Kokumi Substance as an Enhancer of Koku: Biochemistry, Physiology, and Food Science 9819983029, 9789819983025

Citation preview

Motonaka Kuroda Editor

Kokumi Substance as an Enhancer of Koku Biochemistry, Physiology, and Food Science

Kokumi Substance as an Enhancer of Koku

Motonaka Kuroda Editor

Kokumi Substance as an Enhancer of Koku Biochemistry, Physiology, and Food Science

Editor Motonaka Kuroda Institute of Food Sciences & Technologies Ajinomoto Co., Inc. Kawasaki, Kanagawa, Japan

ISBN 978-981-99-8302-5 ISBN 978-981-99-8303-2 https://doi.org/10.1007/978-981-99-8303-2

(eBook)

© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2024 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore Paper in this product is recyclable.

Preface

This book describes biochemistry, physiology, and food science of kokumi substance as an enhancer of koku. Kokumi substance is one of the taste-related kokuenhancers. The definition of koku is described in a book entitled Koku in Food Science and Physiology (Springer Nature LLC, 2019). In this book, koku is defined as total sensations that are sensed through taste, smell (odor), and texture. The fundamental elements of koku were proposed as complexity, mouthfulness, and lingeringness (continuity). As described before, kokumi substance is one of the taste-related koku-enhancing substances. Originally, the word kokumi was made by combining “koku” and “-mi” which means taste in Japanese. Kokumi substance is defined as a taste-related substance which modifies the basic tastes and sensory characteristics like complexity, mouthfulness, and lingeringness (continuity) when added to foods although it is tasteless itself at the concentration tested. In this book, biochemical, physiological and sensory studies on various kokumi substances containing γ-glutamyl-peptides, α-peptides, and lipid-related compounds are described. I hope that information provided in this book shall give an outlook of the definition of “kokumi substance,” as an enhancer of koku, and can be a concise starting book for all interested in biochemistry, physiology, and food science of kokumi substances. Kawasaki, Kanagawa, Japan

Motonaka Kuroda

v

Contents

Part I

General Concept

1

Koku Perception and Kokumi Substances . . . . . . . . . . . . . . . . . . . . Toshihide Nishimura

3

2

Kokumi Substance as an Enhancer of Koku: Its Definition . . . . . . . Motonaka Kuroda

15

Part II

Kokumi γ-Glutamyl-Peptides

3

Biochemical Studies on Kokumi γ-Glutamyl Peptifdes . . . . . . . . . . Motonaka Kuroda

25

4

Kokumi Substances from Garlic; Discovery of Glutathione (GSH; γ-Glu-Cys-Gly) as a Kokumi Substance . . . . . . . . . . . . . . . . Yoichi Ueda and Motonaka Kuroda

33

5

Kokumi Substances in Soybean Seeds . . . . . . . . . . . . . . . . . . . . . . . Masayuki Shibata and Yasuki Matsumura

6

Kokumi Substances in Thai-Fermented Freshwater Fish, “Pla-ra” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Preecha Phuwapraisirisan, Apiniharn Phewpan, Panita Ngamchuachit, Kannapon Lopetcharat, Chirapiphat Phraephaisarn, Corinna Dawid, Thomas Hofmann, and Suwimon Keeratipibul

41

83

7

Identification and Quantification of the Kokumi Peptide, γ-Glu-Val-Gly, in Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 Motonaka Kuroda and Toshimi Mizukoshi

8

Mechanism for Perceiving Kokumi Substances: Involvement of Calcium-Sensing Receptor (CaSR) in the Perception of Kokumi Substances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133 Motonaka Kuroda vii

viii

Contents

9

Molecular Mechanism of Enhancement in Basic Tastes by Kokumi Substances: A Potent Calcium-Sensing Receptor (CaSR) Agonist, γ-Glutamyl-Valinyl-Glycine, Amplifies Sweet-Induced ATP Secretion Via Cell-to-Cell Communication in a Mouse Taste Bud . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 Yutaka Maruyama

10

Enhancement of Combined Umami and Salty Taste by Glutathione in the Human Tongue and Brain . . . . . . . . . . . . . . . 159 Tazuko K. Goto, Andy Wai Kan Yeung, Hiroki C. Tanabe, Yuki Ito, Han-Sung Jung, and Yuzo Ninomiya

11

γ-Glutamyl-Valyl-Glycine (γ-Glu-Val-Gly) and Glutathione (γ-Glu-Cys-Gly) as Kokumi Substances in Rodents . . . . . . . . . . . . . 177 Takashi Yamamoto

12

Effects of the Potent Kokumi Peptide, γ-Glutamyl-Valyl-Glycine, on Sensory Characteristics of Foods and Beverages . . . . . . . . . . . . 187 Motonaka Kuroda

13

Perceptual and Nutritional Impact of Kokumi Compounds . . . . . . . 229 Ciarán Forde and Markus Stieger

Part III 14

Amino Acids, α-Peptides, and Their Related Kokumi Substances

Amino Acids, α-Peptides, and Their Related Kokumi Substances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245 Motonaka Kuroda

Part IV

Lipid-Related Kokumi Substances

15

Biochemical Studies on Lipid-Related Kokumi Substances . . . . . . . 255 Motonaka Kuroda

16

Involvement of GPR120 in Perception of Fatty Oral Sensations in Humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259 Naoya Iwasaki, Seiji Kitajima, and Motonaka Kuroda

Part V 17

Future Prospect

Overview and Future Prospects of Studies on Kokumi Substances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291 Motonaka Kuroda

Contributors

Corinna Dawid Chair of Food Chemistry and Molecular Sensory Science, Technical University of Munich (TUM), Freising, Germany Ciarán Forde Sensory Science and Eating Behaviour, Division of Human Nutrition and Health, Wageningen University, Wageningen, The Netherlands Tazuko K. Goto Oral Diagnosis and Polyclinics, Faculty of Dentistry, The University of Hong Kong, Pok Fu Lam, Hong Kong Department of Oral and Maxillofacial Radiology, Tokyo Dental College, Tokyo, Japan Thomas Hofmann Chair of Food Chemistry and Molecular Sensory Science, Technical University of Munich (TUM), Freising, Germany Yuki Ito Mitsubishi Shoji Foodtech Co., Ltd., Tokyo, Japan Naoya Iwasaki Institute of Food Sciences and Technologies, Ajinomoto Co., Inc., Kawasaki, Kanagawa, Japan Han-Sung Jung Oral Biosciences, Faculty of Dentistry, The University of Hong Kong, Pok Fu Lam, Hong Kong Suwimon Keeratipibul ChulaUnisearch, Chulalongkorn University, Pathumwan, Bangkok, Thailand Seiji Kitajima Institute of Food Sciences and Technologies, Ajinomoto Co., Inc., Kawasaki, Kanagawa, Japan Motonaka Kuroda Institute of Food Research and Technologies, Ajinomoto Co., Inc., Kawasaki, Kanagawa, Japan Kannapon Lopetcharat Nouveau Centric Co. Ltd., Suanluang, Bangkok, Thailand

ix

x

Contributors

Yutaka Maruyama Department of Physiology and Biophysics, University of Miami Miller School of Medicine, Miami, FL, USA Institute of Food Sciences and Technologies, Ajinomoto Co., Inc, Kawasaki, Kanagawa, Japan Yasuki Matsumura Research Institute for Sustainable Humanosphere, Kyoto University, Gokasho, Uji-shi, Kyoto, Japan Toshimi Mizukoshi Research Institute for Bioscience Products and Fine Chemicals, Ajinomoto Co., Inc., Kawasaki, Kanagawa, Japan Panita Ngamchuachit Department of Food Technology, Faculty of Science, Chulalongkorn University, Pathumwan, Bangkok, Thailand Yuzo Ninomiya Section of Oral Neuroscience, Graduate School of Dental Sciences, Kyushu University, Fukuoka, Japan Division of Sensory Physiology, Research and Development Center for Taste and Odor Sensing, Kyushu University, Fukuoka, Japan Toshihide Nishimura Faculty of Nutrition, Kagawa Nutrition University, Sakado, Saitama, Japan Apiniharn Phewpan CPF Research and Development Center Company Limited, Ayutthaya, Thailand Chirapiphat Phraephaisarn Research and Development Office, Betagro Group, Pathumthani, Thailand Preecha Phuwapraisirisan Department of Chemistry, Faculty of Science, Chulalongkorn University, Pathumwan, Bangkok, Thailand Masayuki Shibata Research Institute for Creating the Future, Fuji Oil Holdings Inc., Tsukubamirai-shi, Ibaraki, Japan Markus Stieger Sensory Science and Eating Behaviour, Division of Human Nutrition and Health, Wageningen University, Wageningen, The Netherlands Hiroki C. Tanabe Graduate School of Environmental Studies, Department of Social and Human Environment, Nagoya University, Nagoya, Japan Yoichi Ueda Institute of Food Research and Technologies, Ajinomoto Co., Inc., Kawasaki, Kanagawa, Japan Takashi Yamamoto Department of Nutrition, Faculty of Health Sciences, Kio University, Nara, Japan Health Science Research Center, Kio University, Nara, Japan Andy Wai Kan Yeung Oral Diagnosis and Polyclinics, Faculty of Dentistry, The University of Hong Kong, Pok Fu Lam, Hong Kong

Part I

General Concept

Chapter 1

Koku Perception and Kokumi Substances Toshihide Nishimura

Abstract The perception of “Koku” in food has been associated with deliciousness, but its definition has been vague, leading to its ambiguous usage. In recent years, efforts have been made to clarify the definition of koku perception. It has been discovered that koku perception encompasses the overall sensory experience of taste, aroma, and texture and can be objectively described in terms of three elements: complexity, mouthfulness, and lastingness. Additionally, certain substances have been identified as enhancers of koku perception. This paper provides a definition of koku perception and explains the three elements based on this definition and the substances that enhance koku perception. Furthermore, it explores the role of kokumi substances in koku perception.

1.1

Definition of “Koku Perception”

The term “Koku” is commonly found on the packaging of various food products such as curry, cup noodles, coffee, cocoa, mayonnaise, cocktails, and kimchi. However, the exact perception that the word “Koku” refers to has been unclear due to the lack of a precise definition. In many cases, “Koku” has been used interchangeably with “deliciousness” because people often associate it with the enjoyment of delicious food. However, this is not entirely accurate, as “Koku” is not used to describe fresh foods like fruits, vegetables, or sashimi (raw fish). It is more commonly associated with foods prepared through simmering, aging, fermentation, or those containing fats or oils.

T. Nishimura (✉) Faculty of Nutrition, Kagawa Nutrition University, Sakado, Saitama, Japan e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2024 M. Kuroda (ed.), Kokumi Substance as an Enhancer of Koku, https://doi.org/10.1007/978-981-99-8303-2_1

3

4

1.1.1

T. Nishimura

Koku Perception by Foods

The palatability of food is subjectively determined by various sensory experiences derived from taste compounds, aroma compounds, and the food’s structure (Fig. 1.1). Taste perception involves the five basic tastes: sweet, bitter, sour, salty, and umami, which are triggered by taste compounds. Aroma plays a crucial role in food perception and is experienced through olfaction. There are two types of aroma sensations: orthonasal aroma, perceived before food enters the mouth, and retronasal aroma, perceived during putting food in mouth or after chewing. The latter, released from the food in the mouth, significantly influences the palatability of food. Textural sensations, such as the softness of meat or the creaminess of a sauce, are detected by the somatic senses. Koku perception is a result of the combined effects of taste, aroma, and texture. Therefore, koku perception is objectively defined as the perception induced by these factors. To objectively evaluate koku perception, it can be assessed based on three fundamental elements: complexity, mouthfulness, and lastingness (Figs. 1.2 and 1.3) (Nishimura 2019).

Fig. 1.1 Factors involved in food palatability

Fig. 1.2 Elements in taste and koku attributes




1

Koku Perception and Kokumi Substances

5

Taste stimulation Aroma stimulation Texture stimulation

Fig. 1.3 The definition of Koku

1.1.2

“Koku” and “Deliciousness” Are Not Synonymous

A study on the etymology of the word “koku” reveals two possible roots: one meaning “thickness” and the other referring to the ripeness of grain in Chinese. However, the specific meaning of “Koku” in the context of food perception in Japan has remained unclear. It is important to note that “Koku” and “deliciousness” are not synonymous. While some foods may be rich and delicious, not everyone considers richness to be synonymous with deliciousness. Conversely, some foods can be delicious even without being rich. The association of the term “complexity (richness)” with the flavor of food is rooted in people’s subjective experiences. When a food has a strong flavor, some individuals may perceive it as “too strong” or “too rich,” while others may find it lacking in flavor if it is not rich enough. In many cases, people describe food as “delicious” when it exhibits a level of koku perception that suits their preferences, leading to the misconception that “Koku” and “deliciousness” are equivalent. However, an objective examination reveals that koku perception does not always correspond to deliciousness.

1.1.3

Definition of “Koku Perception”

While fresh foods like fruits, vegetables, and sashimi (raw fish) are not typically associated with richness of flavor, the term “Koku” is commonly used for foods that

6

T. Nishimura

have undergone long cooking processes, aging, fermentation, or those containing fats or oils. Koku perception is elicited by a combination of taste, aroma, and texture stimuli resulting from the composition and structure of the food. For instance, miso soup is considered a Koku food, but when miso is dissolved in hot water without umami seasoning, the soup’s flavor and lastingness are weak. However, when umami seasoning is added, the overall flavor becomes stronger, more expansive, and more persistent, enhancing the sense of full-body; that is “Koku perception.” Pinching one’s nose while eating curry rice or stew, which are typically associated with koku and deliciousness, weakens the flavor due to the reduction in the unique aroma’s complexity, persistence, and mouthfulness. Based on previous findings, the author defines koku perception as complexity, mouthfulness, and lastingness resulting from taste, aroma, and texture components.

1.2

Three Elements Involved in “Koku Perception”

Just as there are five basic tastes such as sweet, sour, bitter, salty, and umami, the three elements of koku perception are defined as “complexity,” “mouthfulness,” and “lastingness.” These elements are collectively referred to as “basic Koku” (Fig. 1.2). Each element has its own intensity of perception, similar to how taste intensity evaluated. For instance, tonkotsu (pork bone) ramen is renowned for its lingering and enduring rich flavor. On the contrary, light soy sauce ramen has a flavor that fades quickly. Hence, there are objective strengths and weaknesses in the persistence of koku perception. Now, let’s explore how each of these factors arises.

1.2.1

Complexity in Koku Perception

When food or its ingredients undergo heat treatment, aging, or fermentation processes, a wide variety of tasting and aromatic substances are produced, resulting in a sense of complexity (richness) in the food (Fig. 1.4). These processes trigger the production of diverse taste and aroma compounds through intrinsic enzymes in the food ingredients, chemical reactions between substances, and the action of microorganisms. The intensity of complexity is believed to be determined by the type and quantity of these substances, which can be controlled by the duration of heat treatment, aging, and fermentation processes. The longer the processing times, the stronger the complexity of the foods with koku (Koku foods). The complexity of a Koku food is closely linked to its characteristics. However, assessing the intensity of complexity through sensory evaluation or compound analysis can be challenging. Therefore, it is proposed to evaluate the intensity of complexity based on the processing period or time in the production of Koku foods.

1

Koku Perception and Kokumi Substances

.a few stimulation .not rich

7

.lots of stimulations .rich

Fig. 1.4 Formation of complexity in koku attributes of foods

1.2.2

Mouthfulness in Koku Perception

Mouthfulness refers to the intensity of sensation derived from the complex stimuli produced by heating processes, aging, and fermenting of foods. In general, the longer the respective processing times, the stronger the mouthfulness, as well as the complexity, of the Koku food. When a Koku food with abundant stimuli is consumed, the overall sensation is perceived more intensely as these stimuli spread throughout the oral cavity. And despite the presence of numerous compounds in Koku foods, many of them exist below their threshold levels. These compounds are also believed to enhance mouthfulness and are referred to as “Kakushi-aji” in Japan. In cases where the mouthfulness of certain Koku foods is weak, compounds with an enhancing effect on mouthfulness can be added to intensify the perception. Kokumi substances such as allin of garlic, PeCSO (propenyl-cysteine sulfoxide) of onions, and Maillard peptides, at below their threshold levels, have been found to enhance the intensity of mouthfulness in Koku foods. These compounds are extracted from food stuffs and produced during processing.

1.2.3

Lastingness in Koku Perception

Lastingness refers to the sensation of unique stimuli from a food persisting in the mouth for an extended period. Lastingness in koku perception can arise from taste, aroma, or texture stimuli. However, if these stimuli linger for too long, they may be perceived as “undelicious” or “persistent,” negatively impacting the overall deliciousness of the food. Appropriate intensity in lastingness contributes to the

8

T. Nishimura

deliciousness of Koku foods. Lastingness can be objectively evaluated by measuring the duration of the stimulus remaining in koku perception. Substances that contribute to lastingness in Koku foods can be naturally produced or extracted from food materials. They can also be added during cooking or food processing. Now, umami compounds and lipids have been identified as substances that enhance lastingness in Koku foods, increasing their perceived intensity.

1.3 1.3.1

Substances Involved in Each Element of Koku Perception Substances Involved in the Complexity of Koku Perception

The substances contributing to the complexity of koku perception are extracted from food ingredients during cooking. They can also be produced through chemical reactions during heating and the action of microorganisms during aging or fermentation processes. These substances include taste compounds such as free amino acids, peptides, organic acids, saccharides, and more. Additionally, aroma compounds play a significant role in expressing the characteristics of foods, and there are also compounds that contribute to viscosity. Hence, a wide range of compounds are involved in the complexity of koku perception and the overall characteristics of Koku foods.

1.3.2

Substances Involved in the Mouthfulness of Koku Perception

In general, the longer the processing times in heating, aging, or fermentation, the stronger the intensity of both mouthfulness and complexity in Koku foods. This is because the substances in foods increase during extended processing, resulting in higher amounts of many substances in the final product compared to the initial ingredients. The increase in substances intensifies the overall sensation in Koku foods, leading to a stronger perception of mouthfulness. In cases where the intensity of mouthfulness in Koku foods appears to be weak, it can be enhanced by adding substances that enhance mouthfulness. As described before, various substances have been reported to enhance the intensity of mouthfulness, such as umami substance, allin from garlic, and trans-S-propenyl-L-cysteine sulfoxide (PeCSO), Maillard peptides, and certain aroma components like phthalides found in celery. The miso soup (50 g/L) without seasonings showed us very weak sensation in the space of our oral cavity, while it has the characteristic flavor and complexity of miso soup. The addition of umami substances to this miso soup can enlarge the intensity of spread of flavor sensation in our oral cavity without changing its characteristic

1

Koku Perception and Kokumi Substances

9

3.5

Intensity of aroma sensation

3 2.5

MSG

2 1.5 1 0.5 0

0

0.1

0.2

0.3

0.4

0.5

Concentration of MSG (%)

Addition of umami subsatance such as Glu enhanced 2.5 times high on the retronasal aroma sensation. Fig. 1.5 Effects of umami substance on the intensity of aroma sensation using a flavored solution

flavor. Yamaguchi et al. reported that umami substances had the effect of flavor enhancer of foods when they were added to foods (Yamaguchi and Kimizuka 1979). Recently, Nishimura et al. examined the mechanism of flavor enhancement by the addition of umami substances to foods. The analysis using model chicken extract clarified that the addition of monosodium glutamate to the extract made the sensation of retronasal aroma 2.5 times higher than no addition (Nishimura et al. 2016b) (Fig. 1.5). This effect became higher; the concentration of added MSG was higher until 0.3%. The addition of MSG at over 0.3% made the sensation of retronasal aroma of the extract weaker, while the intensity of umami taste became stronger. Therefore, umami substances have the strong effect of the mouthfulness in koku perception by enhancing flavor, especially retronasal aroma. Other than umami substances, there have been reports for the substances involved in mouthfulness of the koku perception. In 1990, Ueda et al. (Ueda et al. 1990) found that the addition of a water extract of garlic to soups enhanced the sensation of continuity (lastingness), mouthfulness, and thickness (complexity) in soups. It was also found to have this enhancement effect when it was added to the umami solution composed of 0.05% monosodium glutamate and disodium inosinate. The key compounds in a water extract of garlic were clarified to be allin, S-methyl-L-cysteine sulfoxide, and γ-L-glutamyl-S-allyl-L-cysteine. Furthermore, the addition of alliin at 0.05% (w/v), which had not any aroma and taste, was reported to have the enhancement effect of the soups. Ueda et al. (Ueda et al. 1994) also clarified that PeCSO or its γ-glutamyl peptide (γ-Glu-PeCSO) enhanced the sensation of lastingness, mouthfulness, and complexity in the umami solution by its addition at a

10

T. Nishimura

concentration of 0.02% (w/v). It is also reported that these compounds at this concentration did not cause any aroma and taste in the umami solution. Kuroda et al. (Kuroda et al. 2013) also discovered kokumi peptide, γ-L-glutamylL-valyl-glycine (γ-EVG), from soy sauce, raw scallop, and processed scallop products. Then these kokumi peptides were perceived through a calcium-sensing receptor (CaSR) (Ohtsu et al. 2010). However, there is no clarification of the mechanisms for the sensation of mouthfulness and lastingness by the binding kokumi peptides to CaSR. Dunkel et al. (Dunkel and Hofmann 2009) have reported that addition of nearly tasteless aqueous extract isolated from beans to a model chicken broth enhanced the sensation of mouthfulness and complexity and successively induced long-lasting savory taste on the tongue. They clarified γ-L-glutamyl-L-leucine, and γ-L-glutamyl-L-cysteinyl-β-alanine as key molecules and called them kokumi peptides. Then, they have clarified that γ-L-glutamyl peptides such as γ-glutamyl-Glu, γ-glutamyl-Gly, and so on found in Gouda cheese were key compounds enhancing mouthfulness and lastingness of the matured cheese. Ogasawara et al. (Ogasawara et al. 2006a,b) have found that Maillard peptides with a molecular weight of 1000–5000 from soybean paste enhanced the sensation of lastingness and mouthfulness of flavor in the umami solution and consommé soup when these peptides were added to these solutions. Maillard peptides, which increased during fermentation in the production process of long-ripened miso, were thought to be key substances to enhance the characteristic flavor of miso. Kurobayashi et al. (Kurobayashi et al. 2008) have reported that the addition of three phthalides, such as sedanenolide, 3-n-butylphthalide, and sedanolide, to broth enhanced the umami intensity as well as complexity of chicken broth when these phthalides were added to the broth at a concentration that no distinct orders were detected by sensory evaluation.

1.3.3

Substances Involved in the Lastingness of Koku Perception

Lastingness refers to the sensation of complex stimulation remaining in the oral cavity for an extended period in koku perception. Lastingness in Koku foods is not solely a result of mouthfulness enhanced by abundant stimuli; specific substances play a role in creating lastingness. Umami substances and lipids have already been identified as substances that exhibit this effect (Fig. 1.6). Umami substances themselves leave a sensory stimulus in koku perception. For example, pork sausages with different levels of umami substances were found to have varying degrees of flavor lastingness, with products containing higher levels of umami substances exhibiting stronger lastingness. Fats and oils, despite being tasteless and odorless, also play a role in creating lastingness. They bind nonspecifically to the flavor and aroma components of other ingredients when cooked (Fig. 1.7). In foods that contain a significant amount of fats and oils, the

1

Koku Perception and Kokumi Substances

11

2 2

1

: : :

.

Fig. 1.6 Visualization of mouthfulness and lastingness in Koku perception

*

soups without (

) and with (

*

*

*

*

*

)β

Fig. 1.7 Effect of addition of 0.05% β-sitosterol on the sensory characteristics of Chinese soup

persistence of taste and aroma is perceived during consumption as the aroma gradually releases into the oral cavity from the fats and oils present (Nishimura et al. 2016a; Nishimura and Egusa 2019). This may explain why foods with fats and oils are often perceived as delicious. Overall, koku perception encompasses the three elements of complexity, mouthfulness, and lastingness, as visualized in the figure. The expression “Strong Koku perception with long-lastingness” or “Weak perception with short-lastingness” is recommended to be on the packaging of Koku foods.

12

T. Nishimura

Fig. 1.8 Kokumi substances enhance koku perception

: ⇒Glutathione, γ (

.

)

1 2

Utilizing this definition in the development of Koku foods and cooking foods can be beneficial in food production and cooking.

1.4

Kokumi Substances Enhancing the Intensity of Koku Perception

Even after proposing a definition of “koku,” food packages and information in foodrelated magazines worldwide continue to use different words to describe it, such as kokumi, koku-taste, koku-flavor seasoning, and others related to koku. All these words are thought to be associated with the deliciousness of foods. However, it is possible that each term is not used correctly, which can confuse consumers. In particular, “kokumi taste” is often used as a synonym for koku, even though it has a completely different meaning and should not be used alone. There is a significant reason why “kokumi” is used instead of “koku perception.” As described in the later chapter, in 2010, the receptor protein (CaSR) to which glutathione and γ-glutamylvalylglycine (γ-EVG) bind was identified (Kuroda et al. 2013), and these kokumi substances enhance the intensity of taste in koku foods. Therefore, the term “kokumi” is often used interchangeably with “koku perception.” However, it is important to note that “kokumi” or “kokumi taste” is different from “koku perception” and should not be used as a synonym for it. This is because kokumi substances have an enhancement effect on koku perception only when added to food or solutions at concentrations below their threshold levels (Fig. 1.8). To summarize, kokumi substances such as glutathione and γ-EVG are the enhancer of koku perception in Koku foods. Therefore, these substances should be referred to as “kokumi substances” or “kokumi peptides” rather than “kokumi taste” or “kokumi perception” to avoid confusion with the broader concept of koku perception.

1

Koku Perception and Kokumi Substances

13

References Dunkel A, Hofmann T (2009) Sensory-directed identification of b-alanyl dipeptides as contributors to the thick-sour and white -meaty orosensation induced by chicken broth. J Agric Food Chem 57:9867–9877 Kurobayashi Y, Katsumi Y, Fujita A, Morimitsu Y, Kubota K (2008) Flavor enhancement of chicken broth from boiled celery constituents. J Agric Biol Chem 56:512–516 Kuroda M, Kato Y, Yamazaki J, Kageyama N, Mizukoshi T, Miyama H, Eto Y (2013) Determination of γ-glutamyl-valyl-glycine in scallop and processed scallop products using high pressure liquid chromatography-tandem mass spectrometry. Food Chem 141:823–828 Nishimura T (2019) Definition of “Koku” Involved in Food Palatability. In: Nishimura T, Kuroda M (eds) Koku in food science and physiology. Springer, pp 1–16 Nishimura T, Egusa SA (2019) Umami compounds and fats involved in Koku attributes of Pork Sausages. In: Nishimura T, Kuroda M (eds) Koku in Food Science and Physiology. Springer, pp 47–58 Nishimura T, Egusa AS, Nagao A, Odahara T, Sugise T, Mizoguchi N, Nosho Y (2016a) Phytosterols in onion contribute to a sensation of lingering of aroma, a koku attribute. Food Chem 192:724–728 Nishimura T, Goto S, Miura K, Takakura Y, Egusa AS, Wakabayashi H (2016b) Umami compounds enhance the intensity of reteronasal sensation of aromas from modek chicken soups. Food Chem 196:577–5835 Ogasawara M, Katsumata E, Egi M (2006a) Taste properties of maillard-reaction products prepared from 1000 to 5000 Da peptide. Food Chem 99:600–604 Ogasawara M, Yamada Y, Egi M (2006b) Taste enhancer from the long-term ripening of miso (soybean paste). Food Chem 99:736–741 Ohtsu T, Amino Y, Nagasaki H, Yamanaka T, Takeshita S, Hatanaka T, Maruyama Y, Miyamura N, Eto Y (2010) Involvement of the calcium-sensing receptor in human taste perception. J Biol Chem 285:1016–1022 Ueda Y, Sakaguchi M, Hirayama K, Mijajima R, Kimizuka A (1990) Characteristic flavor constituents in water extract of garlic. Agric Biol Chem 54:163–169 Ueda Y, Tsubuku T, Miyajima R (1994) Composition of sulfur-containing components in onion and their flavor characters. Biosci Biotechnol Biochem 58:108–110 Yamaguchi S, Kimizuka A (1979) Psychometric studies on the taste of monosodium glutamate. In: Filer LJ Jr et al (eds) Glutamic acid: advances biochemistry and physiology. Raven Press, New York, pp 35–54

Chapter 2

Kokumi Substance as an Enhancer of Koku: Its Definition Motonaka Kuroda

Abstract As describe in Chap. 1, Koku is defined as total sensations that sensed through taste, smell (odor), and texture. The fundamental elements of koku were proposed as complexity, mouthfulness, and lingeringness (continuity). Kokuenhancing substances are divided into three groups: taste-related substances, smell-related ones, and texture-related ones. Taste-related koku-enhancing substances consist of tastants and taste-modulators. Regarding koku-enhancing tastants, monosodium glutamate (MSG), an umami substance, was reported to enhance the thickness, complexity, continuity, and mouthfulness when added to various kinds of soups. Similarly, sugar and salt (NaCl) have been reported to enhance these characters when added to foods. Among the taste-modulating substances, the substances which enhance basic taste and flavor characters such as complexity, mouthfulness, and lingeringness when added to foods although they were tasteless at the concentration tested are referred to kokumi substances. In this chapter, the actions of smell (odor)-related koku-enhancing substances are introduced. Phthtalide compounds identified in celery were reported to enhance the thick and lasting flavors when added to chicken broth. Rotundone identified in fruits and spices enhanced the intensities of complex flavor when added to fruits-flavored beverages. Regarding the texture-related koku-enhancing substances, the additional effect of glycogen to reconstituted scallop extract was described, and indicated that it enhanced the continuity, complexity, fullness and mildness.

2.1

Classification of Koku-Enhancing Substances

Koku is defined as total sensations that are sensed through taste, smell, and texture. It has been reported that there are various koku-enhancing substances. Figure 2.1 reveals the classification of various koku-enhancing substances. Koku-enhancing

M. Kuroda (✉) Institute of Food Research and Technologies, Ajinomoto Co., Inc., Kawasaki, Kanagawa, Japan e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2024 M. Kuroda (ed.), Kokumi Substance as an Enhancer of Koku, https://doi.org/10.1007/978-981-99-8303-2_2

15

16

M. Kuroda [Taste-related koku-enhancers]

[Odor-related koku-enhancers]

.Tastants: Basic taste substances (Umami, Sweet, Salt etc)

.Odorants: Pyrazines, Phthalides, Rotundone, Allicin etc.

.Taste-modulating substances: Kokumi substances (glutathione, γ-Glu-peptides, Maillard peptides Alliin, Methylcysteineoxide)

.Odor-modulating substances: Phytosterols etc.

Foods with koku

[Texture-related koku-enhancers] .Texture-modifying substances: Oils, Gelatin, Glycogen, Tropomyosin etc.

Fig. 2.1 Classification of koku-enhancing substances

substances are divided into three groups: taste-related substances, smell-related ones, and texture-related ones.

2.1.1

Taste-Related Koku-Enhancing Substances

Taste-related koku-enhancing materials consist of tastants and taste-modulating substances. Yamaguchi et al. (Yamaguchi and Kimizuka 1979) investigated the effects of several tastants on the flavor of foods using a scoring method from 25 to 50 accessors. They reported that the addition of monosodium glutamate (MSG), one of umami substances, enhanced continuity, mouthfulness, and thickness when added to various kinds of soup such as beef consommé, chicken noodle soup, and chicken cream soup (Fig. 2.2), although aqueous MSG solution does not elicit such sensations. Yamaguchi et al. (Yamaguchi and Kimizuka 1979) also reported that salt (NaCl) enhanced the continuity, mouthfulness, and thickness when added to reduced-salt (0.2% w/w) beef consommé (Fig. 2.3), and sugar enhanced the continuity, mouthfulness, and thickness when added to Bavarian cream (Fig. 2.4). These results suggest that umami, salty, and sweet tastants can act as koku-enhancing substances. There have also been several studies on taste-modulating koku-enhancing substances. The substances which modify the basic tastes and flavor characteristics such as complexity, thickness of taste, continuity, and mouthfulness are proposed to be called kokumi substances (Ueda et al. 1990). Ohsu et al. (Ohsu et al. 2010) reported that kokumi γ-glutamyl peptides are perceived by calciumsensing receptor (CaSR). Therefore, kokumi substances consist of CaSR agonists and others.

2

Kokumi Substance as an Enhancer of Koku: Its Definition

(A)

17

(B)

AROMA

Whole aroma Meaty Vegetable-like

*

BASIC TASTE

Whole taste Salty Sweet Sour Bitter

*

*

*

* *

*

FLAVOR CHARACTER Continuity Mouthfulness Impact Mildness Thickness

* *

OTHER FLAVOR

Spicy Oily Meaty Vegetable-like

*

*

*

* *

*

*

* * * * *

* *

*

*

Fig. 2.2 Additional effect of (A) 0.2% monosodium glutamate (MSG) and (B) twice amount of beef broth on beef consommé. The sensory evaluation was performed by a scoring method comparing with the control beef consommé. -2, certainly weaker than the control; -1, slightly weaker than the control; 0, almost same; +1, slightly stronger than the control; +2, certainly stronger than the control. *, significant at a 95% confidence level

2.1.2

Odor-Related Koku-Enhancing Substances

It has also been considered that various volatile substances can act as kokuenhancing substances. Kurobayashi et al. (Kurobayashi et al. 2008) focused on the volatile compounds in celery which is often used in cooking of a stock or bouillon for the enhancement of a rich and complex flavor, and they indicated that the addition of volatile fraction from celery extract significantly enhanced the intensities of umami and thick, impactful, mild, lasting, satisfied, and clarified flavor. They identified several phthalide compounds as key components responsible for the enhancing effect, and revealed that the addition of single phthalide-skeleton-structure compound such as 3-n-butylphthalide, sedanenolide, and trans/cis-sedanolide at the concentrations below the detection thresholds to chicken broth enhanced the intensities of the sensory characteristics which were enhanced by celery volatile fraction. The results suggest that phthalide compounds act as koku-enhancing substances. Hayase et al. (Hayase et al. 2013) reported that seasoning soy sauces are koku (mouthfulness and continuity of the flavor)-enhancing odor substances. They prepared the aroma fraction by SDE and identified the key components by GC-O/ADA

18 Fig. 2.3 Additional effect of 0.6% sodium chloride (NaCl) on the reduced-salt (0.2%) beef consommé. The sensory evaluation was performed by a scoring method comparing with the control beef consommé. -2, certainly weaker than the control; -1, slightly weaker than the control; 0, almost same; +1, slightly stronger than the control; +2, certainly stronger than the control. *, significant at a 95% confidence level

M. Kuroda

AROMA

Whole aroma Meaty Vegetable-like

BASIC TASTE

Whole taste Salty Sweet Sour Bitter

* *

FLAVOR CHARACTER Continuity Mouthfulness Impact Mildness Thickness

*

Spicy Oily Meaty Vegetable-like

*

*

*

OTHER FLAVOR

Fig. 2.4 Additional effect of 10% sugar on the Bavarian cream. The sensory evaluation was performed by a scoring method comparing with the control beef consommé. -2, certainly weaker than the control; -1, slightly weaker than the control; 0, almost same; +1, slightly stronger than the control; +2, certainly stronger than the control. *, significant at a 95% confidence level

*

* *

*

AROMA

Whole aroma Egg-like Milky

BASIC TASTE

Whole taste Salty Sweet Sour Bitter

* *

FLAVOR CHARACTER Continuity Mouthfulness Impact Mildness Thickness

OTHER FLAVOR Spicy Oily Egg-like Milky

*

* *

* *

* *

*

(aroma extract dilution analysis). They identified three components, 2-acetylfuran, 2-ethylhexanol, and 1-octen-3-ol, as koku-enhancing odor substances from seasoning soy sauces. Among them, the addition of 2-acetylfyuran and 1-onten-3-ol under the intrinsic threshold enhanced koku when added to salty/umami

2

Kokumi Substance as an Enhancer of Koku: Its Definition

19

solution consisting of 0.5% NaCl and 0.3% monosodium glutamate (MSG), or seasoning soy sauce. Nakanishi et al. (Nakanishi et al. 2017) focused on rotundone which exist in various kinds of fruits and investigated the effect of rotundone on the sensory characteristics of four kinds of fruit-flavored beverages (grapefruit, orange, apple, and mango). The beverages were prepared by mixing sugar, citric acid, and aroma compounds reconstituted based on the analytical data reported in the literatures. The addition of rotundone at 5 ppt which is lower than the detection threshold in aqueous solution (8 ppt) enhanced the intensities of complex flavor when added to all kinds of fruits-flavored beverages. These results suggested that rotundone act as kokuenhancing substances in model fruits-flavored beverages.

