oils, more peroxides were generated in corn, sunfower, and rapeseed oils due to the presence of β-carotene (at 50–300 μg addition levels/g) (Zeb and Murkovic 2013). In all oils combined with added β-carotene, there was a marked increase in the free fatty acids content. Steenson and Min (2000) reported that in the dark, the thermal degradation products from β-carotene accelerated soybean oil oxidation. Haila et al. (1996) confrmed the prooxidant activities of lutein and lycopene in model system studies using acylglycerols. In this work, oxidation was monitored by measuring the generation of hydroperoxide formation as PVs. The researchers concluded that the potential prooxidant effects of carotenoids ought to be considered when carotenoids are proposed as a natural color additive to lipid-containing foods. Park et al. (2013) investigated the prooxidative properties of β-carotene at concentrations of 10, 100, and 1,000 μM in chlorophyll- and ribofavin-photosensitized oil-in-water emulsions. When chlorophyll was employed as the photosensitizer, a prooxidant action was observed at all three concentrations tested. However, when ribofavin was used, β-carotene acted as an antioxidant at concentrations of 100 and 1,000 μM. In the study of Ha et al. (2012), β-carotene addition to lard at 1.25 and 2.50 μM signifcantly decreased headspace oxygen levels, while its addition at 0.25 to 2.50 μM signifcantly increased p-anisidine values when the fat was oxidized at 60° C for 60 h; the results here suggesting that β-carotene functioned as a prooxidant. Experiments with DPPH radicals indicated that β-carotene at concentrations greater than 1.25 μM can accelerate the formation of radical-scavenging compounds generated from oxidized lipids. A prooxidant effect of β-carotene in binary blends with ascorbyl palmitate was observed in butter oil acylglycerols. This was observed when an accelerated oxidation test (i.e., Schaal oven conditions) was performed at 60° C (Karabulut 2003). Osborn-Barnes and Akoh (2003) reported the prooxidant activity of β-carotene on lipid oxidation for a structured lipid (i.e., a canola oil/caprylic acid-based oil-in-water emulsion stabilized with 0.5% of sucrose fatty acid ester). The researchers concluded that manufacturers must experiment with β-carotene addition levels before adding it to structured lipid-based products as a functional ingredient; otherwise, lipid oxidation might be accelerated in the product thereby decreasing the product’s shelf-life.

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The addition of higher quantities of β-carotene to faxseed oil leads to a signifcant intensifcation of oil oxidation during storage. After 12 months of cold storage, the oil containing 25 mg β-carotene/100 g was reported to have a concentration of hydroperoxides 2.6 times greater than that of the non-fortifed control oil sample (Shadyro et al. 2020). In a different study, soybean oil containing 50 mg of degraded β-carotene/kg, stored in the dark at 60° C for 24 h, displayed signifcantly higher levels of headspace oxygen depletion compared to the control oil counterpart (Steensen and Min 2000).

11.9.3

VITAMIN C

Ascorbic acid at high concentrations in vitro catalyzes the reduction of free transition-metal ions, especially iron and copper. Reduced iron (i.e., ferrous) ions then can react with H2O2 to form •OH or peroxide ions (Kaźmierczak-Barańska et al. 2020). The prooxidant properties of vitamin C have been confrmed in food models by several researchers. For instance, a system of Cu(II)/ascorbic acid was used as a prooxidant for linoleic acid oxidation by Bakir et al. (2013). In an experiment by Kanner et al. (1997), Cu(II) formed a prooxidant system with ascorbic acid, but only at low metal-ion concentrations. However, this prooxidant effect was enhanced in the presence of added linoleate hydroperoxides. According to Franke et al. (2004), orange juice, which is rich in vitamin C at a concentration of up to 1%, demonstrated oxidative damage to methyl linoleate; yet, when the concentration was increased to 10%, a lesser extent of lipid peroxidation was observed. In the experiments, methyl linoleate oxidation was monitored via the formation of TBARS with CuSO4 as the oxidation inducer. The fndings of Hamre et al. (2010) revealed that ascorbic acid fortifcation resulted in an increase of lipid oxidation in an experimental fsh feed, made directly from marine raw materials. A system of ferric chloride hexahydrate and L-sodium ascorbate was used for catalyzed oxidation of lamb meat homogenates by Luciano et al. (2017) and Valenti et al. (2018). Research on cholesterol oxidation in marinated foods during heating revealed that the addition of 0.02% (w/w) vitamin C reduced the content of cholesterol oxidation products, namely 7a-OH, 7b-OH, 25-OH, and 7-keto, in marinated pork and its purge (Lee et al. 2008). The concentrations of the same cholesterol oxidation products rose, however, when the addition level of vitamin C was increased to 0.1%. Based on EPR studies of Buettner at Jurkiewicz (1993), the researchers explained the results via the formation of dehydroascorbic acid and semi-dehydroascorbic during heating; that is, when the addition level of vitamin C is high, there is promotion of cholesterol oxidation by the last vitamin C derivative.

11.9.4

FLAVONOIDS

The results of numerous studies have demonstrated a prooxidant activity arising from favonoids, but this is dependent upon their concentrations and the reaction

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conditions in question (Rietjens et al. 2003; Kessler et al. 2003; Procházková et al. 2011). Heim et al. (2002) reported that multiple hydroxy groups attached to a favonoid’s ring system, particularly those on the B-ring, signifcantly increased production of •OH via the Fenton reaction. The free hydroxy moiety at position-3 of a favonoid has been implicated in the prooxidant mechanism, whether the catechol group is free or substituted. According to Bayrakçeken et al. (2003), scavenging of ROS by favonoids generates favonoid phenoxyl radicals (Fl–O•) with a lifetime of 200 μs. These reaction products are extremely reactive and subject to further oxidation, thus, forming a favonoid semiquinone radical. Under physiological conditions, this radical may be scavenged by reduced glutathione producing a thiyl radical of glutathione. This thiyl radical may react subsequently with another glutathione molecule to generate a disulfde radical anion that rapidly reduces to O2 and O2•‒ (Galati et al. 1999). Tea polyphenols have been reported to exhibit prooxidant activity, especially in the presence of transition-metal ions (Bouayed and Bohn 2010). At a low iron concentration, (+)-catechin was shown to act as a prooxidant against pectins in an H2O2/Fe2+ model system (Vidot et al. 2020). The prooxidative properties of myricetin, caused by the reduction of O2 to a ROS and Fe3+ to Fe2+, were reported by Chobot and Hadacek (2012). In a lipid oxidation model, Martins et al. (2018) observed a prooxidant effect of polylactic acid flm containing 2% of green tea polyphenolics after 60 days of storage. The results of Tian et al. (2022) indicated that tea polyphenols in model oil-in-water emulsions (pH 7) containing protein may act as either antioxidants or prooxidants: it all depends on their concentration and also on the location of the proteins in the emulsions. As an illustration, the prooxidant activity towards proteins in emulsions with proteins at the interface was observed when 0.04% (w/w) of tea polyphenols was added. At this concentration, endogenous Fe3+ was reduced to Fe2+ and this accelerated the decomposition of lipid peroxides to lipid alkoxyl radicals (LO•). The interfacial proteins were also oxidized by the reaction with LO•.

11.9.5

PROOXIDANT TRANSITION-METAL IONS

The basic prooxidant action of transition-metal ions is oxygen activation either by formation of the peroxyl radical (HO2•) or 1O2 (Nawar 1996): Mn+ + O2 → M(n+1)+ + O2‒ O2‒ – e‒ → 1O2 O2‒ + H+ → HO2• Fe(II) generates •OH in the Fenton reaction: Fe2+ + H2O2 → Fe3+ + •OH + OH‒

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Fe(II) can also react with lipid hydroperoxides (Mozuraitytea et al. 2006): ROOH + Fe2+ → RO• + Fe3+ + OH‒ Several authors used CuSO4 as an initiator of LDL oxidation (Amarowicz and Pegg 2017). Cu(II) also formed a prooxidant system with ascorbic acid (Bakir et al. 2013): Cu2+ + AscH‒ → Cu+ Asc•‒ + H+ The prooxidant activity of iron when used as a whey protein–iron complex in a linoleic acid emulsion was signifcantly reduced as compared to iron salt (Shilpashree et al. 2020). To monitor the oxidative stability, the production of TBARS and the length of the induction period were determined. According to Richards and Li (2004), who evaluated the effects of released iron, lipid peroxides, and ascorbate in trout hemoglobin-mediated lipid oxidation of washed cod muscle, roughly 7% of the iron associated with the trout hemoglobin was released from the heme–protein complex during storage at 2° C. Ascorbate addition (2.2 mmol) was found to inhibit active hemoglobin-mediated lipid oxidation, but it depended on which washed cod preparation was assessed. The combination of ascorbyl palmitate at 400 mg/kg and co-spray-dried heme iron with calcium caseinate at a 1:1 (w/w) ratio was capable to offer protection against oxidation of the palm oil matrix when used as a model for bakery products (Alemán et al. 2016a). The extent of lipid oxidation was characterized by PVs, lipid hydroperoxides content, p-anisidine values, and hexanal concentrations. The same system was effective for bakery products such as cookies and biscuits (Alemán et al. 2016b). The prooxidant effect of iron in fsh oil emulsions, stabilized with phospholipids and Tween, and in liposomes made from phospholipids was reduced by employing less unsaturated phospholipids, specifc amounts of emulsifers, the presence of chloride anions, or xanthan gum (Kristinova et al. 2014). Lipid oxidation, induced by cod and bovine hemoglobin and measured by consumption of dissolved oxygen in a liposome model system, was inhibited by α-tocopherol and astaxanthin (Carvajal et al. 2009). Interestingly, the application of ethylenediaminetetraacetic acid did not provide any protective effect at the levels tested. A packaging flm comprising chitosan and sodium alginate exhibited Cu2+chelating capability in a model oil-in-water emulsion against oxidation (Yu et al. 2022). Bou et al. (2010) investigated the effect of heating oxyhemoglobin (oxyHb) and methemoglobin (metHb) on microsomes, which leads to oxidation. The prooxidant effects of native oxyHb and metHb were similar to and higher than their corresponding oxyHb or metHb when heated at 68 and 90° C. The temperature of 45° C was not effective, but metHb when heated at 68 and 90° C exhibited the same degree of prooxidant activity. Noteworthy is that the prooxidant effect of oxyHb when heated at 68° C was more effective than that of oxyHb when heated at 90° C.

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321

LIPOXYGENASES

Lipoxygenases (LOXs) are a family of non-heme iron-containing dioxygenases. They catalyze the insertion of O2 into PUFAs containing a cis,cis-l,4-pentadiene moiety. This reaction generates lipid hydroperoxides, which can be involved in a lipid oxidation chain reaction (Andreoun and Freussner 2009). Hydroperoxides are unstable and break down to form secondary products of oxidation, many of which have undesirable favor attributes. The degradation of PUFAs by LOXs is a major cause of undesirable off-favor development in legumes (Roland et al. 2017). The technological possibilities of LOX inhibition are important from both economical and nutritional points of view. Thermal treatments, such as hot grinding and blanching, are the most commonly used techniques of LOX inactivation in plant material (Baysal and Demirdöven 2007). Steaming of wheat kernels for 90 s at 15 s intervals decreased the LOX activity by 64% (Poudela and Rose 2018). Chickpeas that were cooked and parched showed a marked drop in LOX activity (Attia et al. 1996). Anthon and Barrett (2003) observed a rapid inactivation of tomato LOX at 60° C. The inactivation kinetics for both enzymes indicate that they would be rapidly inactivated at the cold break target temperature of 60° C. The optimal parameters of superheated steam treatment for thermal inactivation of LOX in soya beans and soya milk were at 9.3 min and 119° C (Chong et al. 2019). According to Kubo et al. (2021), almost complete inactivation of enzymatic activity in soymilk was achieved after 9 min of microwave treatment at 90° C. In food technology, non-thermal treatments have been employed as a method of LOX inhibition. Indrawati et al. (1998) reported that soybean LOX was sensitive to a high-pressure–low-temperature treatment. The researchers noted almost complete inactivation of the enzyme when a pressure of 400 MPa was applied for 10 min at –20° C. A similar fnding was determined for the inactivation of LOXs in carrot juice, with the product being treated with a pressure of 500 MPa for 10 min at either 60 or 70° C (Kim et al. 2001). Min et al. (2003) reported up to 20% residual activity of LOX in tomato juice that had been subjected to high-intensity pulsed electric feld treatment. The inactivation of soybean LOX by pulsed electric feld processing was studied by Li et al. (2008). Residual activity of soybean LOX decreased with an increase in the treatment time, pulse strength, pulse frequency, and pulse width. The maximum inactivation of soybean LOX achieved by pulsed electric feld treatment (i.e., 88%) required 42 kV/cm for 1,036 μs with 400 Hz of pulse frequency and a 2 μs pulse width at 25° C. Riener et al. (2008) showed that the highest level of soymilk LOX inactivation (84.5%) was attainable when frst preheating the milk to 50° C followed by pulsed electric feld treatment (100 μs at 40 kV/cm). The synergistic effect between action of heat and ultrasound on soybean LOX inhibition was reported by Lopez et al. (1994).

11.9.7

FREE FATTY ACIDS

Free fatty acids can exhibit prooxidant characteristics in food systems. An explanation for this observation might be related to the catalytic effect of the carboxylate

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group on the production of free radicals via the decomposition of hydroperoxides formed in the initial stage of autooxidation (Aubourg 2001). According to Mistry and Min (1987), free fatty acids can decrease the surface tension of edible oils and permit an increase in the diffusion rate of oxygen from the headspace into the oil, hence, accelerating lipid oxidation. Free fatty acids also can act as surfactants when an oil contains trace amounts of water. Therefore, lipid hydroperoxides and watersoluble transition-metal ions can migrate to the interface and lead to lipid peroxide decomposition (Kittipongpittaya et al. 2014). Several studies have reported on the prooxidant properties of free fatty acids. To illustrate, such an effect of stearic acid addition to soybean oil was observed by Miyashita and Takagi (1986), Mistry and Min (1987), and Colakoglu (2007). According to Mistry and Min (1987), the prooxidant effect against soybean oil was also exhibited by free oleic, linoleic, and linolenic acids. Fatty acids with a shorter chain length and a higher degree of unsaturation exhibited a prominent prooxidant effect against soybean oil during microwave heating (Yoshida 1993). Paradiso et al. (2010) incorporated 0.25, 0.5, 1, and 3% free fatty acid levels into purifed olive oil, while ensuring that overall lipid composition did not deviate from the specifcations for that of olive oil. Oxidation proceeded under Schaal oven conditions (i.e., bulk oil storage at 60° C for 10 days) and by the Rancimat assay (85° C with an airfow of 20 L/h). The researchers found that low levels of added free fatty acids acted as prooxidants in both test systems. The higher addition of free fatty acids led to lower quantities of oxidized forms of the acylglycerols with respect to the purifed oil. The scientists suggested that such fndings might result from peroxide decomposition of free fatty acids when present in greater quantities. De Leonardis et al. (2016) showed a negative effect of the presence of free fatty acids on the oxidative stability of red palm oil. In this work, the Rancimat method and medium-temperature assay (i.e., with the reaction induced by the radical initiator, 2,2′-azobis[2,4-dimethylvaleronitrile]) were employed to assess red palm oil’s stability against oxidation. A fuorescence study on the prooxidant effect of free fatty acids in cod liver oil was carried out by Aubourg (2001). At 30° C, the researchers determined that shorter chain-length fatty acids (i.e., lauric and myristic acids) imparted a more pronounced prooxidant effect than that of longer chain-length fatty acids (i.e., arachidic and stearic acids). The prooxidant effect of fatty acids was also dependent on each fatty acid’s degree of unsaturation. The aforementioned effect was found in the order of linoleic acid > linoleic acid > oleic acid > stearic acid.

11.9.8

SALT

The prooxidant effect of sodium chloride, hereinafter referred to as salt or NaCl, on lipid oxidation has not been fully elucidated (Mariutti and Bragagnolo 2017). In meat, fsh, and seafood, salt can inhibit the activity of antioxidant enzymes (Hernandez et al. 2002), liberate iron from heme proteins (Kanner et al. 1991), change the integrity of cell membranes, and increase the contact of prooxidant substances with lipids (Rhee 1999). The prooxidant properties of salt in emulsions are likely affected by its

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capability to increase the contact of free iron ions with the lipid phase (Zhou et al. 2021; Liu et al. 2019; Cui et al. 2018) and to increase the catalytic activity of iron ions (McClements and Decker 2000). The prooxidant activity of salt on lipid oxidation of chicken meat was reported by Bragagnolo et al. (2006) (3% NaCl, an EPR study, high hydrostatic pressure for 5 to 30 min); Bragagnolo et al. (2005) (0.5% NaCl, TBARS, determination of pentanal and hexanal levels); Mariutti et al. (2011) (determination of pentanal and hexanal levels, fatty acid composition, cholesterol oxides, storage at –18° C for 90 days); and Gheisari and Motamedi (2010) (2.5% NaCl, TBARS, PV, storage at 4° C for four days). Lin et al. (2015) reported the prooxidant effect of salt in beef (0.5 and 2% NaCl, TBARS, determination of pentanal, hexanal, octanal, and nonanal levels, storage at 4° C for seven days), and Overholt et al. (2016) for pork (1.5% NaCl, TBARS, storage at 4° C for 11 days). According to Shimizu et al. (2009), the addition of a mole of NaCl to yellowtail fsh muscle tissue demonstrated a prooxidant effect. After 20 weeks of storage at –20° C, the mean content of malonaldehyde in the control fsh sample was 2.96 µmol/g tissue, whereas in fsh with 0.3, 0.6, and 0.9 mol of added salt, the malonaldehyde levels were found to be 5.59, 9.73, and 8.42 µmol/g tissue, respectively. For the same fsh species, a similar prooxidant effect for the addition of 0.6 and 0.9 mol of salt was observed after storage at 0° C for seven days (Sakai et al. 2003). The authors reported inhibition of superoxide dismutase activity due to the addition of 0.3, 0.6, and 0.9 mol of salt to the fsh, thereby increasing the extent of oxidation. According to Zhou et al. (2021), the content of hydroperoxides and TBARS in soybean oil bodies’ emulsions containing salt were elevated, indicating that the incorporation of the salt signifcantly reduced the system’s oxidative stability. This prooxidant effect was reported for emulsions at both pH 7 and 3, and for 0.4, 0.8, and 1.2% NaCl contents. The levels of hydroperoxides and TBARS for the systems at pH 7 were greater than those at 3. According to the researchers, the negative surface charge of soybean oil bodies drops at pH 7 and can therefore attract transition-metal ions to come in contact with the lipid phase. The addition of salt (0.4, 0.8, 1.2%) to an oil-in-water emulsion stabilized by Tween 60 promoted lipid oxidation (Liu et al. 2019), as assessed by increased PV and TBARS values. In the authors’ opinion, the salt ions were able to change the arrangement of surface-active molecules on the droplet surface, which affected the contact of the prooxidant substances with the lipid phase. It was noted that NaCl and KCl addition (at 1.0 and 1.6% levels) to an oil-in-water emulsion, stabilized by lecithin, acted prooxidatively, likely due to more iron ions being concentrated at the proximity of the droplet surface (Cui et al. 2018).

11.10

ANTIOXIDANT ACTIVITY AND ITS MEASUREMENT

There are many in vitro assays available to assess the antioxidant activity in foods. First, however, the antioxidant constituents need to be collected in the form of an extract before colorimetric assays can be performed. Phenolic antioxidant assessment assays tend to be grouped according to the chemistry of the reactions involved; that is, it may specifcally pertain to the mechanisms of either HAT (hydrogen atom

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TABLE 11.2 Chemical Methods Used for Determining Antioxidant Activity Assay

Radical/ion measured

HAT assays Azo-initiated chemiluminescence (CL) Photochemiluminescence (PCL)

RO2● O2●‒

Total antioxidant reactivity (TAR) ORACFL

RO2● RO2●

TRAP

RO2●

Crocin or β-carotene bleaching assays UV-Vis

RO2●

Microtiter plate Total oxyradical-scavenging capacity (TOSC) Liposome model systems Low-density lipoprotein (LDL) oxidation models SET assays Cupric reducing antioxidant capacity (CUPRAC) UV-Vis Microtiter plate Ferric-reducing antioxidant power (FRAP) UV-Vis Microtiter plate Mixed-mode assays TEAC UV-Vis Microtiter plate DPPH● UV-Vis Microtiter plate Quantifcation assay Total phenolics content (TPC) UV-Vis Microtiter plate Source: Craft et al. 2012.

RO2● and ●OH RO2● and ●OH RO2● and ●OH

Descriptive reference Alho and Leinonen (1999) Popov and Lewin (1999); Pegg et al. (2007) Campos et al. (1996); Lissi et al. (1995) Huang et al. (2002); Prior et al. (2003); Wu et al. (2004) Wayner et al. (1985); Wayner (1987); Lussignoli et al. (1999) Miller (1971); Kampa et al. (2002); Tanizawa et al. (1983); Tubaro et al. (1998) Mikami et al. (2009) Regoli and Winston (1999); Winston et al. (1998) Roberts and Gordon (2003) Esterbauer (1993); Frankel et al. (1995)

Cu2+  Cu+ [complexed] Apak et al. (2004); Moffet et al. (1985) Ribeiro et al. (2011) Fe3+−TPTZ  Fe2+−TPTZ Benzie and Strain (1996); Pulido et al. (2000); Amarowicz et al. (2004) Firuzi et al. (2005) ABTS●+ Miller et al. (1993); Re et al. (1999) Kambayashi et al. (2009) DPPH● Hatano et al. (1988); Sánchez-Moreno et al. (1998) Fukumoto and Mazza (2000) Mo6+[yellow]  Mo5+[blue] Singleton and Rossi (1965); Folin and Ciocalteu (1927); Singleton et al. (1999) Zhang et al. (2006)

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transfer) or SET (single electron transfer), or be a mixed-mode method pertaining to both (Schaich 2006). Consequently, more than one antioxidant capacity assay should be carried out to evaluate the antioxidant potential of bioactives (e.g., phenolics) in food based on the different mechanisms of antioxidation. Examples of some common in vitro assays used to evaluate the antioxidant activity of, say, a phenolic extract from a food, include the following: DPPH• scavenging activity; ferric reducing antioxidant power (FRAP); ABTS•+ scavenging activity; O2•‒ scavenging activity; reducing power; oxygen radical absorbance capacity (ORACFL); Trolox equivalent antioxidant capacity (TEAC); peroxyl radical (ROO•) scavenging capacity (PSC); cupric reducing antioxidant capacity (CUPRAC); photochemiluminescence (PCL); and coupled oxidation of β-carotene/linoleate. The total phenolics content (TPC) assay is also of importance, as it is typically employed to quantify inherent phenol content, but in fact, is a colorimetric assay related to the redox potential of antioxidant constituents. Examples of some of the antioxidant assays listed above are organized into categories in Table 11.2 based on their mode of action. Craft et al. (2012) and Munteanu and Apetrei (2021) provide more details about the chemistry of these in vitro assays in their respective review articles on assessing antioxidant activity. As Craft et al. (2012) pointed out, one should not expect the results of antioxidant contents or capacity assessed by a HAT assay to be compatible (either quantitatively or qualitatively) with those obtained by a SET assay. The various mechanisms of measurement may present different determinations of antioxidant activity according to the antioxidant composition of the sample in question. As an illustration, Ou et al. (2001) noted that the antioxidant capacities of foods rich in ascorbic acid are underrepresented when the ORACFL assay is performed. Another explanation is that different foods, when analyzed, may enact interferences that have varying magnitudes of effect upon different assays. To this end, Prior et al. (2005) proposed that assessments of SET reactions may be more sensitive to potential interferences than those of HAT reactions. This is because SET reactions often take long periods to reach completion; consequently, interfering substances (such as trace components and metal-ion contaminants) can exert a more pronounced effect on their measurement. Nevertheless, each assay – be it HAT, SET, or mixed – involves the correlation of an antioxidant’s capability to perform in relation to a standard antioxidant compound.

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Verma, B., Hucl, P., and Chibbar, R.N. Phenolic content and antioxidant properties of bran in 51 wheat cultivars. Cereal Chem., 85(4), 544, 2008. Vidot, K., Maury, C., Siret, R., and Lahaye, M. Phenolic compounds limit or promote oxidative degradation of pectin related to iron-H2O2 ratio. LWT Food Sci. Technol., 125, 109324, 2020. Waller, S.B., Madrid, I.M., Hoffmann, J.F., Picoli, T., Cleff, M.B., Chaves, F.C., de Faria, R.O., Meireles, M.C.A.M., and de Mello, J.R.B. Chemical composition and cytotoxicity of extracts of marjoram and rosemary and their activity against Sporothrix brasiliensis. J. Med. Microbiol., 66(7), 1076, 2017. Wang, X., Zheng, X., Kopparapu, N., Cong, W., Deng, Y., Sun, X., and Liu, X. Purifcation and evaluation of a novel antioxidant peptide from corn protein hydrolysate. Process Biochem., 49(9), 1562, 2014. Wayner, D.D.M. 1049 – radical-trapping antioxidants in vitro and in vivo. Bioelectroch Bioener, 18(1–3), 219, 1987. Wayner, D.D.M., Burton, G.W., Ingold, K.U., and Locke, S. Quantitative measurement of the total, peroxyl radical-trapping antioxidant capability of human blood plasma by controlled peroxidation. FEBS Lett., 187(1), 33, 1985. Weidner, S., Amarowicz, R., Karamać, M., and Dąbrowski, G. Phenolic acids in caryopses of two cultivars of wheat, rye and triticale that display different resistance to preharvest sprouting. Eur. Food Res. Technol., 210(2), 109, 1999. Wijesundera, C., Ceccato, C., Fagan, P., and Shen, Z. Seed roasting improves the oxidative stability of canola (B. napus) and mustard (B. juncea) seed oils. Eur. J. Lipid Sci. Technol., 110(4), 360, 2008. Winston, G.W., Regoli, F., Dugas, A.J. Jr., Fong, J.H., and Blanchard, K.A. A rapid gas chromatographic assay for determining oxyradical scavenging capacity of antioxidants and biological fuids. Free Radic. Biol. Med., 24(3), 480, 1998. Wu, X., Gu, L., Holden, J., Haytowitz, D.B., Gebbhardt, S.E., Beecher, G., and Prior, R.L. Development of a database for total antioxidant capacity in foods: A preliminary study. J. Food Compos. Anal., 17(3–4), 407, 2004. Yamamoto, Y. Role of active oxygen species and antioxidants in photoaging. J. Dermatol. Sci., 27, S1, 2001. Yoshida, H. Infuence of fatty acids of different unsaturation in the oxidation of purifed vegetable oils during microwave irradiation. J. Sci. Am. Oil Chem. Soc., 62(1), 41, 1993. Yu, Z., Lu, L., Lu, L., Pan, L., Qiu, X., and Tang, Y. Development and antioxidation of metal ion chelating packaging flm. Food Pack. Shelf Life, 32, 100846, 2022. Yuan, S., Li, X., Jin, Y., and Lu, J. Chocolate consumption and risk of coronary heart disease, stroke, and diabetes: A meta-analysis of prospective studies. Nutrients, 9(7), 688, 2017. Zeb, A. Effects of β-carotene on the thermal oxidation of fatty acids. Afr. J. Biotechnol., 10(68), 15346, 2011. Zeb, A., and Murkovic, M. Characterization of the effects of β-carotene on the thermal oxidation of triacylglycerols using HPLC-ESI-MS. Eur. J. Lipid Sci. Technol., 112(11), 1218, 2010. Zeb, A., and Murkovic, M. Carotenoids and triacylglycerols interactions during thermal oxidation of refned olive oil. Food Chem., 127(4), 1584, 2011. Zeb, A., and Murkovic, M. Pro-oxidant effects of β-carotene during thermal oxidation of edible oils. J. Am. Oil Chem. Soc., 90(6), 881, 2013. Zhang, Q., Zhang, J., Shen, J., Silva, A., Dennis, D.A., and Barrow, C.J. A simple 96-well microplate method for estimation of total polyphenol content in seaweeds. J. Appl. Phycol., 18(3–5), 445, 2006.

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Zhang, T., Li, Y., Miao, M., and Jiang, B. Purifcation and characterisation of a new antioxidant peptide from chickpea (Cicer arietium L.) protein hydrolysates. Food Chem., 128(1), 28, 2011. Zhou, L., Xie, M., Yang, F., and Liu, J. Antioxidant activity of high purity blueberry anthocyanins and the effects on human intestinal microbiota. LWT Food Sci. Technol., 117, 108621, 2020. Zhou, X., Liu, Z., Wang, W., Miao, Y., Gu, L., Li, Y., Liu, X., Jiang, L., Hou, J., and Jiang, Z. NaCl induces focculation and lipid oxidation of soybean oil body emulsions recovered by neutral aqueous extraction. J. Sci. Food Agric., 102(9), 3752, 2021. Żyżelewicz, D., Krysiak, W., Oracz, J., Sosnowska, D., Budryn, G., and Nebesny, E. The infuence of the roasting process conditions on the polyphenol content in cocoa beans, nibs and chocolates. Food Res. Int., 89, 918, 2016.

12

Food Allergens Barbara Wróblewska

CONTENTS 12.1 12.2 12.3 12.4 12.5

Nomenclature of Allergens...........................................................................340 Causes of Food Allergy ................................................................................340 Mechanisms of the Allergic Reaction to Food ............................................. 341 Symptoms and Health Hazards .................................................................... 342 Allergens of Animal and Plant Origin ......................................................... 343 12.5.1 Allergenic Protein Families of Animal Origin ................................ 343 12.5.2 Main Allergens of Animal Origin....................................................344 12.5.2.1 Cow’s Milk Allergens ........................................................344 12.5.2.2 Egg Allergens..................................................................... 345 12.5.2.3 Fish Allergens .................................................................... 345 12.5.2.4 Crustacean Allergens.........................................................346 12.5.2.5 Mollusk Allergens..............................................................346 12.5.3 Protein Families of Plant Allergens..................................................346 12.5.3.1 Prolamin Superfamily........................................................ 347 12.5.3.2 Cupin Superfamily.............................................................348 12.5.3.3 Proflins Superfamily.........................................................348 12.5.3.4 PR-10 Proteins.................................................................... 349 12.5.4 Main Allergens of Plant Origin ........................................................ 349 12.5.4.1 Peanut Allergens ................................................................ 349 12.5.4.2 Soy Allergens..................................................................... 350 12.5.4.3 Nut Allergens ..................................................................... 351 12.5.4.4 Wheat Allergens ................................................................ 352 12.5.4.5 Mustard .............................................................................. 352 12.5.4.6 Sesame ............................................................................... 353 12.5.4.7 Celery................................................................................. 353 12.5.4.8 Lupine ................................................................................ 353 12.6 Methods for Allergen Determination ........................................................... 353 12.6.1 ELISA (Enzyme-Linked Immunosorbent Assay) ............................ 353 12.6.2 Methods Based on DNA Analysis .................................................... 354 12.6.3 Methods Using Mass Spectroscopy.................................................. 354 12.6.4 Biosensors......................................................................................... 354 12.7 Effects of Technological Processes on Food Allergens................................ 355 12.7.1 Thermal Processes............................................................................ 355 12.7.2 Glycation........................................................................................... 355 12.7.3 Lactic Fermentation.......................................................................... 356 12.7.4 Enzymatic Modifcations.................................................................. 356 DOI: 10.1201/9781003265955-12

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12.7.5 Cross-Linking with Transglutaminase ............................................. 357 12.7.6 Pressurization ................................................................................... 357 12.7.7 Ultrasound ........................................................................................ 358 12.8 Prevention of Food Allergy .......................................................................... 359 References.............................................................................................................. 359

12.1 NOMENCLATURE OF ALLERGENS Allergens are most frequently proteins or glycoproteins that induce IgE-mediated reactions in humans. A system of naming allergens was established in 1986 by experts grouped in the Allergen Nomenclature Subcommittee (WHO/IUIS) to help researchers, clinicians, pharmaceutical companies, regulators, and the public properly understand the identity of allergens important for a clinical diagnosis and to ensure compliance for improved safety (Chan et al., 2019). The frst three letters of an allergen are given according to the taxonomic name of the species from which it has been extracted. Next, there is the frst letter of the kind of a given species and an Arabic numeral based on the order of registration in the database. The isoallergens are designated by the addition of two digits after the decimal in the number, and isoforms or variants by the addition of two more digits (e.g. Amb a 1.0101). Since the evolutionary steps involved in generating each new isoform or variant are usually not known, the Committee bases designations on the percent identity of amino acids compared to the frst identifed sequence. Sequences within ∼67% identity to the original allergen are designated as isoallergens, and sequences differing by < 90% identity are isoforms or variants (Chan et al., 2019). Since there is a large number of already registered allergens, in the case a given name is identical to the one already existing, it is necessary to add another letter to the frst part of the name and to establish whether the chemical compound to obtain the status of “allergen” is a sensitizing agent inducing the clinical manifestations in allergic patients. Labeling an allergen as “dominating” or “less signifcant” depends on the number of patients in the studied populations identifed with IgE antibodies specifc to the compound tested. The information concerning allergens is collected and made available in an online database (www.allergen.org). The basic criterion for entering a new allergen into the database is to identify at least fve IgE-dependent patients with a confrmed allergy to a new agent (Pomesa et al., 2018).

12.2 CAUSES OF FOOD ALLERGY Development of food allergy is combined with environmental changes, increased urbanization, global warming, decreased exposure to early-life infections, and lifestyle changes, as well as short breastfeeding period, diet, and early-life exposure to a source of strong food allergens, e.g. peanuts. Food allergy and eczema usually start in the frst year of life initiating the allergic march. Usually, allergic asthma and

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allergic rhinitis induced by exposure to indoor (house dust mites, pets, molds, and cockroaches) and outdoor (mainly pollen) allergens lead to a further increase in food allergies based on IgE cross-reactivity (van Ree, 2021). Current observations point out that sensitization via skin may play an important role in the development of food allergy. The breakdown of the skin barrier in atopic dermatitis can result in epicutaneous sensitization to foods resulting in food allergy. Therefore, flaggrin mutations are perceived as a trigger for promoting the allergic disease and consequent development of food allergy. Numerous other factors, including vitamin D defciency and obesity, have been associated with food allergy, yet these require further exploration (Patel and Volcheck, 2015). Genetic factors are estimated to be responsible for ca. 50% of all allergy cases. The probability of inheriting an allergy when both parents are allergic is 60–70%; when only one parent is allergic, it is 30–40%. There is also a 10–15% likelihood that a child of non-allergic parents will be allergic. The increase in food allergy incidence is related to non-specifc factors of an adjuvant character present in the environment. These comprise ozone, formic aldehyde, SO2, NO, NO2, CO2, tobacco smoke, and household chemicals. They have an irritating effect on the respiratory tract and conjunctival mucosa, which weakens the immune system of the whole body.

12.3

MECHANISMS OF THE ALLERGIC REACTION TO FOOD

There are four basic types of mechanisms that induce allergy: • • • •

type I-anaphylactic and atopic reactions, type II-cytotoxic and cytolytic reactions, type III-Arthus reactions induced by immunological complexes, type IV-delayed cell reaction.

Food allergies are most often caused by the type I mechanism, which refers to an allergic reaction occurring immediately after contact with the allergen (Hugh et al., 2018). Sensitization to food antigens may take place in the gastrointestinal tract, oral cavity, and skin, and occasionally in the respiratory tract (Sampson et al., 2018). In the stomach and intestine, protein allergens are hydrolyzed to peptides and amino acids, which are recognized by the immunological system. Some parts of the antigens remain intact food proteins. So far, no threshold value for a molecular weight has been established that would be decisive for the allergenic character of the compound formed. Digestive content is transported to the mucosa through gut epithelial cells (ECs) and specialized ECs called M cells localized above the Peyer’s patches. Mucosal dendritic cells (DCs) extend dendrites into the gut lumen to uptake the ingested food moieties. After internalization, they are moved to T cell areas of draining lymph nodes, where DCs can interact with naive T cells and present antigens on MHC class II molecules. Several factors play a role in the development of TH2 polarization. IL-4 cytokine secreted from innate lymphoid cells (ILCs), basophils,

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and natural killer T cells is a major player in the further development of type II immune responses. IgE is a fundamental antibody in patients with atopic diseases and a hallmark of allergic sensitization. TH2 lymphocytes and associated cytokines support B-cell proliferation, promote immunoglobulin isotype class-switch recombination toward IgE, and drive their differentiation into antibody-secreting plasma cells. IgE mediates immediate-phase reactions by inducing mast cell and basophil degranulation. After sensitization and re-exposure to the allergen, mast cell–derived mediators such as histamine, prostaglandin, and proteases change the bioavailability of food proteins, whereas ECs upregulate their secretion of TH2-promoting mediators, including thymic stromal lymphopoietin, IL-25, and IL-33 (Sampson et al., 2018). As a result of such reactions, itching rashes, erythema, urticaria, papular rashes, or other forms of allergic response occur. Plant and animal proteins are very diverse in terms of digestibility and epithelial transport. Some plant proteins are rapidly degraded by pepsin (proflins and PR-10), which leads to a reduced IgE-binding capacity. Others, like 2S albumins, nsLTP, ATIs, cereal prolamins, legumins, and vicilins, are resistant to proteolysis and only partially degraded during gastrointestinal digestion. Certain proteins (2S albumins, nsLTP, cereal prolamins, and vicilins) are able to cross the epithelium barrier in a native state, causing an increase in their allergenicity (Costa et al., 2020). Animal allergens (caseins, serum albumins, lipocalins, and serpins) are pepsinresistant. Most animal allergens have a lower IgE-binding capacity after digestion; however, there are some exceptions. In caseins, the aggregates formed can preserve and/or increase immunoreactivity. In parvalbumins, amyloid fbers increase the IgEbinding capacity. In transferrins, the partial protective effect of matrix components preserves their IgE-binding capacity. In lipocalins, the formation of aggregates hampers digestion, changing the mechanism of transport across the epithelium barrier and increasing its allergenicity (Costa et al., 2021).

12.4 SYMPTOMS AND HEALTH HAZARDS Allergic reactions to food are associated with a wide range of symptoms, which can affect the entire body – the skin, the gastrointestinal and respiratory tract, and the cardiovascular system. Common skin reactions include urticaria, angioedema, and erythema. As to the respiratory tract, the adverse symptoms include laryngeal edema, nasal swelling, and bronchospasm. The gastrointestinal tract–related symptoms include nausea, vomiting, abdominal pain, and diarrhea. The IgE-mediated reaction may be limited to local infammation such as oral allergy, which is known as a pollen-food syndrome causing tingling and itching of the mouth and throat. Most often these symptoms are triggered after eating certain fresh fruits and vegetables due to cross-reactions. A common example concerns patients suffering from birch pollen allergy, who may also develop the aforementioned symptoms after eating raw carrots, celery, or apples. The most serious reaction is anaphylaxis, which is very rapid and violent and can be fatal without immediate pharmacological help (Boyce et al., 2010, Anagnostou and Turner, 2019).

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ALLERGENS OF ANIMAL AND PLANT ORIGIN ALLERGENIC PROTEIN FAMILIES OF ANIMAL ORIGIN

Due to the nature of their structure, allergenic animal proteins are classifed into the following families (Hoffmann-Sommergruber and Mills, 2009; Costa et al., 2020). Tropomyosin has been identifed in crustaceans, mollusks, and fsh parasites, Anisakis simplex, as well as in mites and cockroaches. Strong allergenic activity with a high degree of cross-reactivity was found among proteins present in the invertebrate family, where they are considered panallergens – universal proteins responsible for IgE cross-reactivity of related and unrelated allergens. EF-hand domain proteins are calcium ion-binding proteins. All identifed proteins of this family have a characteristic structure of a 12-amino-acid loop and two adjacent 12-amino-acid helixes, which bind calcium ions. The role of these proteins is to induce and transduce “calcium” signals in cellular processes, transport calcium ions, and buffer calcium levels in the cytoplasm. The prototype protein with EF-hand domains is calmodulin, while the other known proteins are parvalbumin, calbindin, and troponin. Parvalbumins of fsh (cod and carp) are unstable as allergens under acidic conditions and degrade during digestion in the stomach. α-parvalbumins are generally not considered allergenic due to their proximity to human homologs. However, some of them were identifed as food allergens in frog (Ran e 1), chicken (Gal d 8), and crocodile (Cro p 2) meat. β-parvalbumins are classifed as complete food allergens. They are both food and respiratory allergens because they can induce sensitization by inhalation during food handling and processing (Costa et al., 2021). Arginine kinases are proteins that have a molecular weight of 40–45 kDa with two polypeptides of 355–357 amino acids organized in an asymmetric monomeric structure. They are found in seafood, cockroaches, and mites. Allergic patients may exhibit systemic symptoms or anaphylaxis. Caseins are proteins present in the milk of all mammals. They have the ability to bind calcium through a phosphoserine-phosphothreonine cluster. Caseins have an amorphous structure. By binding calcium, they increase its level in milk. The different fractions of casein have different functions: casein stabilizes the nanostructure, while S1-casein and S2-casein are the strongest allergens among milk proteins. Caseins are considered major allergens responsible for the development of mild to severe allergic reactions in sensitized individuals and are usually the frst foreign proteins in a neonate’s diet. Other animal protein families include the following: Serum albumins are abundant in the plasma and play various biological functions, including the transport of different molecules such as water, cations (Ca2+/ Na+/K+), fatty acids, hormones, bilirubin, and drugs. They are also involved in the regulation of colloid osmotic pressure in the blood. Their molecules of 60–69 kDa weight and immature primary sequences of 607–608 amino acids are present in the dander, skin, saliva, milk, and meat of different animal species. Lipocalins are small extracellular proteins with 150–250 residues and 17–25 kDa. Their β-barrel structure is stabilized by two disulfde bonds, and depending on the pH can form monomers, dimers, or higher-order oligomers. Proteins that

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belong to lipocalins are abundant in epithelial mucosa and skin, as well as body fuids and secretions. The main representative of the family is β-lactoglobulin. The 3D structure is similar to other proteins of the family, but the sequence similarity is low. These proteins are carriers of lipid molecules, steroids, hormones, and retinoids. Transferrins are glycoproteins rich in sulfur and binding iron. They exhibit antibacterial activity in their iron-free form. Examples are lactoferrin in cow’s milk and egg ovotransferrin. Gal d 3, egg white protein, is classifed as a minor allergen, with the clinical symptoms being mostly associated with urticaria and angioedema. Glycoside hydrolase family 22 is a group of enzymes that catalyze the hydrolysis of a glycosidic bond between carbohydrates, or between a carbohydrate and a noncarbohydrate moiety. The two main food allergens that belong to this group are Gal d 4 (lysozyme C) from hen’s egg and Bos d 4 (α-lactalbumin) from cow’s milk. Allergens from this group are responsible for eliciting respiratory, cutaneous, and gastrointestinal symptoms, and often anaphylaxis in milk-allergic individuals. Inhibitory Kazal-type proteins, including avian ovomucoid, pancreatic secretory trypsin inhibitor, acrosin inhibitor, and elastase inhibitor, have the main biological activity associated with the suppression of several serine proteases. The main allergenic representative of this group is egg ovomucoid. Clinical symptoms of allergic patients are atopic eczema, urticaria, or vomiting. Serpins play a biological role mainly through protease inhibitory activity and control of proteolytic cascades. They are hormone transporters, molecular chaperones, and tumor suppressors. The residual ovalbumin (Gal d 2), a strong allergen of chicken egg white present in infuenza vaccines, can cause reactions in egg-allergic patients.

12.5.2 MAIN ALLERGENS OF ANIMAL ORIGIN 12.5.2.1 Cow’s Milk Allergens Patients allergic to casein (Bos d 8) are usually sensitive to the four basic fractions of this protein: αS1- (Bos d 9), αS2- (Bos d 10), β- (Bos d 11), and κ- (Bos d 12) caseins, representing 40%, 12.5%, 35%, and 12.5% of the casein fraction in milk, respectively. The body’s immunological defense, expressed by the formation of anti-casein IgE, is linked to the presence of homologous amino acid sequences in individual epitopes, which react in a cross-reactive manner. One of the immunoreactive and digestionresistant epitopes is the phosphorylation site present within S1-, S2-, and casein. Three casein peptide sequences (AA: 19–30, 86–103, 141–50) that reacted with the sera of cow’s milk allergy patients have been characterized. These peptides were located in the hydrophobic region of the molecule and were only accessible to the antibodies after casein denaturation. Of the whey proteins, the most potent allergens are β-lactoglobulin (β-lg), Bos d 5, and α-lactalbumin (α-la), Bos d 4. The β-lg protein is found in cow’s milk in two genetic forms, i.e. A and B, differing by mutations at positions 64 and 118. Form A contains aspartic acid and valine, while form B contains glycine and alanine. There are two disulfde bridges and three free thiol groups in the molecule. This

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structure allows for interactions with casein during thermal processes. Lactoglobulin is relatively resistant to acid hydrolysis and protease action and therefore remains largely unhydrolyzed during the transport through the gastrointestinal tract, mainly the intestinal mucosa. The allergenicity of β-lg results from the presence of numerous epitopes, visualized by trypsinization, being the amino acid fragments AA102–124, AA41–60, and AA149–162 identifed in about 90% of patients, AA1–8, AA25–40, and AA92–100 recognized in 58–72% patients, and AA9–14, AA84–91, and AA92– 100 found in 40% of patients. Administration of low doses of antigen, e.g. in the form of selected hydrolyzed β-lg fractions, can induce tolerance. The α-la protein from cow’s milk shows a high similarity (ca. 72%) to α-la of human origin. A stronger antigenic region is the amino acid loop located between residues (60–80):S–S:(91–96). Amino acid sequences with the ability to bind IgE are also located in the hydrophobic region of α-la molecule, i.e. between amino acids 99 and 108, as well as in the amino acid sequence region 17–58 and 108–123. Epitope mapping revealed the presence of four linear IgE-binding epitopes that overlap with three IgG-reactive regions. 12.5.2.2 Egg Allergens Most allergies to eggs affect children between four and fve years of age. The egg white portion (about 10%) is the main source of allergens, of which the most allergenic proteins are ovotransferrin, ovomucoid, ovalbumin, and lysozyme. Usually, 100% of chicken egg–sensitized patients tested show a positive reaction to ovalbumin. Data on the allergenicity of lysozyme are inconclusive. Allergens isolated from egg yolk are the lipoprotein fraction apovitellin and α-livitellin, which can sensitize via the respiratory tract. Allergens of secondary immunological relevance include ovomucin and fosvitin. Egg allergy can also be an occupational disease of workers employed in the sector of egg processing. Therefore, it is recommended to perform environmental monitoring of egg allergen concentrations in the processing plants, on the processing lines, and in the adjacent rooms. 12.5.2.3 Fish Allergens Fish allergies played a historic role in the pioneering research into so-called “abnormal” reactions. A protein isolated from fsh muscle tissue was used by Prausnitz and Kustner to prove the existence of a factor in the blood serum which many years later was defned as immunoglobulin of the IgE class. This protein was the frst allergen to have its amino acid sequence described. The allergen was named Cod M, after the English “cod,” thus initiating a new nomenclature for allergens. In later systematics, the name was changed to Gad c 1 (Gadus – the frst part of cod’s Latin name) and classifed as parvalbumin. Proteins that belong to this group control the movement of calcium ions in and out of the cell. They were found in the muscles of fsh (0.05–0.1%) and reptiles. The presence of a protein similar in structure to Gad c 1 was confrmed in the muscle tissue of carp and pike. The molecular weight of Gad c 1 allergen is 12.3 kDa. The allergen is composed of 113 amino acid residues and one glucose residue. At least fve IgE-binding sites were found in the structure

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of Gad c 1, with the arginine residue at position 75 playing the dominant role. The glucose residue, located next to Cys-18, had no effect on the allergenicity of the whole compound. Trypsin hydrolysis revealed a very active allergenic (AA: 33–44) and a weaker (AA: 88–96) site. Less important fsh allergens include a protein with a molecular weight of 63 kDa and an aldehyde-phosphate dehydrogenase homolog with a molecular weight of 41 kDa. 12.5.2.4 Crustacean Allergens Shrimp allergens are among the best characterized allergens. The frst of these, Antigens I and II, were isolated and described by Hoffman et al. (1981). The antigen is a dimer with a molecular weight of 45 kDa, composed of 189 amino acid residues and 0.5% saccharides. It was isolated from both raw shrimps and chitin-lime carapace. Antigen II was extracted from cooked shrimps as an acidic, heat-stable glycoprotein with a molecular weight of 38 kDa, composed of 341 amino acid residues and 4% saccharides. In a later study, two further allergens, SA-I (m.w. 8.2 kDa) and SA-II (m.w. 34 kDa), were characterized. In 1992, allergens Pen a 1 (m.w. 36 kDa) and Pen i 1 (m.w. 34 kDa) were isolated from cooked prawns (Panaeus aztecus) and Indian white prawn (Panaeus indicus), respectively. By comparing the amino acid composition of Pen a 1, Antigen II, and Sa-II allergens, they were found to be signifcantly similar to each other and classifed as tropomyosin. It has been estimated that ingestion of one or two medium-sized shrimps is capable of stimulating the anaphylactic reaction in allergic individuals. Tropomyosin was described as an allergen of the crawfsh (Panulirus stimpsoni), Pan s 1, and the American lobster (Homarus americanus), Hom a 1. Both of these proteins were cloned, had their sequences examined, and were found to be homologous to the shrimp allergen Pen a 1. Hypersensitivity to crab allergens has been observed mainly in people professionally involved in shellfsh processing. IgE-mediated reactions are mainly triggered by contact with extracts obtained during crab cooking rather than with the raw material. Crab allergens are proteins with molecular weight in the range of 37–42 kDa. 12.5.2.5 Mollusk Allergens The group of edible mollusks is made up of three classes: cephalopods (squid, cuttlefsh, and octopus), bivalves (mussels, clams, razorfsh, winkles, oysters, and scallops), and gastropods (limpets, snails, and abalone). The prevalence of allergic causes in mollusks is estimated at approx. 0.4% levels (in French children, 0.15%, in Spain, 1.6% of food allergies). Tropomyosin is perceived as a major allergen, being responsible for a high cross-reactivity between mollusks, mites, and crustaceans. Lately, new mollusk allergens have been identifed, i.e. actin, enolase, and a putative C1q domain–containing protein (Azofra et al., 2017).

12.5.3 PROTEIN FAMILIES OF PLANT ALLERGENS Due to their diversity, plant-derived food has been organized into several families. They share common structural features such as numerous disulfde bonds or oligomeric structures. Representatives of plant-based allergens have in their structure six or

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eight cysteine residues with Cys Cys and Cys X Cys motifs (X representing every other amino acid), with two additional fragments with the structure of α-amylase and trypsin inhibitors. The structure of prolamin proteins contains an insert repeat domain rich in proline and glutamine residues. Proteins of families with known 3D structures (LTPs, 2S albumin, and α-amylase and protease inhibitors) have mutually similar folds containing packets of four α-helices stabilized by disulfde bonds. Some plant food allergens cross-react with pollen allergens. The incidence and clinical activity of plant allergens are infuenced by the geographical distribution of specifc plant allergen families. PR-10 proteins are very common in the northern part of Europe and in the Alpine regions, while nsLTP affects the Mediterranean population. 12.5.3.1 Prolamin Superfamily Members of this superfamily have a large amount of proline and glutamine residues and share a highly conserved pattern of eight cysteine residues that stabilize their three-dimensional (3D) structure of four α-helices, which form a right-handed super-helix. Prolamine storage proteins rarely cause IgE-mediated reactions. The most common are atopic dermatoses or anaphylaxis co-existing with the ingestion of cereal proteins and simultaneous strenuous exercise: food-dependent, exerciseinduced anaphylaxis (FDEIA). The prolamine superfamily consists of four seed-storage protein families. 2S albumins are found in tree nuts, legumes, cereals, and peanuts, e.g. Sin a 1 (mustard), Ber e 1 (Brazil nut), Jug r 1 (walnut), Ara h 2, and Ara h 6 (peanut). They are monomeric proteins with a molecular weight of 10–18 kDa, usually based on two polypeptide chains connected by disulfde bonds.

Non-specifc lipid transport proteins (nsLTPs) play an important role in protecting plants from fungi and bacteria. The majority of nsLTPs are extracellular proteins located in epidermal tissues surrounding the aerial organs (leaves, fruits, stems). Consequently, a higher allergenic potency is attributed to the peels rather than to the pulps of Rosaceae fruits. nsLTPs are also present in nuts, seeds, vegetables, pollen, and latex. The biological role of nsLTPs is to transport phospholipids between cell membranes. Cereal α-amylase trypsin inhibitors (ATIs) are present in the endosperm of cereal seeds: wheat, barley, rye, corn, and rice. Their biological functions are connected with plant defense mechanisms against parasites, insects, mites, and mammalians. ATIs have a molecular weight of 12–16 kDa. There are four or fve disulfde bonds in their structure that support their inhibitory activity. The major wheat ATI allergens are Tri a 28, Tri a 15, and Tri a 30. ATIs can infuence the organism through inhalation or ingestion, causing occupational allergies like baker’s asthma or atopic dermatitis in children. Tri a 30 (CM3) and Tri a 40 (subunit CM16) were indicated as wheatdependent exercise-induced anaphylaxis (WDEIA) allergens. Cereal prolamins are the major storage proteins of the endosperm of cereal grains, classifed as glutenins and gliadins in wheat, secalins in rye, and hordeins in barley.

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Due to their solubility in alcohol-water solutions, they have been divided into two groups: gliadins (soluble proteins) and glutenins (insoluble proteins). Glutenins are divided into high (HMW) and low molecular weight (LMW) subunits, while the gliadin fraction consists of three types of proteins, namely α/β-, γ-, and ω-gliadins. These cereal prolamins differ in their methionine-cysteine contents and are categorized in sulfur-poor (S-poor) or sulfur-rich (S-rich) groups. In wheat-allergic patients, gliadins (Tri a 19, Tri a 20, and Tri a 21) and glutenins (Tri a 26 and Tri a 36) were found to trigger such clinical symptoms as urticaria, angioedema, erythema, vomiting, persistent cough, respiratory distress, and in the most severe cases, anaphylaxis. The ω-gliadins, and notably ω-5 (Tri a 19), are mainly associated with WDEIA in adults. 12.5.3.2 Cupin Superfamily Proteins of this family have a characteristic β-barrel structure. Cupins have evolved from prokaryotic ancestors to proteins present in fungi, higher plants, and animals. Cupins differ in their structure. Cupins with a single barrel domain are allergens identifed in peppers and oranges. Bicupins contain two β-barrel domains, which are the major components of seeds, legumes, and tree nuts. Based on their sedimentation coeffcient, globulins are classifed as 7/8S (vicilins) and 11S (legumins), which are found in dicotyledonous plants as a source of protein with a high nutritional value. In the cupin superfamily, the following are distinguished: • Vicilins – trimers with a molecular weight of 150–190 kDa, with two subunits in the range of 40–80 kDa, and with a typical subunit of ~50 kDa. Vicilins present two β-barrel core domains, stabilized by non-covalent hydrophobic interactions, hydrogen bonds, and van der Waals interactions. As allergens, they are found in peanuts (Ara h 1), lupine (Lup an 1), peas (Pis s 1), tree nuts (e.g. hazelnut, Cor a 11; walnut, Jug r 2; pistachio, Pis v 3), and Goji berries. • Legumes – multimeric proteins with quaternary structures (360 kDa) as hexamers or as a mix of trimers and hexamers linked by non-covalent interactions. As allergens, they prevail in peanuts (Ara h 3), soybeans (Gly m 6), cashew nuts (Ana o 2), walnuts (Jug r 4), hazelnuts (Cor a 9), almonds (Pru du 6), and Goji berries. • Auxin-binding proteins (ABPs) – a family of dimeric monocupins found in plants like apples and strawberries. ABPs interact with a plant hormone auxin. 12.5.3.3 Proflins Superfamily These proteins of a small molecular weight (12–15 kDa) are ubiquitous in all eukaryotic cells. They are located in the cytosol and their role is to bind actin. Proflins contribute also to cell morphogenesis and division and regulate intercellular transport processes. The amino acid sequence similarity between proflins derived from eukaryotic and plant cells is low, but the sequences of plant proflins are 75% homologous in structure. Proflins occur as pollen sensitizers (Bet v 2, birch allergen), fruit (Pyr c 4, pear; Pru p 4, peach; Mus Xp1, banana), and hazelnut (Cor a

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2) allergens. Therefore, the proflin group is considered as bridge allergens between inhalation and food allergy. Clinical symptoms are mild, mainly observed as oral allergy syndrome (OAS) with rare cases of severe reactions. 12.5.3.4 PR-10 Proteins This is a huge family of proteins related to the birch pollen allergen Bet v 1, causing severe clinical allergy symptoms. Allergens of this group are proteins with 154–160 amino acid polypeptide chains, with high sequence homology. All Bet v 1 homologs have a characteristic amino acid sequence GXGXXG in their structure, called the Rossman loop, which in some proteins can be reduced to GXG and form phosphatebinding loops. This structure ensures the formation of a hydrophobic pocket, which allows the binding of plant steroids. The function of Bet v 1 homologs is attributed to their ribonuclease activity. Allergens that are homologs of Bet v 1 proteins are mainly pollen allergens. The frst sensitization of the organism takes place through inhalation, while the subsequent contact with Bet v 1 homologs from fruit (apples, pears, cherries, kiwi), nuts (e.g. walnuts) and vegetables (carrots, celery, parsley) results in the development of further allergic symptoms. When exposed to these allergens, the symptoms are weak, often confned to the oral area (OAS). Among the severe reactions, anaphylaxis has been observed as a response to the soy allergen, Gly m 4. Bet v 1 homologs are unstable during changes in environment and not very resistant to high temperatures. Less numerous families of allergenic plant proteins are described in the following. Oleosins stabilize spare plant fat bodies. They are found in fat storage organelles known as oleosomes. As allergens, they have been identifed in legumes, nuts, and seeds. Pathogen-resistant proteins are activated by the natural mechanisms of plants when they defend themselves against pathogens or during exposure to abiotic stress. Many identifed allergens belong to the families from this group, e.g. β-1,3-glucanases, chitinases, and thaumatin-like proteins. Plant endochitinases play a minor role in IgE-mediated reactions. Proteins that protect plants against plant pathogens are β-1,3glucanases, involved in pollen ripening, fertilization, fruit ripening, seed germination, and the accumulation of storage substances in the endosperm and cereal grains. They protect the plant from injury, low temperature, ozone, and UV-B radiation. The proteins that are similar in structure to thaumatin are synthesized in plants under biotic and abiotic stress, and in some cases, during fruit ripening. These proteins have a molecular weight of about 20 kDa; they are shaped of unevenly spaced β-harmonicons and are stabilized by eight disulfde bonds. They are resistant to high temperatures and proteolysis. Allergens classifed in this group include proteins found in avocado, banana, fg, kiwi, and chestnut, as well as pollen and latex.

12.5.4

MAIN ALLERGENS OF PLANT ORIGIN

12.5.4.1 Peanut Allergens Peanut allergy affects about 2.9% of the general population, primarily in the USA (4.6 million adults). In EU countries, it is most common in the Netherlands and

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France (Warren et al., 2021). Peanut allergy dominates also in Saudi Arabia – about 20% of allergy sufferers there have this type of hypersensitivity, with cases of anaphylaxis being reported frequently. Allergenic proteins make up about 7–10% of peanut protein. The bestcharacterized allergens are Ara h 1, Ara h 2, and Ara h 3. The allergen Ara h 1 has a molecular weight of 65 kDa, contains four immunodominant epitopes and 20 determinants of lesser immunological signifcance, and is completely heat-resistant. The second, Ara h 2, is a very potent allergen (m.w. 17 kDa), while Ara h 3 is a homolog of 11S protein. Further isolated allergens found in peanuts were designated up to Ara h 17, involving the existing allergen isoforms. Peanut allergy is one of the most severe food allergies which usually is not outgrown. Symptoms can be triggered by tiny amounts of allergens and even manifest as severe anaphylaxis. The threshold dose of arachidonic protein that triggers allergy has not been clearly defned yet. In some patients, a dose of 100 pg of peanut protein can provoke an immune reaction in the form of IgE, while in others such a reaction appears only after the ingestion of 2 mg of protein. Sensitization to peanut allergens varies among populations in different geographical regions. Practically, the only effective protection for people with food allergies caused by peanut consumption is to avoid contact with a potential allergen. Sensitization may occur not only by direct ingestion but also by inhalation. 12.5.4.2 Soy Allergens It is currently estimated that about 0.4% of children and 0.3% of adults are allergic to soy. Among the population of children with a cow’s milk allergy, about 30% of patients are also allergic to soy proteins. Soy proteins are used in raw (grains) and processed (four, fakes, grits, oil, or soy milk) forms, and can be found in foods like cured meat products, sauces, mayonnaise, ready-made dinner dishes, minced meat, margarine, bread, pastries, cakes, sweets, bars, chocolate, ice cream, and dairy desserts. The main soy allergens are: • Gly m1 – m.w. 7 kDa, hydrophobic protein, involved in lipid transport proteins. This protein protects plants from fungal and bacterial infection and facilitates the transport of phospholipids from liposomes to the mitochondria. As an allergen, Gly m1 exists in two isoforms, 1A and 1B. It is mainly an inhalant allergen, causing asthma. • Gly m 2 – m.w. 8 kDa, protein belonging to defensins. This allergen is present in soybean dust, therefore is dangerous for patients with asthma. • Gly m 3 – m.w.14 kDa (proflins). It is thermolabile and easily deactivated during digestion. This protein changes in structure during technological processes (high temperature, hydrolysis, fermentation), which lowers its allergenic potential. • Gly m 4 – m.w. 17 kDa, with biological function corresponding to PR-10 protein. It is mainly responsible for oral allergy syndrome (OAS), causing

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swelling of the tongue and esophagus. The possibility of cross-reactivity between Gly m 4 and cow’s milk casein has been observed. Gly m 5 – the group of proteins known as conglycinin (vicine, 7S globulin). One of the strongest soy allergens. Occurs as a trimer consisting of α, α ′, and β subunits. 7S globulin protein is highly immunogenic and resistant to digestive processes and therefore may induce allergic reactions causing damage to the microstructure of the small intestine. Gly m 6 – the group of proteins known as glycine (legumins, 11S globulins). It occurs as a hexamer or dimer with disulfde bonds in the structure. Since it is resistant to high temperatures, it remains a strong allergen after heat treatment. Sensitization to Gly m 6 – glycine is indicative of allergic reaction to soy lecithin. Immunodominant determinants of this allergen have been identifed as A (AA217–235) and B (AA253–265). Epitope AA1– 23 was found to be specifc for soy, while the others had the ability to bind antibodies from patients suffering also from peanut allergy. Gly m 6 causes a very wide spectrum of clinical symptoms. Gly m 7 – m.w. 67.9 kDa, seed-specifc biotinylated protein. Studies have shown that this protein was a more severe activator of basophilia than the major soy allergen, Gly m 5. Gly m 8 – 2S albumin. It shows high allergenicity due to the structure which determines its higher stability and resistance to digestive tract proteolytic enzymes. 2S albumin is present also in sesame, walnuts, and mustard. Gly m 8 shows signifcant homology with a strong peanut allergen Ara h 2.

Kunitz-type trypsin inhibitor – m.w. 21.5 kDa, belongs to the family of all antiparallel beta-sheet proteins, highly resistant to thermal and chemical denaturation. Cases of anaphylactic shock as a result of food allergy caused by contact with a trypsin inhibitor have been reported. In addition, further studies are ongoing into such soy allergens as Gly m 29 kDa, Gly m Bd30K, Gly m Lectin, Gly m Bd60K, Gly m TI, Gly m oleosin, or Gly m IFR (Rosada et al., 2019). 12.5.4.3 Nut Allergens Among the known tree nuts, walnuts (Corylus avallena) are the most allergenic nuts found in Europe. The frst allergen isolated from walnut was the allergen Cor a 1, with a molecular weight of 17 kDa. Hirschwehr et al. (1992) demonstrated an IgE-mediated reaction in the sera of all patients allergic to this nut species. Several other allergens have also been recognized with molecular weights of 14 kDa – pollen proflin; 9 kDa – most probably belonging to lipid-transfer protein (LTP); 32 kDa – 2S albumin; 35 kDa – legumin; and 47 kDa – a glycoprotein. A protein with a molecular weight of 40 kDa was also isolated from walnut tree pollen, which was identifed as Cor a 9. This protein is classifed as 11S globulin. It is the frst walnut allergen the amino acid sequence of which was known, and, based on the oligonucleotide characterization, its cDNA library was prepared.

