Maize: Cultivation, Uses and Health Benefits : Cultivation, Uses and Health Benefits [1 ed.] 9781620815182, 9781620815144

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Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved. Maize: Cultivation, Uses and Health Benefits : Cultivation, Uses and Health Benefits, Nova Science Publishers, Incorporated, 2012. ProQuest Ebook

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved. Maize: Cultivation, Uses and Health Benefits : Cultivation, Uses and Health Benefits, Nova Science Publishers, Incorporated, 2012. ProQuest Ebook

AGRICULTURE ISSUES AND POLICIES

MAIZE

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CULTIVATION, USES AND HEALTH BENEFITS

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Maize: Cultivation, Uses and Health Benefits : Cultivation, Uses and Health Benefits, Nova Science Publishers, Incorporated, 2012. ProQuest Ebook

AGRICULTURE ISSUES AND POLICIES

MAIZE CULTIVATION, USES AND HEALTH BENEFITS

JOSE C. JIMENEZ-LOPEZ Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

EDITOR

Nova Science Publishers, Inc. New York

Maize: Cultivation, Uses and Health Benefits : Cultivation, Uses and Health Benefits, Nova Science Publishers, Incorporated, 2012. ProQuest Ebook

Copyright © 2012 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works.

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Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Additional color graphics may be available in the e-book version of this book.

Library of Congress Cataloging-in-Publication Data Maize : cultivation, uses and health benefits / editor: Jose C. Jiminez-Lspez. p. cm. Includes index. ISBN:  (eBook) 1. Corn. 2. Corn--Utilization. 3. Corn--Therapeutic use. I. Jiminez-Lspez, Jose C. SB191.M2M255 2012 633.1'5--dc23 2012010684 Published by Nova Science Publishers, Inc. † New York

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CONTENTS Preface Chapter 1

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Chapter 2

vii Opportunities and Constraints in Modifying the Composition of Maize Grain George G. Harrigan and Tyamagondlu V. Venkatesh Plus-Hybrid System in Maize (Zea mays L.) Production: A New Approach Combining the Effect of Cytoplasmic Male Sterility and Xenia for Grain Yield Increase and Nutritional Improvement Jelena Vancetovic, Sofija Bozinovic, Dragana Ignjatovic-Micic, and Ksenija Markovic

Chapter 3

New Perspectives in Maize Breeding Víctor M. Rodríguez, Pedro Revilla, and Bernardo Ordás

Chapter 4

Effect of Genetically Modified Maize on Rhizobacterial Communities Jorge Barriuso and Rafael P. Mellado

Chapter 5

Chapter 6

Chapter 7

Ferulated Arabinoxylans as by-Product from Maize Wet-Milling Process: Characterization and Gelling Capability Ana L. Martínez-López, Elizabeth Carvajal-Millan, Jaime Lizardi-Mendoza, Yolanda López-Franco, Agustín Rascón-Chu, Erika Salas-Muñoz, and Benjamín Ramírez-Wong Extrusion of Quality Protein Maize (Zea mays L.) in Combination with Hard - To-Cook Bean (Phaseolus vulgaris L.) Jorge Ruiz-Ruiz, David Betancur-Ancona, Rolando González, and Luis Chel-Guerrero Corn Tortillas: Physicochemical, Structural and Functional Changes María del Carmen Robles-Ramírez, Areli Flores-Morales, and Rosalva Mora-Escobedo

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Contents

Chapter 8

Nutraceutical Properties of Blue Maize Silverio García-Lara, Janet A. Gutiérrez-Uribe, and Sergio O. Serna-Saldivar

Chapter 9

Molecular Features of Maize Allergens and their Implications in Human Health Jose C. Jimenez-Lopez, Simeon O. Kotchoni, Emma W. Gachomo, Antonio J. Castro-López, María I. Rodríguez-García, and Juan D. Alché

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Index

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PREFACE Maize (Zea mays L.) commonly called corn, field corn, or Indian corn belongs to the Poaceae (Gramineae) family. It grows almost anywhere, although approximately 50% of the world's maize is produced in the USA. It is one of the 3 major grain crops worldwide and extremely important for human and animal consumption (15 to 56% of total daily dietary caloric intake), food processing and other commercial activities in many developed and developing countries. This book aims to provide an up-to-date knowledge of recent developments in maize cultivation; the improvement of maize nutritional values to be used in food preparation and human consumption; and maize health benefits provided by the use of alternative maize varieties. The authors expect this book will provide a compendium of valuable knowledge for anyone interested in agricultural, environmental and food sciences. Chapter 1 - The composition of modern day maize reflects its domestication origins from its wild ancestor, teosinte, and the impact of artificial selection including the development of hybridization technology. Presented in chapter 1 is a compositional comparison of teosinte and maize and review arguments that domestication and breeding technology may have conferred constraints on maize compositional diversity, particularly with respect to starch, protein, and oil. The impact of modern agricultural biotechnology on compositional diversity is reviewed under two separate considerations. First, a review of the scientific literature allows a reasonable conclusion that for agronomic traits, the effect of transgene insertion on composition is generally significantly less than the impact of environmental or germplasm variation on conventional crops. Given constraints that appear to be imposed by domesticcation and artificial selection, as well as the quantitative nature of compositional traits, this result may not be surprising. Second, nutritional enhancement (or biofortification) of maize through modern agricultural biotechnology may have some advantages over conventional breeding for some components by providing a means to more directly target key regulatory steps in the biosynthesis of these components. Chapter 2 - Cytoplasmic male sterility (cms) in maize is used to increase the quality of hybrid seed production and reduce its costs. Sterile hybrids often outyield their fertile counterparts, especially if pollinated by a genetically unrelated pollinator. This fact can be used in modern production to increase grain yield of maize hybrids, and to improve agronomic traits. The combined effect of cytoplasmic male sterility and xenia (the influence of pollinators in the year of pollination) is called the Plus-hybrid effect. Accordingly, Plushybrid system refers to commercial production of two hybrids in the mixture, one of which is male sterile and high yielding, and other is unrelated fertile hybrid pollinator. Potential Plushybrid mixture consists of 75-80% of sterile hybrid and 20-25% of fertile hybrid. In modern

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Jose C. Jimenez-Lopez

agriculture continuously is searched for increased both grain yield and quality of cultivated hybrids. Plus-hybrid system represents one of these attempts. Investigations on Plus-hybrid effect in the world began in the late 20th century through a series of micro-trials and larger strip-trials in several locations and countries. The best Plushybrid combinations had significantly higher grain yield, without compromising quality, than the individual hybrids. The greater the genetic distance between the hybrids combined in the mixture, the expected Plus-hybrid effect is higher (due to the increased effect of xenia). Research conducted in Serbia was done at one location in three years and included two sterile hybrids as mothers and five fertile hybrids as fathers. It turned out that the Plus-hybrid effect had different influence on the investigated traits of the two hybrids, and this effect highly depends on the genotype of the mother. The increase in yield was accompanied with the increase in oil content in one of the sterile hybrids. In addition, health condition of the grain was not affected. It turned out that characteristics of fathers may influence the characteristics of pollinated mothers due to the xenia effects, so the choice of superior fathers increases the profits of the Plus-hybrid effect, also it seems that xenia effects with the same pollinator differ in sterile and fertile version of the same hybrid, and this phenomenon should be further investigated (it could be a kind of interaction between the sterile cytoplasm and xenia, that is different from the Plus-hybrid effect). What is significant is that the Plus-hybrid system can be successfully used to prevent pollination of genetically modified plants (GMO), growing the genetically modified cms maize hybrids mixed with unmodified fertile pollinators. The objective of this study was to determinate both, individual and combined (Plushybrid) effects, of cms and xenia on grain yield, chemical composition and grain health condition of examined hybrids. Although studies related to the Plus-hybrid system are very new, it is believed that this unconventional approach of maize growing could enter the commercial use, which could be the most important significance of this research. The seed production of Plus-hybrid mixture is not more expensive than production of conventional hybrids, and this fact should increase its commercial use. Therefore, results achieved so far are presented and introduce a Plushybrid system of maize production. Chapter 3 - Traditionally maize breeding has been focused on the increase of yield, without taking into account the amount of inputs, such as Nitrogen, herbicides or pesticides provided to the crop. Nowadays, consumers are highly concerned with the detrimental effects of intensive agriculture on environment and food safety. Firstly, the concern for the environment has introduced or has increased the interest for plant breeding under conditions of low inputs. In addition, global climate change has become the priority for plant breeding in many areas of the world. Concerning food safety, eating habits have dramatically changed in the last years increasing the demand for wealthier foods cultivated in a more environmentalfriendly conditions. The nutritional value of maize has been untapped and, with the exception of protein quality, few breeding programs have been developed to increase the nutritional quality of maize and its derivates. The production of new germplasm rich on functional compounds such as flavonoids or isopropanoid-derivates with potential antioxidant properties would open a new field for maize breeders. Sustainable production of maize involves also some new social demands, such as removable sources of energy. Indeed, the world’s oil reserves are limited while the demands for energy in industrial countries are increasing. As a consequence, the search for alternative sources of renewable energies has been intensified over the last few years. Maize, given its high productivity levels compared with other crops,

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Preface

ix

is an excellent candidate for bioethanol production. The use of grain Maize for energy production has some undesirable effects, however this inconvenient could be overtaken using the non profitable cellulosic residue which remains in the field after maize grain is harvested. In addition to the improvement of grain yield, the improvement of the celullosic biomass yield, suitable for biofuel production, probably will became a new objective in several breeding programs. Therefore, this review focuses in the new challenges maize breeders have to face in the next decades to obtain a wealthier and more environmental-friendly maize production suitable for new uses. Chapter 4 - Maize is one of the most important crops worldwide for human and animal consumption. Some genetically modified varieties of maize have been developed and are widely cultured in several countries. Among these transgenic lines, the first to be cultivated and from which more risk environmental studies have been performed was the so-called maize Bt, resistant to the corn borer. Maize tolerant to the herbicide glyphosate constitutes a new generation of transgenic maize that is able to grow in the presence of a wide spectrum herbicide to which it is not naturally resistant, thus allowing effective weed management of the fields seeded with this kind of maize. Soil bacteria are known to affect plant growth and viability and thus play an important role in terrestrial agricultural ecosystems, in particular, the bacterial communities associated to the plant roots as they form part of the rhizosphere. Genetically modified plants can alter these soil bacterial communities; particularly by the use of herbicides, and these alterations can endanger soil fertility and cultivar sustainability. Many techniques have been used to analyse soil bacterial communities, including classic approaches based on the cultivation of viable bacteria, metabolic profiling studies and nucleic acid-based methods. Recently, among these, massive parallel pyrosequencing of the 16S rDNA has aroused great interest, as it has been shown to be very useful in the study of structural changes to the diversity of bacterial communities associated to soil. The objective of this chapter is to review the studies conducted to determine the composition of rhizobacterial communities of genetically modified maize, and how these genetic modifications or associated agricultural practices may alter the structure and functionality of maize rhizobacterial communities. This may be of relevance as regards the potential impact that the alterations of maize rhizobacterial communities may have on effective growth and viability of GM-maize over long cultivation periods. Chapter 5 - Maize bran is a by-product of the commercial maize wet-milling process in Mexico. This process involves nixtamalization, which is an alkali cooking widely used to improve the maize nutritional value. Maize nixtamalization is important in Mexico as half of the total volume of consumed food is maize. This process degrades and solubilizes maize cell wall components allowing bran removal. Due the high volume of maize bran generated in Mexico, it is becoming into a potential source of added-value biomolecules such as ferulated arabinoxylans. This chapter has been focused on the extraction, characterization and gelling capability evaluation of maize bran ferulated arabinoxylans recovered from maize nixtamalization (N-FAX). Yield of ferulated arabinoxylans extracted from maize bran was 18% (w/w) on a dry matter basis (w N-FAX/w maize bran). N-FAX presented a pure arabinoxylan content of 85 % (w/w), a ferulic acid content of 0.25 µg/mg arabinoxylans, an A/X ratio of 0.75, an intrinsic viscosity [] of 268 mL/g and a viscosimetric molecular weight (Mv) of 197 kDa. Gelling capability of ferulated arabinoxylans at different concentrations was investigated. Gels were obtained from this polysaccharide by laccase covalent cross-