2.1.3

Texture-Related Koku-Enhancing Substances

Although the number of studies is limited, several studies have been performed on the texture-related koku-enhancing substances. Watanabe et al. (Watanabe et al. 1990) investigated the effect of glycogen, which are contained in shellfish at high amount, on the sensory characteristics of scallop. They prepared the reconstituted scallop extract as shown in Table 2.1 and investigated the additional effect of scallop glycogen (4.9%, w/w) by sensory evaluation with 10 trained assessors. Although glycogen does not taste itself, the addition of glycogen significantly enhanced the intensities of continuity, complexity, fullness, and mildness, while it did not affect the intensities of basic tastes (Table 2.2). These results suggest that glycogen act as a koku-enhancing substance in scallop. Tomaschunas et al. (Tomaschunas et al. 2013) reported that addition of inulin and citrus fiber, kinds of texture-modifiers, to low-fat Lyon-style sausage increased the intensity of aftertaste meat flavor. Additionally, Liou and Grun (Liou and Grun 2007) reported that addition of fat mimetics such as microparticulated whey protein concentrate, one of texture-modifying agents, and polydextrose powder enhanced thickness. These previous studies suggest that texture-related substances also contribute to koku in foods.

Table 2.1 Composition of reconstituted scallop extract

Component Gly Arg Ala Glu･Na･H2O AMP･Na2 KOH KCl NaCl

Amount (mg/dL) 1925 256 323 179 195 232 109 71

20

M. Kuroda

Table 2.2 Additional effect of glycogen on the reconstituted extract of scallop

Basic taste Sweetness Sourness Saltiness Bitterness Umami Flavor character Continuity Complexity Fullness Mildness

t-value

Probability

1.16 1.45 0.62 1.14 1.71

n.s. n.s. n.s. n.s. n.s.

5.67 5.10 4.59 2.56

** ** ** *

n.s not significant, *p < 0.05, **p < 0.01

2.2

Kokumi Substance; Its Definition

As described in the previous section, kokumi substance is one of the taste-related koku-enhancing substances. Originally, the word “kokumi” was made by combining “koku” and “-mi” which means taste in Japanese. Kokumi substance is defined as a taste-related substance which modify the basic tastes and sensory characteristics like complexity, mouthfulness, and lingeringness (continuity) when added to foods, although it does not have any tastes itself at the dose tested (Ueda et al. 1990). The studies on kokumi substances are gradually increasing. Figure 2.5 shows the number of the literatures obtained by the PubMed database search with a keyword of “kokumi.” Figure 2.5A indicates the number of the literatures published each year, and Fig. 2.5B does the number of cumulative number of literatures. In particular, the number increased fast in the 2020s. However, it is a fact that several kokumi substances in those literatures do not match the condition of a kokumi substance. In this book, the studies on kokumi substances which match the definition will be introduced. In detail, substances with the following characteristics were selected: (1) The substance has sensory effect as kokumi substance such as the enhancement of thickness of taste, complexity, mouthfuleness, and continuity (lastingness) at the concentrations below the threshold in water. (2) The substance was evaluated with a nose clip to avoid cross-modal interactions. Among the kokumi substances studied, γ-glutamyl peptides were investigated on their perspective mechanism which are related to taste sense, as well as the sensory characteristics of which the substances enhance complexity, mouthfulness, and lingeringness at the concentration below the intrinsic threshold. Regarding the other candidate for the kokumi substances, several α-peptides, acyl derivatives of amino acids, and lipid-related compounds match the condition of kokumi substances. Therefore, the studies on these compounds will be introduced in Chap. 15.

2

Kokumi Substance as an Enhancer of Koku: Its Definition

21

(A) Numbers of Literatures

(B) Cumulative Numbers of Literatures

Fig. 2.5 The numbers of the published literatures on “kokumi” found by the database search with PubMed. (A) The numbers of published literatures in each year. (B) The cumulative numbers of published literatures

References Hayase F, Takahagi Y, Watanabe H (2013) Analysis of cooked flavor and odorants contributing to the Koku taste of seasoning soy sauce. J Jpn Soc Food Sci Tech 60:59–71 Kurobayashi Y, Katsumi Y, Fujita A, Morimitsu Y, Kubota K (2008) Flavor enhancement of chicken broth from boiled celery constituents. J Agric Food Chem 56:512–516 Liou BK, Grun IU (2007) Effect of fat level on the perception of five flavor chemicals in ice cream with or without fat mimetics by using a descriptive test. J Food Sci 72:S595–S604 Nakanishi A, Fukushima Y, Miyazawa N, Yoshikawa K, Masuda Y, Kurobayashi Y (2017) Identification of rotundone as a potent odor-active compound of several kinds of fruits. J Agric Food Chem 65:4464–4471 Ohsu T, Amino Y, Nagasaki H, Yamanaka T, Takeshita S, Hatanaka T, Maruyama Y, Miyamura N, Eto Y (2010) Involvement of the calcium-sensing receptor in human taste perception. J Biol Chem 285:1016–1022 Tomaschunas M, Zorb R, Fischer J, Kohn E, Hinrichs J, Busch-Stockfisch M (2013) Changes in sensory properties and consumer acceptance of reduced fat pork Lyon-style and liver sausages containing inulin and citrus fiber as fat replacers. Meat Sci 95:629–640

22

M. Kuroda

Ueda Y, Sakaguchi M, Hirayama K, Miyajima R, Kimizuka A (1990) Characteristic flavor constituents in water extract of garlic. Agric Biol Chem 54:163–169 Watanabe K, Lan H-L, Yamaguchi K, Konosu S (1990) Role of extractive components of scallop in its characteristics taste development. J Jpn Soc Food Sci Tech 37:439–445 Yamaguchi S, Kimizuka A (1979) Psychometric studies on the taste of monosodium glutamate. In: Filer LJ Jr, Garattini S, Kare MR, Reynolds WA, Wurtman RJ (eds) Glutamic acid: advances in biochemistry and physiology. Raven Press, New York, USA, pp 35–54

Part II

Kokumi γ-Glutamyl-Peptides

Chapter 3

Biochemical Studies on Kokumi γ-Glutamyl Peptifdes Motonaka Kuroda

Abstract As described in Chap. 1, kokumi substances are defined as taste-related substances that modify flavor characteristics, such as complexity, mouthfulness, and lastingness (continuity), when added to basic taste solutions or food, but are tasteless themselves at the dose tested. Among the kokumi substances reported to date, γ-glutamyl peptides have been examined in detail. This chapter introduces studies on γ-glutamyl peptides. γ-Glu-Leu, γ-Glu-Val, and γ-Glu-β-Ala in edible beans (Phaseolus vulgaris L.) and γ-Glu-Tyr and γ-Glu-Phe in soybean seeds were identified as kokumi peptides. γ-Glu-Glu, γ-Glu-Gly, γ-Glu-Gln, γ-Glu-Met, γ-Glu-Leu, γ-Glu-Val, and γ-Glu-His in cheese, γ-Glu-Glu, γ-Glu-Gly, γ-Glu-Gln, γ-Glu-Met, γ-Glu-Leu, γ-Glu-Val, and γ-Glu-Val-Gly in soy sauce, and γ-Glu-Val-Gly in some fermented fish seasonings were indicated to act as kokumi peptides.

3.1

Discovery of Glutathione as a Kokumi Substance

Ueda et al. (Ueda et al. 1990) attempted to isolate and identify koku-enhancing components in an aqueous extract of garlic (Allium sativum L). Based on the findings obtained, they identified S-allyl-L-cysteine sulfoxide (alliin) and glutathione (GSH; γ-Glu-Cys-Gly) as potent kokumi substances. Due to its relatively stable and odorless nature, they focused on GSH and examined its sensory characteristics and distribution in various foods. The threshold of GSH in umami solution was markedly lower than that in water solution (Ueda et al., 1997), suggesting its function as a kokumi substance. The quantification of GSH revealed that it was widely distributed in various foods, containing meats, fishes, vegetables, and alcoholic beverages. The details of this study on GSH will be described in the next chapter.

M. Kuroda (✉) Institute of Food Research and Technologies, Ajinomoto Co., Inc., Kawasaki, Kanagawa, Japan e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2024 M. Kuroda (ed.), Kokumi Substance as an Enhancer of Koku, https://doi.org/10.1007/978-981-99-8303-2_3

25

26

3.2

M. Kuroda

Kokumi γ-Glutamyl Peptides in Beans

The research group led by Professor Thomas Hofmann at the Technical University of Munich investigated kokumi substances in various foodstuffs, such as edible beans, cheese, and cooked prawns, using sensory evaluations (sensomics approach). The sensory effects of various compounds isolated from foods were examined by panelists who were trained to assess changes in mouthfulness and complexity using 5 mM GSH in model chicken broth (Dunkel et al. 2007). Dunkel et al. (Dunkel et al. 2007) investigated flavor-modulating compounds in a nearly tasteless aqueous extract of beans (Phaseolun vulgaris L.). They fractionated the extract by gel permeation chromatography and hydrophilic interaction liquid chromatography and then subjected it to a comparative taste dilution analysis. The structures of the isolated compounds were analyzed using CC-MS/MS and 1D/ 2D-NMR. They identified γ-Glu-Leu, γ-Glu-Val, and γ-Glu-Cys-β-Ala (homoglutathione) as kokumi substances in edible beans (Phaseolun vulgaris L.). They indicated that the threshold values of these peptides were markedly lower in salty/umami solution and chicken broth than in water. For example, the threshold of γ-Glu-Cys-β-Ala in salty/umami solution (the taste-modulating threshold) was 0.1 mM, while that in water was 3.8 mM (Dunkel et al. 2007). Shibata et al. (Shibata et al. 2017) investigated key kokumi substances in soybean seeds and identified γ-Glu-Tyr and γ-Glu-Phe. They also reported that the threshold of γ-Glu-Tyr was 0.005 mM in a salty/umami solution, which was markedly lower than that in water (3.0 mM). The details of this study will be described in Chap. 4.

3.3 3.3.1

Kokumi γ-Glutamyl Peptides in Fermented Foods Cheese

Toelstede et al. (Toelstede et al. 2009) focused on the sensory characteristics of mouthfulness and long-lasting taste complexity in a comparative sensory evaluation of matured and young Gouda Cheese. They attempted to isolate and identify the compounds responsible for these sensory characteristics in a water extract of matured Gouda cheese. They fractionated the extract using gel permeation chromatography and analyzed the compounds obtained using HPLC-MS/MS. γ-Glu-Glu, γ-Glu-Gly, γ-Glu-Gln, γ-Glu-Met, γ-Glu-Leu, γ-Glu-Val, and γ-Glu-His were identified in the active fractions. The threshold values of these peptides were markedly lower in the reconstituted Gouda cheese extract solution than in water. For example, the threshold of γ-Glu-His in the reconstituted Gouda cheese extract solution was 0.01 mM, while that in water was 2.5 mM (Toelstede et al. 2009). They also quantified the contents of the above peptides. γ-Glu-Glu, γ-Glu-Gln, and γ-Glu-Met were detected at concentrations higher than the modulating thresholds, which suggested that these three γ-glutamyl peptides act as kokumi substances in

3

Biochemical Studies on Kokumi γ-Glutamyl Peptifdes

27

Gouda cheese. Toelstede and Hofmann (Toelstede and Hofmann 2009) also quantified kokumi γ-glutamyl peptides in various types of cheese. Hillmann et al. (Hillmann et al. 2016) attempted to reconstitute the taste of Parmesan cheese and quantified γ-glutamyl peptides in Parmesan cheese. The findings obtained on kokumi γ-glutamyl peptides in various types of cheese are shown in Table 3.1. Kokumi γ-glutamyl peptides were present at higher concentrations than the modulating threshold values. These findings suggested that kokumi γ-glutamyl peptides, such as γ-Glu-Glu, γ-Glu-Gly, γ-Glu-Gln, γ-Glu-Met, γ-Glu-Leu, γ-Glu-Val, and γ-Glu-His, act as kokumi substances in various types of cheese. The contents of kokumi γ-glutamyl peptides were shown to increase during the ripening of Gouda cheese (Toelstede and Hofmann 2009) and Parmesan cheese (Hillmann et al. 2016) (Table 3.2). Toelstede and Hofmann (Toelstede and Hofmann 2009) focused on the activity of γ-glutamyl transferase (GGT) in several types of cheese and detected it in raw milk and various types of cheese, such as Gouda cheese, ripened goat cheese, Blue Shropshire, and Gruyere Swiss. In addition, Hillmann et al. (Hillmann et al. 2016) reported GGT activity in milk samples treated under various heating conditions. GGT activities in cheese, milk, and microorganism samples are shown in Table 3.3. Toelstede and Hofmann (Toelstede and Hofmann 2009) indicated that Penicillium roquefortii, which was isolated from Blue Shropshire, exhibited GGT activity. They also demonstrated that the incubation of amino acid mixtures of Gln, Glu, Leu, Met, and His with an isolated single strain of P. roquefortii resulted in the formation of γ-glutamyl dipeptides, such as γ-Glu-Glu, γ-Glu-Gly, γ-Glu-Gln, γ-Glu-Met, γ-Glu-Leu, and γ-Glu-Val. These findings suggested that kokumi γ-glutamyl dipeptides were generated from glutamine and amino acids by an enzymatic reaction with GGT (Fig. 3.1). In blue-vein cheese, such as Blue Shropshire, GGT from mold contributes to the formation of kokumi γ-glutamyl dipeptides. In cheese made from raw milk, such as goat cheese and Gruyere Swiss, GGT in raw milk is considered to contribute to the formation of kokumi γ-glutamyl dipeptides. GGT may originate from different sources, such as milk, starter cultures, and mold, depending on the type of cheese. Further studies are needed to clarify the origin of GGT responsible for the formation of kokumi γ-glutamyl dipeptides in each type of cheese.

3.3.2

Soy Sauce

Junger et al. (2022) attempted to construct recombinant soy sauce using known tasteactive compounds, amino acids, organic acids, minerals, sugars, and other components. They also analyzed kokumi γ-glutamyl peptides in soy sauce and indicated that γ-Glu-Leu, γ-Glu-Gln, γ-Glu-Glu, γ-Glu-Gly, γ-Glu-Met, and γ-Glu-His were present at higher concentrations than the taste-modulating thresholds. This finding suggests that these γ-glutamyl peptides act as kokumi substances in soy sauce. Kuroda et al. (Kuroda et al. 2013) investigated the contents of the kokumi tripeptide, γ-Glu-Val-Gly, in Japanese commercial soy sauces. They showed that

Goat's milk N.I.

Cow's milk Semi-hard

27.6 (2.7)

13.9 (2.4)

6.3 (3.2)

6.4 (4.8)

7.1 (2.9)

20.3 (4.2)

2.0 (7.7)

0.3 (16.9) 2.6 (1.8)

Country of origin Main raw material Type of cheese

Kokumi peptides γ-Glu-Glu

γ-Glu-Gln

γ-Glu-Gly

γ-Glu-His

γ-Glu-Leu

γ-Glu-Met

γ-Glu-Phe

γ-Glu-Tyr γ-Glu-Val

18.8 (4.5) 39.6 (2.4)

55.5 (5.7)

164.2 (7.9)

102.6 (3.1)

117.7 (3.7)

12.5 (2.2)

107.1 (2.9)

198.2 (3.0)

Cow's milk Semi-hard

Milner Netherland

7.7 (5.1) 21.3 (4.1)

4.7 (9.0)

6.5 (3.0)

11.9 (7.9)

29.7 (5.9)

21.4 (3.6)

13.3 (4.4)

26.0 (3.5)

White cheese

Cow's milk

Camembert France

71.6 (5.9) 58.7 (6,5) 36.8 (5.6) 32.3 (6.1) 0.3 (8.9) 21.3 (4.3)

71.8 (3.6) 27.2 (4.5) 2.5 (4.9)

Ewe's milk Semihard

Mouton France

13.1 (8.9) 50.7 (5.1)

44.6 (6.9)

41.8 (5.9)

58.2 (8.4)

40.1 (4.9)

6.2 (3.5)

32.9 (8.9)

47.2 (6.8)

Cow's milk Hard

Kernhem Netherland

10.2 (5.6) 13.2 (6.9)

31.7 (6.7)

82.9 (3.8)

58.0 (6.4)

106.6 (5.5)

9.3 (3.4)

94.5 (3.4)

134.7 (4.8)

Semi-hard

Cow's milk

Leerdammer Netherland

32.8 (3.7) 217.7 (4.9)

155.3 (5.9)

119.1 (4.1)

264.0 (4.7)

201.3 (6.7)

n.d.

147.3 (2.5)

599.3 (7.0)

Hard

Cow's milk

Swiss Gruyere Switzerland

10.7 (6.1) 951.8 (3.6)

366.1 (4.7)

458.5 (5.9)

705.1 (9.0)

59.0 (5.0)

108.4 (7.4)

234.1 (3.9)

619.5 (6.0)

Blue cheese

Blue Shropshire United Kingdom Cow's milk

N.I. no information in the reference, n.d. not detected a The numbers indicate the mean values (n = 3), and the numbers in parenthesis indicate the percentage of relative standard deviation (RSD)

243.1 (2.6) 351.0 (4.2) 268.8 (2.3) 212.3 (1.5) 44.6 (2.9) 295.3 (2.1)

768.9 (2.3) 193.5 (2.2) n.d.

Goat cheese N.I.

Gouda Netherland

Table 3.1 Description and contents of kokumi γ-glutamyl-peptides in various kinds of cheesea

1146 (4.2) 200 (5.9) 1290 (7.0)

1055 (5.7) 6204 (6.2) 1296 (4.2) 626 (5.2)

3299 (4.7) 152 (8.3)

Cow's milk Hard

Parmesan Italy

28 M. Kuroda

3

Biochemical Studies on Kokumi γ-Glutamyl Peptifdes

29

Table 3.2 Changes on the contents of kokumi γ-glutamyl-peptides during the ripening of cheese

Kokumi peptides γ-Glu-Glu γ-Glu-Gln γ-Glu-Gly γ-Glu-His γ-Glu-Leu γ-Glu-Met γ-Glu-Phe γ-Glu-Tyr γ-Glu-Val

Gouda Ripening period 4 weeks 0.3 (9.0) 0.6 (12.4) 3.3 (6.9) 0.3 (10.9) n.d. 0.7 (12.2) n.d. n.d. n.d.

Parmesan Ripening period 13 months 1677.0 (7.5) 237.7 (7.7) 586.2 (8.3) 2807.8 (4.3) 1028.8 (8.6) 459.4 (5.6) 526.3 (7.4) 195.9 (4.4) 488.5 (9.1)

44 weeks 27.6 (2.7) 13.9 (2.4) 6.3 (3.2) 6.4 (4.8) 7.1 (2.9) 20.3 (4.2) 2.0 (7.7) 0.3 (16.9) 2.6 (1.8)

24 months 4564.9 (5.7) 210.9 (9.2) 1449.2 (7.4) 8486.9 (7.0) 1804.2 (4.2) 866.2 (5.1) 1595.3 (4.1) 274.7 (5.3) 1785.2 (7.1)

n.d. not detected a The numbers indicate the mean values (n = 3), and the numbers in parenthesis indicate the percentage of relative standard deviation (RSD) Table 3.3 γ-Glutamyl-transferase (GGT) activity in cheese, milk, and microorganism samples Sample Cheese sample (U/g dm) Gouda cheese (made from raw milk) Ripened goat cheese Blue Shroshire Gruyere Swiss Parmesan cheese (13 months ripened) Parmesan cheese (24 months ripened) Parmesan cheese (30 months ripened) Milk samples (U/mL) Milk (raw) Milk (treated at 55°C for 60 min)a Milk (treated at 55°C for 10 min) Milk (treated at 60°C for 10 min) Milk (treated at 65°C for 10 min) Milk (treated at 70°C for 10 min) Milk (treated at 75°C for 60 min) Milk (treated at 100°C for 1 min) Microorganism samples (U/g protein) Penicillium roquefortii Lactobacillus casei Lactobacillus harbinesis a

GGT activitya,b

Reference

3.91 0.12 0.54 2.54 14.6 ± 1.7 15.3 ± 1.4 14.7 ± 1.8

Toelstede and Hofmann (2009) Toelstede and Hofmann (2009) Toelstede and Hofmann (2009) Toelstede and Hofmann (2009) Hillmann and Hofmann (2016) Hillmann and Hofmann (2016) Hillmann and Hofmann (2016)

5.3 ± 0.5 4.5 ± 0.2 4.8 ± 0.04 4.8 ± 0.05 3.4 ± 0.1 1.4 ± 0.03 0.2 ± 0.07 nd

Hillmann and Hofmann (2016) Hillmann and Hofmann (2016) Hillmann and Hofmann (2016) Hillmann and Hofmann (2016) Hillmann and Hofmann (2016) Hillmann and Hofmann (2016) Hillmann and Hofmann (2016) Hillmann and Hofmann (2016)

0.37 nd nd

Toelstede and Hofmann (2009) Hillmann and Hofmann (2016) Hillmann and Hofmann (2016)

The heating conditions of milk for Parmesan cheese production. GGT activity retained under these conditions suggesting that GGT from milk contributes to the formation of kokumi γ-glutamyl peptides b The data are indicated as means or means ± standard deviation (n = 3)

30

M. Kuroda

Fig. 3.1 Scheme of the enzymatic synthesis of kokumi γ-glutamyldipeptides

X (amino acid) Gln NH3

γ-Glutamyltransferase (GGT)

γ-Glu-X all of the soy sauce samples tested contained this peptide at a concentration of 1.6–6.1 mg/L (5.3–20.1 μmole/L). Although the taste-modulating threshold of this peptide was not reported, 16.5 μM (5 ppm) of γ-Glu-Val-Gly was shown to enhance umami and mouthfulness when added to chicken consommé (Kuroda et al. 2015). This finding indicated that the content of this tripeptide in soy sauce is higher than the modulating threshold and also that it acts as a kokumi substance in several types of soy sauces. The details of this study will be described in Chap. 6.

3.3.3

Fermented Fish Seasonings

Kuroda et al. (Kuroda et al. 2012) investigated the contents of the kokumi tripeptide, γ-Glu-Val-Gly, in various types of fish sauces. Among the fish sauces tested, the concentration of γ-Glu-Val-Gly was the highest in Nuoc-Mam (Viet Nam). In commercial Nuoc-Mam samples, γ-Glu-Val-Gly was present at a concentration range of 10.4–12.6 mg/L (34.3–41.6 μmole/L). Miyamura et al. quantified the contents of γ-Glu-Val-Gly in various types of fermented shrimp paste (Miyamura et al., 2014). They showed that γ-Glu-Val-Gly was present at a concentration of 5.2 mg/kg (17.2 μmole/kg) in Indonesian fermented shrimp paste named “Terasi.” As previously described, 16.5 μM (5 ppm) of γ-Glu-Val-Gly enhanced umami and mouthfulness when added to chicken consommé (Kuroda et al. 2015). This finding indicates that this tripeptide acts as a kokumi substance in several types of fermented fish seasonings. The details of this study on the quantification of γ-Glu-Val-Gly in various foods will be described in Chap. 6. The studies described in this chapter reveal that kokumi γ-glutamyl-peptides are distributed in various kinds of foods. Table 3.4 summarizes the structure, threshold, and origin of kokumi γ-glutamyl-peptides.

3

Biochemical Studies on Kokumi γ-Glutamyl Peptifdes

31

Table 3.4 Structure, threshold, and origin of kokumi γ-glutamyl-peptides

Peptide Dipeptides γ-Glu-Val γ-Glu-Leu γ-Glu-Glu γ-Glu-Gly γ-Glu-His γ-Glu-Gln γ-Glu-Met γ-Glu-Tyr γ-Glu-Phe Tripeptides γ-Glu-Cys-Gly (glutathione) γ-Glu-Cys-β-Ala (homoglutathione) γ-Glu-Val-Gly

Threshold (μmole/L) In In water foods

Origin

3300 9400

400a 5.0b

2400 1250 2500 2500 2500 3000 3000

17.5b 17.5b 10.0b 7.5b 5.0b 5.0c 2.8c

Edible beans Edible beans, Gouda cheese, Parmesan cheese Gouda cheese, Parmesan cheese Gouda cheese, Parmesan cheese Gouda cheese, Parmesan cheese Gouda cheese, Parmesan cheese Gouda cheese, Parmesan cheese Soy beans, Parmesan cheese Soy beans, Parmesan cheese

1303 3800

200a 200a

Garlic, scallop, yeast extract Edible beans

66

n.t.

Soy sauce, fish sauce

a

Threshold determined in chicken broth (Dunkel et al. 2007) b Threshold determined in reconstituted matured cheese extract ((Toelstede et al. 2009)) c Threshold determined in salty/umami solution (Shibata et al. 2015)

References Dunkel A, Koster J, Hofmann T (2007) Molecular and sensory characterization of γ-glutamyl peptides as key contributors to the kokumi taste of edible beans (Phaseolus vulgaris L.). J Agric Food Chem 55:6712–6719 Hillmann H, Hofmann T (2016) Quantitation of key tastants and re-engineering the taste of parmesan cheese. J Agric Food Chem 64:1794–1805 Hillmann H, Behr J, Ehrmann MA, Vogel RF, Hofmann T (2016) Formation of kokumi-enhancing γ-glutamyl dipeptides in parmesan cheese by means of γ-glutamyl transferase activity and stable isotope double-labeling studies. J Agric Food Chem 64:1784–1793 Kuroda M, Kato Y, Yamazaki J, Kageyama N, Mizukoshi T, Miyano H, Eto Y (2012) Determination and quantification of γ-glutamyl-valyl-glycine in commercial fish sauces. J Agric Food Chem 60:7291–7296 Kuroda M, Kato Y, Yamazaki J, Kai Y, Mizukoshi T, Miyano H, Eto Y (2013) Determination and quantification of the kokumi peptide, γ-glutamyl-valyl-glycine, in commercial soy sauces. Food Chem 141:823–828 Miyamura N, Kuroda M, Kato Y, Yamazaki J, Mizukoshi T, Miyano H, Eto Y (2014) Determination and quantification of a kokumi peptide, γ-glutamyl-valyl-glycine, in fermented shrimp paste condiments. Food Sci Tech Res 20:699–703 Shibata M, Hirotsuka M, Mizutani Y, Takahashi H, Kawada T, Matsumiya K, Hayashi Y, Matsumura Y (2017) Isolation and characterization of key contributors to the “kokumi” taste in soybean seeds. Biosci Biotech Biochem 81:2168–2177

32

M. Kuroda

Toelstede S, Hofmann T (2009) Kokumi-active glutamyl peptides in cheeses and their biogeneration by Penicillium roquefortii. J Agric Food Chem 57:3738–3748 Toelstede S, Dunkel A, Hofmann T (2009) A series of kokumi peptides impart the long-lasting mouthfulness of matured gouda cheese. J Agric Food Chem 57:1440–1448 Ueda Y, Sakaguchi M, Hirayama K, Miyajima R, Kimizuka A (1990) Characteristic flavor constituents in water extract of garlic. Agric Biol Chem 54:163–169 Ueda Y, Yonemitsu M, Tsubuku T, Sakaguchi M, Miyajima R (1997) Flavor characteristics of glutathione in raw and cooked foodstuffs. Biosci Biotech Biochem 61:1977–1980

Chapter 4

Kokumi Substances from Garlic; Discovery of Glutathione (GSH; γ-Glu-Cys-Gly) as a Kokumi Substance Yoichi Ueda and Motonaka Kuroda

Abstract Garlic has been used in various cuisines worldwide. The unique sensory characteristics of garlic include not only its aroma, but also its flavor, designated as the thickness of taste, complexity, continuity of flavor, and mouthfulness. The present chapter describes the fractionation and identification of the compounds responsible for enhancing the thickness of taste, mouthfulness, and continuity of flavor. The results of fractionation and sensory evaluations revealed that S-allyl-Lcysteine sulfoxide (alliin), cycloalliin, S-methyl-L-cysteine sulfoxide, γ-glutamyl-Sallyl-cysteine, γ-glutamyl-S-allyl-cysteine sulfoxide, cysteine, and glutathione were the compounds responsible for enhancing the flavor characteristics described above. Among these compounds, glutathione has been detected in animals, plants, and microorganisms. Therefore, it is expected to be present in various foods. Highperformance liquid chromatography was performed to quantify glutathione in various foods, such as meat and meat products, chicken, seafood, vegetables, and alcoholic beverages. A sensory evaluation of glutathione revealed a lower threshold in an umami solution than in water. Furthermore, the addition of glutathione at a concentration below the threshold in water to reconstituted model extract enhanced the thickness of taste (complexity), mouthfulness, and continuity of flavor. These results suggest that glutathione acts as a kokumi substance in foods.

4.1

Introduction

Garlic (Allium sativum L) has been used in various cuisines worldwide. Its unique sensory characteristic is its odor, particularly that of crushed or sliced raw garlic. The mechanisms underlying the formation of the representative odorant, allicin (diallylthiosulfonate) from alliin ((+)-S-allyl-cysteine sulfoxide), were investigated by Stoll and Seebeck (1948). Thiosulfate compounds containing allicin are unstable and

Y. Ueda · M. Kuroda (✉) Institute of Food Research and Technologies, Ajinomoto Co., Inc., Kawasaki, Kanagawa, Japan e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2024 M. Kuroda (ed.), Kokumi Substance as an Enhancer of Koku, https://doi.org/10.1007/978-981-99-8303-2_4

33

34

Y. Ueda and M. Kuroda

easily converted to various odorous compounds (Boelens et al. 1971). Odor is an important sensory characteristic when raw garlic is used in cuisine. Garlic is often used as seasoning when cooking. The heating process changes the taste of raw garlic. The water extract of garlic has a weak aroma and sweetness. However, the addition of a small amount of garlic extract to dishes enhances flavor, designated as the thickness of taste or complexity, mouthfulness, and continuity of flavor. These flavor characteristics cannot be explained by the five basic tastes, namely, sweetness, saltiness, sourness, bitterness, and umami. In this chapter, fractionation was performed to identify the compounds in garlic that are responsible for enhancing the thickness of taste, mouthfulness, and continuity of flavor. The sensory characteristics of glutathione (GSH), one of these compounds, and its quantification in various foods have been described herein.

4.2

Identification of Kokumi Substances in Garlic

Ueda et al. (1990) investigated the flavoring effects of a diluted extract of garlic that enhanced the continuity of flavor, mouthfulness, and the thickness of taste when added to an umami solution. They attempted to isolate and identify the key compounds responsible for these effects. They initially prepared an active fraction by cation exchange chromatography using a Duolite C-25 column. The scheme of this preparation is shown in Fig. 4.1. The enhancing effects of this fraction on the continuity of flavor, mouthfulness, and the thickness of taste in an umami solution were exerted by the Duolite C-25 absorbed fraction. The components of the Duolite Garlic 100 kg Heated in an autoclave at 110 ℃ for 40 min Added to 150 L of water, heated at 90 ℃ for 30 min Filtered with cloth

Extract 180 L Adjusted pH to 1.0 with conc. HCl, filtered by celite Put on a Duolite C-25 (H+) column (30 x 100 cm) Washed with 70 L of water Eluted by 300 L of 0.5 M NaOH

Unabsorbed Fr.

Eluate Run through Amberlite IRC-50 (H+) column (12 x 62 cm)

NinhydrinNegative Fr.

Ninhydrin-positive Fr. 136 L Concentrated to 20 L by reverse osmotic membrane Concentrated to 10 L by an evaporator Deodorized by charcoal (300g) and lyophilized

Duolite C-25 absorbed fraction 700 g Fig. 4.1 Preparation of Duolite C-25 absorbed fraction from garlic

4

Kokumi Substances from Garlic; Discovery of Glutathione (GSH; γ-Glu-Cys-Gly. . .

Table 4.1 Content of each sulfur-containing compound in Duolite C-25 absorbed fraction

Compounda Alliin Cycloalliin MeCSO GAC GACSO Glutathione (GSH) Cysteine Methionine

35

Content (%) 16.0 9.2 2.1 5.3 4.7 0.2 3.0 0.1

MeCSO S-methyl-cysteine sulfoxide, GAC γ-glutamyl S-allylcysteine, GACSO γ-glutamyl S-allyl-cysteine sulfoxide

a

Table 4.2 Effect of each sulfur-containing compound in salty/umami solution

Compounda Alliin Cycloalliin MeCSO GAC GACSO Glutathione (GSH) Cysteine Methionine

Enhancement of taste +++ + ++ ++ + +++ + +

Other flavors Garlic-like Leak-like Garlic-like Garlic-like

MeCSO S-methyl-cysteine sulfoxide, GAC γ-glutamyl S-allylcysteine, GACSO γ-glutamyl S-allyl-cysteine sulfoxide

a

C-25 absorbed fraction were analyzed, and the major components identified were Sallyl-L-cysteine sulfoxide (alliin), cycloalliin, S-methyl-L-cysteine sulfoxide (MeCSO), γ-glutamyl-S-allyl-cysteine (GAC), γ-glutamyl-S-allyl-cysteine sulfoxide (GACSO), cysteine (Cys), and GSH (Table 4.1). To compare their sensory effects, each component was prepared, added at 0.2% (w/v) to an umami solution, and changes in the continuity of flavor, mouthfulness, and the thickness of taste were evaluated by a sensory panel. The sensory evaluation was performed by 20 trained assessors and their findings are shown in Table 4.2. Among the compounds tested, alliin and GSH exerted the strongest enhancing effects.

4.3

Assessment of Threshold Values of GSH in Water and Umami Solutions

Since GSH does not have any flavor and is relatively stable, Ueda et al. investigated its sensory characteristics (Ueda et al. 1997). They examined the threshold values of GSH in various systems. Absolute threshold values were measured using the triangle discrimination test by 15 well-trained assessors. The pH of the tested solutions was adjusted to 7.0 by 1 M HCl or NaOH. The findings shown in Table 4.3 demonstrated

36

Y. Ueda and M. Kuroda

Table 4.3 Threshold values of glutathione in water and umami solutions

Solution Water MSG 0.05% MSG 0.80% MSG 3.1% MSG 0.05% + IMP 0.05%

Threshold value (%) 0.04 0.04 0.02 0.01 0.01

that the threshold of GSH was markedly lower in an umami solution (consisting of a solution containing 3.1% MSG and a solution containing 0.05% MSG and 0.05% IMP) than in water. Therefore, although the flavor of GSH was negligible in water, when added to an umami solution, it strongly enhanced the thickness of taste, continuity, and mouthfulness (Ueda et al. 1997). Ueda et al. proposed referring to substances with these properties as kokumi substances. Therefore, as cited in Chap. 1, a kokumi substance is considered to be one of the “Koku”-imparting substances.

4.4

Sensory Characteristics of GSH in Reconstituted Model Extracts

The effects of GSH on the flavor characteristics of reconstituted model beef extract were investigated by a sensory evaluation (Ueda et al. 1997). The composition of reconstituted model beef extract is shown in Table 4.4. The effects of 0.02% (w/v) GSH were evaluated by 20 trained assessors with a scoring method. Assessors were instructed to evaluate samples relative to control samples with scores ranging from 2 (apparently weak) to +2 (apparently strong). The following sensory attributes were used: whole aroma, sweetness, saltiness, bitterness, sourness, umami, the continuity of flavor, mouthfulness, the thickness of taste, and meat-like flavor. The findings of the sensory evaluation (Table 4.5) indicated that the addition of GSH significantly enhanced the intensity of the continuity of flavor, mouthfulness, and the thickness of taste. Ueda et al. (1998) also investigated the sensory effects of GSH on the flavor of reconstituted model scallop extract. The composition of reconstituted model scallop extract is shown in Table 4.6. The effects of 29 mg/100 mL GSH, which reflected its concentration in raw scallop, were examined by 28 trained assessors with a scoring method. Assessors were instructed to evaluate samples relative to control samples with scores ranging from -2 (apparently weak) to +2 (apparently strong). The following sensory attributes were used: whole aroma, sweetness, saltiness, bitterness, sourness, umami, the continuity of flavor, mouthfulness, and the thickness of taste. The findings of the sensory evaluation (Fig. 4.2) revealed that GSH significantly enhanced the intensity of sweetness, umami, the thickness of taste, mouthfulness, and the continuity of flavor. Therefore, GSH appeared to enhance the flavors of beef and scallop.

4

Kokumi Substances from Garlic; Discovery of Glutathione (GSH; γ-Glu-Cys-Gly. . .