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On the basis of the information obtained so far, the recombinant allergen Cor a 1.04 with a low IgE binding was proposed as the basic component of specifc immunotherapy in the allergy to walnut proteins. By studying the epitope structure, it was possible to establish their similarity to the peanut allergen Ara h 3 and soybean, which may be helpful in predicting the cross-reactions. The main allergens of almonds are two proteins identifed as albumin 2S (homologous in structure with the walnut allergen Jug r 1) and conglutin γ (60% homologous with conglutin γ of the seed lupin Lupinus albus and 7S protein of soybean Glycine max). Both almond allergens cross-react with walnut and hazelnut allergens. The strongest Brazil nut allergen is the albumin protein (Ber e 1) with a molecular weight of 9 kDa. This protein has a high content of methionine residues and consists of two subunits. Analyzing the IgE of patients allergic to these nuts, it was found that their serum also reacts with proteins with molecular weights of 25 and 58 kDa, which are allergens of weaker clinical relevance. The major allergen of cashews (Anacardium occidental) is Ana o 1 protein with a molecular weight of 50 kDa. There are 11 linear epitopes in its structure, among which three are immunodominant. Ana o 1 is a protein belonging to the vicinalin family. 12.5.4.4 Wheat Allergens Wheat has a high nutritional value, it is highly palatable and therefore commonly used as a component of bread, pasta, pizza, bulgur, couscous, and beer. Wheat proteins may induce food allergies, both IgE and non-IgE mediated. Sensitization due to contact with wheat components affects mainly people occupationally exposed to cereal allergens, e.g. workers in cereal processing plants and bakeries. The main allergen held responsible for immediate allergic reactions in children or anaphylaxis is Tri a 19 (Ω-5 gliadin). Wheat has been reported as a risk factor for severe anaphylactic reactions upon ingestion and for wheat-dependent, exercise-induced anaphylaxis (WDEIA). In a population of children allergic to wheat, more than 50% have experienced anaphylaxis upon wheat ingestion (Cianferoni, 2016). Such reactions have also been observed in athletes after consumption of products containing wheat proteins, in which Ω-5 gliadin was found. As a result of ongoing studies, the number of wheat allergens being identifed is increasing rapidly. The current knowledge in this respect is gathered in an online database, e.g. www.allergome.org, where 124 wheat proteins of allergenic potential have been listed so far (download date: 9.03.2022). Wheat allergy should not be mistaken for celiac disease, which is a gluten enteropathy. 12.5.4.5 Mustard The major allergens from yellow and oriental mustard seeds are Sinapis alba and Brassica juncea, respectively. They belong to the 2S albumin class of storage proteins which are abundant in seeds. They are small (m.w. of 12 to 15 kDa), basic

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proteins, generally composed of two different polypeptide chains (of about 3–5 and 8–10 kDa) linked by disulfde bridges (Monsalve et al., 2001). 12.5.4.6 Sesame To date, seven sesame allergy components – two 2S albumins (Ses i 1 and Ses i 2), one vicilin-like 7S globulin (Ses i 3), two oleosins (Ses i 4 and 5), and two 11S globulins (Ses i 6 and Ses i 7) – have been registered (Adatia et al., 2017). 12.5.4.7 Celery The most important allergen of celery is Api g 1, consisting of two isoforms Api g 1.0101 and Api g 1.0201. This allergen is a homolog of birch pollen Bet v 1. Allergies to celery are common in northern and central European countries where birch is a popular tree. A recombinant derivative of celery allergen Api g 4 has been developed, which is a proflin and in turn shows a high structural similarity with Bet v 2 of birch pollen. Celery allergens cross-react not only with birch pollen allergens, but also with soy (Gly m 3) in 78%, barley in 75%, and to a lower extent with mugwort and spices. Api g 5, which is a glycoprotein with a molecular weight of about 60 kDa, was also isolated. 12.5.4.8 Lupine Lupine allergens are classifed into different families of proteins, namely globulins (α-, β-, and γ-conglutin), 2S albumins (δ-conglutin), and some minor fractions such as pathogenesis-related (PR)-10 proteins (Lup a 4 and Lup l 4), non-specifc lipid transfer proteins (nsLTP) (Lup an 3), and proflins (Lup a 5) (www.allergome.org/).

12.6 METHODS FOR ALLERGEN DETERMINATION Mandatory allergen labeling by the food industry helps allergic consumers avoid allergenic foods. Allergens may be present in foods as a result of contamination, the use of the same technological lines for the production of similar products, or non-compliance with the storage regime of food ingredients. Therefore, methods for allergen determination must be fast, inexpensive, highly sensitive with a low detection limit, specifc, reliable, and accurate for analyzing amino acid sequences, to allow unambiguous identifcation of protein allergens, especially the so-called hidden ones. Methods used to analyze the trace amounts of proteins and peptides with allergenic properties in the food matrix include immunometric assays and techniques of molecular biology and mass spectrometry.

12.6.1

ELISA (ENZYME-LINKED IMMUNOSORBENT ASSAY)

Immunoassay, especially the ELISA test, is currently one of the most preferred methods today. The tests are commercially available, making it very easy to assess allergenicity. The analysis is performed on a titration microplate in different variants: direct, indirect, or sandwich. The principle of the method is the measurement of the enzymatic activity generated during the reaction between a protein (allergen) and

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a specifc antibody (anti-IgG of animal or human origin specifc to the determined allergen). The analysis is carried out in several steps. In the indirect test, the microplate is coated with an antigen and the non-specifc binding sites are saturated with a blocker, which is usually gelatine, OVA, or BSA. Then, the specifc antibody toward the analyzed protein is administered and, as the last step, the secondary antibody conjugated with an enzyme (e.g. horseradish peroxidase or alkaline phosphatase) is added. The addition of a substrate causes a color reaction, and the intensity of the color in the direct, indirect, and sandwich methods is proportional to the concentration of the allergen tested. In the competitive method, the color intensity upon reaction is inversely proportional to the allergen concentration. ELISA is a rapid method (of about three hours) and easy to perform by a skilled laboratory technician.

12.6.2 METHODS BASED ON DNA ANALYSIS Molecular biology methods (PCR, RT-PCR, PCR-ELISA, PCR-PNA_HPLC, duplex-PCR, multiplex real-time PCR) are used for direct analysis of allergenic components present in food. The PCR allows specifc amplifcation and detection of selected deoxyribonucleic acid (DNA) stretches from species or groups of species that are used in food manufacturing; however, it does not directly detect allergenic proteins. The analysis is based on the segment of the gene encoding the allergenic protein. PCR sensitivity and specifcity strongly depend on the selected DNA stretch of the targeted allergenic food. Thanks to DNA amplifcation of a selected protein fragment, it becomes possible to detect the indicated allergen. PCR analysis is more sensitive and more specifc than the aforementioned ELISA test; however, it is also more time-consuming (approx. six hours). The detection limit is approx. 10 mg/kg (e.g. for almonds, walnuts, soy, or milk), avoiding positive false results and minimizing cross-reactions.

12.6.3 METHODS USING MASS SPECTROSCOPY A protein that is isolated from the food matrix by two-way electrophoresis is pretrypsinized. The molecular mass of the resulting peptides formed after hydrolysis is determined by mass spectrometry (MS). Then, after the analysis of the peptide map spectrum against the online databases, the selected protein can be identifed. This method also allows evaluation of the amino acid sequence and the changes that were involved in post-translational reactions. The main limitation of MS is that this method relies on the access to repositories of protein sequences largely derived from the genome, which are in general scarce in sequences for plant-based allergenic foods.

12.6.4

BIOSENSORS

In biosensors, a signal is produced by a transducer upon recognizing the interaction between a probe or an antibody (receptor) and a target molecule (DNA or protein). Surface plasmon resonance (SPR), applied in optical biosensors, is the most

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commonly used device, which relies on the measurements of the refractive index changes that occur upon binding of the antibody to the target protein. Aptamers are used for the development of sensors due to their high affnity and binding specifcity for the targets, low production cost, and ease of labeling with different reporter molecules (Villa et al., 2020).

12.7 EFFECTS OF TECHNOLOGICAL PROCESSES ON FOOD ALLERGENS Food raw materials are subjected to numerous technological processes which infuence the food matrix, changing its structure and giving new, desirable characteristics to the products obtained. At the same time, the allergenic potential of proteins changes due to their partial unfolding, aggregation, and cross-linking, as well as chemical modifcations such as lactosylation, oxidation, or Maillard reactions. The following sections present the technological processes that have a great impact on the immunoreactive properties of cow’s milk proteins, as examples of industrial infuences on raw materials.

12.7.1 THERMAL PROCESSES Thermal processes are essential during the production of drinking milk and during its diversion to the manufacture of dairy products. High-temperature treatment of the raw material causes chemical changes such as denaturation, aggregation, and Maillard reactions, which infuence the antigenicity of allergenic milk proteins. Caseins are heat-stable due to their micellar shape. They do not have secondary, tertiary, or quaternary structures that can be changed by thermal processes, therefore only a partial reduction in casein allergenicity was noted. Whey globular proteins are more sensitive to a high temperature than casein fractions, which do not lose their antigenic potential after heating at 120° C for 15 min. An increase in the antigenicity of whey proteins was observed after heating them to temperatures above 50° C as a result of structure unfolding and “opening up.” An increase in the antigenicity of whey proteins was observed when heated to a temperature above 50° C. This was a consequence of the destruction or masking of conformational epitopes caused by the formation of new aggregates containing disulfde bonds(Bogahawaththa et al., 2018). Thermal changes in milk proteins depend on many factors, including the chemical composition of milk, the conditions applied during milk processing, and the genetic variant of the cows which the milk was obtained from. Therefore, particular attention should be paid to the quality of milk intended for processing into baby food.

12.7.2

GLYCATION

The reactions of proteins with reducing sugars lead to the formation of glycation end products in the Maillard reaction. Changes to the proteins during glycation depend on the relative ratio of saccharides and proteins, temperature, and reaction time. It has been found that the use of high molecular weight sugars such as carboxymethylene

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dextran, leads to a decrease in the allergenicity of β-lg and epitope masking, resulting in reduced immunogenicity. In turn, glucosamine and chitopentosan induce the formation of new epitopes which increase the allergenic potential of the conjugates.

12.7.3 LACTIC FERMENTATION Lactic fermentation bacteria are characterized by the presence of a very active proteolytic enzyme system. The purpose of lactic fermentation is to obtain an easily digestible milk product with available bioactive peptides. Lactic fermentation bacteria activate cells of the host’s gastrointestinal immune system through direct contact with the whole bacterial cell, its fragment, or metabolites. The properties of selected strains of lactic fermentation bacteria encourage their use in the prevention and treatment of food allergy, thanks to such mechanisms as normalization of intestinal microfora, production of antimicrobial factors, reduction of intestinal mucosal permeability for antigens, decline of antigen penetration, reduction of infammation in the intestines, stimulation of synthesis of secretory IgA antibodies in Peyer’s patches, increase of IgA response to potential allergens, protection of intestinal mucosa against penetration of allergenic proteins, activity of bacterial proteolytic enzymes, stimulation of Th1 type response (i.e. by IL-12, IL-2, INF-γ production), and inhibitory action of IL-4 cytokine, which stimulates IgE synthesis, inhibition of Th2 type response, and stimulation of alimentary tolerance mechanism, mainly by increased production of a transforming growth factor (TGF-β). Studies on the infuence of microorganisms on the human organism, especially on the immune system, emphasize the signifcant role of the microfora living naturally in the human intestine, especially the Lactobacillus genus. The immunostimulating effect of Lactobacillus bacteria results from increased production of INF-γ and lymphokines, and IgA. An increase in phagocyte and lymphocyte activity has also been also observed. Analyzing fermented milk beverages obtained with mesoand thermophilic strains, it was found that the allergenic properties of cow’s milk proteins signifcantly decreased (Wróblewska et al., 2010, 2019, 2020).

12.7.4

ENZYMATIC MODIFICATIONS

Enzymatic hydrolysis is a process used in the production of foods dedicated to children with health problems (e.g. allergies, phenylketonuria, liver disease). The hydrolysis conditions vary depending on the enzymes used (pepsin, Alkalase, papain, trypsin, Protamex, Flavourzyme) and their activity (Wróblewska et al. 2005; Liang et al., 2022). Hydrolysates are more soluble and thermally stable and show greater resistance to changes in medium acidity than the proteins which these hydrolysates were produced from. This is important when applying them to people with health problems, mainly gastrointestinal diseases. To date, no hypoallergenic mixture based on whey, casein, and soy proteins has been produced that would be completely free of allergenic properties. Even peptide mixtures obtained by the hydrolysis of casein with a mass below 500 Da still

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exhibit allergenicity. These peptides should be identifed and inactivated, and the sensory properties, nutritional qualities, and storage stability need to be evaluated in the future. So far, only amino acid mixtures intended for parenteral infusion are non-allergenic.

12.7.5

CROSS-LINKING WITH TRANSGLUTAMINASE

Food products obtained with microbial transglutaminase (m-TG) are considered non-allergenic. During technological processes, the formation of the crosslinking amino acid N-ε-(γ-glutamyl)-lysine (Glu_Lys) is observed. The isopeptides are not hydrolyzed by gastrointestinal enzymes. Casein cross-linked with mTG was digested in vitro in a stimulated human model. However, the mTG-treated wheat product is still immunoreactive. The enzymes responsible for the cleavage of these bonds are γ-glutaminocyclotransferase located in kidneys and γ-glutaminotranspeptidase found in kidneys and intestinal brush border. Treatment of milk proteins with m-TG revealed that the extent of crosslinking decreases in the following order: sodium caseinate > proteins obtained by ultrafltration > milk powder > whey protein isolate. Casein is a particularly favorable substrate for m-TG. Nevertheless, various casein fractions react differently with m-TG. κ-casein and β-casein show a higher affnity for the enzyme than α-casein. This is due to the structure of casein micelles. m-TG does not change the size of the micelles because it catalyzes the formation of crosslinking bonds only within the molecule. Whey proteins are more diffcult to crosslink than casein. Accessibility of β-lactoglobulin and α-lactalbumin for reactions catalyzed by m-TG can be increased by a pre-treatment with reducing compounds that cleave disulfde bonds so that the structure of the proteins “opens up” and new cross-linking sites for m-TG are revealed. Yogurt and kefr made from cross-linked milk show a lower allergenicity than similar products made without m-TG. Moreover, they have more favorable sensory characteristics, including increased palatability (Wroblewska et al., 2009; Wroblewska et al., 2013). Gamma radiation applications The γ-radiation reduces the antigenicity of bovine serum albumin (BSA) and β-lg. The conformational epitope structures of BSA are destroyed under exposure to radiation; however, in the case of sequential structures, the antigenicity may increase. The use of γ-irradiation is useful for food products that cannot undergo any other technological methods that may cause a loss of features determining the nutritional properties and culinary usefulness of the raw materials, e.g. in the case of eggs.

12.7.6 PRESSURIZATION The use of high pressure in the food industry causes denaturation changes and leads to the formation of aggregates, which affect the allergenicity of proteins. Effects on protein allergenicity are non-uniform. It was shown that a pressure of 200–600

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MPa increased the antigenicity of β-lg present in whey protein solution, sweet whey, and milk. On the other hand, it was observed that the hydrolysis of β-lg with different enzymes performed under high pressure allowed a complete reduction of antigenicity.

12.7.7 ULTRASOUND Sonication is a technique used to homogenize flter milk whey, mayonnaise, and fruit juice solutions, tenderize meat, and dehydrate fruits and vegetables. High-intensity ultrasound uses high-energy mechanical waves (20–100 kHz), which can lead to TABLE 12.1 Infuence of Technological Processes on Allergenicity of Different Types of Plant- and Animal-Based Proteins Process

Plant allergens

Animal allergens

1. Glycosylation

Not all proteins after glycosylation become allergens.

Different effects on the IgE binding capacity of various proteins: tropomyosins (increase/ maintain/decrease), arginine kinases (unknown), ovomucoids (maintain/ increase), and caseins (increase).

2. Heat stability

2S Albumins, nsLTP, cereal prolamins, legumins, and vicilins are heat-stable. Proflins and PR-10 proteins are heat-labile.

3. Pressure stability

Most plant allergens are pressurestable; 2S albumins and legumins can be slightly reduced.

4. Light/ radiation Stability

Most plant families are not affected. Exceptions: IgE-binding capacity is increased in cereal prolamins and decreased in proflins and PR-10.

Tropomyosins, parvalbumins, caseins, and ovomucoids are heat-stable proteins. Serum albumins are partially heat-labile/stable proteins. Arginine kinases and other miscellaneous protein families (glycoside hydrolase family 22, transferrin, lipocalins, and serpins) comprise heat-labile proteins. Tropomyosins and parvalbumins are pressure-labile proteins while serum albumins, ovomucoids, and serpins appear pressure-stable. Caseins, glycoside hydrolase family 22, and lipocalins expose a dual behavior toward pressure (most likely pressure-stable). Most protein families are labile, with some exceptions (tropomyosins, parvalbumins, lipocalins, and serpins).

5. Hydrolysis/ fermentation

Fragmentation of proteins – decrease in IgE-binding capacity of 2S albumins, ATIs, legumins, vicilins, proflins, and PR-10.

Source: Costa et al., 2020, 2021.

Changes in protein size; reduced or even impaired IgE-binding capacity of all animal protein families.

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conformational changes in food proteins, and thus affect their allergic reactivity. The ultrasound method was found to reduce the allergenicity of soy proteins and peanut allergens (Ara h 1 and Ara h 2). The impact of different processing methods on raw plant and animal-based materials and protein thereof varies greatly, depending on the type of proteins and food matrix. Examples are shown in Table 12.1.

12.8

PREVENTION OF FOOD ALLERGY

Currently, the only effective method for controlling food allergy is to avoid the food source that is dangerous to the patient. Appropriate training and education of patients and their families is a fundamental part of the preventive management of allergic diseases. The treatment strategy for allergic diseases is based on the patient’s education, control of environmental factors, dietary habits, knowledge of the role of nutrients, limited exposure to allergens, pharmacotherapy, and possibly immunotherapy (IT). In food allergy, no standards for IT have been developed to date. Of great importance in allergic diets is the presence of essential fatty acids, zinc, and vitamin D, which can strengthen the anti-infammatory and antioxidant barrier and promote immune tolerance. Additionally, nutrients such as pre- and probiotics have been shown to benefcially modulate the tolerogenic immune environment (Mazzocchi et al., 2017, Galdeanoa et al., 2019). New forms of immunotherapy are being researched and low-allergenicity raw materials and products are being sought, which could be included in the diet of patients with food allergies.

REFERENCES Adatia, A. et al. Sesame allergy: Current perspectives. Journal of Asthma and Allergy, 10, 141–151, 2017. https://doi.org/10.2147/JAA.S113612. Anagnostou, K. and Turner, P.J. Myths, facts and controversies in the diagnosis and management of anaphylaxis. Archives of Disease in Childhood, 104(1), 83–90, 2019. https://doi.org/10.1136/archdischild-2018-314867. Azofra, E.S. et al. Heterogeneity in allergy to mollusks: A clinical-immunological study in a population from the north of Spain. Journal of Investigational Allergology and Clinical Immunology, 27(4), 252–260, 2017. https://doi.org/10.18176/jiaci.0137. Bogahawaththa, D. et al. In vitro immunogenicity of various native and thermally processed bovine milk proteins and their mixtures. Journal of Dairy Science, 101(10), 8726– 8736, 2018. https://doi.org/10.3168/jds.2018-14488. Boyce, J.A. et al. Guidelines for the diagnosis and management of food allergy in the United States: Summary of the NIAID-sponsored expert panel report. Journal of Allergy and Clinical Immunology, 126(6), 1105–1118, 2010. https://doi.org/10.1016/j.jaci.2010.10.008. Chan, S.K. et al. Keeping AllerGen names clear and defned. Frontiers in Immunology, 10, 2600, 2019. https://doi.org/10.3389/fmmu.2019.02600. Cianferoni, A. Wheat allergy: Diagnosis and management. Journal of Asthma and Allergy, 9, 13–25, 2016. https://doi.org/10.3168/jds.2018-14488. Costa, J. et al. Are physicochemical properties shaping the allergenic potency of plant allergens? Clinical Reviews in Allergy and Immunology, 62(1), 37–63, 2008. https://doi.org /10.1007/s12016-020-08810-9.

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Costa, J. et al. Are physicochemical properties shaping the allergenic potency of animal allergens? Clinical Reviews in Allergy and Immunology, 62(1), 1–36, 2021. https://doi .org/10.1007/s12016-020-08826-1. Galdeanoa, C.M. et al. Benefcial effects of probiotic consumption on the immune system. Annals of Nutrition and Metabolism, 74(2), 115–124, 2019. https://doi.org/10.1159 /000496426. Hirschwehr, R. et al. Identyfcation of common allergenic structures in hazel pollen and hazelnuts: A possible explanation for sensitivity to hazelnuts in patients allergic to tree pollen. Journal of Allergy and Clinical Immunology, 90(6 Pt 1), 927, 1992. Hoffman, D.R. et al. The major heat stable allergen of shrimp. Annals of Allergy, 47(1), 17, 1981. Hoffmann-Sommergruber, K. and Mills, E.N.C. Food allergen protein families and their structural characteristics and application in component-resolved diagnosis: New data from the EuroPrevall project. Analytical and Bioanalytical Chemistry, 395(1), 25–35, 2009. https://doi.org/10.1007/s00216-009-2953-z. https://www.allergome.org date of download 9.03.2022. Hugh, A. et al. Mechanisms of food allergy. Journal of Allergy and Clinical Immunology, 141, 1–19, 2018. https://doi.org/10.1016/j.jaci.2017.11.005. Liang, X. et al. Evaluation of allergenicity of cow milk treated with enzymatic hydrolysis through a mouse model of allergy. Journal of Dairy Science, 105(2), 1039–1050, 2022. https://doi.org/10.3168/jds.2021-20686. Mazzocchi et al.. The Role of Nutritional Aspects in Food Allergy: Prevention and Management. Nutrients, 9, 850. doi:10.3390/nu9080850, 2017. Monsalve, R.I. et al. Allergy to mustard seeds: The importance of 2S albumins as food allergens. Internet Symposium on Food Allergens, 3(2), 57–69, 2001. Patel, B.Y. and Volcheck, G.W. Food allergy: Common causes, diagnosis, and treatment. Mayo Clinic Proceedings, 90(10), 1411–1419, 2015. https://doi.org/10.1016/j.mayocp .2015.07.012. Pomésa, A. et al. WHO/IUIS AllerGen Nomenclature: Providing a common language. Molecular Immunology, 100, 3–13, 2018. https://doi.org/10.1016/j.molimm.2018.03.003. Rosada, T. et al. Soy allergy – what do we know? Alergia Astma Immunologia, 24(3), 119– 125, 2019. Sampson et al.. Mechanisms of food allergy Journal of Allergy and Clinical Immunology 141, 11–9, 2018. van Ree, R. Food allergy: A growing public health problem. Frontiers in Allergy, 10, 2021. https://doi.org/10.3389/falgy.2021.668479. Villa, C. et al. Lupine allergens: Clinical relevance, molecular characterization, crossreactivity, and detection strategies. Comprehensive Reviews in Food Science and Food Safety, 1–29, 2020. https://doi.org/10.1111/1541-4337.12646. Warren, C. et al. Prevalence and characteristics of peanut allergy in US adults. Journal of Allergy and Clinical Immunology, 147(6), 2263–2270.e5, 2021. https://doi.org/10.1016 /j.jaci.2020.11.046. Wróblewska, B. et al. The reduction of cow milk proteins immunoreactivity by two-step enzymatic hydrolysis. Acta Alimentaria, 34(3), 307–315, 2005. https://doi.org/10.1556/ aalim.34.2005.3.13. Wróblewska, B. et al. Infuence of the addition of transglutaminase on the immunoreactivity of milk proteins and sensory quality of kefr. Food Hydrocolloids, 23(8), 2434–2445, 2009. https://doi.org/10.1016/j.foodhyd.2009.06.023. Wróblewska, B. et al. Troszyńska Impact of transglutaminase reaction on the immunoreactive and sensory quality of yoghurt starter. World Journal of Microbiology and Biotechnology, 27(2), 215–227, 2010. https://doi.org/10.1007/s11274-010-0446-z.

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Food Flavors Shwu-Pyng Joanna Chen and Bonnie Sun Pan

CONTENTS 13.1 Introduction ..................................................................................................364 13.2 Sources of Food Flavors ...............................................................................364 13.2.1 Flavors Formed Naturally in Plants..................................................364 13.2.1.1 Spices and Herbs................................................................364 13.2.1.2 Fruits and Vegetables......................................................... 365 13.2.1.3 Algae .................................................................................. 368 13.2.2 Flavors Produced in Animals ........................................................... 369 13.2.2.1 Meats.................................................................................. 369 13.2.2.2 Seafood .............................................................................. 370 13.2.2.3 Flavors Produced by Microbes and Enzymes.................... 371 13.3 Aroma Compounds Classifcation and Chemical Structures ....................... 373 13.3.1 Chemical Structures and Their Odors.............................................. 373 13.3.2 Odor Intensity of Aroma Compounds .............................................. 375 13.4 Aroma Changes during Post-Harvest Storage of Plants............................... 375 13.4.1 Spices and Herbs............................................................................... 375 13.4.2 Fruits and Vegetables........................................................................ 376 13.5 Thermal Reactions and Flavor Compounds Formation ............................... 377 13.5.1 Maillard Reaction ............................................................................. 377 13.5.2 Lipid Oxidation................................................................................. 378 13.5.3 Interaction of Lipids in the Maillard Reaction ................................. 380 13.6 Flavor Industry: A Blend of Art, Science, and Technology ......................... 380 13.6.1 Ingredients for Flavor Creation......................................................... 381 13.6.2 Flavorings for Food Industries.......................................................... 382 13.6.3 Flavor Formulation and Labeling ..................................................... 383 13.7 Flavor Manufacturing and Flavor Delivery Systems.................................... 385 13.7.1 Emulsion Flavors .............................................................................. 385 13.7.2 Powder Flavor ................................................................................... 386 13.7.3 Reaction Flavors and Safety Concerns ............................................. 387 13.7.4 Herbs and Seasonings Blends........................................................... 388 13.8 Food Trends and Future Flavor Industry...................................................... 389 13.8.1 Flavor Applications........................................................................... 389 13.8.2 Plant-Based Meat and Drinks........................................................... 390 13.8.3 Recombinant DNA Technology for Flavor....................................... 391 13.8.4 Flavor Legislation ............................................................................. 392 References.............................................................................................................. 393

DOI: 10.1201/9781003265955-13

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Shwu-Pyng Joanna Chen and Bonnie Sun Pan

INTRODUCTION

Flavor is a critical and essential part of food. It serves as a stimulator of human appetite and an indicator of the quality of food. This chapter reviews and discusses different sources of food favors from natural origins, including herbs, spices, fruits, vegetables, meats, and seafood, and those produced by microbes, enzymes, and thermal reactions. Different aroma compounds’ chemical classes and molecular structures are reviewed for their roles in authentic food characteristics. Important reactions generating aromas such as the Maillard reaction and lipid oxidation are discussed for their contributions to food acceptance. Factors affecting favor quality and carcinogenic compounds generated from thermal processes are covered for ensuring optimum acceptance and safety. The favor industry serving as an accelerator of processed food diversity is a blend of art, science, and technology. The uniqueness, various ingredients used, labeling, manufacturing, and delivery systems are described for a better understanding of their operations. With the growing global trend of plant-based foods and drinks, the favor industry plays an even more important role in providing natural and green ingredients utilizing modern technologies such as recombinant DNA to optimize taste and cost. With the diverse and signifcant role of favors in foods, legislation must be complete and complied with.

13.2 SOURCES OF FOOD FLAVORS 13.2.1 FLAVORS FORMED NATURALLY IN PLANTS Flavor compounds exist in nature. They are developed via photosynthetic and metabolic pathways in plants. The genetic nature of a plant plays an important role in its formation. In plants, aroma compounds can be developed utilizing fatty acids, carbohydrates, terpenes, and amino acids. They can also be derived from precursors involving enzymatic and interactive reactions. Whether compounds are formed directly through metabolic pathways or via precursors (enzymatic or non-enzymatic reactions) after cell rupture depends on the nature of the plant. Environmental impact can further infuence their formation and combination. At different maturity stages, different combinations of volatiles can be formed and infuence the unique favor profle of a crop (Reineccius, 2006a). 13.2.1.1 Spices and Herbs Spices and herbs are aromatic plants. In a broader defnition, spices cover herbs since nearly all plant parts from leaves, aril, barks, berries, buds, bulbs, pistil, kernels, rhizomes, roots, seeds, etc. have been used in foods, health food, and medicinal applications for centuries. Due to the increasing interest in products of natural origin globally, the popularity of natural botanical favors has increased in importance in recent years. Herbs refer to aromatic leaves from herbaceous plants (plants lacking woody stems, except a few). Many, like basil, bay leaf, marjoram, oregano, rosemary, sage,

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peppermint, and more, are widely used in culinary applications because of their favoring aromatic characteristics. They can be used as fresh, dried, or extracts (essential oil and oleoresin). Some of these herbs also exert good antioxidant and antibacterial activities. The former can help preserve food quality and the latter gives health benefts (Ribeiro-Santos et al., 2015). The combination of the volatile aroma compounds is different depending on the varieties and physical states of the same spice (Simon et al., 1999). Using rosemary as an example, the aromas are different in living, picked, and oil extract. The fatty green volatiles, trans-2-hexenal, cis-3-hexenol, and hexanol, are only present in the pickled plant. Estragole, a powerful sweet, herbaceous aroma, is mainly in the living plant, responsible for the fresh herbaceous character. Both living and picked plants are high in myrcene, p-cymene, limonene, and linalool but only a small amount is present in essential oil. Major volatiles identifed in essential oil are α-pinene, β-pinene, eucalyptol, and camphor. These are present in small quantities or none in living or picked plants (Mookherjee et al., 1989). Modern analysis confrmed eight key constituents in essential oil responsible for strong rosemary aroma, α-pinene, 1,8-cineole, camphene, camphor, p-cymene, myrcene, limonene, and β-caryophyllene. Signifcant variations in the chemical composition of rosemary essential oils affect the oil’s functional properties. Myrcene-rich oil is found to associate with the highest antioxidant activity and α-pinene-rich oil has the highest antibacterial activities (OjedaSana et al., 2013). Spices are plants of which only parts are very aromatic, with a few exceptions (e.g., capsicum, fenugreek). They contain relatively high percentages of volatile oil as well as other powerful non-volatiles and/or coloring components. The oils are extracted along with by-products such as oleoresin for further favoring applications. The spice plants mostly require further processing before they can be incorporated into foods. There are hundreds of varieties of spice species/varieties. Spices with characteristic aroma compounds are used widely in food seasonings. Examples are black pepper (piperine, S-3-carene, β-caryophyllene), cinnamon (cinnamaldehyde, eugenol), chili (capsaicin, dihydro capsaicin), coriander (d-linalool, C10-C14-2-alkenals), ginger (gingerol, shogaol, neral, geranial), mustard (allyl thiocyanate), parsley (apiol), vanilla (vanillin), etc. (Peter, 2001). Diversifed sensory characteristics are delivered by these different spices such as sweet (anise), sour (tamarind), bitter (oregano), spicy (ginger), hot (black pepper), sulfury (onion), nutty (sesame), etc. (Raghavan, 2000a). Individually or in combination, the spices make food unique, ethnic, and authentic. They not only deliver taste, aroma, and texture but also color and antioxidative and antibacterial benefts. 13.2.1.2 Fruits and Vegetables Fruit favor compounds are biosynthesized in intact tissues during different ripening stages. Different components such as lipid/fatty acid, (poly)saccharide/carbohydrate, protein/amino acid, and lignin/cinnamic acid are utilized to convert to different aroma compounds. Vegetables in general require non-volatile precursors to be broken down by enzymatic actions when tissues are disrupted before giving their

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typical favor. An overview of the formation pathways and detailed explanations are provided by Reineccius (1994, 2006a). 13.2.1.2.1 Fruits Fruit aroma compounds are diverse. Hundreds exist in fruit in trace amounts less than ppm level but are detectable by human senses. Quantities of over 300 volatiles can be easily identifed from the majority of fruits but maybe less than 20% are critical for characteristic aroma. The diversity of the volatile combination is unique in different fruit varieties. Volatile composition varies according to the ripeness of a fruit and its growing conditions. Many compounds can exist with or without signifcant detection levels depending on the species/status of the fruit and how the volatiles are obtained and analyzed (Jiang and Song, 2010). Many volatiles are found present but without critical characteristic function. Apple: More than 300 volatiles have been identifed in apple aroma including alcohols, aldehydes, acids, ketones, esters, terpenoids, and sesquiterpenes, but only 20–30 contribute signifcantly to the typical apple aroma. Esters are the most abundant compounds responsible for the fruity odor of ripe apples. Esters found commonly in peels among 40 cultivars are butyl acetate, 2-methyl butyl acetate, butyl butyrate, butyl 2-methyl butyrate, hexyl butyrate, hexyl 2-methylbutyrate, and hexyl hexanoate (even-numbered carbon chains including combinations of acetic, butanoic, and hexanoic acid with ethyl, butyl, and hexyl alcohols). Volatile profles show large differences among the cultivars. Compounds like hexanal give a characteristic fresh sweet odor and confer a green aroma. (E)-2-hexenal gives a grassy, leafy, apple odor, estragole gives an anise odor, and α-farnescene gives a green, oily/fatty note. Compounds like ethyl 2-methylbutanoate, 2-methylbutyl acetate, and hexyl acetate contribute to the characteristic Fuji apple aroma (Yang et al., 2021). Banana: Major volatiles in ripe bananas are esters such as amyl acetate, isoamyl acetate, butyl butyrate, amyl butyrate, 3-methylbutyl, two methyl propyl esters of acetate and butyrate, isopentyl acetate, isobutyl acetate, etc. Isoamyl acetate represents the characteristic banana odor along with butyrate and propionate. Volatiles of bananas at the green stage are mainly C6-aldehydes and C6-alcohols. As the fruit ripens, the esters increase and the unsaturated aldehydes and alcohols decrease (Zhu et al., 2018). Citrus: Important citrus fruit species include but are not limited to lemon, orange, mandarin orange, lime, and grapefruit. The volatiles of these fruits are present in the juice and the peel. About 300 volatiles have been identifed including terpenes, hydrocarbons, aldehydes, alcohols, esters, and ketones. Only 20–30 compounds may be important for a typical citrus fruit odor. Esters are important for the characteristic citrus favor. Major esters are ethyl esters of C3 to C6 organic acids such as ethyl butanoate, ethyl 2-methylpropanoate, ethyl 2-methylbutanoate, and ethyl hexanoate that impart strong fruity aromatic notes to juices. The terpene alcohol, linalool, is the most important alcohol that contributes to the distinctly fresh and foral notes. Geraniol is another terpene alcohol that contributes to the foral and fruity notes. Limonene is present in a large quantity but does not directly contribute to citrus character. It acts more like a lifting agent for other volatiles. Aldehydes generally

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provide green, fresh, citrus-like notes. Ketones like carvone provide a fresh minty note. α-ionone and β-ionone provide sweet foral notes. Compounds in trace quantity may contribute to the characteristic fruit aroma. One example is nootkatone which provides a typical grapefruit-like odor. Citral (a mixture of neral and geranial isomers) gives a typical lemon scent (Porat et al., 2016). Strawberry: Over 360 volatiles have been identifed. These compounds are highly diverse, variable, and complex. Their quantities are infuenced by genetic and growing environmental factors. The most frequently identifed are esters, acids, lactones, aldehydes, furans, alcohols, ketones, and terpenoids. Ethyl butanoate, ethyl hexanoate, methyl butanoate, and methyl hexanoate are the most abundant esters important to the strawberry fruity aroma. Lactones like γ-decalactone and γ-dodecalactone give peach-like odor-enhancing sweetness. Aldehydes like 2-hexenal and (Z)-3-hexenal impart a green/fresh aroma. Furanones such as 4-hydroxy-2,5,dimethyl-3(2H)-furanone and furaneol are associated with a sweet aroma. Nonanal and 6-methyl-5-hepten-2-one provide fowery notes (Fan et al., 2021). Melon: There is extensive favor variation among melons due to the highly polymorphic species in nature. Over 240 volatiles including esters, aldehydes, furans, sulfdes, and thiols have been identifed in climacteric melons, such as cantaloupes. About half of the volatiles are esters correlating to fruity/foral/sweet favors. Non-climacteric fruits have longer shelf-life and lower levels of esters. They are associated with fresh, green, grassy alcohols and aldehydes predominantly. Key melon volatiles are ethyl butyrate (fruity), ethyl-2-methyl butyrate (sweet/fruity), ethyl-2-methyl propanoate (foral melon-like), hexanal (green/grassy), (Z,Z)-3,6nonadienol (grassy, boiled leaf-like), (E,Z)-2,6-nonadienal (cucumber/melon), ethyl 3-(methylthio) propionate (fruity/pineapple-like), 2-phenethyl acetate (rosy/foral/ fruity/sweet), (E)-β-ionone (violet/foral/raspberry/woody), and dimethyltrisulphide (cabbage/garlic) (Farcuh et al., 2020; Gonda et al., 2016). 13.2.1.2.2 Vegetables Vegetables do not have a ripening process as climacteric fruits do. The favor formation pathways are different from those of fruits. Aroma comes from precursors during cellular disruption. Crushing of cells allows the mixing of enzymes and substrates resulting in the generation of vegetable volatile compounds. Volatile formation in vegetables can come from nonvolatile precursors, such as linoleic/linolenic acids, thioglucosinolates, cysteine sulfoxides, methyl methionine, via enzymes-catalyzed reactions, and further heating to form different volatile groups (Reineccius, 2006a; Siegmund, 2015). Flavors of allium species (garlic, onion, shallot, leek, and chives) are formed from precursors (sulfur-containing amino acids). Several sulfur-containing volatile compounds not only provide aroma but also inhibit bacteria growth as a vapor (Borlinghaus et al., 2021) and have medicinal and agricultural signifcance. Garlic: When garlic tissues are crushed, the enzyme alliinase is released and catalyzes the formation of different sulfenic acids from the non-volatile/odorless precursor [(+)-S-alk(en)yl cysteine sulphoxide or alliin]. These highly reactive sulfenic acids further react and degrade to thiosulfates allicin (S-allyl-2 propene-1-sulfnathioate)

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which represents the characteristic odor of crushed fresh garlic. Because of its unstable nature, allicin further reacts and converts to different sulfur-containing compounds such as mono-, di-, poly-, cyclic sulfdes, etc. and forms part of the fresh garlic aroma (Abe et al., 2020). Onion: The most important sulfur-containing volatile compound of raw onion identifed is thiopropanal-S-oxide which has a lachrymatory (tear=producing) effect (Jones et al., 2004; Yoshimoto and Saito, 2019). Different physical states yield different key aroma compounds. When onion is freshly cut, key aroma compounds are propyl thiosulfonates. Boiled onion is typifed by propyl and propenyl di- and trisulfdes. Fried onions are typifed by dimethylthiophenes. Brassicas: Sources of health foods and favors, brassicas include turnip, rutabaga, mustard, cabbage, broccoli, and Brussels sprout. Their volatiles contain sulfur components, mainly the bioactive isothiocyanates which are highly pungent. Cooking causes the inactivation of the enzymes leading to a decrease in the distinctive volatiles but an increase in the bioactive isothiocyanates shown in most cultivars (Wieczorek and Jelen, 2019). A few vegetables do contain a typical aroma prior to rupturing the cells. Examples are celery (pathalides), asparagus (1,2-dithiolane-4-carboxylic acid), and bell pepper (2-methyl-3-isobutylpyrazine). 13.2.1.3 Algae There are macroalgae and microalgae. The former is commonly referred to as seaweed which is large, aquatic, and multicellular. Macroalgae can be grouped into three main types: red algae (e.g., nori), green algae (e.g., Ulva spp., sea lettuce), and brown algae (e.g., kelp). The volatile aroma compounds of selected edible macroalgae and microalgae vary with species, life cycle, habitats, geographical origins, and environmental conditions, plus the lability of the chemical compounds during sample preparation, extraction, and analysis. Gaps exist in connecting the key compounds to their sensory characteristics. Macroalgae: Volatiles from commercially important seaweeds (four brown and two red types) include more than 200 types. Hydrocarbons, ketones, aldehydes, alcohols, halogen- or sulfur-containing compounds, acids, esters, furans, and phenols were found in decreasing order. Using wakame (a brown marine algae) as an example, the most abundant hydrocarbons are 4-ethyldecane, pentadecane, and 2-methynonane. Predominant ketones are (Z,E)-3,5-octadien-2-one, 2-propanone, and 2-butanone. Major aldehydes are benzaldehyde, 2-hexenal and hexanal, etc. The most abundant volatiles from nori (a red macroalga) are hydrocarbons: 3-methylundecane, 4-methyldecane, and 2-methylnonane; ketones: (E,E)-3,5octadien-2-one; and aldehydes: hexanal and pentanal. A detailed volatile list has been reviewed by Vilar et al. (2020). A fresh marine green alga (Ulva pertusa) used as a substitute for aonori in Japan, consists of 76 volatile compounds including hydrocarbons, aldehydes, terpenes, alcohols, and sulfuric compounds. Several compounds, 7-heptadecene, hexanal, (E)-2-octenal, (E)-2-nonenal, (Z,E)-2,6,-nonadienal, (E,E)-2,4,-deca-dienal, (Z,Z)8,11-heptadecadienal, (Z,Z,Z)-8,11,14-heptadecatrienal, and (Z)-8-heptadecenal,

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are responsible for the sensory characteristic aroma of the Ulva. Dimethyl sulfde is detected in many seaweeds immediately after crushing them (Sugisawa et al., 1990). Microalgae: It can be cultivated and have great potential for food, feed, nutraceutical, pharmaceutical, and environmental applications (Udayan et al., 2021). Volatiles of microalgae of marine and freshwater species have high levels of sulfuric compounds (dimethyl sulfde, dimethyl trisulfde, and methional), diketones (α- and β-ionone), and aldehydes (2,4-alkadienals and 2,4,6-alkatrienals) to give the fresh algae a seafood-like odor character. Other compounds are alcohols, ketones, and terpenes (Van Durme et al., 2013). Volatiles of marine Crypthecodinium cohnii are high in sulfur compounds (dimethyl sulfde and ethanethiol), esters, and alcohols that give a “sulfur-cabbage, fruity, rosy and boiled potato” aroma. Freshwater Chlorella vulgaris has high levels of ketones and terpene compounds and produces “woody and cereal-like” characteristics (Hosoglu, 2018). Volatiles of selected strains of blue-green algae Spirulina platensis used in novel foods have high concentrations of alkanes such as heptadecane, pentadecane, hexadecane, ketone β-ionone, and alcohol 2,6-dimethylcyclohexanol (Milovanovic et al., 2015). These volatiles have antibacterial effects (Ozdemir et al., 2004).

13.2.2

FLAVORS PRODUCED IN ANIMALS

13.2.2.1 Meats Meat favor is mainly thermally induced by reactions between the non-volatile compounds existing in the lean and fatty tissues. Fresh meats do not have much odor unless they turn rancid or spoiled. The favor compounds of cooked meats consist of hydrocarbons, alcohols, aldehydes, ketones, carboxylic acids, esters, lactones, ethers, furans, pyridines, pyrazines, pyrroles, oxazoles and oxazolines, thiazoles and thiazolines, thiophenes, and other sulfur-containing substances. Sulfur-containing compounds and carbonyl-containing compounds are key volatile components for favor aromatics in meats. These volatile compounds are developed from interactions between amino acids, peptides, reducing sugars, nucleotides, and vitamins, especially thiamine (vitamin B1). Lipids also play an important role in meat favor being distinctive and species-specifc. Pork and chicken contain a higher proportion of unsaturated fatty acids in the triacylglycerols than those of beef or lamb and produce more unsaturated aldehydes volatiles which may differentiate the specifc aromas of meat species (Mottram and Maarse, 1991; O’Sullivan and Kerry, 2012). Feeding meat-producing species different dietary ingredients affects the favor of the meat. Brown, roasted, and umami favors are enhanced when the dried distillers’ grain with solubles (DDGS) replaces cottonseed meal (CSM) and sorghum grain in Dorper lamb feedlot diets. Methyl pyrazine, 2-heptenal, heptanal, and 2-penthyl furan of lamb meat increase with increased DDGS in feed (Hodges et al., 2020). For another example, if chicken feed contains rancid fsh oil, the sensory quality of the chicken meat is lowered. Fish of the same species harvested from a culture pond or wild catch have subtle favor differences due to the difference in feeds. 2-methyl-3-furanthiol (MFT) is a potent meaty favor compound. MFT occurs in a variety of meat products. It is generated from the Maillard reaction between

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carbohydrates and cysteine. It may also be formed from thiamine degradation or enzymatic reactions. MFT is unstable and readily reacts with different components in food systems including forming disulfdes through radical reactions or interacting with melanoidins. MFT also has antioxidant activities that may protect lipid oxidation in meats (Tang et al., 2012). Heme-containing proteins like hemoglobin and myoglobin are naturally occurring in muscle foods. Leghemoglobin (derived from legume + hemoglobin) exists in the legume root nodules including soybean and other nitrogen-fxing plants. It has been used by Impossible Foods™ as an ingredient and created a plant-based hamburger-like product. The uncooked Impossible Burger™ has the red color of fresh meat. When it is cooked, the plant-based burger turns brown and has the aroma, taste, and texture of a beef burger. The meat-like favor results from the chemical reactions involving heme supplied by leghemoglobin, which has a 3-dimensional structure similar to animal myoglobin. Leghemoglobin protein currently is made using recombinant protein technology and has been approved as GRAS (generally recognized as safe) for Impossible Food™ to use in their products (US FDA, 2018). 13.2.2.2 Seafood Fresh fsh odor is perceived in fresh catch. Shortly post-harvest, they develop a fshy smell. Microbial growth causes the biodegradation of proteins to form volatile basic nitrogen compounds (VBN)/total basic nitrogen (TVB-N), mainly ammonia and amines, i.e., trimethylamine (TMA). VBN and TMA are odorous volatiles. Increases in these compounds downgrade the fsh quality. VBN/TVB-N and TMA are generally used as spoilage indicators for hygienic control (Bekhit et al., 2021a, 2021b). The favor compounds of fresh fsh consist of mainly six-, eight-, and nine-carbon alcohols, aldehydes, and ketones. They are derivatives from the unsaturated fatty acids via catalysis by the endogenous lipoxygenases (LOXs) in fshes. The most commonly occurring carbonyl and alcohol compounds, together with their thresholds and aroma, are shown in Table 13.1 which was compiled by Josephson and Lindsay (1986), Lindsay (1990), and Cadwallader (2000). The six-carbon compounds (hexanal, trans-2-hexenal, cis-3-hexenal) provide green plant-like aromas. They are generally found in freshwater fsh but not found in saltwater species. The eight-carbon compounds (1-octen-3-ol, 1-cis-5-octadien3-ol, 1-octen-3-one, 1-cis-5-octadien-3-one) occur in most types of fsh and seafood contributing to heavy plant-like and metallic odors. The eight-carbon alcohols and ketones each have a mushroom or geranium-like aroma, whereas in freshly harvested fsh they contribute to heavy plant-like aromas. The nine-carbon compounds (3,6-nonadienal, 2,6-nonadienal, 3,6-nonadienal) contribute fresh, green, cucumberlike, and melon-like aromas to fsh, similar to those perceived in some vegetables. Lipoxygenases (LOXs) are non-heme iron-containing enzymes that catalyze the oxygenation of polyunsaturated fatty acids in fsh as well as in plants and algae. In aquatic organisms, LOXs are most active in sanguineous tissues such as in fsh gills and blood (Liu and Pan, 2004). LOXs are also present in the ovaries and skin of fsh. Three isozymes, 5-LOX, 12-LOX, and 15-LOX, exist in fsh and shrimp. Their activities vary with species, tissues, and growth stages. The LOX isozymes

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TABLE 13.1 Aroma Compounds and Their Odor Thresholds in Fresh Fish Compound

Concentration range (μg/kg)

Aroma quality

Odor thresholds (μg/I)

Alcohols 1-penten-3-ol (Z)-3-hexen-1-ol 1-octen-3-ol (Z)-1,5-octadien-3-ol (Z)-2-octen-1-ol (E,Z)-2,5-octadien-1-ol (Z)-6-nonen-1-ol (Z,Z)-3,6-nonadien-1-ol Aldehydes Hexanal (E)-2-hexenal (E)-2-octenal (E)-2-nonenal (E,Z)-2,6-nonadienal Ketones 1-penten-3-one 1-octen-3-one

0.5–49 0–0.3 18.6–110 24.8–94.8 6.3–18.8 3.8–24.3 Trace–4.8 Trace–30.1

Grassy, green Green, cut-leaf Mushroomlike Earthy, mushroomlike Fatty, beany Earthy, mushroomlike Green, melon Watermelon, fatty

400 70 1 10 40 10 n.a. 10

8.3–418 0.5–6.3 0–5 Trace–5.8 0.3–17.2

Green, cut-grass Green apple Tallowy, nutty Tallowy, stale Cucumber

4.5 17 3 0.08 0.01

12.2–20.1 0.1–10

Pungent, green Earthy, mushroomlike

1.25 0.005

(Z)-1,5-octadien-3-one

0.1–5

Geraniumlike, metallic

0.001

Source: compiled by Josephson and Lindsay (1986), Lindsay (1990), and Cadwallader (2000).

have a preference for highly unsaturated fatty acids, C22:6 and C20:5 more than C20:4 and C18:3, while C18:2 shows the lowest substrate affnity (Pan and Kuo, 2000). Due to the specifcity and the substrate reactivity of the isozyme on PUFA, different hydroperoxy derivatives are formed and further break down, producing different volatile compounds, thus developing a distinctive seafood aroma. LOXs have signifcant effects on favor formation, either desirable or undesirable. They also play physiological roles in immunity regulation, defense mechanisms, and ovarian development of aquatic organisms (Pan and Kuo, 2000). The contributions of LOXs in seafood favor formation using fresh and cooked shrimp as an example are shown in Figure 13.1. Fresh shrimp is abundant in C8 and C9 alcohols and aldehydes with no shrimp odor, while 5,8,11-tetradecatrien-2-one is formed after cooking, giving a noticeable cooked shrimp aroma (Kuo and Pan, 1991). 13.2.2.3 Flavors Produced by Microbes and Enzymes Fermentation has been practiced over centuries to produce fermented foods like bread and cheese, alcoholic and non-alcoholic beverages, soy sauce, etc., all of which are popular among indigenous peoples and cultures around the world.

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FIGURE 13.1 A favor formation scheme of fresh and cooked shrimp (adapted from Kuo and Pan, 1991).

FIGURE 13.2 Dairy product favor formation.

Lactic acid fermentation can use Lactococcus lactis and Lactobacillus, which belong to an important group of lactic acid bacteria. Traditionally they have been used in dairy, fermented meats and fsh, and fermented vegetables. Lactic acid is the major end product of glucose, while other end products including ethanol, diacetyl, and acetoin together contribute to the favor of the fermented products (de Oliveira, 2014). The overall favor formation mechanism in dairy products is summarized as shown in Figure 13.2. Yogurt made by using mixed starter cultures of Streptococcus thermophilus and Lactobacillus bulgaricus improves the favor produced, better than the single-cultured product, although either strain alone produces lactic acid. Additionally, acetaldehyde and diacetyl are important volatile compounds giving the typical yogurt favor.

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Cheddar cheese consists of at least fve sulfur-containing volatiles, hydrogen sulfde, methanethiol, carbon disulfde, and dimethyl disulfde. Each of them individually may seem unappealing, but in combinations, these compounds give the products their desirable characteristics. Volatile sulfur-containing compounds, i.e., methanethiol, dimethyl disulfde, and dimethyl trisulfde, contribute to a desirable favor of cheddar cheese. The enzyme cystathionine β- and γ-lyase of lactic acid bacteria is able to convert methionine to methanethiol which gives the cheddar cheese aroma. Mixed starter cultures of Brevibacter, Bacilli, and Pseudomonas accelerate the methanethiol production and improve the cheddar cheese favor (McSweeney, 2007). Fermented milk using L. delbrueckii subsp. bulgaricus as starter culture produces acetaldehyde, 3-methyl-butanal, (E)-2-pentenal, hexanal, (E)-2-octenal, and nonanal, which are key odorants with odor activity values (OAV = ratio of the concentration of the compound in sample/its sensory threshold concentration) > 1. Among a total of 86 volatile favor compounds in fermented milk upon completion of fermentation, 17 carboxylic acids, 14 aldehydes, 13 ketones, 29 alcohols, eight esters, and fve aromatic hydrocarbon compounds contribute to its favor profle (Dan et al., 2019). The composition of favor compounds of a product is genuinely complex. Solid-state fermentation and submerged fermentation have been applied to produce biologically active metabolites for food, feed, pharmaceuticals, and industrial chemicals, and have achieved yielding enzymes, antioxidants, organic acids, biosurfactants, and biofuel (Yafetto, 2022). Utilization of solid-state fermentation complimented with submerged fermentation to reuse agro-industrial waste to culture favoring mushrooms may have potential for green economics.

13.3 AROMA COMPOUNDS CLASSIFICATION AND CHEMICAL STRUCTURES 13.3.1

CHEMICAL STRUCTURES AND THEIR ODORS

From natural sources as well as different cooking/processing, many volatile compounds are generated. There are more than 7,000 favor molecules known to exist in nature. These compounds can be chemically classifed according to their functional groups (Reineccius, 2006b). Selections of common classes as examples of odor compounds and their presence in foods are outlined as shown in Table 13.2. These compounds are used in different combinations in favoring formulations for different applications. The aroma of most food favors is made up of hundreds of volatile chemicals. Because of the wide range of structures, thresholds, and characters, there is not much direct correlation between chemical structure and authentic organoleptic properties. Characteristic aroma depends on the combination of a mix of volatile compounds from various chemical classes. A limited number of foods, vanilla (vanillin), banana (isoamyl acetate), and cinnamon (cinnamaldehyde), can be typifed by one single compound. A similar odor may be found in compounds of different

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TABLE 13.2 Common Classes of Odor Compounds and Their Presence in Selected Foods 1

Esters: Isoamyl acetate (fruity, banana)

2a

Terpenes: Linear hydrocarbon: citronellal (citrus-like, lemongrass)

2b

Cyclic hydrocarbon: d-limonene (citrusy, orange/lemon)

3

Aromatic: Benzaldehyde (sweet, foral, spice-like, cherry)

4

Amines: Pyridine (fshy and others)

5

Alcohols: Cis>-hexen-1-ol (grassy green, various)

6

Aldehydes: Anisic aldehyde (foral, sweet, hawthorn, anise, and others) Ketones: Dihydrojasmone (fresh, fruity, jasmine odor with woody/ herbal undertones, also creamy lactonic, fruits like mango/peach and dairy)

7

8

Lactones: γ-decalactone (fruity, peach, creamy, fatty, various)

9

Thiols: Furfuryl mercaptan (roasted coffee, various)

10

Sulfdes: Dimethyl sulfde (cabbage, sulfurous, alliaceous vegetables, marine algae, various bad odors) Others: Example: Diacetyl (α-diketone) (buttery, sweet, creamy, pungent caramel, various)

11

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structures. Compounds of the same structure may exhibit different odor characteristics under different conditions/combinations. The correlation between volatile odor and sensory characteristics has not often been conclusive. Reasons are attributed to poor measurement/identifcation, noise from other compounds/attributes, overlap and variable naming of sensory terminology, unexpected relationships among compounds, and statistical issues (Chambers and Koppel, 2013).

13.3.2

ODOR INTENSITY OF AROMA COMPOUNDS

The odor intensity of aroma compounds can be determined by sensory combining with instrumental methods (GC-O, gas chromatography with olfactometry detector). Odor intensity can be affected by many different factors: release from the food matrix or solvent/carrier base, chemical interactions (duplication/synergistic/antagonistic), panel sensitivity, and data-gathering conditions. Concentration obtained from GC peaks presents a relative quantity of compounds detectable by the conditions set. By sniffng (olfactometry), each compound (or compound combination) exhibits a sensory character. However, a very minor compound in quantity may be a major favor contributor and an abundant compound with a large odor threshold may not play a role to give the food an authentic aroma. To determine the contribution of a chemical compound to a food aroma, odor activity value (OAVs or favor units) is calculated. OAV is the ratio between the concentration of an individual substance in a sample and the threshold concentration of this substance (odor threshold value, the minimum concentration detectable by the human nose). A study of six rice aromas can be used as an example. The 2-acetyl1-1-pyroline with an odor threshold in the air of 0.02 ng/L contributes signifcantly to Jasmine rice aroma with OAV of 191 but less from hexanal (threshold in air 1.1 ng/L, OAV 117) and little from benzaldehyde (threshold in air 85 ng/L, OAV 0.05) (Yang, et al., 2008). The odor intensity of many aroma compounds has been tested and documented in several media including air, water, orange juice, beer, wine, oil, etc. The software Flavor-Base 10th Edition (Leffngwell & Associates, 2015), covering over 4,000 favor chemicals recognized by the US FDA (Food and Drug Administration), FEMA (Flavor Extract Manufacturing Association), GRAS (generally recognised as safe), and EC (European Commission), is available commercially.

13.4 13.4.1

AROMA CHANGES DURING POSTHARVEST STORAGE OF PLANTS SPICES AND HERBS

Herbs can be used fresh or dried. The main aroma characters are from their essential oils. When herbs are harvested at an optimum time (mature and ripe) and used fresh, the aroma is well preserved. The herbs can be dried to extend their shelf-life. These processes may change appearance but aroma quality remains relatively the same unless further grinding or prolonged storage exposure to direct light, heat, and air/ oxygen occurs, which triggers aroma loss and essential oil oxidation.

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Spices mostly require further processing before application. When spices are used immediately and fresh, aroma quality remains stable. For value addition, extraction of essential oil, oleoresin, and derivatives of essential oils and oleoresins are performed (Momin and Jamir, 2021). These processing steps will alter the aroma quality of the spice but will not change the overall characteristics. The aroma quality changes post-harvest depend on the quality of the original materials. Fresh material is harvested at an optimum stage, transported to the processing site immediately, and handled under a clean/sanitary condition to minimize contaminations. High moisture content of fresh material promotes microbial growth and fermentation that adds off-notes. Longer storage without proper packaging material and exposure to light, heat, and air/oxygen will trigger aroma loss and oil oxidation/volatile compound interactions which can be catalyzed by impurities during processing (Hailegeorgis and Anbesse, 2021).

13.4.2

FRUITS AND VEGETABLES

Post-harvest storage of plants affects key aroma compound formation depending on the plant type. Aromatic fruits are affected more than vegetables. These changes largely depend on the biosynthetic pathways during different stages of maturing, ripening, and senescence. Volatile composition changes while maturing and ripening. Metabolic changes occurring during post-harvest storage lead to a general deterioration in favor quality and off-aroma compound formation. Climacteric fruits and non-climacteric fruits follow different traits which relate to ethylene control (Lara, 2010). Storage temperature has a direct impact on volatile quality. Grapefruit stored under moderate-intermediate temperature but not refrigerated conditions promotes nootkatone formation (Pott et al., 2020). The general trend of volatile change during storage is a loss of “green” or “fresh” notes and a concomitant increase in “fruity,” “overripe,” or “musty” aromas. Activation of anaerobic fermentative metabolism due to post-harvest abiotic stress resulting in pyruvate accumulation serves as a substrate for respiration. As a consequence, off-aroma volatiles, namely ethanol, acetaldehyde, and ethyl acetate accumulate, causing fruit aroma quality to decline. Control of the aging process, better monitoring of the process, and optimizing volatile and metabolite generation together achieve optimum fruit quality (Spadafora et al., 2019). Aroma volatiles are mostly developed via precursors. When tissues are broken or damaged, volatile compounds are generated through enzymatic degradation and/or autoxidation reactions of primary and/or secondary metabolites such as amino acids, lipids, glucosinolates, and terpenoids. Loss or change of typical aroma compounds happens during post-harvest storage. The loss or change varies greatly among different vegetables. For example, the characteristic odor of parsley is from the p-mentha-1,3,8-triene, myrcene, 3-sec-butyl-2-methoxypyrazine, myristicin, linalool, (Z)-6-decenal, and (Z)-3-hexenal. During storage, the odor profle of parsley-like and green notes decreases because of losses of p-mentha-1,3,8-triene, myrcene, and (Z)-6-decenal (Husain, 2010).

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13.5 13.5.1

377

THERMAL REACTIONS AND FLAVOR COMPOUNDS FORMATION MAILLARD REACTION

The Maillard reaction has been covered in all food chemistry books. It is one of the most important chemical reactions together with lipid oxidation occurring in foods. The signifcance lies in its role involved in sensory qualities, both browning intensity of color and favor of foods. The industrial application of the Maillard reaction is to produce process favor in model systems, i.e., meaty or beefy favors. The overall scheme of Maillard favor formation is shown in Figure 13.3 which is adapted from Parliament (1989). In the Maillard reaction at the early stage, the key precursors, amines/amino acids and reducing sugars, ensure the subsequent generation of aromas and color. Combinations of different amino acids and reducing sugars react at different rates. Lysine and arginine are amongst the most effective in forming condensation precursors. The aromatic end products produced from the Maillard reaction are generally carbonyl compounds, S-containing compounds, and N-containing heterocyclic compounds. The latter two groups can be perceived at levels as low as 0.001–0.01 mol% (Cerny, 2008). The volatile end products are a combination of various mixtures, depending on the precursors/ingredients, water activity, pH, reaction temperature, and methods of heating, i.e., boiling, baking, frying, or roasting, etc. to give the overall favor notes.

FIGURE 13.3 Aroma compounds formed by Maillard reaction (adapted from Parliament, 1989).

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Muscle foods, which are mostly proteins aside from water and a little sugar, proceed with the Maillard reaction to yield more favor (including taste) compounds than aromas, whereas bakery products having higher sugar content and less protein develop more aromatic molecules and fewer favor molecules. Moreover, the caramelization of sugars in bakery products also develops odor-active compounds and product color. The effects are especially pronounced in drying squid mantle muscle. Different species of squid have different contents of free amino acids. Those squids that consist of high free taurine and proline react with free reducing sugars and appear to have a higher browning intensity than those other squids consisting of other kinds of free amino acids (Tsai et al., 1991). In a model system, each individual amino acid reacts with glucose and attains the Maillard reaction to form the downstream aroma compounds. Cysteine and methionine interacting with glucose produce an umami, meaty, and soy-sauce-like aroma (Wong et al., 2008). The meaty favor formed can be accelerated by the addition of glycine (Zhao et al., 2019) and xylose (Hou et al., 2017). The meaty favor is contributed by molecular weight < 200 Dalton fraction, whereas the browning pigments are polymerized N-containing compounds, melanoidins. A combination of amino acids and sugars can produce different types of aromas, with a stronger note dominating the odor of the mixture. Aromas from the Maillard reaction stimulate positive moods. Aromas generated from glycine/glucose of the Maillard reaction signifcantly decrease negative moods and α-brainwaves of test objects. 2,3-dimethylpyrazine and 2,5-dimethyl-4-hydroxy3(2H)-furanone (DMHF) are the strongest odorants causing the effect (Zhou et al., 2016). Indeed, the Maillard reaction aroma engulfed in the smell system makes the food more enjoyable to the tasters. A comprehensive review of the thermal generation of aromas was published by Parliament (1989). The scope and the intricacies of the Maillard reaction were elucidated by Parker (2015).