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linking of ferulic acid. Large deformation mechanical tests (compression mode) revealed an increase in strength from 45 to 60 N as N-FAX concentration changed from 2 to 4 % (w/v). Lightness (L), redness (a), and yellowness (b) values were registered for N-FAX gels at both concentrations. Significant differences were found for b values, which increased as the polysaccharide concentration augmented from 2 to 4% (w/v) in the gel. Extraction of N-FAX from maize wet-milling process could represent an alternative source of this polysaccharide which is receiving increasing attention for food and non-food applications. Chapter 6 - Maize has high carbohydrate content, but very low protein content and low levels of lysine and tryptophan. These deficiencies have been addressed through development of maize hybrids known as quality protein maize (QPM) in which lysine and tryptophan levels are twice that of normal maize. During storage under high humidity (> 75%) and temperature (> 30 ºC) beans develop the hardening process, which changes its characteristics in a negative way, mainly by increasing cooking time, these features greatly reduce their commercial value. Mixing of QPM and hard-to-cook beans (HTC) can be used to balance the amino acid profile of the resulting product without notably affecting sensory acceptance. Processing by extrusion is of particular interest because it is widely used to incorporate legume seeds into cereals for production of precooked flours, infant food and expanded snacks. These extruded products offer advantages in terms of their sensory characteristics and nutritional properties. Extruded maize products on the market usually provide a 6% of protein, the combination of QPM with HTC yields a product with a higher protein intake (15%), with a better balance of amino acids and appropriate sensory characteristics. Thus extruded products obtained have the potential to be commercially produced in addition to having a better nutritional intake than snack type products on the market. Chapter 7 - Maize (Zea mays L) is the third most important food crop in the word and a major source of energy, protein and other nutrients for both human and livestock. Starch and non-starch polysaccharides (dietary fiber) are the major carbohydrate constituents. Starch is a polymeric mixture of essentially linear (amylose) and branched (amylopectin) α-glucans. Starch owes much of its functionality and physical organization into a granular structure to these macromolecules. In Mexico, and gradually in other countries like the United States, consumption of maize tortillas becomes common. The process to convert maize into tortillas, called nixtamalization, starts when the grain is cooked in lime [Ca(OH)2] to produce nixtamal. The moist nixtamal is stone-ground into a dough (masa), that is further shaped into disks and baked on a hot griddle or gas-fired oven to produce tortillas. Tortillas can be produce from fresh masa using traditional nixtamal, or instant corn flour, which is dehydrated masa. During maize tortillas production, many changes occur, especially in starch, and the changes that this polymer suffers during processing are responsible for the textural and sensorial properties of masa and tortillas. The masa obtained from the nixtamalization process is a network of disperse and soluble starch polymers; starch granules, partially gelatinized in a continuous water phase, supported by free starch granules, other pieces of endosperm and lipids. The reassociation of the starch granules can also significantly affect the rheological properties of products made from masa. On the other hand, reassociation of the starch molecules may occur on cooling. Retrogradation, the ability of starch chains to form ordered structures in pastes, gels, and baked foods during storage, greatly influences the texture and shelf life of these products. In starch-rich products, amylose retrogradation is a rapid process taking only a few hours. Cereal-based foods such a tortillas can contain appreciable amounts of resistant starch (RS) which survives prolonged incubation with amylolitic enzymes. The

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Preface

xi

occurrence of RS has important implications for human bowel physiology. Diets rich in RS produce a larger faecal mass with lower pH and increase daily butyrate production. The presence of fermentable substrates helped in preventing inflammatory intestinal diseases. The study of the reactions leading to the staling of stored tortillas has considerable technological, commercial and health implications, because different physicochemical and structural changes in RS formed during nixtamalization, tortillas elaboration and storage happen. Chapter 8 - The interest in blue corn has increased in recent years guided by the conservation of germplasm, improvement of varieties and landraces, and a desire to learn about its qualities and their characteristics. Mexican pigmented varieties possess great cultural value and are an important source of nutraceuticals. Blue maize has a high concentration of anthocyanins which are phytochemical compounds that have been proven to have antioxidant, anti-inflammatory and hypoglycemic effects. It is expected for blue corn to become an important subject of research to increase its productivity and nutraceutical properties. Nowadays, blue corn is mainly used to manufacture pigmented soft tortillas and fried chips due to its enhanced nutritional and nutraceutical values. The anthocyanins and phenolic compounds associated to blue corns make these kernels promising as raw materials for production of functional foods. Traditional and alternative extrusion cooking processes have been improved to preserve these properties. For this reason, it is expected that blue corns will be highly demanded for the production of products like tortillas, masa flours, corn and tortilla chips, and biopolymers. Mexico is the country with the highest child obesity rate and a high incidence of chronic degenerative diseases among the general population; therefore it is necessary to continue the research and product development based on indigenous sources of functional foods. The consumption of these new products can significantly decrease the incidence of chronic diseases and improve public health. This chapter describes the main phytochemicals associated with corn and their role in preventing chronic diseases and oxidative stress. Furthermore, it presents the nutraceutical properties of transformedpigmented corns into tortillas and their implication on improving health. Novel products with nutraceutical properties based on blue maize are a clear option for the near future. Chapter 9 - Foods from plant origin, particularly nuts and seeds, represent the major source of registered food allergy due to the high variability of plant allergenic molecules, which depends on plant growing conditions, seed/fruit ripening, environmental stresses and/or industrial processing. Maize, one of the major human diet components, is one of the most world widely consumed cereals. It has become therefore one of the major causes of food allergy due to the widespread corn derived food products, which make difficult itsto avoid it. However, detailled biochemical knowledge of maize allergy is lacking. There are several unanswered questions including the symptoms and mechanisms involved in maize allergenic reactions, its prevalence in adults and children, the implicated allergen molecules and the clinical crosssensitization. Therefore, diagnostic tests and maize allergy management constitute a field of great interest. Currently, maize allergen proteins are classified into 20 different families, displaying diverse structures and functions. They are responsible for many IgE cross-reactions between unrelated pollen and plant food allergen sources. The most relevant maize allergen molecules belong to the expansin and the Ole e 1 superfamilies, the panallergen profilin, and the Lipid Transfer Proteins (LTPs), i.e. Zea m 14, the major maize allergen.

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Agricultural biotechnology promises plant food production through genetically modified (GM) crops with improved agronomic characteristics and enhanced consumer benefits. Numerous efforts in plant breeding programs have resulted in producing low-allergen or allergen-null plants that could moderate the allergic response. For this purpose, identification of new genes, and knowledge of allergen protein structures and function is crucial for the understanding of biochemical processes inducing maize allergy. Computational biology and protein modeling are increasingly used to evaluate whether a novel protein correspons to a known allergen, has a potential to become an allergen or could possibly cross-react with another existing allergen. In order to answer the above crucial questions and bring insights into the processes of food allergy, attention in this review is focused on maize allergens protein families, their biochemical, structural and immunological properties, while discussing possible strategies to predict biological consequences of allergen sensitization and cross-reactivity as well as therapies to mitigate maize allergy.

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Chapter 1

OPPORTUNITIES AND CONSTRAINTS IN MODIFYING THE COMPOSITION OF MAIZE GRAIN George G. Harrigan and Tyamagondlu V. Venkatesh Crop Composition Team, Regulatory Product Characterization and Safety Center, Monsanto Company, St. Louis, Missouri, US

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ABSTRACT The composition of modern day maize reflects its domestication origins from its wild ancestor, teosinte, and the impact of artificial selection including the development of hybridization technology. We present here a compositional comparison of teosinte and maize and review arguments that domestication and breeding technology may have conferred constraints on maize compositional diversity, particularly with respect to starch, protein, and oil. The impact of modern agricultural biotechnology on compositional diversity is reviewed under two separate considerations. First, a review of the scientific literature allows a reasonable conclusion that for agronomic traits, the effect of transgene insertion on composition is generally significantly less than the impact of environmental or germplasm variation on conventional crops. Given constraints that appear to be imposed by domestication and artificial selection, as well as the quantitative nature of compositional traits, this result may not be surprising. Second, nutritional enhancement (or biofortification) of maize through modern agricultural biotechnology may have some advantages over conventional breeding for some components by providing a means to more directly target key regulatory steps in the biosynthesis of these components.

INTRODUCTION Past and current choices made in crop domestication and breeding have helped determine food and feed nutrient qualities that serve human needs today. Of over 250,000 plant species, only 7000 are considered as foodstuffs (Khoshbahkt and Hammer, 2008), and even fewer, 150, supply over 90% of all plant food. Three major crops, i.e. maize, wheat, and rice, supply over 66%. The emphasis on these three crops as a food and feed source reflects their desirable

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George G. Harrigan, and Tyamagondlu V. Venkatesh

agronomic and nutritional properties. Here we review the current literature on the composition of maize particularly in the light of key events and processes that have led to its modern day nutritional value and to its leading contribution to the food and feed chain. These processes include domestication from a wild ancestor (teosinte), artificial selection and advances in breeding technologies including hybrid seed development and marker-assisted selection (MAS). Significantly, the reduced diversity in modern maize, and the quantitative nature of yield components as well as crop composition has implications for modifying agronomic and nutritional qualities of maize through modern agricultural biotechnology. First, improving agronomic traits, such as herbicide- or insect tolerance through transgene insertion, has been shown to have a negligible impact on crop composition. Second, improving compositional (nutritional) traits, or enhancing vitamin fortification will require increased understanding of genetic regulation of composition as well as the use multitransgene incorporation strategies. It is also not unreasonable to suggest that, “future maize breeding would perhaps benefit from the incorporation of alleles from maize's wild relatives” (Buckler et al., 2006) by re-incorporating genetic diversity that would have been lost during domestication. Partnering this approach with modern agricultural biotechnology may prove beneficial; integration of the ancestral and the modern may help facilitate improvements in compositional diversity and the nutritional quality of one of the world’s most important crop.