Table 4.4 Composition of the reconstituted model beef extract

Compound Amino acids Threonine Serine Monosodium glutamate Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalaine Lysine HCl Histidine Arginine Taurine Sugars Ribose Mannose Fructose Glucose Nucleotides IMP 2Na 7.5H2O GMP 2Na 7H2O AMP Carbonic acids Pyroglutamic acid Lactic acid (90% w/v) Sodium lactate (50% w/v) Succinic acid Salts NaCl KCl MgCl2 6H2O CaCl2 2H2O KH2PO4 Others Anserine Carnosine Creatine Creatinine

37

(mg/100 mL) 7.1 3.8 6.4 1.7 5.9 20.2 3.6 2.1 1.7 2.9 4.0 2.7 4.6 4.0 3.6 26.0 0.4 0.8 2.1 4.0 44.5 0.8 6.3 136.1 75.0 717.0 10.3 89.3 156.0 110.0 1.5 276.0 16.8 79.0 116.1 60.1

38

Y. Ueda and M. Kuroda

Table 4.5 Effects of glutathione on the flavor characteristics of reconstituted model beef extract

Sensory attributes Whole aroma Sweetness Saltiness Bitterness Sourness Umami Continuity Mouthfulness Thickness Meat-like flavor

Effect of GSH – – – – – – 0.95** 0.85* – 0.70**

*P < 0.01; **P < 0.001, – not signifcant Table 4.6 Composition of the reconstituted model scallop extract

Fig. 4.2 Additional effect of glutathione on sensory characteristics of reconstituted model scallop extract. The bars indicate the 95% confidence intervals

4.5

Compound Glycine Arginine Alanine Glu Na H2O AMP Na2 KOH KCl NaCl

(mg/100 g) 1925 323 256 179 195 232 109 71

Weak -0.5

Strong 0

0.5

1.0

Whole aroma Saltiness Sourness Sweetness Bitterness Umami Thickness of taste Mouthfulness Continuity

Quantitative Analysis of GSH in Various Foods

Ueda et al. (1997) investigated the contents of GSH in various foods. They quantified GSH using HPLC equipped with an amino-column with post-column fluorescent detection by derivatization with N-ethylmaleimide, which selectively reacts with the -SH group (Takahashi et al. 1979). The findings obtained are shown in Table 4.7. GSH was quantified in beef, pork, and chicken and in meat products, such

4

Kokumi Substances from Garlic; Discovery of Glutathione (GSH; γ-Glu-Cys-Gly. . .

Table 4.7 Content of glutathione in various foods

Food Beef fillet meat A Beef fillet meat B Beef marbled meat A Beef marbled meat B Pork (round meat) Chicken (round meat) Ham Liver (Cow) Foie Gras Tuna Scallop A Scallop B Tomato juice Broccoli White wine A White wine B White wine C Red wine A Red wine B Red wine C Sake (rice wine) A Sake (rice wine) B Beer

39

Content (mg/100 g) 20 40 17 13 13 20 7.5 2.4 9.4 5.3 9.6 25 8.2 0.1 13 3.0 7.3 38 3.7 14 0.07 nd 0.16

as ham and foie gras. It was also quantified in seafood, including tuna and scallop, as well as in vegetable foods, such as broccoli and tomato juice. Furthermore, GSH was detected in alcoholic beverages, including wine, rice wine (sake), and beer. Its contents in beef (13–40 mg/100 g), pork (13 mg/100 g), chicken (20 mg/100 g), scallop (10–25 mg/100 g), and some brands of wine (13–38 mg/100 g) were higher than the threshold concentration in umami solutions. Therefore, GSH appears to function as a kokumi substance and plays a role in modulating the flavor of various foods. Ueda et al. (1998) focused on the contents of GSH in seafood. They quantified GSH using ion-paired HPLC equipped with an octadecyl-silica column with postcolumn fluorescent detection by derivatization with N-ethylmaleimide. The findings of the quantitative analysis are shown in Table 4.8. GSH was widely distributed in various fish and other seafood, such as sea urchin, prawn and shrimp, and scallop. The quantification of GSH in foods revealed that this peptide is widely distributed in and contributes to the flavor of various foods.

40 Table 4.8 Content of glutathione in various seafoods

Y. Ueda and M. Kuroda Samples Fishes Bigeye tuna Bluefin tuna Skipjack tuna Chub mackerel Horse mackerel Sardine Yellow tail Coho salmon Flatfish Other seafoods Northen green sea urchin Black tiger prawn Northen shrimp Neon flying squid Japanese flying squid Short-neck clam Scallop

Contents (mg/100 g) 0.81 5.46 2.48 0.29 2.48 6.51 0.17 9.22 2.57 7.36 0.19 4.45 nd nd nd 29.05

References Boelens M, de Valois PJ, Wobben HJ, van der Gen A (1971) J Agric Food Chem 19:984 Stoll A, Seebeck E (1948) About Alliin, the genuine mother substance of garlic oil. Helv Chem Acta 31:189–210 Takahashi H, Nara Y, Meguro H, Tujimura K (1979) A sensitive fluorometric method for the determination of glutathione and some thiols in blood and mammalian tissues by high performance liquid chromatography. Agric Biol Chem 43:1439–1445 Ueda Y, Hibino G, Kohmura M, Kuroda M, Watanabe K, Sakaguchi M (1998) Contents of glutathione in seafoods and its flavor characteristics. Nippon Suisan Gakkaishi 64:710–714 Ueda Y, Sakaguchi M, Hirayama K, Miyajima R, Kimizuka A (1990) Characteristic flavor constituents in water extract of garlic. Agric Biol Chem 54:163–169 Ueda Y, Yonemitsu M, Tsubuku T, Sakaguchi M, Miyajima R (1997) Flavor characteristics of glutathione in raw and cooked foodstuffs. Biosci Biotech Biochem 61:1977–1980

Chapter 5

Kokumi Substances in Soybean Seeds Masayuki Shibata and Yasuki Matsumura

Abstract Certain soy-based foods such as soymilk and the roasted beans improve the taste of other food products by enhancing the sensation of thickness, continuity, and mouthfulness. These features are characteristics of koku, which acts as a taste attribute, additional to the five basic tastes. Although there are reports of kokumi substances being isolated from various edible materials, there have been no reports to date on the kokumi substances of soymilk or soybean. In this study, we applied sensory-guided fractionation and taste-related sensory analysis to isolate and extract the key contributors to koku in processed soybean. In addition, to gain insight into the genetic diversities and molecular mechanisms of accumulation of kokumi substances in soybean seeds, we measured the content of these substances in soybean accessions from Japan and world mini core collections. These studies will lead to the increase of the kokumi substances in soybean seeds and improve the palatability of soy-food.

5.1 5.1.1

General Introduction General Description of Soybean Products

Soybean (Glycine max (L.) Merr.) is a diplodized tetraploid (2n = 40), and belonging to the Soja subgenus under the genus Glycine of the Leguminosae family. The Soja subgenus also contains G. soja, which is a wild species of soybean (Hymowitz 1970). It grows in the fields, hedgerows, and riverbanks in many Asian countries, including China, Korea, Japan, and the former USSR.

M. Shibata (✉) Research Institute for Creating the Future, Fuji Oil Holdings Inc., Tsukubamirai-shi, Ibaraki, Japan e-mail: [email protected] Y. Matsumura Research Institute for Sustainable Humanosphere, Kyoto University, Gokasho, Uji-shi, Kyoto, Japan © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2024 M. Kuroda (ed.), Kokumi Substance as an Enhancer of Koku, https://doi.org/10.1007/978-981-99-8303-2_5

41

42

M. Shibata and Y. Matsumura

Soybean is generally considered as one of the oldest cultivated crops, native to Northern and Central China (Hymowitz 1970). The first mention of soybean appeared in a series of books known as Pen Ts’ao Kong Mu, written by emperor Sheng Nung in the year 2838 B.C., which describes the floral diversity of China. Today, soybean is grown as a commercial crop in over 35 countries. The major producers of soybeans are the United States, Brazil, and Argentina; and the production of soybean in these countries accounts for 80% of the world’s soybean production. The largest consumer of soybean is China, and the consumption of soybean in China accounts for 60% of the world’s soybean consumption (PS&D 2016). Soybean is grown primarily for the production of seeds and is often used in the food and industrial sectors. It is primarily used for oil extraction, which utilizes over 90% of the total soybean produced. Soybean oil is used for the manufacture of margarines, shortenings, and cooking oils in the food industry, and for the manufacture of inks, dyes, and biodiesel in the chemical industry (Sharma and Erhan 2009; Compton and Laszlo 2009). Soybean is therefore recognized as an important source of oil. On the other hand, the consumption of soybean-based foods derived from the whole bean has a long tradition in far eastern Asia. The whole beans were consumed as cooked beans, roasted soy flour, miso, soy sauce, natto, and tempeh, among others. The latter four items are fermented foods. On the other hand, soymilk is prepared from soybean, and most of the soymilk produced is utilized for making tofu. In several countries, including Japan, about 30% of the soybean allocated for consumption is utilized for the production of soybean-based foods, whereas in the United States and Brazil this amounts to only about 10% (Ministry of Agriculture. Forestry and Fisheries database 2016). The aforementioned soy-based foods have been widely used in Japanese cuisines for a long time, and they play an essential role in the food culture of Japan. As shown in Fig. 5.1, about one million ton of soybeans are used for the preparation of soy-based foods (Ministry of Agriculture. Forestry and Fisheries database 2016). Among the soy foods, the highest amount of soybean is utilized for the production of tofu, followed by the production of miso and natto. These soy foods are consumed on a daily basis; however, the production of these soy foods is gradually decreasing in the recent times. This tendency indicates that the soy food markets may have reached a saturation point. Although the utilization of soybean for the manufacture of other soy foods has decreased gradually or has remained the same, the utilization of soybean for the production of soymilk is gradually increasing. Soymilk is primarily consumed as a drink, but its use is increasing for the purpose of cooking in the recent years. According to the Japanese Agricultural Standard (JAS), soymilk is classified into three categories: regular soymilk, blended soymilk, and beverage-containing soymilk (Table 5.1). Among the three categories, the consumption of regular soymilk has been increasing most rapidly in the past 15 years (Fig. 5.2). Without additional components, soymilk has a bland taste and flavor, but it can enhance the palatability when added to other foodstuffs. It is thought that the increase in soymilk production is due to the increasing use of regular soymilk as a seasoning during cooking. In this context,

5

Kokumi Substances in Soybean Seeds

43

Fig. 5.1 Changes in the amount of annual soybean usage utilization for various different soy-based foods. Source: Ministry of Agriculture, Forestry and Fisheries, Food Industry Affairs Bureau Table 5.1 The varieties of soymilk classified according to the JAS

Product Soymilk Blended soymilk Beverage containing soymilk

Containing fruit juice Other

Concentration solid soybean ≥8% ≥6% ≥2% ≥4%

Protein concentration ≥3.8% ≥3.0% ≥0.9% ≥1.8%

the demand for improving the flavor and taste of regular soymilk is presently growing, so as to promote the use of soybean for cooking.

5.1.2

Flavor and Taste Components in Soybean

In addition to soy foods such as soymilk or tofu, which have been part of the traditional diets of east Asia, commercial products of soy proteins, such as soy protein isolate (SPI) or soy protein concentrate (SPC), are also important ingredients of many processed foods. The worldwide production and use of soy protein products have been increasing rapidly in the recent years due to their high nutritional values

44

M. Shibata and Y. Matsumura

Fig. 5.2 Changes in the annual soybean utilization for different varieties of soymilk, classified according to the JAS. Source: Japan Soymilk Association’s database (2016)

and numerous bioactivities. Nevertheless, the use of soybean as food material has been limited in the western world, because of its “grassy/beany” flavor (Krishna et al. 2003). These undesirable flavors are primarily attributed to aldehydes, which are associated with the oxidation of polyunsaturated fatty acids by the lipoxygenase (LOX) present in soybeans. Methods for suppressing LOX activity broadly involves: (1) breeding LOX-free soybeans (Hildebrand and Hymowitz 1981; Hildebrand and Hymowitz 1982; Kitamura et al. 1985; Davies and Nielsen 1986; Kitamura et al. 1983) and (2) denaturing LOX activity by food processing. Thermal processing is very important for improving flavor quality, especially in the latter method, and is commonly employed on an industrial scale. LOX activity is controlled by directly heating the soybean seeds (Mustakas et al. 1969) or heating during soymilk extraction (Wilkens et al. 1967; Mizutani and Hashimoto 2004), which reduces the generation of off-flavors. These methods are used to produce soy foods with less undesirable grassy/beany flavor, which have enhanced the utilization of soybeans and soy foods across the world, especially in the Western countries. Some studies are focusing on the total sugar content of soybean for improving its quality and taste. The total sugar content was found to affect the taste of tofu (Fujino et al. 2012), and the recent breeding studies resulted in the production of the “Sayanami” cultivar, which elevated the total sugar content of soybean (Yamada et al. 1998).

5

Kokumi Substances in Soybean Seeds

45

Although there are numerous studies attempting to improve the undesirable flavor of soybean, few studies directly contribute to improving its palatability. Considering the recent rise in the use of soymilk as a seasoning for cooking, it is important to focus on the new components of soybean or soymilk for enhancing the palatability of food.

5.1.3

Contribution of Kokumi Substances to Food Palatability

There are many components related to taste quality, and umami is an important element which influences the taste quality of food. Acidic amino acids, including glutamic acid, and nucleic acids contribute to the umami taste of food (Ohara 1966). Besides these components, guanylate analogs, amadori compounds of glutamic acid, acidic peptides, pyroglutamylated peptides, and organic acid glucosides also contribute to the umami taste (de Rijke et al. 2007; Festring and Hofmann 2010; Beksan et al. 2003; Noguchi et al. 1975; Arai et al. 1973; Schlichtherle-Cerny and Amadò 2002; Rotzoll et al. 2005). In addition, it is known that MSG and nucleic acids such as IMP or GMP act synergistically in producing the umami taste. Kuninaka (Kuninaka 1960) and Sugita (Sugita 2002) reported that the detection threshold of MSG was markedly lowered in the presence of IMP or GMP (Table 5.2). Besides these components, guanylate derivatives, betaine, pyroglutamylated peptides, organic acid amide, and aromatic amino acids are also known as umami tasteenhancing components (Soldo et al. 2003; Okada et al. 2005; Lioe et al. 2005; Winkel et al. 2008; Festring and Hofmann 2011). Similar to these compounds, a variety of components with different properties work in a synergistic manner, to influence the overall taste as well the individual taste qualities. Although the umami taste is an important factor in improving the taste quality of food, the “koku” attribute is also recognized in Japan as an important factor, contributing to the palatability of food. “Koku” is the unique sensation caused by several stimulations of taste, aroma, and texture, which is expressed as the complexity of tastes in “koku.” Furthermore, this sensation spreads throughout the mouth and contributes to the richness or body of the taste and lingers even after swallowing,

Table 5.2 The taste threshold of umami substances MSG alone IMP alone GMP alone MSG in 0.25% IMP IMP + GMP (1:1 blend) IMP + GMP (1:1 blend) in 0.8% MSG

Detection threshold (g/dL) 1.20 × 10-2 2.50 × 10-2 1.25 × 10-2 1.50 × 10-4 6.30 × 10-3 1.30 × 10-5

46

M. Shibata and Y. Matsumura

resulting in the strong “koku” taste attribute. Therefore, “koku” comprises three factors: complexity, richness (body), and continuity (Nishimura and Egusa 2016). The kokumi substance is one of the “koku”-enhancing materials and is a crucial factor for improving the overall taste quality of food. Kokumi substance is distinct from the tastants of five basic tastes, namely, sweet, salty, sour, bitter, and umami, and is defined by the three functions: thickness (intensity remains after ~5 s of tasting), continuity (intensity lingers ~20 s after tasting), and mouthfulness (the reinforcement of the taste sensation throughout the mouth) (Ohsu et al. 2010). There are several reports on the kokumi taste components. The kokumi substances themselves are almost tasteless, but the savory taste increases upon combining with the five basic taste qualities of food. A similar example has been empirically confirmed by studying soybean soup stock. It is assumed that the kokumi substances of soybean soup stock are water soluble. Such water-soluble components are probably present in soymilk as well and act as a seasoning. Therefore, the identification of kokumi substances in soybean seeds is essential for enhancing the value of soymilk as a seasoning during cooking.

5.1.4

Analytical Methods for Studying Kokumi Substances

To identify the components that contribute to a particular taste, it is a common practice to isolate the key components by combining multiple separation and purification methods and identifying the components thus isolated by using liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) (Dunkel et al. 2007). The taste of food is mainly attributed to five basic tastes, namely, salty, sour, sweet, bitter, and umami. Although the organic acids that contribute to the sourness of food are volatile components, most of the taste components are non-volatile. For this reason, unlike aromatic components which are volatile in nature, taste components are usually analyzed with HPLC (The Japan Society for Analytical Chemistry, Kanto 1985). While Reverse Phase HPLC is used to analyze a wide range of targets, Normal Phase HPLC is used for analyzing saccharides that contribute to the sweetness of food (The Japan Society for Analytical Chemistry, Kanto 1985). Ion-exclusion chromatography is used to analyze minerals that contribute to the saltiness (Hayashi 1995; Small et al. 1975), and ion-exchange chromatography is used to analyze amino acids and peptides that are responsible for the umami taste of food (Moore and Stein 1948). In addition to these methods, HILIC is being recently applied for analyzing hydrophilic components such as hydrophilic saccharides (Benvenuti 2013) or peptides (Jablonski et al. 2012). Although these hydrophilic components have little or no retention on the C18 stationary phases used for Reverse Phase HPLC, HILIC is an alternative chromatographic technique employed for analyzing such components. Since the kokumi substances of soymilk probably consist of water-soluble and hydrophilic components, it is thought that the HILIC

5

Kokumi Substances in Soybean Seeds

47

method is more suitable for analyzing these components than the other analytical methods.

5.1.5

Scope of This Research

Soybean has been an important food material in East Asia including Japan for a long time and recently has been recognized as a useful food material in Western countries, too. The usage of soybean has been expanded to a large number of cooking scenes in Japan (Mameplus 2016). Under the circumstances that soybean is attracting attention as the sustainable source of protein and lipid (de Ron et al. 2017), it is urgently requested to increase the value of soybean as the food material. Especially, it is crucial to improve the palatability of food products containing soybean or soybean extracts, but the research targeting on this point has been limited. Based on these backgrounds, this study was performed to identify the new taste components which are defined as kokumi substances and accumulate the basic findings for promoting the effective use of these components.

5.2 5.2.1

Isolation and Characterization of the Key Kokumi Substances in Soybean Seeds Introduction

Soybeans contain about 20% oil in their seeds, and soybean oil is widely used for cooking, baking, and frying. Considerable research has been focused on these uses, and numerous attempts to improve oil quality and increase oil content have been put forth (Broun et al. 1999; Thelen et al. 2002). On the other hand, soybeans comprises 40% proteins and carbohydrates, which are known to have health benefits (Moure et al. 2006). For example, 7S globulin can reduce serum triglyceride levels (Moriyama et al. 2004), and oligosaccharides such as raffinose and stachyose are utilized by bifido-bacteria to improve the intestinal environment (Hayakawa et al. 1990). In addition to these applications, in many Asian countries including Japan, various types of soy-products such as miso, soy sauce, tofu, tempeh, and soymilk are widely utilized. Miso and soy sauce are produced by fermentation and are used as seasonings. Fermented soy-products, especially soy sauce, abound worldwide. On the other hand, in Japan, roasted soybean seeds are used to make soup stocks for Japanese vegetarian foods. Soybean soup stocks are nearly tasteless, but the savory taste increases upon combination with other soup stocks made from dried kelp or mushrooms, which contain glutamic acid or guanylic acid and are known to impart the umami taste. Recently, in addition to soybean soup stocks, soymilk has also been

48

M. Shibata and Y. Matsumura

widely used as a flavor enhancer, especially in Japanese Nabe (hot pot cooking), Ramen (noodles), and soups. Such products exhibit improved properties such as enhanced thickness, continuity, and mouthfulness. These effects were defined as koku, which is distinct from the five basic tastes (Ueda et al. 1990; Dunkel et al. 2007; Ohsu et al. 2010; Ueda et al. 1994). Specifically, in Japan, koku is a concept that describes a certain mouthfeel and is thought to be a key factor for palatability. Many taste-improving components are present in various food materials such as sulfur-containing peptides in garlic and onions (Ueda et al. 1990, 1994), N(1-carboxyethyl)-6-(hydroxymethyl) pyridinium-3-ol inner salt (alapyridaine) in beef broth (Ottinger and Hofmann 2003), γ-glutamyl peptides in snap beans (Dunkel et al. 2007), Gouda cheese (Toelstede et al. 2009), and Parmesan cheese (Hillmann et al. 2016), and C17-C21 oxylipins exhibiting 1-acetoxy-2,4-dihydroxy- and 1-acetoxy-2-hydroxy-4-oxo motifs in processed avocados (Degenhardt and Hofmann 2010). Although many studies regarding such components have been reported, no data is available regarding the taste-improving components in soymilk or soybeans. In order to isolate and characterize the key taste contributors in soybeans, sensory-guided fractionation and taste sensory analysis were carried out in this study. The structures of the kokumi substances in the water extract of soybean seeds were analyzed by LC-MS. The taste-improving effects of the identified compounds were evaluated quantitatively in basic model experiments.

5.2.2

Materials and methods

Materials γ-Glutamyl-tyrosine and γ-glutamyl-phenylalanine were purchased from BACHEM (Bubendorf, Switzerland). Saccharides and other materials in the control solution were purchased from WAKO Pure Chemical Industries (Osaka, Japan). All solvents were of LC-MS grade and were purchased from WAKO Pure Chemical Industries (Osaka, Japan). Soybeans (Glycine max (L.) Merr.) were provided by Fuji Oil Holdings Inc. Fractionation of Water-Soluble Components from Soybeans Extraction of water-soluble components from soybeans was carried out using following method. Dried soybean seeds (50 g) were autoclaved (120 °C, 5 min) and powdered using a coffee grinder. The resulting powder was dispersed into distilled water (200 mL), and the dispersion was stirred for 60 min. The resulting slurry was centrifuged, and the supernatant was freeze-dried as the aqueous phase of heattreated soybean seeds. This aqueous phase was stored at –20 °C until use.

5

Kokumi Substances in Soybean Seeds

49

Ultrafiltration (UF) was carried out using following method. Freeze-dried materials obtained from the aqueous phase described above (10 g) was dissolved in deionized water and filtered through 10,000 Da cut off membranes on a HIT-1FUSO181 (DAICEN MEMBRANE SYSTEMS, Tokyo, Japan) at room temperature to obtain high- and low-molecular-weight fractions. Two fractions were freeze-dried and stored at -20 °C until use. Gel permeation chromatography (GPC) was carried out using following method. The low-molecular-weight fraction (0.5 g), which exhibited kokumi taste activity, was dissolved in 5 mL deionized water and the pH was adjusted to 4.0 with 1% formic acid. An aliquot of this solution (5 mL) was applied on a 70 × 5 cm glass column (Bio Rad Laboratories, CA, USA) filled with a slurry of BioGel P-2 (Bio Rad). The column was conditioned with water adjusted to pH 4.0 with 1% formic acid. Chromatographic separation was performed at a flow rate of 1.5 mL/min for 24 h. The effluent was monitored at 220 nm using an UV 900 ultraviolet detector (GE Healthcare, Uppsala, Sweden), and divided into five fractions (fractions 1–5) collected using a fraction collector DC-1200 (EYELA, Tokyo, Japan); each fraction was freeze-dried. These samples were subjected to sensory analysis and chromatographic subfractionation. Preparative HPLC using hydrophilic interaction chromatography (HILIC) column was carried out using following method. The freeze-dried GPC fraction-3 (0.1 g) was dissolved in a 1:1 volume ratio mixture solution of water and acetonitrile containing 0.1 v/v% formic acid and separated by HPLC using an HILIC column (XBridge BEH Prep Amide column; 10 × 250 mm i.d., 5 μm; Waters). Preparative HPLC was performed on a Waters MassLynx preparative system (Waters, MA, USA) consisting of a 2535 quaternary gradient module, system fluidics organizer, 515 HPLC pomp, 2767 sample manager, 2998 photodiode array detector, and ACQuity QDa MS detector. Before injection, the sample was filtrated using a membrane filter (0.2 μm); a 1 mL aliquot was injected. Chromatography was performed at a flow rate of 6 mL/min using formic acid (0.1% in water) as solvent A, and acetonitrile containing 0.1% formic acid as solvent B. The gradient was as follows: 95% B for 2 min; 95–85% B from 2 to 22 min; 85–50% B from 22 to 30 min; 50–95% B from 30 to 31 min; 95% B from 31 to 45 min. The effluent was monitored at 220 nm and with an MS (ESI-) detector and separated into 6 fractions. The fractions were collected over 30 runs, and freeze-dried twice for taste dilution analysis and further separation. To further separate the kokumi substances, the active HPLC fractions (fractions 3–4 and 3–5) were rechromatographed in a similar way as described above. The gradient was as follows: 95% B for 0.5 min; 95–85% B from 0.5 to 12 min; 85–50% B from 12 to 20 min; 50–95% B from 20 to 21 min; 95% B from 21 to 30 min. Fractions 3–4 and fractions 3–5 were separated into 8 and 11 fractions, respectively. These fractions were collected over 20 runs, and freeze-dried for the following experiments.

50

M. Shibata and Y. Matsumura

Identification and Quantitative Analysis of Kokumi Substances Ultra performance liquid chromatography-quadrupole-time-of-flight-mass spectrometry (UPLC-Q-TOF-MS) was carried out using following method. The identification of the kokumi substances in the selected fractions was performed as previously described with some modifications (Takahashi et al. 2015). UPLC-MS was performed using a Waters Acquity UPLC system (Milford, MA) coupled to a Xevo QTOF-MS equipped with an electrospray source operated in the negative ion mode (ESI negative mode), with a lock-spray interface for accurate mass measurements. Leucine enkephalin was employed as the lock-mass compound. A solution of this compound (200 ng/mL in 50% acetonitrile, 50% water, and 0.1% formic acid) was infused directly into the MS at a flow rate of 20 μL/min. The capillary, sampling cone, and extraction cone voltages were set at 2700 V, 20 V, and 1 V, respectively. The source and desolvation temperatures were 120 °C and 450 °C, respectively. The cone and desolvation gas flow rates were set at 50 and 800 L/h, respectively. An aliquot of the extracted sample (3 μL) was injected into an Acquity UPLC BEH amide column (column size, 2.1 × 100 mm; particle size, 1.7 μm). Mobile phases A (water and 0.1% formic acid) and B (acetonitrile and 0.1% formic acid) were used. The column temperature was set to 40 °C. The buffer gradient consisted of 75%– 55% B for 0–10 min, 55%–50% B for 10–10.1 min, 50% B for 10.1–15 min, and 75% B for 5 min, at a flow rate of 300 μL/min. In the MS/MS mode, the collision energy was set at 10–40 V using collision energy ramping. Quantitative analysis of γ-glutamyl peptides and oligosaccharides was carried out using the following method. The freeze-dried low-molecular-weight fraction and soybean seed powder were dissolved in 1:1 volume ratio mixture solution of water and acetonitrile containing 0.1% formic acid (25 mg/mL), filtered using a membrane (Vivaspin Turbo 15 VS15T01, Sartorius, Göttingen, Germany), and analyzed by UPLC-Q-TOF-MS/MS using the parameters described above. Quantitative analysis was performed in triplicate by comparing the peak areas of the corresponding mass traces with those of defined standard solutions of each reference peptide and oligosaccharide. Data were acquired with MassLynx software (Waters). External mass calibration was performed following the manufacturer’s provided protocol. Sensory Analysis The sensory analysis was reviewed for adherence to ethical guidelines and approved by the research ethics board at the Graduate School of Agriculture, Kyoto University (H27–3). Seventeen panelists (eight males and nine females, 21–40 years old), who gave informed consent for participating in the sensory tests, were trained in sensory experiments for at least 1 year for familiarity with the applied techniques. Sensory analysis for the identification of kokumi substances was carried out using following method. Sample solutions for sensory analysis were prepared as follows: the freeze-dried aqueous phase extracted from heat-treated soybean seeds (1 g) was dissolved in 10 mL distilled water; the freeze-dried low-molecular-weight fraction

5

Kokumi Substances in Soybean Seeds

51

(0.4 g) and high- molecular-weight fraction (0.6 g) separated by the UF membrane were dissolved in 10 mL of distilled water. GPC fractions (0.1 g) and preparative HPLC fractions (50 mg) were dissolved in 10 mL distilled water. All samples were dissolved in the control solution consisting of monosodium glutamate (0.02 w/v%), inosine monophosphate (0.01 w/v%), and sodium chloride (0.5 w/v%), and adjusted to pH to 6.5 with NaOH. These solutions were then presented in a dual standard discrimination test along with the control solution to the trained sensory panelists. The panelists were asked to judge whether the samples had enhanced the koku attributes as compared to the control solution. To evaluate the taste profile of these samples, time course profiles of taste intensity (thickness; intensity ~5 s after tasting, continuity; intensity ~20 s after tasting) and mouthfulness (the reinforcement of the taste sensation throughout the mouth) were evaluated (Ohsu et al. 2010). Time course profiles of taste intensity were evaluated using a non-graduated 10 cm long linear scale (0 cm: low taste intensity; 10 cm: high taste intensity), and mouthfulness was evaluated using a non-graduated 10 cm long linear scale (0 cm: not detectable; 10 cm: intensity perceived). Each sample (10 mL) was administered orally, and panelists were instructed to keep the sample in their mouths for 20 s, during which the taste intensity was monitored every 5 s. Determination of recognition threshold concentration of koku attribute was carried out using following method. The taste recognition threshold of each substance in water and the control solution (described above) was determined by means of a three-alternative forced-choice test (Frank et al. 2001). Solutions of each substance were presented in the order of increasing concentrations (serial 1:1 dilutions), and panelists were asked to evaluate whether the solutions exhibited enhanced the intensities in koku attributes as compared to the control solution. The taste threshold values of each substance were determined as the concentration at which none of the panelists could distinguish the difference as compared to the control solution. The taste threshold was averaged in three different sessions. The values among individuals and separate sessions differed by no more than one dilution step. Statistical Analysis In each sensory evaluation, differences between groups were compared with Dunnett’s test and one-way analysis of variance (ANOVA) followed by Tukey’s test. p values less than 0.05 and 0.01 were considered statistically significant.

5.2.3

Results and Discussion

Taste-Improving Effects of Soybean Seeds To identify compounds with taste-improving activities in soybeans, the watersoluble extract was prepared. Generally, the water-extract of raw soybean seeds

52

M. Shibata and Y. Matsumura

Fig. 5.3 Sensory analysis of taste sensation of control solution without additives and with aqueous extract of soybeans; fractions were separated using UF membrane. Data are presented as mean ± SE (n = 17), evaluated using a non-graduated 10 cm long linear scale. Asterisks indicate samples showing significant differences (Dunnett’s test) from the control solution: ‫٭‬p < 0.05; ‫٭٭‬p < 0.01

has a grassy smell and astringent taste owing to lipoxygenase activity (Wilkens and Lin 1970; Torres-Penaranda et al. 1998). Therefore, soybean seeds were heat-treated to deactivate lipoxygenase prior to extraction. These heat-treated soybean seeds were powdered, dispersed in water, and kept for 60 min. After centrifugation, the aqueous phase was collected as an extract and freeze-dried. The aqueous phase was dissolved in water or in the control solution, and the taste was evaluated by a sensory panel. When the aqueous phase was dissolved in water, it was nearly tasteless or exhibited a slightly sweet flavor. On the other hand, the addition of the same amount of the aqueous phase to the control solution caused a significant increase in thickness (intensity at ~5 s after tasting), continuity (intensity ~20 s after tasting), and mouthfulness (Fig. 5.3) as compared to the control solution only, without affecting the taste of the control solution. Since such effects are known as the ones of kokumi substance, it was thought that the aqueous phase from soybeans included some kokumi substances. Isolation of the Key Kokumi Substances The aqueous phase extracted from heat-treated soybean seeds contained major soybean proteins (7S globlin, 11S glycinin, and lipophilic proteins), carbohydrates, ash, lipids, and other minor components. In order to determine whether the high- or

5

Kokumi Substances in Soybean Seeds

53

low-molecular-weight fraction contributed to the flavor, the aqueous phase was separated by a UF membrane with a molecular weight cut-off profile of 10,000), carbohydrates were mainly separated into the lowmolecular-weight fraction (< 10,000). Sensory analysis revealed that the dispersion of freeze-dried powder in the high-molecular-weight fraction was almost tasteless, but the dispersion of the low-molecular-weight fraction had a slightly astringent and sweet taste. The sensory effects of the kokumi substance of the powders of the highand low-molecular-weight fractions were also evaluated upon dispersion in the control solution. The low-molecular-weight fraction demonstrated koku-enhancing activity, and the strength of this effect was almost the same as that of the aforementioned aqueous phase, whereas the high-molecular-weight fraction did not influence the taste profile of the control solution (Fig. 5.3). These results suggested that the key kokumi substances were water soluble and had comparatively low molecular weights. Next, the low-molecular-weight fraction was further separated by GPC fractionation, and the effluent was monitored at 220 nm. Five fractions (fraction 1–5, Fig. 5.4a) were separated and individually freeze-dried for sensory evaluations. Fractions 1 and 3 were slightly astringent and fraction 5 demonstrated the umami taste when dispersed in water. The other two fractions were almost tasteless in water. In addition, these fractions were evaluated in the control solution, and only fraction 3 exhibited the koku- enhancing effects (Fig. 5.4b). Faction 3 likely contained peptides, as noted by the UV absorption at 220 nm. This fraction also contained saccharides (data not shown). In order to isolate the kokumi molecules, GPC fraction 3 was further separated by preparative HPLC using an HILIC column because of the high polarity. The effluent was monitored using an MS detector and was separated into 6 fractions (Fig. 5.5a); each fraction was freeze-dried and used for sensory analysis using the control solution as the solvent. As shown in Fig. 5.5b, the koku enhancement was only found in fractions 3–4 and 3–5. To obtain insight into the chemical structures of the kokumi substances, these fractions were further separated into sub-fractions by HPLC using an HILIC column (Fig. 5.6). These fractions were also subjected to sensory analysis as described above, and only fractions 3–4–4 and 3–5–5 exhibited the koku-enhancing effects (Fig. 5.7). As a result, the components of fractions 3–4–4 and 3–5–5 were speculated to be major contributors to the kokumi taste sensation. However, the taste intensity of fraction 3–5–5 was lower than expected (i.e., equal to the intensity of fraction 3–5 (Fig. 5.7b)). Since all of the sub-fractions of fraction 3–5 except for 3–5–5 did not exhibit the koku- enhancement, the effects of combining sub-fraction 3–5–5 with other sub-fractions were investigated. As shown in Fig. 5.7b, fraction 3–5–9 and 3–5–10 combined with fraction 3–5–5 demonstrated a higher enhancement in koku as compared to fraction 3–5–5 alone. These results suggested that there were two kokumi substances in the water extract of soybean seeds: one which imparted the intensities in koku attributers intrinsically, and

54

M. Shibata and Y. Matsumura

Fig. 5.4 Gel permeation chromatogram (a) and sensory analysis of taste sensation of fractions separated by UF membrane (b). Taste intensity is presented as mean ± SE (n = 17), evaluated using a non-graduated 10 cm long linear scale. Asterisks indicate samples showing significant differences (Dunnett’s test) from the control solution: ‫٭‬p < 0.05; ‫٭٭‬p < 0.01

5

Kokumi Substances in Soybean Seeds

55

Fig. 5.5 HILIC-HPLC total ion chromatogram (a) and sensory analysis of taste sensation of fractions separated by GPC fraction 3 (b). Taste intensity is presented as means ± SE (n = 17), evaluated using a non-graduated 10 cm long linear scale. Asterisks indicate samples showing significant differences (Dunnett’s test) from the control solution: ‫٭‬, p < 0.05; ‫٭٭‬, p < 0.01

another which enhanced the intensities in koku attributes when combined with kokumi substances. In this case, the former and latter corresponded to 3–5–5 and 3–5–9 or 3–5–10, respectively. In order to identify the kokumi substances, the chemical structures of these fractions were analyzed.