13.5.2

LIPID OXIDATION

Lipids are responsible for the development of both desirable and undesirable favors in foods. Lipid oxidation can occur in any foods that contain lipids. The rate of lipid oxidation depends on fat content, fatty acids composition, and presence of antioxidants and/or pro-oxidants, i.e., heme pigments. Thus meats and seafood are more susceptible to lipid oxidation than other food categories because of their fat contents and compositions. Lipid oxidation in foods is mainly of three types: 1. Autoxidation: It occurs in the presence of oxygen. Fatty acids form free radicals and hydroperoxides, which break down into low-molecular-weight odor-active compounds. 2. Photooxidation: When food is exposed to UV radiation, singlet oxygen is formed and reacts with polyunsaturated fatty acids (PUFAs) to form hydroperoxides which proceed to autoxidation.

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3. Enzymatic oxidation: The endogenous lipoxygenases catalyze the reaction between PUFAs and molecular oxygen to form hydroperoxides at specifc sites of the PUFA, then break down to low-molecular-weight odor-active compounds following the route of autoxidation. Aldehydes are the most important lipid breakdown products mainly from the cleavage of linoleic acid after peroxidation. They are predominant in favor because of their low odor threshold and relatively large quantities. The fnal aroma depends on the concentration and the olfactory threshold. For example, the most abundant and important aldehyde in chicken favor is 2,4-decadienal of which the threshold is 0.00007 mg/kg followed by hexanal, its threshold being 0.0045 mg/kg (Ho and Chen, 1994). Other lipid oxidation products include n-alkanals, trans-2-alkenals, 4-hydroxy-trans-2-alkenals, and malondialdehyde. Aldehydes also interact with proteins and proceed to the Maillard reaction forming more complex favor mixtures. Thermally processed methods affect the formation of odor-active compounds and the characteristic favor from the same food or seafood materials. For example, raw shrimp has the characteristic volatile compound of 1-octen-3-ol, cooked shrimp has 5E,8Z11Z-tetradecatrien-2-one (Pan and Kuo, 2000), roasted shrimp has 2,6-dimethyl pyrazine (Okabe et al., 2018), hot-air dried shrimp has 3-ethyl-2,5-dimethylpyrazine (Zhang et al., 2020), and shrimp being blanched or pan-fried has 3-methyl-2,4-nonanedione or 4-hydroxy-2,5-dimethyl-3(2H) furanone respectively (Mall and Schirberle, 2016). Deep-fat frying of shrimp develops more aromas due to the high temperature ca. > 180° C and short time ca. 2–3 min; both oil and shrimp or breaded shrimp undergo hydrolytic, oxidative, and pyrolytic processes and the Maillard reaction, making the fnal aroma more complex and rich, more inviting than shrimp thermally processed by other methods. The undesirable odors of foods are generally associated with lipids more than proteins and carbohydrates. Foods rich in polyunsaturated fatty acids (PUFAs) are labile to oxidation. Long-chain PUFAs, specifcally EPA and DHA, consist of fve and six double bonds and have a very short induction time and a rapid propagation rate of autoxidation. Fish and fsh oil are abundant in EPA and DHA, whereas seed oils or vegetable oils are composed mainly of oleic, linoleic, or linolenic acids containing single, two, or three double bonds respectively. The off-favor of fsh oils is due to the oxidized product, 2-trans,4-cis,7-cis-decatrienal, which has a very low threshold and is responsible for the fshy odor. Other aldehydes, 4-cis-heptenal, 2,4-decadienal, hexanal, and octanal, also contribute to the fshy odor (Fugimoto, 1989). Warmed-over favors (WOF): In cooked meats or leftover fsh and poultry that have been refrigerated for 24 hours, or pre-cooked frozen meats refrigerated for a longer time and then reheated, the WOF can be easily perceived. PUFAs in particular are readily oxidized and give the meat deteriorated odor. Wrapping tightly to create an air barrier may reduce the WOF. Cooking or thermal processing of fsh for a prolonged time results in an increase of H2S and intensifes the WOF (Pan et al., 1997).

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Modifcation of the off odor: In fsh oil treated with an extract of lipoxygenases (LOXs) from a green marine macroalga, Ulva conglobate, the LOX-modifed fsh oil conserved 99% of the originally highly unsaturated fatty acids (containing more than three double bonds). The increased total volatile compounds include higher concentrations of the desirable odor-active unsaturated aldehydes, ketones, and alcohols, with odors resembling fresh fsh, yielding odor notes of apple, citrus, melon, fruit, and oyster. These compounds were tentatively identifed (Hu and Pan, 2000). The same LOX-modifcation treatment is applicable to chicken fat to improve the favor of chicken oil (Ma et al., 2004). Immobilization of the algal LOX is able to stabilize the enzyme activity and extend the shelf-life for use to modify the aroma of oils (Tsai et al., 2008).

13.5.3

INTERACTION OF LIPIDS IN THE MAILLARD REACTION

Lipids and the precursors of the Maillard reaction exist in close proximity in most foods. Lipid oxidation and the Maillard reaction are the most important chemical reactions in food systems. During cooking or processing, lipids oxidize to produce desirable aromas consisting of the acyclic compounds of alcohols, aldehydes, ketones, carboxylic compounds, and hydrocarbons. As the process temperature elevates or reaction time extends, the acyclic compounds diminish and the heterocyclic compounds increase, indicating the aldehydes and ketones play roles similar to the reducing sugars and participate in the Maillard reaction (Ho et al., 1989). Deep-fat fried foods are good examples of the interaction of lipids and the Maillard reaction that creates attractive favors that are so irresistible. On the other hand, a fatty fsh like round herring showed that the TBA value of the dried herring decreased during storage. Even the dried fsh smelled rancid and developed an unacceptable VBN level, and the TBA value was as still as low as < 1.0 (Pan et el., 1978; Chen et al., 1978). The reality is that PUFAs have oxidized and formed malonaldehyde which interacts with the amino acids and ensues in the Maillard reaction, thus reducing the malonaldehyde content available for the TBA reagent to react.

13.6

FLAVOR INDUSTRY: A BLEND OF ART, SCIENCE, AND TECHNOLOGY

The favor industry has been known for being unique and secretive. Company size ranges from billion-dollar multinationals to small local garage operations. The entry barrier can be low if the operation is only simple blending. If a company makes its own starting raw materials, uses different delivery systems in applications, optimizes olfactory smell/taste experiences, and strives to manufacture quality products in a sustainable/green way, the operation becomes very complex and requires advanced R&D set-up and capability to deliver the needed favor. Unless a formulation contains proprietary ingredients developed in-house or requires advanced/unique technology, it is diffcult to be patented. Because the

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majority of compounds are commercially and readily available, once a formulation is open, it is subjected to duplication and loses its value. Besides science and technology, creation of an innovative favor still requires a human touch to perfect it. Cumulative experience and knowledge about raw materials and their combination and handling are vital to the industry for long-term success. In large favor companies, each has its own expertise. Companies like Swiss Givaudan/Firmenich and German Symrise operate in the felds of chemical specialties and favor and fragrance blends; McCormick, a US company, specializes in providing spice seasoning blends for snacks and ready-to-eat meals. A recent global food trend is going “natural” as well as nutraceuticals for health and well-being, which impacts favor companies. Mergers and acquisitions happen to expand the expertise of individual favor companies, i.e., Givaudan acquired Naturex which offers natural ingredients. Firmenich acquired Evonik for its CO2 extraction technology to enhance its capability of offering 100% pure natural ingredients. IFF merged with the Dupont Nutrition and Biosciences unit that offers nutraceuticals.

13.6.1

INGREDIENTS FOR FLAVOR CREATION

Ingredients for favoring creation cover a wide range of raw materials: • Natural origin: Essential oils, oleoresins, absolutes, extracts, and fractions from different crops and crop parts. • Aromatic compounds: Naturally extracted, artifcially synthesized. • Reaction favors (savory/smoke favor): Protein/carbohydrate/fat or fatty acid/other food ingredients. • Herbs and spices (seasoning): Single or blend. Each class of raw materials is unique in its function/performance. It can be used alone or in combination at different proportions depending on the creativity of the favorist to achieve the desired favoring effect. Major raw material classes are: • Essential oils: Natural oils typically obtained by distillation or pressing having the characteristic fragrances of the plant or other source from which it is extracted. • Concretes and absolutes: Plant materials extracted with a hydrocarbon solvent to obtain the concretes. The concrete is then extracted with ethanol/ enfeurage to obtain absolutes. • Oleoresins: From ground herbs or spices by extraction with a selected solvent or solvents which must be totally removed from the fnal product by vacuum distillation. • Concentrates: Concentrated fruit and/or other plant juices. • Aromatic chemicals and blends: Assorted volatile chemical compounds that can be sourced naturally or synthetically made.

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• Reaction favor (or thermal process favoring): A product prepared by heating food ingredients and/or ingredients that are permitted for use in foodstuffs or in process favorings (like cooking foods). • Smoke favors: Via pyrolysis by burning hardwoods followed by condensation, distillation, and fractionation. The favoring raw materials for favor industry application and the operation technologies involved are illustrated in Figure 13.4. Natural raw materials can be sourced from animal or plant origin. Many parts of plants are usable. Via extraction, expression, distillation, or other biotransformation, specifc raw material classes are formed. They are then used in combination or with the addition of aroma compounds to create favors for fnal applications.

13.6.2

FLAVORINGS FOR FOOD INDUSTRIES

More and more food products are created to fulfll demands for ease of preparation, handling, longer storage time, and selection diversity. Flavorings help greatly to extend the range and fexibility of food products and processing technologies. Flavoring for industries can be divided in general by application such as beverages, sweet goods, and savory foods. Beverages include carbonated drinks, still drinks in dry forms, or liquids (water, juice, tea, coffee, milk, energy drinks, milk, liquor, etc.). Sweet goods cover confectionery, bakery goods, ice creams, bubble/ chewing gums, oral care, and pharmaceutical products. Savory food products

FIGURE 13.4 Flavoring raw materials for the favor industry.

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include instant/ready-to-serve meals, meats/sausages, seafood, snacks, etc. Because of the wide application range and many different favor groups/types, detailed classifcation, grouping, and categorization help to foster the favor and food industry for effcient communication and collaboration.

13.6.3

FLAVOR FORMULATION AND LABELING

Formulation: Flavor formulations are trade secrets for favor companies. Natural favor formulations can start by using the composition calculated out based on GCMS-O and sensory analyses or from the empirical database that the favor company owns. Once the basic framework is established, it is up to the creativity of an individual to make a favor richer in characteristics like freshness, greenness, ripeness, creaminess, spiciness, fantasy, etc. Although the same favor type is submitted for application evaluation, it can have various ways of formulating it depending on the favorist and the favor company. Flavorists may try to add different supporting ingredients to make the creation closely resemble the natural counterpart yet still be unique. How many and which supporting compounds to add is subject to individual interpretation. Many factors can impact the formulation variations. They could be the favorist’s know-how/knowledge/preference/experience, the number of ingredients involved, the raw material suppliers, the chemical grade of the compounds, and the different ingredient combinations. A favor formulation can contain several ingredients from a couple to hundreds. Each favor company has its own unique collection of raw materials and processing technologies. Formulations for what is offered may vary greatly for the same favor type/direction. Varying percentages of ingredients or addition of non-infuencing chemicals without tipping the overall sensory balance is possible to keep the favor profle unchanged. Since the ingredient proportion changes, the formulation becomes a new creation that is different from the original. The key to a successful favor formulation is to ensure the favored fnal product meets consumers’ satisfaction. A successful favor formulation does not have bad favor interaction with other food ingredients when applied, can withstand the processing conditions required, and can last through the shelf-life of the product without changes of favor profle. Labeling: Aroma compounds can be chemically categorized as natural, natureidentical (NI), and artifcial. Natural refers to the molecule structures being present in nature. Nature-identical means the compounds naturally exist but they are made synthetically. Artifcial refers to those synthetically made chemicals whose molecular structures do not exist in nature. Vanillin (from vanilla bean), an important favoring compound, is a good example. The chemical structure of vanillin exists in nature but that of ethyl vanillin does not. Extracting vanillin from natural sources can be very costly. Synthesizing it by using less expensive raw materials such as plant lignin can bring costs drastically down. Since the synthetically made vanillin has the exact same structure as the natural one, it is a nature-identical compound. Ethyl vanillin (Figure 13.5) differs from vanillin by having an ethoxy group (–O-CH2CH3) instead

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FIGURE 13.5 Chemical structure of vanillin and ethyl vanillin.

of a methoxy group (–O-CH3). Ethyl vanilla has a more powerful vanilla note than vanillin but its structure does not exist in nature, hence it is “artifcial.” Both vanillin and ethyl vanillin are widely used in different favor/food applications. They need to be labeled differently. Global and local legislation defnes “natural” labeling clearly for different natural raw materials. Flavor labels on food products are regulated in many different countries with variations. They used to follow the classifcation of natural, NI, and artifcial. Due to the consumer perception of resisting “artifcial ingredients in foods,” more and more countries are moving away from labeling favor types. The distinction between NI and artifcial is abandoned. A favor is only labeled as “favoring or favoring substances” in the EU, China, and some other countries. The US is using the natural and artifcial distinction. Labeling “natural favoring” requires specifc criteria to be met in both the EU and the US. According to US and EU regulations, a natural favoring label requires exclusively defned natural favoring substances. Increasing novelties of biotechnology to produce natural favor compounds complicates the “natural favoring substances” defnition and they need to be verifed before claiming the natural status. There are different types of natural favoring. FTNF (from the named fruit) means all favoring ingredients in this favor must come from the favors of the named fruit. WONF (with other natural favors) means this natural favor has been added with natural favor ingredients that are not normally associated with the favor origin. For example, a natural cheddar cheese WONF favor must contain all natural favor ingredients but these can be derived from cheddar cheese as well as natural compounds from other natural mozzarella cheese or butter or milk. Orange juice products often contain FTNF favor. This type of favor addition is exempted from labeling. During the orange juice pressing and concentrating process, some key volatile favor components are lost. The complete fruit should consist of juice and peel notes (foral, fruity essences and juicy notes). Understanding favor components in juice and peel can help identify and restore the fnished juice to the correct fresh aroma/taste balance, closely resembling the fruit when it was frst picked from the tree. A favor company installs collection towers at the orange juice production site to collect those evaporating compounds and analyze/fractionate them to achieve this purpose. This “add-back” process is commonly practiced in the orange juice industry.

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FLAVOR MANUFACTURING AND FLAVOR DELIVERY SYSTEMS

Manufacturing: Flavor manufacturing can involve different technologies and operation arrangements as outlined in the following: • Simple compounding/blending: Mixing of liquids or solids into a uniform blend. • Extraction/distillation/fractionation/purifcation: Processing by separation principles to obtain a single or a blend of compounds from a complex mixture. • Emulsifcation and homogenization: Mixing liquids of different solubilities and making the fnal mixture phase uniform/soluble in a desired food product base. • Powder plating: Mixing a liquid with a solid carrier to uniform for powder application. • Spray drying/drum drying/freeze drying: Drying to remove water. • Encapsulation: Embedding compounds into a protective carrier matrix to increase favor stability or control release time. Extrusion by embedding volatile and unstable favors in a glassy carbohydrate matrix is one process; coacervation and cocrystallization are the others (Madene et al., 2006). • Kettle reaction: Heating a blend of ingredients to generate desired favor compounds. • Others: Industrial biotechnology such as fermentation/bioconversion/synthetic biology with enzymes, microbes, yeasts, and genetic manipulation (Sales et al., 2018). A favor company can have one type of setup (e.g., simple blending in small companies) or several combinations depending on the commercial scope and scale of the company. Delivery systems: Flavors are created for different applications with different costs and performance targets. Because favor raw materials and blends are in very concentrated form, dilution is often required before applications. There is a wide range of solvent selections to dilute the concentrated favor to facilitate application. It ranges from ethyl alcohol to isopropyl alcohol, propylene glycol, benzyl alcohol, glycerol water, edible oils/fractionated oils, triacetin, etc. The selection is largely based on the target performance expected. To make dry powder favor, there is an equally wide range of carrier selections to achieve the delivery purpose. The selection ranges from salt to sugar, starch, modifed starch, maltodextrin, gum arabic and or other vegetable gums, microcrystalline cellulose, etc. All the ingredients used in a favor including solvents and carriers must comply with food ingredient regulations.

13.7.1

EMULSION FLAVORS

Flavor is an essential part of a beverage. Citrus-favored beverages are particularly popular. Most compounded favors with solvent carriers can dissolve in beverages.

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Citrus essential oils and compounds (hydrophobic in nature), however, are not soluble in water nor in water-based solvents. Turning the oils into emulsions is a must in order to create clear or slightly cloudy juice drinks. Because the oils are lighter in density compared to water or beverage bases, they foat to the surface soon after addition. Using a weighing agent to increase the density of the oil is required. An emulsifying agent is added after oil weighing to prevent the oil phase from focculating. Emulsifying agents used typically are gum arabic and modifed starch for beverage applications. For baking applications, xanthan gum is used to ensure stability and baking performance. Bakery emulsion avoids using alcohol-based favors to prevent unpleasant alcohol taste and favor “bake-out.” The emulsion needs to be homogenized to reduce the size of the suspending oil droplets in the water base to gain stability. The optimum droplet size has been suggested to be as close to 1 micron as possible (Reineccius, 2006c). Most favor emulsions are oil-in-water emulsions. Typical emulsion formulations contain an oil (citrus, terpene, or vegetable oil), weighing agents such as brominated vegetable oil (BVO), glyceryl abietate (also known as ester gum or glycerol ester of wood rosin), and sucrose acetate isobutyrate (SAIB), gums like gum arabic or xanthan gum, and water. Water-based emulsions may require the addition of preservatives such as sodium benzoate to prevent microbial growth. If the emulsion is not prepared properly, beverages will have creaming or ringing problems (oils foating on the surface). There are modern nanotechnologies to make microemulsions or nano-emulsions to increase favor-oil stability and transparency of liquids and to incorporate bioactive compounds for use in the food matrix. These micro- and nano-emulsions use different sets of stabilizing agents and emulsifers, different from the conventional beverage emulsions (Aswathanarayan and Vittal 2019).

13.7.2

POWDER FLAVOR

Powder favors can be made using plated or conventional spray drying which offers economical solutions with three to six months of stability. Dried powders still have essential oils exposed to ambient oxygen and light. Conventional spray-dried powders are already more stable than simple plated. Citrus favor powders, in particular, oxidize and change favor profle before product shelf life ends. Oxidation generates off odors. To extend the shelf-life of spray-dried powder favors, an advanced encapsulation technique is applied. Such types of encapsulation can vary among companies. The purpose of encapsulation is to wrap the favor oils in a matrix to minimize oxygen contact. Hence, the shelf-life of the favor can be greatly extended (from six months to several years). Firmenich is the leader in favor encapsulation. Its Durarome series (Firmenich Press Release, 2018) uses extrusion technology with a subsequent solvent wash to produce citrus favors that can last up to four years in comparison to six to 12 months of conventional spray-dried/plated products in room temperature storage. Encapsulation offers a great advantage for powder drinks and other food products such as soups and nutritional supplements in lasting favor profle and favor bursting

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quality. Encapsulation technology also applies to products requiring the “controlledrelease” effect for confectionery and pharmaceutical products. Encapsulation can be achieved by spray drying, spray chilling, extrusion, coacervation, and molecular inclusion. Each method requires different processing technology and selection of matrix material to achieve the application purpose (Madene et al., 2006).

13.7.3

REACTION FLAVORS AND SAFETY CONCERNS

Reaction favors: According to the IOFI (International Organization of the Flavor Industry) defnition, a thermal processing favoring is a product prepared for its favoring properties by heating food ingredients and/or ingredients that are permitted for use in foodstuffs or processed favorings. Production guidelines regarding raw materials, ingredients, production, and process are provided in the IOFI Code of Practices (IOFI, 2020) to ensure safety. • Raw materials: A protein nitrogen source; a carbohydrate source; a fat or fatty acids source and other materials permitted for use. • Other ingredients: Flavoring materials; process favor adjuncts (carriers; emulsifers; antioxidants; stabilizers; anticaking agents, etc.). • Production parameters: Temperature does not exceed 180° C; time does not exceed 15 mins at 180° C; pH does not exceed 8; favoring, favoring substances, favor enhancers, and process favor adjuncts shall only be added after processing is completed and cooled down to below 70° C (this temperature is set by users). • Process (company specifc): Kettle reactor, extrusion reactor, etc. Reaction favors result in a complex cascade of volatile aroma compounds because of the Maillard reaction and other reactions such as lipid oxidation derivatives. Depending on the processing condition, hundreds of mouthwatering aroma compounds are generated and perceived as roasted, baked, grilled, meaty, fried, and savory aromas. Under high temperatures, carcinogenic compounds can be formed at the same time. Food safety has been a concern. Production must follow the IOFI guidelines to keep the concentration of unsafe compounds under control. Safety concerns: Reaction favor includes N-containing compounds, e.g., pyrazines, thiazoles, and their derivatives (Figure 13.6) which are responsible for the desirable favor and aroma of cooked foods. Different polycyclic heterocyclic aromatic amines (HAAs) and polycyclic aromatic hydrocarbons (PAHs) may also be formed at ppm level at high temperatures (Knize et al., 1999). HAAs (Turesky, 2007) and PAHs (Sun et al., 2019) are found in food cooked particularly in fame-grilling, frying, and roasting that produce high levels of mutagenic and carcinogenic derivatives. Therefore, these compounds are to be kept at a safe level to comply with safe reaction production guidelines. The Maillard reaction also generates acrylamide (Figure 13.6), a neurotoxin and carcinogen (Virk-Baker et al., 2014). It is formed at high temperatures via the

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FIGURE 13.6 Structure of pyrazine, thiazole, and acrylamide.

interaction of sugar and amino acid-asparagine. Cooking at high temperatures such as frying, roasting, baking, and toasting particularly plant foods such as potato, grain, and coffee yields acrylamide. A tolerable intake level has been defned as 2.6 micrograms per kg body weight (Tardiff et al., 2010). Reaction favors can use hydrolyzed vegetable proteins (HVP) and soy sauces as the ingredients to provide amino sources. Compounds including 3-MCPD (3-monochloro-1,2-propanodiol) and 1,3-DCP (1,3-dichloro-2-propanol) can be identifed in such reaction favors. They are the products of high-temperature reactions during hydrochloric acid protein hydrolysis (Hamlet et al., 2002). For the 3-MCPD and its fatty derivatives, EU Regulation has set a limit on soy sauce and HVP at 20μg/ kg (EU 2020/1322, 2020). The 1,3-DCP has mutagenic activity. Its concentration is generally lower than 3-MCPD. The US, EU, China, and Korea set maximum limits from 0.02 to 1 mg/kg in acid HVP and soy sauce (Kim et al., 2015).

13.7.4

HERBS AND SEASONINGS BLENDS

Herbs and seasoning blends are complex formulations. They are made for various applications such as condiments, cuisines, restaurant dishes, processed meats/ seafood/ready meals, soups, and snacks. The formulations can include ingredients like ground herbs and spices, spice essential oils/oleoresins plated on carriers, spice extracts, favor enhancers, aromatic favorings, salt, sucrose, MSG, dextrose, maltodextrin, milk whey, sodium caseinate, HVP, yeast extract, preservatives, colors, etc. Ingredient selection is up to the creativity of the chef and the favorist. The application dosages tend to be much higher than aromatic favoring and concentrated raw materials like essential oils/oleoresins. Final seasoning blend profles need to resemble the familiar cuisine dishes depending on the chef’s skill and input by working with favorists mimicking the live cooking process. There are many varieties of ethnic spices and seasoning blends such as Chinese fve spices; chili blends (many variations), curry blends (many areas of India N/E/S/W, Southeast Asia, Japan, Middle East, etc.), salsa blends, and pasta sauce blends that originate from famous local cuisines. One variant of the Chinese fve spices blend can have ingredients like star anise, fennel, clove, cassia, Szechwan pepper/black peppercorns, and also ginger, licorice, cardamon, and dried orange peel (Raghavan, 2000b,c). A chef can adjust the composition or add a special touch to create his/her signature blend. Urbanization increases the need for processed ready-meals and snacks which greatly increases the use of herbs and spices seasoning blends.

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FOOD TRENDS AND FUTURE FLAVOR INDUSTRY

The global food trend is going toward a “plant-based” direction by reducing or eliminating meat consumption. Several factors are driving this trend. 1. Environmental reasons: Global warming is changing the world’s landscape. Cow farming is the largest contributor to CO2 emissions. Reducing meat consumption is thus advocated. 2. Sustainability needs: World population increases faster than resource growth. More foods need to be grown effciently. 3. Health and wellness needs: Due to the pandemic, people care more and more about their own health and wellness. Additional reasons for choosing a plant-based diet are animal welfare and religion. A favor industry trend is “going natural” for consumer health and wellness and “going green” with renewable/sustainable ingredients to achieve carbon-neutral/ water-neutral goals. “Going natural” requires sourcing/producing to be cost-effective and supplying natural ingredients in large quantities. This trend has prompted the merger and acquisition of companies producing natural extracts and nutraceuticals as mentioned in section 13.6. To scale up production and achieve cost effciency of natural ingredients, renewable raw materials and biotechnologies utilizing microbial genetic modifcations are areas of research and development focus (Sharma et al., 2020). Flavor companies having big databases (thousands of compounds, raw materials, sensory profles, descriptors, combinations, formulations, and applications in different food matrixes) are using artifcial intelligence (AI) technology to create favors to shorten the time required to deliver a favor. It offers unlimited creativity because unexpected compound combinations can be generated that go beyond human imagination. Examples of using AI favors technology are Firmenich’s grilled beef taste favor for plant-based meat (Firmenich Press Release, 2020) and Givaudan’s launch of AI tools for product development (Givaudan Media, 2021).

13.8.1

FLAVOR APPLICATIONS

Flavorings are in a concentrated format (even with dilution carrier/solvent) and cannot be consumed directly. Flavoring itself has a self-limiting effect. Overdosing does not make it more effective for sensory and cost reasons. Staying at a suggested application safety level is the best solution. Reasons for adding favors can be several. They include but are not limited to: 1. Offer crops/foods with consistent quality: Crop aroma changes after harvest. Further processing promotes more changes. Adding favor to maintain its original aroma quality is essential for keeping the good’s commercial value. 2. Cover off-notes: Crops can be fractionated into different food ingredients. These ingredients such as protein isolates/concentrates and sweeteners are

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used to formulate different kinds of ready-to-eat/drink processed foods. These fractions often carry poor aroma/taste. Adding favors to mask offnotes improves product acceptance. 3. Replace costly ingredients: Compounds obtained naturally can be very costly such as vanillin from natural vanilla. Artifcially made vanillin or vanillin made by microbes using biotechnology is much less expensive and hence a good alternative for cost control and supply stability. 4. Replace raw materials which are diffcult to handle/source: I.e., some raw materials are very unstable and subject to oxidation quickly. Technologies such as encapsulations are used to address these stability issues. 5. Drive consumer preferences: Food products with innovative favors bring more pleasure to the taster. Besides the compatibility of favoring with the food base, stability during/after processing, and consumer acceptance, favoring application requires other criteria for products to be delivered and perform in a competitive market. These criteria include regulation for a specifc country (compounds permitted and dosage limitation), availability of raw materials in a particular area, cost limitation, and allergen declaration. Complying with regulations is a must. Allergen declaration is essential because of liability. There are growing numbers of food allergic outbreaks worldwide. Declarations of favor free of allergens are critical because of fatal situations that have happened during allergen attacks. There are more than ten different allergens required by food companies worldwide to declare in the favorings applied. These allergens include milk, eggs, fsh, shellfsh, crustaceans, wheat, gluten, peanuts, tree nuts, soybeans, sulftes, etc. In addition, religion-related criteria such as halal and kosher certifcations are required. The going greener trend prompts foods/ingredients to shift to natural, organic, and non-GMO directions. Consumers today demand cleaner labels (as few ingredients as possible and known ingredients from grandmother’s cupboard). Flavor companies are asked to ensure their favors are vegetarian (non-animal origin but accepting egg/ dairy) or vegan (non-animal origin and excluding egg/dairy). Fulflling such green requirements also often requires certifcations on raw materials and facilities.

13.8.2 PLANT-BASED MEAT AND DRINKS Plant-based foods and beverages use protein powders fractionated from plants in formulations. These proteins come from grain cereals, pulses, nuts, and others. Naturally, these ingredients carry mild favors even after cooking. Lacking the richness/fullness and fatty sensation of dairy or meat components makes products taste bland, even with no distinction after heat processing. These ingredients often carry lingering bitterness and off-taste. Adding favors, favor enhancers, spices, and seasonings makes products palatable. Examples like plant-based meats without any animal meats require the addition of reaction favors to impart the meaty aroma either roasted, grilled, stewed, etc. Other food additives are added to achieve functional purposes (taste enhancers for adding mouthfeel/complexity, stabilizers

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to bind/hold ingredients together, and leghemoglobin (refer to 13.2.2.1) for beef bloody sensation). Plant-based beverages (soy milk, almond milk, oat milk, peanut milk) can be made by extraction directly, via enzymatic treatment or protein powder blending. To keep a clean label, favors sometimes are not added. Controlling processing conditions may sustain the natural, pleasant plant aroma. Adding natural favors is common practice to increase taste acceptance and create favor differentiation. The favor industry nowadays is striving to deliver natural and performing favors via different technologies at a reasonable cost. Formulating healthy food products and nutraceuticals requires the addition of favors and mouthfeel enhancers to improve acceptability. Sugar or salt alternatives may carry bitter/metallic off-taste. Flavors added modify overall aroma/taste, mask off-taste, and enhance overall palatability. Umami, which delivers a meaty and brothlike taste, and kokumi, which delivers richness, body, and complexity to savory products, enhance the acceptance of plant-based foods, particularly low-salt and low-fat foods (Ahmad and Dalziel, 2020; Ajinomoto Group Global Website, 2022). Flavor companies invest in research to understand the smell and taste receptors hoping to identify the right favor/taste compounds to couple with the receptors that can turn a tasteless favor into a favorful product to achieve the desired sweetness, saltiness, thickness, creaminess, and richness (Gleason-Allured, 2008). Kokumi substances that do not impart a taste by themselves are found to activate calcium receptors on the tongue resulting in enhancing other tastes and aromas with a different sensation than umami. Consumers have high expectations of plant-based foods that make favor formulation even more challenging.

13.8.3

RECOMBINANT DNA TECHNOLOGY FOR FLAVOR

A process by which either DNA was combined from different genomes or foreign DNA was inserted into a genome has been used in the pharmaceutical industry to produce drugs. Subsequently, a genetically engineered Flavr Savr tomato was introduced to the food industry. The tomato modifed with inserted PG antisense DNA ripens without softening and thus prolongs quality (Bruening and Lyons, 2000). The favor industry has been using biotechnology to produce favoring raw materials. Traditional separation techniques (extraction/distillation) are no longer feasible in recovering minute quantities from complex natural sources effciently and economically. Biotransformation utilizing enzymatic microbial conversion has been adopted to increase quantities and reduce costs. These processes have produced many natural aroma chemicals including various aromatic acids, esters, and lactones (Muheim et al., 1998) Recombinant DNA technology has been used to enhance and alter the favor profle of fruits and plants by overexpressing key metabolic enzymes. Applying GM technology to microorganisms to yield useful fermentation products have been

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adopted by many favor companies in recent years. Research on genetic coding techniques, microbial/yeast strains, culture substrates, and fermentation bioreactor conditions has been expanding to generate a higher yield of existing compounds and create new compounds to fulfll supply and cost demand. These favoring substances derived from GM organisms often can meet all the requirements of natural favors and can be labeled as such. The future of producing natural aroma compounds using recombinant DNA technology is surging. Productions of limonene (commonly in citrus oils); nootkatone (key grapefruit compound); vanillin (vanilla); and raspberry ketone (from red raspberry, which is very expensive) are examples. Microbes used historically as hosts for DNA manipulations include E. coli and yeast Saccharomyce and are advancing to many different selections from wild strains (Sales et al., 2018)

13.8.4

FLAVOR LEGISLATION

Flavors are generally classifed under food additives. Regulation of favorings is generally defned separately with different levels of detail according to the legislations governed by each country. International guidelines provide the reference basis for national regulations. Countries use either a negative control system (defning substances prohibited from inclusion), a positive control system (defning substances permitted for use), or a mixed system (providing both permitted and prohibited items for use). Legislations are regularly updated when novel substances and new safety data are available. Differences among countries in regulating favor components cause great diffculties for favor companies operating globally. One favor regulation to ft all countries is not always possible. Legislation on favorings covers several aspects such as favor labeling, compounds permitted/prohibited to use, safe usage levels, processing requirements (thermal reaction and smoke favors), and worker safety precautions. Since favor compounds are very volatile and can easily pollute the environment, factory design and waste discharge are to be regulated. Because favor compounds are fammable, their handling, storage, and transportation requirements are defned for safety. Declarations along with pertinent certifcations for the favor such as diet-related, religion-related, environmental, and sustainable sources are often required by food companies. These can involve terms defned by different laws. Accuracy is respected. The following organizations are important in keeping the favor industries in order: JECFA: Joint Food and Agriculture Organization (FAO) of the United Nations/ WHO Expert Committee on Food Additives The JECFA organizes evaluation of the safety of food additives including favoring agents and provides fnal operating guidelines on Codex Alimentarius International Food Standards for countries/organizations to adopt. This database provides the most recent specifcations for food additives evaluated by JECFA. IOFI: International Organization of the Flavor Industry

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The IOFI is a global association representing the industry that creates, produces, and sells favorings worldwide. Members are major global favor companies. IOFI publishes the GRL (Global Reference List) and Code of Practice and plays an active role to ensure the international trade of products and add value to the food and beverages consumed around the world. The GRL lists favoring materials that are considered safe for their intended use in food by one or more internationally recognized assessment bodies. The Code of Practice is a commitment to producing favor ingredients that are safe for their intended use, covering guidelines for the manufacture, handling, and use of materials for favor applications. FEMA: Flavor and Extract Manufacturers Association This is the US national association of the favor industry. Members include favor manufacturers, favor users, favor ingredient suppliers, and others with an interest in the US favor industry. The FEMA is also a founding member of the IOFI and provides a favor library to the JECFA for safety evaluation. The FEMA developed a program utilizing the US FDA GRAS (generally recognized as safe) concept to evaluate the safety of favoring substances. A list has been published with FEMA GRAS numbers for substances for use by the industry. www.femafavor.org Each individual country has its own regulatory body to govern the favoring industry. In general, the guidelines and systems established by the JECFA, FEMA, and IOFI are adopted with country variations. Following are three varying examples of favor regulation from the EU, China, and the US: 1. EU favoring regulation, (EC) No. 1334/2008 Flavoring Substances. 2. Chinese favoring regulations, China GB 2760-2015 Flavoring Substances; GB 30616-2020 Food Flavor Safety Standard. 3. US favoring regulation, US Code of Federal Regulations, Title 21, Section 101.22, Foods: Labeling of Spices, Flavorings, Colorings, and Chemical Preservatives.

REFERENCES Abe, K., Hori, Y. and Myoda, T., 2020. Volatile compounds of fresh and processed garlic. Experimental and Therapeutic Medicine 19(2): 1585–1593. Ahmad, R. and Dalziel, J., 2020. G protein coupled receptors in taste physiology and pharmacology – A review article. Frontiers in Pharmacology 11: 587664. https://doi.org/10 .3389/fphar.2020.587664. Ajinomoto Group Global Website. http://www.ajinomoto.com >Products and Innovation>Inn ovation>Innovation in Action>Kokumi substances. Assessed May 14, 2022. Aswathanarayan, J.B. and Vittal, R.R., 2019. Nanoemulsions and their potential applications in food industry. Frontiers in Sustainable Food Systems 3: 95. Bekhit, A.E.-D.A., Giteru, S.G., Holman, B.W.B. and Hopkins, D.L., 2021a. Total volatile basic nitrogen and trimethylamine in muscle foods: Potential formation pathways and

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effects on human health. Comprehensive Reviews in Food Science and Food Safety 20(4): 3620–3666. https://doi.org/10.1111/1541-4337.12764. Bekhit, A.E.-D.A., Holman, B.W.B., Giteru, S.G. and Hopkins, D.L., 2021b. Total volatile basic nitrogen (TVB-N) and its role in meat spoilage: A review. Trends in Food Science and Technology 109: 280–302. https://doi.org/10.1016/j.tifs.2021.01.006. Borlinghaus, J., Foerster Née Reiter, J., Kappler, U., Antelmann, H., Noll, U., Gruhlke, M.C.H. and Slusarenko, A.J., 2021. Allicin, the odor of freshly crushed garlic: A review of recent progress in understanding allicin’s effects on cells. Molecules 26(6): 1505. https://doi.org/10.3390/molecules26061505. Bruening, G. and Lyons, J.M., 2000. The case of the FLAVR SAVR tomato. California Agriculture 54(4): 6–7. Cadwallader, K., 2000. Enzymes and favour biogenesis in fsh. In: Seafood Enzymes. Haard, N.F. and Simpson, B.K. Eds. Marcel Dekker, New York, pp. 365–384. Cerny, C., 2008. The aroma side of the Maillard reaction. Annals of the New York Academy of Sciences 1126(1): 66–71. Chambers, E. IV. and Koppel, K., 2013. Association of volatile compounds with sensory aroma and favor: The complex nature of favor. Molecules 18(5): 4887–4905. Chen, H.-C., Kuo, J.D., Chen, S.S. and Pan, B.S., 1978. Quality changes during storage of commercial dried round herring. Food Science 5(2): 12–24. (Taipei). Dan, T., Ren, W., Liu, Y., Tien, J., Chen, H., Li, T. and Liu, W., 2019. Volatile favour compounds profle and fermentation characteristics of milk fermented by Latobacillus delbrueckii sub sp. bulgaricus.b. Frontiers in Microbiology, 18 September. https://doi .org/10.3389/fmicb 2019.02183. de Oliveira, M.N., 2014. Fermented milks and yogurt. In: Encyclopedia of Microbiology, 2nd ed. Moselio Schaechter. Ed. Elsevier, Cambridge England, pp. 908–922. EU Commission Regulation 2020/1322, 2020, Amending Annex (EC) No 1881/2006 Section 4, As regards maximum levels of 3-MCPD, 3-MCPD fatty acid esters and glycidyl fatty acid esters in certain foods. Offcial Journal of the European Union L310/2. Fan, Z., Hasing, T., Johnson, T.S., Garner, D.M., Schweiterman, M.L., Barbey, C.R., Coquhoun, T.A., Sims, C.A., Resende, M.F.R. and Whitaker, V.M., 2021. Strawberry sweetness and consumer preference are enhanced by specifc volatile compounds. Horticulture Research 8(1): 66. https://doi.org/10.1038/s41438-021-00502-5. Farcuh, M., Copes, B., Le-Navenec, G., Marroquin, J., Cantu, D., Bradford, K.J., Guinard, J.X. and Deynze, A.V., 2020. Sensory, physicochemical and volatile compound analysis of short and long shelf-life melon (Cucumis melo L.) genotypes at harvest and after postharvest storage. Food Chemistry: X 8: 100107. Firmenich Press Release Dec 11, 2018, Firmenich Durarome® celebrates 50 years of long lasting, fresh & authentic tastes. https://www.frmenich.com. Home>Taste-andBeyond>Durarome>Articles. Accessed on May 14, 2022. Firmenich Press Release Oct 2, 2020, Firmenich announces the world’s frst AI created favour, www.frmenich.com. Home>Company/newsroom/press release>Taste&Beyond>Feb 20 2020. Accessed May 14, 2022. Fujimoto, K., 1989. Flavor chemistry of fsh oils. In: Flavor Chemistry of Lipid Foods. Min, D.B. and Smouse, T.H. Eds. The Stephen S. Chang Symposium. American Oil Chemists’ Society, Champaign, IL, pp. 190–195. Givaudan Media, 2021, February 18. Givaudan launches ground-breaking AI tools for next generation product development. www.givaudan.com. Home>media>trade media>2021. Accessed on May 14, 2022. Gleason-Allured, J., 2008. Taste receptor research and the discovery and development of new favour materials (Senomyx). Perfumer & Flavourist 33: 36–38.

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Gonda, I., Burger, Y., Schaffer, A.A., Ibdah, M., Tadmor, Y., Katzir, N., Fait, A. and Lewinsohn, E., 2016. Biosynthesis and perception of melon aroma. In: Biotechnology in Flavor Production, 2nd ed., Chapter 11, Daphna Havkin-Fenkel and Nativ Dudai, Eds. John Wiley & Sons, Wiley-Blackwell, Hoboken, NJ. USA, pp. 281–305. Hailegeorgis, A. and Anbesse, D., 2021. Effect of harvesting stage, form and duration of storage on some physico-chemical properties of Korarima [Afframomum (Braun) P.G.M. Jansen] collected from Masha Woreda, Sheka Zone, Southwestern, Ethiopia. Heliyon 7(11): e08271. https://doi.org/10.1016/j.heliyon.2021.e08271. Hamlet, C.G., Sadd, P.A., Crews, C., Velíšek, J. and Baxter, D.E., 2002. Occurrence of 3-MCPD and related compounds in foods: A review. Food Additives and Contaminants 19(7): 619–631. Ho, C.T., Bruechert, L.J., Zhang, Y. and Chiu, E.M., 1989. Contribution of lipids to the formation of heterocyclic compounds in model. In: Thermal Generation of Aromas. Parliment, T., McGorrin, R.J. and Ho, C.T. Eds. ACS Symposium Series 409. American Chemical Society, Washington DC., pp. 105–113. Ho, C.T. and Chen, Q., 1994. Lipids in food favors - an overview. In: Lipids in Food Flavors. Ho, C.T., Hartman, T.G. Eds. ACS Symposium Series 558. American Chemical Society, Washington, DC, pp. 1–14. Hodges, K.M., Kerth, C.R., Whitney, T.R., Wall, K.R., Miller, R.K., Ramsey, W.S. and Woerner, D.R., 2020. Replacing cottonseed meal and sorghum grain with corn dried distillers’ grains with solubles in lamb feedlot diets: Carcass, trainged sensory panel, and volatile aroma compounds traits. Journal of Animal Science 98(6): skaa182. https:// doi.org/10.1093/jas/skaa182. Hosoglu, M.I., 2018. Aroma characterization of fve microalgae species using solid-phase microextraction and gas chromatography-mass spectrometry/olfactometry. Food Chemistry 240: 1210–1218. Hou, L., Xie, J., Zhao, M., Fan, M., Xiao, Q., Liang, J. and Chen, F., 2017. Roles of different initial Maillard intermediates and pathway in meat favour formation for cysteinexylose-glycine model reaction systems. Food Chemistry 232: 135–144. https://www.fda.gov/Food/IngredientsPackagingLabeling/GRAS/NoticeInventory. Grass Notices Inventory>GRAS Notices> Basic Search 737. Accessed May 16, 2022. Hu, S.P. and Pan, B.S., 2000. Modifcation of fsh oil aroma using a macroalgal lipoxygenase. Journal of the American Oil Chemists’ Society 77(4): 343–348. Husain, Q., 2010. Chemistry and biochemistry of some vegetable favours. In: Handbook of Fruit and Vegetable Flavors. Hui, Y.H. Ed. John Wiley & Sons Inc, Hoboken, NJ, USA. Section B, Part VI.32, pp. 573–626. IOFI (International Organization of the Flavor Industry). 2020, Code of Practice for the Flavor Industry, 5th revision, Item 14. Guidelines for the production of thermal process favorings, p. 36, 37. Jiang, Y. and Song, J., 2010. Fruits and fruit favor: Classifcation and biological characterization. In: Handbook of Fruit and Vegetable Flavors. Hui, Y.H. Ed. John Wiley & Sons Inc., Hoboken, NJ, USA, Section A, Participant Journal 1(A), pp. 3–24. Jones, M.G., Hughes, J., Tregova, A., Milne, J., Tomsett, A.B. and Collin, H.A., 2004. Biosynthesis of the favor precursors of onion and garlic. Journal of Experimental Botany 55(404): 1903–1918. Josephson, D.B. and Lindsay, R.C., 1986. Enzymatic generation of volatile aroma coumpounds from fresh fsh. In: Biogeneration of Aromas. Parliment, T.H. and Croteau, R. Eds. American Chemical Society Symposium Series 317, Chapter 17, Washington DC., USA, pp. 201–221.

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Kim, W., Jeong, Y.A., On, J., Choi, A., Lee, J.Y., Lee, J.G., Lee, K.G. and Pyo, H., 2015. Analysis of 3-MCPD and 1,3-DCP in various foodstuffs using GC-MS, toxicological research. Offcial J. Korean Society of Toxicology 31(3): 313–319. Knize, M.G., Salmon, C.P., Pais, P. and Felton, J.S., 1999. Food heating and the formation of heterocyclic aromatic amine and polycyclic aromatic hydrocarbon mutagens/ carcinogens. Advances in Experimental Medicine and Biology 459: 179–193. Kuo, J.M. and Pan, B.S., 1991. Effect of lipoxygenase on formation of cooked shrimp favour compound, 5,8,11-tetradecatrien-2-one. Agricultural and Biological Chemistry 55: 827–848. Lara, I., 2010. Changes in favour-related volatile production during post-harvest handling of apple and pear fruit. In: Fresh Produce 4 (special issue 1). Global Science Books, pp. 76–83. Global Science Books Ltd., UK. Leffngwell & Associates., 2015. Flavor-Base, 10th ed. http://www.leffngwell.com. Accessed May 14, 2022. Lindsay, R.C., 1990. Fish favors. Food Reviews International 6(4): 437–455. Liu, Y.J. and Pan, B.S., 2004. Inhibition of fsh gill lipoxygenase and blood thinning effects of green tea extract. Journal of Agriculture and Food Chemistry 52(15): 4860–4864. Ma, N.T., Chyau, C.C. and Pan, B.S., 2004. Fatty acid profle and aroma compounds of lipoxygenase-modifed chicken oil. Journal of the American Oil Chemists’ Society 81(10): 921–926. Madene, A., Jacquot, M., Scher, J. and Desobry, S., 2006. Flavour encapsulation and controlled release – A review. International Journal of Food Science and Technology + Technology 41(1): 1–21. Mall, V. and Schieberle, P., 2016. Chracterization of key aroma compounds in raw and thermally processed prawns and lobsters by application of aroma extract dilution analysis. Journal of Agriculture and Food Chemistry 64(33): 6433–6442. McSweeney, P.L.H., 2007. Cheese manufacture and ripening and their infuence on cheese favor. In: Improving the Flavour of Cheese. Weimer, B.C. Ed. Woodhead Publishing Series in Food Science, Technolgy and Nutrition, Cambridge England, pp. 1–25. Milovanovic, I., Misan, A., Simeunovic, J., Kovac, D., Jambrec, D. and Mandic, A., 2015. Determination of volatile organic compounds in selected strains of Cyanobacteria. Journal of Chemistry 2015: 1–6. Ch. Momin, M. and Jamir, A.R., 2021. Harvesting, handling and processing of spices. In: Postharvest Management of Horticultural Crops, Chapter 23, Edited by Mitra Surajit, First Edition. Jaya Publishing House, New Delhi, pp. 458–484. Mookherjee, B.D., Wilson, R.A., Trenkle, R.W., Zampino, M.J. and Sands, K.P., 1989. New dimensions in favor research herbs and spices, favor chemistry trends and developments, american chemical society. Symposium Series 388, Chapter 14, pp. 176–187. Mottram, D.S. and Maarse, H., 1991. Volatile compounds. In: Volatile Compounds in Foods and Beverages. Maarse, H. Ed. Marcel Dekker, New York, USA, pp. 107–178. Muheim, A., Hausler, A., Schilling, B. and Lerch, K., 1998. The impact of recombinant DNA technology on the favor and fragrance industry. Perfumer & Flavorist 23: 21–27. O’Sullivan, M.G. and Kerry, J.P., 2012. Sensory and quality properties of packaged meats. In: Advances in Meats, Poultry and Seafood Packaging. Kerry, J.P. Ed. Woodhead Publishing Series in Food Science, Technology and Nutrition, Cambridge England, pp. 86–111. Ojeda-Sana, A.M., Van Baren, C.M., Elechosa, M.A., Juarez, M.A. and Moreno, S., 2013. New insights into antibacterial and antioxidant activities of rosemary essential oils and their main components. Food Control 31(1): 189–195.

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Okabe, Y., Inoue, Y., Kanda, Y. and Katsmata, T., 2018. Odor-active compounds contributing to the characteristic aroma of shrimp cooked whole, including shell and visera. European Food Research and Technology 245: 233–241. Ozdemir, G., Karabay, N.K., Delay, M.C. and Pazarbasi, B., 2004. Antibacterial activity of volatile component and various extracts of Spirulina platensis. Phytotherapy Research: PTR 18(9): 754–757. Pan, B.S., Kuo, J. D. and Chen, C.C., 1978. Quality evaluation of the commercial dried round herring. Food Science 5(2): 1–11. (Taipei). Pan, B.S. and Kuo, J.M., 2000. Lipoxygenases. In: Seafood Enzymes. Haard, N.F. and Simpson, B.K. Eds. Marcel Dekker, New York, Basel, pp. 317–336. Pan, B.S., Tsai, J.R., Chen, L.M. and Wu, C.M., 1997. Lipoxygenase and sulfur-containing amino acids in seafood favor formation. In: Flavor and Lipid Chemistry of Seafoods. Shahidi, F. and Cadwallader, K.R. Eds. ACS Symposium Series 674. American Chemical Society, pp. 64–75. Parker, J.K., 2015. Thermal generation or aroma. In: Flavour Development, Analysis and Perception in Food and Beverage. Parker, J.K., Elmore, J.S. and Methven, L. Eds. Woodhead Publishing Series in Food Science, Technology and Nutrition, Cambridge England. pp. 151–185. Parliament, T.H., 1989. Thermal generation of aromas - An overview. In: Thermal Generation of Aromas. Parliment, T.H., McGorrin, R.J. and Ho, C.T. Eds. ACS Symposium Series 409. American Chemical Society, pp. 4–5. Peter, K.V., 2001. Processing issues and the functional role of spices. In: Handbook of Herbs and Spices, Chapter 1, CRC Press, Woodland Publishing, pp. 1–31.Woodhead Publishing Limited, Cambridge, England; CRC Press LLC., Boca Raton, FL, USA. Porat, R., Deterre, S., Giampaoli, P. and Plotto, A., 2016. The favor of citrus fruit. In: Biotechnology in Flavor Production, Chapter 1, John Wiley & Sons, pp. 1–31. Edited by Daphna-Havkin-Fenkel and Nativ Dudai, 2nd edition, Wiley-Blackwell, Hoboken, NJ, USA. Pott, D.M., Vallarino, J.G. and Osorio, S., 2020. Metabolite changes during postharvest storage: Effects on fruit quality traits. Metabolites 10(5): 187. https://doi.org/10.3390 /metabo10050187. Raghavan, S., 2000a. The functions of spices. In: Handbook of Spices, Seasonings, and Flavorings, Chapter 3. CRC Press, pp. 23–52., CRC Press LLC, Boca Raton, FL, USA. Raghavan, S., 2000b.Trends in the world of spices today and emerging spice blends and seasonings. In: Handbook of Spices, Seasonings, and Flavorings, Chapter 2. CRC Press, pp. 11–22. CRC Press LLC, Boca Raton, FL, USA. Raghavan, S., 2000c. Emerging Spice blends and Seasongings. In: Handbook of Spices, Seasonings, and Flavorings, Chapter 6. CRC Press, pp. 225–302. CRC Press LLC, Boca Raton, FL, USA. Reineccius, G., 1994. Flavor chemistry. In: Source Book of Flavors, 2nd ed., Chapter 3. Chapman and Hall, pp. 62–74. An Aspen Publication, Maryland, USA. Reineccius, G., 2006a. Flavor formation in fruits and vegetables. In: Flavor Chemistry and Technology, 2nd ed., Chapter 4. CRC Press, pp. 73–98. Taylor & Francis Group, Boca Raton, FL, USA. Reineccius, G., 2006b. Classifcation of aroma compounds by molecular structure. In: Flavor Chemistry and Technology, 2nd ed., Chapter 10. CRC Press, pp. 300–303. Taylor & Francis Group, Boca Raton, FL, USA. Reineccius, G., 2006c, Flavor Production - Emulsions in Flavor Chemistry and Technology, 2nd ed., Chapter 13.3. CRC Press, pp. 352–360. Taylor & Francis Group, Boca Raton, FL, USA.

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Ribeiro-Santos, R., Carvalho-Costa, D., Cavaleiro, C., Costa, H.S., Albuquerque, T.G., Casilho, M.C., Ramos, F., Melo, N.R.. and Sanches-Silva, A., 2015. A novel insight on an ancient aromatic plant: The rosemary (Rosmarinus offcinalis L.). Trends in Food Science and Technology 45(2): 355–368. Sales, A., Paulino, B.N., Pastore, G.M. and Bicas, J.L., 2018. Biogeneration of aroma compounds. Current Opinion in Food Science 19(2): 77–84. Sharma, A., Sharma, P., Singh, J., Singh, S. and Nain, L., 2020. Prospecting the potential of agroresidues as substrate for microbial favour production: A mini review. Frontiers in Sustainable Food Systems 4: 18. Siegmund, B., 2015. Biogenesis of aroma compounds: Flavor formation in fruits and vegetables. In: Flavor Development, Analysis and Perception in Food and Beverages, Chapter 7. Woodhead Publishing Series in Food Science, Technology and Nutrition, pp. 127–149. Edited by J.K Parker, J.S. Elmore and L. Methven, Woodhead Publishing, Elsevier Ltd., Cambridge England. Simon, J.E., Morales, M.R., Phippen, W.B., Vieira, R.F. and Hao, Z., 1999. Basil: A source of aroma compounds and a popular culinary and ornamental herb. In: Perspectives on New Crops and New Uses. Janikck, J. Ed. ASHS Press, Alexrandria, VA, pp. 499–505. Spadafora, N.D., Cocetta, G., Cavailuolo, M., Bulgari, R., Dhorajiwala, R., Ferrante, A., Spinardi, A., Rogers, H.J. and Muller, C.T., 2019. A complex interaction between pre-harvest and post-harvest factors determines fresh-cut melon quality and aroma. Scientifc Reports 9(1): Article Number:2745. https://doi.org/10.1038/s41598-019-39196 -0. Sugisawa, H., Nakamura, K. and Tamura, H., 1990. The aroma profle of the volatiles in marine green algae (Ulva pertusa). Food Reviews International 6(4): 573–589. Sun, Y., Wu, S. and Gong, G., 2019. Trends of research on polycyclic aromatic hydrocarbons (PAHs) in food: A 20-year perspective from 1997–2017. Trend in Food Science and Technology 183: 86–98. Tang, W., Jiang, D., Yuan, P. and Ho, C.T., 2012. Flavor chemistry of 2-methyl-3-furanthiol: An intense meaty aroma compound. Journal of Sulfur Chemistry 34(1–2): 38–47. Tardiff, R.G., Gargas, M.L., Kirman, C.R., Carson, M.L. and Sweeney, L.M., 2010. Estimation of safe dietary intake levels of acrylamide for humans. Food and Chemical Toxicology: An International Journal Published for the British Industrial Biological Research Association 48(2): 658–667. Tsai, C.H., Pan, B.S. and Kong, M.S., 1991. Browning behavior of taurine and proline in model and dried squid systems. Journal of Food Biochemistry 15(1): 67–77. Tsai, C.J., Li, W.F. and Pan, B.S., 2008. Characterization and immobilization of marine algal 11-lipoxygenase from Ulva fasciata. Journal of the American Oil Chemists’ Society 85(8): 731–737. Turesky, R.J., 2007. Formation and biochemistry of carcinogenic heterocyclic aromatic amines (HAA) in cooked meats. Toxicology Letters 168(3): 219–227. Udayan, A., Pandey, A.K., Sharma, P., Sreekumar, N. and Kumar, S., 2021. Emerging industrial applications of microalgae: Challenges and future perspectives. Systems Microbiology and Biomanufacturing 1(4): 411–431. US FDA. 2018. GRAS Notice (GRN) No. 737 for Soy Leghaemoglobin Protein Preparation Derived from Pichia pastoris. Van Durme, J., Goiris, K., De Winne, A., DevCoorman, L. and Muylaert, K., 2013. Evaluation of the volatile composition and sensory properties of fve species of microalgae. Journal of Agriculture and Food Chemistry 61(46): 10881–10890. Vilar, E.G., O’Sullivan, M.G., Kerry, J.P. and Kilcawley, K.N., 2020. Volatile compounds of six species of edible seaweed: A review. Algal Research 45(4): 101740.

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Virk-Baker, M.K., Nagy, T.R., Barnes, S.. and Groopman, J., 2014. Dietary acrylamide and human cancer: A systematic review of literature. Nutrition and Cancer 66(5): 774–790. Wieczorek, M.N. and Jelen, H.H., 2019. Volatile compounds of selected raw and cooked Brassica vegetables. Molecules 24(3): 391. Wong, K.H., Aziz, S.A. and Mohamed, S., 2008. Sensory aroma from Maillard reaction of individual and combinations of amino acids with glucose in acidic conditions. Intern. Food Science and Technology 43: 1512–1519. Yafetto, L., 2022. An application of solid-state fermentation by microbial biotechnology for bioprocessing of agro-industrial wastes from 1970–2020: A review and bibliometric analysis. Heliyon 8(3): e09173. https://doi.org/10.1016/j.heliyon.2022.e091. Yang, D.S., Shewfelt, R.L., Lee, K.S. and Kays, S.J., 2008. Comparison of odor-active compounds from 6 distinctly different rice favor types. Journal of Agriculture and Food Chemistry 56(8): 2780–2787. Yang, S., Hao, N., Meng, Z., Li, Y. and Zhao, Z., 2021. Identifcation, comparison and classifcation of volatile compounds in peels of 40 apple cultivars by HS-SPME with GC-MS. Foods 10(5): 1051. Yoshimoto, N. and Saito, K., 2019. S-alk(en)ylcysteine sulfoxides in the genus Allium: Proposed biosynthesis, chemical conversion, and bioactivities. Journal of Experimental Botany 70(16): 4123–4137. Zhang, Q., Ji, H., ,Liu, S. and Gao, J., 2020. Similarity of aroma attributes in hot-air-dried shrimp (Panaeus vannamei) and its different parts using sensory analysis and GC-MS. J. Food Research International (Ottawa Ont.) 137: 109517. https://doi.org/10.1016/j .foodres.2020.109517. Zhao, J., Wang, T., Xie, J., Xiao, Q., Du, W., Wang, Y., Cheng, J. and Wang, S., 2019. Meat favor generation from different composition patterns of initial Maillard stage intermediates formed in heated cysteine-xylose-glycine reaction systems. Food Chemistry 274: 79–88. Zhou, L., Ohata, M. and Arihara, K., 2016. Effects of odor generated from the glycineglucose Maillard reaction on human mood and brainwaves. Food and Function 15(6): 2574–2581. Zhu, X., Li, Q., Li, J., Luo, J., Chen, W. and Li, X., 2018. Comparative study of volatile compounds in the fruit of two banana cultivars at different ripening stages. Molecules 23(10): 2456.

14

The Role of Food Additives Joanna Le Thanh-Blicharz and Jacek Lewandowicz

CONTENTS 14.1 Introduction .................................................................................................. 401 14.2 Additives That Extend Shelf-Life of Food Products ....................................403 14.2.1 Preservatives .....................................................................................403 14.2.2 Acidity Regulators ............................................................................406 14.3 Additives Infuencing Sensory Perception of Food Products .......................408 14.3.1 Sweeteners ........................................................................................408 14.3.2 Flavor Enhancers .............................................................................. 410 14.4 Additives with Structure-Promoting Properties ........................................... 411 14.4.1 Hydrocolloids.................................................................................... 412 14.4.2 Emulsifers ........................................................................................ 413 14.5 Recent Trends in the Use of Food Additives ................................................ 414 14.6 Principles of Safety Assessment of Food Additives ..................................... 415 14.7 International Regulations Governing the Use of Food Additives ................ 416 References.............................................................................................................. 417

14.1 INTRODUCTION According to the Codex Alimentarius, a food additive means any substance not normally consumed as a food by itself and not normally used as a typical ingredient of the food, whether or not it has nutritive value, that is intentionally added to food for a technological (including sensory) purpose in the manufacture, processing, preparation, treatment, packing, packaging, transport, or holding of such food results, or that may be reasonably expected to result (directly or indirectly) in it or its by-products becoming a component of or otherwise affecting the characteristics of such foods. The term technological is crucial as contaminants or substances added intentionally to improve or maintain the nutritional value of the food are not considered additives (Codex Alimentarius CXG 36-1989). However, in the European Union, according to Regulation (EC) No 1333/2008, certain groups of substances that would meet the criteria of the aforementioned defnition, i.e. enzymes and favorings, are not considered food additives, as they are covered by other legislative acts: Regulation (EC) No 1332/2008 (on food enzymes) and Regulation (EC) No 1334/2008 (on favorings and certain food ingredients with favoring properties for use in and on foods). The approach toward the separation of favors and enzymes is consistent throughout the

DOI: 10.1201/9781003265955-14

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literature on the subject and therefore will not be elaborated in this chapter. More information on the use of enzymes in food processing can be found in Chapter 6, whereas Chapter 13 covers food favors. Fulflling the increasing demand for food caused by an ever-growing human population and the continuous development of new types of products would not be possible without the use of appropriate additives. The use of food additives facilitates the preservation of food quality including nutritional as well as sensory aspects, but above all the use of additives is required to ensure shelf-life and/or safety. The main purpose of food additives became the factor that contributed to the classifcation of them in Codex Alimentarius into 27 functional classes: acidity regulator, anticaking agent, antifoaming agent, antioxidant, bleaching agent, bulking agent, carbonating agent, carrier, color, color retention agent, emulsifer, emulsifying salt, frming agent, favor enhancer, four treatment agent, foaming agent, gelling agent, glazing agent, humectant, packaging gas, preservative, propellant, raising agent, sequestrant, stabilizer, sweetener, or thickener. The functional classes are further assigned with overall 90 technological purposes (Codex Alimentarius CXG 36-1989). The classifcation in the European Union listed in Regulation (EC) No 1332/2008 is similar and divides food additives into 26 different types based on the technological function they reveal. The differences include the exclusion of bleaching and carbonating agents as well as the inclusion of modifed starch (function). The United States approach is partially different, as the Substances Added to Food inventory published by the US Food and Drug Administration (FDA) lists substances with overall 38 technical effects that partially overlap with both functional classes and technological purposes listed in Codex Alimentarius and include: anticaking agent or free-fow agent, antimicrobial agent, antioxidant, boiler water additive, color or coloring adjunct, curing or pickling agent, dough strengthener, drying agent, emulsifer or emulsifer salt, enzyme, frming agent, favor enhancer, favoring agent or adjuvant, four treating agent, formulation aid, freezing or cooling agent, fumigant, humectants, leavening agent, lubricant or release agent, malting or fermenting aid, masticatory substance, nonnutritive sweetener, nutrient supplement, nutritive sweetener, oxidizing or reducing agent, pH control agent, preservative, processing aid, propellant, sequestrant, solvent or vehicle, stabilizer or thickener, surface-active agent, surface-fnishing agent, texturizer, tracer, and washing or surface removal agent. Partially the difference in this approach relates to the inclusion of substances used as food ingredients, processing aids, for treatment of water for human consumption, enzymes, and favorings, which are not included in Regulation (EC) No 1332/2008. Nevertheless, the approach toward technological functions of food additives can be different also due to the fact that the use of a single substance can beneft the food product in numerous ways. For example, starch sodium octenyl succinate or acetylated distarch adipate would be usually labeled with a technological function as modifed starch, whereas they can also reveal emulsifying, stabilizing, thickening, or gelling properties as well as being used as a carrier. A more systematic approach toward the classifcation of food additives is the use of the International Numbering System for Food Additives (INS). The INS was frst published in 1989 and is an open list subject to the inclusion of additional additives or the removal of existing ones on an ongoing basis (Codex

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Alimentarius CXG 36-1989). The INS number consists of three to four digits that can be additionally followed by an alphabetical symbol to further characterize different alterations of an additive. Currently, the INS list includes 607 additives (while the EU permits the use of 334) that are listed according to their basic property. Numbers 100–199 are used for colors; 200–299 for preservatives; 300–399 for antioxidants and acidity regulators; 400–499 for thickeners, emulsifers, and stabilizers; 500–599 for acidity regulators and anticaking agents; 600–650 for favor enhancers; 900– 969 for miscellaneous (including 900–915 glazing agents, 916–930 four treatment agents, 940–949 propellants and packaging gases, 950–969 sweeteners); 999–1599 for others that do not ft into previous categories, while 1400–1499 are used for modifes starches. The INS classifcation might be helpful for labeling and fast identifcation of additives by consumers but is far from perfect when it comes to classifcation related to the continuous evolution of the list. Nevertheless, among food properties that are altered by the addition of additives, three main aspects should be mentioned, i.e. extension of shelf-life, structure/texture modifcation, and sensory aspects.