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DOMESTICATION Maize was domesticated from teosinte about 6,500 to 9,500 years ago in central Mexico (Piperno and Flannery, 2001; Matsuoka et al., 2002). The extensive genetic and phenotypic diversity of early maize (Buckler et al., 2006) facilitated its rapid expansion across the Americas and, as quoted by Kingsbury (2010, pg 229) “By the time of Columbus, [Native Americans] had already made more changes to corn than humans had made to any other plant: all the major types had been developed: flint, flour, pop, dent, and sweet.” Numerous agronomic and physiological changes between wild and domesticated plants are known many of which, e.g. increased grain to stem ratio, loss of spontaneous shattering of seed head on ripening and greater uniformity of seed ripening and germination, facilitated human harvest (Kingsbury, 2010). Key genetic loci that drove domestication include teosinte branched 1 (tb1) (Doebley et al., 1995) and barren stalk1 (bs1) (Gallavotti et al., 2004) which were responsible for converting the lateral multiple branches of teosinte into one or two maize ears. Of obvious importance, teosinte glume architecture1 (tga1) (Dorweiler et al., 1993) was “responsible for transforming the hard cupulate fruitcase of teosinte into the uncovered grain of the maize ear, a key step in making teosinte an edible crop” (Buckler et al., 2006). The key change of relevance to this chapter is that of crop composition. The composition of modern day commodity maize grain is, on a dry matter basis, ~72% starch, ~10% protein, ~4.5% oil, and ~2.5% free sugars (OECD, 2002; Watson, 2003). The essential amino acids lysine, tryptophan, and methionine are deficient although Quality Protein Maize (QPM) variants have been developed with improved levels of lysine and tryptophan (Prasanna et al., 2001). Maize grain is also a source, albeit limited, of vitamin A, vitamin E, thiamin, riboflavin, niacin, pantothenic acid, and pyridoxine (OECD, 2002). Biologically available

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Opportunities and Constraints in Modifying the Composition of Maize Grain

3

levels of these vitamins are generally low and breeding efforts to increase their levels are in place (Harjes et al., 2008; Naqvi et al., 2009). SUCROSE sh1 (sucrose synthase)

UDP-GLUCOSE

sh2 (large subunit) *bt2 (small subunit) ADP-GLUCOSE wx1 (granule-bound starch synthase) AMYLOSE

*ae1 (starch branching enzyme IIB) *su1 (debranching enzyme) AMYLOPECTIN

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Figure 1. A schematic of the starch biosynthesis pathway in maize. The six genes depicted were studied by Whitt et al. (2002) and those indicated with an asterisk show strong signs of selection.

Overall, it can be considered that the composition of maize today is both different from but dependent on the composition of the original wild ancestor, teosinte. We now illustrate this by contrasting the known starch and protein content of teosinte to that of maize and by reviewing some of the information on the effect of domestication on gene selection and on protein and starch quality. Starch levels in teosinte lines have been shown to be ~20% less than those of modern maize lines (Flint-Garcia et al., 2009; Watson, 2003). Increased levels of starch in maize can be attributed to selection for yield and seed size, both of which correlate with starch production. Analysis of maize candidate genes has confirmed that key enzymes associated with starch biosynthesis have been targets of selection during crop improvement (See Figure 1) (Whitt et al., 2002). Flint-Garcia et al. (2009) have argued that this “reduction of diversity in starch loci is dramatic” and that greater variability in teosinte genes associated with starch biosynthesis could be exploited in modern breeding programs seeking changes in protein:starch ratios or starch composition in maize. Protein levels in teosinte lines have been shown to be higher than modern maize lines (Flint-Garcia et al., 2009). As is the case for starch, this most probably reflects selection biases for yield and seed size. The qualitative and quantitative expression of seed storage protein has been extensively modified during the domestication process (Flint-Garcia et al., 2009). Teosinte has higher alpha zein content than maize, contains novel alcohol-soluble proteins not present in maize, and some lines appear to have higher levels of methionine, lysine, and tryptophan, which as noted earlier, are deficient in maize. Flint-Garcia et al. (2009) did conclude that there remained sufficient diversity in maize lines to facilitate further

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George G. Harrigan, and Tyamagondlu V. Venkatesh

improvements in amino acid levels but also suggested that genetic variation could be introduced from teosinte to alter levels in selected amino acids such as methionine and lysine. Other researchers have also described the value of expanding the germplasm base by exploiting wild relatives of maize (Wang et al., 2008). The protein and amino acid content of grain from hybrids generated from an elite maize line and a wild relative, Zea mays ssp. mexicana was recently assessed (Wang et al., 2008). It was concluded that some introgression lines showed higher protein levels and a different amino acid profile (including increased lysine) than the parental progenitors. Genetic sequencing studies suggest that 2 to 4% of the maize genome has experienced selection during domestication and/or plant breeding (Wright et al. 2005; Yamasaki et al. 2005) and that this may imply that there is insufficient genetic variation remaining in inbred lines to contribute to improvement in maize composition through traditional methods. Exploiting the genetic variability in teosinte as well as other wild relatives of maize may enhance continued compositional improvement in maize breeding programs (Buckler et al., 2006; Flint-Garcia et al., 2009; Wang et al., 2008).

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LONG-TERM TEMPORAL SHIFTS IN MAIZE COMPOSITION The discussion above suggests that domestication pressure has impacted composition and even reduced the potential for further gains. We now illustrate how artificial selection for desired agronomic qualities can impact composition and also provide examples of direct selection for specific compositional properties such as enhanced oil or protein production. To determine how modern hybrids have impacted grain composition, Scott et al. (2006) grew a set of cultivars that were widely available in different eras from the 1920s through 2001. Levels of starch, protein, and oil in grain composition exhibited clear trends with era, and consequently yield (see Figure 2). The compositional differences between the different era hybrids were however, generally modest and, as pointed out by Scott et al (2006) “While clear trends are observable in grain composition over the course of development of the era hybrids, the magnitude of the changes are small and on the order of magnitude of changes attributed to environmental effects.” In other words, grain composition has been influenced by long-term temporal shifts in the genetic and physiologic make up of maize hybrids, but this change is modest and real-time environmental factors including location, biotic and abiotic stresses, and management practices, appear to contribute more significantly to compositional variability. Whilst selection based on yield has indirect effects on compositional change, direct selection for specific compositional traits has been a breeding focus. Maize lines with high protein or high oil have been developed through the Illinois Long Term Selection Experiment (Dudley and Lambert, 2004; Moose et al., 2004). This experiment was initiated in 1896 using an open pollinated variety, Burr’s White. Selection for oil and protein has been conducted every year for over 100 years. From an initial level of ~4% dw, oil levels of over 20% dw have now been attained in some lines. Correspondingly, protein levels up to ~32% dw have been attained from an initial starting point in Burr’s White of ~11% dw. The value of the new high oil and high protein lines lies in the accumulation of alleles favorable to oil or protein in a single source.

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Figure 2. A schematic from Scott et al. (2006) of changes in starch, protein, and oil levels in maize grain over time. There has been a corresponding increase in yield over the same time-frame. These data were based on analysis of set of cultivars that were widely available in different eras from the 1920s through 2001.

A number of studies have been conducted to identify quantitative trait loci (QTL) that affect kernel composition in maize. Although the number of QTLs is a function of markers used and the numbers of lines evaluated, it is clear that large numbers of QTLs with small effects are evident for oil (>40) and protein (>50) (Dudley et al., 1977, Dudley, 2007). Unfortunately, the high oil and protein lines are associated with some negative agronomic characteristics, including reduced grain yield, kernel size and plant height (Dudley et al., 1977, Dudley, 2007) and it is possible that selection for specific compositional traits may adversely impact agronomic quality. A further example of this is quality protein maize (QPM) which, as we mentioned earlier, contains increased levels of lysine and tryptophan, a change attributable to a decrease in zein protein and a corresponding increase in glutelin (Prasanna et al., 2001). The altered protein and amino acid profile of QPM, attributable primarily to a mutation in the opaque 2 gene, is accompanied by poor agronomic characteristics including kernel softness, poor yield and germination, and increased disease susceptibility. The identification of “modifier” genes and over 40 years of breeding have mitigated some of these adverse effects and QPM varieties have been successfully introduced in many parts of the worlds (Mbuya et al., 2010). In summary, compositional changes when selecting for agronomic traits are likely to be modest and within the ranges expected from environmental factors. Selecting for compositional traits however, may impact agronomic gains that may require mitigation through further genetic analysis and extensive breeding efforts.

THE MODERN BIOTECHNOLOGY ERA Agriculture’s ability to supply an abundance of nutritious foods and feeds to nourish the world’s growing population faces serious challenges (Foresight, 2011). In order to meet challenges, plant breeders will be required to continuously improve agricultural productivity as well as enhance food and feed nutritional quality. As mentioned earlier some researchers consider that there is little or no genetic variation remaining in inbred maize lines to contribute to crop improvement by traditional breeding or gene discovery by genetic analysis (Buckler et al., 2006; Flint-Garcia et al., 2009). The development of methods for the direct introduction of new agronomic traits to produce GM crops has proven to be a powerful tool in

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the hands of breeders and one which has yielded extensive socio-economic benefits (see Brookes and Barfoot [2011] and James [2010] for reviews). However, rigorous pre-market regulatory assessments of GM crops are required (Kalaitzandonakes et al., 2007). Germane to this discussion, comprehensive compositional analyses represent a key component of these pre-market evaluations of GM crops; results from many of these studies have been reviewed to assess the impact of transgene insertion on compositional variability (Harrigan et al., 2010). Compositional analyses typically include the measurement of levels of key nutrients such as protein, storage oil, fiber, amino acids, fatty acids, vitamins, as well as crop-specific anti-nutrients or metabolites such as, for example, gossypol and cyclopropenoid fatty acids in cotton or isoflavones in soybean. Key analytes recommended for evaluation in a number of major crops are listed by the Organization of Economic Cooperation and Development (OECD) in a series of consensus documents that generally form the basis of most compositional studies conducted for regulatory assessments (http://www.oecd.org). The use of multiple geographically separate sites is also required in regulatory assessments to allow compositional studies across a wide range of environmental conditions. Studies incorporating a minimum of five field sites are typical in regulatory assessments, although the European Food Safety Authority (EFSA) has recently mandated a minimum of eight sites (EFSA, 2011). Whilst these rigorous pre-market regulatory assessments consume time and resources (Kalaitzandonakes et al., 2007) and, according to some, have been a major impediment to the development of new and improved crops (Graff et al., 2009; Potrykus, 2010) they have yielded an extensive source of information on the impact of transgene insertion on compositional variation. Overall, it can be concluded that this impact is negligible. We highlight this by reviewing information from i) an assessment of the compositional stability of insect-protected MON 810, one of the first widely cultivated GM maize crops (Zhou et al., 2011) and ii) a recent analysis of GM maize covering a total of four different transgenic traits (Harrigan et al., 2010).

Stability in the Compositional Equivalence of MON 810 The development of insect-protected maize through incorporation of Bacillus thuringiensis (Bt) derived proteins was a key milestone in the widespread adoption of GM technology. The Cry1Ab-containing product MON 810 was introduced in 1998, and although superseded in many countries by newer Bt and multi-trait insect-protected products, MON 810 remains the only GM maize crop grown in Europe. However, within Europe it has also been the subject of national bans inspiring considerable controversy over the scientific rationale for such actions (Ricroch et al., 2010). Zhou et al. (2011) recently reported a meta-analysis of compositional data from a total of 74 maize hybrids and varieties grown over at least nine growing seasons in geographic areas encompassing the U.S., Canada, South America, and Europe. Analytes measured in these studies included ash, carbohydrates by calculation, fat, moisture and protein. A summary of the values of these analytes obtained for MON 810 and conventional maize grain for studies conducted on samples from 2001 to 2009 is shown in Table 1. MON 810 and conventional maize analyte levels were consistently similar, as reflected in values for the results combined from each year.