56

M. Shibata and Y. Matsumura

Fig. 5.6 Total ion chromatograms of fractions 3–4 (a) and 3–5 (b) separated by HILIC-HPLC

Structure Determination of the Kokumi Substances LC-MS and LC-MS/MS analysis of fractions 3–4–4 and 3–5–5, which exhibited koku- enhancement, indicated that these fractions contained different dipeptides. In fraction 3–5–5, an unfragmented deprotonation ion (C14H17N2O6-, [M-H]-, Rt = 1.75 min, m/z = 309.1074) was detected by electrospray negative ionization mass spectrometry (Fig. 5.8a, b). Tandem mass spectrometry (MS/MS) (Fig. 5.8c) revealed [Glu-H2O-H]- (m/z = 128.0356), [Tyr-H]- (m/z = 180.0673), and [Tyr-NH3-H]- (m/z = 163.0405) fragments, and the fragment ions detected in fraction 3–5–5 matched those of the pure γ-glutamyl-tyrosine standard. Similarly,

5

Kokumi Substances in Soybean Seeds

57

Fig. 5.7 Data are presented as means ± SE (n = 17), evaluated using a non-graduated 10 cm long linear scale. (a) Asterisks indicate samples showing significant differences (Dunnett’s test) from the control solution: ‫٭‬p < 0.05; ‫٭٭‬p < 0.01. (b) Within the same evaluation points, values without a common letter were significantly different ( p < 0.05; Tukey’s test)

an unfragmented deprotonation ion (C14H17N2O5-, [M-H]-, Rt = 1.45 min, m/ z = 293.1139) was detected in fraction 3–4–4 by electrospray negative ionization mass spectrometry (Fig. 5.9a, b). MS/MS analysis (Fig. 5.9c) revealed [Glu-H2OH]- (m/z = 128.0363), [Phe-H]- (m/z = 164.0720), and [Phe-NH3-H]- (m/ z = 147.0458) fragments, and the detected fragment ions in fraction 3–4–4 matched those of the pure γ-glutamyl-phenylalanine standard. Fraction 3–5–9 and 3–5–10,

58

M. Shibata and Y. Matsumura

Fig. 5.8 Identification of γ-glutamyl-tyrosine by LC-MS/MS analysis. (a) Extracted ion chromatogram data; (b) full mass data; (c) MS/MS data. Each data point represents standard compound and fractionated sample

which contained kokumi substances, were also analyzed using the same method. Unfragmented deprotonation ions (C18H31O16-, [M-H]-, Rt = 4.37 min, m/ z = 503.1603; C24H41O21-, [M-H]-, Rt = 6.11 min, m/z = 665.2132) were detected in fractions 3–5–9 and 3–5–10 by electrospray negative ionization, respectively (Figs. 5.10a, b and 5.11a, b). MS/MS analysis (8C and 9C) revealed the loss of hexose fragments in fractions 3–5–9 ([M-Hex-H]-, m/z = 341.1082; [M-2Hex-H]-, m/z = 179.0561) and 3–5–10 ([M-Hex-H]-, m/z = 503.1602; [M-2Hex-H]-, m/ z = 341.1076; [M-3Hex-H]-, m/z = 179.0555). Upon comparison to the pure standards, the fragment ions of fractions 3–5–9 and 3–5–10 were identified as raffinose and stachyose, which are known to be contained in soybeans. Based on

5

Kokumi Substances in Soybean Seeds

59

Fig. 5.9 Identification of γ-glutamyl-phenylalanine by LC-MS/MS analysis. (a) Extracted ion chromatogram data; (b) full mass data; (c) MS/MS data. Each data point represents standard compound and fractionated sample

these data, γ-glutamyl-tyrosine and γ-glutamyl-phenylalanine were identified as the kokumi substances in fractions 3–5–5 and 3–4–4, and raffinose and stachyose were identified as kokumi substances which enhance the effect of γ-glutamyl-tyrosine and γ-glutamyl-phenylalanine in fractions 3–5–9 and 3–5–10. Although the presence of γ-glutamyl-tyrosine, γ-glutamyl-phenylalanine, raffinose, and stachyose in soybean seeds has been reported (Morris and Thompson 1962; Hou et al. 2009), this is the first indication that these peptides and oligosaccharides are the kokumi substances in soybean extracts, soymilk, and their derivatives.

60

M. Shibata and Y. Matsumura

Fig. 5.10 Identification of raffinose by LC-MS/MS analysis. (a) extracted ion chromatogram data; (b) full mass data; (c) MS/MS data. Each data point represents standard compound and fractionated sample

Quantitative Analysis of the Kokumi Substances In order to determine the quantitative effect of the kokumi substances in soybean seeds, quantitative analysis of two γ-glutamyl peptides and two oligosaccharides in soybean seeds was carried out; the low-molecular-weight fraction of the soybean water extract separated by a UF membrane with a molecular weight cut-off profile of 1%, mature Ganjang enhanced CT response to glutamate. The authors concluded that altered CT responses to NaCl may be due to the release of calcitonin gene-related peptides from trigeminal fibers near the fungiform taste buds. Identification of the kokumi peptides, as well as the receptor mechanisms, should be further investigated. Concerning trigeminal involvement in sensory expression by kokumi substance, Leijon et al. (2019) recently reported that oral application of kokumi substances including EVG elicited small responses with variable latencies ranging from 2 s to more than 200 s in a very small fraction (0.6%) of trigeminal neurons in mice. The co-application of a CaSR antagonist decreased the magnitude of these responses, indicating the involvement of CaSR. These findings offer suggestive evidence for the involvement of the CaSR in trigeminal neuron responses to kokumi substances. However, further studies are needed to elucidate the contribution of oral texture perceptions to kokumi flavor.

11

γ-Glutamyl-Valyl-Glycine (γ-Glu-Val-Gly) and Glutathione (γ-Glu-Cys-Gly). . .

11.5

185

Conclusion

EVG, at low concentrations that do not elicit a taste of its own, enhances the preference for palatable tastes induced by umami, fatty, and sweet stimuli in rats. GSH also enhances the preference for umami solutions in mice. The taste nerve responses to these stimuli increased upon addition of kokumi substances. These findings, obtained in rodents, are similar to those observed in human sensory tests, suggesting that kokumi substances enhance the palatability of food. However, it is difficult to determine whether animals can detect the sensory effect of kokumi substance by evaluating three main attributes: thickness, mouthfulness, and continuity (lingeringness). Although we should be careful in applying results from animals to humans because of species differences, the results obtained in animal experiments may partially reveal the mechanisms by which EVG and GSH induce thickness, mouthfulness, and continuity (lingeringness) in humans. It is natural to assume that some basic mechanisms may be common between species in searching for and accepting more nutritional and palatable edibles.

References Ahmad R, Dalziel JE (2020) G protein-coupled receptors in taste physiology and pharmacology. Front Pharmacol 11:587664 Bystrova MF, Romanov RA, Rogachevskaja OA, Churbanov GD, Kolesnikov SS (2010) Functional expression of the extracellular-Ca2+sensing receptor in mouse taste cells. J Cell Sci 123: 972–982 Dunkel A, Köster J, Hofmann T (2007) Molecular and sensory characterization of gamma-glutamyl peptides as key contributors to the kokumi taste of edible beans (Phaseolus vulgaris L.). J Agric Food Chem 55:6712–6719 Feng T, Wu Y, Zhang Z, Song S, Zhuang H, Xu Z, Yao L, Sun M (2019) Purification, identification, and sensory evaluation of kokumi peptides from Agaricus bisporus mushroom. Foods 8(2):43 Kuroda M, Miyamura N (2015) Mechanism of the perception of “kokumi” substances and the sensory characteristics of the “kokumi” peptide, γ-Glu-Val-Gly. Flavour 4(1):11 Leijon SCM, Chaudhari N, Roper SD (2019) Mouse trigeminal neurons respond to kokumi substances. In: Nishimura T, Kuroda M (eds) Koku in food science and physiology. Springer, Singapore, pp 171–187 Liu J, Song H, Liu Y, Li P, Yao J, Xiong J (2015) Discovery of kokumi peptide from yeast extract by LC-Q-TOF-MS/MS and sensomics approach. J Sci Food Agric 95:3183–3189 Maruyama Y, Yasuda R, Kuroda M, Eto Y (2012) Kokumi substances, enhancers of basic tastes, induce responses in calcium-sensing receptor expressing taste cells. PLoS One 10:e34489 Matsumura S, Mizushige T, Yoneda T, Iwanaga T, Si T, Inoue K, Fushiki T (2007) GPR expression in the rat taste bud relating to fatty acid sensing. Biomed Res 28:49–55 Mizuta H, Kumamoto N, Ugawa S, Yamamoto T (2021) Additive effects of L-ornithine on preferences to basic taste solutions in mice. Nutrients 13(11):3749 Nishimura T, Kuroda M (eds) (2019) Koku in food science and physiology. Springer, Singapore Ohsu T, Amino Y, Nagasaki H, Yamanaka T, Takeshita S, Hatanaka T, Maruyama Y, Miyamura N, Eto Y (2010) Involvement of the calcium-sensing receptor in human taste perception. J Biol Chem 285:1016–1022

186

T. Yamamoto

Rhyu MR, Song AY, Kim EY, Son HJ, Kim Y, Mummalaneni S, Qian J, Grider JR, Lyall V (2020) Kokumi taste active peptides modulate salt and umami taste. Nutrients 12(4):1198 Salger M, Stark TD, Hofmann T (2019) Taste modulating peptides from overfermented cocoa beans. Agric Food Chem 67:4311–4320 San Gabriel A, Uneyama H, Maekawa T, Torii K (2009) The calcium-sensing receptor in taste tissue. Biochem Biophys Res Commun 378:414–418 Shibata M, Hirotsuka M, Mizutani Y, Takahashi H, Kawada T, Matsumiya K, Hayashi Y, Matsumura Y (2017) Isolation and characterization of key contributors to the “kokumi” taste in soybean seeds. Biosci Biotechnol Biochem 81:2168–2177 Toelstede S, Dunkel A, Hofmann T (2009) A series of kokumi peptides impart the long-lasting mouthfulness of matured Gouda cheese. J Agric Food Chem 57:1440–1448 Ueda Y, Sakaguchi M, Hirayama K, Miyajima R, Kimizuka A (1990) Characteristic flavor constituents in water extract of garlic. Agric Biol Chem 54:163–169 Ueda Y, Tsubuku T, Miyajima R (1994) Composition of sulfur-containing components in onion and their flavor characters. Biosci Biotechnol Biochem 58:108–110 Ueda Y, Yonemitsu M, Tsubuku T, Sakaguchi M, Miyajima R (1997) Flavor characteristics of glutathione in raw and cooked foodstuffs. Biosci Biotechnol Biochem 61:1977–1980 Yamamoto T, Mizuta H (2022) Supplementation effects of a kokumi substance, γ-Glu-Val-Gly, on the ingestion of basic taste solutions in rats. Chem Senses 47:bjac008 Yamamoto T, Watanabe U, Fujimoto M, Sako N (2009) Taste preference and nerve response to 5′-inosine monophosphate are enhanced by glutathione in mice. Chem Senses 34:809–818

Chapter 12

Effects of the Potent Kokumi Peptide, γ-Glutamyl-Valyl-Glycine, on Sensory Characteristics of Foods and Beverages Motonaka Kuroda

Abstract Recent studies demonstrated that kokumi substances, such as glutathione, are perceived via the calcium-sensing receptor (CaSR). Screening with the CaSR assay and sensory evaluations have shown that γ-glutamyl-valyl-glycine (γ-Glu-ValGly) is a potent kokumi peptide. In the present study, the sensory effects of γ-Glu-Val-Gly on chicken consommé, hamburger steak, orange flavored drink, and reduced-fat foods (peanut butter, French dressing and custard cream) were investigated using a descriptive analysis. The addition of γ-Glu-Val-Gly significantly enhanced umami, mouthfulness, and the mouth coating in chicken consommé, and oiliness, umami, the thickness of taste in gravy, the middle and last meaty flavor, and pepper flavor in hamburger steak. The addition of this peptide significantly enhanced sweetness, the thickness of taste, and juiciness in orange flavored drink. In reduced-fat foods, the addition of γ-Glu-Val-Gly significantly enhanced thickness of taste, continuity, aftertaste, and oiliness. Collectively, these results suggest that the kokumi peptide, γ-Glu-Val-Gly, modifies several sensory characteristics in various foods. As a kokumi substance, γ-Glu-Val-Gly has no taste of its own and, thus, may be used to enhance the flavor of both savory and sweet foods.

Abbreviations CaSR FEMA GSH IMP JECFA MSG γ-Glu-Val-Gly

Calcium-sensing receptor Flavor and Extract Manufacturers’ Association Glutathione Inosine monophosphate The joint FAO/WHO expert committee Monosodium glutamate γ-Glutamyl-valyl-glycine

M. Kuroda (✉) Institute of Food Research and Technologies, Ajinomoto Co., Inc., Kawasaki, Kanagawa, Japan e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2024 M. Kuroda (ed.), Kokumi Substance as an Enhancer of Koku, https://doi.org/10.1007/978-981-99-8303-2_12

187

188

12.1

M. Kuroda

Background

Taste and aroma are important factors affecting the flavor of foods. Sweet, salty, sour, bitter, and umami comprise the five basic tastes. Each taste is recognized by specific receptors and is associated with specific neural transmission pathways. However, foods have sensory attributes that cannot be explained by aroma and the five basic tastes alone, such as texture, continuity, complexity, and mouthfulness. As shown in Chap. 4 (Ueda et al. (1990) investigated the flavoring effects of a diluted extract of garlic that enhanced continuity, mouthfulness, and the thickness of taste when added to an umami solution. They identified S-allyl-L-cysteine sulfoxide (alliin), cycloalliin, S-methyl-L-cysteine sulfoxide, cysteine (Cys), and glutathione (GSH) as the compounds responsible for these sensory effects. Ueda et al. also examined the threshold of GSH in various systems, and showed that it was markedly lower in an umami solution (consisting of a solution containing 3.1% monosodium glutamate (MSG) and a solution containing 0.05% MSG and 0.05% inosine monophosphate (IMP) than in water (Ueda et al. 1997). Therefore, GSH only has a minimal flavor in water, but if added to an umami solution, it markedly enhances the thickness of taste, continuity, and mouthfulness (Ueda et al. 1997). Ueda et al. proposed referring to substances with these properties as kokumi substances. Therefore, as cited in Chap. 2 a kokumi substance is considered to be one of the “Koku”-imparting factors. The research group led by Professor Hofmann at the Technical University of Munich investigated kokumi substances in various foodstuffs using sensory evaluation-guided elucidation (a sensomics approach). They examined the sensory effects of various compounds isolated from foods by panelists who were trained using 5 mM GSH in model chicken broth to recognize enhancements in mouthfulness and complexity (Dunkel et al. 2007). Dunkel et al. also isolated γ-Glu-Leu, γ-Glu-Val, and γ-Glu-Cys-β-Ala (homoglutathione) as kokumi substances from edible beans (Phaseolus vulgaris L.). They indicated that the threshold values for these peptides were markedly lower in salty/umami solution and chicken broth than in water. Toelstede et al. (2009) identified γ-Glu-Glu, γ-Glu-Gly, γ-Glu-Gln, γ-Glu-Met, γ-Glu-Leu, γ-Glu-Val, and γ-Glu-His as kokumi substances from matured Gouda cheese. They indicated that the threshold values for these peptides were markedly lower in a reconstituted Gouda cheese extract solution than in water. As shown in Chap. 5, Shibata et al. (2017) examined the key kokumi substances in soybean seeds and identified γ-Glu-Tyr and γ-Glu-Phe as key kokumi peptides. They also reported that the threshold of γ-Glu-Tyr was 0.005 mM in a salty/umami solution, which was markedly lower than that in water (3.0 mM). Kokumi substances, such as GSH, were previously reported to be perceived via the calcium-sensing receptor (CaSR) in humans (Ohsu et al. 2010). The studies described above indicated that GSH and several other peptides activate human CaSR, similar to several γ-glutamyl-peptides, including γ-Glu-Ala, γ-Glu-Val, γ-Glu-Cys, γ-Glu-α-aminobutyryl-Gly (ophthalmic acid), and γ-Glu-Val-Gly.

12

Effects of the Potent Kokumi Peptide, γ-Glutamyl-Valyl-Glycine, on. . .

189

Furthermore, these γ-glutamyl-peptides possess the characteristics of kokumi substances that modify the five basic tastes (particularly sweet, salty, and umami) when added to basic taste solutions or food, even though they have no taste themselves at the concentrations used. The CaSR activity of these γ-glutamyl peptides positively correlated with the sensory activity of kokumi substances, suggesting these γ-glutamyl peptides are perceived via CaSR in humans. γ-Glu-Val-Gly has been identified as a potent kokumi peptide with sensory activity that is 12.8-fold greater than that of GSH (Ohsu et al. 2010). In that study, γ-Glu-Val-Gly enhanced several basic tastes, such as sweetness, umami, and saltiness, at one tenth of the concentration of GSH (Ohsu et al. 2010). Collectively, these findings on various kokumi peptides indicate that γ-Glu-ValGly is a potent kokumi peptide. In this chapter, we describe the sensory effects of γ-Glu-Val-Gly in various food systems.

12.2

Effects of γ-Glutamyl-Valyl-Glycine on Sensory Characteristics of Chicken Consommé

12.2.1

Introduction

Ohsu et al. (2010) previously reported that the addition of 0.01% γ-Glu-Val-Gly to 3.3% sucrose solution, 0.9% NaCl solution, and 0.5% MSG solution significantly enhanced sweetness, saltiness, and umami, respectively. They also showed that the addition of 20 ppm of γ-Glu-Val-Gly (66 μmole/kg) to chicken consommé prepared from commercial chicken consommé powder significantly enhanced the thickness of taste, continuity, and mouthfulness. The sensory evaluation employed was performed with sensory attributes described in a previously reported method (Ueda et al. 1990, 1997). In the present study, we characterized the sensory properties of food with added γ-Glu-Val-Gly using a descriptive sensory analysis of chicken consommé containing γ-Glu-Val-Gly.

12.2.2

Materials and Methods

Materials The γ-Glu-Val-Gly used in the present study was of food additive grade (FEMAGRAS No. 4709; JECFA food flavoring No. 2123), obtained from Ajinomoto Co., Inc. (Tokyo, Japan), and prepared by chemical synthesis as previously reported (Ohsu et al. 2010). Chicken consommé was prepared by a method described by Miyaki et al. (2015). The resulting chicken consommé was freeze-dried (freezing temperature, -24 °C;

190

M. Kuroda

vacuum, 65% in traditional mayonnaise. Therefore, it is generally regarded as a high-fat and highly caloric food. The preparation of low-fat salad dressing, particularly mayonnaise-type dressing, has been extensively examined. Previous studies prepared and evaluated low-fat mayonnaise containing polysaccharide gums (Wedninin et al. 1997; Su et al. 2010), whey protein isolate and pectin (Liu et al. 2007; Sun et al. 2018), mucilage from plants (Aghdaei et al. 2014; BernardinoNicanor et al. 2015; Fernandes and Mellado 2018), microcrystalline cellulose (Grodzka et al. 2005), or an extruded flour paste (Roman et al. 2015) as well as low-fat mayonnaise produced via double emulsions (Yildrim et al. 2016). The preparation and evaluation of French dressing containing a sucrose polyester emulsifier (Mellies et al. 1985) have also been reported. In the present study, we examined the effects of γ-Glu-Val-Gly on the sensory characteristics of reduced-fat French dressing.

212

12.6.2

M. Kuroda

Materials and Methods

Preparation of γ-Glu-Val-Gly γ-Glu-Val-Gly in the present study was of food additive grade (FEMA-GRAS No. 4709; FEMA: Flavor and Extract Manufacturers’ Association; JECFA food flavoring No. 2123; JECFA: Joint FAO/WHO Expert Committee on Food Additives), obtained from Ajinomoto Co. Inc. (Tokyo, Japan), and prepared by chemical synthesis as previously reported (Ohsu et al. 2010). Preparation of Reduced- and Full-Fat French Dressing Raw materials for reduced-fat French dressing (15.0% fat content) and full-fat French dressing (37.5% fat content) are shown in Table 12.14. Each French dressing sample was prepared as follows. Raw materials, except for soybean oil, were mixed by the handy food processer Bamix M-300 (ESGE AG, Switzerland) for 2 min, heated at 85 °C for 10 min, and cooled to room temperature by iced water. Soybean oil was then added, followed by homogenization at 10,000 rpm for 2 min using a laboratory-scale homogenizer (Labolution, Primix Corp., Hyogo, Japan). Prepared French dressing samples were stored in a refrigerator at 4 °C until the sensory evaluation. Sensory Panel In the present study, 29 panelists (17 men and 12 women; 28.8 ± 5.0 years old, mean ± standard deviation) participated in the sensory evaluation. All panelists were employees of the Ajinomoto Shanghai Food Research and Technology Center and were working in food development. All panelists were Chinese and residents of Shanghai city. In addition, all of the panelists passed the sensory panel examination conducted using a previously described method (Furukawa 1977). Twenty panelists (9 men and 11 women; 27.6 ± 3.6 years old, mean ± standard deviation) participated in the sensory evaluation of reduced- and full-fat French dressing. Nineteen panelists (13 men and 6 women; 29.9 ± 5.3 years old, mean ± standard deviation) performed the sensory evaluation to investigate the effects of γ-Glu-Val-Gly on reduced-fat French dressing. Selection of Sensory Attributes Panelists evaluated reduced- and full-fat French dressing samples. A panel leader led the group in discussions on differences and similarities between the samples. Panelists developed 11 attributes: garlic flavor, spice flavor, thickness of taste,

12

Effects of the Potent Kokumi Peptide, γ-Glutamyl-Valyl-Glycine, on. . .

213

Table 12.14 Raw materials for the reduced-fat and full-fat French dressing Materials High-fructose corn syrup Vinegar Soybean oil Salt Hydroxypropyl distarch phosphate Paprica powder Citric acid Xanthan gum Emulsifier (polyoxyethylene sorbitan monostearate) Roast garlic powder Tomato flavor Lemon flavor Food coloring agent Water

Reduced-fat (wt%) 28.20 23.10 15.00 2.20 1.60 1.10 0.40 0.60 0.40 0.40 0.60 0.05 2.40 23.95

Full-fat (wt%) 28.20 23.10 37.50 2.20 1.60 1.10 0.40 0.28 0.40 0.40 0.60 0.05 2.40 1.77

continuity of taste, smoothness, oiliness, viscosity (viscous sensation), sourness, sweetness, the initial taste, and aftertaste. Panelists practiced rating samples according to the list as preparation for data collection. Procedure for the Sensory Evaluation The sensory evaluation was conducted between 10:00 am and 11:30 am in a partitioned booth at 25 °C in an air-conditioned sensory evaluation room. In the evaluation of samples, 15 g of the French dressing sample was mixed with 25 g of cut lettuce, and panelists held the sample in their mouths, evaluated the taste, and rated each attribute. They rinsed their mouths with commercial mineral water between samples. They completed the rating for each attribute on a 3-point linear scale; -1.0: apparently weaker than the control; 0: same as the control; and 1.0: apparently stronger than the control. The combination of samples was randomized and balanced. Human sensory analyses were conducted according to the guidelines of the Declaration of Helsinki, and informed consent was obtained from all panelists. The experimental procedure was approved by the Ethics Board of the Institute of Food Sciences & Technologies, Ajinomoto. Statistical Analysis Statistical analyses were conducted using JMP version 9.0 (SAS Institute, Cary, NC, USA). Data were collected as means ± standard errors and assessed by the paired t-test. Data were considered to be significant at p < 0.05.

214

12.6.3

M. Kuroda

Results and Discussion

Sensory Attributes During the group discussion, panelists listed words, selected attributes, and reached a consensus on which sensation the attribute expressed. They developed 11 attributes: garlic flavor, spice flavor, thickness of taste, continuity of taste, smoothness, oiliness, viscosity (viscous sensation), sourness, sweetness, the initial taste, and aftertaste. The definitions of these sensory attributes are shown in Table 12.15. Sensory Evaluation of Reduced- and Full-Fat French Dressing The results of the sensory evaluation of reduced- and full-fat French dressing samples are shown in Table 12.16. Full-fat French dressing had significantly higher scores for the thickness of taste ( p < 0.05) and smoothness ( p < 0.01). No significant differences were observed in the other attributes examined. Among these attributes, the ones of which probability was less than 0.2 ( p < 0.2) were used for the sensory evaluation on the effect of γ-Glu-Val-Gly. Therefore, spice flavor, the initial taste, and aftertaste were added as sensory attributes (Table 12.17). Effects of the Addition of γ-Glu-Val-Gly on Reduced-Fat French Dressing To clarify the effects of γ-Glu-Val-Gly on the sensory characteristics of reduced-fat French dressing, we evaluated reduced-fat French dressing with the addition of 40 ppm of γ-Glu-Val-Gly. The results of the sensory evaluation are shown in Table 12.18 and Fig. 12.5. The addition of γ-Glu-Val-Gly significantly enhanced the intensity of the aftertaste ( p < 0.05), and slightly increased the intensities of the thickness of taste and initial taste ( p < 0.1). These results demonstrated that the addition of γ-Glu-Val-Gly increased some sensations that were lacking in reducedfat French dressing, suggesting the potential of this peptide to improve the flavor of reduced-fat French dressing. As discussed above, previous studies prepared and evaluated low-fat mayonnaise dressing containing polysaccharide gums (Wedninin et al. 1997; Su et al. 2010), whey protein isolate and pectin (Liu et al. 2007; Sun et al. 2018), mucilage from plants (Aghdaei et al. 2014; Bernardino-Nicanor et al. 2015; Fernandes and Mellado 2018), microcrystalline cellulose (Grodzka et al. 2005), or an extruded flour paste (Roman et al. 2015) as well as low-fat mayonnaise produced via double emulsions (Yildrim et al. 2016). Although many of these studies focused on physicochemical properties, several performed sensory evaluations of low-fat mayonnaise containing fat mimetics or replacers. Liu et al. (2007) investigated the rheological, textural, and sensory properties of low-fat mayonnaise with various fat mimetics, and reported that low-fat mayonnaise (40% fat; full-fat mayonnaise contains 80% fat) containing

12

Effects of the Potent Kokumi Peptide, γ-Glutamyl-Valyl-Glycine, on. . .

215

Table 12.15 Sensory attributes and their definition used in evaluation of the full-fat and reducedfat French dressing Sensory attributes Garlic flavor Spice flavor Thickness of taste Continuity of taste Oiliness Viscosity Smoothness Sourness Sweetness Initial taste Aftertaste

Definition The flavor intensity reminiscent of garlic The flavor intensity reminiscent of spice, especially paprika and garlic The enhancement of taste intensity with maintaining the balance of taste The total taste intensity at 20 s after tasting The oily mouth-coating sensations The degree to which the samples in viscous in mouth from thin to thick The amount in which the sample slides across the tongue One of the basic taste, common to organic acids such as citric acid, acetic acid, and malic acid One of the basic taste, common to sugars such as sucrose, fructose and maltose, and high-intensity sweeteners such as aspartame, sucralose, and advantame The total taste intensity at 1 s after tasting The total taste intensity at 5 s after tasting

Table 12.16 Result of the sensory evaluation of full-fat French dressing comparing to the reducedfat French dressing Sensory attributes Garlic flavor Spice flavor Thickness of taste Continuity of taste Oiliness Viscosity Smoothness Sourness Sweetness Initial taste Aftertaste

Score of sample with γ-Glu-Val-Gly 0.03 ± 0.04 0.05 ± 0.04 0.08 ± 0.03 0.05 ± 0.04 0.05 ± 0.04 0.00 ± 0.04 0.10 ± 0.03 -0.04 ± 0.06 0.03 ± 0.04 0.07 ± 0.04 0.07 ± 0.05

Significance n.s. n.s. * n.s. n.s. n.s. ** n.s. n.s. n.s. n.s.

Data was shown as means ± standard errors n.s. Not significant * p < 0.05, ** p < 0.01

pectin weak gel showed similar scores for odor, texture, taste, and acceptability as full-fat mayonnaise. Aghdaei et al. (2014) demonstrated that sensory scores, such as odor, texture, taste, and mouthfeel, for low-fat mayonnaise (46.8% fat; full-fat mayonnaise contains 78% fat) containing Isfarzeh seed mucilage did not significantly differ from those of full-fat mayonnaise. Furthermore, Su et al. (2009) showed that sensory scores, such as aroma, taste, and greasiness, for low-fat mayonnaise (36.5% fat; full-fat mayonnaise contains 73% fat) containing xanthan gum and guar

216

M. Kuroda

Table 12.17 Sensory attributes and their definition used in evaluation on the effect of γ-Glu-ValGly on the reduced-fat French dressing Sensory attributes Spice flavor Thickness of taste Initial taste Aftertaste Smoothness

Definition The flavor intensity reminiscent of spice, especially paprika and garlic The enhancement of taste intensity with maintaining the balance of taste The total taste intensity at 1 s after tasting The total taste intensity at 5 s after tasting The oily smooth sensation

Table 12.18 Effect of γ-Glu-Val-Gly on the reduced-fat French dressing Sensory attributes Spice flavor Thickness of taste Initial taste Aftertaste Smoothness

Score of sample with γ-Glu-Val-Gly 0.05 ± 0.04 0.07 ± 0.04 0.07 ± 0.04 0.08 ± 0.03 0.01 ± 0.04

Significance n.s. p < 0.1 p < 0.1 p < 0.05 n.s.

Data was shown as means ± standard errors n.s. Not significant Spice flavor 0.2 0.1

Smoothness

0

#

-0.1

Thickness of taste

-0.2

Aftertaste*

#

Initial taste

Fig. 12.5 Effects of adding 40 ppm of γ-Glu-Val-Gly on sensory characteristics of reduced-fat French dressing. The blue line indicates the mean scores for control reduced-fat French dressing. The red line indicates the mean scores for reduced-fat French dressing with 40 ppm of γ-Glu-ValGly. # p < 0.1, * p < 0.05

gum did not significantly differ from those of full-fat mayonnaise. However, in these studies, the effects of a fat replacer or mimetic on sensory characteristics were not examined in detail. Nevertheless, the combination of γ-Glu-Val-Gly and a fat replacer may further improve the sensory quality of reduced- or low-fat salad dressing.

12.6.4

Conclusions and Implications

The effects of the kokumi peptide, γ-Glu-Val-Gly, on the flavor of reduced-fat French dressing were investigated in the present study. The results obtained

12

Effects of the Potent Kokumi Peptide, γ-Glutamyl-Valyl-Glycine, on. . .

217

indicated that the addition of γ-Glu-Val-Gly significantly enhanced the intensity of the thickness of taste, and slightly increased the continuity of taste and aftertaste. These results demonstrated that the addition of γ-Glu-Val-Gly increased some sensations that were lacking in reduced-fat French dressing, suggesting the potential of this peptide to improve the flavor of reduced-fat French dressing.

12.7 12.7.1

Effects of γ-Glutamyl-Valyl-Glycine on Sensory Characteristics of Reduced-Fat Custard Cream Introduction

The effects of γ-Glu-Val-Gly on the sensory characteristics of reduced-fat peanut butter and reduced-fat French dressing were examined in the previous sections. The results obtained indicated that the addition of γ-Glu-Val-Gly (40 ppm; 162 μM) significantly enhanced the intensities of the thickness of taste, and aftertaste. Furthermore, the addition of γ-Glu-Val-Gly enhanced oiliness, defined as an oily mouth coating. These sensations were previously shown to be evoked by the addition of fat-containing food materials, such as a dairy fat emulsion (Flett et al. 2010). As previously discussed, an increase in the prevalence of obesity has spurred the development and commercialization of various reduced-fat foods; however, these foods are generally less palatable than full-fat foods (McClements and Demetriades 1998). Among foods containing dairy cream, the sensory characteristics of low-fat ice cream have been examined, and the findings obtained showed lower scores for flavor and texture-related attributes, such as creaminess and smoothness (Patel et al. 2010; Abdel-Haleem and Awad 2015; Azari-Anpar et al. 2017; de Souza Fernandes et al. 2017; Sharma et al. 2017; Zhang et al. 2018; Guo et al. 2018). A descriptive analysis of low-fat ice cream (4.0% fat; full-fat ice cream contains 10.0% fat) revealed lower scores for thickness, smoothness, creaminess, mouth coating, cooked sugar flavor, milky flavor, condensed milk flavor, and milky aftertaste (Liou and Grun 2007). In the previous section, the addition of γ-Glu-Val-Gly, a potent kokumi peptide, to reduced-fat peanut butter increased the intensities of the thickness of taste, aftertaste, and oiliness. This result suggests the potential of γ-Glu-Val-Gly to improve the flavor of reduced-fat foods. In the present study, we investigated whether the addition of γ-Glu-Val-Gly enhanced the flavor of various reduced-fat foods. We examined the effects of this peptide on the sensory characteristics of reduced-fat custard cream.

218

12.7.2

M. Kuroda

Materials and Methods

Preparation of γ-Glu-Val-Gly γ-Glu-Val-Gly in the present study was of food additive grade (FEMA-GRAS No. 4709; FEMA: Flavor and Extract Manufacturers’ Association; JECFA food flavoring No. 2123; JECFA: Joint FAO/WHAO Expert Committee on Food Additives), obtained from Ajinomoto Co. Inc. (Tokyo, Japan), and prepared by chemical synthesis as previously reported (Ohsu et al. 2010). Preparation of Reduced- and Full-Fat Custard Cream The raw materials for reduced-fat custard cream (3.6% fat content) and full-fat custard cream (12.0% fat content) are shown in Table 12.19. Each custard cream was prepared as follows. Raw materials were mixed by the food processer Bamix M-300 (ESGE AG, Switzerland) for 1 min, heated at 95 °C for 30 min, screened through a mesh, and cooled to room temperature using iced water. Prepared custard cream samples were stored in a refrigerator at 4 °C until the sensory evaluation. Sensory Panel In the present study, 29 panelists (17 men and 12 women; 28.8 ± 5.0 years old, mean ± standard deviation) participated in the sensory evaluation. All panelists were employees of the Ajinomoto Shanghai Food Research and Technology Center and were working in food development. All panelists were Chinese and residents of Shanghai city. In addition, all of the panelists passed the sensory panel examination conducted using a previously described method (Furukawa 1977). Twenty panelists (9 men and 11 women; 27.6 ± 3.6 years old, mean ± standard deviation) participated in the sensory evaluation of reduced- and full-fat custard cream. Nineteen panelists (13 men and 6 women; 29.9 ± 5.3 years old, mean ± standard deviation) performed the sensory evaluation to investigate the effects of γ-Glu-Val-Gly on reduced-fat custard cream. Selection of Sensory Attributes Panelists evaluated reduced- and full-fat custard cream samples. A panel leader led the group in discussions on differences and similarities between the samples. Panelists developed a list of sensory attributes that described the sensory characteristics of the products. They developed nine attributes: thickness of taste, continuity of taste, sweetness, aftertaste, egg flavor, vanilla flavor, cream flavor, viscosity

12

Effects of the Potent Kokumi Peptide, γ-Glutamyl-Valyl-Glycine, on. . .

219

Table 12.19 Raw materials for the reduced-fat custard cream and full-fat custard cream Materials Fresh cream Milk Skim milk Egg yolk (20% sugar added) Sugar Flour Vanilla beans Starch Inulin cream Water

Reduced-fat (wt%) 0.0 0.0 6.0 14.5 8.0 3.0 0.1 0.5 24.3 43.6

Full-fat (wt%) 19.0 45.5 0.0 14.5 10.0 3.0 0.1 0.0 0.0 7.9

(viscous sensation), and oiliness. Panelists practiced rating samples according to the list as preparation for data collection. Procedure for the Sensory Evaluation The sensory evaluation was conducted between 10:00 am and 11:30 am in a partitioned booth at 25 °C in an air-conditioned sensory evaluation room. In the evaluation of custard cream samples, 10 g of a sample was put in the one piece of commercial chou (10 g). Panelists held each piece of chou cream in their mouths, evaluated the taste, and rated each attribute. They rinsed their mouths with commercial mineral water between samples. They completed rating each attribute on a 3-point linear scale; -1.0: apparently weaker than the control; 0: same as the control; and 1.0: apparently stronger than the control. In comparisons between the reducedand full-fat samples, 50% of panelists evaluated the full-fat sample using the reduced-fat sample as the control, while the other 50% evaluated the reduced-fat sample using the full-fat sample as the control. The serving order of samples was randomized and balanced. Human sensory analyses were conducted according to the guidelines of the Declaration of Helsinki, and informed consent was obtained from all panelists. The experimental procedure was approved by the Ethics Board of the Institute of Food Sciences & Technologies, Ajinomoto. Statistical Analysis Statistical analyses were conducted using JMP version 9.0 (SAS Institute, Cary, NC, USA). All data of the sensory evaluation were presented as means ± standard errors. Data were analyzed using the paired t-test. Differences were considered to be significant at p < 0.05.