14.2 ADDITIVES THAT EXTEND SHELF-LIFE OF FOOD PRODUCTS Shelf-life of food products is dependent upon several factors that are infuenced by the following processes: physiological (respiration, transpiration, and ripening); chemical (oxidation, hydrolysis, and Maillard’s reactions); biochemical (native and microbial enzymes); physical (sorption and desorption of gases, retrogradation of starch, and frost damage); and microbiological (bacteria, yeast, and molds). The signifcance of the aforementioned processes is related to various aspects of the food product itself, including its composition, water activity, pH, processing technique, packaging material, as well as preservation technique. Dry products with low water activity will be more prone to oxidative deterioration rather than microbiological. Fermented products with low pH will be more prone to fungal attack rather than bacterial. The theoretical number of different implications is almost infnite as there are numerous ways for food preservation that extend the shelf-life but also can alter the potential cause of spoilage. Among food additives, four groups of substances can be considered as those that help to extend the shelf-life of food products: preservatives, acidity regulators, stabilizers, and antioxidants (including sequestrants). The mechanism of action of stabilizers is strongly related to their emulsifying properties or ability to increase the viscosity of the system, therefore they will be discussed along with additives with structure-promoting properties. Chapter 11 was dedicated to the latter group.

14.2.1 PRESERVATIVES Preservatives are substances used to achieve microbiological stability of food products and can be divided into two subcategories: antiseptics and antibiotics. The frst group consists of rather simple compounds with a molecular mass usually below 200 Da and used at sub-gram per kilogram of product levels, whereas antibiotics are more complex substances that are used at much lower concentrations, below 20

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mg/kg of product. Due to the great importance of antibiotics in healthcare, only two antibiotics that are not considered as effcient for medical purposes are currently assigned with INS codes and can be used as food additives – nisin (234) and natamycin (235). The use of preservatives should only be considered if physical preservation techniques such as drying, pasteurization, sterilization, freezing, or concentration are not suitable or effective. Those include products that cannot be subjected to thermal treatment or that are intended for use after opening for several days or weeks. This relates to a small contribution of preservatives in the global market as only about 5% of additives are used for safety reasons. The average consumption of preservatives in industrialized countries is estimated between 350 and 400 grams annually per capita (Kaplan 2019). While this value may seem relatively low, the acceptable daily intake (ADI) for preservatives is usually below 25mg/kg (JECFA) which equals 2 grams per day for average human body weight. Considering the exposure to preservatives in other fast-moving consumer goods, i.e. cosmetics and detergents, the estimated daily intake may be higher (Le Thanh-Blicharz and Lewandowicz 2020) and presumably exceed ADI. The 2001 UE report on dietary intake of food additives indicated that consumption of sulftes (INS 220–228) and nitrates (INS 249–250) exceeded the ADI levels (Commission of the European Communities 2001). Currently, the use of preservatives (1333/2008) is strictly limited both in terms of groups of food products that they can be added to and in terms of addition levels. Antiseptics are far from perfect additives, not only due to safety reasons. While they extend the shelf-life of food products by inhibiting the growth of microorganisms, their effectiveness may be limited to certain groups (bacteria, yeasts, molds) or even certain strains. The effectiveness of the most commonly used preservatives against microorganisms is presented in Table 14.1. Therefore, in order to achieve proper microbiological stability, often a combination of two additives may be used that provide synergistic effects, i.e. sorbates that are more effective against fungi and benzoates that inhibit bacterial growth. Moreover, to further enhance the safety

TABLE 14.1 Effectiveness of Preservatives against Microorganisms Group

INS

Bacteria

Yeasts

Molds

Sorbates

200–203

*

***

***

Benzoates Parabens Sulftes Nitrates Nisin

210–213 214–219 220–228 249–250 234

** ** ** ** **

*** *** * – –

*** *** * – –

Natamycin

235

–

***

***

Notes: – not effcient, * partially effective, **effective, *** very effective. Source: own work based on Rutkowski et al. (2003).

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of food products, the hurdle of technology may be considered. This includes modifcation of several factors that infuence microbial growth, among others: storage temperature, water activity, or pH value. The proper combination of different factors results not only in the reduction of the required preservative dose but also improves product quality and safety. Sorbic acid is a naturally occurring compound in berries, especially in unripe fruits of Sorbus aucuparia (rowan). Sorbic acid is particularly effective in non-dissociated form, therefore its effectiveness increases with lower pH. It is effcient in pH values between 3 and 6, especially against fungi, but also against certain species of bacteria that among others include: Bacillus, Clostridium, Pseudomonas, Salmonella, and Staphylococcus. Its greatest limitation is related to a lack of effectiveness against lactic acid bacteria. Nevertheless, this property makes sorbates a valuable preservative for fermented food products. Sorbates are considered relatively safe additives as they are metabolized similarly to fatty acids in the process of beta-oxidation. The ADI of sorbates is 25 mg/kg and is the highest among all preservatives used (JECFA), while the permitted dosage for most products is 0.1% (Regulation (EC) No 1333/2008). However, long-term studies on exposure to higher doses of sodium sorbate provided conficting results. Some indicated that sodium sorbate has in vitro a genotoxic effect on lymphocytes, whereas in others this effect was not observed. The controversy regarding the safety of sorbates as food additives resulted in their prohibition in the USA (Le Thanh-Blicharz and Lewandowicz 2020). Benzoic acid and its esters are also naturally occurring compounds in berries at concentration levels of approximately 0.05% (Le Thanh-Blicharz and Lewandowicz 2020). Therefore this amount in the form of benzoic acid and its salts is permitted for use in most applications. However, in a few applications, it can be used at elevated levels as high as 0.2%, while for beverages only 0.02% of benzoic acid can be used (Regulation (EC) No 1333/2008). Benzoates, similarly to sorbates, are most effcient in acidic environments with a pH value below 5.0, with a further increase in effectiveness at lower pH levels. At pH values of 3.5–4.0 it will exhibit an antimicrobial effect at a concentration of 0.08% with action against fungi and most bacteria (not effective against butyric and acetic bacteria). The presence of sulfur dioxide, carbon dioxide, salt, sugar, and sorbates can further increase its effectiveness (Rutkowski et al. 2003). Benzoic acid and its salts are considered relatively safe additives with an ADI of 5 mg/kg. Nevertheless, the use of benzoic acid in some non-alcoholic drinks raises doubts due to the fact that benzoic acid and its salts can react with ascorbic acid creating small amounts of carcinogenic benzene. Moreover, its consumption at elevated levels may result in allergic reactions in asthmatics and allergy sufferers. Parabens or actually esters (methyl, ethyl, or propyl) of p-hydrobenzoic acid and their salts are more effective in higher pH spectrum (3–8) than sorbates and benozates. In the form of esters, they are poorly soluble in water, especially with the increase of alkyl chain length. Their solubility in water is greatly improved in the form of salt derivatives, but as a a consequence, they tend to be easily hydrolyzed. Parabens are of minor importance as far as food technology is considered but are used on a large scale in cosmetics.

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Sulfur dioxide was used in wine production for thousands of years as an agent that prevented the growth of wild yeast and lactic and acetic acid bacteria. It is also effective against molds. Sulfur dioxide and sulftes inhibit the activity of oxidoreductive enzymes, thus stabilizing wine color (preventing browning). For the same reason, sulftes are also used in the production of dried fruits; however, extensive use of them leads to deterioration of quality. In higher concentrations, they reveal cytotoxic and carcinogenic effects. Moreover, they are known to hinder the immune system and be allergens. Products with concentrations of sulfur dioxide > 10 mg/kg in the EU require additional labeling (WE 1169/2011). Sulfur dioxide easily evaporates and after storage, a considerable amount of this gas may be separated from the product, therefore it is recommended to let wine “breathe” after opening. Nitrates(III) and nitrates(V) are used as preservatives as well as color retention agents for meat products. Nitrates are only effective against bacteria and their effciency increases at lower pH. Nitrates(V) do not exhibit direct antibacterial effects but are rather converted by microbial enzymes to nitrates(III). The use of nitrate salts in food technology raises numerous concerns, i.e. they contribute to formation of cancerogenic N-nitrosamines or oxidation of oxyhemoglobin to methemoglobin. The main reason for the approval of nitrates(III) as food additives is related to the inhibition of the growth of Clostridium botulinum, which produces an extremely deadly neurotoxin. Therefore, their use is strictly limited and in the EU nitrates(III) can be used only to for selected meat preparations, and nitrates(V) for pickled herring and sprat as well as selected cheese products (Regulation (EC) No 1333/2008). Nisin is an antibacterial peptide produced by the Lactococcus lactis bacteria. It is effective only against gram-positive bacteria including Bacillus, Clostridium, Lactobacillus, and Listeria (Chena and Hoover 2003). Nisin is used in milk-derived products in doses not exceeding 12.5 mg/kg, mainly in cheese, but is also allowed for use in clotted cream, mascarpone, puddings, and liquid eggs (Regulation (EC) No 1333/2008). Natamycin is an antifungal polyene antibiotic produced by species of the genus Streptomyces. It is effective against most fungi, with minimal inhibitory concentrations in the micromolar range, being signifcantly more effective than sorbates. When applied on the surface of foods, it does not affect their sensory properties (taste, texture, and color), and has prolonged antimicrobial activity (Aparicio et al. 2016), being safe for consumption because its oral absorption is negligible. Nevertheless, in the EU it is used only for surface treatment of cured sausages and ripened cheese (Regulation (EC) No 1333/2008).

14.2.2 ACIDITY REGULATORS Acidity regulators are a group of substances that can be considered as prolonging the shelf-life of food products by inhibition of microbial growth. Nevertheless, they play an important role in the perception of sensory properties, i.e. by stabilization of color, especially in products containing anthocyanins and betalains. Moreover, acidity regulators, especially organic acids, play an important role in shaping the sour taste of numerous food products. In terms of acidity, food products can be divided

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into three categories, with pH < 3.7 – fermented or pickled vegetables, citrus fruits, carbonated beverages, wine; between 3.7 and 4.5 – most fruits, fermented milk products, beer; and > 4.5 – milk, meat, fsh, vegetables, legumes. The 4.5 pH threshold is crucial for food preservation as it allows for pasteurized products to achieve proper microbiological stability, thus excluding the need for sterilization. Moreover, the activity of hydrogen ions is also an inhibitory factor in microbial growth (Rutkowski et al. 2003): < 5.0 putrefying bacteria, < 4.0 butyric acid bacteria, < 2.5 lactic acid bacteria and yeast, < 2.0 molds. The regulation of pH value is not the only factor infuencing the shelf-life of food products. The addition of certain acids and their salts such as acetic, citric, or lactic interact with bacteria cell walls and can effectively inhibit the growth of certain bacteria species, including Listeria monocytogenes, Salmonella typhimurium, Escherichia coli, Aspergillus favus, Penicillium purpurogenum, etc. (Braiek and Samaoui 2021). Most organic acids and their salts are considered safe and do not have an assigned ADI value; those include acetic, lactic, malic, fumaric, citric, and ascorbic. The only exception is tartaric acid with an ADI of 30 mg/kg, but it can also be used according to the quantum satis rule, meaning that it can be used according to good manufacturing practice (GMP) in levels required to achieve the desired technological result, but not more. The addition of organic acids changes the sour taste of food products to a different extent. It is accepted that the sensory perception of common organic acids increases in the order: acetic < lactic < malic < citric < tartaric (Rutkowski et al. 2003). The choice of proper acid may be a diffcult task, while it is relatively easy to set the pH to the designated value with the addition of organic acids and their salts (most of them reveal buffering properties). Their sensory acceptance is different in many products. Acetic acid is commonly used for pickled vegetables, while citric and malic acid as well as their mixtures are well accepted in fruit preserves and fruit-favored products including sweets and beverages. While still common, other organic acids are used to a much lesser extent. Among inorganic acids used in food technology, phosphoric and carbonic play the most important roles. The frst one in its pure form is an important factor in the development of the taste of cola-type beverages, but in form of phosphoric acid salts (sodium, potassium, calcium, magnesium), it is commonly used as a stabilizer and water-binding substance in the production of various milk- and meat-derived products. The maximum permitted dose of phosphoric acid and its salts varies greatly and may be as high as 5% (for beverage whiteners for vending machines) and as low as 0.05% (favored sports drinks), while the most common doses are between 0.1 and 1.0% (Regulation (EC) No 1333/2008). The technological importance of carbonic acid is not directly related to the activity of hydrogen ions, as it is a very weak acid and saturation of water with carbon dioxide will result in a pH of approximately 4.2. Its primary use is related to the addition of a refreshing sparkling taste to beverages. Moreover, in gaseous form, it is used as an element of controlled atmosphere and modifed atmosphere packaging. Carbon dioxide is also used as a solvent for supercritical extraction. The applicability of most salts of carbonic acid is related to their ability to decrease acidity or as raising agents due to degradation into carbon dioxide and water in acidic environments.

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14.3 ADDITIVES INFLUENCING SENSORY PERCEPTION OF FOOD PRODUCTS Sensory attractiveness is a crucial factor determining the market success of every food product. Ongoing globalization and mass customization that currently shapes many consumer expectations create a necessity for continuous improvement as well as the development of new types of food products. This would not be possible without numerous additives that facilitate the sensory design of food products. Table 14.2 links common groups of additives with properties they alter in exemplary products. Apart from the improvement of color, taste, and texture of “classical” food products, sensory additives are the foundation that builds many products with particularly benefcial nutritional properties, i.e. low energy, low fat, and low sodium.

14.3.1 SWEETENERS Sweeteners are sugar substitutes that can be divided into two main categories: substances that have intensively sweetening properties and sugar mimetics. The frst group consists mainly of artifcial sweeteners and their role in food products is limited to the development of sweet taste. The latter consists mainly of sugar alcohols and is usually used for substitution in a 1:1 ratio. Artifcial sweeteners usually do not have an energy value, with some exceptions including aspartame (4 kcal/g) or neohesperidin dihydrochalcone (2 kcal/g). Nevertheless, due to their high sweetness which is at least one level of magnitude higher than that of sucrose (relative sweetness values are presented in Chapter 5), the potential caloric value of artifcial sweetener introduced to the product is negligible.

TABLE 14.2 Sensory Properties Altered by Food Additives Descriptor (sense)

Type of compounds

Main technological function

Exemplary products

Sweet (taste)

Artifcial sweeteners

Sweeteners

Zero calorie beverages

Sour (taste) Salty (taste) Umami (taste)

Acids Carrier Flavor enhancers

Gloss (sight) Shape (sight)

Organic acids Potassium chloride Glutamates, guanylates, inosinates Anthocyanins, carotenoids, caramels Waxes Hydrocolloids

Pickles, beverages Low-sodium products Food concentrates, potato chips Beverages, dietary supplements Jellies, candies Puddings

Texture (touch)

Hydrocolloids

Cold (touch)

Polyols

Color (sight)

Colors Glazing agent Thickeners, gelling agents Thickeners, modifed starches Sweeteners

Low-fat products Candies

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As an example, table sweetener which contains 40 mg of sodium cyclamate and 4 mg of sodium saccharin corresponds to the sweetness of 4,400 mg of sucrose. The blending of sweeteners is a common industrial practice used for the improvement of sensory profles as well as synergy (Schiffman et al. 2007). Blends may mask undesirable favor components of individual sweeteners such as bitter, metallic, or licorice. Blending acesulfame K with aspartame results in approximately 60% higher sweetness, and a synergistic effect is also observed when blending with cyclamates, but not with saccharin. On the other hand, aspartame reveals a synergistic effect with all aforementioned sweeteners. The choice of proper sweeteners or their blend based on their specifcation is an extremely diffcult task as their properties change with the composition or acidity of the product. Therefore, the substitution of sugar with sweeteners should be accompanied by sensory evaluation of various product prototypes. Another issue associated with the use of artifcial sweeteners is related to their low ADI value (Table 14.3). The acceptable intake of acesulfame K (which has relatively high ADI among sweeteners) for average adults is 1,200 mg, which corresponds to the replacement of 180–240 grams of sucrose. This value may seem suffcient as the WHO recommends a reduction of sugar consumption to 10% of total energy value, which corresponds to 50 g for a 2,000 kcal/day diet. Nevertheless, this amount of saccharides is usually present in approximately 2 dm3 of carbonated beverages. This poses a potential threat of consumption in quantities exceeding ADI values in individuals consuming excessive amounts of zero-calorie beverages, further highlighting the necessity of blending sweeteners for synergistic effects. This problem was highlighted in the UE report which indicated the potential exceeding of the ADI by children with regards to acesulfame K (Commission of the European Communities 2001). Sugar alcohols are a group of bulk sugar replacers that theoretically replace sucrose in a 1:1 ratio. This advantage over artifcial sweeteners is associated with

TABLE 14.3 Properties of Artifcial Sweeteners Sweeteners Acesulfame K Aspartame Cyclamic acid and its Na, L, and Ca salts Saccharin and its Na, K, and Ca salts Sucralose Thaumatin Neohesperidine DC Steviol glycosides Neotame Salt of aspartame-acesulfame

INS code

ADI mg/kg

Specifc maximum level mg/kg or dm3

950

15

25–2,500

951 952 954 955 957 959 960a–c 961

40 11 5 5 – 5 4 2

25–6,000 250–1,250 50–3,000 10–2,400 0.5–400 10–400 40–3,300 1–250

962

As for components

110–2,500

Source: own work based on Regulation (EC) No 1333/2008; JECFA.

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TABLE 14.4 Properties of Sugar Alcohols Polyol

Energy value kcal/g

Heat of solution cal/g

Sorbitol

E/INS code 420

2.6

–26

Mannitol Polyglycitol syrup Maltitols Lactitol Xylitol

421 964 965 966 967

1.6 3.0 2.1 2.0 2.4

–29 n/d –19 –14 –36

Erythritol

968

0

–43

Source: own work based on Jamieson (2016).

two major disadvantages. Firstly, their sweetness in relation to sucrose is signifcantly lower; the only exception is xylitol (relative sweetness values are presented in Chapter 5). Secondly, they do exhibit caloric value (Table 14.4.), and in the EU product labeling, it is approximated as 2.4 kcal/g regardless of the real energy value. The only exception is erythritol which is considered calorie free (Regulation (EU) No 1169/2011). These two unique properties made xylitol and erythritol particularly popular as sugar replacers, which should be also associated with the fact that they may be considered a healthier option when compared to artifcial sweeteners as they do not have an assigned ADI value (JECFA). Nevertheless, excessive consumption of products with sugar alcohols may produce a laxative effect, as their lowered energy value is associated with the fact that they are not digested in the human gastrointestinal tract. This property imposes an obligation to label accordingly products with 10% sugar alcohols added (Regulation (EU) No 1169/2011). Along with the sweetening effect, the consumption of polyols is linked with a cooling effect. The cause of this phenomenon is related to the endothermic reaction of their dissolution. The cooling effect is a desired property in the production of chewing gum and some types of candies but may have a negative effect on sensory perception of solid food products such as chocolate or biscuits.

14.3.2 FLAVOR ENHANCERS Flavor enhancers or potentiators are substances that augment the existing taste and/ or odor of a food product. Most of them are linked with umami taste, and those include L-glutamic acid, guanylic acid, and inosinic acid as well as their salts. The most commonly used preparations are sodium salts which are abbreviated as MSG, GMP, and IMP. The main feld of their application includes food concentrates such as soups, sauces and gravies, pasta, and spice blends. Another group is sterilized ready-to-eat products including meat, fsh, vegetables, or fungi. Lastly, favor enhancers play a signifcant role in the sensory evaluation of snacks, such as potato chips. It is estimated that a 0.1–0.8% addition of MSG provides the best

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enhancement of a food’s natural favor. The optimal dose is dependent on product composition as well as the addition of other enhancers. Large synergism is observed between MSG and both IMP and GMP. The synergistic effect between MSG and IMP increases exponentially with their concentration, enabling a signifcant reduction in the required dose in order to achieve a similar technological effect. The mixture containing 95% MSG and 2.5% both IMP and GMP can replace between fve and ten times higher a dose of MSG without changing the favor of the product (Branen et al. 2002). MSG, IMP, and GMP are naturally occurring in many products and are generally recognized as safe additives with no specifed ADI value (JECFA); however, in the EU, their use is limited to 10 g/kg for GMP and to 0.5 g/ kg in case of GMP and IMP. This relates to an ongoing debate over potential undesired symptoms (headache, muscle tightness, general weakness) described by many people after ingestion of excessive amounts of MSG (Yang et al. 1997). On the other hand, the consumption of MSG can be associated with potential health benefts, as it helps to reduce sodium consumption. While MSG contains 12.3% sodium, in general, more than 30% of sodium content in products may be reduced while maintaining a very palatable and acceptable level of taste by using small amounts of MSG (Branen et al. 2002).

14.4

ADDITIVES WITH STRUCTURE-PROMOTING PROPERTIES

Food texture is the sensory and functional manifestation of the structural, mechanical, and surface properties of foods, detected through the senses of vision, hearing, touch, and kinesthetics (Surmacka-Szcześniak 2002). Structure-promoting substances may be into two main categories that shape: • Mechanical and rheological properties (thickeners, gelling agents), • Structural and surface properties (emulsifers, anticaking agents, antifoaming agents, foaming agents). Among additives that help to maintain the desired structure of food products, stabilizers should also be mentioned; however, their role is usually not limited to this technological function only. Stabilizers are substances that make it possible to maintain the physicochemical state of a foodstuff; stabilizers include substances that enable the maintenance of a homogenous dispersion of two or more immiscible substances in a foodstuff; substances that stabilize, retain, or intensify the color of a foodstuff; and substances that increase the binding capacity of the food, including the formation of cross-links between proteins enabling the binding of food pieces into re-constituted food (Regulation (EC) No 1333/2008). In general, stabilizers are used for maintaining the quality of emulsion systems and the term emulsifer and stabilizer may be used interchangeably in many cases. Nevertheless, emulsions as well as other colloidal systems such as suspensions may be stabilized by increasing the viscosity of the system where the action of thickeners is necessary. For the aforementioned reason, many stabilizers are used primarily for their thickening or emulsifying capabilities.

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14.4.1 HYDROCOLLOIDS Substances with thickening and gelling capabilities are referred to as hydrocolloids, which is related to the fact that they are dispersible in water, forming a colloidal solution. Based on their chemical composition hydrocolloids are divided into polysaccharides or proteins. The latter source is usually not considered a food additive, but rather a food component, and these include gelatine, soy proteins, whey proteins, gluten, or albumin (Rutkowski et al. 2003). Polysaccharide hydrocolloids can be further divided based on their origin: plant (pectin, cellulose, starch), seaweed (agar, alginates, carrageenans), and microbiological (gellan, xanthan). Hydrocolloids are polymers and their technological properties are greatly affected by molecular structure. Among the most important aspects infuencing the applicability, solubility (in cold or hot water), gelling capability, and rheological properties should be mentioned. While the solubility and gelling properties can be characterized quite easily and indisputably, the characterization of rheological properties in relation to performance is an extremely diffcult task. Hydrocolloid solutions are non-Newtonian fuids that in general are characterized by pseudoplastic and thixotropic behavior. This poses a challenge to the industry as the choice of the right additive and its dose has to be determined experimentally and even minor changes to the formulation may cause undesired interactions. Hydrocolloids are often blended for possible synergistic actions of biopolymers applied as thickeners. However, the observed effects are multidirectional, and this makes it impossible not only to defne specifc technological recommendations but even to establish a coherent set of conclusions. Natural polysaccharide hydrocolloids are, mostly, generally recognized as safe with no specifed ADI value (JECFA). Commonly used ones include alginic acid and its salts (E400–404) extracted from a certain genus of brown algae; agar (E406) extracted from a certain genus of red algae, Rhodophyta; carrageenan (E407) extracted from certain genera of red algae, which is further divided into three commercial classes based on its properties: kappa, iota, and lambda; locust bean gum (E410) produced by grinding seeds of the carob tree; guar gum (E412) produced by grinding seeds of guar beans; gum arabic (E414) which is dried sap of acacia trees; xanthan gum (E415) which is a product of sugar fermentation by Xanthomonas campestris; and gellan gum (E418) which is an anionic polysaccharide produced by the bacterium Sphingomonas elodea. Special technological importance is also attributed to modifed polysaccharides that are produced based on pectins, cellulose, and starch. Pectins are the main component of cell walls found in terrestrial plants. Commercial pectin preparations are primarily produced from apple pomace and citrus fruit peels. Pectins are classifed based on the degree of esterifcation (DE) into high methoxyl (DE > 50%) or low methoxyl (DE ≤ 50%). Both types have distinct properties and thus different industrial applications. High methoxyl pectin forms gel in solutions with a high concentration of soluble solids and acid medium. They are generally used in the production of jellies, sweets, and desserts. Low methoxyl pectins form gels over a wider pH range, while the presence of calcium ions or multivalent cations is required for this purpose (Freitas et al. 2021).

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Cellulose is the most abundant biopolymer that is not soluble in water in its pure form, which limits its applicability in food technology to ballast material. The typical water-soluble derivatives of cellulose include INS 461 methyl cellulose (MC), INS 463 hydroxypropyl cellulose (HPC), INS 464 hydroxypropyl methyl cellulose (HPMC), and INS 466 carboxy methyl cellulose (CMC) (Li et al. 2018). Although cellulose derivatives are obtained by means of chemical modifcation, they are considered safe with no specifed ADI values (JECFA). Among notable cellulose applications, reduced-calorie products, sauces, ice cream, food concentrates, bakery products, as well as coatings should be mentioned. Starch is the most versatile biopolymer used in food technology, what can be partially attributed to the biodiversity of raw material used for its production. The versatility of starch can be further improved by means of enzymatic, physical, and chemical modifcation. The frst two types of derivatives are still considered food ingredients. Enzymatic modifcation is mostly limited to hydrolysis to produce glucose while physical modifcation (by means of hydrothermal treatment) is performed to obtain cold water swelling starch preparations. Chemical modifcation of starch leads to the production of additives with numerous technological properties; for this reason in the EU a separate technological function, i.e. modifed starches, was adopted (1333/2008). Among noticeable modifcation methods, the oxidation process (INS 1403 and 1404) leads to depolymerization, which is accompanied by gelling capability. Acetylation (INS 1420) when used as a standalone modifcation process causes only minor changes that result in the stabilization of rheological properties. Cross-linking (INS 1412 and 1422) of starch leads to improvement of thickening capabilities (Lewandowicz et al. 2022). Starch sodium octenyl succinate (INS 1450) is mainly used as an emulsifer due to its excellent ability to lower surface tension (Prochaska et al. 2007). Modifed starches are considered safe additives with no specifed ADI value (JECFA) and can be used according to good manufacturing practice and the quantum satis rule. Modifed starches have a wide range of applications including sauces, dressings, emulsions, puddings, jellies, reducedcalorie products, and food concentrates.

14.4.2 EMULSIFIERS Food emulsions are complex dispersed systems that are thermodynamically unstable and have to be stabilized by adding amphiphilic molecules. An emulsifer is a single chemical or mixture of components having the capacity of promoting emulsion formation and short-term stability by interfacial action (Prochaska et al. 2007). Among classifcation methods of emulsifers, the hydrophilic–lipophilic balance (HLB) proposed by Griffn (1949) is the most commonly used. The HLB value is based on the molecular ratio of the hydrophilic and lipophilic parts of the molecule and gives results on a scale of 0–20, where 0 corresponds to a completely hydrophobic and 20 to a completely hydrophilic molecule. Anti-foaming agents have an HLB of 1–3, water-in-oil emulsifers are characterized by an HLB between 3 and 6, while oil-inwater emulsifers have a wider range of HLB between 8 and 16. Commonly used food additive emulsifers in the EU include lecithin (E322), found in 14% of all food

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products, as well as mono- and diacylglycerols of fatty acids (E471), present in 7% of foods. These are followed by hydrocolloids (used as emulsifers) such as guar gum in 6%, xanthan gum in 5%, carrageenan in 4%, and celluloses in 2% of foods (Cox et al. 2021). The most notable applications include the production of margarine or ice cream with the assistance of mono- and diacylglycerols of fatty acids as well as chocolate products emulsifed by soy or sunfower lecithin.

14.5 RECENT TRENDS IN THE USE OF FOOD ADDITIVES In the initial period of industrial food production at the beginning of the 19th century, additives were used mainly in the form of colors in order to mislead the consumer. This dishonest approach toward the use of additives left an enormous burden on food technology. Nowadays the use of additives is strictly regulated in all developed countries, but the consumer approach toward them is still negative. Generally, consumers would like to buy additive-free products, but those with additives are preferred as being more convenient. Numerous variables are related to the acceptance of food additives and those include knowledge of regulation, trust in regulators, preference for natural products, and perceptions of risk and beneft (Bearth et al. 2014). The globalized market and constant development in the processing of food contributed to a growing distance and knowledge gap between consumers and food manufacturers (Asioli et al. 2017). This led to the creation of the clean label trend, a response that simplifes the understanding of a product by the consumer. The origin of clean labels dates back to 2007 and is linked with the publication of McCann and co-authors that suggested a possible link between the ingestion of food additives and hyperactive behavior in children (McCann et al. 2007). To date, there is no legal defnition of a clean label or what clean label products are. The simplest defnition of a clean label describes it as a trend in product labeling that aims to place clear and understandable information on product packaging, along with a reduction of food additives for its production. In a broader sense, clean label product packaging should indicate the lack of artifcial or chemical-sounding ingredients. Therefore, no consensus regarding the defnition of a clean label can be made as it is subject to consumers’ interpretations and perceptions of what chemical-sounding ingredients may be. However, some sources indicate that consumer expectations toward clean labels overlap with the terms natural, organic, free from additives/preservatives, less processed, and transparent information on the ingredients (Grant et al. 2021). The lack of a legal defnition of clean labels poses two major problems for the industry as well as indirectly for the consumers. The question that arises is how to indicate that the label (and presumably the composition) of the product was “cleaned.” Manufacturers use numerous different types of pictograms that are usually green colored with a clean label slogan; moreover, indications that the product is natural, low processed, or has no preservatives/additives are also made. The second issue relates to understanding what type of product change may be considered label cleaning. In general, it can be assumed that the complete removal of food additives from product composition meets the consumer’s understanding of the term clean

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label. Unfortunately, this is often not possible without the deterioration of product quality and/or safety. However, many consumers may perceive the replacement of synthetic additives with naturally derived ones also as label cleaning. A common example includes the replacement of synthetic sweeteners such as acesulfame K or aspartame with plant-sourced steviol glycosides. The fact that all of these substances have an ADI value, and their doses are limited, is of secondary importance. This practice can be further exploited in the form of a similar procedure, greenwashing, by replacement of chemical-sounding food additives like modifed starches (with no ADI specifed) with more natural-sounding food additives (with specifed ADI value) like quillaia extract (Le Thanh-Blicharz and Lewandowicz 2020). Consumers on average are willing to pay a premium for clean labels/fewer ingredients in food products (Grant et al. 2021), thus raising the demand for products that can substitute food additives in clean label applications. Many alternatives are sought after to replace or reduce the use of food additives, which usually meet various technological obstacles.

14.6 PRINCIPLES OF SAFETY ASSESSMENT OF FOOD ADDITIVES Prior to approval, substances that are considered possible food additives have to undergo detailed characterization and safety assessment. The initial stage includes a description of chemical composition, methods of production, acceptable levels of impurities, as well as methods for their determination. These data are published in Joint FAO/WHO Expert Committee on Food Additives (JECFA) or European Food Safety Authority (EFSA) reports, and constitute a framework for further quality assessment. The safety assessment covers several aspects including acute and chronic exposure toxicity, carcinogenesis, neurotoxicity, irritation and allergic infammation tests, teratogenesis, and reproductive toxicity. The toxicity evaluation is performed on animals; small rodents (mice and rats) are most commonly used. In exceptional cases, other animals are used, including primates. The epidemiological trials as well as case studies are only a valuable addition. The most commonly published acute toxicity parameter – LD50 (median lethal dose) indicating the death of 50% of test subjects – is of low signifcance as substances evaluated as potential food additives in general should not be considered toxic. The goal of the safety assessment of food additives is to determine the dose that can be consumed throughout the whole life and cause no potential harmful effects. This relates to chronic toxicity assessment and its no-observed-adverseeffect level (NOAEL), commonly defned as the highest experimental point that is without adverse effects. At the time of the frst JECFA meeting, it was recognized that the amount of an additive used in food should be established with “an adequate margin of safety to reduce to a minimum any hazard to health in all groups of consumers.” During the second meeting, it was indicated that “some margin of safety is desirable to allow for any species difference in susceptibility, the numerical differences between the test animals and the human population exposed to the hazard.” As a consequence, an ADI value was based on NOAEL with a safety factor of 100, meaning that ADI is 100 times lower than NOAEL (WHO 1987). ADI values are

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generally published in JECFA reports; however, in the EU, the EFSA coordinates the work of its own panels aimed at the evaluation of ADI values. The European Commission has set up a program for the years 2010–2020 for successive re-evaluation of approved food additives (Regulation (EU) No 257/2010). The results of the reevaluation were published gradually from scientifc opinions in the EFSA Journal. The sole determination of an ADI value is not suffcient to assure the safety of the whole population. The eating habits of consumers may vary greatly, while abnormal consumption of certain types of products or groups of products could possibly lead to systematic exceeding of an ADI value. Therefore population trials that asses the levels of consumption as well as those that track abnormal consumption patterns are necessary in order to ensure food safety.

14.7 INTERNATIONAL REGULATIONS GOVERNING THE USE OF FOOD ADDITIVES The framework for international regulations regarding the use of food additives is included in the Codex Alimentarius. Standards published within the Codex Alimentarius are developed by the Codex Alimentarius Commission (CAC), a body established in 1961 by the Food and Agriculture Organization of the United Nations (FAO), which was later joined by the World Health Organization (WHO) in 1962. The CAC is advised by another organ, namely the Joint FAO/WHO Expert Committee on Food Additives (JECFA) which was founded earlier in 1956. Codex Alimentarius international food standards are recognized by the World Trade Organization (WTO) as a point of reference in the resolution of international disputes regarding the safety of food. Nevertheless, Codex Alimentarius standards are not legal acts formally applicable in countries that are members of the CAC. Historically, the frst legal regulation with regard to food additives was introduced in 1962 in the European Economic Community and referred only to colors. The Council Directive on the approximation of the rules of the Member States concerning the coloring matters authorized for use in foodstuffs intended for human consumption used the currently used E-number classifcation for the frst time. This approach was later refected in the International Numbering System adopted by the CAC for the frst time in 1989 (Codex Alimentarius CXG 36-1989). However, being assigned an INS number by the CAC does not confrm safety or legal permission to use a substance as a food additive, as in the case of the EU and E-number. The Codex Alimentarius standards are only a framework, and many countries impose regulations that differ to some extent from the information contained in CAC publications. These differences may include crucial aspects such as the INS, ADI, or approval. While many countries rely on the INS (including the EU, Australia, New Zealand, and China), it is not used in the USA. The US regulations also differ in other aspects, i.e. cyclamates approval; while the CAC allows cyclamates to be used as a sweetener in various products and so do the EU, Australia, and New Zealand, they are listed in the FDA Substances Added to Food Inventory as prohibited. Thus, it seems that the idea of a globally harmonized system has failed so far (Kaplan 2019).

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REFERENCES Aparicio, J.F., Barreales, E.G., Payero, T.D., Vicente, C.M., de Pedro, A., Santos-Aberturas, J., 2016. Biotechnological production, and application of the antibiotic pimarici: Biosynthesis and its regulation. Applied Microbiology and Biotechnology 100, 61–78. https://doi.org/10.1007/s00253-015-7077-0 Asioli, D., Aschemann-Witzel, J., Caputo, V., Vecchio, R., Annuziata, A., Naes, T., Varela, P., 2017. Making sense of the “clean label” trends: A review of consumer food choice behavior and discussion of industry implications. Food Research International 99(1), 58–71. https://doi.org/10.1016/j.foodres.2017.07.022 Bearth, A., Cousin, M.-E., Siegrist, M., 2014.The consumer’s perception of artifcial food additives: Infuences on acceptance, risk and beneft perceptions. Food Quality and Preference 38, 14–23. https://doi.org/10.1016/j.foodqual.2014.05.008 Braïek, O.B., Smaoui, S., 2021.Chemistry, safety, and challenges of the use of organic acids and their derivative salts in meat preservation. Biomaterials for Food Preservations 2021, 6653190. https://doi.org/10.1155/2021/6653190 Branen, A.L., Davidson, P.M., Salminen, S., Thorngate, III, J.H., 2002. Food additives. Marcel Dekker Inc. Chen, H., Hoover, D.G., 2003. Bacteriocins and their food applications. Comprehensive Reviews in Food Science and Food Safety 2(3), 82–100. https://doi.org/10.1111/j.1541 -4337.2003.tb00016.x Codex alimentarius: Class names and the international numbering system for food additivesCXG 36-1989. Adopted in 1989. Revised in 2008. Amended in 2018, 2019, 2021. Codex alimentarius: General standard for food additivescodex stan 192–1995 adopted in 1995. Revision 1997, 1999, 2001, 2003, 2004, 2005, 2006, 2007, 2008, 2009, 2010,2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019. Commission of the European Communities., 2001. Report from the commission on Dietary Food Additive Intake in the European Union. Brussels. https://eur-lex.europa.eu/ LexUriServ/LexUriServ.do?uri=COM:2001:0542:FIN:EN:PDF Commission Regulation (EU) No 257/2010 of 25 March 2010 setting up a programme for the re-evaluation of approved food additives in accordance with Regulation (EC) No 1333/2008 of the European Parliament and of the Council on food additives. Offcial Journal of the European Union, L 80/19. Cox, S., Sandall, A., Smith, L., Rossi, M., Whelan, K., 2021. Food additive emulsifers: A review of their role in foods, legislation and classifcations, presence in food supply, dietary exposure, and safety assessment. Nutrition Reviews 79(6), 726–741. https://doi .org/10.1093/nutrit/nuaa038 Grant, K.R., Gallardo, R.K., McCluskey, J.J., 2021. Consumer preferences for foods with clean labels and new food technologies. Agribusiness 37(4), 764–781. https://doi.org/10 .1002/agr.21705 Griffn, W.C., 1949. Classifcation of surface-active agents by “HLB”. Journal of the Society of Cosmetic Chemists 1, 311–326. Jamieson, P., 2016. Reducing added sugars with polyols. Food Technology Magazine (IFT) 70(11), 42–48. JECFA: Evaluations of the Joint FAO/WHO Expert Committee on Food Additives (JECFA). Available online at https://apps.who.int/food-additives-contaminants-jecfa-database/ Kaplan, D.M., 2019. Encyclopedia of food and agricultural ethics. Springer, Dordrecht. https://doi.org/10.1007/978-94-024-1179-9 Le Thanh-Blicharz, J., Lewandowicz, G., 2020. Dodatki do żywności – korzyści i zagrożenia. In: Szkodliwe substancje w żywności Pochodzenie. Działanie. Zagrożenia zdrowotne.

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pod red. Naukową: a. Witek, Z.E. Sikorski. Wydawnictwo Naukowe PWN, S.A. Warszawa, s. 229–242. ISBN 978-83-01-21299-5. Lewandowicz, J., Le Thanh-Blicharz, J., Szwengiel, A., 2022. The effect of chemical modifcation on the rheological properties and structure of food grade modifed starches. Processes 10(5), 938. https://doi.org/10.3390/pr10050938 McCann, D., Barrett, A., Cooper, A., Crumpler, D., Dalen, L., Grimshaw, K., Kitchin, E., Lok, K., Porteous, L., Prince, E., Sonuga-Bar, E., Warner, J.O., Stevenson, J., 2007. Food additives and hyperactive behaviour in 3yearold and 8/9yearold children in the community: A randomised, double-blinded, placebo-controlled trial. The Lancet 370(9598), 1560–1567. https://doi.org/10.1016/S0140-6736(07)61306-3 Polesca Freitas, C.M., Reis Coimbra, J.S., Lauriano Souza, V.G., Superbi Sousa, R.C., 2021.Structure and applications of pectin in food, biomedical, and pharmaceutical industry: A review. Coatings 11(8), 922. https://doi.org/10.3390/coatings11080922 Prochaska, K., Kędziora, P., Le Thanh, J., Lewandowicz, G., 2007. Surface activity of commercial food grade modifed starches. Colloids and Surfaces B: Biointerfacesvol 60(2), 187–194. https://doi.org/10.1016/j.colsurfb.2007.06.005 Regulation (EU) No 1169/2011 of the European Parliament and of the Council of 25 October 2011on the provision of food information to consumers. Offcial Journal of the European Union, L 304/18. Regulation (EC) No 1332/2008 of the European Parliament and of the Council of 16 December 2008 on food enzymes. Offcial Journal of the European Union, L 354/7. Regulation (EC) No 1333/2008 of the European. Parliament and of the Council of 16 December 2008 on food additives. Offcial Journal of the European Union, L 354/16. Regulation (EC) No 1334/2008 of the European Parliament and of the Council of 16 December 2008 on favourings and certain food ingredients with favouring properties for use in and on foods. Offcial Journal of the European Union, L 354/34. Rutkowski, A., Gwiazda, S., Dąbrowski, K., 2003. Kompendium dodatków do żywności. Konin: Hortimex. Schiffman, S.S., Sattely-Miller, E.A., Bishay, I.E., 2007. Time to maximum sweetness intensity of binary and ternary blends of sweeteners. Food Quality and Preference 18(2), 405–415. Surmacka-Szcześniak, A., 2002. Textureis a sensory property. Food Quality and Preference 13(4), 215–225. https://doi.org/10.1016/S0950-3293(01)00039-8 WHO., 1987. Principles for the safety assessment of food additives and contaminants in food. Environmental Health Criteria 70. https://www.inchem.org/documents/ehc/ehc/ehc70 .htm Yang, W.H., Drouin, M.A., Herbert, M.,Mao, Y., Karsh, J., 1997. The monosodium glutamate symptom complex: Assessment in a double-blind, placebo-controlled, randomized study. Journal of Allergy and Clinical Immunology 99(6 Part 1), 757–762. https://doi .org/10.1016/S0091-6749(97)80008-5 Ya-Yu, Li, Wang, B., Ma, M.-G., Wang, B., 2018. Review of recent development on preparation, properties, and applications of cellulose-based functional materials international. Journal of Polymer Science 2018, 8973643. https://doi.org/10.1155/2018/8973643

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Food Safety Agata Witczak and Kamila Pokorska-Niewiada

CONTENTS 15.1 Introduction .................................................................................................. 419 15.2 Harmful Substances Generated during Food Production and Storage ........ 421 15.3 New Food Safety Problems: Micro- and Nanoplastics in Foods.................. 428 15.4 Food Safety Control...................................................................................... 429 References.............................................................................................................. 431

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INTRODUCTION

According to the Food and Agriculture Organization of the United Nations (FAO), food safety is an important element of food security, which is defned as every inhabitant of the globe having physical and economic access to food. Food safety is a signifcant public health problem, and with increasing numbers of environmental pollutants, providing consumers access to safe food is becoming increasingly diffcult. While governments globally are making every effort to improve food quality, the incidence of foodborne diseases remains a signifcant health concern in both developed and developing countries. The World Health Organization (WHO) estimates that over 600 million people become sick annually from eating food contaminated with bacteria, parasites, viruses, toxins, and chemicals. As many as 1.8 million people die each year from diarrheal disease, and most of these cases can be attributed to contaminated food or water. It is estimated that over 200 known diseases are foodborne, and they can be especially severe in infants, young children, the elderly, and people with a variety of medical conditions. Despite no evidence that COVID-19 transmission is associated with food, the pandemic has increased worldwide focus on food safety issues such as hygiene, antimicrobial resistance, zoonoses, the impact of climate change, pest resistance, and food adulteration. Food poisoning with bacteria such as Staphylococcus aureus, Salmonella, and Bacillus cereus was common in the 1960s; however, from the 1970s, many viruses were found to be transmitted not only by water droplets but also by food. Occurrences of bacteria became increasingly common, including those from the genera Campylobacter, which causes diarrhea, and Yersinia, which causes fu-like symptoms. In the 1980s and 1990s, the new disease listeriosis, caused by infection with the bacterium Listeria monocytogenes, became widespread. Currently, hepatitis A and diseases caused by intestinal pathogens are widespread. The most common symptoms of infection with intestinal pathogens include abdominal pain, diarrhea, and extraintestinal symptoms such as arthritis, nausea, vomiting, and fever. Humans DOI: 10.1201/9781003265955-15

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ingest intestinal pathogens through the consumption of sprouts, leafy vegetables, and berries, and sometimes by drinking contaminated water. Pathogens that cause gastrointestinal infections include bacteria (e.g., Clostridioides diffcile which produces toxins A and B, Campylobacter spp., pathogenic Escherichia coli, Salmonella spp., Yersinia enterocolitica, and Vibrio cholerae), viruses (e.g., rotavirus A, adenovirus, astrovirus, noroviruses, and sapovirus), and parasites (e.g., Cryptosporidium spp., Cyclospora cayetanensis, Entamoeba histolytica, and Giardia lamblia). Microorganisms are not the only cause of foodborne diseases. Many health problems can also be caused by undesirable chemical compounds in foods that are a consequence of widespread environmental contamination. Toxic compounds are released into the environment mainly by industrial processes and agricultural practices, the consequences of which are the presence of many undesirable chemicals in the human food chain. Particular attention is now being paid to risks associated with the indiscriminate use of pesticides. Another important problem is the inappropriate application of crop protection measures which most often results from disregarding proper use recommendations (e.g., incorrect preparation of working solutions, non-compliance with spraying dates, intervals between successive sprayings, or exceeding the maximum annual total dose of the active substance), which pose risks to human and animal health. The magnitude of the exposure of the human population to pesticides is diffcult to estimate. The most common health consequences include gastrointestinal disorders such as loss of appetite, nausea, dysgeusia, and vomiting, as well as neurological disorders, headache, dizziness, balance disorders, and hyperactivity (Gilden et al. 2010). The chronic effects of pesticides consumed with food on human health are not well understood, but there is increasing evidence of their carcinogenicity and genotoxicity, and the possibility of disrupted hormonal function and effects on the immune system. Pesticides are commonly found in human tissues, some of which are described as immunosuppressants while others as procarcinogens (Kasmani et al. 2014). Given continued human population growth, the scales of pesticide use and industrial waste discharge are likely to increase over the next several decades. The consequences can be serious, especially in developing countries. They use very large amounts of chemicals, often in an uncontrolled manner. In addition, the resistance of inhabitants to disease and poisoning is lowered due to malnutrition, which mainly affects many millions of children. Proper food preparation can prevent most foodborne diseases. Washing and peeling vegetables and fruits are the most common preliminary processing methods applied in domestic settings and food processing facilities. Surface residues of many pesticides can be removed with varying effciency by washing using only water or aqueous solutions of certain substances such as sodium chloride or acetic acid (Witczak & Abdel-Gawad 2018). Numerous studies report that improper culinary or technological processing and storage can result in the formation of toxic compounds in food, including acrolein, nitrosamines, and mycotoxins. The most dangerous risks associated with most xenobiotics are carcinogenicity, teratogenicity, and endocrine-disrupting effects. These include mycotoxins, among which afatoxins (AF) and ochratoxin A (OTA) pose the

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greatest risk to human health, and afatoxin B1 (AFB1) is identifed as the strongest known hepatocarcinogenic factor (Tola & Kebede 2016). Afatoxins, produced by mold fungi of the family Aspergillus, are most often found in improperly stored cereal grains, legume seeds, nuts, dried fruits (fgs, raisins), vegetable oils, and spices (pepper, paprika, nutmeg, ginger, turmeric). Other threats in food include anthropogenic compounds such as industrial and agricultural environmental pollutants (e.g., PCBs, heavy metals, pesticides), chemical compounds that are added intentionally to processed foods (e.g., preservatives, nitrates (III)), and by-products (e.g., dioxins, dibenzofurans) created in foods during various combustion processes. Currently, the problem of food contamination is an important issue for food processing companies. The feld-to-table concept enables monitoring products by tracing them from the moment raw materials are harvested through all production stages. The shortcomings of modern food production include, inter alia, the extended path from raw materials to fnished products which results from globalization and multi-stage food production in, for example, ready meals. The content of some groups of toxic compounds, such as pharmaceuticals, pesticides, and preservatives can be avoided or reduced to acceptable levels if safety systems are properly implemented by hazard analysis and critical control points (HACCP); good manufacturing practices (GMP); good hygienic practices (GHP); and standards of the British Retail Consortium (BRC), which, in 2019, rebranded as Brand Reputation through Compliance of Global Standards (BRCGS), and the International Organization for Standardization (ISO). The production of safe food can also be facilitated by introducing increasingly stringent standards regarding permissible levels of toxic compounds in various food types. Consumer health safety with regard to food consumption can also be infuenced by educating consumers about potential hazards in both raw and processed food products and also providing consumers with reliable information on food composition.

15.2

HARMFUL SUBSTANCES GENERATED DURING FOOD PRODUCTION AND STORAGE

The progressive contamination of the natural environment and intensifed food production are the main sources of the contamination of food products and the raw materials for their production. With the further development of the food processing industry, it is becoming an ever greater challenge for public health authorities to protect consumer health from the risks of exposure to hazardous substances produced during food processing. Improper technological processes and culinary thermal processing create chemical compounds in many food products that are potentially carcinogenic and mutagenic. Processing foods by heating at particularly high temperatures, frying, grilling, drying, and fermenting can create undesirable hazardous substances that can include, inter alia, polycyclic aromatic hydrocarbons (PAHs), heterocyclic amines (HAA), acrylamide, nitrosamines, 5-hydroxymethylfurfural (5-HMF), ethyl carbamate,

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furan, 3-monochloropropane-1,2-diol esters (3-MCPDE), glycidyl fatty acid esters (GE), and 4-methylimidazole (4-MEI). Many substances have carcinogenic properties following metabolic activation. Some of them (N-nitrosamines) require the presence of precursors that occur naturally in foods or are added to them artifcially, while others are formed when food is stored improperly. There are also chemical compounds that can stimulate cancers only when they react with other carcinogens (Rywotycki 2002). These substances occur in both plant and animal foods. Some of the harmful compounds generated during food processing are presented in the following. Polycyclic aromatic hydrocarbons are a group of chemical compounds in which carbon and hydrogen atoms form two or more conjugated aromatic rings. These compounds in food mainly derive from environmental pollution and some technological food preservation processes such as smoking, frying, and grilling. PAHs are formed not by heating products themselves, but from the pyrolysis of fat dripping from products onto fres. As much as 20 μg of PAHs can be detected in 1 kg of charred meat. They are also found in products such as peanuts, coffee, refned plant oil, spinach, and cereals. The toxic effects of PAHs depend on the manner and duration of exposure. Short-term effects include skin and eye irritation, nausea, vomiting, and infammation. Long-term effects can include lung, bladder, and gastrointestinal cancers, kidney and liver damage, cataracts, and also genetic mutations, cell damage, and mortality associated with the cardiopulmonary system (Sampaio et al. 2021). PAH content in foods is regulated by law. According to Commission Regulation (EU) No 835/2011 of 19 August 2011 amending Regulation (EC) No 1881/2006 as regards maximum levels of polycyclic aromatic hydrocarbons in foodstuffs, the maximum levels of PAHs in food should be as low as reasonably achievable (the ALARA principle). Limits for benzo(a)pyrene and the sum of four PAHs are set for the following food categories: oils and fats; cocoa beans and derived products; smoked meat and smoked meat products; smoked fsh muscle meat and smoked fshery products; food for infants and small children; and processed cereal-based foods. Heterocyclic aromatic amines belong to the group of compounds composed of ring systems that include nitrogen atoms and free amino groups attached to them. HAAs form when food, especially that with a high protein content, is heat treated. The type of amine formed depends on the temperature at which the food is processed. Amino acid and protein pyrolysis products are formed at temperatures higher than 300° C, while quinoline, quinoxaline, and pyridine derivatives are formed at lower temperatures (150–200° C). Consequently, these compounds are found in fried and roasted meats and fried fsh. HAAs are potent carcinogens caused by the Maillard reaction. It is a non-enzymatic browning reaction that occurs between amino acids and reducing sugars during the processing or cooking of food at high temperatures. Of the 20 heterocyclic aromatic amines identifed in heat-treated food (meat and fsh), nine amines are classifed as carcinogens in humans, including 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline (IQ); 2-amino-3, 4-dimethyl-3H-imidazo[4,5-f]quinoline (MeIQ); 2-amino-3,8-dimethyl-3H-imidazo [4,5-f]quinoxaline (MeIQx); 2-amino-1-methyl-6-phenyl-1H-imidazo[4,5-b]pyridine (PhIP); 2-amino-9H-dipyrido[2,3-b]indole (AαC); 2-amino-3-methyl-9H-dipyrido[2

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,3-b]indole (MeAαC); 2-amino-6-methyl-dipyrido[1,2-α:3',2'-d]imidazole (Glu-P-1); 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Tryp-P-1); and 3-amino-1-methyl5H-pyrido[3,4-b]indole (Tryp-P-2). Free amino acids, protein, creatinine, reducing saccharides, and nucleosides are the main precursors involved in the production of polar and non-polar HAAs (Iwasaki & Tsugane 2021). In 2004, IQ, MeIQ, 8-MeIQx, and PhIP were listed as carcinogenic to humans by the US National Toxicology Program (NTP 11th Report on Carcinogens 2004). There are no other legal regulations on permissible HAA levels in food. Acrylamide is formed in plant-based foods, including those containing potatoes and grains, as a result of the Maillard reaction that occurs mainly between free asparagine and reducing sugars (mainly glucose and fructose). Acrylamide is also formed through the enzymatic decarboxylation of asparagine, without the participation of the reducing sugar or glycerol contained in fats, during baking and frying. A thermal treatment temperature exceeding 120° C is the factor that plays an important role in the formation of acrylamide in foods (Mogol & Gokmen 2016). In addition to temperature, the asparagine, reducing sugars, pH, and moisture of products and the antioxidants in vegetables and grains are also signifcant. Acrylamide is classifed as a toxic substance that poses a serious threat to health. Acrylamide is a mutagen and a carcinogen, a reproductive toxin, and an eye irritant, and it can cause allergic skin reactions. A small amount of acrylamide is excreted in the urine, but about 90% is metabolized in the body. One of the hepatic metabolic pathways of hepatic cytochrome P450 2E1 monooxygenases converts acrylamide to the epoxide derivative glycidamide. Glycidamide is a reactive metabolite of acrylamide that is highly mutagenic. Plant extract additives in various types of food can be a good way to reduce acrylamide content. Acrylamide formation is limited by favonoids which are a group of functionally important plant components present in herbs, vegetables, and fruits. Vitamin C can also be added to foods; research has demonstrated that the less stable the antioxidant compound is, the greater the decrease in acrylamide content, e.g., vitamin C reduced acrylamide content by 63.4% and ferulic acid by 66.2% (Ou et al. 2010). The current law in the European Union is Commission Regulation (EU) 2017/2158 of 20 November 2017 establishing mitigation measures and benchmarks for the reduction of the presence of acrylamide in food by limiting the availability of substrates, selecting technological processes that do not facilitate the formation of acrylamide, and increasing awareness among food business operators at all stages of the production chain. The 3-MCPD and 2-MCPD (2-monochloropropane-1,2-diol) esters and glycidyl fatty acid esters are food processing contaminants that form at high temperatures used during oil refning (> 200° C), especially in the deodorization stage. The presence of 3-MCPD esters and accompanying GEs were found in higher amounts in refned vegetable oils and products obtained using them. Under appropriate conditions, they can be de-esterifed and free 3-MCPD and glycidol are released, which are undesirable compounds in foods because of their toxic properties. Free 3-MCPD is considered to be a potentially carcinogenic compound (group 2b) that mainly negatively affects the functions of the excretory system, especially the kidneys, and

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the male reproductive system, while glycidol is a carcinogen (group 2) and mutagen (group 3) and is harmful to reproduction (group 2). The carcinogenicity of glycidol mainly affects the central nervous and male reproductive systems (IARC 2000). Dried spicy and sweet products with salt and fat (inter alia, crispbreads, salty crackers, and cookies) contain elevated levels of 3-MCPD (EFSA 2016). In 2018, the European Union established maximum limits for glycidyl esters in oils/fats and infant formulas ([EU] 2018/290 of 26 February 2018), and from 1 January 2021, regulations came into force on the sum of free 3-MCPD fatty acid esters and 3-MCPD (Commission Regulation [EU] 2020/1322 of 23 September 2020) amending Regulation (EC) No 1881/2006 as regards maximum levels for 3-monochloropropanediol (3-MCPD), 3-MCPD fatty acid esters, and glycidyl esters of fatty acids in certain foods. N-nitrosamines are derivatives of secondary dialkyl-, diaryl-, or alkylarylamines, or secondary cyclic amines. Nitrates (III) in food are hydrogenated to hydrogen oxide under acidic conditions and the resulting hydrogen oxide reacts with other nitrate (III) molecules to form nitrogen anhydride after dehydration. Nitrogen anhydride donates a nitroso group to amines in food to create N-nitrosamines. The formation of nitrosamines depends on a number of factors, including amine alkalinity, environmental pH, and temperature. Primary aliphatic and aromatic amines do not form nitro compounds at low pH or temperatures, while secondary amines are formed faster and easier the less alkaline the amine is. Increased pH and temperatures facilitate the nitrosation of tertiary aliphatic amines. While amounts of nitrosamines in raw products are negligible, fried and smoked products contain the most (Bedele et al. 2016). Preparing grilled dishes with cured meats is disadvantageous. This is because of its content of saltpeter (from which nitric oxide is formed) the main task of which is to combine with myoglobin and hemoglobin to form nitrosomyoglobin and nitrosohemoglobin. These compounds protect meat from losing its desired pink-red color during heating, which happens when uncured meat products are heated and a brown color develops. However, nitric oxide also reacts with secondary amines found in food (mainly in fsh and meat) to form nitrosamines, which are highly carcinogenic compounds. The factor that accelerates this reaction is the high temperature of the grill hearth, which, during grilling, can exceed even 200° C. The most common nitrosamines that humans are exposed to in foods are N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosopyrrolidine (NPYR), N-nitrosopiperidine (NPIP), and N-nitrosodibutylamine (NDBA). Nitrosamines are also formed by microbiological and enzymatic changes that occur, inter alia, in meat, fsh, cheese, cereal products, tea, and wine (Park et al. 2015). N-nitrosamines are classifed as carcinogens (IARC and US EPA) of the gastrointestinal tract, lungs, kidneys, and tongue. Moreover, they have genotoxic, mutagenic, and teratogenic properties. The six-carbon aldehyde 5-hydroxymethylfurfural is a heterocyclic derivative of furan that contains both aldehyde and alcohol functional groups. It is found in many foods, and it occurs during the thermal processing of food. Technological procedures

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such as baking, frying, pasteurization, or cooking lead to the formation of signifcant amounts of HMF. The synthesis of this compound is particularly effective in acidic environments and in the presence of simple sugars. Although a range of studies has been conducted on HMF, its impact on the human body has not been clearly established. 5-HMF is an irritant to the eyes, upper respiratory tract, and skin, and it has carcinogenic, hepatotoxic, and nephrotoxic properties. 5-HMF occurs in, inter alia, malt, coffee, vinegar, honey, and fruit juice. The content of 5-HMF is considered an indicator of quality because high levels of it indicate overheating during processing or inadequate storage conditions. For example, the concentration of 5-HMF is considered to be a measure of honey freshness since concentrations of it tend to increase with overheating or aging. The limit for 5-HMF in honey was set by the Codex Alimentarius Commission at ≤ 40 mg/kg (or ≤ 80 mg/kg in tropical honey) (Shapla et al. 2018). In addition to honey standards, there are similar standards set out in the juice industry, where the concentration of 5-HMF in fruit juice should be in line with recommendations of the International Federation of Fruit Juice Processors (IFFJP) at 5–10 mg/L (or 25 mg/kg), while the European Union limit for the content of 5-HMF in juices for children is 20 mg/kg (EC 1881/2006). With the aim of reducing the formation of 5-HMF or removing 5-HMF from food, UV radiation, vacuum treatment, microwave heating, and favan-3-ol-like compounds are added to food products (Qi et al. 2018). Ethyl carbamate (EC, urethane) is an ethyl ester of carbamic acid. It can form in stone fruit spirits by chemical reactions involving, among others, cyanides, hydrocyanic acid, and cyanogenic glycosides. It is found in many fermented foods and alcoholic beverages such as cheese, bread, yogurt, wine, whiskey, soy sauce, etc. Ethyl carbamate has been identifed most likely as a carcinogen and a probable health risk to people who regularly consume large amounts of alcoholic beverages. EC cellular metabolism is associated with oxidative stress (OS) and DNA damage. EC formation can be accelerated, inter alia, by heat or thermal treatment, transition metals, storage conditions, pH, and UV radiation (Gowd et al. 2018). According to the European Food Safety Authority (EFSA) opinion “Ethyl Carbamate and Hydrocyanic Acid in Food and Beverages – Scientifc Opinion of the Panel of Contaminants” (EFSA 2007), which assesses the margins of exposure to ethyl carbamate, this substance poses a potential health risk to consumers who consume stone fruit spirits. In 2016, the Commission (EU) introduced Recommendation 2016/22 of 7 January 2016 on the prevention and reduction of ethyl carbamate contamination of stone fruit spirits and stone fruit marc spirits, repealing Recommendation 2010/133/EU. Furan and its derivatives (2-methylfuran, 2-ethylfuran, 2-pentylfuran, 2,5-dimethylfuran, 2-butylfuran, and 2,3-benzofuran) occur in heat-treated foods and beverages and affect the sensory properties of food. The formation of furan is facilitated primarily by the Maillard reaction of reducing sugars, but also by the thermal degradation of some amino acids, and thermal oxidation of ascorbic acid, polyunsaturated fatty acids, and carotenoids. Furan can form in some foods when heated for cooking, pasteurizing, and preserving (Han et al. 2017). The level of furan in foods is infuenced by many factors, including temperature, pH, product storage

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conditions, and the presence or absence of inhibitors and activators. At temperatures around 200° C, furan levels increase with increasing temperature; however, temperatures exceed 200° C, furan concentrations are independent of temperatures. Frying (150–200° C) produces higher levels compared to baking. Furan occurs at increased levels in, inter alia, chips, dried fruits, and corn crisps. Commission Regulation (EC) No 1881/2006 sets maximum levels for non-dioxinlike PCBs, dioxins, and dibenzofurans, and the sum of dioxins, dibenzofurans, and dioxin-like PCBs in certain foodstuffs. 4-methylimidazole (4-MEI) is an undesirable by-product that can form during the interaction of ammonia with reducing sugars in class III (ammonia caramel) and class IV (sulfte ammonia caramel) caramelization processes. 4-MEI is toxic in humans and animals. It is present in elevated amounts in many beverages (e.g., cola, root beers, iced teas, coffee, and whiskey) and in food products that contain caramel color type IV (Mehri et al. 2020). Because of health risks related to potential carcinogenic effects, in 2011 the EFSA recommended reducing the level of these compounds and developing research on the nature of the formation of these ingredients during caramel production processes (EFSA 2011). Mycotoxins of the genera Aspergillus, Fusarium, and Penicillium are products of the secondary metabolism of fungi, and they frequently cause contamination of foods and feeds. While Aspergillus and Penicillium species often grow on foods and fodders during storage, Fusarium species can infect crops such as wheat, barley, and maize in the feld and reproduce in the plants. To date, more than 300 mycotoxins have been identifed and reported; however, only a few regularly contaminate foods and fodders. These include afatoxins (AF), ochratoxins (OT), fumonisins, patulin, zearalenone (ZEA), and trichothecenes, including deoxynivalenol (DON) and T-2 toxin. Many mycotoxins are not easily eliminated during food processing because they are resistant to heat treatments and physical and chemical processing. Fodder contamination can also pose additional risks to food safety since it is possible for mycotoxins to be transferred into animal products such as milk, meat, and eggs, which leads to humans consuming them. Many strategies for controlling the presence of mycotoxins in various foods have been proposed; however, there are no clear solutions (Alshannaq & Yu 2017). Mycotoxins pose health risks because they can cause serious diseases. For example, afatoxins produced by Aspergillus species can cause afatoxicosis, a life-threatening form of acute intoxication that can cause liver damage. There is also evidence suggesting that afatoxins are genotoxic and might have more longterm health effects, such as liver cancer. Ochratoxins, which are produced by both Penicillium and Aspergillus species, lead to kidney damage and are implicated as causative agents of kidney cancer in animals, and they also adversely affect fetal development and the immune system. Recent research also suggests that ochratoxin A might be associated with autism in children. Fusarium, another major fungus responsible for mycotoxin production, produces fumonisin, a common corn contaminant that is associated with esophageal cancer. Maximum levels of afatoxins in foodstuffs are set in Commission Regulation (EU) No 165/2010 of 26 February 2010, amending Regulation (EC) No 1881/2006,

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while those of ochratoxin A are set in Commission Regulation (EU) No 105/2010 of 5 February 2010, amending Regulation (EC) No 1881/2006. Histamine, a biogenic amine found naturally in food, is also produced during fermentation and maturation processes and when foods are stored inappropriately or spoil. The longer a given food is stored or matured, the higher the content of histamine in it is, but other factors also infuence its content in foods. For fsh, these include the species, freshness, transport conditions, and refrigeration temperatures, and histamine is responsible for the characteristic smell of rotten fsh. Scombrotoxin poisoning, also known as histamine poisoning, continues to be a food safety issue in the fsh industry. Consumption of improperly handled fsh belonging to the families Scombridae (such as tuna or mackerel), Clupeidae (sardines, herring), or Engraulidae (anchovies) can lead to acute illness with symptoms similar to those of an allergic reaction or a Salmonella infection. The bacterial and enzymatic formation of histamine from histidine is associated with product storage temperature, the standard of which is below 4° C. Histamine formation can occur, however, at all stages of the food production chain (Tortorella et al. 2014). Various methods are used to control the formation of biogenic amines, including histamine, in the food processing industry, including adding starter cultures to reduce the quantities of histamine formed, irradiation, chemical additives of various functions (citric acid, D-sorbitol, potassium, sodium sorbate, etc.) and preservatives, innovative packaging methods in modifed atmospheres, and smart packaging. Rapidly cooling raw materials, freezing, gutting and deheading perishable fshes, and heat treatments are also essential in reducing the possibility of histamine formation (FAO/WHO 2012; Commission Regulation [EU] No 1019/2013). The conditions of harvesting and storing agricultural crops and irregularities that can occur during the technological processing of products can signifcantly infuence contamination levels in raw materials, semi-fnished products, and fnal products. Most often, human exposure to toxins results from the inadvertent ingestion of natural toxins, heavy metals (e.g., methylmercury, arsenic), and environmental pollutants such as veterinary drugs in water, pesticides that are used improperly, substances used to maintain hygiene, and food additives that are used improperly or in excess. Food safety begins on the farm with initial production, i.e., seed choices, how and where to grow crops, and crop protection methods and products. This is also important with regard to animal fodder. Products that are consumed raw like fruits and vegetables and especially when not peeled or washed in clean water can transmit pathogens and residues of hazardous chemicals, mainly pesticides, to consumers. Simple measures such as washing and peeling can sometimes reduce the risk associated with chemicals on food surfaces (while proper storage can prevent certain toxins from forming or reduce their quantities). Peeling potatoes reduced residual chlorpropham by 91–98% from an initial concentration of 3.8 µg/g in individual tubers (Lentza-Rizos & Balokas 2001). During long-term storage (3–36 months), cereal grains are stored at ambient temperatures in bulk grain silos, where insecticides can be applied after harvest to reduce losses caused by storage pests, which means that grain-based foods can be sources of insecticide residues in diets. A study

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of grain to which post-harvest insecticides were applied revealed that chemical residues decreased rather slowly, and residues of more lipophilic materials tended to remain on seed coats, although some migrated to the bran and embryo, which contain high levels of triacylglycerols. Conversely, malathion content in corn kernels and beans decreased over 12 months of storage by 64% and 47%, respectively (Lalah & Wandiga 2002). Residues of veterinary drugs, including antibiotics, can be detected in food products of animal origin such as meat, milk, and eggs. The occurrence of these residues can be caused, inter alia, by non-compliance with drug withdrawal periods, fodder contamination with excreta from treated animals, and the application of unlicensed antibiotics. Antibiotic residues in animal-derived foods can cause many health problems in humans; for example, immunopathological effects, carcinogenicity (e.g., sulfamethazine, oxytetracycline, furazolidone), mutagenicity, nephropathy (e.g., gentamicin), hepatotoxicity, reproductive disorders, bone marrow toxicity (e.g., chloramphenicol), and allergies (e.g., penicillin). These residues can also contribute to the transmission of antibiotic resistance from animals to humans. Microbial and chemical contamination is diffcult to detect without testing, so it usually goes unnoticed until products are consumed. Improving analytical and diagnostic methods will permit detecting as yet unknown threats to human health and causes of disorders.