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Table 1. Proximate analyses of grain from MON 810 and conventional maize hybrids harvested in 2001 to 2009

Year

Substancea

2001

MON 810 Conventional

2002

MON 810 Conventional

2003

MON 810 Conventional

2004

MON 810 Conventional

2005

MON 810 Conventional

2006

MON 810 Conventional

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2007

MON 810 Conventional

2008

MON 810 Conventional

2009

MON 810 Conventional

Combined

MON 810 Conventional

Ash Mean (Range) 1.39(1.16 1.62) 1.48 (1.34 1.71) 1.23 (1.07 1.40) 1.42 (1.26 1.56) 1.24 (1.04 1.45) 1.20 (1.05 1.46) 1.22 (1.03 1.41) 1.17 (1.01 1.36) 1.46 (1.31 1.64) 1.45 (1.31 1.61) 1.25 (1.12 1.38) 1.18 (1.07 1.26) 1.44 (1.01 1.84) 1.31 (1.06 1.46) 1.26 (1.06 1.44) 1.33 (1.20 1.43) 1.27 (1.07 1.42) 1.26 (1.05 1.37) 1.30 (1.01 1.84) 1.31 (1.01 1.71)

Carbohydrates by Calculation Mean (Range) 86.22 (83.65 87.90) 84.11 (79.80 85.55) 86.97 (85.72 88.81) 84.80 (82.13 86.50) 85.97 (85.25 86.95) 86.32 (85.21 87.68) 86.56 (85.26 87.57) 86.93 (85.73 88.18) 86.70 (85.27 88.15) 86.84 (85.64 87.57) 85.79 (84.61 86.41) 86.67 (85.38 87.71) 84.34 (81.04 88.21) 85.12 (82.46 87.75) 86.22 (81.85 87.84) 86.39 (83.24 88.64) 84.81 (83.36 86.05) 84.94 (82.97 86.56) 85.92 (81.04 88.81) 85.68 (79.80 88.64)

Fat Mean (Range) 3.69 (3.03 4.54) 3.66 (2.99 4.73) 2.93 (1.97 3.75) 3.32 (2.76 4.30) 3.97 (3.02 4.88) 3.79 (3.31 4.40) 3.96 (3.43 4.35) 3.77 (3.27 4.44) 3.80 (3.40 4.25) 4.06 (3.66 4.47) 3.64 (3.17 4.51) 3.21 (2.66 4.48) 4.7 (3.31 5.90) 4.33 (2.99 5.51) 3.63 (3.18 4.90) 3.59 (2.77 4.82) 3.96 (3.05 4.97) 4.00 (3.11 4.74) 3.82 (1.97 5.90) 3.77 (2.66 5.51)

Moisture Mean (Range) 11.58 (9.57 17.40) 9.14 (7.86 10.40) 11.56 (6.98 15.10) 9.01 (7.66 10.40) 11.16(9.50 12.60) 10.91(8.82 12.95) 8.50 (7.29 9.69) 8.27 (7.56 9.13) 10.96 (8.88 12.45) 11.19 (9.16 12.80) 8.38 (7.60 9.15) 8.33 (7.54 9.38) 9.28 (7.50 11.45) 9.65 (7.53 13.10) 12.74 (11.94 - 13.25) 13.01 (12.38 - 14.03) 13.36 (12.06 - 15.52) 13.59 (12.24 - 15.76) 10.98 (6.98 17.40) 10.42 (7.53 15.76)

Protein Mean (Range) 8.70 (7.60 10.30) 10.76 (9.42 13.70) 8.85 (6.98 15.10) 10.46 (9.15 12.06) 8.82 (7.62 10.06) 8.69 (7.33 9.78) 8.27 (7.17 9.01) 8.14 (7.40 9.03) 8.04 (7.02 8.83) 7.63 (6.79 8.33) 9.31 (8.62 10.31) 8.92 (8.33 10.04) 9.49 (7.41 11.65) 9.22 (7.64 10.75) 8.88 (7.54 11.88) 8.69 (6.91 11.46) 9.96 (8.33 11.34) 9.80 (8.13 11.14) 8.95 (6.9815.10) 9.24 (6.79 13.70)

a

Units in % dry weight except moisture which is in % fresh weight. Values for 2000-2001, 2004, 20072009 are based on 12 MON 810 and conventional hybrids, 2005 and 2006 values are based on 7 and 6 hybrids respectively. With the exceptions of the 2001 and 2002 studies, and in part for 2007, MON 810 and conventional hybrids were grown at the same location.

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Figure 3. Boxplots showing protein data in conventional (reference) maize hybrids and MON 810 (See Table 1 for details).

For example, protein mean values for MON 810 and conventional maize were 8.95% and 9.24% dw, respectively; corresponding values for fat were 3.82% and 3.77% dw (Table 1). For all components, the combined data showed that differences in relative magnitudes in mean values, with respect to the control, were small and ranged from 0.28% (carbohydrates by calculation) to 5.37% (moisture). The similarity in the distribution of values is shown graphically for protein (Figure 3). The fact that the assessment presented here highlighted no meaningful differences between MON 810 over multiple generations and the control also attests to the compositional stability of MON 810 and the lack of effect of transgene insertion at each generation. Importantly, this observation is consistent with (Coll et al., 2008, Coll et al., 2009, Coll et al., 2010a, Coll et al., 2010b) proteomic and transcriptomic observations that showed near-identical protein profiles in MON 810 and comparable non-GM varieties, as well as a “lack of repeatable differential expression patterns between MON 810 and comparable commercial varieties of maize”. It is also consistent with data on the stability of the MON 810 transgene in maize (La Paz et al., 2010).As pointed out by Sanahuja et al. (2011) in their extensive review of commercial applications of B. thuringiensis “Bt transgenic crops have been overwhelmingly successful and beneficial, leading to higher yields and reducing the use of chemical pesticides and fossil fuels”. Others have concluded (Park et al., 2011) that GM crops support the “three traditional pillars of sustainability” which are economic, environmental and social. As reiterated here, Zhou et al. (2011) demonstrated that these benefits have been associated with consistently maintained compositional equivalence in GM crops.

Lack of Impact of Transgene Insertion in GM Maize Products with Agronomic Traits The high degree of compositional parity observed between MON 810 and the conventional comparators within and across growing seasons is quite noteworthy given the

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Opportunities and Constraints in Modifying the Composition of Maize Grain

known natural variability in levels of the evaluated components. However this observation extends to a range of GM maize products. Harrigan et al. (2010) reviewed the composition of four different GM maize products, MON 87460 (water-use efficient), MON 89034 (insectprotected), MON 88017 (insect-protected) and NK603 (herbicide-tolerant). These products were grown in a variety of geographies and over a range of growing seasons (see Table 2). Overall, a total of 2,350 (number of sites × number of compositional components) statistical comparisons between the GM varieties and their corresponding conventional controls were conducted. Of these, 91.5% were not significantly different (p 75%) and temperature (> 30 ºC) beans develop the hardening process, which changes its characteristics in a negative way, mainly by increasing cooking time, these features greatly reduce their commercial value. Mixing of QPM and hard-to-cook beans (HTC) can be used to balance the amino acid profile of the resulting product without notably affecting sensory acceptance. Processing by extrusion is of particular interest because it is widely used to incorporate legume seeds into cereals for production of precooked flours, infant food and expanded snacks. These extruded products offer advantages in terms of their sensory characteristics and nutritional properties. Extruded maize products on the market usually provide a 6% of protein, the combination of QPM with HTC yields a product with a higher protein intake (15%), with a better balance of amino acids and appropriate sensory characteristics. Thus extruded products obtained have the potential to be commercially produced in addition to having a better nutritional intake than snack type products on the market.

*

Corresponding author: Dr Luis Chel-Guerrero. Email: [email protected].

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Jorge Ruiz-Ruiz, David Betancur-Ancona, Rolando González et al.

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INTRODUCTION Maize (Zea mays L.) plays a vital role in human and animal nutrition in many developed and developing countries. Indeed, maize grain accounts for 15 to 56% of total daily dietary caloric intake in numerous developing countries. This is particularly the case in Africa and Latin America, where animal protein is scarce and/or expensive and consequently unavailable to large portions of the population. However, cereal proteins have poor nutritional value for monogastric animals such as humans due to low levels of essential amino acids, including lysine, tryptophan and threonine. Cereal proteins contain an average of about 2% lysine, or less than one-half the concentration recommended for human nutrition by the Food and Agriculture Organization [1]. In human nutrition, lysine is the most important limiting amino acid in the maize endosperm protein, followed by tryptophan. This shortfall has been addressed mainly by supplementing maize grain with essential amino acids produced by bacterial fermentation. Though quite expensive, this approach works well for animal feeds. In addition, amino acids are commonly lost in foods processed from grain meals such as maize. This nutritional challenge has been addressed by using genetic enhancement in which essential amino acids are either incorporated into grain proteins or their levels increased [2]. An outstanding accomplishment of genetic enhancement strategies has been the creation of quality protein maize (QPM). People who depend on maize for their intake of energy, protein and other nutrients benefit immensely from incorporating QPM into their diets. Generally, QPM protein contains 55% more tryptophan, 30% more lysine and 38% less leucine than normal maize. It also offers better biological value (the amount of absorbed nitrogen needed to provide the necessary amino acids for different metabolic functions), with 80% for opaque-2 maize (o2) protein compared to only 45% for normal maize protein. Only 37% of common maize protein intake is utilized compared to 74% of the same amount of o2 maize protein. A minimum daily intake of approximately 125 g of o2 maize can guarantee nitrogen equilibrium, something impossible to attain with consumption of even twice this amount of normal maize; stated differently, 24 g of normal maize per kg of body weight are required for nitrogen equilibrium whereas only about 8 g of QPM are required. The nitrogen balance index for skim milk is 0.80 and that for o2 maize protein is 0.72, showing that QPM’s protein quality is 90% that of milk [2]. Due to their high protein content, legumes are one of the most important food sources in developing countries. Dry beans are a major source of dietary protein, dietary fiber, starch, vitamins and certain minerals. Unlike cereals, however, dry beans can deteriorate as a function of time and storage conditions, especially the high temperature and humidity conditions prevalent in the tropics. Degradation in dry beans includes increased cooking time, texture and flavor deterioration and loss of nutritional value. Known as the hard-to-cook (HTC) defect, this phenomenon and any possible changes in antinutrient compound levels caused by it have received little attention. Effective utilization of HTC beans in human nutrition requires pretreatment to remove or eliminate these undesirable attributes [3]. Cereal flours and starches are extruded to produce expanded snack foods, ready-to-eat cereals and dry pet foods. Extrusion is a high-temperature, short-time process in which food material is cooked by a combination of pressure, temperature and mechanical shear. Among other results, this can cause starch gelatinization, protein denaturation, inactivation of native enzymes (which cause food deterioration during storage), inactivation of antinutrient factors

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Extrusion of Quality Protein Maize …

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and reduction of microbial counts in the final product [4]. In general, a high expansion index, low bulk density and a firm texture are desirable qualities for extruded products. These physical characteristics are governed by feed material properties and extrusion cooking parameters [5]. Very little data is currently available on extrusion processing of QPM-legume flour blends. The present study objective was to determine the most appropriate extrusion conditions for a QPM/HTC bean blend for providing adequate physical and nutritional characteristics in production of an expanded snack type product.