220

12.7.3

M. Kuroda

Results and Discussion

Sensory Attributes During the group discussion, panelists listed words, selected attributes, and reached a consensus on which sensation the attribute expressed. Panelists developed seven attributes: thickness of taste (enhanced taste intensity while maintaining the balance of taste), continuity of taste (taste intensity assessed 20 s after tasting), sweetness, aftertaste (total taste intensity of all flavor notes within the sample 5 s after tasting), egg flavor, vanilla flavor, and oiliness. The definitions of these sensory attributes are shown in Table 12.20. Sensory Evaluation of Reduced- and Full-Fat Custard Cream The results of the sensory evaluation of reduced- and full-fat custard cream samples are shown in Table 12.21. Full-fat custard cream had significantly higher scores for aftertaste ( p < 0.05), vanilla flavor ( p < 0.01), and cream flavor ( p < 0.01) than reduced-fat custard cream. It also had slightly higher scores for the thickness and continuity of taste ( p < 0.1). No significant differences were observed in sweetness, bitterness, smoothness, or the viscous sensation between reduced- and full-fat samples. We considered fat to enhance sensory characteristics, such as the thickness of taste, continuity, aftertaste, vanilla flavor, and cream flavor, in custard cream. In other words, the thickness of taste, continuity, aftertaste, vanilla flavor, and cream flavor were the main sensory functions of fat in custard cream. Sensory attributes and their definitions are shown in Table 12.22. Effects of γ-Glu-Val-Gly on Reduced-Fat Custard Cream To clarify the effects of γ-Glu-Val-Gly on the sensory characteristics of reduced-fat custard cream, we evaluated reduced-fat custard cream with the addition of 40 ppm of γ-Glu-Val-Gly. The results of the sensory evaluation are shown in Table 12.23 and Fig. 12.6. The addition of γ-Glu-Val-Gly significantly enhanced the intensity of the thickness of taste ( p < 0.05), and slightly increased those of the continuity of taste and aftertaste ( p < 0.1). These results demonstrated that the addition of γ-Glu-Val-Gly increased some sensations that were lacking in reduced-fat custard cream, suggesting the potential of this peptide to improve the flavor of reduced-fat custard cream. Among foods containing dairy cream, the sensory characteristics of low-fat ice cream have been examined, and the findings obtained revealed lower scores for flavor and texture-related attributes, such as creaminess and smoothness (AbdelHaleem and Awad 2015; Azari-Anpar et al. 2017; de Souza Fernandes et al. 2017; Sharma et al. 2017; Zhang et al. 2018). The abilities of various types of fat replacers,

12

Effects of the Potent Kokumi Peptide, γ-Glutamyl-Valyl-Glycine, on. . .

221

Table 12.20 Sensory attributes and their definition used in evaluation of full-fat and reduced-fat custard cream Sensory attributes Thickness of taste Continuity of taste Sweetness Aftertaste Egg flavor Vanilla flavor Cream flavor Viscosity Oiliness

Definition The enhancement of taste intensity with maintaining the balance of taste The total taste intensity at 20 s after tasting One of the basic taste, common to sugars such as sucrose, fructose and maltose, and high-intensity sweeteners such as aspartame, sucralose, and advantame The total taste intensity at 5 s after tasting The flavor intensity reminiscent of egg yolk The flavor intensity reminiscent of vanilla The flavor intensity reminiscent of cream The degree to which the samples in viscous in mouth from thin to thick The oily mouth-coating sensation

Table 12.21 Result of the sensory evaluation of full-fat custard cream comparing to the reduced-fat custard cream

Sensory attributes Thickness of taste Continuity of taste Sweetness Aftertaste Egg flavor Vanilla flavor Cream flavor Viscosity Oiliness

Score of full-fat model 0.09 ± 0.05 0.10 ± 0.05 -0.03 ± 0.06 0.12 ± 0.06 0.09 ± 0.06 0.16 ± 0.05 0.18 ± 0.06 -0.01 ± 0.09 0.09 ± 0.06

Significance p < 0.1 p < 0.1 n.s. p < 0.05 n.s. p < 0.01 p < 0.01 n.s. n.s.

Data was shown as means ± standard errors n.s. Not significant Table 12.22 Sensory attributes and their definition used in evaluation on the effect of γ-Glu-ValGly on the reduced-fat custard cream Sensory attributes Thickness of taste Continuity of taste Aftertaste Vanilla flavor Cream flavor

Definition The enhancement of taste intensity with maintaining the balance of taste The total taste intensity at 20 s after tasting The total taste intensity at 5 s after tasting The flavor intensity reminiscent of vanilla The flavor intensity reminiscent of cream

such as sago powder (Patel et al. 2010), barley flour and barley β-glucan (AbdelHaleem and Awad 2015), resistant starch and maltodextrin (Azari-Anpar et al. 2017), cassava derivatives (de Souza Fernandes et al. 2017), citrus fiber (Zhang et al. 2018), citrus pectin (Zhang et al. 2018), and nano-bacterial cellulose/soy protein isolate complex gel (Guo et al. 2018), to improve the sensory characteristics

222

M. Kuroda

Table 12.23 Effect of γ-Glu-Val-Gly on the low-fat custard cream Sensory attributes Thickness of taste Continuity of taste Aftertaste Vanilla flavor Cream flavor

Score of sample with γ-Glu-Val-Gly 0.086 ± 0.033 0.070 ± 0.032 0.063 ± 0.036 0.026 ± 0.043 0.071 ± 0.042

Significance p < 0.05 p < 0.05 p < 0.1 n.s. n.s.

Data was shown as means ± standard errors n.s. Not significant

Thickness of taste 0.2 0.1

Mildness

*

0 -0.1

#

Continuity of taste

-0.2

#

Cream Flavor

Aftertaste

Vanilla Flavor

Fig. 12.6 Effects of adding 40 ppm of γ-Glu-Val-Gly on sensory characteristics of reduced-fat custard cream. The blue line indicates the mean scores for control reduced-fat custard cream. The red line indicates the mean scores for reduced-fat custard cream with the addition of 40 ppm of γ-Glu-Val-Gly. # p < 0.1, * p < 0.05

of low-fat ice cream have been tested. Patel et al. (2010) reported that the sensory characteristics, such as flavor, body and texture, and the melting quality, of low-fat mango ice cream (2.4% fat; full-fat mango ice cream contains 10.0% fat) containing sago powder did not significantly different from those of full-fat mango ice cream. Azari-Anpar et al. (2017) showed that the use of resistant starch and maltodextrin improved sensory characteristics, such as color, texture, and flavor. de Souza Fernandes et al. (2017) demonstrated that cassava bagasse and maltodextrin improved sensory characteristics, such as flavor, color, appearance, texture, and taste. Zhang et al. reported no significant differences in scores for appearance, flavor, taste, and smoothness between reduced-fat ice cream (45% fat decrease) containing citrus pectin and full-fat ice cream; however, the score for the mouth coating of reduced-fat ice cream containing citrus pectin was significantly lower than that of full-fat ice cream (Zhang et al. 2018). Guo et al. (2018) found that the texture profiles, such as hardness, springiness, chewiness, cohesiveness, and resilience, of low-fat ice cream containing nano-bacterial cellulose/soy protein isolate complex gel did not significantly differ from those of full-fat ice cream. However, in these studies, the effects of a fat replacer on sensory characteristics were not examined in detail.

12

Effects of the Potent Kokumi Peptide, γ-Glutamyl-Valyl-Glycine, on. . .

223

A descriptive sensory analysis of low-fat ice cream showed that low-fat ice cream (4.0% fat; full-fat ice cream contains 10.0% fat) had lower scores for thickness, smoothness, creaminess, mouth coating, cooked sugar flavor, milky flavor, condensed milk flavor, and milky aftertaste (Liou and Grun 2007). In that study, low-fat ice cream containing fat mimetics, such as microparticulated whey protein concentrate and polydextrose powder, was prepared to improve its flavor and texture, and the findings obtained revealed that scores for thickness, smoothness, creaminess, cooked sugar flavor, and condensed milk flavor did not significantly differ from those of full-fat ice cream. However, the milky flavor and milky aftertaste of low-fat ice cream containing fat mimetics were still lower than those of full-fat ice cream (Liou and Grun 2007). Based on the results shown in this section, γ-Glu-Val-Gly may be used to improve the flavor of other reduced-fat foods containing fat mimetics or replacers. To assess this possibility, the combined effects of γ-Glu-Val-Gly and fat replacers need to be examined, and this investigation is now underway in our laboratory.

12.7.4

Conclusions and Implications

The effects of the kokumi peptide, γ-Glu-Val-Gly, on the flavor of reduced-fat custard cream were investigated in the present study. The results obtained indicated that the addition of γ-Glu-Val-Gly significantly enhanced the intensity of the thickness of taste, and slightly increased the continuity of taste and the aftertaste. Therefore, the addition of γ-Glu-Val-Gly increased some sensations that were lacking in reduced-fat custard cream, suggesting the potential of this peptide to improve the flavor of reduced-fat custard cream.

12.8

General Discussion

In this chapter, the effects of the kokumi peptide, γ-glutamyl-valyl-glycine (γ-Glu-Val-Gly) on the sensory characteristics of foods and beverages, such as chicken consommé, hamburger steak, orange flavored drink, reduced-fat peanut butter, and reduced-fat French dressing, were evaluated using a descriptive analysis. The addition of this peptide enhanced umami, mouthfulness, and mouth coating in chicken consommé, and oiliness, umami, the thickness of taste in gravy, the middle and last meaty flavor, and pepper flavor in hamburger steak. Furthermore, the addition of this peptide significantly enhanced sweetness, the thickness of taste, and juiciness in orange flavored drink. Moreover, the addition of γ-Glu-Val-Gly significantly decreased the intensities of mouth drying in hamburger steak. The additional effect of this tripeptide on reduced-fat foods was also investigated. The addition of γ-Glu-Val-Gly significantly increased the intensities of thickness of taste,

224

M. Kuroda

aftertaste, and oiliness in reduced-fat peanut butter, and significantly increased the intensities of aftertaste in reduced-fat French dressing. The results of sensory evaluations are summarized in Table 12.24. The addition of γ-Glu-Val-Gly to savory foods, such as chicken consommé, and hamburger steak significantly increased the intensity of umami, while the addition of this peptide to a sweet beverage (orange flavored drink) significantly increased the intensity of sweetness. Interestingly, the addition of γ-Glu-Val-Gly enhanced sensory characteristics related to complexity, namely, the thickness of taste in hamburger steak, orange-flavored drink, and reduced-fat peanut butter and umami in chicken consommé. Yamaguchi and Kimizuka (1979) reported that the addition of monosodium glutamate (MSG) to beef consommé or chicken soups (chicken consommé, chicken noodle soup, and cream of chicken soup) enhanced continuity, mouthfulness, and the thickness of taste. These results suggest that MSG, an umami substance, increases the intensity of not only umami (the taste of a MSG aqueous solution), but also those of continuity, mouthfulness, and the thickness of taste, which are koku-related attributes. Yamaguchi and Kimizuka (1979) also demonstrated that the addition of sugar to caramel custard or Bavarian cream increased the intensities of continuity, mouthfulness, and the thickness of taste, suggesting that sugar (sucrose), a sweetener, increased the intensity of not only sweetness, but also those of continuity, mouthfulness, and the thickness of taste, which are koku-related attributes. Based on these findings, enhancements in sensory characteristics related to complexity and the thickness of taste in savory and sweet foods and beverages may be attributed to the enhancement in umami or sweetness. Therefore, γ-Glu-ValGly, a kokumi substance, enhanced koku-related attributes, such as mouthfulness Table 12.24 Summary of the effect of γ-Glu-Val-Gly on the sensory characters of foods and beverage

Chicken consomme

Hamburger steak

Orange flavored drink

Reduced-fat peanut butter

Reduced-fat French dressing Reduced-fat custard cream

Enhanced characteristics Umami Mouthfulness Mouth-coating Umami Oiliness Thickness of taste in gravy Meaty flavor (middle to lasting) Pepper flavor Sweetness Thickness of taste Juiciness Thickness of taste (middle to lasting) Aftertaste Oiliness Aftertaste Thickness of taste Continuity of taste

Suppressed characteristics

Mouth-drying

12

Effects of the Potent Kokumi Peptide, γ-Glutamyl-Valyl-Glycine, on. . .

225

and the thickness of taste, in savory and sweet foods because it has no taste itself at the concentrations used in the sensory evaluation. As shown in Table 12.24, the addition of γ-Glu-Val-Gly significantly increased the intensities of the mouth coating in chicken consommé, and oiliness, the definition of which is an oily mouth-coating, in hamburger steak and reduced-fat peanut butter. These results suggest the potential of γ-Glu-Val-Gly to enhance the mouth coating in liquid and solid foods as well as in savory and sweet foods. Although the viscosity of chicken consommé did not significantly change following the addition of 5 ppm of γ-Glu-Val-Gly (data not shown), an enhancement in the mouth coating was observed. Although the physical properties of hamburger steak and Brioche with and without γ-Glu-Val-Gly were not measured, they did not appear to be affected by the addition of γ-Glu-Val-Gly at the concentrations tested (20 ppm in hamburger steak and 30 ppm in Brioche). Therefore, the enhancement in the mouth coating appears to be due to a change in oral conditions; however, further studies are warranted to elucidate the mechanisms responsible for this enhancement. The addition of γ-Glu-Val-Gly significantly decreased the intensity of mouth drying in hamburger steak. Previous studies suggested that sweet and umami solutions increased the secretion of saliva more than water (Sasano et al. 2010). Materials containing umami substances and solutions have been used in the treatment of dry mouth (Sasano et al. 2010). In the present study, γ-Glu-Val-Gly enhanced umami in hamburger steak and sweetness in Brioche. Furthermore, as described in Chap. 8, γ-Glu-Val-Gly enhanced sweetness and umami when added to a sucrose solution and MSG solution, respectively. Based on previous findings, the decrease in mouth drying appears to be related to the increase in the secretion of saliva evoked by enhancements in umami and sweetness. However, further studies are required to clarify the mechanisms underlying the change in mouth drying. The studies described in this chapter demonstrate the potential of the kokumi peptide, γ-Glu-Val-Gly, to enhance the flavor of various foods and beverages, including savory and sweet foods. The potential of this peptide needs to be investigated in more detail in a study on the perceptive mechanism of kokumi substances. Acknowledgments I sincerely thank Dr. Yuzuru Eto and Mr. Naohiro Miyamura of Ajinomoto Co., Inc. for their encouragement and continued support of this work. I thank Ms. Sharon McEvoy and Dr. Dawn Chapman of the National Food Laboratory LLC for their cooperation and valuable discussions. I also thank Dr. Chinatsu Kasamatsu and Dr. Hiroya Kawasaki for their valuable suggestions on sensory evaluations. I thank Mr. Hiroaki Nagasaki, Mr. Tomohiko Yamanaka, Mr. Fusataka Kenmotsu, Mr. Takashi Miyaki, Mr. Takaho Tajima, Mr. Shuichi Jo, Mr. Keita Sasaki, and Ms. Takako Hirose of Ajinomoto Co., Inc., for their assistance. I am grateful to the panelists who participated in sensory evaluations.

References Abdel-Haleem AM, Awad RA (2015) Some quality attributes of low fat ice cream substituted with hulless barley flour and barley β-glucan. J Food Sci Technol 52:6425–6434

226

M. Kuroda

Aghdaei SSA, Aslami M, Geefan SB, Ranjbar A (2014) Application of Isfarzeh seed (Plantago ovate L.) mucilage as a fat mimetics in mayonnaise. J Food Sci Technol 51:2748–2754 Amelia I, Drake MA, Nelson B, Barbano DM (2013) A new method for the production of low-fat Cheddar cheese. J Dairy Sci 96:4870–4884 Arocas A, Sanz T, Varela P, Fiszman SM (2010) Sensory properties determined by starch type in white sauces: effects of freeze/thaw and hydrocolloid addition. J Food Sci 75:S132–S140 Azari-Anpar M, Khomeiri M, Ghafouri-Oskuei H, Aghajani N (2017) Response surface optimization of low-fat ice cream production by using resistant starch and maltodextrin as a fat replacing agent. J Food Sci Technol 54:1175–1183 Bernardino-Nicanor A, Hinojosa-Hernandez EN, Juarez-Goiz JMS, Montanez-Soto JL, RamirezOrtiz ME, Gonzalez-Cruz L (2015) Quality of Opuntia robusta and its use in development of mayonnaise like product. J Food Sci Technol 52:343–350 Dawid C, Hofmann T (2012) Identification of sensory-active phytochemicals in asparagus (Asparagus officinalis L.). J Agric Food Chem 60:11877–11888 de Souza Fernandes D, Leonel M, Del Bem MS, Mischan MM, Garcia ÉL, Dos Santos TPR (2017) Cassava derivatives in ice cream formulations: effects on physicochemical, physical and sensory properties. J Food Sci Technol 54:1357–1367 Dunkel A, Koster J, Hofmann T (2007) Molecular and sensory characterization of γ-glutamyl peptides as key contributors to the kokumi taste of edible beans (Phaseolus vulgaris L.). J Agric Food Chem 55:6712–6719 Fernandes SS, Mellado MMS (2018) Development of mayonnaise with substitution of oil or egg yolk by the addition of chia (Salvia Hispanica L.) mucilage. J Food Sci 83:74–83 Flett KL, Duizer LM, Goff D (2010) Perceived creaminess and viscosity of aggregated particles of casein micelles and κ-carrageenan. J Food Sci 75:S255–S261 Furukawa H (1977) The sensory test and the selection of panellists. In Proceedings of the 7th symposium sensory inspection, p 111 Grodzka K, Maciejec A, Krygier K (2005) Attempt to apply microcrystalline cellulose as a fat replacer in low fat mayonnaise emulsions. Zywnosc 12:52–61 Guo Y, Zhang X, Hao W, Xie Y, Chen L, Li Z, Zhu B, Feng X (2018) Nano-bacterial cellulose/soy protein isolate complex gel as fat substitutes in ice cream model. Carbohydr Polym 198:620– 630 Liou BK, Grun IU (2007) Effect of fat level on the perception of five flavor chemicals in ice cream with or without fat mimetics by using a descriptive test. J Food Sci 72:S595–S604 Liu H, Xu XM, Guo SD (2007) Rheological, texture and sensory properties of low-fat mayonnaise with different fat mimetics. LWT Food Sci Technol 40:946–954 McClements DJ, Demetriades K (1998) An integrated approach to the development of reduced-fat food emulsions. Crt Rev Food Sci Nutr 38:511–536 Mellies MJ, Vitale C, Jandacek RJ, Lamkin GE, Glueck CJ (1985) The substitution of sucrose polyester for dietary fat in obese, hypercholesterolemic outpatients. Am J Clin Nutr 41:1–12 Miyaki T, Kawasaki H, Kuroda M, Miyamura N, Kouda T (2015) Effect of a kokumi peptide, γ-glutamyl-valyl-glycine, on the sensory characteristics of chicken consommé. Flavour 4:17 Miyamura N, Jo S, Kuroda M, Kouda T (2015) Flavour improvement of reduced-fat peanut butter by addition of a kokumi peptide, γ-glutamyl-valyl-glycine. Flavour 4:16 Ohsu T, Amino Y, Nagasaki H, Yamanaka T, Takeshita S, Hatanaka T, Maruyama Y, Miyamura N, Eto Y (2010) Involvement of the calcium-sensing receptor in human taste perception. J Biol Chem 285:1016–1022 Patel AS, Jana AH, Aparnathi KD, Pinto SV (2010) Evaluating sago as a functional ingredient in dietetic mango ice cream. J Food Sci Technol 47:582–585 Pimentel TC, Cruz AG, Prudencio SH (2013) Influence of long-chain inulin and Lactobacillus paracasei subspecies paracasei on the sensory profile and acceptance of a traditional yogurt. J Dairy Sci 96:6233–6624 Roman L, Martinez MM, Gomez M (2015) Assessing of the potential of extruded flour paste as fat replacer in O/W emulsion: a rheological and microstructural study. Food Res Int 74:72–79

12

Effects of the Potent Kokumi Peptide, γ-Glutamyl-Valyl-Glycine, on. . .

227

Sasano T, Satoh-Kuriwada S, Shoji N, Sekine-Hayakawa Y, Kawai M, Uneyama H (2010) Application of umami taste stimulation to remedy hypogeusia based on reflex salivation. Biol Pharm Bull 33:1791–1795 Sasano T, Satoh-Kuriwada S, Shoji N, Iikubo M, Kawai M, Uneyama H, Sakamoto M (2014) Important role of umami taste sensitivity in oral and overall health. Curr Pharm Des 20:2750– 2754 Scharbert S, Holzmann N, Hofmann T (2004) Identification of the astringent taste compounds in black tea infusions by combining instrumental analysis and human bioresponse. J Agric and Food Chem 52:3498–3508 Schwarz B, Hofmann T (2009) Identification of novel orosensory active molecules in cured vanilla beans (Vanilla planifolia). J Agric and Food Chem 57:3729–3737 Sharma M, Singh AK, Yadav DN (2017) Rheological properties of reduced fat ice cream mix containing octenyl succinylated pearl millet starch. J Food Sci Technol 54:1638–1645 Shibata M, Hirotsuka M, Mizutani Y, Takahashi H, Kawada T, Matsumiya K, Hayashi Y, Matsumura Y (2017) Isolation and characterization of key contributors to the “kokumi” taste in soybean seeds. Biosci Biotech Biochem 81:2168–2177 Su H-P, Lien C-P, Lee T-A, Ho J-H (2010) Development of low-fat mayonnaise containing polysaccharide gums as functional ingredients. J Sci Food Agric 90:806–812 Sun C, Liu R, Liang B, Wu T, Sui W, Zhang M (2018) Microparticulated whey protein—pectin complex: a texture-controllable gel for low-fat mayonnaise. Food Res Int 108:151–160 Toelstede S, Dunkel A, Hofmann T (2009) Series of kokumi peptides impart the long-lasting mouthfulness of matured Gouds cheese. J Agric Food Chem 57:1440–1448 Tomaschunas M, Zorb R, Fischer J, Kohn E, Hinrichs J, Busch-Stockfisch M (2013) Changes in sensory properties and consumer acceptance of reduced fat pork Lyon-style and liver sausages containing inulin and citrus fiber as fat replacers. Meat Sci 95:629–640 Ueda Y, Sakaguchi M, Hirayama K, Miyajima R, Kimizuka A (1990) Characteristic flavor constituents in water extract of garlic. Agric Biol Chem 54:163–169 Ueda Y, Yonemitsu M, Tsubuku T, Sakaguchi M, Miyajima R (1997) Flavor characteristics of glutathione in raw and cooked foodstuffs. Biosci Biotech Biochem 61:1977–1980 Wedninin K, Aaby K, Edris A, Ellekjaer MR, Albin R, Bergensahl B, Johansson L, Willers EP, Solheim R (1997) Low-fat mayonnaise: influences of fat content, aroma compounds and thickeners. Food Hydrocol 11:87089 Yamaguchi S, Kimizuka A (1979) Psychometric studies on the taste of monosodium glutamate. In: Filer LJ Jr, Garattini S, Kare MR, Reynolds WA, Wurtman RJ (eds) Glutamic acid: advances in biochemistry and physiology, vol 54. Raven Press, New York, pp 35–54 Yildrim M, Sumnu G, Sahin S (2016) Rheology, particle-size distribution, and stability of low-fat mayonnaise produced via double emulsions. Food Sci Biotechnol 25:1613–1618 Zhang H, Chen J, Li J, Wei C, Ye X, Shi J, Chen S (2018) Pectin from citrus canning wastewater as potential fat replacer in ice cream. Molecules 23(4):E925. https://doi.org/10.3390/ molecules23040925

Chapter 13

Perceptual and Nutritional Impact of Kokumi Compounds Ciarán Forde and Markus Stieger

Abstract The chapter first reviews the impact of kokumi compounds on sensory perception, then highlights opportunities to use koku sensations to support product reformulation and to impact energy perception and finally provides an outlook on research gaps and future directions. Many desirable sensory characteristics are enhanced by the addition of kokumi compounds to foods which can have a positive effect on consumer acceptance and product liking. Kokumi compounds impact not only the flavor of foods but also mouthfeel properties that are often associated with the presence of fat and calories. Adding kokumi compounds to reduced calorie soups and broths can enhance the perceived sensory intensity for sensations linked to calorie perception and enhance desirable sensory qualities linked to hedonic appeal. This raises the possibility that enhancement of sensory intensity with kokumi compounds could be applied to support reformulation efforts such as reduced fat or reduced calorie products by impacting how consumers perceive the energy content through sensory properties. Currently findings are limited to measuring expectations of fullness, and would need to be further confirmed through controlled feeding trials to quantity the impact of koku enhancement on energy intake and postmeal satiety to assess whether this could be a viable strategy to support long-term reductions in energy intake.

13.1

Introduction

The taste of food cannot be described by the five basic tastes alone, as there is often an additional complexity that goes beyond simple combinations of sweet, sour, salty, bitter, and umami. For example, mature Cheddar cheese differs from younger Cheddar cheese in primary tastes, but also has distinctive differences in sensory characteristic such as flavor continuity, complexity, and mouthfulness (Drake et al. C. Forde (✉) · M. Stieger Sensory Science and Eating Behaviour, Division of Human Nutrition and Health, Wageningen University, Wageningen, The Netherlands e-mail: [email protected]; [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2024 M. Kuroda (ed.), Kokumi Substance as an Enhancer of Koku, https://doi.org/10.1007/978-981-99-8303-2_13

229

230

C. Forde and M. Stieger

2005; Muir et al. 1997; Rehman et al. 2000; Urbach 1993). Many foods that have been aged, fermented, or cooked at low temperatures for long time have distinctive desirable perceptual properties linked to their flavor and mouthfeel complexity. Although widely recognized in Western cultures, these sensations are poorly defined or described, yet they have been recognized as “full taste” in Japan for many years. The first identification of compounds to produce the koku sensation was described in 1990 by Ueda et al. (1990) who studied the flavoring effects of a water extract of garlic that increased continuity, mouthfulness, and thickness when it was added to umami solutions [monosodium glutamate (MSG), inosine-5′-monophosphate (IMP)] and broths (Chinese style broth and curry flavored broth). The key compounds responsible for this effect were identified as sulfur-containing compounds such as S-allyl-cysteine sulfoxide (alliin) and glutathione (GSH) (Ueda et al. 1990). These compounds distinctively impacted not only flavor complexity and duration, but also contributed to mouthfeel properties such as in-mouth thickness and mouthfulness (Ueda et al. 1990). The compounds responsible for the koku sensation (termed kokumi compounds) are often produced through heating, fermentation, and aging of a product, which leads to the formation of tri-peptides that alone have low taste activity, but when combined with other tastant compounds can produce an intense flavor response. Kokumi compounds have been isolated and characterized in fermented foods such as kimchi, beer, and fish sauce; aged foods such as aged cheese, salami, wine, and dried scallops; and in slow-cooked foods such as consommés and meat broths. Differences in flavor perception and intensity in these products can be obtained by varying the treatment conditions (e.g., time, heat, fermentation) which can modify the concentration of the formed kokumi compounds (Nishimura 2019). For example, a simple chicken stock can be prepared by boiling chicken meat and bones with various vegetables (e.g., onion, carrots, leeks, celery). Variations in the cooking duration of the chicken soup result in the formation of kokumi compounds at different concentrations since the Maillard reaction between free amino acids and sugars is influenced by cooking duration. To date more than 50 chemical compounds have been identified from a range of different sources (natural and synthetic) that differ in chemical composition and potencies that cause koku sensations. Unlike the basic tastes, there has been confusion on the description and definition of the koku sensation. This has led to a lack of clarity in its perceptual description, since kokumi compounds do not elicit a distinct taste quality of their own, but rather enhance the complexity, continuity, and mouthfulness of other taste sensations. Though not defined by the same criteria as other taste primaries, perception of kokumi compounds is mediated by an independent taste receptor system and is perceived via the calcium-sensing receptor (CaSR) (Briand and Salles 2016; Maruyama and Kuroda 2019). Some of the most potent kokumi compounds are tri-peptides, of which γ-glutamyl-valyl-glycine (γ-Glu-Val-Gly) is one of the most potent compounds. As with rare sugars and low-calorie sweeteners, kokumi compounds can enhance the sensory complexity and intensity of a mixture without adding significant amounts of calories. As such, kokumi compounds could be used to enhance the body and mouthfeel properties often associated with the presence of

13

Perceptual and Nutritional Impact of Kokumi Compounds

231

fat and energy, and potentially be applied to support reductions in energy density or fat content in foods. Beyond what is sensed in the oral cavity, taste stimuli are likely to play an important role in moderating metabolic kinetics and gastric emptying throughout the alimentary canal. Kokumi tri-peptides potentially play a role in signalling the presence of specific nutrients on the gut-brain axis. The current chapter first reviews the impact of kokumi compounds on sensory perception, then highlights opportunities to use koku sensations to support product reformulation and to impact energy perception and finally provides an outlook on research gaps and future directions.

13.2

Impact of Kokumi Compounds on Sensory Perception

The term kokumi originates from the Japanese words “koku” meaning full and “mi” meaning taste, and is often used interchangeably in conversational Japanese to describe a range of foods as “palatable” (Nishimura 2019). The direct translation of kokumi remains elusive for many English speakers as there is not an equivalent single concept or lexicon for its usage in English. The koku sensation implies a tactile and somatosensory component in addition to the enhancement of taste intensity perception. The original koku sensation was first defined as a rich, mouthful, thick, and delicious sensation (Ueda et al. 1990). Kokumi compounds tend to act as enhancers of other taste qualities such as sweet, salty, and umami, rather than eliciting any specific taste of their own (Briand and Salles 2016; Maruyama and Kuroda 2019; Zhao et al. 2016; Kuroda and Miyamura 2015; Maruyama et al. 2012). Kokumi compounds have diverse sources, chemical structures, and potencies. A quantitative comparison between kokumi compounds showed marked differences in their koku enhancement potential (Ohsu et al. 2010). Currently, the most potent kokumi peptide identified to date is γ-Glu-Val-Gly. The point of subjective intensity equivalence in a salty-umami solution was estimated to be 12.8 times stronger for γ-Glu-Val-Gly compared with Glutathione (GSH) (i.e., 0.01% γ-Glu-Val-Gly solution had an average perceived koku intensity equivalent to a 0.128% GSH solution) (Ohsu et al. 2010). Compounds that elicit a koku sensation have only a slight flavor of their own in water, yet they have been shown to markedly increase perceived thickness, continuity, and mouthfulness when added to an umami solution or other foods (Ueda et al. 1997, 1994). The umami compound MSG can give koku in that it contributes to mouthfeel, intensity, and continuity, yet kokumi compounds cannot provide the same savory taste as umami in isolation (see review on umami by Yamaguchi and Ninomiya (1998)). Unlike umami, it has been demonstrated that kokumi compounds do not have a distinct taste of their own (Ueda et al. 1990, 1994, 1997; Kuroda and Miyamura 2015; Kuroda et al. 2012) although many kokumi compounds have subtle taste and odor qualities (Kurobayashi et al. 2019). Unlike umami, kokumi compounds arise from a wide range of different chemical compounds and sources, whereas umami is primarily associated with salts of glutamic

232

C. Forde and M. Stieger

acid [i.e., monosodium glutamate (MSG), disodium 5′-guanylate (GMP), inosine5′-monophosphate (IMP), etc.] (Toelstede et al. 2009; Amino et al. 2016). A kokumi compound is often produced from pre-cursors through heating, fermenting, or aging a product. For example, hydrolyzed soy protein is used to produce “Maillard peptides” by heating (Ogasawara et al. 2006). These “Maillard peptides” have been shown to increase overall taste intensity and give longer taste continuity and mouthfulness in a chicken consommé broth and salty-umami solution (sodium chloride and MSG) in comparison to control samples (Xu et al. 2018). Similarly, a study on taste-active peptides in bovine bone marrow extract found that kokumi compounds enhanced the taste activity of these peptides after Maillard reaction (Xu et al. 2018). Interestingly, 8 out of the 12 produced peptides have been shown to enhance koku sensations when added to beef broths (Xu et al. 2018). Fermentation and aging of certain peptides also influences the expression of kokumi compounds and the koku sensation (Toelstede et al. 2009). A comparative quantitative analysis between a Gouda cheese ripened for 4 weeks versus a Gouda cheese matured for 44 weeks revealed significant differences in perceived mouthfulness, taste intensity, and continuity (Toelstede et al. 2009). The enhancement of koku sensation of the matured cheese was linked to increased concentrations of key kokumi compounds comprising γ-L-glutamyl dipeptides (Toelstede et al. 2009). Similar enhanced koku sensations have been observed in dried herring that underwent a longer drying time which increased concentrations of creatinine compounds (Azad Shah et al. 2013). In fermented sourdough breads, similar effects and compounds have been reported (Tang et al. 2017). The concentration of kokumi compounds can also differ by cultivar type or by food processing condition. Raw onions cultivated from different regions in Japan contained different amounts of kokumi peptides (cycloalliin ranged from 5.6 mg/100 g of onion bulb in Nagano onions to 11.4 mg/100 g in Kagawa onions) (Ueda et al. 1994). When added into umami solutions, these kokumi peptides extracted from different types of onions (raw and cooked) gave different characteristics of koku mouthfeel and flavor, but the panellists were not able to recognize sweet taste qualities in the solutions (Ueda et al. 1994). These findings suggest that the koku sensation was independent from the sweetness of cooked onions (Ueda et al. 1994). For scallops, the concentration of the kokumi tripeptide γ-Glu-Val-Gly depended on the scallop processing conditions (Kuroda et al. 2012). Raw scallops contained 0.08 μg/g γ-Glu-Val-Gly, whereas dried scallops contained 0.64 μg/g and scallop extracts 0.77 μg/g γ-Glu-Val-Gly (Kuroda et al. 2012).

13.2.1

Kokumi Perception and Hedonic Responses

In order for koku to be classified as a basic taste quality, there are a number of specific criteria that must be fulfilled before the sensation can be categorized as a new “taste” sensation (Keast and Costanzo 2015; Mattes 2011; Hartley et al. 2019; McBurney and Gent 1979; Kurihara 2015). Although the proposed criteria vary

13

Perceptual and Nutritional Impact of Kokumi Compounds

233

considerably, perceptual salience and uniqueness of the taste quality and consensus on its description are necessary for a novel sensation to be categorized as a basic taste (Keast and Costanzo 2015; Mattes 2011; Hartley et al. 2019; McBurney and Gent 1979; Kurihara 2015). The relationship between individual measures of taste function (i.e., taste thresholds and suprathreshold intensity perception) from tasting a range of dissolved chemical stimuli can be determined with precision [e.g., detection threshold, recognition threshold, and suprathreshold intensity perception between the five basic taste qualities (Webb et al. 2015)]. This has led to proposed novel “tastes” for compounds linked to the perception of fatty acids (Running et al. 2015), carbohydrate (Lim and Pullicin 2019), metallic (Lawless et al. 2004), and more recently, kokumi (Rhyu et al. 2020; Feng et al. 2016). The uniqueness of kokumi compounds is that some compounds such as γ-Glu-Val-Gly are consistently undetectable in low concentrations when diluted in water (Kuroda and Miyamura 2015; Ohsu et al. 2010; Kuroda and Harada 2004) whereas some kokumi compounds (i.e., GSH, γ-Glutamyl Peptides such as γ-Glu-Val, γ-Glu-Leu and β-Alanyl Dipeptides) are detectable in comparison to water control solutions (Ueda et al. 1997) due to a slight sour taste (Lee et al. 2010; Dunkel and Hofmann 2009), bitter taste (Shibata et al. 2018a, b), and/or astringency (Shibata et al. 2018a; Dunkel et al. 2007; Yang et al. 2017). These compounds are detectable as astringent in aqueous solutions (Lee et al. 2010). Similarly, kokumi compounds such as creatine, creatinine, and yeast extract compounds are barely detectable in terms of oral intensity perception when diluted in water (Shah et al. 2010; Liu et al. 2015). By contrast kokumi peptides from beef bone marrow are perceivable due to their weak sourness intensity (Xu et al. 2018). However, adding kokumi compounds (i.e., alliin, xycloalliin, MeCSO, GAC, GACSO) into an umami mixture of MSG-IMP or umami-salty (MSG-IMP-NaCl) or sweet-salty-umami (sucrose-NaCl-MSG) mixtures significantly reduced the detection (Ueda et al. 1997) and recognition thresholds (Dunkel et al. 2007) and significantly enhanced the rated koku intensity (Ueda et al. 1990, 1994, 1997; Kuroda and Miyamura 2015; Ohsu et al. 2010; Amino et al. 2016) in comparison to control-base solutions. These threshold concentrations and perceived koku sensations were similar when kokumi peptides were added to a model soup-base chicken broth (Kuroda and Miyamura 2015; Ohsu et al. 2010; Dunkel et al. 2007; Yang et al. 2017), Chinese and curry flavored soups (Ueda et al. 1990), beef broth (Ueda et al. 1997; Xu et al. 2018), Japanese fish-based noodle soups (Azad Shah et al. 2013), reduced-fat cream (Kuroda and Miyamura 2015), and commercial soy sauce (Yang et al. 2017), where their addition was characterized by reduced detection and recognition thresholds and enhanced rated koku intensity, but not basic taste intensity. Descriptive sensory profiling of chicken consommé containing γ-Glu-Val-Gly showed that umami taste, mouthfulness, and mouth-coating sensations were more intensive than in control consommé (Miyaki et al. 2015) implying that the kokumi peptide γ-Glu-Val-Gly could be used to enhance and improve the flavor of chicken consommé. These sensory qualities have been associated with the perception of higher expected fullness and a higher caloric content in a food (see Sect. 13.3).