15.3

NEW FOOD SAFETY PROBLEMS: MICROAND NANOPLASTICS IN FOODS

According to defnitions adopted by the EFSA, microplastics are a heterogeneous mixture of various materials, mainly polypropylene, polyethylene, polystyrene, nylon, polyester, and acrylic, in various shapes of particles including fbers, ellipsoids, granules, shots, and fakes that range in size from 0.1 μm to 5 mm. Whether they are primary (washing synthetic clothes, car tire abrasion, city dust, microbeads in personal care products, ship hull cleaning materials, industrial cleaning products) or secondary (e.g., degraded macroplastics, PET [polyethylene terephthalate] bottles, fshing gear parts, fshing nets, etc.), microplastics are found on the sea surface, in water columns, on seabeds, on shorelines, and in both aquatic and terrestrial organisms. As plastics degrade further, nanoparticles with a size range of 1–1000 nm are formed. Plastic particles found in fshes contain previously adsorbed chemicals such as dyes, phthalates, plasticizers, retardants, POP residues, heavy metals, etc. The most common way for the nanoplastics in seafood, fsh, and contaminated water to enter the human body is with foods. Experiments have demonstrated that the content and transfer of nanoplastics in the marine trophic chain is one of the sources of human exposure to these plastics. Brewing tea in plastic bags has also been confrmed to facilitate the release of micro- and nanoparticles into the drink (Leslie et al. 2022). Bottled water has also been found to have increased plastic particle content, which might be caused by exposure to UV radiation and temperature during long periods of shelf storage. Apart from expiry dates, there are currently no offcial criteria to exclude bottles with the highest residual plastic content from

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the market. As long as no pathogenic microorganisms develop in bottled water, it cannot be recalled. The result of plastic particles in the air, food, and water is that they are detected in human blood. The (PET) polymer used in the production of beverage bottles, food packaging, and fabrics, has been detected in the blood, along with polystyrene, which is used in the production of disposable tableware and food containers, polystyrene, and polyethylene. Once nanoplastics are inside the human body, they cross tissue barriers and reach all the organs with the blood. Studies on erythrocytes indicate that polystyrene nanoparticles with a carboxyl group (NP-PSCOOH) are capable of being absorbed and penetrating into red blood cells. Recently, they have also been found in lung alveoli (Jenner et al. 2022). In conclusion, the mass use of nanomaterials in various areas of life, including the manufacture of everyday products (cosmetics, clothing) and in many sectors of the consumer market (electronics, the automotive industry, the food industry, agriculture) poses a risk of the uncontrolled release and accumulation of plastics in the environment. Despite attempts to assess the risks of introducing nanoparticles into the environment and to test their toxicity to living organisms, these issues remain poorly understood. Among other things, this is because of the unique properties of nanomaterials in relation to traditional materials, which creates many problems in tracking what happens to them in the environment and in foods. This topic remains open and much is still unknown regarding it, and little is known about long-term health effects or those that could arise in the distant future.

15.4 FOOD SAFETY CONTROL Ensuring the quality and safety of food requires thorough control, which is possible thanks to creating food quality and food safety management systems. The FAO and the WHO are making efforts worldwide to ensure food safety by addressing a wide range of issues supporting global food safety and protecting consumer health. The FAO is addressing food safety issues throughout the food supply chain, while the WHO is working with the public health sector to reduce the burden of foodborne diseases. Both organizations have joint programs on food standards (Codex Alimentarius), providing scientifc advice (JECFA – the Joint FAO/WHO Expert Committee on Food Additives; JEMRA – the Joint FAO/ WHO Expert Meetings on Microbiological Risk Assessment; JMPR – the Joint Meeting on Pesticide Residues; JEMNU – the Joint FAO/WHO Expert Meetings on Nutrition), and emergency responses (INFOSAN). Food safety practices are monitored by food technologists, microbiologists, veterinarians, and toxicologists throughout the food chain, and implementing Codex Alimentarius food standards helps to protect consumer health. The FAO/WHO Codex Alimentarius Commission establishes international, science-based standards, guidelines, and codes of conduct that help ensure food safety and quality consistently and transparently. It covers all food-related issues: contamination, hygiene practices, labeling, additives, inspection and certifcation, nutrition, and residues of veterinary drugs and pesticides. National governments adopt and implement international standards with local regulations.

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Food quality and safety management systems include controlling foods at all stages of production from producer to consumer. The most commonly used systems for ensuring and maintaining food safety include: good manufacturing practice (GMP), good hygienic practice (GHP), agricultural good practice (GAP), the hazard analysis and critical control point system (HACCP), the ISO 9001 standard, Brand Reputation through Compliance of Global Standards (BRCGS) standards, and the International Featured Standards (IFS) Food standard. GMP and GHP are two basic systems aimed at maintaining proper hygiene control of the work environment and technological processes applied in food industry companies. GMP ensures food safety in food processing facilities through inspections of plant equipment, implementing personal hygiene practices, food plant design and construction, and plant site maintenance, sanitation, plant sanitary conditions, and production and process control during food production. GHP is the frst step in hygiene management and is typically applied throughout the food supply chain. GHP covers hygiene management in the food supply chain, employee personal hygiene practices, sanitation, pest control, and the prevention of physical and chemical contamination. The areas studied under GHP include initial production, plant design and equipment, equipment maintenance and sanitation, employee personal hygiene, transport conditions, product information, and training. HACCP focuses on ensuring the health safety of food and is applied widely in European Union countries. This system is based on identifying food hazards, the potential risk of their occurrence, and their impact on all stages of production and distribution. The HACCP system is based on seven principles: conducting hazard analyses, determining critical control points (CCPs), establishing critical values, determining how to monitor CCPs, establishing corrective actions, establishing verifcation procedures, and developing system documentation. Control points can include, for example, raw materials intended for production, employees on production lines, technical equipment in plants, or specifc procedures. There are two critical control points in this system: CCP1 for points where hazards can be eliminated, and CCP2 for points where only mitigation is possible. The ISO 9001 standard introduced elements of management into the food safety assurance system, which includes food safety policy, planning and identifying competencies and responsibilities of the safety system and safe products, manageriallevel competencies and responsibilities, plant infrastructure management and human resources, information fow, document archiving systems, control of the safety management process, validation, and improving and updating the system. In turn, in 1998 in Great Britain, the BRC Global Standard for Food Safety was developed in response to emerging crises and reduced confdence in food safety in the food industry. The main goal of the BRC Global Standard for Food Safety standard is the possibility of uniformly assessing companies that produce food supplied to retail chains. This standard allows the supplier, its management system, production conditions, and product safety to be assessed by a competent auditor. The international safety standard IFS Food was developed by trade organizations from Germany, France, and Italy. This standard is dedicated to suppliers of products for retail chains under their own brands.

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The international IFS Food safety standard is dedicated to suppliers of products for retail chains under their own brands. The IFS Food standard approved by the Global Food Safety Initiative is accepted by retail chains operating in Germany, France, Italy, and the USA. The most harmonized food safety management system is the ISO 22000 standard of 2005. It is based on practices mentioned above (GMP, GHP), ISO 9001, and the HACCP system and includes, inter alia, requirements for interactive communication that is necessary for identifying hazards, managing food safety systems through planning activities, monitoring the effectiveness of activities after their implementation, determining the competencies and responsibilities of management and personnel, training to ensure appropriate hygienic environments throughout food chains, preventing crisis situations, and ensuring product traceability throughout the processing. Adopting and implementing food safety management systems based on HACCP/ ISO 22000 and quality management systems based on ISO 9001 helps the food processing industry to maintain food quality and ensure the safety of consumer health.

REFERENCES Alshannaq, A., Yu, J.H. Occurrence, toxicity, and analysis of major mycotoxins in food. Int. J. Environ. Res. Public Health 2017, 14(6):632. https://doi.org/10.3390/ijerph14060632 Bedele, W., Sindelar, J.J., Milkowski, A.L. Dietary nitrate and nitrite: Benefts, risks, and evolving perceptions. Meat Sci. 2016, 120:85–92. https://doi.org/10.1016/j.meatsci.2016 .03.009 EFSA 2007. Opinion of the Scientifc Panel on Contaminants in the Food chain on a request from the European Commission on ethyl carbamate and hydrocyanic acid in food and beverages, EFSA J. 2007, 551:1–44. EFSA 2011. EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS); Scientifc Opinion on the reevaluation of caramel colours (E 150a,b,c,d) as food additives. EFSA J. 2011, 9(3): 2004, 103. https://doi.org/10.2903/j.efsa.2011.2004. EFSA 2016. EFSA CONTAM Panel (EFSA Panel on Contaminants in the Food Chain), 2016. Scientifc opinion on the risks for human health related to the presence of 3- and 2-monochloropropanediol (MCPD), and their fatty acid esters, and glycidyl fatty acid esters in food. EFSA J. 2016;14(5): 4426, 159. https://doi.org/10.2903/j.efsa.2016.4426. Gilden, R.C., Huffing, K., Sattler, B. Pesticides and health risks. J. Obstet. Gynecol. Neonatal Nurs. 2010, 39(1):103–110. https://doi.org/10.1111/j.1552-6909.2009.01092.x Gowd, V., Su, H., Karlovsky, P., Chen, W. Ethyl carbamate: An emerging food and environmental toxicant. Food Chem. 2018, 248:312–321. https://doi.org/10.1016/j .foodchem.2017.12.072 Han, J., Kim, M.K., Lee, K.G. Furan levels and sensory profles of commercial coffee products under various handling conditions. J. Food Sci. 2017, 82(11):2759–2766. https://doi.org /10.1111/1750-3841.13933 IARC 2000. Monographs on the Evaluation of Carcinogenic Risk to Humans. Some Industrial Chemicals Summary of Data Reported and Evaluation, IARC 77: 41–148 Iwasaki, M., Tsugane, S. Dietary heterocyclic aromatic amine intake and cancer risk: Epidemiological evidence from Japanese studies. Genes Environ. 2021, 43(1):33. https://doi.org/10.1186/s41021-021-00202-5

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Jenner, L.C., Rotchell, J.M., Bennett, R.T., Cowen, M., Tentzeris, V., Sadofsky, L.R. Detection of microplastics in human lung tissue using μFTIR spectroscopy. Sci. Total Environ. 2022, 831:154907. https://doi.org/10.1016/j.scitotenv.2022.154907 Kasmani, R., Othieno-Abinya, N.A., Singh Riyat, M.T., Kiarie, G.W., Wanzala, P. Environmental and occupational factors associated with chronic myeloid leukemia: A case-control study. J. Afr. Cancer 2014, 6(4):194–201. https://doi.org/10.1007/s12558 -013-0297-2 Lalah, J.O., Wandiga, S.O. The effect of boiling on the removal of persistent Malathion residues fromstored grains. J. Stored Prod. Res. 2002, 38(1):1–10. https://doi.org/10 .1016/S0022-474X(00)00036-9 Lentza-Rizos, C., Balokas, A. Residue levels of chlorpropham in individual tubers and composite samples of postharvest-treated potatoes. J. Agric. Food Chem. 2001, 49(2):710–714. https://doi.org/10.1021/jf000018t Leslie, H.A., Van Velzen, M.J.M., Brandsma, S.H., Vethaak, A.D., Garcia-Vallejo, J.J., Lamoree, M.H. Discovery and quantifcation of plastic particle pollution in human blood. Environ. Int. 2022, 163:107199. https://doi.org/10.1016/j.envint.2022.107199 Mehri, F., Nazari, F., Fasihi, Z., Kobarfard, F. Quantifcation of 4-Methylimidazol in NMRI mice plasma and cerebrospinal fuid (CSF) by using liquid chromatography tandem mass spectrometry. Iran. J. Pharm. Res. 2020, 19(4):143–150. https://doi.org/10.22037 /ijpr.2020.112406.13740 Mogol, B.A., Gokmen, V. Thermal process contaminants: Acrylamide, chloropropanols and furan. Curr. Opin. Food Sci. 2016, 7:86–92. https://doi.org/10.1016/j.cofs.2016.01.005 Ou, S., Shia, J., Huanga, C., Zhanga, G., Tenga, J., Jianga, Y., Yanga, B. Effect of antioxidants on elimination and formation of acrylamide in model reaction systems. J. Hazard. Mater. 2010, 182(1–3):863–868. https://doi.org/10.1016/j.jhazmat.2010.06.124 Park, Je, Seo, Je, Lee, Jy., Kwon, H. Distribution of seven N-nitrosamines in food. Toxicol. Res. 2015, 31(3):279–288. https://doi.org/10.5487/TR.2015.31.3.279 Qi, Y., Zhang, H., Wu, G., Zhang, H., Wang, L., Qian, H., Qi, X. Reduction of 5 hydroxymethylfurfural formation by favan-3-ols in Maillard reaction models and fried potato chips. J. Sci. Food Agric. 2018, 98(14):5294–5301. https://doi.org/10.1002/jsfa .9068 Rywotycki, R. The effect of selected functional additives and heat treatment on nitrosoamine content in pasteurized pork ham. Meat Sci. 2002, 60(4):335–339. https://doi.org/10.1016 /S0309-1740(01)00138-3 Sampaio, G.R., Guizellini, G.M., da Silva, S.A., de Almeida, A.P., Pinaff-Langley, A.C.C., Rogero, M.M., de Camargo, A.C., Torres, E.A.F.S. Polycyclic aromatic hydrocarbons in foods: Biological effects, legislation, occurrence, analytical methods, and strategies to reduce their formation. Int. J. Mol. Sci. 2021, 22(11):6010. https://doi.org/ 10.3390/ ijms22116010 Shapla, U.M., Solayman, M., Alam, N., Khalil, M.I., Gan, S.H. 5- hydroxymethylfurfural (HMF) levels in honey and other food products: Effects on bees and human health. Chem. Cent. J. 2018, 12(1):35. https://doi.org/10.1186/s13065-018-0408-3 Tola, M., Kebede, B. Occurrence, importance and control of mycotoxins: A review. A. Rev. Food Sci. Technol. 2016, 2(1). https://doi.org/10.1080/23311932.2016.1191103 Tortorella, V., Masciari, P., Pezzi, M., Mola, A., Tiburzi, S.P., Zinzi, M.C., Scozzafava, A., Verre, M. Histamine poisoning from ingestion of fsh or scombroid syndrome. Case Rep. Emerg. Med. 2014:482531. https://doi.org/10.1155/2014/482531 Witczak, A., Abdel-Gawad, H. Application od acetic acid solution for the reduction of pesticide residue contents in fruits and vegetables. In Acetic acids: Advances in research and applications. M. Szymczak and O. Topuz. Eds. Nova Science Publishers, Inc., New York, 2018, 97–112.

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Probiotics and Prebiotics in Food Edyta Malinowska-Pańczyk

CONTENTS 16.1 Introduction .................................................................................................. 433 16.2 Probiotics ...................................................................................................... 433 16.2.1 Defnition and Regulations ............................................................... 433 16.2.2 Criteria for Identifcation/Selection of Probiotics............................. 434 16.2.3 Characteristics of Probiotics............................................................. 435 16.2.4 Health Benefts of Probiotics ............................................................ 436 16.2.5 Hazards ............................................................................................. 437 16.2.6 Probiotic Food .................................................................................. 439 16.3 Prebiotics ...................................................................................................... 441 16.3.1 Defnition .......................................................................................... 441 16.3.2 Health Benefts of Prebiotic Consumption ....................................... 441 16.3.3 Types of Prebiotics............................................................................ 442 16.3.3.1 Carbohydrate-Based Prebiotics.......................................... 442 16.3.3.2 Non-Carbohydrate Prebiotics ............................................ 451 References.............................................................................................................. 453

16.1

INTRODUCTION

Nowadays, food is not only used to satisfy hunger and is a source of nutrients, but is also considered a factor that directly affects human health. Consumers are looking for high-quality products that contain bioactive ingredients that affect the proper functioning of the body and good mood. Functional foods, which must exhibit health benefts when consumed as part of a balanced diet, are becoming increasingly popular among health-conscious individuals. Probiotics and prebiotics may be functional components of such foods.

16.2

PROBIOTICS

16.2.1 DEFINITION AND REGULATIONS Probiotics are “live microorganisms that, when administered in adequate amounts, confer a health beneft on the host.” This current defnition was frst provided in 2002 by a working group of the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO) and slightly grammatically revised DOI: 10.1201/9781003265955-16

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in 2014 by the International Scientifc Association for Probiotics and Prebiotics (ISAPP) (FAO/WHO 2002; Hill et al. 2014). This defnition is applicable to dietary supplements and drugs as well as microorganisms found in food. The increase in the consumption of probiotics and the growth of their global market necessitates the need for a regulatory framework that establishes a uniform framework that is strictly adhered to by all manufacturers. In the European Union, probiotic foods and supplements are subject to the Nutrition and Health Claims Regulation 1924/2006. The European Food Safety Authority (EFSA) oversees the verifcation of probiotic health claims that are typically labeled, and issues Qualifed Presumption of Safety (QPS) for various bacterial strains. This list is compiled from historical data and from regular knowledge monitoring and literature reviews. The QPS focuses on the healthy consumer population, it does not take into account the potential risks that probiotics may cause in vulnerable human populations (pregnant women, infants, people with weakened immune systems, or people with short bowel syndrome). EFSA excluded all health claims for probiotics, explaining that achieving a healthy gut microbiota is not a recognized health beneft. In contrast, the US Food and Drug Administration has adopted the term “generally recognized as safe” (GRAS). A microorganism may be GRAS only if recognition of its safety is based on expert opinions authorized to assess the safety of the substance. For encapsulated probiotics in the food industry, only capsule materials approved for human consumption are permitted.

16.2.2 CRITERIA FOR IDENTIFICATION/SELECTION OF PROBIOTICS The qualifcation of a bacterial strain as a probiotic is possible after meeting several very important criteria. First, the probiotic strain must be properly identifed and named according to the currently valid nomenclature based on the International Code of Nomenclature. An updated list of prokaryotic names can be found online at: www.bacterio.net (Binda et al. 2020). Sequencing of 16S ribosomal DNA is one of the recommended methods for identifcation, although currently, the “gold standard” for microbial identifcation is genome-wide sequencing, including extrachromosomal elements, which allows not only identifcation at the species and strain level, but also facilitates the search for the presence or absence of risk. Phenotypic characterization of the strain is recommended, although not a prerequisite: ability to survive in appropriate body sites, production of lactic acid or other short-chain fatty acids (SCFA), adhesion to mucus or intestinal epithelial cells, interaction with human immune cells, resistance to digestive enzymes, bile salts, or acid, antimicrobial activity by competitive exclusion, and production of bacteriocins or hydrogen peroxide. Knowledge regarding these characteristics may be helpful in interpreting the mechanisms of observed clinical fndings and useful in initial screening strategies. Another requirement for the qualifcation of a strain as a probiotic is the confrmation that it will be safe for its intended use in food or as a dietary supplement. This assessment is done on a case-by-case basis (strain, use) according to the safety requirements set by the national/regional regulatory authority. Where historical data exist on the safe, specifc use of the microorganism, these can assist in the overall

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assessment of this condition. When such data are not available, they are determined by conducting appropriate phase 1 studies. The safety assessment shall be designed to ensure that the strain does not possess: a) acquired resistance genes to antimicrobials or other known virulence factors, b) genes encoding known virulence factors, c) transferable antibiotic resistance genes, d) the ability to form biogenic amines and D-lactic acid, e) hemolytic activity, and f) bile salt hydrolase activity (Binda et al. 2020). Another criterion that a strain must meet to qualify as a probiotic is confrmation of its health-promoting properties in at least one human clinical trial conducted in accordance with generally accepted scientifc standards. A strain that has passed the steps described above must remain viable in suffcient quantities in the product at an effective dose throughout the shelf life (Binda et al. 2020). An effective dose of a probiotic according to the ISAPP leafet (https://isappscience.org/isapp-releases-new -infographic/) is from 108 to 5 × 1010 colony forming units (CFU).

16.2.3 CHARACTERISTICS OF PROBIOTICS Many bacteria affect the microbial balance in the gut, so only species and strains of bacteria that have a proven positive effect on the host can be chosen as probiotics. The most common probiotics include the species Lactobacillus, Bifdobacterium, Lactococcus, and Enterococcus. Some species, such as Lactobacillus delbrueckii subsp. bulgaricus or Streptococcus salivarius subsp. thermophilus, are not normally part of the gut microbiota but are nevertheless counted as probiotics. Their effect on the microbial balance of the gut is weak because they lack colonization properties. However, these strains can be considered probiotics due to their ability to produce lactase which leads to a reduction in symptoms associated with abnormal lactose digestion, and because of this beneft. Zhang et al. (2021) divided probiotics into three groups: • traditional probiotic lactic acid bacteria (LAB), • non-LAB, • next-generation probiotics (NGPs). LAB are non-spore-forming, Gram-positive bacteria that ferment saccharides, usually to lactic acid. They are commonly found in the environment and host body (fermented foods, soil, body surface, intestines) and play an important role in regulating the gut microbiota. Lactobacillus and Bifdobacterium are two major genera widely used in fermented products. These are mostly species: Lactobacillus acidophilus, Lactobacillus amylovorus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsoni, Lactobacillus gallinarum, L. delbrueckii, L. delbruecki subsp. bulgaricus, L. delbrueckii subsp. lactis, Lacticaseibacillus rhamnosus (syn. Lactobacillus rhamnosus), Lacticaseibacillus casei (syn. Lactobacillus casei), Lacticaseibacillus paracasei (Lactobacillus paracasei), Lactiplantibacillus plantarum (syn. Lactobacillus plantarum), Limosilactobacillus reuteri (Lactobacillus reuteri), Levilactobacillus brevis (syn.

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Lactobacillus brevis), Limosilactobacillus fermentum (syn. Lactobacillus fermentum), Ligilactobacillus salivarius (syn. Lactobacillus salivarius), Sporolactobacillus inulinus, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis, Leuconostoc mesenteroides subsp. dextranicum, Pediococcus acidilactici, S. salivarius subsp. thermophilus, Enterococcus faecalis, and Enterococcus faecium. LAB are generally associated with the order Lactobacillales (type Firmicutes); however, bacteria of the genus Bifdobacterium (phylum Actinobacteria) are often grouped with them. Bifdobacterium are Gram-positive anaerobic rods that also produce lactic acid as a major product of carbohydrate metabolism. Probiotic strains include the species Bifdobacterium bifdum, Bifdobacterium animalis, Bifdobacterium animalis subsp. lactis (Bifdobacterium lactis), Bifdobacterium longum, Bifdobacterium longum subsp. infantis (syn. Bifdobacterium infantis), Bifdobacterium adolescentis, Bifdobacterium breve. The second group is non-Lab bacteria that exhibit probiotic activity against their hosts. This group includes: Saccharomyces cerevisiae var. boulardii, Weizmannia coagulans (syn. Bacillus coagulans), Bacillus subtilis, Bacillus cereus, Sporolactobacillus inulinus, Clostridium butyricum, Escherichia coli Nissle 1917, and Propionibacterium freudenreichii. The benefcial effects of these microorganisms have been very well understood. It is likely that this group could include many strains of microorganisms that have not yet been studied or are even considered pathogenic. In recent years, through the development of sequencing and anaerobic culture methods, potentially new commensal bacteria have been discovered that are closely associated with improved health. These newly isolated bacteria with no long history of use are called NGPs. Most NGPs are derived from the intestines of healthy humans and include Akkermansia muciniphila, Faecalibacterium prausnitzii, Eubacterium hallii, and Roseburia spp. With continued improvements in culture methods, NGPs have been characterized in recent years, but the feld is still evolving as 70% of the gut microbiota is left uncultured.

16.2.4

HEALTH BENEFITS OF PROBIOTICS

Probiotics are a priori non-pathogenic, meaning that they should never cause or potentiate any disease in humans, regardless of the source of their intake, i.e., food or over-the-counter supplements. The term “health beneft” (often stated on the label) has no clear medical meaning. The situation is further complicated because of the multiplicity of microorganisms used as probiotics and the differences that occur not only between types of bacterial species but also between strains of the same species. Therefore, it can be assumed that probiotics will have different health benefts, but they can also have adverse effects (when inappropriately administered). The health benefts of consuming probiotics are related to both the presence of live microorganisms in foods and the bioactive components released into foods as byproducts of the fermentation process, so-called postbiotics, which include lactic acid and other organic acids, SCFA, vitamins, peptides, bacteriocins, enzymes, and immune system signaling substances.

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Consumption of probiotics is believed to be benefcial, especially in the prevention and treatment of gastrointestinal infections and diseases. Probiotics compete with pathogenic bacteria for binding to intestinal epithelial cells, and they strengthen the intestinal barrier. They also inhibit pathogen growth by secreting antimicrobial peptides and stimulate the body to produce immunoglobulins and enhance phagocytosis and increase natural killer cell activity. Probiotic bacteria also prevent or relieve infammation and help alleviate symptoms of lactose intolerance by producing the enzyme β-D-galactosidase (lactase). Therapeutic uses of probiotics also include preventing urinary tract infections, relieving constipation, protecting against traveler’s diarrhea, treating hypercholesterolemia, protecting against colon and bladder cancer, and preventing osteoporosis and food allergies. Consumption of probiotic bacteria prevents obesity, type II diabetes, and cardiovascular disease while stimulating the immune system. Some health-promoting properties are observed in most probiotic microorganisms, while others are observed at the species level or attributed only to strains (Figure 16.1). Many commercially available probiotic food and supplements typically contain different bacterial strains belonging to different species. This is due to the belief that the presence of several strains will have a wider range and greater effectiveness of benefcial effects and that their effects will be additive or even synergistic. Studies that have compared single-species probiotics with multi-species probiotics have shown that such claims are not yet clear and should be further investigated. Thus, the hypothesis that in some cases there may be antagonistic effects between different probiotic species should also be considered.

16.2.5

HAZARDS

The paradigm of apparent safety and effcacy of probiotics has become controversial due to the lack of results from long-term clinical trials clarifying their effcacy, safety, and toxicity to humans. Consumers may react in different and often unanticipated ways to any drug, dietary supplement, or food, including probiotics (Žuntar et al. 2020). Additionally, probiotics, as living microorganisms, grow and colonize the intestines, and theoretically, under certain circumstances, can cause adverse events in the host and pose a serious health problem (Lerner et al. 2019). There is also a lack

FIGURE 16.1 Distribution of health-promoting properties among probiotics (redrawn from Hill et al. 2014 with permission).

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of data – even in animal models – on pharmacological and toxicological interactions regarding the dose-response relationship of the probiotic. Survival of probiotics in the host may be variable depending on differences in their manufacturing and formulation (food, dietary supplements). Knowledge of the colonization properties of microorganisms is one of the most important factors to assess safety. In addition, probiotics may have adverse effects per se, especially in critically ill, intensive care unit patients, after surgery, and especially in immunocompromised individuals (Table 16.1). There is also a reasonable concern about pharmacological drug interactions with probiotics, which may have clinical relevance. This issue has so far only been studied in an animal model. Probiotics decreased or increased the bioavailability of drugs and xenobiotics in animals, their pharmacokinetics (absorption, distribution, metabolism, and elimination), and pharmacodynamics and toxicity (response to treatment and adverse effects). For some drugs, increased bioavailability has been associated with a risk of causing serious and life-threatening adverse effects, while decreased bioavailability has led to ineffective drug action. Such studies in humans have not yet been published. The safety of probiotics should also be determined by evaluating the blood-brain barrier, as in recent years it has been shown, for example, that a high abundance of probiotic A. muciniphila has been reported in Alzheimer’s, Parkinson’s, and multiple sclerosis patients, suggesting that it is necessary to study the permeability of the blood-brain barrier to probiotics (Zhang et al. 2021). Therefore, the decision to consume probiotics should be preceded by an individualized assessment of the consumer’s nutritional status and general health, their medication use, and the specifcity of the probiotic strains (Žuntar et al. 2020). Probiotics

TABLE 16.1 Examples of the Adverse Effects of Probiotics on Consumers Probiotic

Risk group

Consequences

S. cerevisiae var. boulardii

• Patients with impaired function of the immune system • People in critical condition

Fungal infections related to S. cerevisiae

Lactobacillus sp.

• • • • • • •

Liver abscess Lung infection Bacteremia Endocarditis (23% mortality)

L. acidophilus, L. casei, L. salivarius, L. lactis, B. bifdum, and B. lactis Lactobacillus and Bifdobacterium

Elderly people Patients with liver disease Diabetics Cancer patients and after transplant Immunocompromised patients People with heart defects People with pancreatitis

• Patients with short bowel syndrome • Infants

Intestinal ischemia leading to death (16%) Lactic acidosis, “brain fog” (cognitive impairment)

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(if taken in critical quantities) can also carry demonstrable toxicological consequences and may even lead to death. The effect of probiotics on nutrients and consumer health is an interdisciplinary topic, very broad, and in many respects still well unrecognized. For each probiotic or probiotic mixture, such recognition should be individually established regarding their role, safety, and toxicity effects, gut microbial balance, etc. The one-size-fts-all criterion should absolutely not apply here. The risks associated with probiotic consumption are also linked to safety concerns arising from undefned quality standards and manufacturing procedures, especially for dietary supplements. Evaluation of probiotic products should include controls for pathogenicity, infectivity, virulence, and metabolic activity.

16.2.6 PROBIOTIC FOOD Probiotic foods are classifed as functional foods. The term may be used for a food product only if there is a proven health beneft from the presence of well-defned and characterized live microorganisms in the food product that goes beyond any nutritional properties of the food matrix. Therefore, the terms fermented food and probiotic food cannot be used interchangeably. In 2021, ISAPP published a consensus statement on fermented foods. This document provided the differences between probiotics, fermented foods, and probiotic fermented foods (Marco et al. 2021). The frst group includes products (without a specifc format) that may use the name “probiotic” on the label (a defnition was given earlier) along with a statement of what health benefts its consumption provides, such as helping to strengthen natural defenses or helping to enhance well-being. The second group is fermented foods, which are products that have been obtained by the desired growth of microorganisms and enzymatic conversion of food ingredients. For these products, evidence of health benefts is not required. If the product does not contain live microorganisms because they have been inactivated in subsequent stages of the production process, then it is classifed as a food made by fermentation. If live microorganisms are present in the product, then the packaging may say “contains live and active cultures.” The third group is probiotic fermented products. It has been divided into two subgroups: foods fermented by and/or containing probiotic(s) for which strain-specifc evidence has been identifed, and foods fermented by and/or containing probiotic(s) without strainspecifc evidence. For the frst subgroup, a claim identical to the frst group may be used on the packaging, while only the claim “contains probiotics” may appear in the second subgroup. This means that at least one strain that meets the probiotic criteria has been confrmed in the product because it belongs to a well-studied species that is known to provide health benefts through the principle of “co-benefts.” This claim is based on the knowledge that certain species of bacteria are continuously active in the human body and have retained essential properties associated with improved health (Marco et al. 2021). The food matrix plays an important role in the benefcial effects of probiotics on host health. Probiotics can lead to changes in product composition by producing bioactive compounds such as vitamins, antioxidants, and biologically active peptides, potentiating the health-promoting effect. About 40 strains are currently

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used in the food industry, the most common being LAB. A food product can be considered probiotic when it contains at least 106 CFU/cm3 or g, throughout its shelf life. The survival of probiotics in a food product depends on many factors. First, it depends on the acidity of the environment, the presence of saccharides, salts, antimicrobial substances, oxygen, and water content. In addition, the population size of probiotics in the product is infuenced by conditions during processing and storage. Designing probiotic products, therefore, requires establishing strain compatibility with the food matrix and using them at the appropriate stage of production and storage conditions. The most popular probiotic foods are fermented milk products, which are an important part of people’s diets in many countries and have a long tradition of consumption. One of the oldest products with proven health-promoting properties is kefr, whose consumption is attributed to the longevity of Caucasian people, and koumiss. Other fermented milk beverages containing probiotic bacteria include yogurt, ayran, and yakult, among others. Fermentation of milk leads to its preservation, improves its nutritional value and sensory properties, and in addition is an inexpensive technology. Fermented milk products may contain one or more probiotic strains. Consumption of fermented milk beverages brings a few benefts to the human body, including prevention of gastrointestinal diseases, obesity, diabetes, cancer, infections, and allergies (Zhang et al. 2021). In recent years, the food industry and researchers have been developing technologies to obtain probiotic products in other than milk food matrices, such as probiotic beverages based on fruit juices or plant milk (Zuntar et al. 2020; Ziarno and Cichońska 2021). Fruit juices are a rich source of nutrients such as dietary fber, mineral salts, vitamins, and polyphenols. The health-promoting potential of these products can be enhanced by the addition of probiotic bacteria. The problem in obtaining probiotic fruit juices is the deterioration of the taste of this raw material and the poor survival of microorganisms, which is due to the high acidity, presence of oxygen, insuffcient nitrogen sources (proteins, peptides, amino acids), and lack of oligosaccharides. These limitations can be reduced by the addition of prebiotics or vitamins or microencapsulation of microorganisms. The market for plant-based beverages (e.g., based on legumes: soybeans, beans, broad beans, lentils, chickpeas, peas) is a rapidly growing sector of the food industry due to the increasing popularity of milk substitutes. Fermentation of this raw material not only improves taste, favor, and overall consumer acceptance, but also leads to products with increased nutritional value, high antioxidant potential, and microbiological safety. The production process of fermented probiotic plant beverages is usually completed when the product reaches a pH of about 4.2–4.5, which usually takes 12 to 24 hours. Most commonly, bacterial strains of the genera Lactobacillus, Lactococcus, Streptococcus, and Bifdobacterium are used to obtain these products. Their nutritional value increases due to the reduction of oligosaccharides (mainly stachyose and raffnose), tannins, protease inhibitors, and phytic acid. At the same time, the bioavailability of iron, calcium, and zinc increases. During the fermentation process, the amount of FAA, e.g., GABA, increased. Microorganisms also produce B vitamins, e.g., folic acid, ribofavin, B12, niacin, and pyridoxine.

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PREBIOTICS DEFINITION

The offcial defnition proposed by ISAPP consensus in 2017 states that a prebiotic is “substrate that is selectively utilized by host microorganisms conferring a health beneft.” The earlier description of a prebiotic as an indigestible food ingredient that benefcially affects the host by selectively stimulating and/or activating one or a limited number of bacterial species already residing in the colon, limited the healthpromoting effect of a prebiotic to the gastrointestinal tract only. Considering the new reports, prebiotics may also have a benefcial effect in parenteral sites such as the skin or the vagina. For this reason, the new defnition has been formulated to indicate that the site of action of prebiotics may be any microbial community inhabiting the human body, where they promote homeostasis and/or alleviate specifc disease states associated with microbial imbalance (Gibson et al. 2017). Dietary prebiotics differ from dietary fber in that the latter stimulates the growth of many different groups in the gut microbiota ecosystem. A substance can therefore be considered a dietary prebiotic when: (a) it is resistant to the acidic pH of the stomach; (b) it is not hydrolyzed by endogenous enzymes and is not absorbed in the upper gastrointestinal tract; (c) it should not be absorbent in the gastrointestinal tract; (d) it is metabolized by the microbiota; and (e) it selectively stimulates the growth and/or activity of one or a limited number of bacterial species related to health and well-being. Although there are currently no offcial dietary recommendations for the daily intake of prebiotics in healthy individuals, it is believed that only a daily intake of at least 3 g or more results in benefcial effects.

16.3.2

HEALTH BENEFITS OF PREBIOTIC CONSUMPTION

Prebiotics can indirectly or directly positively affect the health of the consumer. In the frst case, they maintain a healthy gut microbiota by stimulating the growth of appropriate groups of microorganisms. Sometimes the metabolites formed during fermentation are a substrate for other species or may act antagonistically to certain microorganisms. Fermentation products of prebiotics, mainly SCFAs – acetic, propionic, butyric – as well as L-lactic acid, cause a decrease in intestinal pH from 6.5 to 5.5, which contributes to a change in the composition of the intestinal microbiota population. The growth of Gram-negative species, e.g., of the genus Bacteroides and E. coli, are inhibited, while butyrate-producing Gram-positive bacteria related to Eubacterium rectale increase in numbers and occupy niches that have formed after inhibition of Gram-negative bacteria (Duncan et al. 2009). This process is called the butyrogenic effect. Butyric acid also affects intestinal epithelial growth and is preferentially used by colonocytes as an energy source, which helps them fght infammation and carcinogenesis. Propionic acid interferes with and inhibits cholesterol production by the host liver, thereby lowering plasma levels of this sterol and reducing the risk of cardiovascular disease complications (Teferra 2021). Since SCFAs can enter the bloodstream through enterocytes, prebiotics can affect

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not only the gastrointestinal tract but also distant organs. Peptidoglycan, produced also during fermentation, can stimulate the body’s innate immune system against pathogens. Prebiotics have a direct effect on the consistency of the stool and prevent diarrhea and constipation. They also support the immune system, eliminate excess substances such as glucose and cholesterol, stimulate the absorption and production of B vitamins, and control obesity and osteoporosis. It is assumed that prebiotics do not lead to serious or life-threatening side effects. The benefcial effects of prebiotics on human health are observed after consumption of approximately 2.5 to 10 g per day. However, both such doses of prebiotics and much higher doses, 40–50 g per day, can sometimes cause bloating, osmotic diarrhea, and cramping, respectively. This means that prebiotics in therapeutic doses can also lead to mild discomfort. The severity of symptoms depends on the chain length of the prebiotic and the dose taken. Compounds with shorter chains may cause more side effects. Probably shorter molecules are fermented and metabolized more rapidly, primarily in the proximal portion of the colon, while those with longer chains are fermented later and more slowly in the distal (Davani-Davari et al. 2019).

16.3.3

TYPES OF PREBIOTICS

There are many types of dietary prebiotics, most of which are carbohydrate-based. The best-known prebiotics are indigestible oligosaccharides: fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), mannan-oligosaccharides (MOS), xylooligosaccharides (XOS), and isomalto-oligosaccharides (IMO); and inulin and lactulose. Prebiotic effects have also been demonstrated for gentio-oligosaccharides (GnOS) and arabino-oligosaccharides (AOS). Other substances, such as polyphenols, polyunsaturated fatty acids (PUFAs), and conjugated linoleic acid, may also have prebiotic properties. A breakdown of the nutrients whose prebiotic effects have already been confrmed or reported and their distinction from fber and other nonprebiotic substances is shown in Figure 16.2 (Gibson et al. 2017). 16.3.3.1 Carbohydrate-Based Prebiotics 16.3.3.1.1 Fructo-Oligosaccharides (FOS) FOS are composed of fructose monomers linked by β-(2⟶1) or β-(2⟶6) glycosidic bonds and terminally attached glucose. FOS are not digested in the small intestine but are metabolized in the colon to SCFAs, L-lactates, and other bioactive molecules benefcial to human health. Consumption of FOS leads to lower levels of cholesterol, triacylglycerols, phospholipids, and blood glucose; reduces blood pressure; and improves calcium and magnesium absorption, potentially increasing bone mineral density. This group of oligosaccharides also inhibits the production of reductases, enzymes contributing to cancer. The intake of FOS decreases genotoxin production and β-glucuronidase activity, which generates carcinogens in the intestine and thus regulates colon cancer incidence. FOS is also useful in controlling infammatory bowel diseases such as Crohn’s disease

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FIGURE 16.2 Classifcation of nutrients into prebiotic and non-prebiotic (redrawn from Gibson et al. 2017).

and ulcerative colitis. They also benefcially stimulate the growth of specifc bacteria in the intestine, including bifdobacteria and lactobacilli, while reducing populations of Bacteroides, Fusobacterium, and Clostridium sp. (Singh et al. 2017). According to various sources, the caloric value of FOS is 1–2 kcal/g. Consumption of large amounts of FOS, 20 to 30 g per day, can cause bloating, especially in people with lactose intolerance. Larger amounts – 44 g for men and 49 g for women (per day) – cause diarrhea. For this reason, it is recommended to consume 2 to 12 g of FOS daily (Sridevi et al. 2014). FOS naturally occur in many fruits and vegetables. The richest sources of FOS are listed in Table 16.2. Less than 0.1% FOS is contained in fruits such as apples, blueberries, cantaloupe, gooseberries, pears, kiwi, and rhubarb; and vegetables such as asparagus, beans, celery, eggplant, endive, and potatoes. These prebiotics were not detected in ginger, tomato, or zucchini (Campbell et al. 1997). The law treats these oligosaccharides as an ingredient, not as an additive. Their amount in infant formula ranges from 0.6 to 0.8 g/dm3. The concentration of FOS in the plant raw material is insuffcient for a prebiotic effect, so they are synthesized. There are different methods to produce FOS. One of these is the acid hydrolysis of inulin using hydrochloric or sulfuric acid, which requires expensive post-process removal. A better option is to use citric or phosphoric acid, which have GRAS status, for this purpose. FOS can also be produced by biotechnological processes using β-fructofuranosidases (e.g., inulinases that hydrolyze inulin, levanases that degrade levanine) or fructosyltransferases (FTases) that transfer the fructosyl unit of sucrose to different acceptors, generating products of different molecular weights depending on the specifcity of

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TABLE 16.2 FOS Content in Selected Fruits and Vegetables FOS content (%) in selected Fruit Banana Blackberry Grape Muskmelon Orange Plum Raspberry Watermelon

Vegetable 0.18–1.09 0.12 0–0.11 0.12 0.28 0.20 0.15 0.30

Artichoke (globe) Chicory root Chinese chive Garlic Carrot Jerusalem artichoke Leek Lettuce Onion Shallot Peas Radish

2.18 2.1 0.1 1.03 0.14–0.22 28.62 0.48 0.79 1.34–3.2 5.29 0.07–0.84 0.3

Source: Campbell et al. 1997.

the enzyme (Singh et al. 2017). The source of FTases can be microorganisms, both bacteria (B. subtilis, Bacillus macerans, Arthrobacter oxydans, Microbacterium laevaniformans, S. salivarius) and fungi (Aureobasidium pullulans, Aspergillus niger, Aspergillus foetidus, Penicillum citrinum, S. cerevisiae) (Hurtado-Romero et al. 2020). Purifed enzymes and whole cells of microorganisms (usually A. niger and A. pullulans) are used in the production of FOS preparations. FTases result in the formation of 1-kestose (1 × glucose and 2 × fructose, kestotriose), nystose (1 × glucose and 3 × fructose, kestotetraose), and fructofuranosyl nystose (1 × glucose and 4 × fructose, kestopentaose). Due to their organoleptic properties, FOS are widely used in the food industry as a substitute for sugar and fat. In most food products, 1 g of fat can be replaced by approximately 0.35 g of FOS. These oligosaccharides lower the freezing point and serve as a binder; they are added to dairy products, dressings, frozen desserts, and meat products, among others. FOS infuence the rheological properties of dough, enhancing its elasticity and stability. 16.3.3.1.2 Inulin Inulin is a linear fructan containing fructose monomers linked by β-(2⟶1) glycosidic bonds. This confguration means that it is not digested by endogenous digestive enzymes. This saccharide has unique prebiotic, technological, and functional properties. This saccharide has unique prebiotic, technological, and functional properties, which change depending on the DP. Inulin as a soluble dietary fber is fermented in the large intestine to SCFA and leads to an increase in fecal mass and motility. It also has a positive effect on the lymphoid tissue associated with the gut, which in

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turn helps control resistance to pathogen translocation and colonization. It inhibits the proliferation of pathogens such as E. Coli, Campylobacter jejuni, Salmonella enteritidis, and Clostridium perfringens. Inulin has also been shown to have antioxidant properties and anti-infammatory effects, play a role in lowering blood lipid and cholesterol levels, and increase mineral salt absorption. It also has a positive effect on enteroendocrine functions and is involved in the regulation of hormones related to appetite and satiety. This prebiotic is safe to consume and there are no reports of toxicity in animals or humans. However, intake of inulin at 20–30 g per day causes gastrointestinal intolerance. Inulin is found in the bulbs, roots, and tubers of about 45,000 plant species. The richest source of inulin is chicory and Jerusalem artichoke (15–21 g/100 g), followed by garlic (9–16 g/100 g) and leek (3–10 g/100 g). Smaller amounts of inulin, from 1 to 7 g/100 g, are contained in artichoke, rye, wheat, barley, onion, and bananas (Waqas and Summer 2019). In plants, inulin occurs as a mixture of oligo and polysaccharides (up to 100 units), where it acts as a storage material. DP value of inulin is an important factor affecting its functional properties. It depends on the plant species from which inulin is isolated, climatic conditions, growing season, and harvest and storage time (Waqas and Summer 2019). Inulin, both low and high DP, is legally classifed as a food or food ingredient. It has GRAS status in the USA. The application of this prebiotic has grown over the years. On an industrial scale, it is obtained by hot water extraction from plant material, most commonly from the roots of chicory or Jerusalem artichoke. The extraction is performed at 80–85° C and lasts 60–90 min at pH 6.8. Maintaining pH is an important factor to obtain inulin with high DP. Lower pH, < 6, leads to hydrolysis of this polysaccharide. Inulin can also be effciently extracted with organic solvents. Methanol, ethanol, acetone, or acetonitrile can be used for this purpose. Although the highest yields are obtained using the latter two, ethanol is nevertheless the best choice because of its GRAS status (Waqas and Summer 2019). Low DP inulin dissolves quite well in water and has about 30–35% of the sweetness of sucrose, so it can be used as a substitute for sucrose and at the same time lead to lower caloric content of the product. Their caloric content is 1–2 kcal/g. On the other hand, high DP inulin fractions are poorly soluble in water and relatively viscous, and thus can fnd use as fat replacements in low-fat dairy products. They also form microcrystals in milk or water to give products a creamy texture, giving the sensory experience created when fat is ingested. Because of these properties, they are used in confectionery, to thicken ice cream, spreads, fllings, sweets, and salad dressings. 16.3.3.1.3 Galacto-Oligosaccharides GOS are a mixture of compounds made from lactose, containing between two and eight saccharide units. One of these units is terminal glucose, and the other saccharide units are galactose and disaccharides containing two galactose units (Torres et al. 2010). GOS are divided into two subgroups: GOS with excess galactose at C3, C4, or C6 and GOS produced from lactose by enzymatic transglycosylation. This type of GOS is also referred to as trans-galacto-oligosaccharides or TOS.

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These prebiotics modulate the microbiota in the colon by promoting the proliferation of bifdobacteria and lactobacilli and inhibiting the growth of bacteroides. They can bind pathogenic bacteria such as Salmonella sp. or enteropathogenic E. coli, preventing their colonization in the colonic epithelium and inhibiting symptoms of gastrointestinal infection. GOS improve calcium and magnesium metabolism and have a benefcial effect on stool consistency and defecation frequency. The caloric value of GOS is 1–2 kcal/g. Consumption of these prebiotics poses does not pose a risk to the consumer, but excessive consumption, above 20 g per day, can lead to osmotic diarrhea. GOS can be obtained by acid hydrolysis of lactose, which produces a complex mixture of disaccharides and trisaccharides, linked by α- and β-glycosidic bonds. Due to the lack of product specifcity and the extreme conditions during this process, it is not used to produce GOS on an industrial scale. An effcient way to synthesize GOS is enzymatic catalysis from lactose using glycosyltransferases or glycoside hydrolases. However, these enzymes, despite their high regio- and stereo-selectivity, are also not used for industrial production of GOS due to their unavailability, prohibitive prices of commercial enzyme preparations, and the need to use specifc sugar nucleotides as substrates. Currently, GOS are produced using the catalytic activity of retaining glycoside hydrolases, even though these enzymes are less stereoselective. The conversion of lactose to GOS by β-galactosidases is a kinetically controlled reaction through a competition between hydrolysis and transgalactosylation (Vera et al. 2016). The yield for obtaining GOS depends on various factors. In general, the highest yields are obtained from lactose solutions with concentrations between 30 and 40%, at elevated temperatures that promote lactose solubilization and enhance transgalactosylation over hydrolysis. Effcient production of GOS occurs in environments with reduced water activity (Torres et al. 2010). Whole cells of microorganisms producing this enzyme or purifed preparations can be used for GOS biocatalysis. β-galactosidases are extracted from a variety of microorganisms, including molds (Aspergillus oryzae), yeast (Sterigmatomyces elviae), and bacteria (Bifdobacterium and Lactobacillus genera). Depending on the enzyme source, different types of GOS are formed, varying in amount, DP, and glycosidic bonds. Recombinant β-galactosidases are also available and are considered a better alternative than the native ones due to high production yield, easy purifcation, and better enzymatic stability, among other reasons. GOS are a natural component of human milk, so their use in infant formula (nutrition) and infant foods is intended to have a bifdogenic effect and provide multiple health benefts, such as improving bowel frequency, lowering stool pH, and stimulating bifdobacteria and lactobacilli. These products typically contain 6.0 to 7.2 g/dm3 GOS. These compounds are not hydrolyzed in saliva, so they can be used as low-cariogenic sugar substitutes, and they also have a very low effect on blood glucose levels. Due to their stability at low pH and the ability to form clear solutions, GOS is also added to beverages such as juices and fruit drinks. These compounds have a neutral, slightly sweet taste and therefore do not affect the sensory properties of the

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fnal product. They are also often added to fermented milk drinks such as yogurt, buttermilk, or milk-based drinks. The addition of GOS to yogurt (before or after fermentation) makes the gel structure smoother and creamier than yogurt without this ingredient. In addition, the lactic bacteria involved in the fermentation process do not metabolize GOS, so it ends up in the large intestine unchanged. GOS do not degrade during dough fermentation and baking, so they are a very common functional additive in bread and most other high-fber, low-calorie baked goods. In these products, they maintain proper moisture content and improve taste and texture. Due to their health-promoting properties, GOS can be an important food ingredient for the elderly and hospitalized people (Sangwan et al. 2011). 16.3.3.1.4 Human Milk Oligosaccharides (HMO) HMO are the third most abundant component in human milk. Colostrum contains 20–23 g HMO per dm3 and mature milk 12–15 g HMO per dm3, which exceeds the total concentration of milk proteins in this food. These compounds are mainly composed of fve monosaccharides: glucose, galactose, N-ethylglucosamine, fucose, and sialic acid. These monosaccharides form compounds that differ in the amount and type of individual monomers. Currently, more than 150 different HMO structures have been characterized. All HMOs at the reducing end contain lactose, which can be elongated by the addition of lacto-N-biose disaccharides or N-acetyllactosamine. They can be modifed with sialic acid and/or fucosylated. The structure of the HMO often determines its functions. The quantitative and qualitative composition of HMO varies between women but remains constant during lactation in the same woman (Bode 2020; Wiciński et al. 2020). HMO are not hydrolyzed in the stomach and by pancreatic enzymes as well as the brush border in the small intestine. Approximately 99% of the HMO reach the distal small intestine and colon intact, where they are metabolized by microorganisms or excreted in the feces. The remaining 1% is absorbed where it affects tissues and organs other than the intestines. The main function of HMO is primarily to shape a healthy microbiota in a growing body. In the large intestine, they are mainly fermented by bifdobacteria, which metabolize them to SCFA, predominantly acetic acid. It lowers the pH of the intestinal contents, inhibiting the growth of pathogenic bacteria (Nolan et al. 2020). Some HMO also have antimicrobial and anti-adhesive properties – they mimic receptors on the surface of epithelial cells, thus preventing the attachment of pathogens, including viruses, bacteria, and protozoa, to the host surface. Thus, they inhibit the proliferation of undesirable microorganisms and the development of the disease (Bode 2020). HMO can also directly modulate the immune response in an infant by acting on cells of the mucosa-associated lymphoid tissue and at the systemic level. Sialated HMO infuence the maturation of T lymphocytes and contribute to the prevention of allergies. Some HMO can now be obtained on an industrial scale, where microbes that synthesize HMO from monosaccharides are used to produce them. Synthetic HMO are added to infant formula (Bode 2020).

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16.3.3.1.5 Mannan-Oligosaccharides MOS are short-chain saccharides composed of three to ten mannose residues. They can be bonded by both α-(1⟶6) and β-(1⟶4) glycosidic bonds. α-MOS are obtained from the hydrolysis of mannan from yeast cell walls, while β-MOS are obtained from plant mannans (Kumar Suryawanshi and Kango 2021). The prebiotic effect of MOS is to promote the growth of LAB and bifdobacteria, which ferment them mainly to acetic acid and lower the pH of the colon, thus inhibiting the growth of enteropathogenic bacteria. In addition, MOS directly activate and interact with macrophages, trigger the production of pro-infammatory mediators, and increase mucin expression to attenuate the harmful effects of pathogens. MOS have been shown to have therapeutic effects on infammatory bowel syndrome and Crohn’s disease. Recent reports also suggest that MOS exert anticancer effects. They effectively inhibit the proliferation of HT29, Caco-2, HepG2, MCF-7, A549, and Hela cancer cells. MOS may play a key role as an ingredient with antioxidant properties in functional foods (Jana et al. 2021). Although small amounts of MOS are found in the backup and structural material of plant raw materials, on an industrial scale they are obtained by acidic, alkaline, or enzymatic hydrolysis of mannans derived from yeast (most commonly S. cerevisiae) or plant galactomannans or glucomannans. Galactomannan is a backup material in coconut endosperm, guar gum, and tara gum, while glucomannan is found in konjac tubers and some orchid species. Mannans are readily degraded to oligomers in aqueous solutions heated for 3 to 30 min at 180–230° C under elevated pressure. MOS can be produced from agro-wastes (palm kernel cake, copra meal) by enzymatic hydrolysis of the mannans they contain using endo-1,4-β-mannanase. These enzymes are produced by bacteria (Bacillus clausii, B. subtilis, Bacteroides fragilis) and molds of the genus Aspergillus sp. (Jana et al. 2021). 16.3.3.1.6 Xylo-Oligosaccharides (XOS) XOS are linear oligosaccharides composed of two to ten subunits, mainly D-xylose, linked by β-(1⟶4) glycosidic bonds. The prebiotic properties of XOS depend on the structure, presence of other saccharide residues in the chain, and DP. Like other prebiotics, they stimulate the growth of lactobacilli and bifdobacteria and the formation of SCFAs and lactates. This effect is greater than that of FOS and GOS. These prebiotics inhibit the growth of pathogens, including Clostridium diffcile and C. perfringens, and prevent the adhesion of Listeria monocytogenes cells to the intestinal epithelium. Consumption of XOS has also been found to lower blood cholesterol levels and improve the absorption of mineral salts. In addition, they exhibit antioxidant activity and the ability to lower glycated hemoglobin, as well as low-density lipoprotein and apolipoprotein B, which has positive effects on obesity and type 2 diabetes (Palaniappan et al. 2021). XOS occur naturally in honey, fruits, vegetables, and bamboo shoots. However, their amount is at a very low level, making this raw material unsuitable for their effcient production. The raw material for the preparation of XOS is hemicellulose consisting mainly of xylans, linear chains composed of D-xylose residues linked by

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β-(1⟶4) glycosidic bonds and highly branched heteropolysaccharides with substitutions in different side chains by L-arabinose, D-galactose, acetyl groups, and glucuronic acidic moieties. XOS can be obtained by chemical, physical, and enzymatic methods. Acidic or alkaline extraction leads to the formation of impurities, undesirable compounds (e.g., hydroxymethylfurfural), and a large amount of monosaccharides, and is also a cause of equipment corrosion. On the industrial scale, XOS is produced from corn cobs by enzymatic hydrolysis carried out by endoxylanases derived mostly from Aspergillus sp. The XOS obtained by this method are mainly xylobiose, xylotriose, and xylotetraose. Bacteria (Bacillus sp.) and actinomycetes (Streptomyces sp.) can also be the source of these enzymes (Palaniappan et al. 2021). Like other prebiotics, XOS is considered safe for consumption. The recommended daily intake is 1.4–2.8 g (maximum 12 g) without causing gastrointestinal disturbance. Due to their physicochemical properties, such as stability over a wide pH range (2.5–8.0), thermostability, sweet taste (about 30% the sweetness of sucrose), and low calories, they are functional food additives. They can be used in dairy products and a variety of baked goods as sugar and fat replacements and favor enhancers. Products to which XOS has been added have better texture and sensory properties than products without this additive. These substances have also been shown to act as cryoprotectants (Corim Marim and Gabardo, 2021). 16.3.3.1.7 Isomalto-Oligosaccharides (IMO) IMO are a mixture of oligosaccharides made of glucose residues linked by bonds, primarily α-(1⟶6) and α-(1⟶4), α-(1⟶2), and α-(1⟶3) glycosidic bonds. DP ranges from 2 to 8. This group of compounds includes isomaltose, kojibiose, or nigerose (DP = 2) and panose, isopanose, isomaltotriose, or isomaltotetraose (DP ≥ 3) (Singh et al. 2017). The prebiotic status of IMOs is not clear, as they may be partially digested in the gastrointestinal tract before reaching the colon. Although there is no conclusive evidence that they are selectively utilized by species inhabiting the colonic microbiota, they are thought to stimulate the growth of lactobacilli and bifdobacteria, A. municiphila and Roseburia sp., which metabolize them to SCFAs. This promotes the formation of moist fecal matter and facilitates bowel movements. IMOs administered with polyphenols prevent metabolic disorders and infammation resulting from a high-fat diet. The recommended IMO intake is 2.5–10 g per day. Small amounts of IMO naturally occur in honey and some fermented foods, such as sake and soy sauce. The IMO concentration in these products is very low, so it is not economically feasible to isolate them. Therefore, the industrial production of IMO is based on enzymatic processes. They are obtained in an enzymatic reaction from starch (corn, potato, tapioca, and others) using various enzymes that lead to the liquefaction (pullulanases, α-amylases) or saccharifcation (β-amylases) of the substrate, followed in the next step by the production of IMOs by α-transglucosidases. The source of these enzymes can be plants or microorganisms, including bacteria of the genus Bacillus sp. or molds Aspergillus sp. (Basu et al. 2016). IMOs are widely used in foods and foodstuffs (e.g., functional foods and beverages), specifc formulations for different consumer groups (e.g., infant formulas), and

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in dental products as natural non-cariogenic sweeteners. As a food and beverage additive, IMO are used as sugar replacements, to increase fber content, as an ingredient that contributes to improving the organoleptic profle of the product (Singla and Chakkaravarthi 2017). They are most commonly an ingredient in nutritional bars, dietary supplements, bakery and confectionery products, beverages, salad seasonings and dressings, frozen desserts and dairy mixes, hard and soft candies, meat and nut products, processed fruit and vegetable products, and functional foods. Expanding the potential use of IMO in the food and feed industry continues to be the focus of much research and development. The emergence of clinical evidence supporting the health benefts of IMOs will increase their commercial value. 16.3.3.1.8 Other Oligosaccharides Some oligosaccharides including AOS and GnOS are still under investigation for their prebiotic potential. The former consists of arabinose subunits linked by α-(1⟶5) or α-(1⟶2) and α-(1⟶3) glycosidic bonds with DP = 2–15. They are obtained by depolymerization of arabinan using controlled acid or enzymatic hydrolysis. A rich source of arabinan is beet pulp, from which preparations of these oligosaccharides are currently obtained on an industrial scale. AOS have been shown to selectively stimulate the growth of two species of bifdobacteria: B. adolescentis and B. longum (Holck et al. 2011). GnOS, on the other hand, are composed of gentiobiose molecules with DP = 3–8 linked by α-(1⟶6) and α-(1⟶3) glycosidic bonds. They can be effciently produced by partial hydrolysis of the lichen polysaccharide, pustulan, or by chemical synthesis. The latter method is multistep and not very cost-effective. Enzymatic catalysis using alternansucrases is also a useful method to produce GnOS, which allows the formation of defned oligosaccharides with high regio- and stereo-selectivity (Kothari and Goyal 2015). GnOS exhibit bifdogenic activity and selectively inhibit cell proliferation of colon cancer cell lines. They also possess immunomodulatory activity as they induce the production of cytokines IL-4, IL-12, and TNF- α (Ispirli et al. 2019). They can be used in foods as a sweetener with a low glycemic index, and because of their bitterness, they are useful as favor enhancers for some beverages. 16.3.3.1.9 Lactulose Lactulose is a disaccharide composed of galactose and fructose. It is not broken down by gastrointestinal enzymes and does not increase blood glucose levels. The absorption rate of lactulose is less than 1% of the administered dose. This disaccharide is metabolized by the saccharolytic microbiota in the proximal colon, not only by probiotics but also by commensals. Lactulose is not a selective bifdogenic agent, and the actual effect of lactulose on the composition of the gut microbiota depends on the individual characteristics of the patient and the dose. The recommended intake of 10–20 g per day results in an increase in bifdobacteria, lactobacilli, and Streptococcus sp. in healthy individuals and a decrease in Bacteroides sp., Clostridium sp., Eubacterium sp., and coliforms.