MATERIALS AND METHODS Materials White dent quality protein maize (S00TLWQ-TO) was provided by the Centro de Investigaciones Científicas de Yucatán, and Phaseolus vulgaris seed was obtained from the 2004 harvest in the state of Yucatan, Mexico.

Flour Preparation

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The grains were processed in a roller mill and a pneumatic separator to separate the hull and germ. Final particle size ranged from 0.351 to 0.208 mm. Flour moisture content was measured to establish the amount of water to be added to adjust moisture content to required levels. The maize and bean flours were blended at a 60/40 (w/w) ratio in quantities sufficient to produce 500 g of blended flour for each treatment.

Extrusion and Physical Evaluation The flour blend (FB) was extruded using a monoscrew extruder with the following specifications: pressure and temperature sensor; two heating zones; screw with a 4:1 compression ratio and measuring 3.5 mm diameter x 20 mm long. A 32 model was used to evaluate extrusion conditions. Factors and levels were temperature (155, 170 and 185 °C) and moisture content (15.5, 17.5 and 19.5%), with a feed rate of 200 g/min at a fixed screw speed of 130 rpm. Evaluated physical parameters included expansion index (EI), density, resistance to compression (RC) and specific mechanical energy (SME). The expansion index (EI) was measured as described by Gujska and Khan [6]. Density was determined following Wang et al. [7]. Resistance to compression (RC) was determined according to Park et al. [8]. Torque and mass output values were used to calculate specific mechanical energy consumption (SME) according to González et al. [9].

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Chemical Composition Proximate composition was determined using Association of Official Analytical Chemists (AOAC) [10] methods: moisture (Method 925.09); ash (Method 923.03); crude fat (Method 920.39); crude protein, using a 6.25 nitrogen-protein conversion factor (Method 954.01); and crude fibre (Method 962.0 9). Carbohydrate content was estimated as nitrogenfree extract (NFE).

Protein Evaluation

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In vitro protein digestibility was determined following Hsu et al. [11], using a multienzymatic solution containing trypsin, chymotrypsin and peptidase. Apparent in vitro digestibility (Y) was determined using the equation: Y = 210.464 - 18.103 X, where, X = protein suspension pH immediately after digestion with the multi-enzymatic solution for 10 min. Amino acid analysis was done according to Alaiz et al. [12], using reverse-phase high performance liquid chromatography with spectrophotometric detection at 280 nm. Tryptophan was measured by HPLC with spectrophotometric detection at 280 nm according to Yust et al. [13]. Available lysine content was measured at 475 nm, according to Hurrel et al. [14]. Lys levels were determined by the B-A difference. In both tubes, the mmols of bound colouring = (40 mL/1000 mL) (100/weight of sample) (0.146), where 0.146 = Lys conversion factor.

Calculated Protein Efficiency Ratio (cPER) The cPER was calculated following the applicable AOAC [10] method, employing the in vitro digestibility value and the amount (g) of amino acid (AA) /100 g protein for Lys, Met + Cys, Thr, Ile, Leu, Val, Phe + Tyr and Trp. This assumes that the Cys and Tyr values in the Met + Cys and Phe + Tyr combinations do not surpass 50% of the total of their respective combinations.

Starch Evaluation Total starch (TS) was quantified by adapting the method of Tovar et al. [15] using 4 mol/L KOH to guarantee starch solubilization. Hydrolysis was done using thermostable amylase and amyloglucosidase. Reactivate glucose oxidase/peroxidase was used for colorimetric measurement of glucose, and concentration was read at 500 nm. Available starch (AS) was quantified as for TS, but without adding 4 mol/L KOH. Resistant starch (RS) was calculated by the difference between TS and AS: RS = TS AS. In vitro starch digestibility was quantified according to Holm et al. [16], based on the reducing power of the maltose released by the action of pancreatic amylase through 3,5dinitrosalicylic acid.

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Dietary Fibre Evaluation Total dietary fibre (TDF) was measured using the gravimetric enzymatic method proposed by Prosky et al. [17], with thermostable -amylase, protease and amyloglucosidase. Insoluble dietary fibre (IDF) was measured using the gravimetric enzymatic method proposed by Prosky et al. [17]. Soluble dietary fibre (SDF) was calculated by the difference between TDF and IDF: SDF = TDF - IDF.

Statistical Analysis Statistical treatment of the extrusion process results was done by multivariate analysis following Johnson and Wichern [18]. An analysis of variance (ANOVA) and a means comparison were applied to establish differences using the LSD method, regression analysis and surface response, according to Montgomery [19]. Nutritional changes were processed using central tendency and dispersion measurements. All analyses were done with the Statgraphics 5.0 program.

RESULTS

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Extrusion Process and Physical Evaluation The multivariate analysis results indicated that both temperature and moisture significantly (P < 0.05) affected the expansion index (EI), density, and specific mechanical energy (SME), but not resistance to compression (RC). Factor interaction had no effect (P > 0.05) on any of the response variables. The regression analysis for each response variable showed an adequate fit of the experimental values to first-order polynomial models that allow description of the IE, density and SME as a function of the significant factors. The mathematical models indicate that these behaviors are represented by Eqs. 1-3.

Expansion Index Temperature (X1) and moisture (X2) significantly (P > 0.05) affected extrudate EI. Both factors negatively affected variable response (Eq. 1) in that the EI increased as both factors decreased. Maximum expansion was produced with extrusion at 155 °C and 15.5% moisture content (E155) (Table 1). Eq. (1) EI = 1.86 – 0.26 (X1) – 0.16 (X2), r2 = 0.98 This can be explained by the fact that when materials are forced through an extruder die their water content vaporizes, and the simultaneous vapor flash-off expands their starch content, producing a porous, sponge-like structure in the extrudate. Extrudate EI is closely

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linked to the size, number and distribution of air cells within the material. Higher material moisture content creates a lubricating effect in the extruder barrel which lowers friction and increases vaporization of superheated water as the extrudate exits the die, both of which can interfere with product expansion [20, 21].

Density Temperature and moisture significantly (P < 0.05) affected density, although the effect was negative for temperature and positive for moisture (Eq. 2). This coincides with Balandrán-Quintana et al. [22], who reported a 20% reduction in density in bean extrudates when moisture was decreased from 25 to 20%. Compared to products made only with maize, those including legume flour have higher protein content, which can also influence density since friction and shear during extrusion cause extensive interlacing between proteins and lead to their texturization: high protein-content extrudates are denser and more rigid. Eq. (2) Density = 299.55 – 29.21 (X1) + 56.605 (X2), r2 = 0.95

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Resistance to Compression The RC values were not statistically different (P > 0.05), with values ranging from 54.9 to 51.9 N (Table 1). This physical parameter was influenced more by raw material characteristics and composition than by process temperature and moisture, meaning the normal relationship of higher RC as the EI increases was not observed here [23]. This is a function of hardness, expressed as the maximum break force by compression, which reflects alveolar wall resistance and the number of alveoli per unit of height. These characteristics vary widely depending on material composition and degree of protein denaturalization. The latter leads to greater interaction with other components and, even when extrudate protein content remains unchanged, can produce more rigid and denser products that are more resistant to breaking.

Specific Mechanical Energy Both temperature (X1) and moisture (X2) had a significant (P < 0.05) and negative influence on SME (Eq. 3). The SME values tended to decrease as temperature and moisture content increased, with the lowest SME values observed at the highest levels for both factors (185 °C and 19.5% moisture content). The SME required to produce an extrudate was lower in blends processed at high temperature with high moisture content. Higher temperature facilitated the transformation from solid flow to viscoelastic flow, and higher moisture content produced a lubricating effect, resulting in less energy use. Starch gelatinization is positively influenced by SME during extrusion. The higher the SME, the higher the degree of gelatinization since

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mechanical energy favors gelatinization by promoting rupture of intermolecular hydrogen bonds [24]. Degree of gelatinization, in turn, is directly related to product expansion [23], which agrees with the present results in that the extrudates with the highest SME had the highest EI. Eq. (3) SME = 442.0 – 63.0 (X1) – 73.8 (X2), r2 = 0.99 Of the tested extrusion conditions, those at 155 °C (E155) or 170 °C (E170) and 15.5% moisture content produced optimum physical characteristics (Table 1). These products were selected to evaluate extrudate nutritional characteristics.

Nutritional Evaluation Chemical Composition Proximate composition analyses of the flour blend (FB) and extrudates (Table 2) showed that moisture content after extrusion dropped to 8.9% in the E155 and 8.6% in the E170. This is to be expected since a portion of the water vaporizes during extrusion. Table 1. Extrudate expansion index (EI), density, resistance to compression (RC) and specific mechanical energy (SME)

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Sample 1 2 3 4 5 6 7 8 9 10 11 a-f

Temperature (ºC) 155 155 155 170 170 170 170 170 185 185 185

Moisture content (%) 15.5 17.5 19.5 15.5 17.5 17.5 17.5 19.5 15.5 17.5 19.5

Density (g/cm3)

EI a

2.10 2.03abc 1.94bc 2.06ab 1.90cd 1.85cd 1.82cd 1.68de 1.66de 1.54ef 1.40f

abc

307.61 381.20cd 436.23d 237.17a 271.71abc 308.64abc 301.34abc 360.22bcd 261.02ab 339.78abcd 348.98abcd

RC (kgf) 5.48a 5.38a 5.39a 5.29a 5.58a 5.46a 5.62a 5.50a 5.45a 5.45a 5.60a

SME (J/g) 564a 513b 433c 502b 440c 452c 443c 344d 454c 378d 300e

Different superscripts in the same column indicate statistical difference (P200 °C) and screw speed (>300 rpm) during extrusion can cause lipids degradation. Also, fatty acids in the material can form complexes with amylose, making it more difficult to extract crude fats for quantification. The temperatures (155 to 185 °C) and screw speed (130 rpm) used in the present study, however, were below these levels and probably had no effect on these parameters. Indeed, no differences were observed in crude fat content between the FB and the extrudates (E155 and E170). Neither were differences noted in crude fibre content between the FB and extrudates (E155 and E170). Rabe [26] mentioned, however, that insoluble and soluble fibres are redistributed after extrusion, producing thermomechanical transformations that will not appear in a proximal determination of crude fibre due to the technique’s low sensitivity. Mineral components resist the high temperatures, pressures and mechanical forces of extrusion. In an evaluation of extrusion of QPM at 130, 150 and 170 °C, at a screw speed of 300 rpm and 14% moisture content, Diaz [23] reported no differences between flour and extrudate ash content. This agrees with the present results in which ash content in the FB was unmodified in the E155 and E170 (Table 2).

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In Vitro Protein Digestibility The digestibility values for E155 (80%) and E170 (79%) (Table 3), were similar to the 83% reported for P. vulgaris L. extrudates (160 °C, 22% moisture content) [22], and the 82% reported for a 50/50 (w/w) maize/Lima bean blend [21]. The increased digestibility observed in E155 and E170 may be due to two phenomena caused by extrusion: 1) protein denaturalization, which may increase exposure of sites susceptible to enzymatic activity [27]; and 2) inactivation of trypsin and chymotrypsin inhibitors, leading to improved digestibility [28].