234

C. Forde and M. Stieger

Similarly, a distinctive feature of the koku sensation is the temporality imparted by the addition of kokumi compounds as revealed through Time-Intensity sensory profiling (Ogasawara et al. 2006). This has been characterized by an increase in the continuity of savory sensations, longer and more intense flavors, and greater flavor after-tastes. For example, Maillard reaction peptides obtained by enzymatic hydrolysis of soybean protein followed by fractionation have been shown to enhance the flavor and umami sensation and increased the continuity and mouthfulness in umami solutions and consommé soups (Feng et al. 2016; Liu et al. 2016). Similarly, the addition of a mixture of creatine and creatinine to Japanese noodle soups resulted in enhancement of perceived thickness, mouthfulness, and continuity (Azad Shah et al. 2013). Many desirable sensory characteristics are enhanced by the addition of kokumi compounds to foods which can have a positive effect on consumer acceptance and product liking. The sensory characteristics and consumer acceptability of beef soup with added glutathione Maillard reaction products were compared to soups with added glutathione and MSG (Hong et al. 2010). Glutathione Maillard reaction products and glutathione enhanced beef flavor of soups compared to MSG. Glutathione Maillard reaction products increased other flavors such as green onion, garlic, and boiled egg white flavor. Beef soups containing MSG were preferred and soups with reduced Glutathione Maillard reaction products were the least preferred because of their pronounced metallic and astringent notes. While glutathione Maillard reaction products enhanced both desired and undesired flavor notes equally, additional studies sought to optimize Maillard reaction conditions to generate Glutathione Maillard reaction products without undesirable flavors. In a beef broth (Hong et al. 2010, 2012), Glutathione-xylose Maillard reaction products enhanced the perception of beef flavor intensity. Again, these compounds also enhanced other undesirable flavor notes such as sulfur and chestnut flavors, suggesting a general flavor enhancement by glutathione-xylose Maillard reaction products. The combination of glutathione-xylose Maillard reaction products and MSG displayed a synergistic effect on flavor enhancement of beef stocks indicating that MSG is needed to produce the specific desirable koku enhancement. Salty, umami, and sweet tastes impacted liking of the beef broths the strongest.

13.3

Opportunities for Product Reformulation and Energy Content Reduction Using Koku Sensations

There has been widespread research interest on the impact of savory taste intensity on food intake, satiety, and calorie perception. Previous research has shown that savory taste enhancement using MSG can increase liking and enhance post-meal satiety for vegetable soups in overweight and obese adults (Carter et al. 2011; Masic and Yeomans 2014; Miyaki et al. 2016). Similarly, the addition of MSG to vegetable soups has been shown to decrease subsequent energy intake among a cohort of

13

Perceptual and Nutritional Impact of Kokumi Compounds

235

women with overweight and obesity (Miyaki et al. 2016) and MSG has been shown to increase satiety in adults, although the results have been inconsistent across studies. Addition of MSG/IMP to low-energy soup preloads increased appetite during ingestion and enhanced post-ingestive satiety (Masic and Yeomans 2014). Supplementation of a chicken broth with MSG reduced hunger and desire to snack, but did not affect later energy intake (Carter et al. 2011). Taken together these studies suggest a complex interaction between savory taste intensity and quality, and consumers perception of satiety and later snack intake, with equivocal findings that savory intensity can promote and reduce calorie consumption. A meta-analysis of seven cross-sectional studies demonstrated that regular consumption of savory soups and broths was inversely associated with risk of obesity, though it remains unclear which mechanism is driving these observational effects (Kuroda and Ninomiya 2020). Kokumi compounds impact not only the flavor of foods but also mouthfeel properties that are often associated with the presence of fat and calories. For example, umami taste, mouthfulness, and mouth-coating sensations were found to be more intense in a chicken consommé containing γ-Glu-Val-Gly kokumi peptides compared to a control consommé without kokumi tri-peptides (Miyaki et al. 2015). These sensory qualities are often associated with the presence of fat, higher calories levels, and greater expected fullness. Hence, when these sensations are imparted using the low calorie but flavor intense kokumi tri-peptides, it creates a potential opportunity to either enhance the expected fullness of a low calorie food, or conceal a reduction in calories to mimic the sensory properties of a full-caloric version of a food, without a loss of hedonic appeal. Adding kokumi compounds to reduced calorie soups and broths can enhance the perceived sensory intensity for sensations linked to calorie perception and enhance desirable sensory qualities linked to hedonic appeal. This raises the possibility that enhancement of sensory intensity with kokumi compounds could be applied to support reformulation efforts such as reduced fat or reduced calorie products by impacting how consumers perceive their energy content through their sensory properties. The sensory appeal of a food may be compromised during calorie reformulation as reductions of fat or carbohydrate content may result in a loss of mouthfeel, flavor quality, intensity, and duration. Consumers often report that lower calorie or lower fat versions of foods lack sensory intensity, have thin and unappealing mouthfeel properties, and deliver an insipid flavor profile, when compared to their full calorie equivalent products. Preliminary evidence suggests that koku enhancement may offer an effective strategy to support fat reduction and sustained appeal of reduced energy foods. The addition of the kokumi tri-peptide γ-Glu-Val-Gly enhanced the intensities of thick flavor, aftertaste, and oiliness in low fat peanut butter, thereby increasing sensations that were often lacking in the reduced-fat version of peanut butter (Miyamura et al. 2015). In addition to reduced fat peanut butter, koku enhancement using γ-Glu-Val-Gly was also found to enhance the perceived intensity, continuity, mouthfulness, and thick flavor for a series of reduced-fat custard creams (Kuroda and Miyamura 2015). In both cases, the koku enhancement elicited

236

C. Forde and M. Stieger

a greater sensory intensity and duration that was linked to increased hedonic appeal for the reduced calorie versions of the test products. These preliminary findings suggest that koku enhancement could play a role in food reformulations to enhance the sensory perception and hedonic appeal of reduced energy foods. Koku enhancement may also be expected to influence consumer perception of the expected calorie density and satiating properties of foods that vary in this savory/koku dimension. In a study to test this proof of principal, researchers developed a series of beef broths that varied in their savory and koku intensity to test the impact this had on perceived calories, expected fullness, and prospective portion selection (Tang et al. 2020). The energy content of these broths was equivalent, yet they differed considerably in their sensory intensity and koku sensations of mouthfulness, continuity, and savory intensity. Importantly, consumers perceptions of the calorie content and expected fullness differed across the savory- and kokumi-enhanced broths (Fig. 13.1), with the broths that were higher in beef flavor, savoriness, body thickness, mouth-coating, and flavor aftertaste were positively associated with higher calorie ratings and greater expected fullness (Fig. 13.2) (Tang et al. 2020). Conversely, a higher sourness intensity was negatively associated with calorie and fullness expectations (Fig. 13.2). These findings suggest that savory and koku enhancement may not only promote higher sensory intensity and hedonic appeal, but may also enhance the expected calories and satiating properties of lower-calorie foods by playing on our learned associations between these cues and fullness (Tang et al. 2020). Promoting the intensity and duration of sensory cues typically associated with beliefs of greater satiety could potentially be used to support the development of products that maintain sensory appeal and eating enjoyment, while supporting greater fullness per calorie consumed. Currently findings are limited to measuring expectations of fullness, and would need to be further confirmed through controlled feeding trials to quantity the impact of koku enhancement on energy intake and post-meal satiety to assess whether this could be a viable strategy to support long-term reductions in energy intake.

13.4

Outlook: Research Gaps and Future Directions for Koku and Health

Research to date has demonstrated the potential application of savory and koku enhancement to support sensory and hedonic enhancement of low-calorie or reduced fat foods, and in promoting expectations of calories and fullness through the enrichment of these sensory cues. There may be opportunities to extend this application further in the future, with the emergence of new categories of products that aim to mimic the savory properties of meats. Plant-based meat alternatives play an important role in supporting consumers to make the transition to new protein sources and reduce our reliance on animal production by providing meat enthusiasts an

13

Perceptual and Nutritional Impact of Kokumi Compounds

237

Fig. 13.1 Mean (± SEM) Calorie Rating (a) and Expected Fullness (b) of broth, and Portion Selection of chicken rice (c) and noodles soup (d) to accompany broth, across eight savory enhanced broths in increasing calorie rank. Different letters indicate a significant difference at p < 0.05, using Bonferroni corrected comparisons to compare broth samples, where “a” always represents the smallest value. Sample abbreviations are G50Y; Glutathione +50% yeast extracts, GSH; reduced Glutathione, K; Kokumax-100-yeast extract, NaCL; Sodium Chloride, MI; Monosodium glutamate (MSG) + inosine mono-phosphate (IMP), YMI, Yeast extract with 50% reduced glutathione, + Mono-sodium glutamate + inosine mono-phosphate, GMI; reduced Glutathione, MSG, IMP and KMI; Kokumax-100 Yeast Extract + MSG, IMP. Figure was adapted from (Tang et al. 2020) (https://doi.org/10.1016/j.foodqual.2020.103897) and used with permission

opportunity to consume plant-based meat alternatives (PBMAs) that mimic the texture and flavor profile of animal meat. Over the past decade, there has been a proliferation of these PMBA products, with rising demand for better quality products of plant origin to mimic the sensory properties of meat. Several significant hurdles remain in capturing consumer appeal for these PMBA products as many current meat replacers either contain excessive amounts of salt or have flavor profiles that fail to closely mimic the quality of meat flavor (Tso et al. 2020; Tso and Forde 2021). An interesting, and as yet unexplored opportunity, that might tackle both issues could be the use of kokumi compounds to enhance savory intensity and mimic the

238

C. Forde and M. Stieger

Fig. 13.2 Partial least squares regression (PLSR) of the descriptive sensory profiling data of eight broth samples on (a) Calorie Rating, (b) Expected Fullness and (c) Portion Selection of Noodles Soup to accompany broth, across the eight broths. *** indicates a significance of p < 0.001, ** indicates p < 0.01, * indicates p < 0.05 and ^ refers to reaching significance with p < 0.10. Figure was adapted from (Tang et al. 2020) (https://doi.org/10.1016/j.foodqual.2020.103897) and used with permission

flavor release profile of animal meat products. Kokumi compounds have been reported to enhance both saltiness and umami intensity (see Sect. 13.2), two important sensations contributing to meat flavor, and can mimic the experience of fat during consumption by increasing the mouthfulness, mouth coating, and continuity.

13

Perceptual and Nutritional Impact of Kokumi Compounds

239

Future studies are needed to explore the potential application of koku enhancement to further boost the perceptual properties and sensory appeal of plant-based meat alternatives, without the need for excessive salt addition. A majority of kokumi compounds identified to date are tri-peptides, especially γ-glutamyl and leucyl peptides (see Sect. 13.2). These compounds are typically found in fermented foods, yeast extracts, and protein hydrolysates, and have kokumi threshold concentrations (modulating threshold) in the order of 0.2–2.0 mM. Recently, a different class of thiamine-derived compounds having a kokumiimparting ability were identified (Brehm et al. 2019, 2020). These thiamine-derived compounds were identified in commercial process flavors but also in several (roasted) meats. Several of these thiamine-derived compounds have unprecedentedly low kokumi threshold concentrations (0.035–0.08 mM) and are potentially highly potent savory and koku enhancers. Their natural occurrence in roasted meat and low threshold concentrations make these compounds interesting targets for applications in meat alternatives. Furthermore, thiamine is cheap and readily available in food-grade form, facilitating easy and cost-effective synthesis of thiaminederived kokumi compounds. Future studies are now needed to explore the formation pathways of these thiamine-derived kokumi compounds to maximize yield by optimizing the reaction conditions, and simultaneously explore their sensory properties and potential application as flavor enhancers in plant-based meat alternatives. Kokumi compounds impact not only the flavor of foods but also mouthfeel properties which are often associated with the presence of fat and calories. The role of koku enhancement in food reformulations to support energy or fat reduction remains a poorly understood area, despite the clear potential for these compounds to support sustained hedonic appeal of lower energy or fat versions of certain foods. Most studies to date have focused on koku enhancement only in liquid food systems, often showing koku enhancement in low viscosity savory broths (Li et al. 2022). Rheological and tribological properties of liquid foods are known to impact mouthfeel sensations such as mouthfulness, thickness, creaminess, smoothness, and lingering of oral coatings, and these sensory properties are often associated with both fat and energy content. Our understanding of the interplay between rheological and tribological properties of liquid foods and the impact of koku enhancement is limited and it is unclear how successfully these koku enhanced calorie perceptions remain in semi-solid and solid foods. A food’s texture properties may interact synergistically or antagonistically with koku enhancement and further research is needed to explore the opportunity to apply kokumi compounds to support fat and calorie reduction in solid and semi-solid food systems. Future studies are now needed to determine systematically the effect of food texture properties on koku sensation using a range of kokumi compounds, and demonstrate the efficacy of this approach to support calorie reduction in semi-solid and solid foods. Finally, there could be potential to reduce some of the calories in a food and compensate for any associated drop in hedonic appeal through the use of kokumi compounds which enhance desirable sensory qualities (see Sect. 13.3). Savory and kokumi enhancers could be used to increase satiating properties by drawing on learned associations between these sensory cues and appetite and fullness.

240

C. Forde and M. Stieger

Promoting the intensity and duration of sensory cues typically associated with beliefs of greater satiety could potentially be used to support the development of products that maintain their sensory appeal and eating enjoyment, while supporting greater fullness per calorie consumed. Studies to date have only focused on consumers expectations and perceptions of calorie content, and measured the expected rather than the experienced fullness of these foods. Future studies should now extend these findings to acute human feeding trials and long-term clinical trials to assess the potential impact of koku enhancement in supporting long-term reductions in caloric intake without a loss of satiety and sustained sensory appeal. Preliminary findings to date suggest a variety of potential perceptual and health benefits for koku enhancement that now require further testing to confirm these applications to support food reformulation, and the protein transition to reduce the current global reliance on products of animal origin.

References Amino Y, Nakazawa M, Kaneko M, Miyaki T, Miyamura N, Maruyama Y et al (2016) Structure– CaSR–activity relation of kokumi γ-glutamyl peptides. Chem Pharm Bull 64(8):1181–1189 Azad Shah AKM, Ogasawara M, Kurihara H, Takahashi K (2013) Effect of drying on creatine/ creatinine ratios and subsequent taste of herring (Clupea pallasii) fillet. Food Sci Technol Res 19(4):691–696 Brehm L, Frank O, Jünger M, Wimmer M, Ranner J, Hofmann T (2019) Novel taste-enhancing 4-Amino-2-methyl-5-heteroalkypyrimidines formed from thiamine by Maillard-type reactions. J Agric Food Chem 67(50):13986–13997 Brehm L, Frank O, Ranner J, Hofmann T (2020) Quantitative determination of thiamine-derived taste enhancers in aqueous model systems, natural deep eutectic solvents, and thermally processed foods. J Agric Food Chem 68(22):6181–6189 Briand L, Salles C (2016) Taste perception and integration. Flavor:101–119 Carter BE, Monsivais P, Perrigue MM, Drewnowski A (2011) Supplementing chicken broth with monosodium glutamate reduces hunger and desire to snack but does not affect energy intake in women. Br J Nutr 106(9):1441–1448 Drake M, Yates M, Gerard P, Delahunty C, Sheehan E, Turnbull R et al (2005) Comparison of differences between lexicons for descriptive analysis of Cheddar cheese flavour in Ireland, New Zealand, and The United States of America. Int Dairy J 15(5):473–483 Dunkel A, Hofmann T (2009) Sensory-directed identification of β-alanyl dipeptides as contributors to the thick-sour and white-meaty orosensation induced by chicken broth. J Agric Food Chem 57(21):9867–9877 Dunkel A, Köster J, Hofmann T (2007) Molecular and sensory characterization of γ-glutamyl peptides as key contributors to the kokumi taste of edible beans (Phaseolus vulgaris L.). J Agric Food Chem 55(16):6712–6719 Feng T, Zhang Z, Zhuang H, Zhou J, Xu Z (2016) Effect of peptides on new taste sensation: Kokumi-review. Mini-Rev Org Chem 13(4):255–261 Hartley IE, Liem DG, Keast R (2019) Umami as an ‘alimentary’ taste. A new perspective on taste classification. Nutrients 11(1):182 Hong JH, Jung DW, Kim YS, Lee SM, Kim KO (2010) Impacts of glutathione Maillard reaction products on sensory characteristics and consumer acceptability of beef soup. J Food Sci 75(8): S427–S434

13

Perceptual and Nutritional Impact of Kokumi Compounds

241

Hong JH, Kwon KY, Kim KO (2012) Sensory characteristics and consumer acceptability of beef stock containing the glutathione-xylose Maillard reaction product and/or monosodium glutamate. J Food Sci 77(6):S233–S239 Keast RS, Costanzo A (2015) Is fat the sixth taste primary? Evidence and implications. Flavour 4(1):1–7 Kurihara K (2015) Umami the fifth basic taste: history of studies on receptor mechanisms and role as a food flavor. Biomed Res Int 2015:1 Kurobayashi Y, Fujiwara S, Matsumoto T, Nakanishi A (2019) Koku attribute-enhancing odor compounds. In: Koku in food science and physiology. Springer, pp 73–83 Kuroda M, Harada T (2004) Fractionation and characterization of the macromolecular meaty flavor enhancer from beef meat extract. J Food Sci 69(7):542–548 Kuroda M, Miyamura N (2015) Mechanism of the perception of “kokumi” substances and the sensory characteristics of the “kokumi” peptide, γ-Glu-Val-Gly. Flavour 4(1):11 Kuroda M, Ninomiya K (2020) Association between soup consumption and obesity: a systematic review with meta-analysis. Physiol Behav 225:113103 Kuroda M, Kato Y, Yamazaki J, Kageyama N, Mizukoshi T, Miyano H et al (2012) Determination of γ-glutamyl-valyl-glycine in raw scallop and processed scallop products using high pressure liquid chromatography–tandem mass spectrometry. Food Chem 134(3):1640–1644 Lawless HT, Schlake S, Smythe J, Lim J, Yang H, Chapman K, Bolton B (2004) Metallic taste and retronasal smell. Chem Senses 29(1):25–33 Lee SM, Jo Y-J, Kim Y-S (2010) Investigation of the aroma-active compounds formed in the Maillard reaction between glutathione and reducing sugars. J Agric Food Chem 58(5): 3116–3124 Li Q, Zhang L, Lametsch R (2022) Current progress in kokumi-active peptides, evaluation and preparation methods: a review. Crit Rev Food Sci Nutr 62(5):1230–1241 Lim J, Pullicin AJ (2019) Oral carbohydrate sensing: beyond sweet taste. Physiol Behav 202:14–25 Liu J, Song H, Liu Y, Li P, Yao J, Xiong J (2015) Discovery of kokumi peptide from yeast extract by LC-Q-TOF-MS/MS and sensomics approach. J Sci Food Agric 95(15):3183–3194 Liu P, Huang M, Song S, Hayat K, Zhang X, Xia S, Jia C (2016) Sensory characteristics and antioxidant activities of Maillard reaction products from soy protein hydrolysates with different molecular weight distribution. Food Bioprocess Technol 5:1775–1789 Maruyama Y, Kuroda M (2019) Mechanism of Kokumi substance perception: role of calciumsensing receptor (CaSR) in perceiving Kokumi substances. In: Nishimura T, Kuroda M (eds) Koku in food science and physiology. Springer, Singapore Maruyama Y, Yasuda R, Kuroda M, Eto Y (2012) Kokumi substances, enhancers of basic tastes, induce responses in calcium-sensing receptor expressing taste cells. PLoS One 7(4):e34489 Masic U, Yeomans MR (2014) Umami flavor enhances appetite but also increases satiety. Am J Clin Nutr 100(2):532–538 Mattes RD (2011) Accumulating evidence supports a taste component for free fatty acids in humans. Physiol Behav 104(4):624–631 McBurney DH, Gent JF (1979) On the nature of taste qualities. Psychol Bull 86(1):151 Miyaki T, Kawasaki H, Kuroda M et al (2015) Effect of a kokumi peptide, γ-glutamyl-valylglycine, on the sensory characteristics of chicken consommé. Flavour 4:17 Miyaki T, Imada T, Hao SS, Kimura E (2016) Monosodium L-glutamate in soup reduces subsequent energy intake from high-fat savoury food in overweight and obese women. Br J Nutr 115(1):176–184 Miyamura N, Jo S, Kuroda M, Kouda T (2015) Flavour improvement of reduced-fat peanut butter by addition of a kokumi peptide, γ-glutamyl-valyl-glycine. Flavour 4:16 Muir DD, Banks JM, Hunter EA (1997) A comparison of the flavour and texture of Cheddar cheese of factory or farmhouse origin. Int Dairy J 7(6–7):479–485 Nishimura T (2019) Definition of “Koku” Involved in Food Palatability. In: Koku in food science and physiology. Springer, pp 1–16

242

C. Forde and M. Stieger

Ogasawara M, Katsumata T, Egi M (2006) Taste properties of Maillard-reaction products prepared from 1000 to 5000 Da peptide. Food Chem 99(3):600–604 Ohsu T, Amino Y, Nagasaki H, Yamanaka T, Takeshita S, Hatanaka T et al (2010) Involvement of the calcium-sensing receptor in human taste perception. J Biol Chem 285(2):1016–1022 Rehman S-U, Banks J, Brechany E, Muir D, McSweeney P, Fox P (2000) Influence of ripening temperature on the volatiles profile and flavour of Cheddar cheese made from raw or pasteurised milk. Int Dairy J 10(1–2):55–65 Rhyu MR, Song AY, Kim EY, Son HJ, Kim Y, Mummalaneni S, Qian J, Grider JR, Lyall V (2020) Kokumi taste active peptides modulate salt and umami taste. Nutrients 12(4):1198 Running CA, Craig BA, Mattes RD (2015) Oleogustus: the unique taste of fat. Chem Senses 40(7): 507–516 Shah A, Ogasawara M, Egi M, Kurihara H, Takahashi K (2010) Identification and sensory evaluation of flavour enhancers in Japanese traditional dried herring (Clupea pallasii) fillet. Food Chem 122(1):249–253 Shibata M, Hirotsuka M, Mizutani Y, Takahashi H, Kawada T, Matsumiya K et al (2018a) Diversity of γ-glutamyl peptides and oligosaccharides, the “kokumi” taste enhancers, in seeds from soybean mini core collections. Biosci Biotechnol Biochem 82(3):507–514 Shibata M, Hirotsuka M, Mizutani Y, Takahashi H, Kawada T, Matsumiya K et al (2018b) Thermal treatment of soybean seeds can improve the quality of soymilk by enhancing the extraction efficiency of “Kokumi” taste components. Food Sci Technol Res 24(6):1111–1119 Tang KX, Zhao CJ, Gänzle MG (2017) Effect of glutathione on the taste and texture of type I sourdough bread. J Agric Food Chem 65(21):4321–4328 Tang CS, Tan VWK, Teo PS, Forde CG (2020) Savoury and kokumi enhancement increases perceived calories and expectations of fullness in equicaloric beef broths. Food Qual Prefer 83:103897 Toelstede S, Dunkel A, Hofmann T (2009) A series of kokumi peptides impart the long-lasting mouthfulness of matured gouda cheese. J Agric Food Chem 57(4):1440–1448 Tso R, Forde CG (2021) Unintended consequences: nutritional impact and potential pitfalls of switching from animal-to plant-based foods. Nutrients 13(8):2527 Tso R, Lim AJ, Forde CG (2020) A critical appraisal of the evidence supporting consumer motivations for alternative proteins. Foods 10(1):24 Ueda Y, Sakaguchi M, Hirayama K, Miyajima R, Kimizuka A (1990) Characteristic flavor constituents in water extract of garlic. Agric Biol Chem 54(1):163–169 Ueda Y, Tsubuku T, Miyajima R (1994) Composition of sulfur-containing components in onion and their flavor characters. Biosci Biotechnol Biochem 58(1):108–110 Ueda Y, Yonemitsu M, Tsubuku T, Sakaguchi M, Miyajima R (1997) Flavor characteristics of glutathione in raw and cooked foodstuffs. Biosci Biotechnol Biochem 61(12):1977–1980 Urbach G (1993) Relations between cheese flavour and chemical composition. Int Dairy J 3(4–6): 389–422 Webb J, Bolhuis DP, Cicerale S, Hayes JE, Keast R (2015) The relationships between common measurements of taste function. Chemosens Percept 8(1):11–18 Xu X, You M, Song H, Gong L, Pan W (2018) Investigation of umami and kokumi taste-active components in bovine bone marrow extract produced during enzymatic hydrolysis and Maillard reaction. Int J Food Sci Technol 53(11):2465–2481 Yamaguchi S, Ninomiya K (1998) What is umami? Food Rev Intl 14(2–3):123–138 Yang J, Sun-Waterhouse D, Cui C, Dong K, Wang W (2017) Synthesis and sensory characteristics of kokumi γ-[glu] n-phe in the presence of glutamine and phenylalanine: glutaminase from bacillus amyloliquefaciens or aspergillus oryzae as the catalyst. J Agric Food Chem 65(39): 8696–8703 Zhao CJ, Schieber A, Gänzle MG (2016) Formation of taste-active amino acids, amino acid derivatives and peptides in food fermentations–a review. Food Res Int 89:39–47

Part III

Amino Acids, α-Peptides, and Their Related Kokumi Substances

Chapter 14

Amino Acids, α-Peptides, and Their Related Kokumi Substances Motonaka Kuroda

Abstract In the last decade, the number of studies published on kokumi peptides has increased and many novel kokumi peptides have been introduced. Kokumi substances are defined as taste-related substances that modify flavor characteristics, such as complexity (thickness of taste), mouthfulness, and lastingness (continuity), when added to basic taste solutions or food, but are themselves tasteless at the added doses. Among the kokumi peptides and related compounds reported to date, compounds that match the definition of a kokumi substance are discussed in this chapter. Several α-peptides and N-acyl-amino acids have been reported to have lower threshold values in food than in water in sensory evaluations with nose-clips. Therefore, these α-peptides and N-acyl-amino acids may be regarded as kokumi substances.

14.1

Introduction

The chemical structures, perceptive mechanisms, and sensory characteristics of kokumi γ-glutamyl-peptides were described in the previous chapter. In the last decade, the number of studies published on kokumi substances has increased and many novel kokumi substances have been introduced. As indicated in Chap. 2, kokumi substances are defined as taste-related substances that modify the five basic tastes as well as flavor characteristics, such as complexity (thickness of taste), mouthfulness, and lastingness (continuity), when added to food systems, but are tasteless themselves at the added doses. Among the kokumi substances reported to date, compounds that match the definition of kokumi substances are discussed in this chapter. Substances with the following characteristics were selected: (1) A substance that exerts the sensory effects of a kokumi substance, such as enhancing the thickness of taste, complexity, mouthfulness, and continuity (lastingness), at concentrations below the threshold in water, and (2) a substance evaluated with a nose M. Kuroda (✉) Institute of Food Research and Technologies, Ajinomoto Co., Inc., Kawasaki, Kanagawa, Japan e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2024 M. Kuroda (ed.), Kokumi Substance as an Enhancer of Koku, https://doi.org/10.1007/978-981-99-8303-2_14

245

246

M. Kuroda

clip to avoid cross-modal interactions. Although the perceptive mechanisms of most substances remain unclear, several α-peptides and N-acyl-amino acids are considered to have the properties of kokumi substances. The structures and sensory characteristics of these kokumi substances are described in this chapter.

14.2

Kokumi α-Peptides

Liu et al. (2015) investigated kokumi peptides obtained from a yeast extract by fractionation followed by a sensory evaluation. They fractionated a commercial yeast extract paste by ultra-filtration, gel permeation chromatography, and liquid chromatography and identified them using Quadruple-TOF-MS/MS. Fractions and isolated compounds in water and in blank chicken broth were subjected to sensory evaluations with a nose clip to prevent cross-modal interactions with odorants. Four γ-glutamyl peptides were identified as kokumi peptides: γ-Glu-Cys-Gly (glutathione), γ-Glu-Leu, γ-Glu-Val, and γ-Glu-Tyr, and six α-peptides: Leu-Lys, Leu-Gln, Leu-Ala, Leu-Glu, Leu-Thr, and Ala-Leu. The thresholds of these peptides were lower in chicken broth than in water (Table 14.1). These findings suggested that these ten peptides were kokumi peptides. Among them, Leu-Lys, Leu-Gln, Leu-Ala, Table 14.1 Structure, threshold, and origin of kokumi α-peptides Peptide Leu-Lys Leu-Gln Leu-Ala Leu-Glu Leu-Thr Ala-Leu Val-Pro-Ala Asp-Trp-Pro Tyr-Gly-Asp-Gly Lys-Asp-Gln-Pro Ans-Gly-Gly-Leu-Gln Asp-Gly-Phe-Pro Glu-Ser-Leu-Pro-Ala-Leu-Pro Glu-Val-Gly-Tyr-Gly-Tyr Met-Thr-Thr-Phe-The-Trp a

Threshold (μmol/L) In water In foods 2400 1200a 1200 600a 3100 400a 2400 300a 1300 700a 6200 1500a 700 90b 770 290b >2000 310b >2000 230b >2000 160b 420 250c 771 273c 166 162c 354 279c

Chicken broth Model broth c Partial basic taste recombinate of soy sauce b

Origin Yeast extract Yeast extract Yeast extract Yeast extract Yeast extract Yeast extract Cocoa beans Cocoa beans Cocoa beans Cocoa beans Cocoa beans Soy sauce Soy sauce Soy sauce Soy sauce

Reference Liu et al. (2015) Liu et al. (2015) Liu et al. (2015) Liu et al. (2015) Liu et al. (2015) Liu et al. (2015) Salger et al. (2019) Salger et al. (2019) Salger et al. (2019) Salger et al. (2019) Salger et al. (2019) Jünger et al. (2022) Jünger et al. (2022) Jünger et al. (2022) Jünger et al. (2022)

14

Amino Acids, α-Peptides, and Their Related Kokumi Substances

247

Leu-Glu, Leu-Thr, and Ala-Leu were identified as kokumi α-peptides; however, only the threshold values of these peptides for taste-modulating activities in chicken broth were described. Although the taste quality of chicken broth with the fraction obtained by gel permeation chromatography was described as enhanced mouthfulness and complexity, that of chicken broth with each of the proposed kokumi peptides was not indicated. Therefore, more detailed investigations on sensory quality with sensory evaluations are warranted. Salger et al. (2019) examined kokumi peptides in overfermented cocoa beans. The aqueous extract of overfermented cocoa beans was fractionated by sequential solvent extraction, medium-pressure liquid chromatography, and preparative highpressure liquid chromatography and subjected to sensory evaluations. The structures of the isolated peptides were elucidated by ultra-performance liquid chromatography-time-of-flight-mass spectrometry (UPLC-ToF-MS) screening, liquid chromatography-MS/MS (LC-MS/MS), and customized chemical syntheses. Sensory evaluations identified five peptides that enhanced complexity and mouthfulness in food at concentrations lower than the threshold in water (intrinsic thresholds) as kokumi peptides: Val-Pro-Ala, Asp-Trp-Pro, Tyr-Gly-Asp-Gly, Lys-Asp-Gln-Pro, and Asn-Gly-Gly-Leu-Gln (Table 14.1). The lowest threshold concentration in food systems was measured for Val-Pro-Ala (90 μmol/L). Jünger et al. (2022) investigated the taste-modulating peptides of soy sauce by fractionation using solid-phase extraction with C18 resin and identification by ultra-performance liquid chromatography-ToF-MS/MS. The peptides identified in taste-modulating fractions were chemically synthesized and subjected to sensory evaluations. The threshold values of these peptides were measured in water (intrinsic threshold) and in partial taste recombinants of soy sauce (modulating threshold) with a nose clip. Sensory evaluations identified four peptides: Asp-Gly-Phe-Pro, Glu-SerLeu-Pro-Ala-Leu-Pro, Glu-Val-Gly-Tyr-Gly-Tyr, and Met-Thr-Thr-Phe-The-Trp, as kokumi peptides in soy sauce (Table 14.1). Intrinsic taste threshold concentrations ranged between 166 and 771 μmol/L and taste-modulating threshold concentrations between 162 and 279 μmol/L. The modulating threshold of each peptide was lower than the intrinsic threshold. Several salt- and umami-enhancing α-peptides were also identified in soy sauce. Enhancements in mouthfulness and complexity by these peptides were not described. However, since Yamaguchi and Kimizuka (1979) indicated that monosodium glutamate (umami substance) and salt enhance flavor characteristics, such as continuity, mouthfulness, and complexity, when added to foods, umami- and salt-enhancing peptides may have a positive impact on these sensory characteristics in food systems. Further studies using sensory evaluations on these peptides in other food systems are expected. Several α-peptides may act as kokumi substances. Since the perceptive mechanisms of these kokumi α-peptides currently remain unclear, further studies are warranted.

248

14.3

M. Kuroda

Kokumi N-Acyl-Amino Acids

Christa et al. (2022) investigated kokumi substances in the traditional Korean fermented dish kimchi. They fractionated the methanol/water extract of kimchi by ultrafiltration, gel permeation chromatography, and high-performance liquid chromatography. The thresholds of the compounds obtained in water and in a model broth were examined by a sensory evaluation with the duo-trio test wearing nose clips. The structures of the isolated compounds were analyzed by chemical synthesis and LC-MS/MS. N-Acyl-amino acids derived from lactic acid, such as N-L-lactoylL-glutamic acid, N-L-lactoyl-L-glutamine, N-L-lactoyl-L-aspartic acid, and N-Llactoyl-L-asparagine, and N-acylated amino acids derived from succinic acid, including N-succinyl-glycine, N-succinyl-L-alanine, N-succinyl-L-glutamic acid, N-succinyl-L-glutamine, N-succinyl-L-valine, and N-succinyl-L-phenylalanine, were identified as kokumi substances in the kimchi extract. Intrinsic thresholds ranged between 1754 and 8460 μmol/L, while those in the model broth were between 0.017 and 0.373 μmol/L. The threshold of each compound in the model broth (modulating threshold) was lower than the intrinsic threshold (Table 14.2). The quantitation of these compounds and sensory evaluations of the effects of a mixture of these compounds revealed that these compounds directly contributed to the overall taste of kimchi in natural concentrations. Hammerl et al. (2017) examined the secondary metabolites of L-tyrosine in yeast using a differential off-line HPLC-NMR approach. They also investigated the taste and taste-modulating activities of the compounds identified. Measurements of the thresholds of the compounds obtained in water and in the model broth were conducted by sensory evaluations with the duo test using nose clips. Several Table 14.2 Structure, threshold, and origin of kokumi N-acyl amino acids N-Acyl amino acid N-L-Lactoyl-L-Glu N-L-Lactoyl-L-Gln N-L-Lactoyl-L-Asp N-L-Lactoyl-L-Asn N-Succinyl-L-Gly N-Succinyl-L-Ala N-Succinyl-L-Glu N-Succinyl-L-Gln N-Succinyl-L-Val N-Succinyl-L-Phe N-(1-Oxododecanyl)-L-Tyr N-(1-Oxomyristyl)-L-Tyr N-(1-Oxoparmityl)-L-Tyr N-(1-Oxooleoyl)-L-Tyr N-(1-Oxo-dodecanyl)-L-Tyr

Threshold (μmol/L) In water In model broth 1754 157 6434 237 2711 361 4886 373 4732 256 4498 48 1935 17 4245 51 2971 61 8460 287 627 537 480 145 672 160 627 183 446 217

Origin Kimuchi Kimuchi Kimuchi Kimuchi Kimuchi Kimuchi Kimuchi Kimuchi Kimuchi Kimuchi Yeast Yeast Yeast Yeast Yeast

Reference Christa et al. (2022) Christa et al. (2022) Christa et al. (2022) Christa et al. (2022) Christa et al. (2022) Christa et al. (2022) Christa et al. (2022) Christa et al. (2022) Christa et al. (2022) Christa et al. (2022) Hammerl et al. (2017) Hammerl et al. (2017) Hammerl et al. (2017) Hammerl et al. (2017) Hammerl et al. (2017)

14

Amino Acids, α-Peptides, and Their Related Kokumi Substances

249

N-acyl-L-tyrosines, such as N-(1-oxododecanyl)-L-tyrosine, N-(1-oxomyristyl)-L(1-oxooleoyl)-L-tyrosine, and tyrosine, N-(1-oxoparmityl)-L-tyrosine, (1-oxododecanyl)-L-tyrosine, were identified as kokumi substances. Their intrinsic thresholds ranged between 446 and 672 μmol/L, while those in the model broth were between 145 and 537 μmol/L. The threshold of each compound in the model broth (modulating threshold) was lower than the intrinsic threshold (Table 14.2). These studies revealed that several N-acyl-amino acids may act as kokumi substances. The perceptive mechanisms of these compounds have not yet been clarified; therefore, further studies are warranted.