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Bacteria ferment lactulose to SCFA and L- and D-lactic acid, thus acidifying the intestinal contents. During fermentation, hydrogen is also produced, which has biological effects: it increases colonic motility (shortens intestinal transit time, increases stool volume) and exhibits antioxidant and anti-infammatory properties benefcial to consumer health. Monthly supplementation of 25 g per day lowers serum triacylglycerols. Lactulose reduces systemic ammonia levels by prebiotic stimulation of bacterial growth and by inhibiting amino acid deamination, reduces protein absorption in the small intestine, and likely also inhibits amino acid fermentation in a dose- and individual-dependent manner (Ruszkowski and Witkowski 2019). Lactulose may also be used to stimulate calcium and magnesium absorption. Lactulose does not occur naturally in food. However, the dissolved salt system of milk consisting mainly of chlorides, phosphates, citrates, carbonates, and bicarbonates of potassium, sodium, calcium, and magnesium is a buffered solvent that promotes the formation of lactulose from lactose during heat treatment. The formation of lactulose in milk depends on the temperature and time of processing and the pH of the milk. Large amounts of this disaccharide are formed at the high temperatures used during in-container sterilization and UHT processing. The International Dairy Federation (IDF) and the European Union have proposed lactulose as a chemical indicator to distinguish UHT milk from in-container sterilized milk. Both international bodies suggested 600 mg of lactulose per L as the upper limit for UHT milk. Lactulose is added to infant formula at 0.5% to stimulate the growth of bifdobacteria to the extent observed in breastfed infants. The addition of this disaccharide to yogurt improves its functional properties. Yogurt containing lactulose enhances the intestinal transit of healthy individuals and acts as a mild laxative, increasing the amount of feces. The addition of lactulose to yogurt shortens the incubation period in the production of yogurt containing L. acidophilus and B. bifdum. It also signifcantly affects the population size of bifdobacteria during refrigerated storage. Supplementation of yogurt with lactulose may be a satisfactory way to maintain live probiotic cells in yogurt above the suggested minimum of 107 CFU/g. The presence of lactulose during soymilk fermentation signifcantly increases isofavone aglycon content (Olano and Corzo 2009). Lactulose is obtained by isomerization of aldoses to ketoses at neutral or basic pH (Lobry de Bruyn-Alberd van Ekenstein rearrangement). Different basic catalysts (calcium hydroxide, alkali hydroxides, sodium sulfate, ammonia, or basic organic compounds such as tertiary amines) can be used for this purpose. This method provides a low yield and is tedious due to the diffculty in purifying lactulose from the unwanted by-products formed (monosaccharides, aliphatic aldehydes, furan derivatives). This disaccharide can also be obtained enzymatically using β-galactosidase from A. oryzea, Sulfolobus solfataricus, or Pyrococcus furiosus. The substrate in this reaction is lactose as the glycoside donor and fructose as the acceptor. 16.3.3.2 Non-Carbohydrate Prebiotics Prebiotic potential has also recently been proposed for polyphenols and polyunsaturated fatty acids. More than 95% of dietary intact polyphenols accumulate in the

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colon, where they are converted by microorganisms to various bioactive compounds. Bacteroides sp., some species of the genus Clostridium, Eubacterium limosum, and Eggerthella lenta produce aromatic SCFA derivatives such as phenylacetate and phenylbutyrate from polyphenols. Other metabolites that are formed during the bioconversion of phenols in the colon are 3-OH-phenylacetate, 3-phenylpropionate, 3-(4-OH-phenyl)-propionate, and a range of aromatic acids that have an unsaturated side chain: transcinnamic acid, p-coumaric acid, caffeic acid, 4-hydroxybenzoic acid, vanillic acid, pyrogallol, 4-hydroxyhippuric acid, urolithin A, pyrocatechol, protocatechuic acid, gallic acid, and phloroglucinol (Thilakarathna et al. 2018). These compounds prevent or alleviate the symptoms of various diseases – cancer, infammation of the nervous system, and diabetes. They have also been found to affect the activity of enzymes involved in DNA and histone methylation, acetylation, and phosphorylation – methyltransferases, acyltransferases, and phosphotransferases – thus having the ability to reprogram the epigenome, which turns gene expression on and off (Bhat and Kapilal 2017). PUFAs or their conjugates may also be potential prebiotics. The evidence for prebiotic properties is incomplete and still needs confrmation. The results of studies that have partially proved this hypothesis have so far been conducted on a limited basis, in vitro or in an animal model, and mainly concern linoleic acid (LA) and the taxon Lactobacillus. Lipid absorption occurs in the small intestine. It was observed that when dietary LA was supplied in excess, the number of lactobacilli in the small intestine increased and converted LA to 10-hydroxy-cis-12-octadecenoic acid (HYA). This compound reduces lipid absorption and improves intestinal peristalsis, promotes the production of the intestinal hormone glucagon-like peptide-1 responsible for stimulating glucose-induced insulin secretion, and improves glucose homeostasis; it also inhibits the growth of Helicobacter pylori and Helicobacter suis. The health-promoting benefts of PUFAs metabolites, both LA and alpha-linolenic acid, may be much greater by the fact that the enzymes leading to the metabolism of these compounds (myosin-cross-reactive-antigen) are widely conserved in bacteria. The role of the resulting metabolites in the host is so far poorly understood and its understanding will help to strengthen the concept of the prebiotic action of PUFAs (Rinninella and Costantini 2022).

16.4 CONCLUSIONS Probiotics and prebiotics are functional components of food products that positively affect consumer health. This benefcial effect involves improving gastrointestinal function by regulating the gut microbiota, enhancing the immune system, mediating the endocrine system, modulating triacylglycerols metabolism, improving dietary calcium bioavailability, and inhibiting colon carcinogenesis. Probiotic and prebiotic effects may be additive or synergistic. The effects of probiotic microorganisms and prebiotic substances on the human body are highly variable and need to be confrmed in humans in appropriate nutritional studies based on sound mechanistic hypotheses. Additionally, both groups of ingredients affect the technological properties of food products. In particular, prebiotics are interesting as ingredients that

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allow the creation of new low-calorie and low-fat products with interesting sensory and textural properties.

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Mood Food Maria H. Borawska and Sylwia K. Naliwajko

CONTENTS 17.1 Dietary Amino Acids and Neurotransmitters in the Brain .......................... 457 17.2 Sweets and Brain Function ........................................................................... 458 17.3 Food Lipids and the Human Mood...............................................................460 17.4 The Effect of Vitamins and Mineral Compounds on Mood ........................ 461 17.5 Ethyl Alcohol and Human Mood..................................................................464 References..............................................................................................................465

17.1

DIETARY AMINO ACIDS AND NEUROTRANSMITTERS IN THE BRAIN

The brain is unique in the sense that it is a large complex of neural systems, all of which must interact to form a complete, functioning system that determines behavior. Mood is likely to result from the interaction of multiple, semi-independent, neural circuits working together in harmony. Additionally, complex interactions between neural systems are required for the maintenance of appetite, sleep, weight stabilization, and interest in sexual activity. Several studies suggest that brain functions, including mood, respond to changes in nutrients (Strasser et al., 2016). Neurotransmitters (signal particle transfers of information between neurons) or neuromodulators are synthesized from compounds that are essential dietary constituents. In the brain, there are important biogenic amines crucial for mood: serotonin (5-hydroxytryptamine), norepinephrine (NE), dopamine (DA), and amino acids like gamma-aminobutyric acid (GABA), glutamic acid, glycine (co-agonist of glutamate), and N-methyl-D-aspartic acid (NMDA) (connected with receptor NMDA), as well as peptides – enkephalins and endorphins. Serotonin, DA, and NE are formed from tryptophan and tyrosine, respectively. GABA is synthesized from glutamic acid and evokes effects similar to serotonin. Serotonin is the key neurotransmitter linked to emotional and motivational aspects of human behavior. A precursor of serotonin is L-tryptophan. Eating foods high in L-tryptophan – meat, fsh, cheese, eggs, cocoa, bananas, pineapples, plums, nuts, seeds, and grains – leads to increased concentration of serotonin in the enteric nervous system located in the gastrointestinal tract, but not necessarily in the brain. Serotonin cannot effciently pass the blood-brain barrier and the transport of tryptophan into the brain depends on the presence of other large neutral amino acids (Stone et al., 2013). When we eat meals high in carbohydrates, tryptophan has an advantage

DOI: 10.1201/9781003265955-17

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over other amino acids; it crosses the blood-brain barrier, and the level of serotonin in the brain can increase. Dopamine (DA) is one of the major neurotransmitters in the brain in rewardmotivated behavior and is involved in mood regulation (Chung et al., 2014). Disturbances in the dopaminergic system have been implicated in the etiology of several mental and neurological disorders. When we eat a high-protein meal (e.g., meat), there is more tyrosine than tryptophan and it crosses the blood-brain barrier and increases the synthesis of DA. Tyramine (4-hydroxyphenethylamine), synthesized from tyrosine also, and phenylethylamine (PEA) are two endogenous biogenic amines that act as sympathomimetic compounds, and at physiological doses, they may act to potentiate dopaminergic and noradrenergic neurotransmission. Tyramine appears in foods such as aged wines, chocolate, cheese, various meats, marinated fsh, and yeast extracts (Yılmaz et al., 2021). It is metabolized by monoamine oxidase. Previously used for treating depression, monoamine oxidase (MAO) inhibitors had interaction with foods high in tyramine – a hypertensive crisis “cheese syndrome.” Today more selective MAO inhibitors are used for the treatment of depression (Deftereos et al., 2012). In interactions between neurotransmitters and their receptors, the regulation of behavioral complexity relies on second messenger signals within cells like G proteins and on cell membrane receptors to relay information from the extracellular environment to the interior of the cell. Current research focuses on the gut–brain axis and suggests an important role for the gut microbiota in infuencing brain development, behavior, and mood in humans (Strandwitz, 2018). The microbiota may interact with the nervous system through the modulation of major neurotransmitters (serotonin, DA, GABA, noradrenaline) and other neurotransmitters, including histamine, neuropeptides, and endocannabinoids (Strasser et al., 2016; Stasi et al., 2019). The gut microbiome interacts with diet to affect mental disorders, but the ways are imperfectly understood. Some nutritional factors, like dietary fber, antioxidant vitamins, and n-3 fatty acids can support the development of the gut microbiome. On the other hand, some factors, like high-fat and high-sugar diets, can lead to leakiness of the gut epithelium, resulting in the release of infammatory factors and penetration of gut fora in the intestinal wall, which can further increase the risk of depression via alterations in signaling pathways leading to the brain (Bremner et al., 2020).

17.2 SWEETS AND BRAIN FUNCTION Sweets are the group of food for which human beings have always had some inborn preferences because the majority of sweet fruits or eatable parts of plants found in the natural environment are not poisonous. There is no clear empirical support for a link between glucose variability and mood in adults with type 1 and type 2 diabetes (Muijs et al., 2020). A new, systematic review of interventional studies shows limited effects of glucose ingestion on mood and no positive effect of sucrose on mood in short-term interventions (van de Rest et al., 2018). Today most researchers conclude that mood variations and sugar rush after the consumption of carbohydrates

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are a myth (Mantantzis et al., 2019). In the group of sweets, we may include the following things: honey, sweets products like candies, jellies, and marmalades, candied fruit, sweets made of cacao, seed pulp, and caramel mass, for example, halvah, sesame snaps, grillage candies, as well as biscuits, wafers, gingerbread biscuits, etc. Moreover, we often forget that refned sugar (sucrose) is an important ingredient in plenty of beverages, jams, puddings, and some candied fruits. Sucrose and simple sugars derived from eaten sweets cause a sudden growth of the glucose level in the blood and, as a result, an increase in the insulin level. Glucose enters the cells and induces plenty of metabolic changes, including the production of ATP in mitochondria and free radicals. Moreover, insulin simplifes the process of acquiring essential amino acids from the blood into cells; however, tryptophan in the blood rises, which simplifes its penetration through the blood-brain barrier into the CNS. It is the basic material in the synthesis of the neurotransmitter of serotonin in the brain and it has an infuence on our mood and ability to calm down. The ingestion of aspartame can elevate levels of phenylalanine and aspartic acid in the brain. These compounds can inhibit the synthesis and release of neurotransmitters, dopamine, norepinephrine, and serotonin, which are known regulators of neurophysiological activity. Negative mood (irritability) and depression episodes in students were more frequent after a high (25 mg/kg body weight/day) compared to a lower (10 mg/kg body weight/day) aspartame consumption, but other studies did not provide evidence for changes in mood or behavior. The effects of aspartame on mood changes are still controversial (Choudhary and Lee, 2018). Even 25 g of glucose taken orally improves the ability to acquire new knowledge and remember new information, no matter if we take it before or during learning. Glucose helps remembering and learning not only through delivering energy to cells but also through insulin in CNS as it can directly infuence insulin receptors (Duarte et al., 2012). There is no correlation between the growth of glucose levels and remembering. This correlation is not proportional to the acquired amount of sugar. Bigger amounts do not cause an increase in this effect. Special attention should be paid to things made of cacao grains (Theobroma cacao), nut pulp (halvah), and almond pulp (nougat), which, as far as their nutritional value is concerned, include a lot of fat, especially halvah, and phosphorus, calcium, magnesium, or vitamins from the group B. Chocolate made of cacao pulp, cacao fat, castor sugar, and, depending on the additional ingredients, nuts, vanilla, milk powder, coffee, cinnamon, etc. contain about 300 active substances. Polyphenols present in chocolate, especially favonoids (mainly catechins), indicate four times stronger action as antioxidants than in green tea (Stark et al., 2005). Other good sources of favonoids include citrus fruits, berries, onions, parsley, legumes, and red wine. They have a wide range of biological effects including anti-infammatory, anti-allergic, and anti-cancer activity. Eating, especially sweets, causes the release of endogenous opiates having not only an analgesic effect but also an infuence on mental illness and mood. It has been proven that giving young babies water with sugar stops their cries caused by a prick of a needle. Chocolate, chocolate bars, and other chocolate products play the most important part in the process of addiction to sweets. Chocolate is not only a source

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of energy from carbohydrates and fats, but it also contains plenty of pharmacologically known substances, such as alkaloids: salsolinol and xanthines. Salsolinol stimulates dopaminergic receptors D2 and D3 and releases ß-endorphins. Xanthines, such as tyramine and phenethylamine, are commonly used for their effectiveness as mild stimulants, bronchodilators, and stimulating amines. It is very dangerous when we use concurrently an MAO inhibitor drug. Tyramine is also a known trigger of migraine attacks in people who are sensitive to tyramine. Methylated xanthine derivatives include caffeine (1,3,7-trimethylxanthine) and theobromine (3,7-dimethylxanthine). These alkaloids inhibit phosphodiesterase and blockade the adenosine of A1 and A2 receptors. Adenosine is a modulator of CNS neurotransmission and its modulation of dopamine transmission through A2A receptors has been implicated in the effects of caffeine. Other, lesser-known substances included in chocolate are anandamide (deriving from the Indian word “ananda,” meaning “bringing calm”) and its two analogs which are natural agonists of cannabinoid receptors CB1 and CB2. They belong to the so-called cannabinoids, whose central effects include disruption of psychomotor behavior, intoxication, stimulation of appetite, antinociceptive actions (particularly against pain of neuropathic origin), anti-emetic effects, short-term memory impairment, and possibly improvement of mood (we do not feel that the time is “running away”). Properties of cannabinoids that might be of therapeutic use include analgesia, muscle relaxation, immunosuppression, anti-infammation, anti-allergic effects, sedation, stimulation of appetite, anti-emesis, lowering of intraocular pressure, bronchodilation, neuroprotection, and antineoplastic effects. Cannabinoids cause psychical relaxation and positive mood, i.e., the effects that may, to some extent, be similar to those caused by small amounts of ethanol. The new concept of the “mood pyramid” of cacao and chocolate (Tuenter et al., 2018) refers to a broad spectrum of effects ranging from general effects related to the favanols through more specifc effects of methylxanthines and minor alkaloids to more specifc activities related to minor constituents such as salsolinol and anandamide. It can be concluded that addiction to sweets, so diffcult to fght, also depends on our preference for sweet taste and infuence on CNS: it improves mood and ability of learning and lowers aggression, but may also evoke dependency and degrade memory. We may eat small amounts of sweets, preferably one or two pieces of dark bitter chocolate with an estimated low glycemic index = 22.

17.3

FOOD LIPIDS AND THE HUMAN MOOD

Fats, especially unsaturated fatty acids n-6 (from plant oils, nuts, and seeds) and n-3 (obtained from marine or plant sources), are very important in the diet because they build covers of neurons. Brain and nervous tissue membrane lipids contain a particularly high proportion of arachidonic acid (AA) and docosahexaenoic acids (DHA) and low concentrations of their 18-carbon precursors. The soft part of the brain and the retina are very rich in DHA, which is essential for proper neural functioning and vision. Even small changes in the concentration of these acids or in proportion to the

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main phospholipids, cholesterols, and esters may have considerable infuence on the functioning and structure of receptors’ tissues. AA and DHA, no matter if they act as renewed transmitters per se, play an important role in regulating and conducting the signals. There is some evidence to support the use of n-3 fatty acids in the treatment of mood disorders and conditions characterized by a high level of impulsivity and aggression and personality disorders (Bozzatello et al., 2016). The recent appreciation that two G-protein-coupled receptors, metabotropic glutamate and cannabinoid, are trans-synaptically linked by a small lipid messenger has profound implications, both for the control of synaptic transmission and for new therapeutic strategies. Some symptoms of schizophrenia can be related to disturbances in the synthesis and release of the endogenous cannabinoid of anandamide, the amide of AA, and ethanolamine. Oleum with cannabidiol (CBD) is the major non-psychomimetic compound derived from cannabis. CBD was shown to potentially exhibit anti-epileptic, anti-oxidant, anti-infammatory, anti-psychotic, anxiolytic, and anti-depressant properties but larger, randomized, placebo-controlled trials are needed to evaluate the therapeutic potential of CBD (Elsaid et al., 2019).

17.4

THE EFFECT OF VITAMINS AND MINERAL COMPOUNDS ON MOOD

In the most important group of vitamins, the lack of which is related to the disability of brain functions, we may include B1, B6, folic acid, and B12. In counteraction of depression and impeding peroxidative changes in brain lipids and protein, an important role is played by the following vitamins: C, E, and A. Most of the vitamin B1 (thiamine) in the body is present as diphosphate (TDP) at 90%, with approximately 10% as thiamine triphosphate (TTP) in the brain and other neural tissues and consistent with a cholinomimetic effect. Thiamine functions in prime interconversions of sugar phosphates and in decarboxylation reactions with energy production from α-keto acids and their acyl CoA derivatives, which are catabolically derived from carbohydrates and amino acids. Clinical signs of defciency in B1 include nervous symptoms (mental confusion, peripheral paralysis, muscular weakness, ataxia, edema [wet beriberi], or muscle wasting [dry beriberi]) and cardiovascular system symptoms (an enlarged heart and tachycardia). Dietary thiamine for people in a large part of the world is obtained from either unrefned cereal grains or starchy roots and tubers. Other natural sources of thiamine are meat organs, pork fesh, nuts, and legumes. Thiamine is unstable in alkaline solution and is removed from cereals by refning and being highly ground. In raw fsh, shellfsh, ferns, and some bacteria there are thiaminases, which hydrolytically destroy the vitamin in the gastrointestinal tract. In addition, there also exist heat-stable, antithiamine factors which are found in ferns, tea, betel nuts, and a large number of plants, vegetables, and the tissue of some animals. There are six major compounds of vitamin B6; pyridoxal (PL) respective 5’-phosphate derivative (PLP) is a coenzyme for more than 100 enzymes, which primarily include enzymes involved in amino acid metabolism and in glycogen

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metabolism. PLP is a cofactor for decarboxylases that are involved in neurotransmitter synthesis. The clinical symptoms of B6 defciency from CNS are depression and confusion, epileptiform convulsions, and other clinical signs including infammation of the tongue, lesions of the lips and corners of the mouth, seborrheic dermatitis, and microcytic anemia. Vitamin B6 and nicotinamide-containing supplements taken in low doses between meals can quickly improve depressed mood in young adults with severe sub-clinical depression (Tsujita et al., 2019). Rich sources of vitamin B6 are meat, fsh, potatoes, bananas, and pulses. Both low folate and low vitamin B12 status have been found in studies of depressed patients, and a relation between depression and low levels of the two vitamins is found in the studies of the general population (Coppen and Bolander-Gouaille, 2005). Folic acid is important in the synthesis of tetrahydrobiopterin, which is a co-factor for the hydroxylation of phenylalanine and tryptophan and is a rate-limiting step in the synthesis of DA, NA, and serotonin. There is no particularly good source of folate, with the exception of liver. Fresh vegetables are usually considered to be a good source of folate but only in a high intake. Cobalamin (vitamin B12) is involved in many aspects of physiological functioning, including red blood cell production and DNA and myelin synthesis. Lower B12 levels have been noted to be associated with increased overall psychological distress and with specifc mood states, including depression, anxiety, and irritated mood (Tiemeier et al., 2002). Folate and vitamin B12 are involved in homocysteine metabolism. Reducing homocysteine methylation to methionine can lead to an inhibition of methylation reactions involved in the synthesis of DNA, proteins, phospholipids, and neurotransmitters, including dopamine, norepinephrine, and serotonin in the brain. Folate and B12 defciency can cause elevated homocysteine levels which may be associated with depression (Bremner et al., 2020). Sources of vitamin B12 are animal meats and fsh or animal products such as eggs and milk products. Fruits, vegetables, and grain products are devoid of vitamin B12 because only microorganisms can produce it. Having a low level of 25-hydroxyvitamin D has been hypothesized to play a role in the etiology of depression or clinically relevant depressive symptoms or in changes in mood scores, but new fndings do not support it (Okereke et al., 2020). Among the micronutrients, the most important factors affecting the functioning of the brain and human mood are zinc, iron, and selenium. Zinc is a critical nutrient for the development of the central nervous system. The meta-analysis by Li et al. (2017) indicates the signifcant inverse associations between dietary zinc intake and the risk of depression. Zinc status correlates with mental health in elderly people (Markiewicz-Żukowska et al., 2015). The role of zinc in human nutrition has been well known for a long time. Zinc is essential for normal fetal growth and development and also for milk production during lactation, and it is extremely necessary during the frst year of life when the body is growing rapidly. This microelement is known to act on more than 300 enzymes by participating in their structure or in their catalytic and regulatory functions. It is a structural ion of biological membranes, closely related to protein synthesis. Zinc plays the role in gene expression and endocrine function and participates in DNA and RNA synthesis and cell division. Zinc is intimately linked to bone metabolism, thus, zinc has a positive

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infuence on growth and development. Zinc also enhances vitamin D’s effects on bone metabolism through the stimulation of DNA synthesis in bone cells. In brain tissue, zinc, in the ionic form (Zn2+), is present primarily in synaptic vesicles of glutamatergic neurons. Zinc plays a primary role in synaptic transmission and serves as an endogenous neuromodulator (Krall et al., 2021). Zinc supplementation is considered an adjuvant therapy in the treatment of major depressive disorder because it enhances the effciency of pharmacotherapy (Szewczyk et al., 2018). The largest amount of well-absorbable zinc is found in food of animal origin, particularly in oysters and meat. Vegetables and seeds have lower bioavailability of Zn. The effciency of absorption depends on many factors including body size, level of dietary zinc, and presence of antagonistic substances in the diet, such as calcium, phytate, or chelating agents. The major inhibitor of zinc absorption is dietary phytic acid which is present in many cereal grains. Zinc is connected with copper in the infuence on the converse of neurotransmitters in the brain. Copper is involved in the function of several cellular enzymes; it is required for infant growth, host defense mechanisms, bone strength, red and white cell maturation, iron transport, cholesterol and glucose metabolism, myocardial contractility, and brain development. The highest content of copper is in grain products, but the main source of copper in diets is usually vegetables. Another microelement, important for our health and mood, is iron. Iron has several vital functions in the body: as a carrier of oxygen from the lungs to the tissues, as a transport medium for electrons within cells, and as an integrated part of important enzyme reactions in various tissues. A very large number of iron-containing enzymes has been described and they play key roles, not only in oxygen and electron transport but as signal-controlling substances, in some neurotransmitter systems in the brain, for example, the dopaminergic and serotonin system (Yu and Chang, 2019; Kim and Wessling-Resnick, 2014). The mechanism of absorption depends on the kinds of iron in the diet: haem iron and non-haem iron. There are two separate types of receptors for the form of iron on the mucosal cells. Factors enhancing iron absorption are ascorbic acid, meat, fsh, seafood, and certain organic acids. Factors inhibiting iron absorption are phytates, iron-binding phenolic compounds, calcium, and soy protein. Haem iron in meat and meat products constitutes about 1–2 mg, or 5–10%, of the daily iron intake in most industrialized countries. Selenium status modifes mental functions. Low selenium status leads to depressed mood, while high dietary or supplementary selenium improves mood (Benton and Cook, 1990; Ferreira de Almeida et al., 2021). Low selenium status is associated with a signifcantly increased incidence of depression, anxiety, confusion, and hostility, and with senility and cognitive decline in elders. Subjects with head or neck cancers, urinary tract cancers, or rheumatoid arthritis also have depleted concentrations of selenium in the blood (Borawska et al., 2004). The brain selenium level in Alzheimer’s patients is only 60% of that in control groups. In the human body, selenium is bound to a range of selenoproteins. However, only two of them are present in the plasma. Most of this element in plasma, about 60–80%, is associated with selenoprotein P. This protein concentration is associated with the selenium nutritional status in humans. Extracellular glutathione peroxidase (GSHPx)

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is another protein, containing almost 30% of plasma selenium. This can be easily explained by the selenium accumulation in plants, in the form of organic compounds, originating in the inorganic selenite and selenate present in fertilizers. The element is mostly built into the amino acid methionine, instead of the sulfur atom, resulting in selenomethionine formation, which can be nonspecifcally incorporated into human or animal proteins. Selenocysteine follows another metabolic pathway being utilized in the synthesis of selenoproteins. Inorganic forms of selenium, such as selenate and selenite, directly enter the pool from which all selenium is used for the synthesis of selenoproteins, and the excess is excreted. Thus, GSHPx is predominantly regulated by the levels of selenocysteine or inorganic selenium species. Most forms of selenium salts and organic selenocompounds are easily absorbed from the gastrointestinal tract. More than 90% of selenomethionine is absorbed in the small intestine through the Na+-dependent neutral amino acid transport system. Little is known about selenocysteine intestinal absorption. Both selenocysteine and selenomethionine, in particular, have better bioavailability in humans than inorganic selenium species, which are commonly used as selenium supplements. Selenium bioavailability from grains or vegetables, in which selenomethionine is a predominant form, reaches 85 to 100%, and from animal food, containing mostly selenocysteine, about 50%. Alimentary studies show that the selenium status is highly dependable on dietary sources, particularly on their protein content. Therefore, animal food with its high protein content could be regarded as a vast source of this element. Several studies report poultry, liver, eggs, and fsh to be abundant in selenium, in contrast to plant foods. Nevertheless, selenium content in foods can be considerably reduced by food processing. Frying and roasting cause greater selenium losses than boiling. Boiling results in volatilization which may account for a 40% decrease in the selenium content of selected foods.

17.5

ETHYL ALCOHOL AND HUMAN MOOD

Drinking alcohol in most cases is associated with inducing positive feelings like pleasure or reducing negative feelings like uncertainty and tension. However sometimes it can lead to an increase in negative emotions or it can have no direct effects on mood at all (Lloyd and Rogers, 1997; Ray and Hutchison, 2004). Drinking alcohol affects many mental and perceptual processes and motor skills. Consumption of alcohol infuences women to a greater degree than men, in part because the same amount of ethanol produces lower blood alcohol concentrations for the latter. Consuming alcohol appeared to reduce the power of a stressful emotional stimulus to alter mood. However, alcohol intake did not affect each mood to the same degree (Agabio et al., 2016). Alcohol may affect mood differentially (from elation to depression), and it depends on doses and genetic predisposition. In small amounts, alcohol appears to improve people’s mood and may release inhibitions that will make them feel more sociable, making it a way to add to the fun in social drinking situations. On the other hand, when we use alcohol often we evoke dependence, faster in women than in men. Development of alcohol use disorder often involves

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heavy drinking to high levels of intoxication that leads to loss of control, compulsion to consume more, and negative effects of abstention (Koob and Colrain, 2020). Chronic heavy alcohol consumption deactivates vitamins and adversely affects both macronutrients and micronutrients. The effects of alcohol intake on mood, behavior, and cognition may be partly mediated by biological changes related to a defciency in these ingredients of food.

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Krall R.F., Tzounopoulos T., Aizenman E. 2021. The function and regulation of zinc in the brain. Neuroscience 457:235–258. https://doi.org/10.1016/j.neuroscience.2021.01.010 Li Z., Li B., Song X., Zhang D. 2017. Dietary zinc and iron intake and risk of depression: A meta-analysis. Psychiatry Res. 251:41–47. https://doi.org/10.1016/j.psychres.2017.02 .006 Lloyd H.M., Rogers P.J. 1997. Mood and cognitive performance improved by a small amount of alcohol given with a lunchtime meal. Behav. Pharmacol. 8(2–3):188–119. Mantantzis K., Schlaghecken F., Sünram-Lea S.I., Maylor E.A. 2019. Sugar rush or sugar crash? A meta-analysis of carbohydrate effects on mood. Neurosci. Biobehav. Rev. 101:45–67. https://doi.org/10.1016/j.neubiorev.2019.03.016 Markiewicz-Żukowska R., Gutowska A., Borawska M.H. 2015. Serum zinc concentrations correlate with mental and physical status of nursing home residents. PLOS ONE 10(1):e0117257. https://doi.org/10.1371/journal.pone.0117257 Muijs L.T., Racca C., de Wit M., Brouwer A., Wieringa T.H., de Vries R., Serné E.H., van Raalte D.H., Rutters F., Snoek F.J. 2020. Glucose variability and mood in adults with diabetes: A systematic review. Endocrinol. Diabetes Metab. 4(1):e00152. https://doi .org/10.1002/edm2.152 Okereke O.I., Reynolds C.F. 3rd, Mischoulon D., Chang G., Vyas C.M., Cook N.R., Weinberg A., Bubes V., Copeland T., Friedenberg G., Lee I.M., Buring J.E., Manson J.E. 2020. Effect of long-term vitamin D3 supplementation vs placebo on risk of depression or clinically relevant depressive symptoms and on change in mood scores: A randomized clinical trial. JAMA 324(5):471–480. https://doi.org/10.1001/ jama.2020.10224 Ray L.A., Hutchison K.E. 2004. A polymorphism of the mu-opioid receptor gene (OPRM1) and sensitivity to the effects of alcohol in humans. Alcohol. Clin. Exp. Res. 28(12):1789– 1795. https://doi.org/10.1097/01.alc.0000148114.34000.b9 Stark T., Bareuther S., Hofmann T. 2005. Sensory-guided decomposition of roasted cacao nibs (Theobroma cacao) and structure determination of taste-active polyphenols. J. Agric. Food Chem. 53(13):5407–5418. https://doi.org/10.1021/jf050457y Stasi C., Sadalla S., Milani S. 2019. The relationship between the serotonin metabolism, gutmicrobiota and the gut-brain axis. Curr. Drug Metab. 20(8):646–655. https://doi.org/10 .2174/1389200220666190725115503 Stone T.W., Darlington G. 2013. The kynurenine pathway as a therapeutic target in cognitive and neurodegenerative disorders. Br. J. Pharmacol. 169(6):1211–1227. https://doi.org /10.1111/bph.12230 Strandwitz P. 2018. Neurotransmitter modulation by the gut microbiota. Brain Res. 1693(Pt B):128–133. https://doi.org/10.1016/j.brainres.2018.03.015 Strasser B., Gostner J.M., Fuchs D. 2016. Mood, food, and cognition: Role of tryptophan and serotonin. Curr. Opin. Clin. Nutr. Metab. Care 19(1):55–61. https://doi.org/10.1097/ MCO.0000000000000237 Szewczyk B., Szopa A., Serefko A., Poleszak E., Nowak G. 2018. The role of magnesium and zinc in depression: Similarities and differences. Magnes. Res. 31(3):78–89. https://doi .org/10.1684/mrh.2018.0442 Tiemeier H., van Tuijl H.R., Hofman A., Meijer J., Kiliaan A.J., Breteler M.M. 2002. Vitamin B12, folate, and homocysteine in depression: The Rotterdam Study. Am. J. Psychiatry 159(12):2099–2101. https://doi.org/10.1176/appi.ajp.159.12.2099 Tsujita N., Akamatsu Y., Nishida M.M., Hayashi T., Moritani T. 2019. Effect of tryptophan, vitamin B 6, and nicotinamide-containing supplement loading between meals on mood and autonomic nervous system activity in young adults with subclinical depression: A randomized, double-blind, and placebo-controlled study. J. Nutr. Sci. Vitaminol. (Tokyo) 65(6):507–514. https://doi.org/10.3177/jnsv.65.507

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Tuenter E., Foubert K., Pieters L. 2018. Mood components in cocoa and chocolate: The mood pyramid. Planta Med. 84(12–13):839–844. https://doi.org/10.1055/a-0588-5534 Van de Rest O., van der Zwaluw N.L., de Groot L.C.P.G.M. 2018. Effects of glucose and sucrose on mood: A systematic review of interventional studies. Nutr. Rev. 76(2):108– 116. https://doi.org/10.1093/nutrit/nux065 Yılmaz C., Gökmen V. 2021. Perspective on the formation, analysis, and health effects of neuroactive compounds in foods. J. Agric. Food Chem. 69(45):13364–13372. https:// doi.org/10.1021/acs.jafc.1c05181 Yu P., Chang Y.Z. 2019. Brain iron metabolism and regulation. Adv. Exp. Med. Biol. 1173:33– 44. https://doi.org/10.1007/978-981-13-9589-5_3

18

Mutagenic and Carcinogenic Compounds in Food Agnieszka Bartoszek and Serhii Holota

CONTENTS 18.1 Introduction ..................................................................................................469 18.2 Mechanisms Involved in Carcinogenic Transformation Induced by Food Components......................................................................................... 470 18.3 Metabolic Activation of Genotoxic Food Components and Mechanism of DNA Adduct Formation: Evaluation of Cancer Risk and Classifcation of Carcinogens ....................................................................... 473 18.4 Food Mutagens and Carcinogens ................................................................. 478 18.4.1 Introduction ...................................................................................... 478 18.4.2 Mycotoxins ....................................................................................... 479 18.4.3 Nitrosamines..................................................................................... 481 18.4.4 Mutagens in Thermally Processed Foods ........................................ 482 18.5 Other Diet-Related Risk Factors................................................................... 488 18.6 Concluding Remarks .................................................................................... 492 References.............................................................................................................. 493

18.1

INTRODUCTION

The term mutagen embraces chemical and physical factors that induce changes in the genetic code. A carcinogen, less frequently referred to as a cancerogen to avoid associations with only one type of cancer – carcinoma – is any substance or other factor (e.g. ionizing radiation) that promotes carcinogenesis or by analogy cancerogenesis, the formation of cancer. Such factors are ubiquitous in human surroundings; they may be of natural origin or derived from industrial chemical processes. Currently, the list of agents that are or may be human carcinogens according to the International Agency for Research on Cancer (IARC) includes 534 substances (Monographs.iarc.fr., 2022), many of which are also found in food. Among factors increasing the risk of cancer, dietary components play a particularly important role because of daily exposure over the whole lifespan. Mutagens and carcinogens found in foodstuffs can be divided depending on the source of origin into four categories. The frst one encompasses natural compounds, such as mycotoxins or certain plant metabolites. The second category, considered the DOI: 10.1201/9781003265955-18

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most impactful dietary cancer risk factors, includes compounds arising during food storage and processing. The latter category will be discussed in most detail further in this chapter. The substances that enter foodstuffs and potable water as a result of environmental pollution (e.g. heavy metals) or agriculture chemicalization (e.g. pesticides) constitute another group. To the fourth category belong food additives, substances that in contrast to those formerly mentioned are added to foodstuffs purposefully. In addition, food is a source of nutrients as well as non-nutritive components that may trigger endogenous biological processes, which increase or decrease the susceptibility of human cells to cancer transformation. Recent estimates suggest that environmental exposures and lifestyle are responsible for 90–95% of human cancers, among which the major risk factors are diet (30–35%) and cigarette smoking (25–30%) (Anand et al., 2008). Diet as a prevailing environmental variable related to cancer risk was frst proposed by Doll and Peto based on epidemiological observations (1981). Further population studies indicated that cancer incidence varies between world regions, changes sharply (usually increases) with the civilization development of societies, and, in immigrants even within the same generation, achieves the level observed in the country of settlement rather than the country of origin. The current large-scale research concerning different ethnic populations, with the application of genomic technologies, is aimed to establish the correlations between genetic variability, environmental exposure, lifestyle, and the risk of oncological diseases. For instance, the results of the study that compared cancer incidence trends among Japanese in Japan and Japanese and Caucasians in Hawaii showed that diet in childhood and adolescence was decisive, but cumulative cancer incidence could be also modulated at the later life stages and lead to the adoption of the host population’s cancer risk (Mascarinec and Noh, 2004). Oncological diseases have always occurred in human populations, but only currently have reached an epidemic scale, especially in industrialized countries. According to the GLOBOCAN 2020 data produced by IARC, worldwide, an estimated 19.3 million new cancer cases and almost ten million cancer deaths were registered in the year 2020 (Sung et al., 2021). Behind these statistics, there is not only human suffering, but a substantial economic burden as well. On the world scale, the costs of medicines for cancer and related supportive care amounted to US$ 133 billion in 2017 compared to US$ 90.9 billion in 2012, and the annual compound growth rate for cancer medicine spending was 7.9% according to World Health Organization (WHO) data (2018). Considering the aforementioned statistics, it is not surprising that the presence of mutagens and carcinogens in food as well as other diet-related factors increasing the likelihood of cancer development not only arouses particular interest among consumers but is also of concern for medical and governmental institutions.

18.2 MECHANISMS INVOLVED IN CARCINOGENIC TRANSFORMATION INDUCED BY FOOD COMPONENTS The majority of established mutagenic and carcinogenic food components belong to genotoxins. Genotoxic agents are defned as factors that chemically modify nucleic

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acids and therefore induce DNA lesions which may become heritable changes in the nuclear and extranuclear genetic material of somatic or reproductive cells. Such genotoxic alterations stem from covalent modifcations of nucleotides, but also from induction of fusions, breaks, or abnormal segregation of chromosomes. The longest-recognized role of genotoxins in carcinogenesis is described by the socalled “theory of somatic mutations,” the prevailing paradigm in cancer research for the past 50 years (Basu, 2018). According to this concept, genotoxic lesion results from covalent binding of an active form of a mutagen with DNA. The most frequent sites of modifcation are nucleobases, guanine in particular. The presence of modifed nucleotide, that is, of DNA adduct, prevents proper pairing of complementary nucleobases (adenine with thymine and guanine with cytosine), and thus obstructs the fdelity of DNA replication. As a result, the nucleotide sequence may be misread by cellular replicative machinery, which increases the probability of incorporation of the wrong nucleotide into the daughter strand. Therefore, if during replication, the promutagenic lesion, such as an adduct, is not removed by DNA repair mechanisms, it will become fxed as a mutation, i.e. alteration of the genetic code. The change in nucleotide sequence in a gene may in turn lead to an altered sequence of amino acids in proteins. Mutation can be indifferent to cells, but it becomes relevant when the function of a crucial protein is deregulated. In this way, DNA adducts make the cell initiate carcinogenic transformation, especially when the nucleotide sequence and thus the function of so-called oncogenes become affected. The somatic mutation theory assumes that even single exposure to a genotoxin can initiate cancer development. Carcinogenesis, however, is a multistage process, which includes a sequence of events that take place at the molecular, cellular, and tissue levels. The classic multistage carcinogenesis is a stepwise process divided into three phases: initiation (as a result of genotoxic lesion), promotion (growth and transformation of the initiated cell), and progression (invasion of other tissues). This scenario assumes that cancer develops over a long time period, during which somatic mutations accumulate in a single cell causing changes in phenotype, fnally leading to the transformation of a normal cell into a cancer cell capable of metastasis (Basu, 2018). This simplifed view of carcinogenesis still remains vital in cancer research, mostly because in some animal models used to detect carcinogens, the neoplastic transformation indeed follows the route of development it proposes. Initiation involves mechanisms causing DNA damage and leading to somatic mutations as described earlier. The promotion step, believed to be still reversible, is much slower than initiation and results in the accumulation of precancerous mutated cells due to their uncontrolled cell divisions. The progression step is the fnal, irreversible stage of carcinogenesis, during which accelerated tumor growth and metastasis to other tissues occur. This presented model of carcinogenesis provides a convenient framework, but as already mentioned, is very simplifed. In 2000, Hanahan and Weinberg proposed a set of processes that they called “hallmarks of cancer,” which described in their opinion the six most important features of cancer cells. These researchers pointed out that despite signifcant differences between various types of cancer, the transformed

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cells show some similar aberrant properties that are not observed in normal cells. After ten years, the same researchers published the article entitled “Hallmarks of Cancer: The Next Generation,” in which they expanded the list of features of cancer cells by adding two more (Hanahan and Weinberg, 2011). The complete list of hallmarks of cancer includes: genome instability and mutation, sustaining proliferative signaling, evading growth suppression, activating invasion and metastasis, enabling replicative immortality, inducing angiogenesis, resisting cell death, tumorpromoting infammation, deregulating cellular energetics, and avoiding immune destruction. This new perception of cancer development does not exclude the former hypotheses as the important pieces in the puzzle. For instance, induction of certain mutations by genotoxins may be necessary to incur such changes in gene expression that will specifcally alter the function of the cell, e.g. its angiogenic potential. However, other molecular mechanisms, in particular epigenetically evoked alterations in gene expression, became considered at least equally important. The major mechanism of epigenetic regulation of gene expression involves DNA methylation of promoter regions rich in CpG pairs. The methyl group is introduced into the C5 position of cytosine. The methylation occurs symmetrically also on the other strand of the double helix and is maintained after replication. DNA methylation of the promoter region silences the expression of the gene. If the gene is important for controlling cell proliferation, blockage of its expression will make cells more liable to uncontrolled growth, which is one of the hallmarks of cancer. Such epigenetic silencing of tumor suppressor genes was observed in a number of cancers (Kazanets et al., 2016). However, it is important to note that the accessibility of methyl donors is a prerequisite for the maintenance of DNA methylation patterns. Thus, the abundance of folic acid or methionine in the diet may play a decisive role in epigenetically controlled carcinogenesis. This new perspective opens for food components, be they nutrients, non-nutritional or detrimental substances, or a variety of mechanisms by which they may impact carcinogenesis. Polychlorinated biphenyls (PCBs), the frequent environmental contaminants of food and potable water, may serve here as a good example. They can form DNA adducts and thus may cause mutations infuencing genetic code. However, PCBs are also xenoestrogens and therefore may stimulate the growth of hormone-dependent cancers, breast cancer in particular. On top of it, as observed for many toxicants, these compounds can induce oxidative stress in cells, which is associated with the overproduction of reactive oxygen species (ROS). Some ROS are important signaling molecules, so their overabundance may impair redox-dependent signaling pathways. Moreover, ROS may also change DNA methylation patterns as enzymatic demethylation relies on oxidative processes. In unfortunate cases, this may activate the expression of epigenetically silenced oncogenes. In this chapter, mainly genotoxic mutagens and carcinogens arising as a result of food processing will be described because on the one hand, these substances are believed to be the major dietary cancer risk factors whose mechanism of action is relatively well understood, and on the other hand, they represent health hazard that can be readily reduced by changing food storage and/or preparation technologies. However, it should be borne in mind that the impact of diet on cancer risk

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depends on many factors and genotoxicity is only one of them, not necessarily the most important.

18.3 METABOLIC ACTIVATION OF GENOTOXIC FOOD COMPONENTS AND MECHANISM OF DNA ADDUCT FORMATION: EVALUATION OF CANCER RISK AND CLASSIFICATION OF CARCINOGENS In general, the majority of carcinogens, including those found in foods, do not possess mutagenic or carcinogenic properties per se. In order for these properties to become manifested, metabolic activation in a human organism is required. Upon the route of metabolic transformation(s), poorly reactive original compounds become converted into electrophilic metabolites capable of binding with nucleophilic centers in DNA. Therefore, in literature, the name promutagen or procarcinogen is often used to describe the compounds that must be activated by cellular enzymes to ultimately become genotoxic mutagens and carcinogens. Metabolic activation of carcinogens involves various enzymatic systems collectively named phase I enzymes. The most important is cytochrome P450 complex, consisting of several different isoenzymes, particularly active in the liver. Other phase I enzymes include peroxidases, quinone reductases, epoxide hydrolases, or sulfotransferases. Their variety refects the diversity of chemical structures of compounds that may enter the human organism and must be prepared for further reactions. The phase I enzymes catalyze the attachment of polar groups to increase the water solubility of foreign compounds so as to facilitate their excretion or to prepare normal metabolites for further transformations. Unfortunately, in the case of promutagens and procarcinogens, such modifcations result in the formation of electrophilic derivatives capable of covalent modifcation of nucleic acids. One could argue that the activation of genotoxins is an undesirable side effect of metabolic pathways, which were developed in the course of evolution to eliminate unwanted or harmful substances and to improve the utilization of nutrients. The enzymatic activation of promutagens and procarcinogens is most frequently followed by the processes involving phase II enzymes. These enzymes, responsible for the ultimate metabolism of toxic metabolites, activated mutagens, and carcinogens in this number, catalyze conjugation reactions with charged endogenous compounds such as glutathione, glucuronic acid, or others. The conjugation reactions catalyzed respectively by glutathione-S-transferases or glucuronyltransferases generate more polar metabolites that cannot diffuse across membranes, and may, therefore, be transported in a controlled way. However, phase I enzymes are sometimes also responsible for detoxifcation. It even happens that activation and detoxifcation run in parallel and are catalyzed by the same enzymatic system. For instance, epoxidation of benzo[a]pyrene by cytochrome P450 at the 7,8-position results in the formation of a carcinogenic metabolite, while in position 4,5 it produces an inactive derivative excreted readily from the organism. Next, there are given some examples of well-established metabolic activation pathways for a few classes of genotoxic

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compounds found in food along with the major products of reaction of their main activated metabolites with DNA, more precisely with guanine, which is the preferred site of binding of electrophilic intermediates. Metabolic activation of afatoxin B1 belonging to the class of mycotoxins is catalyzed by cytochrome P450 (Figure 18.1). The enzymatic conversion of this compound can follow many pathways; however, only the epoxidation at the 8,9-position produces the ultimate carcinogen. This metabolite binds to the N7 position of guanine giving an unstable adduct 8,9-dihydro-8-(N7-guanyl)-9-hydroxy-afatoxin B1, which either undergoes spontaneous depurination or rearranges to a stable 8,9-dihydro-8-(2,6-d iamino-4-oxo-3,4-dihydropyrimid-5-yl-formamide)-9-hydroxy-afatoxin B1 following the opening of the imidazole ring (Wakabayashi et al., 1991). The formation of nitrosamines in the reaction of amines with nitrites under acidic conditions in the stomach can be considered as non-enzymatic activation of amines present in food (Figure 18.2). Nitrosamines undergo further metabolism, catalyzed enzymatically by cytochrome P450, involving hydroxylation (Li and Hecht, 2022). The hydroxylated derivative is unstable and in a series of spontaneous reactions gives rise to methyl carbocation which alkylates guanine in the position O6, hence, in the site taking part in the formation of hydrogen bonds with complementary base – cytosine – in DNA.

FIGURE 18.1 position N7.

Metabolic activation of afatoxin B1 by adduct formation with guanine at

FIGURE 18.2 Nitrosamine formation from secondary amine and nitrate(III), its activation to methyl carbocation, and DNA adduct formation.

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475

Metabolic activation of benzo[a]pyrene consists of three enzymatic reactions (Figure 18.3). First, the formation of epoxide at the 7,8-position, is catalyzed by cytochrome P450; epoxidation at the 4,5-position results in detoxifcation of this compound. Then, epoxide hydrolase converts the epoxide into 7,8-dihydrodiol, which is subsequently oxidized to 7,8-diol-9,10-epoxide. The formation of four different diastereoisomers is feasible, among which anti-9,10-epoxide derived from (–)-7,8-dihydrodiol is by far the most carcinogenic (Dipple and Bigger, 1991). In DNA, this derivative reacts most frequently with guanine in such a way that the positions 10 of benzo[a]pyrene and N7 of guanine become linked together. Aromatic compounds substituted with amino groups, e.g. heterocyclic aromatic amines (HAAs) present in protein-rich food products, are usually activated by cytochrome P450 to hydroxylamines. This type of metabolism (Figure 18.4) is observed in the case of compounds represented by 3-methyl-3H-imidazo[4,5-f]chinoline2-amine (IQ). Hydroxylamine derivatives may bind with DNA directly in position C8 of deoxyguanosine. However, usually, they initially undergo sulfation or acetylation catalyzed by phase II enzymes, respectively by sulfotransferases (SULT) and/or N-acetyltransferases (NAT), which may result in HAA detoxifcation, but may lead to the covalent modifcation of DNA or proteins by these highly unstable esters. The formation of dG-C8-HAA adducts runs with the concomitant formation of nitrenium ion as an intermediate product. In the case of some carcinogenic amines, DNA adducts can arise also by linkage of the amine group in the N2 position of deoxyguanosine and the C5 atom of the HAA heterocyclic ring (Turesky and Vouros, 2004). The possibility of DNA adduct formation resulting from binding between the N6 atom of an amine group in deoxyadenosine and the C5 atom of HAA has been reported as well (Jamin et al., 2007). The mentioned enzymatic systems implicated in the metabolism of carcinogens may be the reason for the different susceptibility of humans to cancer. Therefore, the genes coding enzymes responsible for the biotransformation of carcinogens are included in the list of cancer-related genes. Importantly, many dietary compounds can infuence various phase I and II enzymes by induction or inhibition. For example,

FIGURE 18.3 One of the metabolic pathways of benzo[a]pyrene leading to the formation of the most carcinogenic metabolite 7,8-diol-anti-9,10-oxide.

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FIGURE 18.4 Metabolic activation of the heterocyclic aromatic amine IQ to the electrophilic nitrenium ion and the formation of IQ adducts with purines present in DNA. Following the cytochrome P450 catalyzed activation step, further activation is the result of N-acetyltransferases (NAT) and sulfotransferases (SULT).

cytochrome P450 isoenzyme CYP1A2 (phase I) activity may be induced by polycyclic aromatic hydrocarbons present in grilled or smoked foods and inhibited by naringenin a polyphenol found in grapefruit. Similarly, the phase II enzymes – glutathione-S-transferases – may be induced by many non-nutrient phytochemicals, dietary lipids, and ROS. Current research attempts to relate genetically correlated sensitivity and environmental exposures, including dietary impact, to individual cancer risk (Guengerich, 2000). To complicate the picture even more, a growing amount of data indicates that DNA adducts can mimic epigenetic markers and disrupt the proper function of the epigenome as a result. For instance, DNA methylation pattern recognition can be impaired by genotoxins via multiple mechanisms affecting subsequently gene expression. There are some suggestions that chemical carcinogens act primarily as epigenome disruptors, whereas mutations are secondary events that occur at later stages of cancer development when genome-protecting mechanisms have already been deregulated (Lewandowska and Bartoszek, 2011). Food products contain thousands of compounds – some of nutritive value, naturally occurring nonnutritive components, and also numerous additives, substances formed during processing, or pesticide residues. Their identity often is not known, let alone the identity of their active metabolites. Moreover, culinary processing may substantially alter the initial chemical composition of food items. Nonetheless, the safety of food components is of utmost importance for human health protection,

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including cancer risk assessment, and hence must be performed. In response to these challenges, short-term reliable and inexpensive tests were developed in which genotoxic properties are most frequently used as a property to monitor. Since cancer risk is associated with exposure to chemical substances and is thought to stem from their ability to induce mutations, mutagenicity is used in the frst-line assessment of the carcinogenic potential of food components. Such ability can be detected with the aid of bacteria whose culturing is easy, quick, and economical. In the case of bacterial mutagenicity tests, it is assumed that the factors capable of damaging bacterial DNA can interact in a similar way with the DNA of higher organisms. The method most widely used to evaluate the mutagenic activity of chemicals and their mixtures is the Ames test (Ames et al., 1975). It utilizes mutant strains of Salmonella typhimurium unable to synthesize histidine, thus dependent on an outer source of this amino acid. The reverse mutation in the appropriate gene makes the bacteria histidine-independent. The frequency of reverse mutations increases in the presence of mutagenic factors. To mimic the metabolic activation of mutagens, typical for mammalian cells but often absent in bacteria, a microsomal fraction (usually isolated from rat liver) is added concomitantly with the substance studied. Currently, an array of Salmonella strains is available which enable not only the evaluation of the overall mutagenic activity of a given compound but also the type of mutation it induces. Moreover, the techniques of genetic manipulation offered by modern molecular biology allowed the construction of bacterial strains expressing various animal and human genes coding enzymes implicated in the activation of chemical mutagens and carcinogens. The evaluation of carcinogenicity, that is the ability of substances to induce cancers, is performed in animals, mainly in mice and rats. In these experiments, animals, either inoculated with a specifc type of cancer cells or made liable to spontaneously develop cancers, are administered a range of doses of potential carcinogens. The highest of them correlates with the maximum tolerated dose (MTD) that does not cause severe weight loss or other life-threatening signs of toxicity. As a result of such studies, the lowest dose is determined at which cancer formation is still observed. The next level below is assumed not to have a biological effect, the so-called “no effect level.” This value, divided by a safety factor, correcting for the difference in sensitivity between animals and humans, of either 100 or 1.000, is considered the acceptable daily intake (IARC, 1987). Nowadays, a variety of animal carcinogenesis models have been constructed with the aid of genetic manipulations, which enables us to assess the carcinogenic potential of evaluated substances as regards the development of specifc tumors. The usage of short-lived species like rodents to estimate carcinogenic effects in a long-lived species such as the human is perceived as just an initial warning that a given substance may be not safe and requires more in-depth consideration. The dietary cancer risk for humans is estimated based on epidemiological studies in which often thousands of volunteers participate and which cover the period of several years of observations to pinpoint the association between diet composition and cancer incidence. The most valued are meta-analyses where statistical elaboration of pooled data integrating different primary studies is performed. Currently, the latter approach involving the systematic review of evidence-based risk assessment is recommended to evaluate health benefts/risks associated with the consumption of

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TABLE 18.1 Carcinogens Classifcation According to the Strength of Evidence of Their Ability to Induce Cancer Group Classifed as

Basis

Carcinogenic to humans

There is enough evidence to conclude that it can cause cancer in humans.

Probably carcinogenic to humans Possibly carcinogenic to humans Possible human carcinogen

Unclassifable as to carcinogenicity in humans

There is strong evidence that it can cause cancer in humans, but at present, it is not conclusive. There is some evidence that it can cause cancer in humans but at present, it is far from conclusive. There is limited evidence that it can cause cancer in animals in the absence of human data, but at present, it is not conclusive. There is no evidence at present that it causes cancer in humans.

Probably not сarcinogenic to humans

There is strong evidence that it does not cause cancer in humans

IACR

US EPA

1

A

2A

A1

2B

A2

2B

C

3

D

4

E

particular foods as well as to formulate dietary recommendations (Gonzales-Barron and Butler, 2011). The appearing data derived from all aforementioned types of scientifc risk assessments are used to classify carcinogens according to the strength of evidence and to formulate recommendations. The two most respected institutions that monitor and publish regularly updated lists of substances that pose or may pose a cancer risk to humans are already mentioned: the IARC (International Agency on Research for Cancer, based in Europe) and the EPA (Environment Protection Agency, based in the USA). Table 18.1 presents the strategies for carcinogenic risk assessment that have been executed by these two institutions for decades. The formulation of “hallmarks of cancer” prompted the discussion on implementing this novel view of carcinogen characterization in the assessment of cancer risk factors. Indeed, the EPA’s Integrated Risk Information System Program and the US National Toxicology Program are already introducing the use of the ten key cancer characteristics to conduct a systematic literature search focused on relevant endpoints to make better use of the results of current mechanistic studies performed with the aid of modern advanced technologies applied for evaluations of carcinogenicity (Smith et al., 2016).

18.4 FOOD MUTAGENS AND CARCINOGENS 18.4.1

INTRODUCTION

Food mutagens and carcinogens mostly are a result of inappropriate food storage and processing and thus represent readily avoidable cancer risk factors. Although

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diet may impact carcinogenesis in many ways, for risk assessments, genotoxic food components remain the most useful indicator because of the well-established methods that allow us to estimate their impact on oncological transformation. Table 18.2 presents the best-recognized foodborne mutagens and carcinogens along with information on their mutagenic strength and the site of tumor induction. In the following description, a large group of mutagenic and genotoxic substances of plant origin was omitted. These potential carcinogens belong to a variety of classes of chemical compounds, e.g. hydrazine derivatives, favonoids, alkenylbenzenes, pyrrolizidine alkaloids, phenolics, saponins, and many other known and unknown compounds. Plants produce these toxins to protect themselves against fungi, insects, and animal predators. For example, cabbage contains at least 49 natural pesticides and their metabolites, a few of which were tested for carcinogenicity and mutagenicity, some of which turned out positive. However, there is no evidence that plant-based food increases cancer risk. In contrast, epidemiological studies demonstrate that phytochemicals found in edible plants exhibit numerous activities preventing carcinogenesis.

18.4.2 MYCOTOXINS Mycotoxins are highly toxic compounds naturally produced by molds, mostly in the genera Aspergillus, Penicillium, and Fusarium. These compounds are regarded as the most dangerous contamination arising during the storage of numerous food commodities, especially corn, peanuts, and cereal products. Tropical and subtropical climates are locations where contamination with mycotoxins occurs most frequently

TABLE 18.2 Mutagenicity and Carcinogenicity of Most Common Genotoxic Food Components

Compound

Exemplary sources

Afatoxin B1

Corn

N,N-dimethylnitrosamine Benzo[a]pyrene Trp-P-2 Glu-P-1 MeIQ MeIQx PhIP

Cured meat Smoked fsh Grilled meat and fsh

Furan

Jarred fruit and vegetable preserves

Meat broth, fried meat Fried meat

Mutagenicity determined in Ames test (revertants/µg) 6,000 0 970 104,200 49,000 661,000 145,000 931 0

Tumors in target tissue of rodents Liver Bladder Skin, lung Liver, small intestine, colon Liver, stomach, lung, colon Colon, breast Liver

Notes: MeIQ – (2-Amin-3,4-dimethylimidazo[4,5-f]quinoline); MeIQx – (2-Amin-3,8dimethylimidazo[4,5-f]quinoxaline; PhIP – (2-Amin-1-methyl-6-phenylimidazo[4,5-b]pyridine).

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because of often poor food harvesting and storage practices. The chemical structures of the most notorious mycotoxins detected in food and feed are presented in Figure 18.5, while their prevalent dietary sources and impact on cancer development are in Table. 18.3. The best-studied genotoxic mycotoxins (Figure 18.5), for which carcinogenic properties were also demonstrated, include afatoxins and sterigmatocystin, inducing liver cancers, and ochratoxin A, implicated in the development of kidney cancers in experimental animals. Mycotoxins may also enter dairy products if milking animals are fed with mold-contaminated feed. Moreover, afatoxin and ochratoxin may be secreted also into milk by human consumers of contaminated food, which poses health risks to newborns (Agriopoulou et al., 2020). In the case of patulin and nivalenols, their ability to damage DNA was demonstrated (Paterson and Lima, 2013). Other common food contaminants with the ability of tumor induction in laboratory animals are fumonisins, among which fumonisin B1 occurs most frequently. Fumonisin B1 is not genotoxic; its mode of action involves apoptotic necrosis, atrophy, and consequent abnormal regeneration of target organs (Stockmann-Juvala, 2008). Table 18.3 presents the sources and carcinogenic risks associated with mycotoxins representing frequent food contaminants. Afatoxin B1 is the most carcinogenic mycotoxin and, based on the available toxicological and epidemiological data, has been classifed as a human hepatocarcinogen (IARC, 1987). It is estimated that in the world, afatoxins are the cause of 25,000 to 155,000 liver cancer cases yearly (Pitt and Miller, 2017). Ochratoxin A is a major contaminant of grain and cereal products in North America and Europe. The protection of agricultural commodities against mold infection is very diffcult. Some expectations were raised by initial reports, which in the United States and other countries associated the Bacillus thuringiensis (Bt) toxin gene expression in GMO crops with reduced fumonisin, deoxynivalenol, and zearalenone contamination and, to a lesser extent, reduced afatoxin contamination in harvested maize kernels. However, subsequent feld results were inconsistent; fumonisin reduction by Bt expression was confrmed, but the effect on afatoxin remained inconclusive (Abbas et al., 2013).

FIGURE 18.5 contaminants.

Chemical structures of mycotoxins that are the most common food

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TABLE 18.3 The Documented Genotoxic, Mutagenic, and Carcinogenic Effects of Some Mycotoxins Contaminating Foods Exemplary occurrence in foodstuffs

Mutagenicity and carcinogenicity and related effects

Afatoxins

Nuts, dried fruits, milk, cereal products

Mutagenicity, carcinogenicity, genotoxicity (DNA adducts formation, induction of DNA lesions, inhibition of DNA repair)

Sterigmatocystin

Wheat four, spices, fruit preserves Grains and cereal products, coffee, milk and dairy products, dried fruits Fruits and fruit juices, bakery, sausages Wheat, barley, and corn four, pasta

Carcinogenicity, genotoxicity (DNA adduct formation) Mutagenicity, carcinogenicity, genotoxicity (DNA adducts formation, induction of DNA singlestrand breaks) Mutagenicity, genotoxicity (induction of DNA–DNA crosslinks, DNA adducts formation) Genotoxicity (induction of DNA damage)

Wheat, beans

Mutagenicity, genotoxicity (induction of DNA damage)

Compound

Ochratoxin A

Patulin Deoxynivalenol Nivalenol

Source: Paterson and Lima, 2013.