Amino Acid Profile The essential amino acids content of the FB and the E155 exceeded that of a FAO [1[reference protein, except in Trp, which was 18% below the reference level (Table 4). Both extrudates had amino acids contents below that of the FB, although the E170 exhibited the greatest decrease, with a 63.37% sulphur amino acid level versus the reference. This is similar to the 10% decrease in sulphur amino acids reported by Diaz [23] for QPM extrudates (130 °C, 20% moisture content, 300 rpm screw speed). Ilo and Berghofer [29] evaluated Cys, Trp, Met, Arg and Lys levels after extrusion of maize flour at 180 °C, 13% moisture content and 57 rpm, and reported losses of 10, 15, 17, 19 and 43%, respectively. Temperature, moisture content and material residence time in the extruder all affect amino acids loss. In the present study, screw speed was fixed at 130 rpm and moisture content was constant at 15.5%, meaning thermomechanical treatment intensity was responsible for the observed decreases in amino acids.

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Table 3. Protein nutritional parameters of raw flour blend (FB) and extrudates (E155 and E170) Component In vitro protein digestibility (%) Available lysine (g/16 g N) cPER

FB 76 ± 0.05 5.2 ± 0.02 1.62 ± 0.01

E155 80 ± 0.03 4.3 ± 0.07 1.55 ± 0.03

E170 79 ± 0.01 3.8 ± 0.05 0.94 ± 0.04

Calculated protein efficiency ratio = cPER.

Table 4. Amino acid content (g/100 g protein) of raw flour blend (FB) and extrudates (E155 and E170) Amino acid Isoleucine Leucine Lysine Methionine + Cysteine Phenylalanine + Tyrosine Threonine Tryptophan Valine Histidine

FB 3.7 9.1 6.0 3.0 8.3 4.9 0.9 5.3 3.4

E155 3.6 9.0 5.8 2.7 8.2 4.8 0.9 5.1 3.2

E170 3.4 8.9 5.6 1.9 8.0 5.0 0.8 4.9 3.1

FAO/WHO 2.8 6.6 5.8 2.5 6.3 3.4 1.1 3.5 1.9

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Table 5. Changes (%) in starch and fibre contents in the raw flour blend (FB) and extrudates (E155 and E170) Component Total starch Available starch Resistant starch In vitro starch digestibility Total dietary fibre Insoluble dietary fibre Soluble dietary fibre

FB 49.1 ± 0.03 41.3 ± 0.07 7.8 ± 0.01 12 ± 0.02 27.5 ± 0.05 23.1 ± 0.01 4.4 ± 0.02

E155 48.8 ± 0.04 46.2 ± 0.04 2.6 ± 0.02 89 ± 0.05 17.0 ± 0.09 13.6 ± 0.02 3.5 ± 0.04

E170 48.9 ± 0.09 46.9 ± 0.01 2.1 ± 0.09 92 ± 0.08 15.0 ± 0.06 11.6 ± 0.03 3.4 ± 0.02

Available Lysine Available Lys content decreased 17.3% in the E155 and 26.9% in the E170 (Table 3). Ilo and Berghofer [29] reported a 43% Lys decrease in maize extrudates. Like the other amino acids, the decrease in Lys observed here resulted from the thermomechanical treatment, and was, therefore, more pronounced (26.9%) at 170 °C. The Maillard reaction has been indicated as the main cause of lysine reductions. In the present case, it is possible that low-moisture, high-temperature extrusion led to starch degradation, producing reducing sugars[30], simultaneously modifying protein structure and exposing reactive sites, all of which favor browning reactions [31]. The lysine ε-amino group has been referred to as a major reactant in the Maillard reaction [27], which may explain the effect of extrusion on this particular amino acid. The high temperatures (155 and 170 °C) and moisture content (15.5%) used here may

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have favored Maillard reactions. The presence of Maillard reactions in the extrudates was corroborated by their color, which varied from light yellow to dark brown due to the melanoidins generated by this type of reactions. It is also possible that non-enzymatic browning reactions occurred during extrusion of the maize-bean flour blend, affecting lysine retention in the extrudates.

Calculated Protein Efficiency (cPER) The extrusion process lowered cPER by 4% in the E155 and by 42% in the E170 compared to the FB (Table 3). In a study of extrudates (190 °C, 18.5% moisture content, 100 rpm) of HTC bean/rice blends (25/75 (w/w)), Steel et al. [4] reported decreases of 8% in cPER compared to the raw material. They concluded that this reduction in protein nutritional value was caused by the thermal treatment. This is similar to the present results, particularly in the observed decreases in sulphur amino acids and Lys.

Total Starch (TS)

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Total starch values were similar in the FB, E155 and E170 (Table 5). These levels are lower than the 62.9% reported by Pérez et al. [21] for a 50/50 QPM/Lima bean blend. Molecular degradation in starches occurs as a function of temperature, moisture and screw speed since increases in these parameters produce severe starch degradation and lower-weight molecules. The studied extrusion conditions (150 and 170 °C, 15.5% moisture content, 130 rpm) were not extreme enough to cause this degradation, explaining the similar starch contents in the FB and extrudates (E155 and E170).

Available Starch (AS) Available starch increased from 84% of total starch in the FB to 94.7% in the E155 and 95.9% in the E170 (Table 5). These values are similar to the 97.4% reported by Betancur et al. [32] for maize starch, and higher than the 80.7% reported by Pérez et al. [21] for an extrudated QPM/Lima bean blend (50/50 (w/w )). Starch loses its crystalline structure due to cooking during extrusion, leaving its molecules available for hydrolysis [4]. This would explain the increased AS values in the two extrusion treatments, which both had higher AS than the FB.

Resistant Starch (RS) The FB had a RS content of 15.9% of TS, higher than the 10.8% RA reported by Pérez et al. (2006) for an unprocessed QPM/Lima bean blend (50/50 (w/w)). This value decreased to 5.3% in the E155 and to 4.3% in the E170, which are higher than the 2.6% reported by Pérez et al. [21] for the same blend as above. The higher RS in the FB in the present study was

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probably due to the 40% HTC bean proportion in the blend. Bean starch granules are more resistant to hydrolysis by digestive enzymes because of their C-type diffraction pattern structural characteristics. This pattern is a mixture of rhomboid and hexagonal crystals, the latter of which are indigestible by enzymes due to their compact supramolecular packing [4]. Depending on process conditions, resistant starch can form during extrusion; at low moisture contents (15-20%) molecules are less mobile, favoring formation of hydrogen bridges between contiguous chains. Screw speed during processing is also important because it determines material residence time, with longer residence times favoring RS formation. High temperatures (180-200 °C) can also increase RS formation [33]. In the present study, however, RS content decreased with extrusion, probably because the conditions (150 and 170 °C, 15.5% moisture content, and 130 rpm) did not meet the moisture content, residence time and temperature requirements for greater RS production. They did cause cooking of the material, however, which agrees with the observed increases in extrudate AS (E155 and E170).

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In Vitro Starch Digestibility As expected, the starch in the FB was not gelatinized, meaning it was practically indigestible by -amylase action. The resulting 12% digestibility value (Table 5) was similar to the 10% reported by Betancur et al. [32] for maize, Lima and velvet bean starches. This low digestibility is caused by the crystalline structure of starch, which protects the glucoside bonds and limits enzyme hydrolytic action. Once gelatinized, however, this crystalline structure is lost, leaving the molecules open for hydrolysis, which breaks the glucoside bonds and increases digestibility. This is what occurred in the present study as digestibility increased to 89% in the E155 and to 92% in the E170. These values are similar to the 92% reported for maize starch and >84% for Lima bean starch [32].

Total Dietary Fbre (TDF) The TDF content in the FB was higher (Table 5) than the 14.46% reported by Pérez et al. [21] for a QPM/Lima bean blend (50/50 (w/w)). Extrudate TDF was similar to the 17.2% reported by Martín-Cabrejas et al.[33] for HTC bean extrudates (140 °C, 25% moisture content, and 130 rpm). Compared to the FB, processing conditions modified TDF content in the extrudates by 38% (E155) and 44% (E170). The modification observed in TDF probably occurred through degradation into low molecular weight fragments since extrusion causes considerable solubilization of dietary fibre components, particularly pectic polymers and cellulose.

Insoluble Dietary Fbre (IDF) The IDF in the FB accounted for 84% of TDF (Table 5), a percentage similar to the 80% reported for HTC bean (P. vulgaris L.) flour [3]. In the E155 extrudate it accounted for 80%

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of TDF and in the E170 for 78%, values similar to those reported for extrudates from HTC bean flours (83 and 79%; 160 °C, 25% moisture content, and 130 rpm) [3]. Compared to the FB, IDF in the E155 decreased by 26%, and that in the E170 by 34%. This reduction was caused by solubilization of insoluble fibre components, particularly arabinose and uronic acid, as well as degradation of pectic polymers and cellulose.

Soluble DietaryFfibre (SDF) Soluble dietary fibre in the FB accounted for 16% of TDF (Table 5), which is lower than the 20% reported for SDF in HTC bean (P. vulgaris L.) flour [3]. The SDF fraction accounted for 20.6% of TDF in E155 and 22.7% in E170, which are higher than SDF values reported for HTC bean flours extruded at 140 °C (17% SDF) or 160 °C (21% SDF) (25% moisture content, 130 rpm) [3]. Increased SDF can be caused by release of the soluble fraction from hemicellulose as a result of heating, which coincides with the highest SDF being observed here in E170.

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CONCLUSION Extrudates with adequate physical characteristics were produced from a blend of QPM and HTC bean (60/40 (w/w)) flours. Of the tested extrusion conditions, those at 155 or 170 °C, 15.5% moisture content and 130 rpm screw speed produced optimum physical characteristics: the highest EI values (2.1 at 155 °C and 2.06 at 170 °C); lowest densities (307.6 1 g/cm3 at 155 °C and 237.17 g/cm3 at 170 °C); and highest SME values (564 J/g at 155 °C and 502 J/g at 170 °C). Combination of the maize with the legume provided the blend a protein content of almost 15%, resulting in extrudates with good nutritional quality. In vitro protein digestibility increased after extrusion, although the extrudates’ amino acid content decreased versus the flour blend, lowering cPER values, especially in the extrudate produced at 170 °C. Total starch content in the flour blend and extrudates was unmodified. Both available starch content and in vitro starch digestibility increased due to extrusion. Total dietary fibre and IDF decreased in the extrudates compared to the flour blend, although SDF increased. Using a blend of flours from QPM and a legume of no commercial value (HTC bean), extrusion at 155 °C, 15.5% moisture content and 130 rpm screw speed produced an extrudate with adequate physical and nutritional characteristics. Extruded products obtained from quality protein maize in combination with hard-to-cook bean presented adequate physical characteristics, as a good expansion index, low density and crunchy texture. These properties are associated with market acceptance of these products. Besides having the potential to be commercially produced, the extrudates have nutritional advantages compared to traditional commercial snack type products such as high protein intake, good amino acid balance, low fatty content and an appropriate soluble/insoluble dietetic fiber ratio. Such products would meet even the current Mexican requirements to be considered suitable in terms of their nutritional intake to be consumed in elementary schools.