14.4

Candidates for Amino Acids and Peptide-Related Kokumi Substances

As described before, substances with the following characteristics were selected for examination as candidate kokumi substances: (1) a substance that exerts sensory effects, such as enhancing the thickness of taste, complexity, mouthfulness, and continuity (lastingness), at concentrations below the threshold in water (intrinsic threshold), and (2) a substance evaluated with a nose clip to avoid cross-modal interactions. Sensory evaluations without nose-clips showed that several types of amino acids, α-peptides, and peptides modified by the heating process enhanced mouthfulness, continuity (lastingness), and complexity (thickness of taste) when added to foods or umami/salty solutions at concentrations below the intrinsic thresholds. Among these compounds, some were considered to be involatile and chemically stable. Since it is possible that flavor enhancements by these involatile and stable compounds occurred through the sense of taste, these compounds are introduced as candidate kokumi substances in this chapter.

14.4.1

Amino Acids

Ohsu et al. (2010) reported that the addition of L-histidine (12.9 mM) to salty/umami solution significantly enhanced the thickness of taste (taste intensity sensed 5 s after tasting). Since the intrinsic threshold of L-histidine was previously reported to be 48.0 mM (Dunkel and Hofmann 2009), L-histidine was considered to act as a kokumi substance. L-Histidine was identified as an agonist of CaSR (Conigrave et al. 2000); therefore, enhancements in the thickness of taste were suggested to be caused by the activation of CaSR. Shah et al. (2010) investigated the components responsible for the flavor enhancement in Japanese traditional dried herring (migaki-nisin). They fractionated an aqueous extract of dried herring by dialysis, gel permeation chromatography, and high-performance liquid chromatography (HPLC). They finally indicated that the

250

M. Kuroda

addition of the fraction consisting of creatine and creatinine significantly enhanced the thickness of taste, mouthfulness, and continuity when added to Japanese noodle soup. A mixture of authentic creatine and creatinine (total concentration of 0.01%) at a ratio of 5:95 (weight basis) also enhanced the thickness of taste, mouthfulness, and continuity. Shah et al. (2013) also examined the sensory effects of creatine and creatinine. The addition of creatine at a concentration of 0.76 mM to Japanese noodle soup significantly enhanced the intensity of mouthfulness, while the addition of creatinine at a concentration of 0.88 mM significantly enhanced mouthfulness and continuity. Since the intrinsic thresholds of creatine and creatinine were previously reported to be 85 and 18 mM, respectively (Kranz et al. 2018), creatine and creatinine were suggested to act as kokumi substances in Japanese noodle soup. However, the mechanisms underlying flavor enhancements by creatine and creatinine remain unclear and, thus, warrant further studies.

14.4.2 α-Peptides Xu et al. (2018) investigated kokumi peptides in Maillard reaction products of the enzymatic hydrolysate of bovine marrow extract (BBME). They fractionated BBME by ultrafiltration, gel filtration chromatography, and reversed-phase HPLC, and identified eight types of kokumi peptides. Among these, one peptide, Cys-ProArg, had a lower threshold in beef broth (0.84 mM) than that in water (1.67 mM). Cys-Pro-Arg has been reported to enhance mouthfulness and complexity when added to beef broth at a concentration close to the modulating threshold (0.84 mM), suggesting that Cys-Pro-Arg acts as a kokumi substance.

14.4.3

Peptides Modified by the Heating Process

Maillard Peptides (Peptides Modified by Heating with Xylose) Ogasawara et al. (2006a) focused on unique flavor characteristics, such as the mouthfulness and continuity of long-term ripened miso (soybean paste). They fractionated the water-soluble fraction of miso ripened for 20 months by ultrafiltration, and revealed that the addition of the 1000–5000 Da fraction to a salty/umami solution significantly enhanced mouthfulness and continuity. Based on the findings showing that these fractions were colored and fluorescent, Maillard-reacted peptides were responsible for enhancements in mouthfulness and continuity. Ogasawara et al. (2006b) prepared model Maillard peptides (Maillard reaction products prepared from peptides) by heating a soy protein enzymatic hydrolysate with xylose and obtaining the 1000–5000 Da fraction by ultrafiltration. The addition of these Maillard peptides to an umami solution and consommé soup at a concentration below the intrinsic threshold value significantly enhanced mouthfulness and

14

Amino Acids, α-Peptides, and Their Related Kokumi Substances

251

continuity. These findings suggest that these Maillard peptides have the properties of kokumi substances. However, since Maillard peptides (Maillard-reaction products prepared from a soy protein hydrolysate and xylose) are a mixture of many types of compounds, further chemical characterization is needed. Maillard-Reacted Products from Tropomyosin and Collagen Kuroda and Harada (2004) attempted to characterize the flavor modifier from commercial beef extract. They focused on the macromolecular fraction (Mw >5000 Da), which enhanced continuity, mouthfulness, and the thickness of tase (complexity) when added to the lower-molecular fraction of beef extract, but had a weak aroma and tastes in water. Fractionation of the macromolecular fraction by anion-exchange chromatography, metal ion chelate chromatography, and gel filtration chromatography provided two fractions that were responsible for the enhancements in mouthfulness, continuity, and the thickness of taste (complexity). Partial enzymatic hydrolysis and a structure analysis of peptide fragments indicated that the precursor proteins of both active fractions were tropomyosin and collagen. The macromolecular fraction obtained from the heated products (heated in the lowermolecular fraction of beef broth) of bovine tropomyosin and bovine collagen significantly enhanced mouthfulness, continuity, and the thickness of taste (complexity) when added to a model stew. These findings suggested that the Maillardreacted products from tropomyosin and collagen had the properties of kokumi substances, similar to the Maillard peptides described above. Since these products are a mixture of many types of compounds, further chemical characterization and clarification of the underlying perceptive mechanisms are expected.

References Christa P, Dunkel A, Krauss A, Stark TD, Dawid C, Hofmann T (2022) Discovery and identification of tastants and taste-modulating N-acyl amino acid derivatives in traditional Korean fermented dish kimchi using a sensomics approach. J Agric Food Chem 70:7500–7514 Conigrave AD, Quinn SJ, Brown EM (2000) L-Amino acid sensing by the extracellular Ca2+sensing receptor. Proc Nat Acad Sci 97:4814–4819 Dunkel A, Hofmann T (2009) Sensory-directed identification of β-alanyl dipeptides as contributors to the thick-sour and white-meaty orosensation induced by chicken broth. J Agric Food Chem 57:9867–9877 Hammerl R, Frank O, Hofmann T (2017) Differential off-line LC-NMR (DOLC-NMR) metabolomics to monitor tyrosine-induced metabolome alterations in Saccharomyces cerevisiae. J Agric Food Chem 65:3230–3241 Jünger M, Mittermeier-Kleβinger VK, Farrenkopf A, Dunkel A, Stark T, Somoza V, Dawid C, Hofmann T (2022) Sensoproteomic discovery of taste-modulating peptides and taste re-engineering of soy sauce. J Agric Food Chem 70:6503–6518 Kranz M, Viton F, Smarro-Menozzi C, Hofmann T (2018) Sensomics-based molecularization of the taste of pot-au-feu, a traditional meat/vegetable broth. J Agric Food Chem 66:194–202

252

M. Kuroda

Kuroda M, Harada T (2004) Fractionation and characterization of the macromolecular meaty flavor enhancer from beef extract. J Food Sci 69:542–548 Liu J, Song H, Liu Y, Li P, Yao J, Xiong J (2015) Discovery of kokumi peptide from yeast extract by LC-Q-TOF-MS/MS and sensomics approach. J Sci Food Agric 95:3183–3194 Ogasawara M, Yamada Y, Egi M (2006a) Taste enhancer from the long-term ripening of miso (soybean paste). Food Chem 99:736–741 Ogasawara M, Katsumata T, Egi M (2006b) Taste properties of Maillard-reaction products prepared from 1000 to 5000 Da peptide. Food Chem 99:600–604 Ohsu T, Amino Y, Nagasaki H, Yamanaka T, Takeshita S, Hatanaka T, Maruyama Y, Miyamura N, Eto Y (2010) Involvement of the calcium-sensing receptor in human taste perception. J Biol Chem 285:1016–1022 Salger M, Stark TD, Hofmann T (2019) Taste modulating peptides from overfermented cocoa beans. J Agric Food Chem 67:4311–4320 Shah AKMA, Ogasawara M, Egi M, Kurihara H, Takahashi K (2010) Identification and sensory evaluation of flavor enhancers in Japanese traditional dried herring (Clupea pallasii) fillet. Food Chem 122:249–253 Shah AKMA, Ogasawara M, Egi M, Kurihara H, Takahashi K (2013) Effect of drying on creatine/ creatinine ratios and subsequent taste of herring (Clupea pallasii) fillet. Food Sci Tech Res 19: 691–696 Xu X, You M, Song H, Gong L, Pan W (2018) Investigation of umami and kokumi taste-active components in bovine bone marrow extract produced during enzymatic hydrolysis and Maillard reaction. Int J Food Sci Tech 53:2465–2481 Yamaguchi S, Kimizuka A (1979) Psychometric studies on the taste of monosodium glutamate. In: Filer LJ Jr, Garattini S, Kare MR, Reynolds WA, Wurtman RJ (eds) Glutamic acid: advances in biochemistry and physiology. Raven Press, New York, pp 35–54

Part IV

Lipid-Related Kokumi Substances

Chapter 15

Biochemical Studies on Lipid-Related Kokumi Substances Motonaka Kuroda

Abstract Lipid-related kokumi substances have been the focus of recent studies. Among the kokumi substances reported to date, several lipid-related compounds were found to have lower threshold values in food than in water. The chemical and sensory characteristics of these lipid-related kokumi substances are described in this chapter. Several octadecadien-12-ynoic acids were shown to have lower threshold values in food than in water. Several oxylipins from thermally processed avocado also had lower threshold values in food than in water. GPR120 agonists, including fatty acids, such as oleic acid and linoleic acid, and synthetic potent agonists enhanced the fatty mouth-coating sensation, which is similar to lastingness, when added to food, but did not evoke this effect themselves.

15.1

Introduction

In the last decade, the number of studies published on kokumi substances has increased and many novel kokumi substances have been introduced. As indicated in Chap. 2, kokumi substances are defined as taste-related substances that modify the five basic tastes as well as flavor characteristics, such as complexity (thickness of taste), mouthfulness, and lastingness (continuity), when added to food systems, although they are tasteless at the added doses. Among the kokumi substances reported to date, compounds that match the definition of kokumi substances are discussed in this chapter. Substances with the following characteristics were selected. (i) The substance exerts the sensory effects of a kokumi substance, such as enhancing the thickness of taste, complexity, mouthfulness and continuity (lastingness), at concentrations below the threshold in water. (ii) The substance is evaluated with a nose clip to avoid cross-modal interactions. Although the perceptive mechanisms of some substances remain unclear, several lipid-related compounds were considered to have the properties of kokumi substances. M. Kuroda (✉) Institute of Food Research and Technologies, Ajinomoto Co., Inc., Kawasaki, Kanagawa, Japan e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2024 M. Kuroda (ed.), Kokumi Substance as an Enhancer of Koku, https://doi.org/10.1007/978-981-99-8303-2_15

255

256

M. Kuroda

The structures and sensory characteristics of these kokumi substances are described herein.

15.2

Alkyne-Containing Fatty Acids and Their Derivatives

Mittermeier et al. (2018) identified kokumi substances in golden chanterelles (Cantharellus cibarius). They fractionated the methanol/water extract of chanterelles by ethyl acetate extraction and medium-pressure liquid chromatography with an octadecyl-silica column and identified them using LC/MS and 1D/2D-NMR. Sensory evaluations were conducted with nose clips to avoid cross-modal interactions with odorants, and their thresholds in water (intrinsic threshold) and the basic taste recombinant of golden chanterelles (modulating threshold) were measured by the triangle discrimination test. The following seven compounds were identified as kokumi compounds: 14,15-dehydrocrepenynic acid methyl ester, 14,15-dehydrocrepenynic acid ethyl ester, 14,15-dehydrocrepenynic acid, (10E,14Z)-9-hydroperoxy-10,14-octadecadien-12-ynoic acid, (10E,14Z)-9hydroxy-10,14-octadecadien-12-ynoic acid, (10E,14Z)-9-oxo-10,14-octadecadien12-ynoic acid, and (9Z,15E)-14-oxo-9,15-octadecadien-12-ynoic acid. The modulating thresholds of these compounds were lower than the intrinsic thresholds, as shown in Table 15.1. Three isolated octadecadienoic acids, (10E,14Z)-12-hydroxy10,14-octadecadienoic acid, (9Z,11Z)-14,18-dihydroxy-9,11-octadecadienoic acid, and (9Z,11Z)-14,17,18-trihydroxy-9,11-octadecadienoic acid, did not exhibit tasteTable 15.1 Structure, threshold, and origin of kokumi octadecadieen-12-yl-acid and their derivatives

Compound 14,15-Dehydrocrepenynic acid methyl ester 14,15-Dehydrocrepenynic acid ethyl ester 14,15-Dehydrocrepenynic acid (10E,14Z)-9-Hydroperoxy-10.14octadecadien-12-ynoic acid (10E,14Z)-9-Hydroxy-10.14octadecadien-12-ynoic acid (10E,14Z)-9-Oxo-10.14octadecadien-12-ynoic acid (9Z,15E)-14-Oxo-9,15-octadecadien12-ynoic acid (9Z,15E)-14,17,18-Trihydroxy-9,15octadecadien-12-ynoic acid

Threshold (μmol/L) In In basic taste water recombinate 648 32 512

59

531

105

536

38

320

69

639

79

228

19

>1000

59

Origin Golden chanterelles Golden chanterelles Golden chanterelles Golden chanterelles Golden chanterelles Golden chanterelles Golden chanterelles Golden chanterelles

Reference Mittermeier et al. (2018) Mittermeier et al. (2018) Mittermeier et al. (2018) Mittermeier et al. (2018) Mittermeier et al. (2018) Mittermeier et al. (2018) Mittermeier et al. (2018) Mittermeier et al. (2018)

15

Biochemical Studies on Lipid-Related Kokumi Substances

257

modulating activity. Therefore, an acetylene moiety appears to be necessary for the properties of kokumi substances.

15.3

Oxylipins

Degenhardt and Hofmann (2010) examined kokumi compounds in thermally processed avocado. The puree of heat-treated avocado was fractionated by solvent extraction and reversed-phase liquid chromatography, and isolated compounds were identified by LC/MS/MS and NMR. The thresholds of the compounds obtained in water and in the model broth were assessed by sensory evaluations with the threealternative forced-choice test wearing nose clips. Based on the findings of LC/MS/ MS and NMR as well as sensory evaluations, 1-acetoxy-2,4-dihydroxyheptadeca16-ene, 1-acetoxy-2,4-dihydroxyheptadeca-16-yne, 1-acetoxy-2-hydroxy-4oxoheptadeca-16-ene, 1-acetoxy-2-hydroxy-4-oxoheptadecane, 1-acetoxy-2hydroxy-4-oxooctadeca-12-ene, 1-acetoxy-2-hydroxy-4-oxoheneicosa-5,12,15-triene, 1-acetoxy-2,4-dihydroxyheneicosa-12,15-diene, and 1-acetoxy-2-hydroxy-4oxoheneicosa-12,15-diene were identified as kokumi substances in thermally processed avocado. Intrinsic thresholds ranged between 27 and 313 μmol/L, while those in the model broth were between 2 and 17 μmol/L. The threshold of each compound in the model broth (modulating threshold) was lower than the intrinsic threshold, as indicated in Table 15.2.

15.4

GPR120 Agonists

Iwasaki et al. (2021) investigated the effects of GPR120 agonists on the fatty after orosensation, which is similar to lastingness and a preferable characteristic of dietary fat in humans. The findings obtained indicated that the potent GPR-120 agonist, TUG-891, enhanced the fatty after orosensation when added to a vegetable oil emulsion, and all GPR120 agonists tested enhanced the fatty after orosensation when added to a low-fat food system, but did not evoke it in aqueous solution or mineral oil emulsion at any of the concentrations tested. In addition, sensory activity positively correlated with GPR120 activity. Therefore, GPR120 appears to be involved in the perception of the fatty after orosensation in humans. A detailed investigation on GPR120 agonists will be described in the next chapter. Lipid-related kokumi substances were introduced in this chapter. Apart from GPR120 agonists, the perceptive mechanisms of lipid-related kokumi substances (alkyne-containing fatty acids and oxylipins) remain unclear; therefore, further studies are needed.

258

M. Kuroda

Table 15.2 Structure, threshold, and origin of kokumi oxylipins

Compound 1-Acetoxy-2,4dihydroxyheptadeca-16-ene

Threshold (μmol/L) In model In water broth 2 9

1-Acetoxy-2,4dihydroxyheptadeca-16-yne

27

5

1-Acetoxy-2-hydroxy-4oxoheptadeca-16-ene

88

11

1-Acetoxy-2-hydroxy-4oxoheptadecane

313

17

1-Acetoxy-2-hydroxy-4oxooctadeca-12-ene

70

5

1-Acetoxy-2-hydroxy-4oxoheneicosa-5,12,15-triene

70

2

1-Acetoxy-2,4dihydroxyheneicosa-12,15diene 1-Acetoxy-2-hydroxy-4oxoheneicosa-12,15-diene

92

2

121

8

Origin Thermally processed avocado Thermally processed avocado Thermally processed avocado Thermally processed avocado Thermally processed avocado Thermally processed avocado Thermally processed avocado Thermally processed avocado

Reference Degenhardt and Hofmann (2010) Degenhardt and Hofmann (2010) Degenhardt and Hofmann (2010) Degenhardt and Hofmann (2010) Degenhardt and Hofmann (2010) Degenhardt and Hofmann (2010) Degenhardt and Hofmann (2010) Degenhardt and Hofmann (2010)

References Degenhardt AG, Hofmann T (2010) Bitter-tasting and kokumi-enhancing molecules in thermally processed avocado (Persea Americana Mill.). J Agric Food Chem 58:12906–12915 Iwasaki N, Sakamoto K, Kitajima S, Maruyama Y, Kuroda M (2021) GPR120 agonists enhance the fatty orosensation when added to fat-containing system, but not evoke it by themselves. Physiol Behav 234:113387 Mittermeier VK, Dunkel A, Hofmann T (2018) Discovery of taste modulating octadecadien-12ynoic acids in golden chanterelles (Cantharellus cibarius). Food Chem 269:53–62

Chapter 16

Involvement of GPR120 in Perception of Fatty Oral Sensations in Humans Naoya Iwasaki, Seiji Kitajima, and Motonaka Kuroda

Abstract The fatty acid transporter, CD36, and fatty acid receptor, GPR120, have recently been shown to play a role in the gustatory perception of fatty acids in humans. However, limited information is currently available to show that agonists of CD36 and GPR120 evoke fatty oral sensations to dietary fat in humans. Therefore, the involvement of GPR120 agonists in dietary fat perception in humans was investigated herein. An emulsion prepared from vegetable oil evoked a stronger fatty after orosensation, an oily mouth coating sensed 5–10 s after tasting, which is similar sensory characteristics to lastingness in koku, than that prepared from mineral oil; however, the physical properties of both emulsions, such as viscosity, particle distribution, interfacial tension, the contact angle, frictional load, and ζ-electric potential, were similar. The potent GPR120 agonist, TUG-891, enhanced the fatty after orosensation when added to the emulsion prepared from vegetable oil, but not to that from mineral oil. All GPR120 agonists tested enhanced the fatty after orosensation when added to a low-fat food system, whereas they did not evoke any fatty sensation in aqueous solution at the concentrations tested in the food system, and sensory activity positively correlated with GPR120 activity. These results suggest that GPR120 agonists enhanced the fatty after orosensation in humans when added to vegetable oil or a low-fat food system, but did not evoke it by themselves. In addition, to examine the sensory effects of GPR120 agonists on fat-containing foods, changes in the sensory characteristics of normal-fat whipped cream following the addition of the potent GPR120 agonist, TUG-891, were investigated using a descriptive analysis. Trained panelists selected 12 attributes from sensory words collected in evaluations of whipped cream with fat contents of 35 and 47%. The addition of 27.4 μM TUG-891 to normal-fat whipped cream significantly enhanced the intensity of the mouth coating (initial, middle and last, and longlasting), thickness of taste, and denseness, and also significantly decreased the score for melting speed in the mouth. Sensory scores for these attributes in normal-fat whipped cream were equivalent (mouth coating) or close (thickness of

N. Iwasaki · S. Kitajima · M. Kuroda (✉) Institute of Food Sciences and Technologies, Ajinomoto Co., Inc., Kawasaki, Kanagawa, Japan e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2024 M. Kuroda (ed.), Kokumi Substance as an Enhancer of Koku, https://doi.org/10.1007/978-981-99-8303-2_16

259

260

N. Iwasaki et al.

taste, denseness, and melting speed in the mouth) to those of full-fat whipped cream. Therefore, GPR120 agonists appear to possess some of the functions of fat in dairy cream.

16.1

16.1.1

GPR120 Agonists Enhance the Fatty After Orosensation When Added to a Fat-Containing System, but Do Not Evoke It by Themselves in Humans Introduction

Dietary fat is an important macronutrient and the second most important energy source. It is perceived by texture (Drewnowski 1992), olfactory (Ramirez 1993) and post-digestive mechanisms (Greenberg and Smith 1996). However, based on research conducted in the last two decades, dietary fat is now known to be perceived by a gustatory mechanism in rodents (Takeda et al. 2001; Fukuwatari et al. 2003; Hiraoka et al. 2003; Laugerette et al. 2005; Cartoni et al. 2010). Three molecules expressed in taste-bud cells have been proposed as the candidates responsible for the perception of fat in rodents: the fatty acid transporter CD36 (Cartoni et al. 2010; Fukuwatari et al. 1997) and the G protein-coupled receptors for the fatty acids, GPR40 and GPR120. Although the main constituent of dietary fat is triglycerides, they are easily hydrolyzed to free fatty acids by the strong lipase activity of the saliva of rodents (Kawai and Fushiki 2003). Therefore, these three candidates may be the systems perceiving dietary fat in rodents. Previous studies reported that knockout mice for CD36, GPR40, and GPR120 showed weaker preferences for fat than wildtype mice (Laugerette et al. 2005; Cartoni et al. 2010; Scrafani et al. 2007). Rodents generally exhibit a strong preference for fatty acids as well as triglycerides (Cartoni et al. 2010; Adachi et al. 2014). Based on these findings, dietary fat is considered to be perceived by CD36, GPR40, or GPR120 after its hydrolysis to fatty acids in rodents (Cartoni et al. 2010). The effects of TUG-891, a synthetic potent GPR120 agonist, on the preference of rats for dietary fat was examined (Murtaza et al. 2020), and the findings obtained using the two-bottle choice test revealed that rats exhibited a stronger preference for 0.3% xanthan gum solution with TUG-891 than for control 0.3% xanthan gum aqueous solution (Murtaza et al. 2020). Candidates for the perception of fat have been investigated in humans, but not as extensively as in rodents. Simons et al. (2010) used CD36-specific antibodies to localize this molecule in human foliate and circumvallate papillae. Galindo et al. (2012) also conducted an expression analysis of GPR40 and GPR120 in human gustatory tissues, and revealed that GPR120 was expressed in human gustatory and non-gustatory epithelia, whereas GPR40 was not. These findings suggested the involvement of CD36 and GPR120 in the gustatory perception of fatty acids in humans. Previous studies indicated that humans have the ability to perceive fatty acids (Galindo et al. 2012; Chale-Rush et al. 2007a, b; Stewart et al. 2010; Godinot et al. 2013; Running et al. 2015). Galindo et al. (2012) described the taste of fatty

16

Involvement of GPR120 in Perception of Fatty Oral Sensations in Humans

261

acids, the “fatty taste,” using an aqueous emulsion of 1 mM oleic acid as reference material. Running et al. demonstrated that medium- and long-chain fatty acids have a bitter and astringent taste sensation that is distinct from the five basic tastes (sweet, sour, salty, bitter, and umami) using perceptual mapping techniques (Running et al. 2015). The relationship between sensitivity to fatty acids and obesity-related parameters, such as BMI, has been examined using an epidemiological approach (ChaleRush et al. 2007b; Stewart et al. 2011; Newman et al. 2013). However, few studies have successfully demonstrated that fatty acids or agonists of candidates for the oral fatty acid transporter and receptor (CD36 and GPR120) had fatty oral sensations that were sensed when dietary fat was orally applied in humans. In the food industry, many types of low-fat foods have been developed to meet the requirements of low-calorie foods. Low-fat foods are generally accepted to be less palatable than full-fat foods. The sensory characteristics of low-fat foods score less than those of full-fat foods. A previous study showed that low-fat sausages scored lower for juiciness, greasiness, aftertaste, and overall flavor (Tomaschunas et al. 2013). Furthermore, low-fat peanut butter scored lower for thickness, the continuity of taste, smoothness, and oiliness (an oily mouth coating sensation) (Miyamura et al. 2015). In the present study, we focused on GPR120 and investigated the effects of GPR120 agonists, including fatty acids, on the perception of the fatty orosensation, which is related to the sensory characteristics of dietary fat and is weaker in low-fat foods than in full-fat foods in humans.

16.1.2

Materials and Methods

Materials TUG-891, (Z)-4-(3-ethyl-4-(2-chlorophenyl)thiazol-2(3H)-ylideneamino) benzenesulfonic acid (IRM-31), and 3-(4-((5-chloro-2,2-dimethyl-2,3dihydrobenzofuran-7-yl)methoxy)phenyl-2-methylpropanoic acid (GDX-13) were used as synthetic GPR120 agonists. TUG-891 was chemically synthesized using a previously described method (Shimpukade et al. 2012). IRM-31 was synthesized according to Xiahui et al. (2010). GDX-13 was synthesized according to Fang et al. (2010). Linoleic acid and oleic acid of food grade were obtained from Sigma Aldrich Co. (St. Louis, MO, USA). The structure of GPR120 agonists tested in the present study was shown in Fig. 16.1. Mineral oil of food grade was purchased from Kaneda Co., Inc. (Tokyo Japan). Canola oil was a gift from J-Oilmills Corp. (Tokyo, Japan) and was obtained just after the refining process, packed with nitrogen gas, and stored at -20 °C until used.

262

N. Iwasaki et al.

Fig. 16.1 Chemical structure of GPR120 agonists tested in this study. (a) TUG-891; (b) IRM-31; (c) GDX-13; (d) linoleic acid; (e) Oleic acid

Preparation of Samples for Sensory Evaluations Preparation of Emulsions for Comparisons of Mineral and Vegetable Oils The process to compare the orosensation of mineral and vegetable oils was optimized by preparing emulsions with similar physical properties, such as viscosity, particle distribution, interfacial tension, the contact angle, frictional load, and ζ-electric potential. The optimized preparation method was as follows. Mineral oil of food grade or canola oil was suspended in distilled water at 5.0% (w/w) and soy lecithin (J-Oilmills Corp.) was added at 5.0% (w/w). The suspensions obtained were passed 20 times through the Shirasu Porous Glass Emulsifying Connecter PC30N (As One Corp., Tokyo, Japan). Physical properties, such as viscosity and particle size distribution, did not significantly change during storage at room temperature for 72 h (data not shown). Sensory evaluations of emulsions were conducted within 24 h of their preparation.

16

Involvement of GPR120 in Perception of Fatty Oral Sensations in Humans

263

Preparation of Emulsions for the Evaluation of the GPR120 Agonist, TUG891 Emulsions for the evaluation of GPR120 agonists were prepared by the method reported by Galindo et al. (2012). Briefly, 10.0% (w/w) of mineral oil or canola oil, 5.0% (w/w) of Gum Arabic (CREO International Corp, Tokyo, Japan), and 0.01% (w/w) of the disodium salt of ethylene diamine tetraacetic acid of food additive grade (Chelest Corp., Osaka, Japan) were mixed and emulsified using the ultrasonic emulsifier, Sonifier 450D (Emerson Japan Ltd., Kanagawa, Japan) for 12 min with cooling on ice. Physical properties, such as viscosity and particle size distribution, did not significantly change during storage at room temperature for 72 h (data not shown). Sensory evaluations of emulsions were conducted within 24 h of their preparation. Samples for the Evaluation of Low-Fat Food Systems Commercial low-fat Ranch dressing (Fat: 23%) was purchased from Hidden Valley Food Products Co. (Oakland, CA, USA). GPR120 agonists were added at adequate concentrations and used in sensory evaluations. The GPR120 agonists used were TUG-891, IRM-31, GDX-13, linoleic acid, and oleic acid. Sensory Evaluations General Methods In the present study, trained panelists (aged between 26 and 54 years) participated in sensory evaluations. All panelists are employees of Ajinomoto Co., Inc. and work at the Institute of Food Sciences & Technologies. They have more than 3 years’ experience of developing food products or seasoning materials, more than 2 years’ experience of participating in descriptive analyses of foods, and more than 2 years’ experience of sensory evaluations of low- and full fat foods. Sensory evaluations were conducted between 10:30 and 11:30 a.m. or between 2:00 and 3:00 p.m. in a partitioned booth at 23 °C in an air-conditioned sensory evaluation room. Evaluations was performed under normal fluorescent light. In one session, panelists generally evaluated two samples consisting of the control sample and test sample, and two sessions (once in a.m. and once in p. m.) at most were conducted in 1 day. Samples were served in transparent, brown-colored glass cups or transparent plastic cups coded with random three-digit numbers. All evaluations were performed with the wearing of a nose-clip. Human sensory analyses were conducted in accordance with the Declaration of Helsinki, and informed consent was obtained from all panelists. The experimental protocol was approved by the Ethics Board of the Institute of Food Sciences & Technologies, Ajinomoto.

264

N. Iwasaki et al.

Sensory Evaluation of Emulsion Samples Ten milliliters of emulsion samples were served in transparent, brown-colored glass cups. Twelve panelists (n = 12; nine men and three women) were instructed to hold a 10-ml sample, which was at room temperature, in their mouths for 10 s, expectorate, and rate the intensity of the fatty after orosensation, a sensation like a mouth coating sensed 5–10 s after tasting. The fatty after orosensation was rated on a scale of 0–100 points. Panelists evaluated distilled water and noted it as an intensity of 0 points (no fatty after orosensation), 50 points was a strong fatty after orosensation, and 100 points was a very strong fatty after orosensation. Panelists were instructed to rinse their mouth five times before evaluating the next sample.

Measurement of the Point of Subjective Equivalent (PSE) in Low-Fat Dressing A negative control was prepared by mixing 99.7 g of low-fat dressing with 0.3 g of distilled water. Low-fat dressing with GPR120 agonists was prepared by mixing 99.7 g of low-fat dressing with 0.3 g of the preparations of GPR120 agonists. PSE was assessed using a previously described adjustment method (Mattes and Lawless 1985; Pangborn and Braddock 1989) with a slight modification. The evaluation was performed with a nose-clip. Six panelists (five men and a woman) performed this evaluation twice; therefore, the total trial number was 12 (n = 12). They were instructed to evaluate the intensity of the fatty after orosensation of low-fat dressing with each GPR120 agonist at various concentrations and compare it with that of low-fat dressing with 13 μM TUG-891. If the fatty after orosensation of the sample was stronger than that with 13 μM TUG-891, dressing with a lower concentration of the tested GPR120 agonist was prepared and applied to the evaluation. If the fatty after orosensation of the sample was weaker than that with 13 μM TUG-891, dressing with a higher concentration of the tested GPR120 agonist was prepared and applied to the evaluation. Sample preparations and evaluations were repeated until the intensity of the fatty after orosensation of dressing with the tested GPR120 agonist was the same as that of dressing with 13 μM TUG-891. PSE was assessed by calculating the average value.

Sensory Evaluation of Low-Fat Dressing with GPR120 Agonists A negative control and sample low-fat Ranch dressing were prepared as described above. TUG-891 was added to low-fat dressing at 13 μM. The other GPR120 agonists, IRM-31, GDX-13, linoleic acid, and oleic acid, were added at concentrations of PSE to 13 μM TUG-891 (Fig. 16.5). Approximately 20 g of dressing samples was served in transparent plastic cups, and the panelists were instructed to hold one teaspoon of dressing, which was at room temperature, in their mouth for 10 s, expectorate, and rate the intensity of the fatty after orosensation. The evaluation

16

Involvement of GPR120 in Perception of Fatty Oral Sensations in Humans

265

was conducted using a paired comparison test. Panelists rated the fatty after orosensation at 0.1 point units on a scale of -2 to +2. They also evaluated the control dressing sample and noted it as an intensity of 0 points. Ratings were defined as follows: -2, markedly weaker than the control; -1, weaker than the control; 0: the same as the control; +1, stronger than the control; +2, markedly stronger than the control. In this evaluation, six panelists (five men and a woman) performed evaluations twice; therefore, the total trial number was 12 (n = 12). Panelists evaluated the GPR120 agonist-added sample using the negative control as a standard (0 pt.) in one session, and evaluated the negative control using the GPR120 agonist-added sample as the standard (0 pt.) in the other session. Panelists were instructed to rinse their mouth five times between sample evaluations.

Sensory Evaluation of GPR120 Agonists in Aqueous Solution or a Suspension of Mineral Oil The sensory evaluation was conducted with a nose-clip. GPR120 agonists were prepared at the concentration of PSE to 13 μM TUG-891. Due to their low solubilities in water, linoleic acid and oleic acid were evaluated in emulsions prepared using mineral oil as described above. Twenty milliliters of aqueous solution or emulsions with GPR120 agonists was served in transparent plastic cups, and panelists were instructed to hold an adequate amount of solution in their mouths for 10 s, expectorate, and rate the intensity of the fatty after orosensation. The evaluation was conducted using a paired comparison test. Panelists rated the fatty after orosensation intensity at 0.1 point units on a scale of -2 to +2. They also evaluated the control sample and noted it as an intensity of 0 points. In the evaluation of TUG-891, IRM-31, and GDX-13, distilled water was used as a negative control. In the evaluation of linoleic acid and oleic acid, a mineral oil emulsion was used as a negative control. Points were defined as follows: -2, markedly weaker than the control; -1, weaker than the control; 0: the same as the control; +1, stronger than the control; +2, markedly stronger than the control. In this evaluation, six panelists (five men and one woman) performed evaluations twice; therefore, the total trial number was 12 (n = 12). Panelists evaluated the GPR120 agonist-added sample using the negative control as the standard (0 pt.) in one session, and evaluated the negative control using the GPR120 agonist-added sample as the standard (0 pt.) in the other session. Panelists were instructed to rinse their mouth five times between sample evaluations. Measurement of Physical Properties of Emulsion Samples The static viscosity of emulsion samples was measured using the static rheometer MCR-301 (Anton-Parr Gmb, Germany) at 25 °C. The distribution of particles in emulsion samples was measured using the laser diffraction scattering particle size

266

N. Iwasaki et al.

distribution analyzer LA-920 (Horiba Co., Ltd., Kyoto, Japan). Samples were diluted in distilled water and analyzed. The interfacial tension of emulsion samples was measured by the hanging drop method using the contact angle meter Drop Master DM701 (Kyowa Interface Science, Inc., Saitama, Japan). An 18-G Teflon-processed needle was used in measurements. The contact angle of emulsion samples was measured by the droplet method using the contact angle meter Drop Master DM701 (Kyowa Interface Science, Inc., Saitama, Japan). In measurements, the 18-G Teflon-processed needle and a silicon court for the bottom with a thickness of 1 mm and hardness of 10° were used in measurements. The frictional load of emulsion samples was measured by the creepmeter RE-3305C (Yamaden Ltd., Tokyo, Japan) with a 20 N load cell for the sliding test. A silicon court with a thickness of 1 mm and hardness of 10° was used at the bottom and the surface of the plunger (ZX-2). An emulsion sample (20 μL) was dropped on the bottom, and the plunger was slid at 1 mm/sec for 30 min. The vertical load was 0.5 N. The frictional load of each sample was assessed by calculating the average load at a sliding distance of 5 to 20 mm. The ζ-electric potential of emulsion samples was assessed using Zetasizer Nano ZS (Marvern Instruments Ltd., Marvern, UK). A DTS 1070 cell (Marvern Instruments Ltd.) was used for measurements. Measurement of Minor Components of Canola Oil Free fatty acids, monoacylglycerides, and diacylglycerides were quantified by gas chromatography using a DB-23 capillary column (0.25 mm i. d. × 30 m; thickness of 0.25 μm; Agilent Technologies, USA) with a flame ion detector (FID). The temperature program used was Temp. (°C): 50–50–170–210 (0–1–13–46.33 min.). The injection was conducted in the splitless mode, helium was used as the carrier gas, and the gas flow rate was 1.5 ml/min. Sterols were quantified by gas chromatography using a DB-1 capillary column (0.25 mm i. d. × 15 m; 0.25 μm membrane thickness; Agilent Technologies, USA) with FID. The temperature program used was Temp. (° C): 240–280 (0–13.33 min). The injection was conducted in the split mode (split ratio = 1:30), helium was used as the carrier gas, and the gas flow rate was 1.0 ml/ min. Assay of GPR120 Activity Using HEK-293 Cells The full-length cDNA of human GPR120 (accession #NM_001195755) or Gaq (accession #NM_002072) was constructed in the expression vector pcDNA3.1(+) and transiently transfected into HEK-293 cells. Briefly, cDNA was diluted with Invitrogen Opti-MEM medium (Thermo Fisher Scientific, Waltham, MA), mixed with Invitrogen Lipofectamine 2000 (Thermo Fisher Scientific), and poured onto HEK-293 cells grown at a submaximal concentration. After being cultured for 24 h in a 96-well plate, cells were incubated with staining solution to measure

16

Involvement of GPR120 in Perception of Fatty Oral Sensations in Humans

267

intracellular calcium ions using Calcium Assay Kit Express (Molecular Devices LLC, San Jose, CA), diluted with assay buffer (20 mM HEPES, 146 mM NaCl, 1 mM MgSO4, 1.39 mM glucose, 1 mM CaCl2, 2.5 mM probenecid, and 0.1% fatty acid-free bovine serum albumin), for 45–60 min. After staining, the stimulant solution containing the test compound prepared in the assay buffer was added to each well, and measurements were conducted using the fluorometric imaging plate reader FDSSμCELL (Hamamatsu Photonics K. K., Hamamatsu, Japan). By measuring the fluorescence value (Ex. 480 nm, Em. 540 nm) before and after the stimulation, the change in the intracellular free calcium ion concentration via the GPR120 receptor by the addition of the stimulant was quantitatively investigated. The measurement and analysis of fluorescence data were performed using the software attached to FDSSμCELL, and ΔF/F values were calculated according to the eq. F/F = (Maximum fluorescence value after the stimulation - Minimum fluorescence value after stimulation)/Fluorescence value before the stimulation. EC50 values were calculated using non-linear curve fitting in GraphPad Prism 7.0 (GraphPad Software Inc., San Diego, CA). Data are expressed as the mean ± SEM of three separate experiments. Statistical Analysis Statistical analyses were conducted using JMP version 9.0 (SAS Institute, Cary, NC, USA). Data were collected as the means ± standard error. Data of the physical properties were analyzed using the Student’s t-test. Data obtained from the evaluation of emulsion samples were analyzed using a two-way analysis of variance (ANOVA) with the factors of samples and subjects. Data obtained from the addition test of GPR120 agonists to low-fat dressing and aqueous solution or mineral oil emulsions were analyzed by a three-way ANOVA with the factors of samples, subjects, and evaluation order. Data were considered to be significant at p < 0.05.