18.4.3 NITROSAMINES A number of nitroso compounds, N-nitrosamines among them, are potent carcinogens. Nitrosamines are found in numerous food products, especially cured meats and smoked fsh, but also in beer, soya sauce, and water as well as in some medications or cosmetics. The substantial amounts of nitrosamine precursors, nitrates, in particular, contain some leafy and root vegetables, e.g. lettuce or red beetroots. The most recent comprehensive review by Li and Hecht summarizes current knowledge on the occurrence and mode of action of these compounds (Li and Hecht, 2022). Ingested nitrosamines are mainly formed during food processing or result from nitrosation of amines present in food by nitrates(III) or nitrates(V) reduced to nitrates(III) by microorganisms inhabiting the mouth cavity. Nitrates(III) are also released endogenously by macrophages from arginine. Under acidic conditions of the stomach, the nitrosation of food amines by nitrates(III) occurs, giving rise to N-nitrosamines. Animal studies suggest, however, that in vivo formation of nitrosamines does not occur to a signifcant extent, and from a cancer risk perspective, much more signifcant seem to be pre-formed N-nitroso compounds consumed in e.g. cured meat or fsh (WCRF/IACR, 1997). The structures of the most common food nitrosamines are presented in Figure 18.6. The consumption of both nitrosamines and their precursors in food products is supposedly increasing the risk of colorectal cancers (Loh et al., 2011). However, the

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FIGURE 18.6

Chemical structures of nitrosamines in most abundant in food products.

presence of nitrates(III) is now perceived as having negative and positive impacts on food safety and human health. On the one hand, in many countries, a correlation between stomach, liver, and colorectal cancers, induced probably by nitrosamines, and the amount of nitrates(III) consumed is observed (Li and Hecht, 2022). On the other hand, nitrates(III) inhibit the growth of Clostridium botulinum, thus reducing the risk of food contamination by botulinum toxins. Moreover, under the acidic conditions of the stomach, hence where they are involved in the formation of carcinogenic nitrosamines, nitrates(III) are capable of neutralizing carcinogens formed as a result of protein pyrolysis. Most recent insights point also to nitrate-rich vegetables as an effective alternative for increasing dietary nitrate intake, which is nowadays associated with cardiovascular health and ergogenic benefts (Van der Avoort et al., 2018). Since the presence of nitrosamines is mainly a consequence of food processing and vegetable cultivation, changes in technology may lead to a considerable decrease in amounts of these compounds in food products, thereby diminishing the risk of cancer. Nonetheless, they are likely to remain components of preserved foods since an alternative to nitrates as curing agents and microbiological preservatives has not been found so far. It has been learned, though, that the formation of carcinogenic nitrosamines during thermal processing, for example, frying, of cured meats can be largely inhibited by the addition of antioxidants, e.g. ascorbate, alpha-tocopherol, or polyphenolic plant antioxidants (Cantwell and Elliott, 2017).

18.4.4

MUTAGENS IN THERMALLY PROCESSED FOODS

In the 1960s, with the advent of bacterial mutagenicity tests and experimental animal models of chemical carcinogenesis, the detection of specifc chemical carcinogens in the human diet became plausible. The demonstration that processing of proteinaceous foods under normal cooking conditions leads to the formation of compounds that promote mutagenesis came as a big surprise at that time. Mutagens were found in grilled and fried meat and fsh and methanol extracts of their charred parts, in smoke condensates produced while cooking these foods, and also in heated, purifed proteins as well as amino acids. Two decades later, the formation of genotoxins in thermally processed plant foods was also confrmed. Currently, it has been generally acknowledged that food processing is the major dietary source of mutagens and carcinogens (Koszucka and Nowak, 2019). Polycyclic aromatic hydrocarbons (PAHs) containing a system of condensed aromatic rings are formed as a result of incomplete combustion of organic matter. PAHs

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are associated with an elevated risk of cancers in various tissues. Around 70 different such compounds have been identifed in foodstuffs; the most abundant are benzo[a] pyrene (B[a]P) and benzo[a]anthracene (B[a]A), present in the greatest amounts in cooked or smoked meat products (Smith et al., 2001). The chemical structures of these two strong carcinogens are shown in Figure 18.7, while their occurrence in food products is in Table 18.4. In food, PAHs are produced mostly during heating, especially open-fame heating, such as grilling of meat. Under such conditions, fat from meat drips onto a hot surface, e.g., hot coal during grilling, and is incinerated. The smoke from the fat pyrolysis containing PAHs is adsorbed on the meat. The levels of these compounds that can potentially be produced are relatively large: the surface of a two-pound “well-done” steak was reported to contain an amount of B[a]P equivalent to that found in the smoke from 600 cigarettes (Pariza, 1982). In the case of smoked meat and fsh, smoke used during processing may also be a source of carcinogenic PAHs. In addition, a number of food products contain measurable amounts of these hydrocarbons resulting from environmental pollution, e.g., fsh caught in heavily industrialized regions (Sampaio et al., 2021).

FIGURE 18.7 Chemical structures of the most important food carcinogens belonging to PAH.

TABLE 18.4 The Contents of Benzo[a]pyrene and Benzo[a] anthracene in Popular Food Products and Cigarette Smoke Content [ng/g] Food product

Benzo[a]pyrene

Benzo[a]anthracene

Fresh vegetables

2.8–25.5

0.3–43.6

Plant oils Tea Smoked fsh Smoked ham Sausage Grilled pork

0.4–1.4 3.9 0.83 3.2 12.5–18.8 8.0

0.8–1.1 2.9–4.6 1.9 2.8 17.5–26.2 4.5

Cigarette smoke

20.0–25.0

20.0–35.0

Source: Smith et al., 2001.

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Although PAHs belong to environmental pollutants formed in numerous industrial processes and during the combustion of fuels, the initial WHO estimates, confrmed repeatedly by subsequent determinations, suggested that over 90% of human exposure to PAHs results from food consumption, while only about 0.9% is inhaled and 0.1–0.3% enters human organisms with potable water (WHO, 1984). Accordingly, to diminish human exposure to these strong carcinogens, the European Union issued the document (Commission Regulation No 1327/2014) regarding maximum levels of PAHs and lowered their allowed level from initial 5 to 2 ng/g in traditionally smoked meat and meat products and traditionally smoked fsh and fshery products, i.e. main PAH dietary sources. Heterocyclic aromatic amines (HAAs) are formed mainly during thermal processing of many kinds of foods, especially foods containing much protein (Chen et al., 2020). Moreover, some studies showed that these compounds may also arise endogenously in the human organism as the presence of compound designated IQ (2-amino-3-methylimidazo[4,5-f]-quinoline) was detected in both meat consumers as well as vegetarians (Scott at al., 2007). HAAs are extremely strong mutagens (Table 18.5) with the exception of harman and norharman, which do not possess exocyclic amine group and are not mutagenic, but they potentiate genotoxicity and mutagenicity of other HAAs (Totsuka et al., 2004). Animal studies demonstrated that prolonged feeding with thermally treated meat containing HAAs induced the formation of tumors in several organs (Zheng and Lee, 2009). Though human studies seem less conclusive, nonetheless positive association between the dietary intake of meat mutagens and colorectal adenoma risk was revealed in a meta-analysis of human epidemiological studies (Gongora et al., 2019). More than 25 different food-derived HAAs have been isolated to date and are divided into two groups depending on the temperature of food processing (Chen et al., 2020). The products of amino acids and protein pyrolysis, accordingly referred to as “pyrolytic HAAs” (Figure 18.8, top structures), are produced in temperatures

TABLE 18.5 The Content of Most Common Thermic HAAs in Different Meats Submitted to Thermal Processing Average content in processed meat (ng/g) Food product

Type of processing

PhIP

MeIQx

Fish

Frying

9.11

2.05

Chicken Beef Pork

Frying Frying Frying

6.06 5.27 9.20

0.46 3.33 2.39

Pork

Baking

2.20

0.23

Source: Puangsombat et al., 2012.

Mutagenic and Carcinogenic Compounds

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FIGURE 18.8 The most common HAAs generated during heat processing of meat.

higher than 300° C. Therefore, they are detected mainly in the surface layers of meat and fsh subjected to open fame broiling. The other type of HAAs is generated in protein-rich foods baked at 150–200° C and is called “thermic HAAs” (Figure 18.8, bottom structures). They are found in the crust of fried or broiled meat and fsh as well as in fried and baked meats and heated meat extracts. Thermic HAAs are derivatives of quinoline, quinoxaline, and pyridine generated in the reaction of creatine or creatinine with amino acids and sugars. All the reactants are thus natural constituents of meat. Some of the latter type of HAAs belong to the strongest foodborne mutagens known and are carcinogenic in rodents; the compound IQ was additionally shown to be carcinogenic in nonhuman primates (Adamson, 1990). As shown in Table 18.5, compound 2-amine-1-methyl-6-phenyl-1H-imidazo[4,5b]pyridine (PhIP) is the most prevalent of HAAs in the human diet. Daily consumption of HAAs may reach about 9 ng/kg/day (Bogen and Keating, 2001). These amounts ingested by humans may not be suffcient to induce cancers by themselves. At least such a conclusion can be drawn from comparison with animal intakes. However, their combination with other environmental factors implicated in neoplastic transformation may increase the risk. For example, it was found that dietary polyenoic fat, e.g. corn oil used for frying meat patties, signifcantly enhanced PhIP mammary carcinogenesis in rats and it was suggested that PhIP initiates carcinogenic process while dietary fat serves as a promoter (Ghoshal and Snyderwine, 1993). Moreover, the estrogenic activity of PhIP was revealed and postulated to play a role in the etiology of breast cancer (Lauber et al., 2004). Particularly worrying are the results of experiments performed in rats, which demonstrated that PhIP was passed via the liver to the breast and was secreted into the milk of lactating

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animals. The newborn pups received a dose suffcient to induce tumors. Such a route of exposure may also exist in other mammals, including humans in whom the enzymes secreted by the mammary gland are able to metabolize these compounds, thus human newborns may be exposed to activated HAAs from early life (Gorlewska-Roberts et al., 2004). Acrylamide (AA) until the year 2000 was regarded solely as a product of the chemical industry and based on animal studies classifed as a probable human carcinogen (IARC, 1994). Therefore, its detection in persons not occupationally exposed to this compound came as a surprise (Bergmark, 1997). Further studies revealed that AA is formed during heat processing of foods with high starch content such as potatoes, baked goods, cereals, nuts, as well as coffee and cocoa. Fast foods such as potato crisps, French fries, and sweet bakery products are particularly abundant sources of AA (Table 18.6) and unfortunately also very much liked by children. AA is formed as a result of a heat-induced reaction of amino groups of asparagine in peptides and proteins with carbonyl groups of reducing sugars such as glucose and fructose, hence concurrently with the formation of so-called Maillard products giving foods color and favor. The prolonged heat-processing on the one hand decreases AA concentration due to the thermal instability of this compound, but on the other hand, the thermal degradation of triacylglycerols present often in substantial amounts in starchy foods leads to the formation of acrolein that may be converted to AA (Pundir et al., 2019). The carcinogenic properties of AA result from the genotoxicity of its metabolite – glycidamide (Figure 18.9), which as demonstrated in vitro and in vivo, displays the ability to form several DNA adducts (Gamboa da Costa et al., 2003). This is also a clastogenic AA derivative that induces structural alterations in chromosomes (Wang

TABLE 18.6 Acrylamide Content and Estimated Maximum Daily Consumption of Acrylamide with Specifc Class of Food Products in Europe Based on Data for Poland

Food product Bread

Content of acrylamide (µg/kg)

Children 1–6 years old

Children and adolescents 7–18 years old

Adults 19–96 years old

(g or dm3/person/day)

35–110

260

900

1,600

Corn fakes Potato crisps French fries Confectionary products

70–1,190 110–3,640 65–800 50–670

150 200 180 270

100 250 720 580

400 200 540 1,200

Roasted coffee

230–700

–

600

1,450

Source: Mojska et al., 2010.

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FIGURE 18.9 AA metabolic activation and one of the possible adduct structures of formed metabolite with guanosine.

TABLE 18.7 The Content of Furan Detected in Some Food Products Content of furan (µg/kg) Food product Cereal products Meat products Canned soups containing meat Jarred or canned vegetables Ground coffee Baby foods

Average

Maximum

14

168

17 88 12 1,113

39 125 74 5,938

25

215

Source: Bakhija and Appel, 2010.

et al., 2010). AA and glycidamide can covalently modify also hemoglobin, which is regarded to be responsible for the neurotoxicity of these carcinogens (Klauing, 2008). The discovery that common food products rich in polysaccharides, hence nutritionally very important and consumed in substantial amounts, contain high amounts of probable human carcinogens was alarming and triggered the large-scale assessment of its impact on cancer risk. The results of these studies did not confrm unequivocally the increased incidence of cancers in people consuming AA with diet. Nonetheless, epidemiological studies suggested associations between AA consumption and kidney cancer formation (Lipworth et al., 2012) as well as increased mortality of breast cancer patients (Olsen et al., 2012). Furan is another genotoxic compound proposed by European Food Safety Authority (EFSA) as a food carcinogen (Fromberg et al., 2009). It occurs in a variety of foods such as coffee and canned and jarred foods, including baby foods containing meat and various vegetables. Signifcant amounts of furan were also detected in toasted baked goods or coffee (Table 18.7). The variety of foodstuffs in which furan was detected suggests that there are probably multiple mechanisms for its formation. Five chemical routes are considered which point to the thermal degradation of such typical food components as saccharides, amino acids, polyenoic fatty acids (PUFAs), ascorbic acid, or carotenoids as furan precursors (Vranova and Ciesarova, 2009).

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FIGURE 18.10 Metabolic activation of furan leading to DNA adduct formation and protein covalent modifcation.

Based on the results of studies in rodents in which the development of liver cancers after furan ingestion was observed, this compound was classifed as a probable human carcinogen. From available data, it appears that possible human exposures match the doses that produced carcinogenic effects in animals. This in vivo carcinogenicity of furan results from the genotoxic potential of its metabolite cis-2butene-1,4-dial (BDA) formed from parent compound upon oxidation catalyzed by cytochrome P450 and capable of binding with deoxynucleosides (Figure 18.10) (Byrns et al., 2006). The demonstrated toxicity of furan prompted WHO recommendations in which the maximum tolerated daily dose of 2 mg/kg body mass was set. In this context, it is important to note that food is not the only route of penetration into human organisms; furan may be also inhaled during the heat processing of food. Particular attention should be paid to the exposure of young children because of low body mass and thus increased risk.

18.5

OTHER DIET-RELATED RISK FACTORS

A number of epidemiological studies indicated that high consumption of fat contributes to the development of some types of cancers in humans. Carcinogenic effects are ascribed also to a high-calorie and protein-rich diet. Animal studies suggest that all the mentioned risk factors come into play after initiation of tumorigenesis, thus at the promotion stage in a multistage carcinogenesis model. Their mode of action relies on the increased production of reactive oxygen species (ROS). ROS are generated in the organism as a result of normal metabolism and play important physiological roles. However, ROS are also produced in the human organism in response to toxicants and infections or during infammatory processes. Similarly, a diet rich in nutrients increases the intensity of metabolic processes, hence also oxygen radical production. When ROS occur in excess, the redox homeostasis of the organism becomes impaired leading to the condition known as oxidative stress. This state may jeopardize normal cellular functions in many ways. Oxidative stress may be also induced by exposure to toxic xenobiotics such as food mutagens or carcinogens or environmental pollutants.

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Oxygen radicals and non-radical ROS are implicated in the induction of endogenous oxidative damage of macromolecules including the formation of so-called oxygen DNA adducts (examples of such adducts resulting from hydroxylation of nucleobases are given in Figure 18.11) and protein carbonyl derivatives (Rani and Gupta, 2015). This type of lesion is believed to play a signifcant role in the process of aging and the variety of degenerative age-related disorders, including cancer. The genotoxicity of DNA oxygen adducts is only one of the mechanisms by which oxidative stress promotes carcinogenesis. Most of all, the excess of ROS triggers or affects a variety of biological processes that may result in the carcinogenic transformation of cells, e.g. impairs signaling pathways controlling cell growth (Liou and Storz, 2010). Moreover, oxidative modifcation of DNA was proposed to mimic epigenetic marks in the genome (Lewandowska and Bartoszek, 2011), which deregulates gene expression as well as causes genome instability. Therefore, dietary plant antioxidants capable of scavenging ROS, described in another chapter of this book, are believed to display preventive properties decreasing the risk of cancer and other non-infectious chronic diseases associated with oxidative stress. Animal studies showed that also calorie and protein restriction markedly inhibits both carcinogenesis and accumulation of endogenous oxidative damage (Longo and Fontana, 2010). In addition, although fats do not display mutagenic activity per se, some of their constituents, such as cholesterol and unsaturated fatty acids, are easily oxidized during thermal processing giving rise to reactive molecules, which in turn may trigger a chain reaction of lipid peroxidation. Lipid peroxidation concerns both ingested fatty acids as well as the oxidative degradation of endogenous membrane lipids by ROS and generates a large variety of breakdown products (Figure 18.12), including alkanes, aldehydes, ketones, alcohols, furans, and others, some of which are genotoxic; others are known to impair cellular signaling pathways (Eckl and Bresgen, 2017). Aldehydes constituting the fnal stage of lipid peroxidation are particularly dangerous because of their relatively high stability, genotoxic potential, and the ease of absorption from diet. Therefore, in humans, the genotoxicity of oxidized PUFA

FIGURE 18.11

DNA bases hydroxylation sites under the action of ROS.

FIGURE 18.12 The mutagenic end products of lipid peroxidation and hydrolysis.

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appears to come mainly from aldehydes, which form characteristic exocyclic DNA adducts (Medeiros, 2009). Another important mechanism by which fat modulates carcinogenesis – some researchers claim the most important – involves its interference with the synthesis of prostaglandins and leukotrienes as well as the development of so-called insulin resistance which in turn stimulates the proliferation of cells (colonic epithelium cells in particular). The thermal processing of fats gives rise to the formation of yet another type of probable carcinogen belonging to chloropropanols, that is, 3-monochloropropane1,2-diol (3-MCPD) and its derivative 1,3-dichloro-2-propanol (1,3-DCP), although other derivatives may be also of interest. These substances (Figure 18.12) are formed in heated oils in substantial amounts, even up to 15 mg/kg. The mechanism involves the initial hydrolysis of triacylglycerols to fatty acids and glycerol and the subsequent chlorination of the hydroxyl group in glycerol (Arisseto et al., 2013). Although the determination of the levels of chloropropanols in food is an active feld of analytical chemistry, their actual impact on human cancer risk is far from being established. Another class of foodborne substances that have been postulated to infuence the frequency of cancer development and which have been drawing attention among nutritionists encompasses environmental pollutants (Figure 18.13). The most important are heavy metals and xenobiotics such as derivatives of dioxin, dibenzofuran, chlorinated biphenyls, and residues of pesticides (Biziuk and Bartoszek, 2005). These compounds display several activities regarded as important for cancer development. Dioxins and biphenyls were shown to form DNA adducts. Some are able to induce oxidative stress in the organism. On top of it, they belong to so-called xenoestrogens which are raising much interest because of, on the one hand, their substantial abundance and, on the other hand, the high incidence of breast and prostate hormone-dependent carcinogenesis. Xenoestrogens are penetrating into the human organism with food and they mimic or change the activity of estrogens produced endogenously. To these compounds whose ability to promote the development of estrogen-dependent cancers has been experimentally documented belong, among others, polychlorinated biphenyls (PCBs) formed during drinkable water chlorination, pesticide residues, 1,1’-(2,2,2-trichloro-1,1-ethanediyl)bis(4-chlorobenzen e) (DDT) in particular, and some components of plastics used for food packaging, especially bisphenol A (Wang et al., 2021). In the case of DDT and certain PCBs, their association with breast cancer incidence was evidenced based on humanderived biological material. These substances are extremely stable and persist in the

FIGURE 18.13 carcinogens.

The structures of the most notorious probable environmental food

Mutagenic and Carcinogenic Compounds

491

environment for many years, even in countries where DDT has been banned long ago. As soon as the association between exposure to environmental xenoestrogens and breast cancer risk was proposed, it was estimated that the decreased exposition to these compounds would decline the frequency of breast cancer by 20%, that is, by 36,000 cases in the USA alone (Davis et al., 1993). Recent experimental and epidemiological studies concluded that xenoestrogens are an important contributor to the development and progression of cancers. Interestingly, the meta-analysis of 18 clinical trials on the application of phytoestrogens, i.e. plant-derived secondary metabolites exhibiting estrogen-like activity, this time in the prevention or treatment of different cancers, provided evidence-based data confrming their benefts in cancer prophylaxis. The ongoing research on foodderived xenoestrogens, both detrimental and benefcial, will help to identify environmental infuences on hormone-related carcinogenesis to guide risk identifcation and the development of preventive strategies (Wang et al., 2021). The substances that by defnition should be safe but turned out to cause mutagenicity in Ames tests and tumors in vivo, thus probably increasing human cancer risk, are food additives. These mostly synthetic compounds were considered non-toxic until 2002 when the results of American studies were published documenting that 40% of additives induced cancers in more than one animal model. Extrapolation of these data suggested that around 1,000 substances used by the American food industry could pose some carcinogenic risks (Johnson, 2002). Also, very recent evaluations have indicated the carcinogenic potential of such a common food additive as titanium dioxide (E171) used to provide whiteness and opacity in a variety of food categories, including bakery products, soups, broths, sauces, salads, or processed nuts. This compound accumulates in the body and shows the potential to induce immunotoxicity as well as genotoxic effects such as DNA strand breaks and chromosomal damage. Although no experiments confrming unequivocally TiO2 carcinogenicity have been conducted so far, the European Food Safety Authority (EFSA) Panel based on available research data has concluded that this food additive can no longer be considered safe (Younes et al., 2021). Perhaps the most surprising, and by many considered the most controversial, was the decision of the IARC in 2015 to classify meat as a human carcinogen. This decision was based on the results of numerous epidemiological studies, which showed a statistically relevant association between the consumption of red meat and cancer incidence. The intake of red meat has been classifed as a probable human carcinogen (Group 2A in IARC classifcation), and the consumption of processed meat as a human carcinogen (Group 1 in IARC classifcation) (IARC, 2015). There are several individual components, including the formation of genotoxic compounds during heat processing or as a result of fat oxidation described in this chapter, that add up to the carcinogenic risk ascribed to meat. However, it should be clarifed that this risk, according to estimations, becomes increased by 18%, while in the case of cigarette smoking, the cancer incidence is elevated by 2,500%. Nonetheless, American Institute for Cancer Research (AICR) estimations must be taken seriously since they predict an additional fve to six new diseases per every 100 diagnosed cancer cases.

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18.6 CONCLUDING REMARKS Human daily dietary choices are a major modifable human cancer risk factor. These choices may cause increased consumption of food products that contain substances exhibiting mutagenic and carcinogenic potential, and thus may induce the transformation of normal somatic cells into cancerous cells, enhancing in consequence the consumer’s health risk. These compounds are occurring in food as a result of microbial contaminations (mycotoxins produced by molds), are generated from natural food components upon processing (e.g. heterocyclic aromatic amines formed during thermal treatment of meat), penetrate into foodstuffs due to environmental exposure (pesticides used to protect crops), but also sometimes are added to foods purposefully (some food additives). The ingestion of each of these classes of carcinogens with the diet can be vastly diminished by avoiding the consumption of moldy foods, choosing culinary processing that does not promote the formation of genotoxins, selecting organic foods more expensive but with a lower load of environmental contaminants, and avoiding highly processed foods packed with additives. However, the most recommended approach is to incorporate in daily routines such cancer-preventive measures as a diet rich in plant foods providing anticarcinogenic substances and regular physical activity. Both are tirelessly advocated by the WCRF/AICR (World Cancer Research Fund/American Institute for Cancer Research) with its regularly issued Expert Reports; the most recent one, Third Expert Report on Diet, Nutrition, Physical Activity and Cancer: a Global Perspective, was published online in 2018 and has been freely available at www .dietandcancerreport.org since then (WCRF/AICR, 2018). It has been widely accepted among food scientists and nutritionists that genotoxic components represent only a minor dietary cancer risk factor. The attention has been redirected more to other diet-related impacts on human susceptibility to these diseases. Among those increasing the carcinogenic risk directly involving foodstuffs are lower fruit and vegetable intake, greater intake of red and processed meat, greater intake of dairy foods, greater intake of alcohol, and, usually resulting from inappropriate nutrition, greater body fatness (WCRF/AICR, 2018). However, as can be noticed, none of the mentioned factors may be easily transformed into quantifable fgures that could support regulatory science-based efforts and be used for legal guarantees directed to food producers and ensuring food safety. In contrast, the established methodologies and availability of a wide array of cellular and animal models represent reliable and quantitative measures of genotoxicity and carcinogenicity testing. Therefore, despite criticism that only one of the hallmarks of cancer is taken into account in these evaluations (i.e. mutations and genome instability), the approach proposed by the IACR and EPA’s continuously updated classifcations, which are traditionally based mainly on chemical carcinogenesis studies, does not seem to lose its relevance. Just the opposite, the constant need for quick assessments of cancer risk associated with novel foods, evolving culinary processes, or modern dietary supplements requires short-term and well-established methodology to ensure an effcient legal framework.

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Non-Nutritive Bioactive Compounds in Food of Plant Origin Barbara Kusznierewicz

CONTENTS 19.1 Introduction .................................................................................................. 497 19.2 Secondary Plant Metabolites ........................................................................ 498 19.2.1 Phenolic Compounds ........................................................................ 499 19.2.2 Nitrogen- and/or Sulfur-Containing Compounds............................. 505 19.2.2.1 Betalains ............................................................................ 505 19.2.2.2 Purine Alkaloids ................................................................ 510 19.2.2.3 Glucosinolates.................................................................... 514 19.2.2.4 Sulfoxides........................................................................... 519 19.2.3 Terpenoids......................................................................................... 522 19.3 Conclusion .................................................................................................... 529 References.............................................................................................................. 530

19.1

INTRODUCTION

Many epidemiological investigations have shown the close relationship between diet and health, reporting that high consumption of plant foods and beverages has protective activity against chronic diseases, including cardiovascular and neurodegenerative diseases, cancer, and diabetes, which are classifed as non‐communicable diseases. Therefore, the World Health Organization and the Food and Agriculture Organization recommend a minimum of 400 g of fruit and vegetables per day for the prevention of chronic diseases as well as for the prevention and alleviation of several micronutrient defciencies. The therapeutic applications of various plants have aroused great interest among researchers, especially in the last decade, because food is no longer perceived only as a source of nutrients, but also as a source of non-nutrients important for health. Non-nutritive phytochemicals do not have energy and building values and are not essential substances, however, they are compounds with “auxiliary” functions that have a positive effect on human health, development, and well-being. Contrary to the vitamins traditionally considered important for human well-being, non-nutrients do not bring immediate health effects right after consumption; only their long-term presence in small doses in the diet is important in the prevention of chronic diseases. The effect of non-nutrients on the human body is also not as fully understood as in the DOI: 10.1201/9781003265955-19

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case of vitamins and minerals. So far, there is a lack of information on their bioavailability or the recommended dose, which on the one hand will provide a therapeutic effect and, on the other hand, will not exceed the acceptable daily requirement. Plants have the ability to synthesize a wide variety of organic compounds, which are usually classifed as primary and secondary metabolites. Primary metabolites are substances that are responsible for basic functions related to photosynthesis, respiration, plant growth, and development. Representatives of this group of metabolites are saccharides, lipids, nucleotides, amino acids, and organic acids. The remaining phytochemicals that are not directly necessary for the development of the organism are secondary metabolites. Originally, after detecting the presence of substances classifed as secondary metabolites, it was considered that they were by-products of plant metabolism and had no biological function. Although the role of some of these compounds is still unknown, most of the known substances are involved in the interactions of plants with the external environment. For centuries, the extraordinary biological activity of plant secondary metabolites has led to their widespread use as drug components and for therapeutic purposes, and in recent years has also been gaining more and more importance in nutritional prevention. This chapter will provide an overview of the main classes of dietary plant secondary metabolites, including their structures, sources, the impact of process treatments, and the current knowledge of their health-promoting effects.

19.2

SECONDARY PLANT METABOLITES

All living cells ultimately have similar primary metabolites. However, plants also produce various organic molecules via metabolic pathways derived from the pathways used to produce primary metabolites, which are known as plant secondary metabolites, which have unique carbon skeleton structures, are present at much lower concentrations, and are restricted to specifc groups of organisms. The role of these compounds, however, is not as important as the primary metabolites in fulflling the basic functions of the cell/organism, but nevertheless, they play an important role in ensuring the continued existence of organisms in the ecosystem. The biosynthesis and profles of plant secondary metabolites may vary depending on the species, morphological part, stage of development as well as ecological and geographical conditions. An important factor infuencing the level of these compounds in the plant is also stress, which can be both biotic (bacteria, fungi, insects, nematodes) and abiotic (temperature, humidity, salinity, shading, UV radiation, presence of heavy metals or injury). Secondary metabolites confer a multitude of adaptive and evolutionary advantages to the plants by performing specialized functions and playing a role in providing quality of life to the producer. Furthermore, they are associated with plant color, taste, and scent. Often, secondary metabolites are associated with defenserelated, antifeedant, and insect-attractant and repellent functions. Secondary metabolites can be broadly classifed into three main groups based on their structure and the metabolic pathways responsible for their biosynthesis: phenolic compounds, nitrogen and/or sulfur-containing compounds, and terpenoids (Figure 19.1). These phytochemicals are derived from the main primary pathways

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FIGURE 19.1 A simplifed scheme of the biochemical pathways conducting the synthesis of secondary metabolites in plants. Natural products are grouped into phenolic compounds, N-/S-containing compounds, and terpenes (E4P, erythrose 4-phosphate; G3P, glyceraldehyde 3-phosphate; PEP, phosphoenolpyruvate; PYR, pyruvate; DOX5P, deoxyxylulose 5-phosphate).

(glycolysis, the tricarboxylic citric acid cycle, the pentose phosphate pathway, aliphatic and aromatic amino acids, and the shikimic acid pathway).

19.2.1 PHENOLIC COMPOUNDS Phenolic compounds are characterized by a chemical structure that includes at least one aromatic ring substituted with one or more hydroxyl groups which are responsible for the strong reducing properties of polyphenols. This group of secondary metabolites is synthesized from shikimic/phenylpropanoid and the phenylpropanoid–acetate–malonate pathways and encompasses a large group of monomeric and polymeric phenols and polyphenols. Currently, more than 8,000 phenolic structures are known, and more than 500 are found in plant foods and are considered dietary polyphenols. The diversity of polyphenols increases signifcantly as they exist in the form of glycosides substituted with one or more sugars. The most common sugar moiety is glucose, but there are also others such as galactose, rhamnose, arabinose, xylose, or glucuronic and galacturonic acids. Several classes of phenolics have been categorized on the basis of their basic skeletons: C6 (simple phenols, benzoquinones), C6–C1 (hydroxybenzoic acids and aldehydes), C6–C2 (acetophenones, phenylacetic acids), C6–C3 (hydroxycinnamic acids, coumarins, phenylpropanes, chromones), C6–C4 (naphthoquinones), (C6-C3)2 (lignans), C6–C1–C6 (xanthones), C6–C2–C6 (stilbenes, anthraquinones), C6–C3–C6 (favonoids), (C6–C3–C6)2,3 (bi-, trifavonoids, proanthocyanidin dimers, trimers), (C6–C3)2 (lignans, neolignans), (C6–C1)n (hydrolyzable tannins), (C6–C3)n (lignins), (C6)n (catechol melanins, phlorotannins), and (C6–C3–C6)n (condensed tannins) (Figure 19.2).

500 Schematic presentation of the classifcation of phenolic compounds along with basic structures and exemplary representatives of

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FIGURE 19.2 each class.

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The main sources of phenolic compounds in diet are fruits, vegetables, and beverages such as tea, coffee, wine, and fresh fruit juices. The polyphenol content is estimated at around 200–300 mg per 100 g of fresh weight in grapes, apples, pears, cherries, and blueberries. Their presence is also high in products made from these fruits. About 100 mg of polyphenols are found in a glass of red wine, a cup of tea, or coffee. Polyphenols can also be found in cereals, dried legumes, and chocolate (Rahman et al., 2022). It is estimated that favonoids make up about two-thirds of the polyphenols in the diet, with the remaining one-third being phenolic acids, which are in second place (Koch, 2019). Table 19.1 lists the sources of the most common dietary phenolic compound classes and their estimated levels of content. In addition to the variety or species, the content of phenolic compounds in plants is infuenced by many different factors. These may include maturity, growing conditions, processing, and storage. Undoubtedly, environmental and climatic factors (soil type, insolation, rainfall) or agricultural (type of cultivation: greenhouse, feld, biological, hydroponic) have the greatest impact on the content of these compounds in plants. Exposure to sunlight has a signifcant impact on the content of most favonoids. The degree of maturity associated with sunlight also determines the proportion of most phenolic compounds. In most cases, the concentration of phenolic acids decreases during the ripening of the fruit, while the content of anthocyanins increases. Many phenolic substances, especially phenolic acids, are directly involved in the plant’s response to various stress factors: these compounds may be involved in the lignifcation process during the repair of damaged tissues or, due to their antimicrobial properties, their biosynthesis may be increased during plant infection. There are also few reports showing that organically grown vegetables contain more polyphenols than those grown under “stress-free” conditions from conventional or hydroponic crops. This fact has been documented for strawberries, blackberries, and maize. Due to the complexity of the topic, it is diffcult to unequivocally estimate the infuence of a single environmental factor on the content of secondary metabolites. This is due to the diffculty of monitoring climate change, the severity of stress factors, and other variables that the plant experiences during the growing season. Moreover, these factors can act synergistically, antagonistically, or additively, which further complicates the analysis of their infuence. However, easier to track are the factors the plant is exposed to after harvesting. For example, storage conditions and time have a profound effect on the oxidation of polyphenols. Oxidation reactions result in the formation of various derivatives of compounds, which affects the quality of products, mainly causing their sensory changes. Such reactions, on the one hand, may be desirable, as in the case of black tea, and, on the other hand, may reduce the attractiveness of the product, as in the case of browning fruit. Undoubtedly, the longer the storage time, the greater the loss of phenolic compounds in the product can be expected. In the case of wheat four, six-month storage resulted in a 70% loss of phenolic acids, and in the case of apples, nine months of storage resulted in a decrease in the content of proanthocyanidins and quercetin by 60%. Low-temperature storage reduces the rate of degradation of phenolic substances. Despite signifcant losses during storage, phenolic compounds are a group of antioxidants considered more stable than vitamin C.

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TABLE. 19.1 The Sources of the Most Common Dietary Phenolic Compounds Classes and Their Estimated Levels of Content Class of phenolics

Source and content

Flavonoids Flavonols

Flavan-3-ols Flavones Isofavones Flavanones Anthocyanins

Non-favonoids Hydroxybenzoic acids Hydroxycinnamic acid

Stilbenes Lignans Hydrolyzable tannins Condensed tannins

Onion – 347 mg/kg, kale – 321 mg/kg, broccoli – 102 mg/kg, red cabbage – 5 mg/kg, tomato – 8 mg/kg, strawberry – 21 mg/kg, apple – 36 mg/kg, apricot – 25 mg/kg Black tea – 1,726 mg/kg, green tea – 1,358 mg/kg, milk chocolate – 30 mg/kg, dark chocolate – 140 mg/kg, cacao – 440 mg/kg, red wine – 61–179 mg/dm3 Red pepper – 11 mg/kg, parsley – 36 mg/kg, celery – 7 mg/kg Soya bean – 1,421 mg/kg, white bean – 1.2 mg/kg, chickpea – 1.2 mg/kg, red lentil – 0.2 mg/kg, pea – 0.2 mg/kg Orange juice – 265–491 mg/dm3, lemon juice – 131–290 mg/dm3, grapefruit juice – 146–642 mg/dm3, tangerine juice – 230–440 mg/dm3 Chokeberry – 148 mg/kg, black elderberry – 138 mg/kg, blackberry – 30 mg/kg, black currant – 48 mg/kg, cranberry – 14 mg/kg, raspberry – 9 mg/kg, strawberry – 2–4 mg/kg, apple – 0.1–1.2 mg/kg, red cabbage – 32 mg/kg, red onion – 5 mg/kg, eggplant – 9 mg/kg Strawberry – 7–12 mg/100 g, blueberry – 11–32 mg/100 g, raspberry – 23–25 mg/100 g, apple – 0.3–7.2 mg/100 g, basil – 41 mg/100 g d.w., thyme – 37 mg/100 g d.w., clove – 3,160 mg/100 g d.w. Strawberry – 12–17 mg/100 g, blueberry – 1–9 mg/100 g, raspberry – 3–5 mg/100 g, apple – 2–24 mg/100 g, basil – 1690 mg/100 g d.w., oregano – 4,310 mg/100 g d.w., rosemary – 1,703 mg/100 g d.w., sage – 2,735 mg/100 g d.w., thyme – 873 mg/100 g d.w. Red wine – 3–88 mg/dm3, white wine – 1–13 mg/dm3, rhubarb – 1–31 mg/g d.w. Flax seeds – 82–371 mg/100 g d.w., strawberry – 1.6 mg/100 g d.w., cranberry – 1.5 mg/100 g d.w., carrot – 0.5 mg/100 g d.w., broccoli – 0.4 mg/100 g d.w. Pomegranate juice – 1,770 mg/L, cloudberry – 160–240 mg/100 g, raspberry – 250–260 mg/100 g, strawberry – 0.8–18 mg/100 g, Italian nuts – 57 mg/100 g Cranberry – 419 mg/100 g, chokeberry – 664 mg/100 g, grape seeds – 3,532 mg/100 g d.w., apple – 126 mg/100 g, hazelnut – 501 mg/100 g, cinnamon – 8,108 mg/100 g d.w.

The method of culinary processing also signifcantly infuences the polyphenol content in food. Even when peeling vegetables and fruits, a signifcant loss of these compounds can be expected due to the fact that their concentration in the skin is much higher than in the fesh. Boiling, heating with microwaves, or frying also contributes to the degradation of polyphenols. More conservative is steaming, which reduces the polyphenol “washing” to water or fat. The industrial processing of fruit and vegetables also has a negative effect on the phenolic content of the raw material.

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The loss of this group of phytochemicals takes place during the peeling and grinding processes, during which the cells are damaged and enzymes are released, which transform polyphenols into brown pigments with different degrees of polymerization. This undesirable process can take place, for example, in the production of jams and fruit purees. In the production of fruit juices, the clarifcation and preservation stages also carry the risk of loss of many favonoids. The pectolytic enzymes used during clarifcation can hydrolyze many polyphenol structures, and pasteurization degrades them further. Table 19.2 shows examples of research results on the effect of food processing on polyphenol content and antioxidant activity. The broad spectrum of biological activity of polyphenols determined both in in vitro and in vivo systems has already been extensively described in numerous scientifc papers, including antioxidant, anti-infammatory, antidiabetic, anti-tumor, cardioprotective, anti-obesogenic, and anti-degeneration activities (Koch, 2019). Some of these described properties can be used in the prevention and treatment of civilization diseases. Unfortunately, the current knowledge about the biological properties of this group of phytochemicals is still insuffcient to formulate specifc recommendations for the general public, or for groups of people with an increased risk of developing particular civilization diseases. Most of the evidence on the biological properties of polyphenols presented in the literature comes from in vitro studies or from experiments in animal models, which were often carried out with higher doses of pure substances than those available in the diet. Only the results of epidemiological studies especially with large populations can confrm the possible preventive action of phenolic compounds against various civilization diseases. Currently, when analyzing the results of such studies to date, it is possible to state only in the case of cardiovascular diseases the protective effect of polyphenol consumption (Arts & Hollman, 2005). For cancer, neurodegenerative diseases and brain dysfunction, there is still mainly evidence from in vitro and in vivo modeling studies. However, the results of these studies provide information on the possible mechanisms of action of phenolic compounds. They indicate, frst of all, the importance of antioxidant, anti-infammatory, and anti-angiogenic properties, initiation of apoptosis, induction of cell differentiation, modulation of the activity of enzymes of the frst and second phase of detoxifcation, interaction with growth factors and sex hormones, and inhibition of the proliferation of neoplastic cells. Table 19.3 presents some of the health-promoting properties of selected groups of phenolic substances found during in vivo tests. Apart from having a positive effect, phenolic compounds can also have a negative effect on the human body. Some data indicate that polyphenols act not only as antioxidants, but also, in suffciently high concentrations and under certain conditions, as pro-oxidants. Green tea polyphenols have been shown to produce signifcant amounts of hydrogen peroxide when consumed in higher concentrations (e.g., when chewing tea leaves) (Lambert, et al., 2007). Although the relatively high level of plant polyphenols in the diet and additionally the presence of transition metal ions indicate the possibility of their activity as pro-oxidants, no signifcant pro-oxidative activity of these compounds after absorption from the gastrointestinal tract has been recorded so far. However, in the case of polyphenols that are to be added to fortifed

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TABLE 19.2 Exemplary Results of Research on the Effect of Food Processing on the Content of Polyphenols and Antioxidant Activity Sample

Treatment

Effects on polyphenols content

Thermal processing Onions Asparagus Onion bulbs Onion bulbs Zucchini, beans, carrots

Boiling (60 min) Boiling (60 min) Boiling (60 min) Boiling (30 min), frying, or roasting Cooking in different volumes of water

Kale Eggplant fruit

Blanching Microwaving (10 min)

Apple juice Cherry tomatoes

Pasteurization (hightemperature/short-time) Domestic cooking

Cassava

Different cooking methods

Blueberry

Baking

Apricots

Domestic and industrial canning

Peanuts

Retorting

Jujube fruits

Vacuum-microwave (480, 120 W) and hot air (70, 60, and 50° C) drying Drying in different temperatures Drying at 50° C (48 h), 65° C (20 h), 130° C (2 h)

Cocoa beans Raspberry, boysenberry, redcurrant, blackcurrant Tomato Pumpkin four

Oven drying (70° C, 36 h) Oven drying (60° C, 24 h)

20.6% loss in total favonol content 43.9% loss in total favonol content 44–53% degradation of quercetin glucosides < 44% degradation of quercetin glucosides Smaller water volumes yielded lower phenolic concentrations in the surrounding water than cooking in larger volumes 51% decrease in polyphenol content Highest total phenolic level and antioxidant capacity 32.3% loss in total polyphenols Increase in bioavailability of naringenin and chlorogenic acid The highest total phenolic content in steaming, followed by microwaving and boiling No changes in the total polyphenolic, 42% loss in anthocyanins, 23% increase in chlorogenic acid, 36% and 28% increase in favanol dimers and trimers 13–47% and 2–33% loss in total phenolics in industrial and domestic conditions, respectively Loss in resveratrol, caffeic acid, and catechin content, increase in genistein content The greatest loss in total phenolic content during hot air drying at 70° C Total polyphenol content decreased with an increase in temperature Drying at 65° C was the best method in terms of the total polyphenol content

Two-fold higher bioavailability of total polyphenols Increase in the content of phenols, their bioavailability, and antioxidant activity (Continued)

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TABLE 19.2 (CONTINUED) Exemplary Results of Research on the Effect of Food Processing on the Content of Polyphenols and Antioxidant Activity Sample

Treatment

Kale

Freezing

Raspberries

Freezing (–30° C)

Strawberries

Quick and slow freezing

Biochemical processing Whole grain Fermentation sorghum Pigeon pea, kidney bean Tea

Fermentation (4 days)

Papaya

Pickling

Soya beans

Pickling

Fermentation

Mechanical processing Onion Peeling, trimming, and chopping Peach Peeling Green tea Superfne grinding Brazilian green propolis

Superfne grinding

Effects on polyphenols content 3% and 7% loss in polyphenol levels and antioxidative activity, respectively No changes in antioxidant capacity and phenolic level Lower levels of monomeric anthocyanins in slow-frozen strawberries Increase in catechin, gallic acid, and quercetin content and decrease in total favonoid, tannin, and phenolic content Increase in the free soluble phenol content and decrease in the bound phenolics content Decrease in monomeric and increase in oligomeric catechin content 68% and 66% loss in total phenolic and favonoid content, respectively 47% and 42% increase in total phenolic and favonoid content, respectively > 39% loss in favonoid content 13–48% loss in total phenolic content Decrease in catechins, increase in antioxidant activity Increase in total phenolic content and antioxidant activity

Source: Arfaoui, 2021.

or functional foods or included in dietary supplements, particular care should be taken when determining the dose, taking into account, e.g., the structure–activity relationship, matrix effect, bioavailability, metabolism, and antioxidant activity.

19.2.2

NITROGEN- AND/OR SULFUR-CONTAINING COMPOUNDS

19.2.2.1 Betalains Betalains are an unusual class of pigments that are found in certain families within the Pentapetalae order Caryophyllales, where they replace the more common anthocyanins. The biosynthesis of these two groups of pigments is mutually exclusive and it is not possible for anthocyanins and betalains to exist simultaneously in the same

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TABLE 19.3 Health Benefts of Selected Groups of Phenolic Substances Confrmed by in Vivo Studies Anthocyanins/berry fruits Improvement of eyesight

Human studies confrm the improvement of myopia after consuming a high dose of pure anthocyanins.

Improving the cognitive and protective functions of the brain Diabetes and obesity prevention Gastroprotective properties Prevention of cardiovascular diseases

Animal studies show that consumption of anthocyanins or berry extracts reduces damage from cerebral ischemia and supports memory.

Antioxidant properties

Anticarcinogenic properties

Flavonols/quercetin Anticarcinogenic properties Anti-obesity properties

Antidiabetic activity

Reduction in blood glucose levels and in adipose tissue and body weight in anthocyanin-fed animals. Prevention of ethanol-induced hemorrhagic damage to the gastric mucosa, inhibition of Helicobacter pylori growth by consuming chokeberry. In vivo animal studies show a reduction in total cholesterol and LDL levels during chokeberry juice consumption by rats. Decrease in the levels of triacylglycerols, total cholesterol, and the surface of atherosclerotic plaques in mice after administration of black rice extract with a high concentration of anthocyanins. Reduction of peripheral vascular resistance in rats fed with blackcurrant extract. Consumption of a commercially available chokeberry preparation in combination with statin therapy for six weeks inhibited infammation as measured by a reduction in isoprostane and oxidized LDL levels in the blood in patients with ischemic heart disease. In a group of healthy volunteers, after daily consumption of blueberry juice for four weeks, a decrease in cell oxidative damage and an increase in the concentration of reduced glutathione were noted. The consumption of blackcurrant preparation increased peripheral blood fow and decreased muscle fatigue of healthy volunteers after routine typing work. Daily drinking of chokeberry juice by cyclists reduced oxidative damage and supported the endogenous antioxidant system. Consuming blueberries for several weeks lowered the level of lipid peroxidation products in heavy smokers. Consumption of blueberry anthocyanin decreased the number of intestinal adenomas in the mouse model. In rats, consumption of blueberries reduced the number of esophageal tumors induced and decreased the biomarkers of induced colon cancer. Treatment with quercetin reduced the tumor volume in a mouse with induced colon and breast cancers and increased animal survival. Diet enriched with quercetin in mice fed a high-fat diet for nine weeks caused signifcant reduction of liver and white adipose tissue weight, decrease in hepatic lipid accumulation, and reduction of the size of lipid droplets in the animal epididymal fat pads. Oral supplementation with quercetin for three weeks in Swiss albino alloxan-induced mice signifcantly decreased fasting blood glucose and markers of liver and kidney damage, increased antioxidant enzymes, decreased TBARs, and increased GLUT4 expression levels. (Continued)

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TABLE 19.3 (CONTINUED) Health Benefts of Selected Groups of Phenolic Substances Confrmed by in Vivo Studies Anthocyanins/berry fruits Proanthocyanidins Anti-infammatory properties Prevention of cardiovascular diseases Skin whitening properties Properties alleviating the symptoms of menopause Flavanones Cardioprotective properties

Supporting the treatment of diabetes mellitus Supporting the treatment of osteoporosis Antiproliferative and anticarcinogenic properties

Stilbenes/resveratrol Anti-aging properties

Anticarcinogenic properties

Inhibition of infammation, vomiting, and pain in women suffering from chronic pancreatitis after taking grape seed extract for a year. A decrease in total cholesterol, LDL, and HDL fractions in patients with hypercholesterolemia during long-term consumption of grape seed extract. A decrease in the level of lipid peroxidation products in heavy smokers after one month of consuming grape seed extract. Prevention of skin hyperpigmentation in women suffering from melanoderma after six months of use of proanthocyanidins. Lowering fuid retention in the body of premenopausal women after consuming a mixture of blueberry and cranberry extracts.

In studies on rats, supplementation of a high-cholesterol diet with naringenin aided the metabolism of cholesterol by lowering its level in the blood and liver, reducing the amount of triacylglycerols, and increasing the level of HDL fraction in the blood. Additionally, it decreased the concentration of lipid peroxidation products in the blood and liver and increased the level of superoxide dismutase and glutathione peroxidase. In human studies, consuming favanones for 24 weeks reduced the levels of triacylglycerols and several apolipoproteins and improved the VLDL/LDL ratio in the blood. Animal studies have shown that the administration of naringin and hesperidin improved the glucose and lipid profle by regulating liver metabolism. The consumption of naringenin and hesperidin resulted in a reduction in bone loss and a reduction in the level of liver lipids in animal model studies. The administration of unnatural favanones as well as naringenin and naringin resulted in tumor growth inhibition in a mouse model. Dietary supplementation with naringenin protected against azoxymethane-induced abnormal intestinal crypt foci in rats by inhibiting proliferation and triggering apoptosis. Administration of resveratrol increased the lifespan of nematodes, fruit fies, and honey bees, and delayed the loss of locomotor and cognitive abilities caused by aging in fsh. Consumption of resveratrol by mice on a high-calorie diet increased their life expectancy and reduced the symptoms of aging: reduced proteinuria, infammation, and apoptosis in blood vessels, increased aortic fexibility, increased motor skills, inhibition of cataracts, and increased bone density. Studies using animal models have shown the prevention or suppression of various cancers: skin, breast, prostate, digestive system, and lung. (Continued)

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TABLE 19.3 (CONTINUED) Health Benefts of Selected Groups of Phenolic Substances Confrmed by in Vivo Studies Anthocyanins/berry fruits Prevention of cardiovascular diseases Diabetes prophylaxis Isofavones Relief of menopausal symptoms

Improving the functions of the cardiovascular system Prevention of osteoporosis

The results of animal studies showed that resveratrol is effective in hypertension, ischemia, post-reperfusion syndrome, and heart attacks, where, through various mechanisms, resveratrol reduced the risk of developing cardiovascular diseases. Studies in rodents have demonstrated the ability of resveratrol to lower glucose and/or insulin levels and promote insulin sensitivity. Clinical trials show that supplementation with soy protein or genistein reduces the incidence of hot fashes in menopausal women. A positive effect was also observed in the skeletal system. Genistein supplementation increased bone mineral density and decreased the concentration of bone resorption markers at levels comparable to hormone therapy. Isofavones also have a positive effect on the circulatory system. Soy and soy products regulate lipoprotein metabolism, raise HDL cholesterol, lower LDL and VLDL levels, as well as platelet activity and aggregation, and improve vascular reactivity. Genistein has an affnity for β-estrogen receptors and can stimulate or inhibit them. These properties are important in the prevention of breast cancer in women and prostate cancer in men.

Source: Espin et al., 2007; Kasiotis et al., 2013; Koch, 2019.

plant. Although plants that accumulate betalains also have some enzymes responsible for the synthesis of favonoids, they can produce signifcant amounts of some favonoids and, in some cases, proanthocyanidins. The uniqueness of betalains is due to their N-heterocyclic nature determined by betalamic acid which is the main precursor in their biosynthesis. These water-soluble nitrogen-containing pigments can be divided into two major structural groups, red-violet betacyanins and yelloworange betaxanthins. In the case of betacyanins, the reaction of betalamic acid with cyclo-DOPA leads to the synthesis of the aglycone, betanidin, which usually forms connections with glucose and sometimes additionally with glucuronic acid, and may also be further modifed by aliphatic and aromatic acid esterifcation. Four structural types of betacyanins have been reported: betanin-type, amaranthin-type, gomphrenin-type, and bougainvillein-type (Figure 19.3). These structures differ by the attachment of glucosyl groups to the oxygen atoms in the o-position on the cyclodopa moiety. The betanin-type group has a hydroxyl attached to the C6 carbon and a glucosyl or derivative on the C5 carbon, whereas the gomphrenin-type group possesses a hydroxyl attached to the C5 carbon and a glucosyl or derivative on the C6 carbon. The amaranthin-type group has the glucuronyl-glucosyl moiety or derivative

Non-Nutritive Bioactive Compounds

FIGURE 19.3 occur.

509

Representative betalain structures along with exemplary plants in which they

linked to the C5 carbon. The bougainvillein-type group may possess a diglucosyl moiety or derivative linked to the C5 carbon or to the C6 carbon of carboxylated or decarboxylated betacyanins. In the case of betaxanthins, they are formed by the synthesis of betalamic acid with an amino acid or an amine. The saccharide substituents in betalains are typically glucose, glucuronic acid, and apiose, and the acid residues most commonly are malonic, 3-hydroxy-3-methyl-glutaric, caffeic, p-coumaric, and ferulic acid. The most known edible sources of betalains in the Caryophyllales are red beetroots (Beta vulgaris L.), grainy or leafy amaranth (Amaranthus sp.), fruits of the cacti Opuntia sp., Eulychnia sp., and Hylocereus sp., and the colored Swiss chard (B. vulgaris L. ssp. cicla). Less common sources include the tubers of ulluco (Ullucus tuberosus Caldas) and the bloodberries (Rivina humilis L.) (Sadowska-Bartosz & Bartosz, 2021). One of the most common and rich sources of betalains is red beet. It contains two major betalain pigments, red betanin and yellow vulgaxanthin I. Betacyanins constitute approximately 65–95% of beetroot pigments, the remaining 5–35% being betaxanthins (Delgado-Vargas et al., 2000; Kusznierewicz et al., 2021). The stability of betalains depends on many factors, such as the pigment content in the raw material, the degree of glycosidation or acylation, the type of plant matrix, the content of chelating substances, temperature, pH, oxygen, light, and water

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activity. Apart from the initial pigment concentration and the betalain structure, the pH and water activity have the greatest infuence on the stability of these dyes. In the case of red beet, the color of betalains does not change at a pH of 4 to 7, but these compounds are very sensitive to high temperatures. Therefore, it is recommended to process the beet at pH = 4 and to store it at low temperature beforehand. The use of such treatments allows the reduction of losses of betacyanins during high-temperature treatment. More information on the stability of betalains during processing can be found in Chapter 10. Dietary betalains may play an important role in maintaining human health because of their many benefcial activities. Many studies carried out in in vitro systems have confrmed the high antioxidant potential of betalain. The antioxidant activity of betacyanins and betaxanthins is three to four times higher than that of vitamin C (Cai et al., 2003). In in vitro studies, betalains also show the ability to protect against oxidative stress, demonstrated against the LDL cholesterol fraction and cell membrane lipids (Allegra et al., 2005). This group of phytochemicals also has the ability to increase the activity of phase II enzymes and inhibit the expression of ICAM1 and endothelial cells. In vivo studies have additionally confrmed the antioxidant effect of betalains in the human body, as evidenced by the reduction in the level of lipid oxidation and LDL cholesterol in the blood of the subjects. The results of other studies have additionally demonstrated the antimalarial and hepatoprotective activity of betalains (Moreno et al., 2008). So far, most of the literature reports on the health-promoting properties of betalains have focused on their high antioxidant activity. However, there is still little research in humans to reliably determine the mechanisms of action of these compounds and their metabolites in protection against chronic degenerative diseases. On the other hand, there are many scientifc reports, examples of which are listed in Table 19.4, which indicate a high healthpromoting potential of betalains and constitute recommendations and suggestions for further, more detailed research. 19.2.2.2 Purine Alkaloids The most well-known secondary metabolites synthesized from nucleotides are purine alkaloids. Purine alkaloids occur in nearly 100 species in 13 orders of the plant kingdom. Initially, they were considered to be a waste end-product in plants, although it was also hypothesized that they play a role in chemical defenses and have an allelopathic function (Ashihara & Crozier, 1999). Recent research using transgenic plants has provided convincing evidence for the chemical defense theory (Sano et al., 2013). Methylxanthines, such as caffeine (1,3,7-trimethylxanthine), theobromine (3,7-dimethylxanthine), and theophiline (1,3-dimethylxanthine) are classifed as purine alkaloids and they occur in tea, coffee, cacao, and a number of non-alcoholic beverages (Figure 19.4). Caffeine is undoubtedly the most frequently consumed methylxanthine in the diet. Roasted coffee beans and tea leaves are the primary sources of dietary caffeine. Caffeine is also found in kola nuts, cocoa beans, yerba mate, and guarana berries. It is estimated that the richest source of caffeine in the diet is coffee (71%), followed by

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TABLE 19.4 Exemplary Health Benefts of Betalains and Plant Extracts Rich in Betalains Activity

Research conclusion

Antimicrobial activity

Beetroot pomace inhibited the multiplication of Salmonella typhimurium, Staphylococcus aureus, Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa, Citrobacter freundii, and Citrobacter youngae. Betalain-rich extracts of amaranth exhibited dose-dependent antimalarial activity in an in vivo mouse model assay.

Anticancer activity

Numerous experiments demonstrated cytotoxicity of betalains and betalaincontaining plant extracts toward different cancer cell lines (e.g., Caco-2, HepG2, K562, B16F10, MCF-7, HCT-116). Oral administration of red beet extract to mice decreased signifcantly the number of papillomas in the skin after topical tumor induction, reduced the number of mice with adenomas (by 60%) and the number of tumors in affected animals (by 30%) after induction of lung tumors, inhibited also the formation of skin tumors induced by UV radiation or chemical treatment, decreased the incidence of liver tumors by 60%, and reduced the splenomegaly. Rats fed betalain-rich beet crisps had signifcantly lowered blood serum glucose level, atherogenic index, isovaleric acid level, cecal weight, and body weight. Administration of red pitahaya fruit to hypercholesterolemic rats lowered serum lipids and total cholesterol, apparently due to enhancement of bile acids excretion. Administration of betalain-rich extracts of amaranth to diabetic rats decreased the levels of blood cholesterol, triglycerides, and LDL, and increased the level of HDL. Body weight, body mass index (BMI), and LDL-cholesterol were decreased in obese human volunteers who consumed freeze-dried red beet leaves for four weeks. Animal experiments showed that betanin is effective in the treatment of steatohepatitis, upregulating the peroxisome proliferator-activated receptor (PPAR)-α, downregulating the sterol regulatory element-binding protein (SREBP)-1c, and modifying adipokine levels and lipid profle. Betalain-rich red beet juice decreased hepatic toxicity caused by N-nitrosodiethylamine and carbon tetrachloride in the rat. Extracts of opuntia fruit and amaranth protected the liver from damage by carbon tetrachloride and stimulated its recovery in rat experiments. In the rat model of Parkinson’s disease, red beet protected against behavioral changes and ameliorated oxidative stress. Betacyanins from common purslane reversed the learning and memory impairments induced by D-galactose in mice. Betanin protected against trimethyltin-induced neurodegeneration in mice, including improvement of spatial learning and memory defcits, showing also anxiolytic properties. Beetroot juice lowers systolic and diastolic blood pressure, which was confrmed in clinical studies with volunteers drinking 500 cm3 of beetroot juice daily and in 68 people with hypertension after drinking 250 cm3 of beet juice daily for two weeks. Betanin decreased markers of glycemia in rats and ameliorated diabetic cardiac fbrosis as well as can effectively suppress renal fbrosis in diabetic nephropathy and may slow down the progression to end-stage renal disease by regulating TGF-β signaling pathway.

Anti-lipidemic effects

Hepatoprotective effects

Neuroprotective effects

Cardiovascular effects Effects in diabetes

Antiinfammatory effects

Oral administration of beetroot juice attenuated isoproterenol-induced cardiac dysfunction and structural damage by reducing oxidative stress, infammation, and apoptosis in the heart. Betanin induced the transcription of antioxidant genes through Nrf2 and, simultaneously, suppressed the pro-infammatory NFk-B pathways, thus alleviating endothelial damage and atherogenesis.

Source: Sadowska-Bartosz & Bartosz, 2021.

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FIGURE 19.4 Structures of the most common dietary purine alkaloids, together with exemplary content (%) in various plant sources (Ashihara et al., 2017).

soft drinks (16%) and tea (12%). The average caffeine consumption from all sources amounts to about 70 mg/person per day (Heckman et al., 2010). The caffeine content in raw coffee can signifcantly vary, depending on many factors, among which the most important are the origin and cultivar, Arabica or Robusta. Raw Arabica has an average of 0.8 to 1.8% caffeine (on a dry basis), while Robusta contains about twice as much from 2.0 to 3.0% (Ashihara et al., 2017). Besides genotype and geographic origin, other environmental factors can infuence caffeine accumulation. For example, light exposure is required for the synthesis of caffeine, although its optimal level is very low. The high-altitude location of the coffee plant also had a positive effect on the caffeine content of the raw beans. Commercially available coffee blends are characterized by high variability in caffeine content, mainly depending on the type of coffee used, but also on the degree of roasting or grinding of the beans. Roasting coffee is one of the most important steps in coffee processing due to the pronounced chemical, physical, structural, and sensory changes that give it world-renowned properties. During this process, the coffee beans are exposed to high temperatures for a period of time which can vary considerably depending on the type of roaster, geographic origin, variety, characteristics of the coffee beans, and the sensory properties desired. In the case of caffeine, despite its high sublimation temperature (178° C), it is reduced by evaporation as it is pulled by the water vapor. This phenomenon is also favored by the increase in the solubility of caffeine in water as a function of temperature. In general, roasting causes a reduction in caffeine content of 30% (Franca et. al., 2005). Moreover, important microstructural changes occurring during roasting can drive an additional loss in caffeine. The high temperature reached during roasting causes bursts accompanied by popping sounds. During popping phenomena, caffeine is easily detectable in the roasting gas because it is emitted during seed fracturing (Severini et al., 2017). In addition to the roasting process, the way in which the coffee is prepared has a very important infuence on the fnal amount of caffeine in a cup of coffee. In different

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geographic areas, coffee infusions can change signifcantly in the preparation of espresso, American coffee, French coffee, Turkish coffee, etc. Therefore, the fnal caffeine content in the coffee is infuenced by many factors, such as the type of contact of water with the ground coffee, extraction time, the ratio of the mass of ground roasted coffee to water, the volume of the extract, water temperature, steam pressure in the case of espresso coffee, and the type of fltration and cooking process. Figure 19.5 shows examples of the caffeine content in coffee prepared in various ways, compiled for comparison with other popular caffeinated beverages (van Dam et al., 2020). Caffeine’s consumption, absorption, metabolism, and physiological and functional effects are infuenced by many different exogenous and endogenous factors, such as age, gender, hormonal status, diet, smoking, drug intake, and genetic predisposition. Complete absorption of caffeine occurs approximately 45 minutes after ingestion, with peak levels in the blood occurring within 15 minutes to two hours. Caffeine spreads throughout the body and crosses the blood-brain barrier. In the liver, caffeine is metabolized by cytochrome P-450 (CYP) enzymes – particularly CYP1A2. The main metabolite of caffeine is paraxanthin (84%), and smaller amounts of

FIGURE 19.5 An example of the caffeine content in a typical portion of commonly consumed beverages (content may vary, particularly with different brands) (van Dam et al., 2020).