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[10] [11] [12]

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F. A. O. Agrostat, Food Balance Sheets, F. A. O., Rome, Italy, 1992. Prasanna, B. M., Vasal, S. K., Kassahun; B., Singh, N. N. (2001). Quality protein maize. Current. Science., 81(10), 1308-1319. Martín-Cabrejas, M., Jaime, L., Karanja, C., Downie, A. J. (1999). Modifications to physicochemical and nutritional properties of hard-to-cook beans (Phaseolus vulgaris L.) by extrusion cooking. Journal of Agricultural and Food Chemistry, 47(3), 11741182. Steel, J. C., Sgarbieri, V. C., Jackix, H. M. (1995). Use of extrusion technology to over come undesirable properties of hard-to-cook dry beans (Phaseolus vulgaris L.). Journal of Agricultural and Food Chemistry, 43(9), 2487-2492. Ali, Y., Hanna, M. A., Chinnaswamy, R. (1996). Expansion characteristics of extruded corn grits. Food Science and Technology-LWT, 29: 702-707. Gujska, E., Khan, K. (1990). Effect of temperature on properties of extrudates from high starch fractions of navy, pinto and garbanzo beans. Journal of Food Science, 55(2), 466-469. Wang, W., Klopfenstein, C. F., Ponte, J. (1993). Effects of twin-screw extrusion on the physical properties of dietary fiber and other components of whole wheat bran and on the baking quality of the wheat bran. Cereal Chemistry, 70(6), 707-711. Park, J., Rhee, K. S., Rhee, K. C. (1993). Single-screw extrusion of defatted soy flour, corn starch and raw beef blends. Journal of Food Science, 58(1), 9-19. González, R., Torres, R., Degreef, D. (2002). Extrusión-cocción de cereales. Boletin, 36(2), 104-115, SBCTA, Campinas. Association of Official Analytical Chemists (AOAC). (1997). Official methods of analysis (15th ed.). Arlington, VA: Association of Analytical Chemists. Hsu, H., Vavak, D., Satterlee, L., Miller, G. (1977). A multienzyme technique for estimating protein digestibility. Journal of Food Science, 42(5), 1269-1279. Alaiz, M., Navarro, J. L., Vioque, E., Vioque, G. (1992). Amino acid analysis by high performance liquid chromatography after derivatization with diethyl ethoxymethylenemalonate. Journal of Chromatography, 591(1-2), 181-186. Yust, M. M., Pedroche, J., Girón-Calle, J., Vioque, J., Millán, F., Alaiz, M. (2004). Determination of tryptophan by high-performance liquid chromatography of alkaline hydrolysates with spectrophotometric detection. Food Chemistry, 85(2), 317-320. Hurrel, R. F., Lerman, P., Carpenter, K. J. (1979). Reactive lysine in food-stuffs as measured by rapid dye-binding procedure. Journal of Food Science, 44(4), 221-227. Tovar, J., Björck, I., Asp, N. G. (1990). Starch content and alpha-amylolysis rate in precooked legume flours. Journal of Agricultural and Food Chemistry, 38(9), 18181823. Holm, J., Björck, I., Drews, A., Asp, N. G. (1986). A rapid method for the analysis of starch. Starch/Stärke, 38, 224-226. Prosky, L., Asp, N., Schweitzer, T., Debris, S., Furda, I. (1989). Determination of insoluble, soluble and total dietary fiber in food and food products: interlaboratory study. Journal of Association of Official Analytical Chemists, 71(5), 1017-1023.

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[18] Johnson, A. R., and Wichern, D. W. (1992). Applied multivariate statistical analysis. Englewood Cliffs, NJ, US: Prentice Hall. pp. 241-271. [19] Montgomery, D. (2004). Diseño y análisis de experimentos. México, D. F. Editorial LimUS S. A. de C. V. pp. 218-276. [20] Kokini, J., Lai, L., Chedit, L. (1992). Effect of starch structure on starch rheological properties. Food Technology, 46(6), 124-139. [21] Pérez, N. C., Cruz, E. R., Chel, G. L., Betancur, A. D. (2006). Effect of extrusion on nutritional quality of maize and Lima bean flour blends. Journal of the Science of Food and Agriculture, 86(14), 2477-2484. [22] Balandrán-Quintana, R. R., Barbosa-Cánovas, G. V., Zazueta-Morales, J. J., AnzaldúaMorales, A., and Quintero-Ramos, A. (1998). Functional and nutritional properties of extruded whole pinto bean meal (Phaseolus vulgaris L.). Journal of Food Science, 63(1), 113-116. [23] Diaz, A. L. (2003). Food quality and properties of quality protein maize. MS thesis, Texas AandM University, College Station, TX, US. [24] Gropper, M., Moraru, C. I., Kokini, J. L. (2002). Effect of specific mechanical energy on properties of extruded protein-starch mixtures. Cereal Chemistry, 79(3), 429-433. [25] Asp, N. G., and Björck, I. (1989). Nutritional properties of extruded foods. In C. Mercier, P. Lindo, and J. M. Harper (Eds.), Extrusion cooking (pp. 398-415). St. Paul, MN, US: American Association of Cereal Chemists, Inc. [26] Rabe, E. (1999). Effect of processing on dietary fiber in foods. In S. Cho, L. Prosky, M. L. Deher (Eds.), Complex carbohydrates in foods. New York, NY, US: Marcel Dekker. (pp. 395-409). [27] Camire, M. E. (2002). Chemical and nutritional changes in food during extrusion. In M. N. Riaz (Ed.), Extruders in food applications. Boca Raton, FL: CRC Press. (pp. 127148). [28] Alonso, R., Aguirre, A., Marzo, F. (2000). Effects of extrusion and traditional processing methods on antinutrients and in vitro digestibility of protein and starch in faba and kidney beans effect of extrusion cooking on digestibility. Food Chemistry, 68(2), 159-165. [29] Ilo, S., and Berghofer, E. (2003). Kinetics of lysine and other amino acids loss during extrusion cooking of maize grits. Journal of Food Science, 68(2), 231-241. [30] Pham, C. B., Del Rosario, R. R. (1984). Studies on the development of texturized vegetable products by extrusion process. II. Effects of extrusion variables on the available lysine, total and reducing sugars. Journal of Food Technology, 19: 549-559. [31] Mauron, J. (1990). Influence of processing on protein quality. Journal of Nutritional Science and Vitaminology, 36: S57-S69. [32] Betancur-Ancona, D., Segura-Campos, M. R., Chel-Guerrero, L. A., Dávila-Ortíz, G. (2011). Structural and some characteristics of Velvet bean (Mucuna prureins) and Lima bean (Phaseolus lunatus) starches. Starch/Stärke, 63, 475-484. [33] Martín-Cabrejas, M., Esteban, R. M., Perez, P., Maina, M., Waldron, K. W. (1997). Changes in physicochemical properties of dry beans (Phaseolus vulgaris L.) during long-term storage. Journal of Agricultural and Food Chemistry, 45(8), 3223-3227.

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In: Maize: Cultivation, Uses and Health Benefits Editor: Jose C. Jimenez-Lopez

ISBN: 978-1-62081-514-4 © 2012 Nova Science Publishers, Inc.

Chapter 7

CORN TORTILLAS: PHYSICOCHEMICAL, STRUCTURAL AND FUNCTIONAL CHANGES María del Carmen Robles-Ramírez1, Areli Flores-Morales2, and Rosalva Mora-Escobedo1,* 1

Instituto Politécnico Nacional, Escuela Nacional de Ciencias Biológicas, México 2 Instituto Tecnológico del Altiplano de Tlaxcala. México

ABSTRACT

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Maize (Zea mays L) is the third most important food crop in the word and a major source of energy, protein and other nutrients for both human and livestock. Starch and non-starch polysaccharides (dietary fiber) are the major carbohydrate constituents. Starch is a polymeric mixture of essentially linear (amylose) and branched (amylopectin) αglucans. Starch owes much of its functionality and physical organization into a granular structure to these macromolecules. In Mexico, and gradually in other countries like the United States, consumption of maize tortillas becomes common. The process to convert maize into tortillas, called nixtamalization, starts when the grain is cooked in lime [Ca(OH)2] to produce nixtamal. The moist nixtamal is stone-ground into a dough (masa), that is further shaped into disks and baked on a hot griddle or gas-fired oven to produce tortillas. Tortillas can be produce from fresh masa using traditional nixtamal, or instant corn flour, which is dehydrated masa. During maize tortillas production, many changes occur, especially in starch, and the changes that this polymer suffers during processing are responsible for the textural and sensorial properties of masa and tortillas. The masa obtained from the nixtamalization process is a network of disperse and soluble starch polymers; starch granules, partially gelatinized in a continuous water phase, supported by free starch granules, other pieces of endosperm and lipids. The reassociation of the starch granules can also significantly affect the rheological properties of products made from masa. On the other hand, reassociation of the starch molecules may occur on cooling. Retrogradation, the ability of starch chains to form ordered structures in pastes, gels, and baked foods during storage, greatly influences the texture and shelf life of these products. In starch-rich products, amylose retrogradation is a rapid process taking only a few hours. Cereal-based foods such a tortillas can contain appreciable amounts of resistant starch *

Corresponding author: Dr Rosalva Mora-Escobedo. E-mail: [email protected], [email protected].

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M. del Carmen Robles-Ramírez, A. Flores-Morales, and R. Mora-Escobedo (RS) which survives prolonged incubation with amylolitic enzymes. The occurrence of RS has important implications for human bowel physiology. Diets rich in RS produce a larger faecal mass with lower pH and increase daily butyrate production. The presence of fermentable substrates helped in preventing inflammatory intestinal diseases. The study of the reactions leading to the staling of stored tortillas has considerable technological, commercial and health implications, because different physicochemical and structural changes in RS formed during nixtamalization, tortillas elaboration and storage happen.

Keywords: Maize tortillas; physicochemical changes; structural changes; functional properties

NIXTAMALIZATION Nixtamalization is the alkaline boiling process of corn that has been practiced in Latin America since the pre-Columbian era, and from which a dough, called “masa”, is obtained and used to prepare tortillas (disc of masa cooked in a griddle) , instant flour, corn and tortilla chips, tostadas, atole, tamales and other regional dishes. Nixtamalization has been transmitted from generation to generation and is still carried out as in ancient times. The term comes from the Nahuatl word nixtli, ashes, and tamalli, masa (dough) (Paredes et al., 2008).

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1. HISTORY The oldest evidence of the existence of maize is still controversial. A recent study demonstrates that the earliest securely dated remains of maize in Mexico are macrofossils from the Mexican Southern Highlands that date to at least 6200 years old. It supports the hypothesis that people of that place domesticated this important crop plant and later diffused its cultivation to groups living in the valley of the Balsas River and coastal lowlands (Sluyter and Dominguez, 2006). The maize derived from teosinte; a grass to which it is genetically closely related. Teosinte is native to the Central Valley of Mexico, from where it spread across America. After thousands of years, the primitive maize became domesticated and its cultivation made possible the flowering of the great pre-Columbian cultures (Brites et al., 2007). Maize was an essential piece in Mayan and Aztec civilizations and had an important role in their religious ideas, festivities and nutrition. At the end of the fifteenth century, after the discovery of the American continent, maize was introduced in Europe through Spain (FAO, 1993). Portuguese sailors and Arabic traders played an important role in the spread of maize cultivation in Asia and Africa (Desjardins and McCarthy, 2004). From maize cultivation, people, applying to the mother plant their agriculture knowledge, conceived the first villages and the cities that were, for a time, the most populous of the world (Esteva, 2003). Mesoamerican people’s food was based on corn, beans, amaranth, chia, pumpkin, cocoa and agave. With them, a great variety of dishes and drinks were prepared giving rise to an original and complex culinary knowledge (Esteva, 2003). The ancient process of nixtamalization was first developed in Mesoamerica, where maize was originally cultivated. There is no precise date when the technology was developed, but

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typical household equipment for making nixtamal out of maize is known on the south coast of Guatemala at dates between 1200-1500 B.C. Probably, people began to prepare tortillas mixing the dry powder from the milling of maize with water and then boiling, but they noted that the product decayed easily and acquired a bad odor (due to germ oil oxidation), so they had to use the fire to break the grains by cooking and pull the tip where the germ is. Later, they discovered that the ashes from the firewood used to cooking the food would be of great utility because of the ash (sodium and potasium oxide), mixed with water, becomes an alkaline solution that softens and destroys the husk from the grain (Véles, 2004). The ancient Aztec and Mayan civilizations developed nixtamalization using lime (calcium hydroxide) and ash (potassium hydroxide) to create alkaline solutions. The association of man with the maize led him to create a variety of tools to corn cultivation and the grain preparation, conservation and storage (Figure 1). Large clay pots for the nixtamal, volcanic stone “metate” to grind it by hand, and the griddles to cook the tortillas, were invented (Esteva, 2003). For at least one third of Mexican people, maize today remains the hub of everyday life. In the fields, as in the home, the attention of the plot, preparing corn, dough and tortillas, the conservation of harvesting, shelling the cobs, animal feeding, daily food, festivals and rituals, all are related to corn (Esteva, 2003).