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theobromine (12%) and theophylline (4%) are also produced. These compounds are further metabolized to uric acid and eventually excreted in the urine (Nehlig, 2018). The half-life of caffeine in adults is usually between 2.5 and 4.5 hours but can vary widely from person to person. Smoking signifcantly speeds up the metabolism of caffeine, reducing the half-life by up to 50%, while oral contraceptives can double the half-life of caffeine. Pregnancy signifcantly reduces caffeine metabolism, especially in the third trimester, when the half-life of caffeine may be up to 15 hours. The activity of caffeine-metabolizing enzymes is partly hereditary. For example, a variant in the gene encoding CYP1A2 is associated with higher plasma caffeine levels and a lower paraxanthine-to-caffeine ratio (refecting slower caffeine metabolism). People with a genetically slower caffeine metabolism tend to compensate for this with a lower habitual caffeine intake than people without this genetic predisposition. In addition, drugs from different classes of drugs (including several quinolone antibiotics, cardiovascular drugs, bronchodilators, and antidepressants) can slow caffeine clearance and increase its half-life, mainly because they are metabolized by the same liver enzymes. Similarly, caffeine can affect the effects of various medications, and clinicians should consider possible interactions between caffeine and drugs during therapy (van Dam et al., 2020). According to various literature data, caffeine exhibits a wide range of biological activities, which, depending on many factors (including dose), may have a positive or negative effect on the human body (Table 19.5). Much evidence suggests that consuming caffeinated coffee does not increase the risk of cardiovascular disease and cancer. In fact, consuming three to fve standard cups of coffee a day is consistently associated with a reduced risk of several chronic diseases. However, high caffeine consumption can have various negative effects, and it is recommended to limit 400 mg of caffeine per day for adults who are not pregnant or breastfeeding, and 200 mg per day for pregnant and lactating women. The current evidence does not support recommending caffeine or coffee consumption for disease prevention but suggests that for adults moderate coffee or tea consumption may be part of a healthy lifestyle. 19.2.2.3 Glucosinolates Glucosinolates (GLs) are nitrogen- and sulfur-containing plant secondary metabolites. This group of phytochemicals plays a vital role in the physiology and protection of plants against several environmental stresses. Their presence has been reported almost exclusively in the order Capparales, which contains 15 families including Brassicaceae, Capparaceae, and Caricaceae. More than 200 different types of GLs have been studied, and 30 of them are identifed in Brassica crops. GLs are organic anions with a chemical structure consisting of a β-D-glucopyranose residue linked via a sulfur atom to a (Z)-N-hydroximinosulfate ester and a variable R group derived from one of eight amino acids (Figure 19.6). GLs can be classifed based on their precursor amino acids and the types of modifcation of the R group. Compounds derived from alanine, leucine, isoleucine, methionine, or valine are classifed as aliphatic GLs, those derived from phenylalanine or tyrosine are classifed as aromatic GLs, and those derived from tryptophan belong to the indolic GLs. The R groups of most GLs are highly modifed, especially those

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TABLE 19.5 Health Effects of Caffeine Consumption According to the Organ System Human organ

Effect of caffeine

Brain

• Due to its structural similarity to adenosine, caffeine has the ability to connect and block adenosine receptors in the brain, thus delaying fatigue and the desire to sleep. Thanks to this, it increases mental effciency and alertness. • Side effects of caffeine at very high levels of intake include anxiety, restlessness, nervousness, dysphoria, insomnia, excitement, psychomotor agitation, and rambling fow of thought and speech. Toxic effects are estimated to occur with intakes of 1.2 g or higher, and a dose of 10 to 14 g is thought to be fatal. • Coffee and caffeine consumption have been associated with reduced risks of depression and suicide in several cohorts in the United States and Europe. • Caffeine can contribute to pain relief when added to commonly used analgesic agents such as nonsteroidal anti-infammatory drugs and paracetamol. • Prospective cohort studies in the United States, Europe, and Asia have shown a strong inverse association between caffeine intake and the risk of Parkinson’s disease.

Cardiovascular system

• In people who have not consumed caffeine before, the consumption of caffeine in a short time raises the level of adrenaline and blood pressure. Tolerance to effects develops within a week but may be incomplete in some people. • Caffeine is widely used as a treatment for apnea of prematurity in infants and slightly improves lung function in adults. • Caffeine may prevent hepatic fbrosis through adenosine receptor antagonism because adenosine promotes tissue remodeling, including collagen production and fbrinogenesis. In line with this observation, caffeine metabolites reduce collagen deposition in liver cells, caffeine inhibits hepatocarcinogenesis in animal models, and a randomized trial showed that consumption of caffeinated coffee reduces liver collagen levels in patients with hepatitis C. • Coffee consumption has been associated with a reduced risk of gallstones and of gallbladder cancer, with a stronger association for caffeinated coffee than for decaffeinated coffee, suggesting that caffeine may play a protective role. • High doses of caffeine may have a diuretic effect, but the usual moderate intake will not signifcantly alter hydration status. • Caffeine intake reduces insulin sensitivity in the short term. This may refect an inhibitory effect of caffeine on storage of glucose as glycogen in muscle and may partly result from increased epinephrine release. However, consumption of caffeinated coffee (four to fve cups per day) for up to six months does not affect insulin resistance.

Lungs Liver

Kidneys and  urinary tract

Endocrine  system

Reproductive  system

• In prospective studies, higher caffeine intake has been associated with lower birth weight and a higher risk of pregnancy loss.

Source: van Dam et al., 2020.

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FIGURE 19.6 Classifcation of glucosinolates supplemented with structures and content (µmol/100 g f.w.) of the most common representatives of each class.

derived from methionine. Most R groups are elongated with one or more methylene groups. Both elongated and non-elongated R groups undergo a wide variety of transformations, including hydroxylation, O-methylation, desaturation, glycosylation, and acylation. GLs are present in varying amounts in roots, leaves, shoots, and seeds. Plants of the Brassicaceae family most often contain a dozen or so GLs, the structure of which is shown in Figure 19.6, and those present at the highest concentration levels are sinigrin, glucoiberin, and glucobrassicin. However, the amount and composition of these compounds in individual plants vary depending on the species, the cultivar tested, the stage of development, and the climatic conditions of the cultivation. Plants containing GLs always possess the β-thioglucosidase known as myrosinase (MYR), creating a so-called substrate-enzyme defense system. In Brassica plant tissues, MYR is probably located mainly in myrosin cells, the distribution of which differs in individual parts of plants, and also depends on the stage of development and plant species. GLs are found in the vacuoles near the myrosin cells. The enzyme and GLs come into contact only after the cells are damaged and then the hydrolysis reaction can begin (during the cutting, mixing, chopping, and chewing of the plant). Myrosinase catalyzes the hydrolysis of thioglucoside bonds of GLs, resulting in the formation of an unstable intermediate product – thiohydroximate-O-sulfonate,

Non-Nutritive Bioactive Compounds

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which, depending on the reaction environment and the presence of additional protein factors, can be converted to isothiocyanates (ITCs), thiocyanates, nitriles, or epithionitriles. Subsequently, unstable ITCs containing a β-hydroxyl group or an indole ring undergo spontaneous conversion to oxazolidine-2-thiones and indole compounds, respectively (Figure 19.7). The specialized glucosinolate-myrosinase system is often referred to as the “mustard oil bomb” that plants have developed to repel herbivores and defend themselves against various pathogens and insects. Moreover, it is believed that the bioactive degradation products of GLs also have various health-promoting functions, making them useful compounds for human consumption. However, GLs degradation products are also strongly associated with the aroma and taste of Brassica crops, as many of them provide spicy and bitter favors. This sensory characteristic is often a limitation in the development of GLs enriched foods due to the negative impact it can have on consumer acceptance. Brassica plants are considered to be the main source of GLs in the human diet. These vegetables are very perishable, so their commercialization requires prior industrial processing to extend their shelf-life while maintaining good overall quality and product safety. Some of these vegetables are eaten cooked, while others are commonly eaten as raw vegetables added to salads, smoothies, or other dishes. But even for the raw ones, minor processes are required at both industrial and domestic levels, such as cutting, shredding, or squeezing. All these processes and treatments affect the stability of GLs and their decomposition products, the activity of myrosinase, and the denaturation of ESP proteins. The conditions of transport, storage, and cooking have a huge impact on the level of GLs in Brassicaceae vegetables. About 70–80% of these compounds are lost in vegetables during storage and transport (Vallejo et al., 2003). The storage of shredded cabbage reduces the concentration of methionine-derived GLs and increases the content of their indole forms (Verkerk et al., 2001). Similar relationships were also observed during the storage of broccoli

FIGURE 19.7 Scheme of the course of the glucosinolates hydrolysis along with examples of the most common breakdown products (ESM, epithiospecifer modifer protein; ESP, epithiospecifer proteins; NSP, nitrile-specifer proteins; MYR, myrosinase; TFP, thiocyanateforming protein).

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(Hansen et al., 1995). During culinary processing, when grinding raw vegetables, a signifcant part (about 80%) of the methionine-derived GLs can be converted into nitriles and isothiocyanates. The proportions between the formed nitriles and isothiocyanate depend on the genotype of the plants. During cooking, both myrosinase and epithiospecifer proteins are degraded. Mild cooking conditions such as steaming broccoli for less than three minutes denature epithiospecifer proteins. Under these conditions, however, small amounts of endogenous myrosinase remain intact, which contributes to a proportional increase in the amount of isothiocyanates. Further boiling or heating of broccoli for another ten or 20 minutes causes the gradual denaturation of the remaining myrosinase, thus preventing the degradation of glucosinolates and the formation of isothiocyanates (Verkerk et al., 2010). The health benefts of consuming Brassica vegetables can only be expected if the intact GLs are hydrolyzed to their reactive products, especially isothiocyanates, in a reaction effectively mediated by plant myrosinase. Therefore, proper processing of such plants should minimize the losses of both GLs and plant myrosinase. Conventional, long-term cooking of Brassica vegetables has a negative effect on the functioning of the glucosinolate-myrosinase system due to thermal degradation and leaching into the water of both the substrate and the enzyme. Therefore, the following is recommended: • short-term cooking or blanching, preferably short steaming for a few minutes; • use of alternative technologies such as HPP combining mild temperature and high pressure; • mixing the thermally treated products with an active myrosinase source (e.g., mustard sauce). In conclusion, the preservation of a high level of GLs and active myrosinase during the processing of brassica plants are goals to obtain the highest quality health-promoting products. This approach requires adjusting industrial processing parameters and increasing consumer awareness to avoid losses during home cooking. Isothiocyanates are considered to be the most important derivatives of GLs in the prevention of cancer, as well as in the case of other diet-related chronic diseases. A diet rich in Brassica plants can reduce the risk of the most commonly diagnosed cancers, such as breast, prostate, lung, stomach, and pancreatic cancer. Initially, it was shown that isothiocyanates reduce the activity of phase I enzymes, contributing to the activation of carcinogens while stimulating the action of phase II enzymes responsible for the detoxifcation of xenobiotics. Then, the infuence of isothiocyanates on a number of parallel processes occurring in the cell was proved by inhibiting or stimulating the activity of transcription factors. This ability of isothiocyanates contributes to the prevention of infammation and the activation of signaling pathways regulating not only detoxifcation mechanisms but also the cell cycle and apoptosis (Srivastava & Singh, 2004; Zhang, 2010). The chemopreventive properties of isothiocyanates are revealed at many levels of the body’s functioning, hence their multidirectional health-promoting potential. So far, most studies

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on anticarcinogenic properties have been carried out for broccoli, and in particular, for the isolated form of sulforaphane isothiocyanate resulting from the breakdown of glucoraphanin. Sulforaphane has been shown to act as an indirect antioxidant by inducing the so-called antioxidant enzymes and also causes cell cycle arrest and apoptosis (Rose et al., 2005). The results of several studies have provided evidence about the ability of sulforaphane to induce apoptosis in a number of cancer cell lines, such as leukemia, prostate cancer, and breast cancer. Moreover, sulforaphane has a high potential to combat Helicobacter pylori, bacteria responsible for the formation of gastric cancer (Fahey et al., 2002). Apart from sulforaphane, which is undoubtedly the best-described isothiocyanate in the literature, other compounds from this group of GLs degradation products may also be responsible for the anticarcinogenic properties of Brassica plants. The breakdown products of glucoiberin, sinigrin, and progoitrin have also been found to protect human and animal cells from carcinogenesis. These compounds can exhibit sulforaphane-like biological activity both through the induction of phase II detoxifcation enzymes and the inhibition of phase I enzymes (Nilsson et al., 2006). Apart from aliphatic isothiocyanates, also aromatic isothiocyanates and indole derivatives have become the subject of research on the chemopreventive potential of Brassicas. Indole-3-carbinol, a degradation product of glucobrassicin and benzyl and phenylethyl isothiocyanate, derived from two aromatic GLs, glucotropaeoline and gluconasturcin, respectively, may be responsible for the selective induction of apoptosis in cancer cells and enhance the preventive and/or therapeutic effect of other types of GLs degradation products against various types of cancers (Zhang & Talalay, 1994). Isothiocyanates formed from indole GLs are unstable and spontaneously hydrolyze to indole-3-carbinol. This compound may condense in the acidic environment of the stomach to form new derivatives. Despite the toxicity of these compounds, their preventive action against breast cancer and cervical cancer has also been proven (Singh et al., 2021). Despite the documented pro-health properties of bioactive phytochemicals of Brassica plants, the knowledge about them is not yet complete. In light of current knowledge, isothiocyanates are the compounds with the most effective pro-health properties among the products of GLs degradation. Their presence in the human diet seems to play an important role in the chemoprevention of civilization diseases, especially cancer. 19.2.2.4 Sulfoxides A characteristic feature of Allium vegetables is the fact that they contain unique sulfur compounds that give them a specifc smell and are responsible for their biological activity. Intact tissues of these plants usually do not emit odors; therefore, it has been hypothesized that the volatile garlic compounds arise from non-volatile precursors during mechanical damage to the plant. For the frst time in 1947, in the laboratory of scientists Stoll and Seebeck, the best-known precursor of volatile organic sulfur compounds of the Allium genus, namely S-allyl-l-cysteine sulfoxide, commonly known as alliin, was isolated and identifed. Alliin is the starting compound of the main substances responsible for the smell of crushed or cut garlic. In later years, further sulfoxides were identifed in the onion tissues as precursors of volatile sulfur

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compounds. Isoalliin is the major sulfoxide present in undamaged onion tissue and is responsible for the formation of tear-inducing compounds. Methiin, propiin, and small amounts of alliin were also found in the onion (Figure 19.8). Sulfoxides are present in plants of the genus Allium only until their tissues remain intact. When plant cells are mechanically damaged by cutting, chewing, crushing, etc., the cysteine sulfoxides degrade into highly odorous thiosulfnates. This reaction is catalyzed by the enzyme alliinase (EC 4.4.1.4), which is located in vacuoles in intact tissue and is separated from the cysteine sulfoxides present in the cytoplasm. Released from damaged tissue, alliinase in the presence of pyridoxal phosphate catalyzes the transformation S-alk(en)ylcysteine sulfoxides into alk(en)nylsulfenic acids, pyruvic acid, and ammonia. Subsequent condensation of the resulting alk(en) ylsulfenic acids leads to the formation of thiosulfnates. The decomposition of sulfoxides is fast. For example, the most abundant alliin in garlic is completely degraded to form diallyl thiosulfnate (allicin) within 10–60 seconds. The sulfenic acids and thiosulfnates formed during damage to the tissue of Allium plants are reactive intermediates from which a signifcant amount of various aromatic sulfur compounds is formed (Figure 19.9). They are formed already at room temperature during plant homogenization and may undergo various transformations depending on the conditions and duration of the reaction. As in the case of Brassica vegetables, also in Allium plants, transport and storage reduce the content of volatile sulfur precursors. This is most likely due to the intensifcation of the respiratory processes associated with weight loss, during which S-alk(en)ylcysteine sulfoxides are consumed by plants as a source of nitrogen and

FIGURE 19.8 Structures and content (mg/100 g f.w.) of S-alk(en)ylcysteine sulfoxides in selected Allium species (Kubec et al., 1999; Kubec et al., 2000).

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FIGURE 19.9 Degradation of alliin in garlic (A) and isoalliin in onion (B) and examples of formed organosulfur derivatives (AM, allyl mercaptan; AMDS, allyl methyl disulfde; AMS, allyl methyl sulfde; DADS, diallyl disulfde; DAS, diallyl sulfde; DATS, diallyl trisulfde; DPDS, dipropyl disulfde; DPS, dipropyl sulfde; DPTS, dipropyl trisulfde; LF, lachrymatory factor; PMDS, propyl methyl disulfde; PMS, propyl methyl sulfde; PMTS, propyl methyl trisulfde; SAC, S-allyl cysteine; SAMC, S-allylmercapto-l-cysteine).

sulfur nutrients. Similarly, dehydration, pickling, cooking, frying, freezing, and pasteurization reduce the content of volatile organosulfur compounds, thus reducing the intensity of the smell of these products. The degree of loss of these substances depends on the duration and conditions of individual processes. When cooking Allium plants, high temperatures have little effect on the stability of the sulfoxides. However, on the other hand, it inactivates allinase, which prevents enzymatic degradation of sulfur precursors. In the case of pickling, the process environment negatively affects both the sulfoxides and the activity of the enzyme itself. As is well known, heat treatment of onions is effective in reducing the lachrymatory factor. This is due to the transformation of the isoalliin of the propanthial-S-oxide precursor into inactive cycloalliin. According to some sources, the amount of tear gas produced when the onion is cut and crushed can be reduced by acidifying the onion to a pH of about 3.9, e.g., with a citric acid solution. The smell of fresh and hightemperature-treated onions is very different. The headspace analysis of both types of onions indicates the presence of mainly alk(en)yl thiosulfnates in the fresh raw material, while in the cooked onion the smell is determined by propyl and propenyl di- and trisulfdes (Boelens et al., 1971). On the other hand, the presence of propyl mercaptan is responsible for the sweet taste of cooked onion. The use of higher and higher temperatures during the processing of onions results in an increase in the intensity of secondary and quaternary reactions, resulting in dimethylthiophenes

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responsible for the characteristic smell of fried onions. The main ingredients formed during the frying of onions, according to Kimura et al. (1990), are 2,4-dimethylthiophene, propyl methyl trisulfde, and allyl propyl trisulfde. The use of Allium plants by humans has a very long history dating back to ancient Egypt. The representatives of this group of plants most often found in the human diet are garlic, onion, leek, or chives. Allium vegetables have a wide range of biological activities, including antibacterial and antifungal properties that have been known for centuries, as well as stimulating the cardiovascular and immune systems. In addition, in recent years, reports about the antioxidant and anticarcinogenic activity of these plants have been appearing more and more often. Most of the professional literature and research concerns garlic as a vegetable from the Allium family with the greatest health-promoting potential. It describes the multidirectional activities of garlic phytochemicals, including: • inhibition of the activity of adenosine deaminase, ensuring a high concentration of adenosine, which determines the renewal of ATP and reduces the overload of the heart muscle, improving blood fow through the coronary vessels; • induction of glutathione peroxidase activity, thus reducing the risk of reactive oxygen species responsible for the peroxidation of cell membrane lipids and the oxidation of unsaturated fatty acids; • decreased adhesion of excited granulocytes to the epithelium of blood vessels and aggregation of platelets; • lowering the level of cholesterol as well as triacylglycerols and phospholipids in the blood serum; • high antibacterial, antifungal, and antiviral activity; • regulation of the composition of the intestinal microfora; • regulation and support of the humoral and cellular immune response, which improves the body’s immune mechanism (especially in people over 60 years of age); • chemopreventive effects, including the inhibition of DNA adduct formation, the inhibition of mutagenesis by blocking metabolism, through its free-radical scavenging, or by decreasing cell proliferation and tumor growth. Examples of health-promoting effects of garlic observed during clinical trials are presented in Table 19.6.

19.2.3 TERPENOIDS Terpenoids are fat-soluble compounds that constitute one of the largest and structurally diverse groups of plant metabolites with over 50,000 different structures detected so far. This class of natural products is derived from the mevalonate pathway, which is active in the cytosol, or from the plasticidal 2-C-methyl-D-erythriol 4-phosphate pathway. Terpenoids are composed of many structural units of isoprene (2-methylbuta-1,3-diene – C5H8). Based upon the number of isoprene units, these

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TABLE 19.6 Examples of Health-Promoting Effects of Garlic Observed During Clinical Trials Activity

Research conclusion

Antioxidant activity

• In diabetic patients, after 30 days of supplementation with 3.6 g of garlic daily, the activity of superoxide dismutase, catalase, and glutathione peroxidase increased. • Meta-analysis of clinical trials demonstrated that garlic supplementation modulates oxidative stress markers, including total antioxidant capacity and malondialdehyde. • In a randomized double-blind placebo-controlled nutritional intervention, garlic extract intake at 400 mg/day for three months enhanced antioxidant status, reducing the cardiovascular risk in obese patients.

Antimicrobial and antiviral activities

• A randomized double-blind controlled clinical trial found an inversed relation between Streptococcus mutans, Lactobacilli species, and Candida albicans and garlic with lime-containing mouth rinses in children with severe early childhood caries. • Allicin exhibits a wide spectrum of activity against both gram-positive and gram-negative bacteria, including Helicobacter pylori, Escherichia, Salmonella, Staphylococcus, Klebsiella, Proteus, and Bacillus, leading to interactions with groups of enzymes such as alcohol dehydrogenase, thioredoxin reductase, or RNA polymerase. • The antifungal activity of garlic was observed in Candida albicans, but also against strains of the genus Cryptococcus, Trichophyton, Epidermophyton, and Microsporum. • Preclinical studies have shown that garlic has potential antiviral activity against a variety of viruses pathogenic to humans, animals, and plants by, inter alia, blocking viral entry into host cells, inhibiting viral RNA polymerase, reverse transcriptase, DNA synthesis, and immediate early gene transcription. It has also been shown that the alleviation of viral infection is associated with the immunomodulatory effects of garlic and its organosulfur compounds. • Clinical trials have demonstrated the prophylactic effects of garlic in preventing widespread viral infections in humans by enhancing the immune response. • A double-blind randomized clinical trial showed a signifcant reduction of infammatory cytokines, such as interleukin 6, C-reactive protein, and erythrocyte sedimentation rate when garlic extract was administered at 400 mg twice a day for eight weeks in peritoneal dialysis patients. • The consumption of aged garlic at a dose of 2.6 g per day for 90 days increased the activity of immune cells and decreased infammation in obese adults. • Garlic supplementation increased microbial richness and diversity and improved infammation condition in patients with uncontrolled hypertension. • After four months, the consumption of garlic extract raised HDL and lowered LDL and cholesterol levels in 23 hyperlipidemic patients. • In a randomized, double-blind, placebo-controlled trial, 10.8 mg per day of garlic powder for 12 weeks reduced the triacylglycerol concentration in healthy volunteers. • 400 mg garlic and 1 mg allicin two times daily reduced cholesterol and LDL levels, showing protective effects in a single-blind study with 150 hypercholesterolemia patients.

Antiinfammatory properties

Lipid-lowering effect

(Continued)

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TABLE 19.6 (CONTINUED) Examples of Health-Promoting Effects of Garlic Observed During Clinical Trials Activity Anticarcinogenic activity

Cardiovascular effects

Effects on diabetes

Antiosteoarthritis potential

Research conclusion • The FDA’s evidence-based review system showed no reliable evidence for the relation between garlic and a reduced risk of gastric, breast, and lung cancer. • Credible evidence for an association between garlic intake and colon, prostate, esophageal, larynx, oral, ovary, and renal cell cancers has been reported. • Randomized double-blind factorial trial on garlic highlighted a decreased appearance of precancerous gastric lesions or gastric cancer. • Long-term consumption of garlic, garlic supplements, or garlic with vitamins reduced gastric cancer, precancerous gastric lesions, and mortality rate. • The intake of garlic supplements of 0.60–3.65 kg per year for two years was signifcantly associated with decreased risk of colorectal adenoma, which is a precursor of colorectal cancer. • Epidemiological studies conducted in the Chinese population found a signifcant inverse relation between consumption of raw garlic or garlic components 8.4 g or 33.4 g per week for seven years and lung cancer. • Garlic can signifcantly reduce the risk of atherosclerosis, hypertension, diabetes, hyperlipidemia, myocardial infarction, and ischemic stroke thanks to the synergistic effects of its nutritional and phytochemical components. • Randomized trial performed with aged garlic extract on adipose tissue surrogates for coronary atherosclerosis progression reported a decrease in coronary atherosclerosis growth by reducing epicardial, pericardial, periaortic, and subcutaneous adipose tissues. • Aged garlic extract at 2.4 g per day prevented atherosclerosis process by developing microcirculation in patients. • Aged garlic extract at 6 g daily for 12 weeks reduced the levels of lipoprotein B and raised HDL levels, showing cardioprotective effect in patients with mild hypercholesterolemia. • Meta-analysis suggested that garlic has a cardioprotective effect by decreasing serum total cholesterol and triglycerides in patients with mild hypercholesterolemia. • Preclinical studies showed that garlic’s active organosulfur compounds reduced hyperglycemia by improving the antioxidant status in circulation of diabetic rats. • Meta-analyses demonstrated that garlic may decrease lipid profle and glucose parameters such as fasting blood glucose concentrations and hemoglobin A1c in diabetic patients. • Garlic supplement at 1 g per day was effective in symptom relief in overweight or obese women with knee osteoarthritis after 12 weeks of administration. • The consumption of a garlic tablet at a dose of 500 mg twice daily for 12 weeks has shown anti-infammatory and analgesic effects in obese or overweight women with osteoarthritis of the knee. • Garlic supplementation twice daily for eight months modulated cytokine production and reduced osteoporosis in postmenopausal women.

Source: Ansary et al., 2020; Rouf et al., 2020.

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have mainly classifed into hemiterpenoids (C5), monoterpenoids (C10), sesquiterpenoids (C15), diterpenoids (C20), triterpenoids (C30), tetraterpenoids (C40), and polyterpenoids (C > 40) (Figure 19.10). The so-called isoprene rule states that all terpenoids are derived from the ordered, head-to-tail joining of isoprene units. A head-to-tail fusion is the most common; however, non-head-to-tail condensation of isoprene units also occurs. Head-to-head fusions are common among triterpenoids and carotenoids, while some compounds are formed by head-to-middle fusions (e.g., irregular monoterpenoids) (Ludwiczuk et al., 2017). In addition to being in the form of terpene hydrocarbons, terpenoids mainly exist in the form of various oxygen-containing derivatives, including alcohols, aldehydes, carboxylic acids, ketones, esters, and glycosides. Terpenoids include compounds that are involved in both the primary and secondary metabolism of plants. Gibberellin, cytokinins, abscisic acid, and brassinosteroids are hormones implicated in plant growth and development, while carotenoids and compounds containing an isoprenyl side chain, such as chlorophyll, phylloquinone, plastoquinone, and tocopherols, are involved in photosynthesis. However, most terpenoids are secondary metabolites that play a key role as pollinating attractants and in plant defense against herbivores and microbial pathogens (Borrelli & Trono, 2016). Hemiterpenoids with a single isoprene unit are the simplest of the terpenoids (C5). The most common hemiterpene, isoprene, is secreted from the leaves of many trees and herbs. Other known hemiterpenoids found in plants are, for example, angelic and isovaleric acids and isoamyl alcohol. Terpenoids consisting of ten carbon atoms (two isoprene units) are monoterpenoids, which can be divided into three subgroups: acyclic, monocyclic, and bicyclic. Common examples of aliphatic monoterpenoids are myrcene, citral, and linalool. The monocyclic types include limonene, thymol, and menthol. The representatives of the bicyclic monoterpenoids are thujone, pinene, and borneol. Another group of compounds that also belong to monoterpenoids are the iridoids, which usually, but not invariably, occur as glycosides. A characteristic feature of these compounds is the skeleton in which the six-membered ring containing the oxygen atom is fused to a cyclopentane ring (iridane skeleton). They can be divided into four main groups: nonglycosidic, glycosides, secoiridoids, and bis-iridoids. The most frequently mentioned iridoids in the therapeutic context include gentiopicroside, sweeroside, loganin, and oleuropein. They can be found in some raw fruits such as olives, cornelian cherry, honeysuckle berries, and cranberries. Sesquiterpenoids are another major class of terpenoids. They are composed of three isoprene units (C15) and exist in many different forms including linear, monocyclic, bicyclic, and tricyclic. Additionally, the sesquiterpenes include lactones which chemically differ from other sesquiterpenoids by the presence of the γ-lactone system. The most characteristic examples of compounds belonging to each of the mentioned groups are presented in Figure 19.10. Another class of terpenoids is diterpenoids. It is a chemically heterogeneous group of compounds with a C20 carbon skeleton based on four isoprene units. Depending on their core skeleton, they can be divided into acyclic (phytane), monocyclic (retinol), bicyclic (labdane, halimane, clerodane), tricyclic (abietane, pimarane, cassane,

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Classifcation of terpenoids with examples of representatives of particular classes and their occurrence.

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FIGURE 19.10

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rosane, podocarpane, chinane, vouacapane), tetracyclic (kaurane, trachylobane, aphidicolane, stemodane, stemarane, beyerane, atisane, scopadulane, gibberellane), macrocyclic (cembrane, taxane, daphnane, tigliane, ingenane, jatrophane), and other miscellaneous forms. The main source of diterpenoids in the diet can be beverages such as coffee or tea; however, additionally, they can be provided in products based on ginkgo, sage, or stevia. Sesterterpenoids consist of a 25-carbon backbone (fve isoprene units) and they are a relatively rare branch of the terpenoid family, which widely exists in plants, microorganisms, marine organisms, and some insects. Triterpenoids are another class of terpenoids composed of 30 carbon atoms, polymerized to form six isoprene units. Based on their chemical structures, triterpenoids can be grouped into linear, monocyclic, dicyclic, tricyclic, tetracyclic, and pentacyclic forms that are mainly derived from squalene. Sterols are also triterpenes which are based on the cyclopentane perhydrophenanthrene ring system. In the case of plant sterols, they are referred to as “phytosterols.” Examples of these are sitosterol, stigmasterol, and campesterol, which are widely distributed in higher plants. Triterpenoids occur naturally both in the free state and in combination with sugars forming glycosides and esters. In particular, triterpenoid saponins are glycosides consisting of a sugar moiety (glycon) and a triterpenoid component (aglycone). A signifcant amount of triterpenoid saponins are found in foods such as beans, soybeans, spinach, lentils, and oats. They can also be found in ginseng and yerba mate. Tetraterpenoids are composed of 40 carbon atoms comprising eight isoprene units. The most common tetraterpenoids in the plant world are carotenoids, which are natural fat-soluble pigments. Carotenoids can be categorized into two classes, xanthophylls (which contain oxygen) and carotenes (which are purely hydrocarbons and contain no oxygen). Carotenoids give the characteristic color (yellow, orange, or red) to pumpkins, carrots, corn, tomatoes, salmons, lobsters, and shrimps. Combined forms of carotenoids occur, especially in fowers and fruits of higher plants, and they are usually xanthophylls esterifed with fatty acid residues, e.g., palmitic, oleic, or linoleic acids. Carotenoid glycosides are usually very rare. In the case of higher plants, the best known is the water-soluble crocin – a gentiobiose derivative of the unusual apocarotenoid crocetin (C20). Crocin and crocetin are found in crocus fowers and are responsible for the yellow color of the saffron. The most characteristic representatives of carotenoids are shown in Figure 19.10; however, additional information on these dyes can also be found in Chapter 10. Herbs and spices are rich sources of essential oils, which include monoterpenes. However, before they reach the consumer, these products are often subjected to a treatment mainly consisting of drying. Air drying and/or elevated temperature are most often used for this purpose. Such conditions are responsible for signifcant changes in the content and composition of essential oils, mainly resulting in volatilization or degradation of monoterpenes. More conservative in relation to essential oils are freeze-drying or drying with the use of microwaves or infrared. Blanching and cooking further exacerbate the loss of monoterpenes in herbs and spices. Carotenoids are another group of terpenoids commonly found in the diet. Tomatoes are a rich source of these compounds, especially lycopene, which is important

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for health. About 20 different carotenoids have been identifed in tomatoes and, depending on the species, growing conditions, and degree of fruit maturity, lycopene typically accounted for 70–90% of the total carotenoids present, corresponding to 3–5 mg/100 g in tomatoes. The tomato is an unusual plant material because, in this case, the processing may have a positive effect on its health-promoting properties. In fresh products, 95% of lycopene is in the trans form, while when exposed to light and elevated temperature or as a result of chemical reactions, it may be isomerized to the cis form, the bioavailability of which in the human body is higher. Therefore, the body is able to absorb more lycopene from fried tomatoes, tomato sauce, soup, and ketchup than from fresh tomatoes. Cooking tomatoes increases the bioavailability of the lycopene present in them additionally by releasing it from the cell matrix to the lipid phase of food, in which it is dissolved. The bioavailability of lycopene consumed together with β-carotene is higher than the bioavailability of lycopene alone (Tan et al., 2010). Due to the diversity of chemical structures, terpenoids exhibit a wide spectrum of biological activities. There are reports in the literature on their antimicrobial, antifungal, antiparasitic, antiviral, antiallergic, antispasmodic, antidiabetic, anti-infammatory, and immune-stimulating properties (Wagner & Elmadfa, 2003; Ludwiczuk et al., 2017, Masyita et al., 2022). One of the most important groups of terpenes from the point of view of health protection is monoterpenes, mainly contained in essential oils present in the greatest amount in citrus fruits and spices. The results of epidemiological studies suggest that this group of compounds may be helpful in the prevention and therapy of neoplastic diseases. Among the monoterpenes found in the diet, limonene and peryllium alcohol are especially considered to be compounds with high chemopreventive potential. These substances can inhibit the growth of many cancers in animal models, including colon, pancreatic, liver, lung, skin, and breast cancer. The activity of these monoterpenes may be revealed in the initiation phase of carcinogenesis by preventing the interaction of carcinogens with DNA or during the promotion phase by inhibiting the growth of neoplastic cells and their migration. The chemopreventive and therapeutic properties of monoterpenes are also visible in the further stages of the carcinogenesis process and include the induction of apoptosis and disruption of the mechanisms regulating the function of neoplastic cells. Other monoterpenes, e.g., carveol, carvone, sobrelol, and uroterpenol, also show anticarcinogenic activity, especially in the case of breast cancer, and in the case of pancreatic cancer, such activity is shown by geraniol and fernesol. The therapeutic use of monoterpenes is also associated with their antimicrobial activity, which has been observed in relation to gram-positive and gram-negative bacteria and fungi. Gram-positive bacteria are more sensitive to monoterpenes than gram-negative bacteria. The mechanism of the antimicrobial action of monoterpenes is determined, inter alia, by their lipophilic nature. These compounds increase the permeability of the cell membrane, cause changes in the position of proteins, and induce disturbances in the respiratory chain. Such an effect was observed in the case of tea oil terpenes in relation to gram-positive and gram-negative strains isolated from human skin, mouth, and upper respiratory tract, and in the case of Staphylococcus

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aureus strains. Other terpenoid compounds, such as bakuchiol, α-pinene, linalool, champene, geraniol, 1,8-cineole, α-phellandrene, 3-carene, p-cymene, perillyl alcohol, bornyl acetate, and citral isomers, also have been reported to yield inhibitory effects on the growth of microorganisms (Masyita et al., 2022). Carotenoids are another group of terpenoids with high biological activity. According to a report by the American Cancer Research Institute and the World Cancer Research Foundation, food products containing this group of compounds are likely to have preventive effects against cancers of the larynx, throat, mouth, and lungs. The results of epidemiological studies indicate that the consumption of tomatoes and their preserves has a positive effect on the reduction of the incidence of certain neoplastic diseases, especially prostate cancer (Campbell et al., 2004). The results of many studies conducted both in in vitro and in vivo systems indicate different biological activities of carotenoids, including antioxidant activity, regulation of gene functions, infuence on intercellular communication, hormonal and immune modulation, and detoxifcation of carcinogens. These mechanisms, among others, underlie the preventive properties of carotenoids against cancer, cardiovascular diseases, osteoporosis, and other chronic diseases (Rao & Rao, 2007).

19.3 CONCLUSION The relationship between fruit and vegetable intake and reductions in risk for many major health problems is strongly supported in many research studies (Zurbau et al., 2020; Jiang et al., 2020; Głąbska et al., 2020; Wang et. al., 2021). Therefore, the increase in the consumption of food of plant origin is reported as one of the important factors in the prevention of non-communicable diseases. For this reason, the World Health Organization recommends eating at least 400 g (i.e., fve portions) of fruit and vegetables per day, excluding potatoes, sweet potatoes, cassava, and other starchy roots (WHO, 2003). These recommendations additionally emphasize the advantage of consuming plant-based food products in their natural form over dietary supplements obtained from the same raw materials. The strategy of increasing the consumption of vegetables and fruits should therefore include education and increasing consumer awareness as well as implementing the production of high-quality food products of plant origin. This creates new challenges for the food industry, which they should take into account in their activities: (i) identifcation of food plants with a high content of bioactive substances that have the potential to be grown on a larger scale and constitute a predictable source of raw material for the food industry, (ii) designing a range of various food products based on such selected edible plants, taking into account specifc varieties, and (iii) development of a production technology that allows preserving not only nutritional values but also biologically active ingredients so that the fnal product shows the health-promoting effects observed for the raw material. Therefore, obtaining vegetable and fruit products, which can be expected to have a health-promoting effect, requires departing from the schemes commonly used in production, where the quality of the fnal product was determined by sensory values, microbiological safety, and production cost. The main goal of modern fruit and

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vegetable processing should be, frst of all, the consumer’s health and the production of food with high health quality and desired sensory characteristics.

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of Agricultural and Food Chemistry, 51(10), 3029–3034. https://doi.org/10.1021/ jf021065j van Dam, R. M., Hu, F. B. & Willett, W. C. (2020). Coffee, caffeine, and health. The New England Journal of Medicine, 23(383), 369–378. https://doi.org/10.1056/nejmra1816604 Verkerk, R., Dekker, M. & Jongen, W. M. F. (2001). Post-harvest increase of indolyl glucosinolates in response to chopping and storage of Brassica vegetables. Journal of the Science of Food and Agriculture, 81(9), 953–958. https://doi.org/10.1002/jsfa.854 Verkerk, R., Knol, J. J. & Dekker, M. (2010). The effect of steaming on the glucosinolate content in broccoli. Acta Horticulturae, 867, 37–46. https://doi.org/10.17660/actahortic .2010.867.3 Wagner, K.-H. & Elmadfa, I. (2003). Biological relevance of terpenoids: Overview focusing on mono-, di- and tetraterpenes. Annals of Nutrition and Metabolism, 47(3–4), 95–106. https://www.jstor.org/stable/48507009 Wang, D. D., Li, Y., Bhupathiraju, S. N., Rosner, B. A., Sun, Q., Giovannucci, E. L., Rimm, E. B., Manson, J. E., Willett, W. C., Stampfer, M. J. & Hu, F. B. (2021). Fruit and vegetable intake and mortality: Results from 2 prospective cohort studies of US men and women and a meta-analysis of 26 cohort studies. Circulation, 143(17), 1642–1654. https://doi .org/10.1161/circulationaha.120.048996 World Health Organization. (2003). Diet, nutrition and the prevention of chronic diseases: Report of a Joint WHO/FAO Expert Consultation. WHO Technical Report Series, No. 916. Geneva. Zhang, Y. (2010). Allyl isothiocyanate as a cancer chemopreventive phytochemical. Molecular Nutrition and Food Research, 54(1), 127–135. https://doi.org/10.1002/mnfr.200900323 Zhang, Y. & Talalay, P. (1994). Anticarcinogenic activities of organic isothiocyanates: Chemistry and mechanisms. Cancer Research, 54, 1976–1981. Zurbau, A., Au‐Yeung, F., Mejia, S. B., Khan, T. A., Vuksan, V., Jovanovski, E., Leiter, L. A., Kendall, C. W. C., Jenkins, D. J. A. & Sievenpiper, J. L. (2020). Relation of different fruit and vegetable sources with incident cardiovascular outcomes: A systematic review and meta‐analysis of prospective cohort studies. Journal of the American Heart Association, 9(19), e017728. https://doi.org/10.1161/JAHA.120.017728

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Analytical Methods Used for Assessing the Quality of Food Products Widiastuti Setyaningsih

CONTENTS 20.1 Introduction .................................................................................................. 535 20.2 Analytical Methods for Food Quality Control ............................................. 536 20.2.1 Standardization................................................................................. 536 20.2.2 Sensorial, Physical, and Chemical Characterization in Foods......... 537 20.2.2.1 Sensory Characteristics...................................................... 537 20.2.2.2 Hidden Characteristics....................................................... 538 20.2.3 Authentication................................................................................... 539 20.2.4 Adulteration ......................................................................................540 20.2.5 Food Safety....................................................................................... 541 20.3 Selecting Appropriate Analytical Methods .................................................. 543 References.............................................................................................................. 547

20.1

INTRODUCTION

People require foods that meet their dietary needs for a productive and healthy life. The degree of food preference is defned by certain standards, viz., intrinsic and extrinsic quality factors (Figure 20.1). The former refers to product conformity, safety requirements, and nutritional considerations, while marketing defnes the extrinsic factors (Linnemann et al., 2006). Therefore, manufacturers establish a number of analytical methods to determine the quality profle, including sensory attributes of food products, thus achieving the aforementioned quality factors. For example, to maintain product conformity, operating variables affecting the quality of fnished goods must be monitored by real-time analytical tools during food processing to produce food that complies with standards. Producing food with good quality begins with the proper selection of raw materials on the basis of specifcations defned during the research and development by food manufacturers. The materials used should meet the authenticity provisions. Later, it is also necessary for the processors to carry out quality control along the production process and even for food regulators to do so in the markets. The absence of risk factors in foods also needs to be considered because any contamination could

DOI: 10.1201/9781003265955-20

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FIGURE 20.1

Widiastuti Setyaningsih

Intrinsic and extrinsic quality factors of food products.

disrupt the production process and spoil the food quality. Numerous analytical methods are available to assist food manufacturers in controlling product quality.

20.2 ANALYTICAL METHODS FOR FOOD QUALITY CONTROL In maintaining the high quality of food products, some specifcations defned by the industry must be fulflled throughout the raw material screening, production process, distribution, and market display. Provided the products are intended to be recognized internationally, the standard specifcations shall comply with the international organization for standardization (ISO) or other global regulations. These specifcations defne food standardization, grading, and authenticity, while as a control against adulteration and confrming safety, appropriate analytical methods are developed to check the compatibility of the products with the standard specifcations.

20.2.1 STANDARDIZATION Food industries set the upper and lower specifcations to establish the quality standard of raw, semi-fnished, and fnished products. Reliable analytical methods are applied to determine the quality markers (viz., target analytes) indicating the standard conformity. When the testing results of the markers are within the specifcations, the materials or products meet the defned standards. However, the methods must also be sensitive, and in some cases where the specifcations are very tight, the standardized products can be sorted into several grades. The grading provides options for consumers to select a higher quality of the standard products. Generally, quality parameters of food products are defned by sensory (appearance, texture, favor, and mouthfeel) and some hidden characteristics. The latter include nutritive value, safety (toxicity and harmless adulterants), and functional properties. Various analytical techniques have been applied to evaluate the sensory and hidden characteristics, from classical to instrumental methods such as spectroscopy and chromatography (Table 20.1).

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TABLE 20.1 Methods or Instruments to Assess the Quality Parameters of Selected Foods Quality parameters Physical and Volume sensory Density Texture Conductivity

Moisture content Dispersibility Morphology

Safety

Functional properties

Sample matrices Beverage, yogurt

Measuring the ratio of weight to volume Compressing or stretching the sample The voltage drop of two electrodes in a probe immersed in the sample Gravimetrically by oven Karl Fisher titration Spray drying Scanning electron microscopy (SEM) Microfuidic rheometers Electronic nose, GC-O

Fruit, oil Fruit, snack bar, pastry Beverage, fruit, water

Bread, fruit Honey, date Yogurt Yogurt, powder drink

Sour, spicy, salty, sweet, metallic, bitter, and umami Volatile compounds

Electronic tongue

Sugars Organic acids Phenolic compounds

HPAE-PAD HPLC-RI HPLC-PDA

Additives Amino acids Protein Lipids Mycotoxins Heavy metals PAH

HPLC-MS, HPLC-DAD HPLC-FD Kjeldahl Soxhlet HPLC-FD ICP-MS GC-MS

Milk, sauce Wine, edible fower, olive oil Water, wine, paneer cheese, snack bar, juice Chocolate, wine, rice, fsh oil, edible fower Coffee, spirulina Fruits, wine Olive oil, rice, edible fower, red fruit oil Powder drink Meat, tempeh, cereal Meat, milk Chocolate, milk Chili, peanut, spice Fish, water Smoked product, water

Antioxidant activities

DPPH, FRAP, ORAC, β-carotenelinoleic acid assay

Snack bar, fruit, olive oil, tempeh

Viscosity Aroma

Nutritive value

Methods/instruments Liquid displacement

GC-MS

20.2.2 SENSORIAL, PHYSICAL, AND CHEMICAL CHARACTERIZATION IN FOODS 20.2.2.1 Sensory Characteristics In food industries, the evaluation of sensory characteristics is performed by a group of panelists. Because this approach is laborious, subjective, and cannot be calibrated, objective measurements using analytical instruments are available as an

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alternative to increase precision, specifcally when the routine production volume is enormous. Electronic tongue and nose have been applied as an attempt to replace the panelists for sensory analysis in various foods and beverages such as red and white wines, mineral water, snack bars, and cheese. Both methods employed distinguished biosensors identical to human sensing systems to detect and quantify the marker chemical compounds. Gas sensors, including metal oxide semiconductors, operated at 150–400° C, are applied to determine the alcohols, organic acids, sulfates, alkanes, esters, aldehydes, and ketones. Less energy is required when utilizing conducting polymer sensors for similar analytes. Other advanced methods such as computer vision or image processing are also suitable alternatives to control the sensory quality of food. These methods are performed by acquiring, processing, and analyzing images from food samples using software to identify visual sensory attributes such as color and surface texture. This method is a non-contact optical sensing approach, the interpretation and decisions are based on software, so there is a slight possibility for biased results. 20.2.2.2 Hidden Characteristics Enhanced quality attributes in food that beneft human health are demanding for the current food market. Hidden characteristics of food, i.e., nutritive value, functional properties, and safety, are potential aspects to attract the consumer. A particular population requires a proper formulation of the nutritional composition. In contrast, products with specifc bioactive compounds are essential to those that need additional health effects. Besides, food safety is the primary aspect of avoiding health detriment that will be further discussed in a later section. Nutritive value in food is described by micro- and macronutrients. The determination of macronutrients such as protein, fat, and carbohydrate is also known as proximate analysis. The Kjeldahl and Soxhlet methods are the most common approach for protein and lipid analysis, respectively, while the total carbohydrates are approximately calculated by subtracting protein, fat, ash, and moisture content from the total sample weight. These methods merely evaluate the total content of the food components. Determination of the fractions of each macronutrient, along with micronutrients, requires advanced analytical techniques, which generally apply sample preparation (hydrolysis, fractionation, extraction, and purifcation). For example, chromatography methods are frequently used to measure fructooligosaccharides (high-pressure liquid chromatography-refractive index, HPLC-RI), fat-soluble vitamins (supercritical fuid chromatography-mass spectroscopy, SFC-MS), amino acids (high-pressure liquid chromatography-fuorescence detection, HPLC-FD), phenolic acids (high-pressure liquid chromatography-diode array detection, HPLC-DAD), and fatty acids (gas chromatography-fame ionization detection, GC-FID). Although micronutrients are considered trace compounds in foods, many of these signifcantly contribute to functional properties. The methods for evaluating the properties are selected on the basis of mechanisms of action of the active compounds in the food system. For instance, the activity of the antioxidant compounds as radical scavengers can be defned by measuring the reaction with an organic

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(2,2-diphenyl-1-picrylhydrazyl-DPPH assay), peroxyl (oxygen radical absorbance capacity-ORAC assay), or luminol-derived radical (total radical-trapping antioxidant parameter-TRAP assay).

20.2.3 AUTHENTICATION The authenticity of foods is commonly defned based on geographical origin, variety, or genetics. The differences in these aspects provide deviations in food quality. The involvement of analytical methods in identifying the authenticity of foods may rely on specifc key-marker compounds or based on non-target compounds as fngerprints. For example, in the case of proving the authenticity of wines from various regions, the products can be classifed based on specifc minerals composition. The mineral content of wine from China is different from Spanish wine. Moreover, within the same country, wines from Dobrogea and Moldova, which are two regions of Romania, can be distinguished based on the presence of Mn, Sr, Ag, Co, and Cr as markers (Popîrdă et al., 2021). For the same purpose, minerals analysis is useful for authentication tests on coffee drinks (Arabica varieties) from Africa, South America, Central America, Asia, and Oceania. Identifying minerals content is suitable for establishing chemical descriptors to distinguish the product origin between the countries or continents as differences in climate, soil type, fertilizer composition, and environmental conditions vary the minerals levels in the beans. Cuban (Central America) coffee showed the lowest content of Mg, Na, and P compared to that from other geographical origins. Coffee from South America has the highest mineral concentration except for Ca, while that from Central America has the lowest concentration, except for Mn. Atomic absorption spectroscopy (AAS) is a standard method for mineral analysis. However, due to its limitations, researchers used high-resolution continuum source atomic absorption spectrometry (HR-CS-AAS) as a form of revolutionary innovation (Oliveira et al., 2015). Inductively coupled plasma-optical emission spectrometry (ICP-OES) is frequently applied to determine macro elements such as Al, B, Ca, Fe, K, Mg, Na, P, S, and Zn. The ICP coupled plasma mass spectroscopy (MS) is used for trace elements quantifcation, including As, Ba, Be, Bi, Cd, Co, Cr, Cs, Cu, Ga, Li, Mn, Ni, Pb, Rb, Se, Sr, U, and V. Combining both methods, the authentication of Korean pork covering the cities of Suncheon, Naju, Chungju, Gangjin, and Yongin can be distinguished from the USA, Germany, Austria, Netherlands, and Belgium as exporting countries. The validation process with multi-element analysis improves the accuracy in determining geographic origin because their level and composition are associated with several factors such as type of feed and drinking water apart from the pigsty environment (Kim et al., 2017). In addition to minerals, profling the bioactive compounds is an alternate approach to predicting the quality of plant-derived products. For example, authentication of macroalgae harvested from different islands in Indonesia relies on the database of phenolic compounds identifed and quantifed using ultrahigh performance liquid chromatography-photo diode array (UPLC-PDA). Moreover, a faster method

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for macroalgae authentication can be achieved by analyzing the fngerprints of the spectroscopic data acquired from visible spectrum (VIS) and near-infrared spectroscopy (NIRS) using chemometrics. Additionally, by establishing a calibration set that defnes the relationship between spectroscopic results with chromatographic data, the method allows the quantifcation of the target analytes (Mutiarahma et al., 2021). Food authentication testing is not only aimed at validating the truth of the product as claimed on the labels or at fnding out the characteristics in different regions. For Muslims, the analysis of halal authentication is of more signifcant concern to avoid market manipulation of beef meat with pork. The difference between the two types of meat can be distinguished using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Additionally, the multiplex PCR approach can distinguish the meat origin of the pig, camel, sheep, donkey, goat, cow, and chicken in one single reaction. These PCR methods provide excellent sensitivity and specifcity, but the process is more complex than commonly used food analysis techniques such as chromatography.

20.2.4

ADULTERATION

Adulteration assessment for raw materials and fnished products is a major concern for industries and consumers to avoid food fraud. The most frequent practice of food fraud is manipulating raw materials with cheaper and lower-quality substitutes. Some of the purposes of adulteration are to increase volume, enrich texture, intensify color, and increase bulk to receive greater profts. Adulteration of dairy commodities occurred by mixing cow’s milk with water, coconut milk, synthetic chemicals, or four solution. Meanwhile, fsh oil can be adulterated using cheaper vegetable oils such as palm or coconut oil. A similar approach is applied to other commodities, including powdered coffee, chocolate, and processed foods like wine, meatballs, and cheese. Differences between labels and actual compositions of products due to food adulteration could impact a severe public health concern. The increasing occurrence of food fraud notably threatens human health, especially for routine consumption products such as milk for babies, oil, honey, sugar, juice, and other essential ingredients. The impacts that arise after consuming adulterated foods are very diverse, ranging from simple (diarrhea, nausea, vomiting, and small bumps) to severe symptoms (asthma, liver disorders, loss of vision, and cardiac arrest). The detection of food adulteration is challenging, especially when the adulterants have similar characteristics to those of the food components. Melamine powder that once adulterated baby milk powder to boost the protein content can not be detected using the Kjeldahl method since both materials contain nitrogen. Hence, advanced analytical methods must be applied to determine melamine in baby milk powder by employing chromatography (HPLC-DAD, HPLC-MS, and GC-MS), spectrometry (Mid-Fourier-transform infrared [Mid-FTIR], NIR hyperspectral imaging) or immunoselective assays (enzyme-linked immunosorbent assay [ELISA]). Figure 20.2 presents currently available analytical methods covering conventional and advanced techniques, destructive and direct detections, and complex or practical preparations

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FIGURE 20.2 Analytical methods for the detection of food fraud. Notes: electronic-nose (E-nose), electronic-tongue (E-tongue), electrical impedance spectroscopy (EIS), infrared (IR), laser-induced breakdown spectroscopy (LIBS).

used to assist food surveillance, from those suitable for laboratory scale to the industrial automated system. Advanced technologies such as HPLC and GC are widely used to determine the key marker compounds to detect adulterants. These technologies provide individual detection with remarkable selectivity and sensitivity. As one example, the HPLCDAD method effciently detects honey adulteration by rice syrup based on the presence of 2-acetyl furan-3-glucopyranoside. However, the methods require sample preparation and are considered a time-consuming measurement. Hence, besides optical and electrical techniques, non-destructive spectroscopy methods (FTIR, near-infrared, and Raman imaging) appear as alternatives. Along with technology that is constantly developing, there are smart devices that work effectively and effciently, namely artifcial intelligence (AI) and the Internet of Things (IoT). Detecting adulterated food with an AI system requires data that includes vision-based models (size, shape, color, and texture) and other parameters such as viscosity, pH, temperature, humidity, and chemical composition (Goyal et al., 2022). Apart from detecting counterfeiting on the basis of the acquired data, the system provides product classifcation to assist quality grading development.

20.2.5 FOOD SAFETY Besides the taste and nutritional value that people expect from consuming food, the quality of food is also priorly determined by its safety. However, a typical food that is safe for one person is not necessarily safe for another. Regular food consumption becomes unsafe for people who have allergies, unusual metabolism, or certain

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diseases. Some contaminants in foods that are unsafe for consumption can be divided into three groups: (i) physical contaminants (hairs, glasses, plastics, metals, stones, and wood), (ii) biological and microbiological contaminants (pathogenic bacteria, viruses, fungi, parasites, and genetically modifed organisms), and (iii) chemical contaminants (pesticides, toxins, heavy metals, and allergens). In addition to being harmful to human health, contaminants reduce the availability of raw materials and interfere with production, resulting in low product quantity and quality. Thus, a food safety objective (FSO) is established as a strategy for controlling product safety by manufacturers and distributors. Controlling food safety can be conducted by various methods based on the contaminant materials. For physical contaminants, the general approach used is a visual inspection by humans or detection by instruments – for example, magnets to detect the metals, while x-ray technology to detect bones and glasses. Detecting and removing physical contaminants such as stones, wood, and plastics can be done using screens, sieves, or flters. Basically, these methods do not require any pretreatment and are able to detect or even eliminate physical contaminants in food immediately. Advanced technology such as focal plane array (FPA)-based micro-FTIR spectroscopy and micro-Raman spectroscopy can detect micro- and nanoplastics in mussels (Vinay Kumar et al., 2021). Surface-enhanced Raman spectroscopy (SERS), which uses silver colloid as the active substrate, can also identify nanoplastics (100 nm) in pure and seawater (Lv et al., 2020). However, these spectroscopic methods require pretreatment, such as purifcation. There are also numerous methods for detecting biological and microbiological contaminants. Primarily, the detection is based on culture-dependent and independent methods. The formerly mentioned method measures the growth and death rate of organisms (bacteria) by inoculating and incubating them. This conventional method is time-consuming for only detecting one type of organism. In contrast, the culture-independent methods with molecular-based systems provide faster results while detecting more than one type of organism. These advanced methods utilize PCR, recombinase polymerase amplifcation (RPA), ELISA, biosensors, and other instruments such as spectrometry, fow cytometry, and chromatography. PCR is a gold standard for biological and microbiological detection. PCR can amplify the specifc microbial DNA sequence to detect microorganisms in foods, such as Toxoplasma gondii and Cyclosporacayetanensis in strawberry fruit; Escherichia coli, Salmonella enteritidis, and Listeria monocytogenes in romaine lettuce juice extract; and Coxiella burnetii in Minas artisanal cheese. Without complex instrumentation, the RPA method can rapidly detect Salmonella strains in tomatoes, cabbage, broccoli, chicken, and eggs. Meanwhile, ELISA measures antibodies, antigens, proteins, and glycoproteins of biological and microbiological contaminants. The ELISA method is widely used to detect E. coli and Salmonella in animal-derived products. Portable near-infrared spectroscopy (NIRS) has been reported to predict microbial parameters in pork during storage (Prado et al., 2011). The advantages of this analytical method are simple, portable, non-destructive, fast, and robust. Therefore, NIRS is suggested for on-site monitoring and quality control of various food products.

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In evaluating the presence of chemical contaminants in food, many methods have been developed, especially to detect food allergens, toxins, and heavy metals. The widely used conventional methods such as chromatography and spectrometry are still recommended as reference methods. In contrast, modern methods such as loop-mediated isothermal amplifcation (LAMP), microfuidic technology, and sensor-based methods can be practical alternatives for more rapid analysis. LAMP can detect mycotoxins such as afatoxins and fumonisin in rice grains and maize. Several pesticides, namely thiabendazole, thiram, endosulfan, and malathion, can be detected in strawberries using a flter-based microfuidic SERS sensor. Other detections are developed with nucleic acid-based, enzyme-based, and polymerbased sensors relying on specifc interactions between the chemical contaminants in food and the receptor. Additionally, some sensors based on acoustic waves assist in detecting pathogens and spoilage in food. Other chemical sensors that are also frequently used include potentiometric and bioelectric sensors. A new tool to identify genetically modifed organisms (GMOs) is available, namely GmoDetector. Its database contains 64 transgenic elements and 76 GMOspecifc events from 251 singular GM events of food so that it can detect and identify various GMOs in food. GmoDetector can accurately analyze several samples in one sample set, thus considered an effcient analytical tool (Chen et al., 2021).

20.3 SELECTING APPROPRIATE ANALYTICAL METHODS Maintaining the sensory and biological properties of foods requires a continuous system to guarantee the quality of products. Deterioration of food products occurs not only after the food is ready for consumption but also due to inappropriate selection of raw materials, uncontrolled production process, or improper storage and product handling. A number of analytical methods are available for selection to assist quality control in food production; each method has advantages and disadvantages. It is necessary to choose the most appropriate method that fts the purpose of the analysis. Conventional methods such as proximate analysis are convenient despite being laborious in sample preparation. They are appropriate when used for sorting and grading. However, as less selective, the methods are lacking effcient to utilize for quality inspection or testing the authenticity of the food. An instrument-based technique such as chromatography is less portable and requires skilled technicians. These methods generally have excellent selectivity and sensitivity for multi-analytes in a single run, especially when the methods are integrated with chemometrics. These instrumental methods are still suitable for testing in most situations, but not with onsite inspection. Table 20.2 compiles selected instrument-based techniques to address some quality issues related to food control. In the current era, many researchers use computer systems and artifcial intelligence in developing analytical methods such as sensors and image processing technology. These approaches acquire data rapidly and accurately. The employed tools are portable and thus suitable for on-site quality control inspection. Although rapid analysis is promising, the newly developed methods need further research to enrich the system database for compound identifcation and quantifcation.

544

TABLE 20.2 Instrument-Based Techniques Applied in Food Quality Control Metabolomics approach No

Sample

Issue addressed

Targeted compounds

Non targeted compounds

Analytical technique

Chemometrics

References

Fish

Assessment of quality changes in tilapia fllets due to thermal processing (boiling, steaming, and air frying)

Screening of 249 metabolites

UHPLC-QTOF-MS/MS

PCA and OPLS-DA

The untargeted metabolomics differentiate thermal processing of tilapia fllets. The processes affect mainly fats, amino acids, and nucleotides as essential quality indicators

(Li et al., 2021)

2

Chicken meat

Measuring the quality of chilled chicken meat during storage

Screening of 63 metabolites

UHPLC-ESIQ-TOF-MS/ MS

PLS-DA

(Wen et al., 2020)

3

Rice

Authentication of aromatic and regular rice grains

Screening of 51 odor-active compounds

HS-SPME GC×GCTOF/MS

PCA

Amino acids, nucleosides, nucleotides, organic acids, and sugars are closely related to changes in meat quality Eight volatile compounds are selected as key markers to distinguish aromatic and nonaromatic rice varieties

(Setyaningsih et al., 2019)

(Continued)

Widiastuti Setyaningsih

Results

1

Metabolomics approach No

Sample

Issue addressed

Targeted compounds

Non targeted compounds

Analytical technique

Chemometrics

4

Honey

Wild honey adulteration

ATR-FTIR spectro-scopic data

ATR-FTIR

PCA-DA and PLS

7

Patin fsh oil

Fish oil adulteration with palm oil

Ion mobility sum spectrum (IMSS)

HS-GC-IMS

LDA and PLS

9

Wheat, corn, dried fg, dried coffee beans

Afatoxins detection (food safety)

ELISA and HPLC-FD

N/A

Afatoxin B1, B2, G1, and G2

Results Measurement of spectra combined with the chemometric enables the detection of wild honey adulterated by sugars A fngerprint based on IMSS allows detection of the presence of palm oil in patin fsh oil within a few minutes ELISA results strongly correlate with HPLC-FD data and both methods are suitable for afatoxins determination in studied samples

References (Riswahyuli et al., 2020)

Methods for Food Quality Assessment

TABLE 20.2 (CONTINUED) Instrument-Based Techniques Applied in Food Quality Control

(Putri et al., 2020)

(Omar et al., 2020)

545

(Continued)

546

TABLE 20.2 (CONTINUED) Instrument-Based Techniques Applied in Food Quality Control Metabolomics approach No 10

Sample

Issue addressed

Fish and water

Prediction of Hg2+ (food safety)

Targeted compounds Mercury (Hg2+)

Non targeted compounds

Analytical technique SERS

Chemometrics GA-PLS, ACO-PLS, SPA-PLS

Results SERS coupled with multivariate calibration effectively quantifes the contamination of Hg2+ in fsh and water samples

References (Hassan et al., 2021)

Widiastuti Setyaningsih

Notes List of abbreviations: - Ultra-high performance liquid chromatography-quadrupole time-of-fight mass spectrometry (UHPLC-QT-TOF-MS) - Ultra-high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-fight mass spectrometry (UHPLC-ESI-Q-TOF-MS) - Partial least squares discriminant analysis (PLS-DA) - Headspace solid-phase microextraction (HS-SPME) coupled with two-dimensional gas chromatography coupled with time-of-fight mass spectrometry (GC × GC-TOF/MS) - Attenuated total refectance-Fourier transform infrared (ATR-FTIR) - Principal component analysis-discriminant analysis (PCA-DA) - Headspace-gas chromatography-ion mobility spectroscopy (HS-GC-IMS) - Linear discriminant analysis (LDA) - Enzyme-linked immunosorbent assay (ELISA) - Surface-enhanced Raman scattering (SERS) - Genetic algorithm-partial least square (GA-PLS) - Ant colony optimization-partial least squares (ACO-PLS) - Successive projections algorithm-partial least squares (SPA-PLS)

Methods for Food Quality Assessment

547

The analytical techniques in the selection must be compatible with the sample matrices and target analytes. Some criteria must be considered, including method validation (accuracy, precision, sensitivity, selectivity, robustness, and ruggedness) and operating conditions (scale of operation, analysis time, availability of equipment, and cost) in screening the most appropriate method.

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