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2. MAIZE PLANT Botanically, maize (Zea mays) belongs to Poaceae (Gramineae) family. It is an annual plant whose fast growth allows it to reach a height of about 2.5 m, although some wild varieties reach up 7 m. Its reproduction is by cross-pollination; the female flower (ear or cob) and the male flower (tassel) are found in separate places on the plant. Long leaves surround the stem from which the cob arises, one on each stalk. The grain is a caryopsis (a fruit with the pericarp fused with the seed coat) which develops in the cob; each ear is 10-25 cm long and has about 300 to 1 000 kernels, in a variable number of rows (12 to 16) (Brites et al., 2007).

Figure 1. Huasteca Civilization. Diego Rivera mural. National Palace, Mexico.

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M. del Carmen Robles-Ramírez, A. Flores-Morales, and R. Mora-Escobedo

Figure 2. World total production of the major cereals (USDA, 2011).

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Maize is cultivated in diverse environments. According to the photoperiod sensitivity, the maize varieties are classified in two types: tropical maize and temperate maize. Maize also has been classified in different varieties according to the grain morphology and endosperm composition: Amylylacea (flour corn), Everta (pop corn), Identata (dent corn), Indurada (flint corn), Saccharata (sweet corn), Ceratina (waxy corn), among others. Maize plant has suffered evolutionary changes that have resulted in a great variability in the plant height, number of ears per plant, maturation cycles, number and weight of grains, among many other characteristics. This has permitted the development of hybrids with improved characteristics like higher yield and resistance to diseases and pests, and improved nutritional value. Biotechnology research aimed at improving maize is concentrated in the International Center for Maize and Wheat Improvement (CIMMYT) located in El Batán, Mexico (Brites et al., 2007).

3. PRODUCTION Maize, a main source of food for both humans and animals, is grown in more countries than any other crop (USDA, 2011). It is currently the cereal most produced in the world (872.4 million tons), surpassing both wheat and rice (Figure 2). The versatile plant can flourish in climates as diverse as the arid desert plains of the southwestern United States and the high Andean mountain plains of Ecuador and Peru. The temperate plains of the United States provides some of the best growing conditions for maize in the world, making the United States the world's top corn producer (U.S. Grain Council, 2010). According to data of Foreign Agricultural Service of USDA, the United States cultivated 40 percent of the world's corn up July of fiscal year 2011/2012, producing 342.2 million metric tons. Other major corn producing countries in 2011 include: China (170 million metric tons); European Union (59.2 million metric tons); Brazil (55 million metric tons); Argentina (26 million metric tons); Mexico (24 million metric tons); and India (21 million metric tons). United States is also the leading exporter and the country that participates more in the world trade of this cereal. The main consumers are China, Brazil and Mexico (USDA, 2011), with being Mexico the main importer of Latin America (Brites et al., 2007).

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4. NIXTAMALIZED CORN PRODUCTS Tortillas are a daily staple in Mexico and Central America, where they provide 50% of the calories in the diet. There is also an increased use of tortillas all over the United States as the popularity of Mexican food is growing. The products made from maize have become very popular in other countries of America and Europe (Paredes-López and Mora-Escobedo, 1983; Gutiérrez-Dorado et al., 2008). The two quintessential nixtamalized snacks, tortilla chips and corn chips, are positioned in second place in sales in the world, after potato chips, and represent a significant income in the United States (Paredes et al., 2008). Factories have been started in Australia, India, China, Korea and other countries (Bello-Perez et al., 2002). According to official numbers, Mexico is the country with the highest per capita consumption of tortillas with about 120 kg tortilla per inhabitant per year. Maize-tortilla chain itself represents 1% of gross domestic product, with an economic impact of about 7 million dollars a year. In this country, more than 50% of domestic production of white maize is for human consumption, which is ingested in the form of tortilla (made from nixtamalized corn) and other traditional products as atoles, tamales, pozole, etc. (SIAP-SAGARPA, 2007).

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4.1. Corn Grain Corn grain represents about half of the dry matter of plant (FAO, 1993). As mentioned above, the grain is a fruit composed by a pericarp that encloses a single seed. This type of fruit in which the pericarp is not opening at maturity, is characteristic of cereals and known as caryopsis. Grains are arranged in an ear or cob to which are attached by means of a lower appendix or tip-cap. An ear contains from 300 to 1000 kernels whose weight can vary between 190-300 g/1000 grains, depending on its genetics, the environment and farming practices.

Figure 3. Cob and grain structure.

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The main parts of corn kernel are the endosperm (80-85%), pericarp (5-6%) and germ (10-12 %) (Figure 3). The pericarp covering the grain consists of a group of layers tightly adhered to each other: epidermis, mesocarp and the layers of cross and tubular cells. The outer surface of the epidermis is permeated with wax which prevents moisture loss during grain development. The mesocarp is composed of thick-walled elongated cells that form a compact structure. By contrast, the inner layers of transverse and tubular cells show numerous intercellular spaces. Due to those features, there is a line of weakening between the mesocarp and the inner layers of pericarp along which the pericarp is easily separated from the rest of the grain (Brites et al., 2007). The endosperm is a continuous tissue with an outer zone, rich in proteins and oil, named aleurone. In the rest of the tissue, called starchy endosperm, two regions are also distinguished. The area near the aleurone, called vitreous endosperm, is of hard consistency and high protein content, while the inner zone, known as floury endosperm is opaque (Hoseney, 1991). The starch is mainly found in the elongated cells of the endosperm, packaged in granules of 5-30 μm, and embedded in a continuous protein matrix. Protein matrix is thin in the floury endosperm and does not fully cover the starch granule which takes on a round shape. This endosperm is opaque due to large amount of air spaces. On the other hand, the vitreous endosperm is very compact, without air spaces; it has a thicker wall forming polyhedral granules due to the compression that they suffer (Hoseney, 1991; Watson, 1991). The maize germ contains low starch content but is rich in oil and proteins. In this part of the grain, there are enzymes capable of degrading endosperm starch, which will serve as nutrient of the embryo during germination (Brites et al., 2007). There are various colors of grains: white, yellow, blackish, bluish-gray, purple, red, orange and green, although the most common are white and yellow. According to Watson (1991), there are five general classes of maize based on the grain characteristics: dent, flint, sweet, popcorn and flour corn. The top of dent corn (Zea mays L. subsp. mays Indentata Group), is floury; the hydration of this area causes a slight collapse during maturation that produces a depression in the crown and the characteristic dent appearance. Its texture is soft and the specific weight is low; it has high tendency to break during harvesting, transport and storage, which facilitates the attack of insects and fungi. This kind of maize has high starch content, while its protein content is low. This corn is the most commercially important and the one preferred to be processed by wet milling and animal feed. Flint maize (Zea mays L. subsp. mays Indurata Group) has higher hardness due to the presence of a large volume of vitreous endosperm, which provides greater mechanical strength during transport and silage. This corn has the major protein content due to its more vitreous endosperm. Besides, it has higher oil and carotene content than dent corn. This type of maize is the ideal to be processed by dry milling in the flour production. The popcorn (Zea mays L. subsp. mays Everta Group) has the very hard vitreous endosperm, with only a small fraction of starch enclosed in a dense and tough pericarp. When this grain is heated, the moisture trapped in the floury part of endosperm is expanded and explodes, creating the popcorn. The endosperm of floury corn (Zea mays L. subsp. mays Amylacea Group) is almost totally floury and opaque. This kind of corn is very susceptible to rotting and attack by worms and insects because of its soft structure.

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It is widely used by human consumption in Mexico. The sweet corn endosperm (Zea mays L. subsp. mays Saccharata Group) is made up mainly by sugar, with low starch content.

4.2. Grain Composition The main parts of corn differ in their chemical composition (Table 1). Pericarp is characterized by its high fiber content which is mainly composed by hemicelluloses, cellulose and lignin. The endosperm is noted by its high starch content, and the germ is rich in oil, proteins and minerals. Due to the great genetic diversity of the species, by nutritional and technologic improvement, as well as the influence of environment on the cultivation, there is a high variability in the chemical composition of grain. This variability is due in part to the different proportions of the grain components, whose distribution can vary in the different types of maize.

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4.2.1. Proteins After starch, proteins are the next chemical component of higher importance in grain. In common varieties, the protein content can range between 6 and 18%. The majority of protein is found in the endosperm. Maize proteins are formed by at least five different fractions according to the Osborne pattern (Hoseney, 1991): albumins and globulins (≈5%), prolamines (≈44%), glutelins (≈28%) and residual protein (≈17%), these amounts can vary depending on the corn type and the method of separation used. Zein is the major protein fraction in the grain constituting 44-79% of the total protein of endosperm. It is alcoholsoluble and can be extracted in the presence or absence of a reducing agent. Zein belongs to the prolamine group; its primary structure, its ability to bind via disulfide bonds, its location in the protein bodies and increased deposition in the vitreous endosperm, suggest that this protein is responsible for the hardness of the corn grain (Lawton and Wilson, 2003). 4.2.2. Amino Acids Maize proteins are defected in two essential amino acids: lysine and tryptophan. Because the endosperm represents between 70 and 80% of the grain weight, the essential amino acid content is a reflection of the amino acid content in the protein of the endosperm, in spite of the fact that the amino acid pattern of germ is higher and better balanced (FAO, 1993). Zein fraction (the major protein in endosperm) is deficient in lysine and tryptophan and has high leucine content. The leucine excess antagonizes the utilization of isoleucine and can thus produce the deficiency of this one (Brites et al., 2007). QPM (Quality Protein Maize) varieties have been developed which contain approximately twice the content of lysine and tryptophan in the endosperm than the common maize (Mendoza-Elos et al., 2006). 4.2.3. Lipids The lipids in maize grain are principally in the germ and its content varies between 3 and 18 %, depending on genetic aspects (Brites et al, 2007). The germ contains 84% of the lipids of the grain, and the remaining 16% is in the endosperm. The lipids of maize flour have low saturated fatty acids, mainly palmitic (13%) and stearic acids (4%); and, on the contrary,

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maize lipids has high content of unsaturated fatty acids: linoleic (50%), oleic (35%) and linolenic (