16.1.3

Results

Physical Properties of Emulsion Samples Prepared from Mineral or Vegetable Oil The physical properties of emulsions prepared from mineral or vegetable oil were measured, and the results obtained are shown in Fig. 16.2. Figure 16.2a shows that the shear viscosity of samples was similar between emulsions from mineral oil and vegetable oil. Figure 16.2b shows the distribution of particle sizes in emulsion samples, and reveals no significant difference between emulsions from mineral oil and vegetable oil. Figure 16.2c and d show interfacial tension and contact angles, respectively, and no significant differences were observed between emulsions from mineral oil and vegetable oil. Figure 16.2e shows the frictional load of emulsions

268

N. Iwasaki et al.

Fig. 16.2 Physical properties of emulsions prepared from mineral oil and vegetable oil. (a) Static viscosity; mean data (n = 3) are indicated. The horizontal axis indicates the shear rate and the vertical axis indicates shear viscosity. Closed circles indicate the emulsion from vegetable oil; closed triangles indicated the emulsion from mineral oil. (b) Distribution of particle sizes; the horizontal axis indicates the particle size and the vertical axis indicates the distribution ratio. Red bar, emulsion from vegetable oil; blue bar, emulsion from mineral oil. (c) Interfacial tension; the vertical axis indicates the interface tension. Data are shown as means ± standard errors (n = 3). (d) Contact angle; mean data (n = 3) are shown. The horizontal axis indicates time and the vertical axis indicates the contact angle. Closed circles, emulsion from vegetable oil; closed triangle, emulsion from mineral oil. (e) Frictional load; the vertical axis indicates the frictional load. Data are shown as means ± standard errors (n = 3). (f) ζ-electric potential; the vertical axis indicates the ζ-electric potential. Data are shown as means ± standard errors (n = 3). n.s. Not significant

from mineral oil and vegetable oil. The frictional load of the emulsion from vegetable oil was slightly higher than that from mineral oil. Figure 16.2f shows the results of the ζ-electric potential, which was similar between the emulsions from mineral oil and vegetable oil. Contents of Minor Components in Canola Oil Canola oil contained free fatty acids, diacylglycerides, and phytosterols at contents of 0.05, 1.4, and 0.78 g/100 g, respectively. Among free fatty acids, only oleic acid

16

Involvement of GPR120 in Perception of Fatty Oral Sensations in Humans

269

was quantified, and its content was 0.05 g/100 g. These results suggest that the contents of these minor components were low, and, thus, the content of triacylglycerides was assumed to be higher than 97%. Sensory Evaluation of Emulsions Prepared from Mineral Oil and Vegetable Oil The preliminary sensory evaluation of emulsions indicated that the sensory characteristic that differed the most between emulsions from mineral oil and vegetable oil was the “fatty after orosensation,” an oral sensation similar to an oily mouth coating that is sensed 5–10 s after holding a sample in the mouth. The results in Fig. 16.2 showed no significant differences in physical properties between the emulsions obtained from vegetable oil and mineral oil, suggesting that this orosensation of the vegetable oil emulsion was due to taste, not texture. Therefore, the intensity of the fatty orosensation was compared in the sensory evaluation of emulsions prepared from mineral oil and vegetable oil, and the results obtained are shown in Fig. 16.3. The fatty after orosensation score was significantly higher for the emulsion from vegetable oil than for that from mineral oil ( p < 0.001). No significant interaction was observed between samples and subjects. This result indicates that despite their similar physical properties, vegetable oil had a stronger fatty after orosensation, which was related to the organoleptic characteristics of dietary fat, than mineral oil. Effects of the GPR120 Agonist, TUG-891 on Emulsions Prepared from Vegetable Oil and Mineral Oil The effects of the GPR120 agonist, TUG-891 on the fatty after orosensation of emulsions are shown in Fig. 16.4. Figure 16.4a shows the additive effects of TUG-891 (13 μM) on the emulsion prepared from vegetable oil. The fatty after Fig. 16.3 Results of a sensory evaluation of emulsions prepared from mineral oil and vegetable oil. The vertical axis indicates the score of the intensity for the fatty orosensation, defined as an orosensation similar to an oily mouth coating sensed 5–10 s after tasting. Data are shown as means ± standard error (n = 12)

270

N. Iwasaki et al.

Fig. 16.4 Effects of the GPR-120 agonist, TUG-891 on the intensity of the fatty orosensation of emulsions. The vertical axis indicates the score of the intensity of the fatty orosensation, defined as an orosensation similar to an oily mouth coating sensed 5–10 s after tasting. 0 pt., not sensed; 50 pt.; strong; 100 pt., very strong. (a) Effects of 13 μM TUG-891 on emulsions prepared from vegetable oil. (b) Effects of 13 μM TUG-891 on emulsions prepared from mineral oil. Data are shown as means ± standard error (n = 12). n. s. Not significant

orosensation was significantly enhanced by the addition of 13 μM TUG-891 ( p < 0.01). Figure 16.4b shows the effects of TUG-891 on the fatty after orosensation of the emulsion from mineral oil. The fatty after orosensation was not significantly altered by the addition of TUG-891. In both evaluations, no significant interaction was observed between samples and subjects. Therefore, TUG-891, a potent GPR120 agonist, enhanced the fatty after orosensation of the emulsion prepared from vegetable oil, but not that from mineral oil. PSE of GPR120 Agonists in Low-Fat Dressing The PSE of each GPR120 agonist is shown in Table 16.1. The PSE of GDX-13, IRM-31, linoleic acid, and oleic acid to 13 μM TUG-891 were 38, 60, 3780, and 9093 μM, respectively. Therefore, the ratios of the sensory intensity of GDX-13, IRM-31, linoleic acid, and oleic acid to TUG-891 were 0.34, 0.22, 0.0036, and 0.0024, respectively. These results suggest that GPR120 agonists, including synthetic agonists and fatty acids, enhanced the fatty after orosensation when added to low-fat dressing. Effects of GPR120 Agonists on the Fatty After Orosensation of Low-Fat Dressing The intensity of the fatty after orosensation of GPR120 agonists was evaluated at a PSE concentration to 13 μM TUG-891. The concentrations tested of TUG-891,

16

Involvement of GPR120 in Perception of Fatty Oral Sensations in Humans

Table 16.1 Point of subjective equivalent (PSE) and relative sensory intensity of various GPR-120 agonists against TUG-891

Compound TUG-891 GDX-13 IRM-31 Linoleic acid Oleic acid

PSE (μM) 13 38 60 3780 9093

271

Relative sensory intensity 1 0.34 0.22 0.0036 0.0014

IRM-31, GDX-0013, linoleic acid, and oleic acid were 13, 60, 38, 3780, and 9093 μM, respectively. The additive effects of various GPR120 agonists on the fatty after orosensation of low-fat Ranch dressing are shown in Fig. 16.5a. The results obtained indicated that all GPR120 agonists significantly enhanced the intensity of the fatty after orosensation when added to low-fat dressing ( p < 0.01 for TUG891, linoleic acid, and oleic acid; p < 0.05 for IRM31 and GDX-13). In all evaluations, no significant interactions were observed among samples, subjects, and evaluation orders. Sensory Evaluation of GPR120 Agonists in Aqueous Solution or the Emulsion from Mineral Oil The intensity of the fatty after orosensation of GPR120 agonists was evaluated at a PSE concentration to 13 μM TUG-891. The concentrations tested of TUG-891, IRM-31, GDX-0013, and linoleic acid were 13, 60, 38, 3780, and 9093 μM, respectively. Linoleic acid and oleic acid were evaluated in the emulsion prepared from mineral oil. The results of the sensory evaluation of aqueous solution are shown in Fig. 16.5b. At the concentrations shown in Fig. 16.5b, TUG-891, IRM-31, and GDX-13 had little tastes, while linoleic acid and oleic acid had slight astringency. None of these agonists significantly increased the intensity of the fatty after orosensation when evaluated in aqueous solution and emulsions from mineral oil. In all evaluations, no significant interactions were observed among samples, subjects, and evaluation orders. In the preliminary evaluation, we investigated the tastes of the agonists in aqueous solution or the mineral oil emulsion at higher concentrations. TUG-891 (26 μM) had a slight bitterness. TUG-891 (52 μM), IRM-31 (90 μM), and GDX-13 (76 μM) were bitter, while linoleic acid (7200 μM) and oleic acid (12,320 μM) were strongly astringent and bitter. Any fatty sensation related to dietary fat, including the fatty after orosensation, was not sensed in any of these samples. These results indicate that the GPR120 agonists did not evoke the fatty after orosensation by themselves. Measurement of GPR120 Activity The GPR-120 activity of each GPR-120 agonist is shown in Fig. 16.6. The EC50 value of each GPR-120 agonist was calculated using this assay system. The EC50

272

N. Iwasaki et al.

Fig. 16.5 Effects of various GPR-120 agonists on the fatty orosensation. GPR120 agonists were added at the concentration of PSE to 13 μM TUG-891. The vertical axis indicates the score of the intensity for the fatty orosensation, defined as an orosensation similar to an oily mouth coating sensed 5–10 s after tasting. -2, markedly weaker than the control; -1, weaker than the control; 0: the same as the control; +1, stronger than the control; +2, markedly stronger than the control. (a) Effects on low-fat dressing. (b) Effects in aqueous solution (TUG-891, IRM-31, and GDX-13) and emulsions prepared from mineral oil (linoleic acid and oleic acid). Data are shown as means ± standard error (n = 12). * p < 0.05, ** p < 0.01

0.5

OA

'F/F

0.4

LA TUG-891

0.3

IRM_31

0.2

GDX-13

0.1 0.0

-2

-1

0

1

2

3

log (PM)

Fig. 16.6 GPR-120 activity of various GPR-120 agonists. The horizontal axis indicates the concentration of agonists, and the vertical axis indicates fluorescent responses. Open circles, oleic acid; closed black squares, linoleic acid; closed green triangles, GDX-13; closed blue squares, IRM-31; closed red circles, TUG-891

values of TUG-891, IRM-31, GDX-13, linoleic acid, and oleic acid were 4.5, 2.5, 3.2, 36.6, and 406.6 μM, respectively. This result indicates that the synthetic agonists, TUG-891, IRM-31, and GDX-13 were stronger agonists than naturally occurring agonists, such as linoleic acid and oleic acid.

16

Involvement of GPR120 in Perception of Fatty Oral Sensations in Humans

273

Relationship Between Sensory Activity and GPR-120 Activity The relationship between the sensory activity and GPR-120 activity of various GPR-120 agonists is shown in Fig. 16.7. GPR-120 activity measured by the cellbased assay positively correlated (r = 0.943, p = 0.013) with sensory activity from the sensory evaluation of low-fat dressing.

16.1.4

Discussion

In the present study, we investigated the effects of GPR120 agonists on the fatty after orosensation in humans, and demonstrated that GPR120 agonists enhanced the fatty after orosensation when added to a fat-containing system, but did not evoke it by themselves. We initially investigated differences in oral sensations between emulsions prepared from vegetable oil (edible triacylglycerides) and mineral oil (n-alkane oil). To minimize the textural effect, the condition of the emulsion preparation was adjusted such that physical properties, including viscosity, were similar in the emulsions prepared from vegetable oil and mineral oil. Figure 16.2 suggests that the emulsions prepared from mineral oil and vegetable oil had similar physical properties. The sensory evaluation of emulsions prepared from mineral oil and vegetable oil revealed that the emulsion from vegetable oil had a stronger fatty after orosensation, which was related to the organoleptic characteristics of dietary fat, than that from mineral oil (Fig. 16.3). Although we cannot deny that the panelists may have discriminated differences in the physical properties of emulsions and also that the fatty after orosensation defined in the present study was due to the texture of oil, because of similarities in the physical properties of emulsions from vegetable oil and mineral oil, it may have been due to a mouthfeel sensation other than texture. However, more detailed studies are needed to clarify these possibilities. Furthermore, since all sensory evaluations were performed with nose-clips, the fatty after orosensation did not appear to be due to a retronasal aroma. Taste is generally Fig. 16.7 Relationship between GPR-120 activity and the sensory activity of various GPR-120 agonists. The horizontal axis indicates GPR120 activity (EC50 value), and the vertical axis indicates sensory intensity (PSE). (a) Oleic acid; (b) linoleic acid; (c) TUG-891; (d) GDX-13; (e) IRM-31

274

N. Iwasaki et al.

defined as a chemical sense that is perceived by taste receptor cells (Kurihara 1987; Chandrashekar et al. 2006; Roper and Chaudhari 2017). Since sufficient evidence to show that the fatty after orosensation in the present study was perceived by taste receptor cells has not yet been obtained, we used the words “fatty after orosensation” not “fatty after-taste” in the present study. Detailed studies on the perceptive mechanism of the fatty after orosensation are needed. The analysis of components present in canola oil revealed that the main component was triacylglycerides, with a content higher than 97%. As mentioned before, oleic acid was present in canola oil at a concentration of 0.05 g/100 g; therefore, its concentration in the emulsion was 0.0025% (88 μM). This concentration was markedly lower than that tested in the sensory evaluation (0.275%, 9093 μM; Fig. 16.5). In supplementary experiments (data not shown), we also investigated the sensory effects of oleic acid, diacylglycerides, and phytosterols at their levels in canola oil using the mineral oil emulsion system, and found that the fatty orosensation was not significantly altered by the addition of these components. Therefore, the fatty orosensation was considered to be elicited by triacylglycerides in canola oil. To clarify the sensory effects of GPR120 agonists in humans, the effects of adding TUG-891, a potent GPR120 agonist, to the emulsions prepared from vegetable oil and mineral oil were examined. Figure 16.4 shows that TUG-891 enhanced the fatty after orosensation when added to the emulsion prepared from vegetable oil, but not that from mineral oil. Since the results in Fig. 16.3 suggested that the emulsion from mineral oil did not elicit a fatty after orosensation, TUG-891 did not evoke the fatty after orosensation by itself. In addition, TUG-891 appeared to enhance the fatty after orosensation, which is related to the organoleptic characteristics of dietary fat, only when added to the emulsion from vegetable oil. The PSE of each GPR120 agonist was measured, and the effects of the GPR120 agonists at PSE were assessed in low-fat dressing. As shown in Fig. 16.5a, all GPR120 agonists enhanced the intensity of the fatty after orosensation when added to low-fat dressing, while each GPR120 agonist did not have a fatty taste at this concentration (Fig. 16.5b). These results suggest that none of the GPR120 agonists tested evoked the fatty taste by themselves. To clarify the relationship between enhancements in the fatty after orosensation and GPR120 activity, the EC50 value of each agonist was measured using a cellbased assay. Some of the EC50 values obtained were different from those previously reported. For example, previous studies reported mean EC50 values of linoleic acid for GPR120 of 14.99 μM (Moore et al. 2009), 5.34 μM (Christiansen et al. 2015), and 5.08 μM (Christiansen et al. 2015), while that obtained in the present study was higher at 36.6 μM. HEK cells were used in the GPR120 assay in all studies. Therefore, the differences observed in EC50 values were attributed to variations in the G protein used in the assay. Endogenous G proteins in HEK cells were used in previous studies (Moore et al. 2009; Christiansen et al. 2015), while Gα16gust44 was used in another study (Galindo et al. 2012). In the present study, the Gαq protein was transfected and used in the assay. A recent study reported that the Gαq protein functions in the transduction of signals from GPR120 (An et al. 2016). Therefore, we decided to use the Gαq protein in the GPR120 assay. Furthermore, regarding linoleic

16

Involvement of GPR120 in Perception of Fatty Oral Sensations in Humans

275

acid and oleic acid, since the response was not saturated, EC50 was calculated using curve-fitting software. This is considered to be one reason for the differences observed in EC50 values. Regarding PSE, to the best of our knowledge, the sensory effects of synthetic GPR120 agonists (TUG-891, IRM-31, and GDX-13) have not yet been examined. A previous study revealed that the taste threshold of oleic acid was higher than that of linoleic acid when evaluated in gum-based emulsions (Running and Mattes 2015), suggesting that humans are more sensitive to the taste of linoleic acid than that of oleic acid. In the present study, the PSE values of linoleic acid and oleic acid were 3780 and 9093 μM, respectively. This result indicated that linoleic acid was approximately 2.5-fold stronger than oleic acid when evaluated in low-fat dressing. Despite differences in the evaluation systems, the present results were consistent with previous findings. The relationship between the sensory activity (PSE value) and GPR120 activity (EC50 value) of GPR120 agonists was investigated. Figure 16.6 shows that GPR120 activity positively correlated with sensory activity. These results suggest that GPR120 contributed to enhancements in the fatty after orosensation. Lin et al. recently demonstrated that the addition of hexadecenoic acid (palmitic acid) at 0.2% (w/w) to potato chips increased the intensity of fattiness (Lin et al. 2020). Since palmitic acid is a GPR120 agonist (Adachi et al. 2014), this increase in fattiness was attributed to a mechanism involving GPR120. In the present study, the following results were obtained from a human sensory analysis and cell-based GPR-120 assays: (1) a fatty after orosensation was sensed in an emulsion from vegetable oil, but not that from mineral oil. (2) TUG-891, a potent GPR120 agonist, enhanced the fatty after orosensation when added to an emulsion from vegetable oil, but not that from mineral oil. (3) All GPR120 agonists tested enhanced the fatty after orosensation when added to low-fat food. (4) None of the GPR120 agonists evoked the fatty after orosensation in aqueous solution or the emulsion from mineral oil. (5) The enhanced intensity of the fatty after orosensation positively correlated with GPR120 activity. These results indicate that the fatty after orosensation was evoked by triacylglycerides in vegetable oil, and GPR120 agonists enhanced the fatty after orosensation when added to vegetable oil-containing systems, but did not evoke the fatty after orosensation by themselves. These results suggest that GPR120 contributed to enhancing the perception of the fatty orosensation. The hypothesis of the perception of the fatty after orosensation in humans is shown in Fig. 16.8. The perceptive mechanism of triacylglycerides in humans currently remains unclear. Furthermore, the role of another candidate for fat taste perception, CD36, has not yet been elucidated. Therefore, further studies are needed to clarify the mechanisms underlying the perception of the fatty after orosensation in humans. A marked difference has been reported in organoleptic properties between lowand high-fat foods using a descriptive sensory analysis. For example, low-fat yogurt has less creaminess (Pimentel et al. 2013), and low-fat Cheddar cheese has a weaker milkfat flavor and brothy flavor than full-fat Cheddar cheese (Amelia et al. 2013). In addition, low-fat ice cream was reported to have less thickness, smoothness, and creaminess and a weaker mouth coating and milky/cooked sugar flavor than full-fat ice cream (Liou and Grun 2007). In the present study, we focused on the fatty

276 Fig. 16.8 Hypothetical scheme for the perception of dietary fat in humans. The fatty orosensation is evoked by triacylglycerides in dietary fat via an unknown system. GPR120 agonists enhance the fatty orosensation by activating GPR120 coupled with the Gαq protein

N. Iwasaki et al.

Gαq PLC

orosensation, which is defined as an orosensation similar to an oily mouth coating that is sensed 5–10 s after tasting. We showed that the addition of GPR120 agonists may modulate sensory characteristics other than the fatty orosensation. The sensory characteristics of low-fat foods containing GPR120 agonists are now being investigated using a descriptive sensory analysis in our laboratory. In addition, a consumer preference test of low-fat foods containing GPR120 agonists will be conducted. The results obtained will be published elsewhere in the future.

16.1.5 Conclusions We herein investigated the effects of GPR120 agonists on the fatty after orosensation, which is related to the preferable characteristics of dietary fat in humans. The results obtained indicated that the potent GPR-120 agonist, TUG-891, enhanced the fatty after orosensation when added to a vegetable oil emulsion, and all GPR120 agonists tested enhanced the fatty after orosensation when added to a low-fat food system, although did not evoke the fatty after orosensation in aqueous solution or mineral oil emulsion at any concentrations. In addition, sensory activity positively correlated with GPR120 activity. These results suggest that GPR120 is involved in the perception of the fatty after orosensation in humans.

16

Involvement of GPR120 in Perception of Fatty Oral Sensations in Humans

16.2 16.2.1

277

Effects of the Potent GPR120 Agonist, TUG-891, on Sensory Characteristics of Whipped Cream Introduction

Dairy cream is a thick white fluid made from milk that is rich in fat and protein. It is used in the production of various food products, such as ice cream and whipped cream. Fat in cream plays a key role as a structural agent, aiding in the stabilization of the air phase (Goff et al. 1999), and creates characteristic sensory qualities that are expected in ice cream (Méndez-Velasco and Goff 2012). Consumer preference tests on ice cream with various fat contents (0–10%) showed that higher fat contents were associated with improvements in sensory quality and increases in overall preference (Zheng et al. 1997). Therefore, milk fat contributes to the sensory qualities of dairy foods made from cream. Recent studies revealed that dietary fat is perceived by a gustatory mechanism in rodents (Takeda et al. 2001; Fukuwatari et al. 2003; Hiraoka et al. 2003; Laugerette et al. 2005; Cartoni et al. 2010). Immunostaining studies (Cartoni et al. 2010; Fukuwatari et al. 1997) and behavioral studies using knock-out mice (Laugerette et al. 2005; Cartoni et al. 2010; Scrafani et al. 2007) reported that the fatty acid transporter, CD36, and fatty acid receptors, GPR40 and GPR120, were responsible for fat taste perception in rodents. In humans, an immunostaining study demonstrated that CD36 localized to foliate and circumvallate papillae in humans (Simons et al. 2010). An expression analysis of GPR40 and GPR120 showed that human gustatory and non-gustatory epithelia expressed GPR120, but not GPR40 (Galindo et al. 2012). These findings suggest roles for CD36 and GPR120 in fatty acid taste perception by humans. However, there is limited evidence to support fatty acids or CD36 and GPR120 activators evoking a fatty oral sensation when humans taste dietary fat. We previously examined the role of GPR120 in fat perception in humans (Iwasaki et al. 2021) and demonstrated the following: (1) The addition of TUG-891, a potent GPR120 agonist, to emulsions from vegetable oil and mineral oil resulted in a stronger fatty after orosensation in the former only. (2) The addition of all of the GPR120 agonists examined to a low-fat food increased the intensity of the fatty after orosensation. (3) The fatty after orosensation was not evoked by an aqueous or mineral oil emulsion containing GPR120 agonists. (4) A positive correlation was observed between the sensory intensities of GPR120 agonists and the activity of GPR120. These findings showed that the intensity of the fatty after orosensation, a similar oral sensation to an oily coating of the mouth 5–10 s after tasting, was enhanced by GPR120 agonists. The fatty after orosensation was the focus of our previous study because it was the most prominent difference between control and GPR120 agonist-treated samples. Nevertheless, GPR120 agonists also alter sensory characteristics, including the thickness of taste, denseness, and initial mouth coating (sensed 0–2 s after tasting). A descriptive sensory analysis revealed that organoleptic properties markedly differed between low- and full-fat dairy foods.

278

N. Iwasaki et al.

Previous studies reported that low-fat yogurt was less creamy (Pimentel et al. 2013) and that the milk and brothy flavors of Cheddar cheese were weaker in the low-fat versus full-fat version (Amelia et al. 2013). Furthermore, low-fat ice cream was less creamy and had a weaker vanilla flavor and aftertaste (Roland et al. 1999), and it was also reported to be less thick, not as smooth or creamy, had a less intense mouth coating, and weaker milky/cooked sugar flavor (Liou and Grun 2007). Therefore, the present study was performed to examine changes in the sensory characteristics of normal-fat whipped cream following the addition of the potent GPR120 agonist, TUG-891, using a descriptive sensory analysis.

16.2.2

Materials and Methods

Materials and Preparation of Samples A previously described method was used to chemically synthesize TUG-891 (Shimpukade et al. 2012). The chemical structure of TUG-891 is shown in Fig. 16.1. Milk with 3.6% fat was purchased from JA Tochigi (Tochigi, Japan), and milk with 4.0% fat (“Toku-nou 4.0”) and lard were from MEGMILK Snow Brand Co., Ltd. (Tokyo, Japan). Jersey milk (fat content: not less than 4.4%) was obtained from Okunakayama Kogen Milk Co., Ltd. (Iwate, Japan). Peanut butter was from Sonton Co., Ltd. (Tokyo Japan). Normal-fat dairy cream (fat content: 35%) and full-fat dairy cream (fat content: 47%) were purchased from Takanashi Milk Co., Ltd. (Kanagawa, Japan). Whipped cream was prepared using the Kitchen Aid cooking mixer KSM51 (FMI Corp., Osaka, Japan). Normal- and full-fat cream were whipped until their densities reached 0.45–0.55 g mL-1. A negative control was prepared for the sensory evaluation by mixing 99.95 g of normal-fat whipped cream with 0.05 g of food additive-grade ethanol (95%). Normal-fat whipped cream with 10 ppm (27.4 μM) TUG-891 was prepared by mixing 99.95 g of normal-fat whipped cream with 0.05 g of 2000 ppm (w/w) TUG-891 ethanolic solution. Sensory Evaluation Panelists and Ethics Approval Eleven trained panelists (age range, 26–54 years; all men) performed sensory evaluations in the present study. Each panelist is employed by the Ajinomoto Group and has more than 3 years’ experience in the development of food products or seasoning materials and conducting sensory evaluations of low- and full-fat foods. Human sensory analyses were performed in accordance with the Declaration of Helsinki, and all panelists provided informed consent. Since TUG-891 is not a food additive, the safety of this compound for human use was assessed using the concept of the threshold of toxicological concerns (TTC) (Munro et al. 2008). The TTC value

16

Involvement of GPR120 in Perception of Fatty Oral Sensations in Humans

279

of TUG-891 per person was set as 180 μg day-1. The experimental protocol was designed to ensure that the amount of TUG-891 for the evaluation did not exceed the TTC value. Furthermore, all sensory evaluations were conducted using the spit-out method, namely, panelists were instructed to hold a sample in their mouth, expectorate, and evaluate. The Ethics Board of the Institute of Food Sciences & Technologies, Ajinomoto Co., Inc. approved the experimental protocols used in the present study.

Selection of Sensory Attributes Panelists assessed the attributes for the sensory evaluation as follows. Each panelist evaluated normal- and full-fat whipped cream with a focus on differences between the two samples, and then extracted and listed words describing the sensory characteristics that differed between the two. The panel leader led the group in a discussion of differences between and similarities in normal- and full-fat whipped cream. A list of sensory attributes that focused on differences in the sensory characteristics of the two samples was developed. During the session, panelists defined 12 sensory attributes shown in Table 16.2. The time of the sensation was defined as follows: initial, from 0 to 2 s after holding the sample in the mouth; middle and last, from 2 to 10 s after holding the sample in the mouth; long-lasting, from 10 to 20 s after holding the sample in the mouth. The scores of the reference samples, normal- and full-fat whipped cream, were also assessed in a discussion by panelists. A scale from 1.0 to 5.0 points was used. Regarding all attributes, the score of normal-fat whipped cream (control) was set as 3.0 points, and each score was defined as follows: 1.0, markedly weaker than the control; 2.0, weaker than the control; 3.0, the same as the control; 4.0, stronger than the control; 5.0, markedly stronger than the control. In addition, the scores of the reference samples in Table 16.2 and full-fat whipped cream were assessed based on consensus (Table 16.2).

Panel Training After the definition of sensory attributes, panelists participated in a training session. They practiced rating each reference sample as well as normal- and full-fat whipped cream. The scores for normal-fat whipped cream were set as 3.0 points for all attributes. The scores for each attribute of full-fat whipped cream are shown in Table 16.2. A training session was conducted before the sensory evaluation on the day of data collection. Panelists evaluated normal- and full-fat whipped cream, and practiced rating both samples according to the scores shown in Table 16.2.

280

N. Iwasaki et al.

Table 16.2 Definition and reference samples for the descriptive attributes of whipped cream

Sensory attributes Initial raw milk flavor Middle and last raw milk flavor Middle and last heated milk flavor Initial sweetness

Middle and last sweetness Long-lasting sweetness Middle and last thickness of taste

Initial dairy-fat like mouth coating Middle and last dairyfat like mouth coating Long lasting dairy-fat like mouth coating Denseness

Definitions The flavor intensity reminiscent of raw milk sensed at 0–2 s after tasting The flavor intensity reminiscent of raw milk sensed at 2–10 s after tasting The flavor intensity reminiscent of heated milk sensed at 2–10 s after tasting One of the basic taste, common to sugar. Sweetness sensed at 0–2 s after tasting One of the basic taste, common to sugar. Sweetness sensed at 2–10 s after tasting One of the basic taste, common to sugar. Sweetness sensed at 10 s after exprorating The degree to which the flavor characters of the sample are harmonized, balanced, and blend well together as opposed to being spiky or striking out The degree to which there is a leftover residues, a dairy fat-like coating sensed at 0–2 s after tasting The degree to which there is a leftover residues, a dairy fat-like coating sensed at 2–10 s after tasting The degree to which there is a leftover residues, a dairy fat-like coating sensed at 10 s after exprorating The degree of compactness of cross section

Normalfat whipped cream 3.0

Full-fat whipped cream 4.0

Milk with 3.6% fat at room temp. (3.5)

3.0

3.5

Jersey milk at room temp. (5.0)

3.0

3.3

8% sucrose solution (3.5)

3.0

3.0

8% sucrose solution (2.5)

3.0

3.5

8% sucrose solution (2.5)

3.0

3.5

Milk with 4.0% fat at room temp. (4.0)

3.0

4.0

Non-whipped cream with fat 35% at room temp. (4.0), non-whipped cream with fat 47% at room temp. (5.0) Non-whipped cream with fat 35% at room temp. (4.0), non-whipped cream with fat 47% at room temp. (5.0) Non-whipped cream with fat 35% at room temp. (4.0), non-whipped cream with fat 47% at room temp. (5.0) Peanut butter (5.0)

3.0

3.5

3.0

3.6

3.0

3.6

3.0

4.0

Reference samples and intensity Milk with 3.6% fat at room temp. (4.0)

(continued)

16

Involvement of GPR120 in Perception of Fatty Oral Sensations in Humans

281

Table 16.2 (continued)

Sensory attributes Speed of melting

Definitions The degree to which there is a leftover residues, a slick, powdery or fatty coating or film in the mouth that is difficult to clear

Reference samples and intensity Lard at room temp (n.s.)

Normalfat whipped cream 3.0

Full-fat whipped cream 2.5

Sensory score of the normal-fat whipped cream was set at 3.0 points in all attributes n.s. Not scored The scale from 1.0 to 5.0 points was used. For all attributes, the score of normal-fat whipped cream (control) was set at 3.0 points, and each score was defined as follows: 1.0, extremely weaker than the control; 2.0, weaker than the control; 3.0, same as the control; 4.0, stronger than the control; 5.0, extremely stronger than the control

Sensory Evaluation of Whipped Cream with TUG-891 Panelists entered a partitioned booth maintained at a temperature of 23 °C in an air-conditioned sensory evaluation room between 10:30 and 11:30 a.m. and between 2:00 and 3:00 p.m. and conducted sensory evaluations under normal fluorescent light. Two evaluations were performed in each session. In the evaluation, panelists examined the negative control and test samples. In one session, panelists assessed negative control whipped cream (whipped cream without TUG-891) and whipped cream with TUG-891 as the test sample. The negative control and whipped cream with TUG-891 were prepared as described above. Whipped cream samples (approximately 15 g) were kept at 10 °C in a refrigerator and given to panelists in transparent plastic cups. Panelists held one teaspoon of the whipped cream sample in their mouth for 10 s, expectorated, evaluated for more 10 s, and then rated the intensity of each sensory attribute on a 15-cm line scale with points from 1.0 to 5.0. Samples were assessed in comparison with the negative control. After each sample evaluation, panelists rinsed their mouth with tap water five times. Statistical Analysis Statistical analyses were performed with JMP version 9.0 (SAS Institute, Cary, NC, USA). Data are shown as means ± standard deviation. The paired t-test was used to examine data obtained from the descriptive analysis. The significance of difference was set at p < 0.05.

282

16.2.3

N. Iwasaki et al.

Results and Discussion

Selection of Sensory Attributes A total of 115 words were extracted from the evaluations of normal- and full-fat whipped cream conducted by the panelists. During the selection of sensory attributes, the panelists developed the 12 sensory attributes shown in Table 16.2. These attributes consisted of three flavor attributes, seven taste and mouthfeel attributes, and two texture attributes. Definitions of the attributes and reference samples were also developed by discussions and are shown in Table 16.2. In the present study, the time of the sensation was defined as follows: initial, from 0 to 2 s after holding the sample in the mouth; middle and last, from 2 to 10 s after holding the sample in the mouth; long-lasting, from 10 to 20 s after holding the sample in the mouth. An initial fresh milk flavor, middle and last milk flavor, and middle and last heated milk flavor were selected and defined as flavor attributes. Initial sweetness, long-lasting sweetness, middle and last thickness of taste, initial mouth coating, middle and last mouth coating, and long-lasting mouth coating were selected and defined as the attributes related to taste and mouthfeel. Denseness and melting speed in the mouth were selected and defined as texture attributes. Sensory Evaluation of Normal-Fat Whipped Cream with TUG-891 The results of the sensory evaluation of normal-fat whipped cream and normal-fat whipped cream with 10 ppm (27.4 μM) TUG-891 are shown in Table 16.3 and Fig. 16.9. No significant differences were observed in the sensory scores for each attribute between normal-fat whipped cream (control whipped cream) and the negative control. The scores for each attribute of full-fat whipped cream are also shown in Table 16.3 and Fig. 16.9 as reference scores. The addition of 10 ppm (27.4 μM) TUG-891 significantly increased the scores of the middle and last thickness of taste ( p < 0.05), initial mouth coating ( p < 0.05), middle and last mouth coating ( p < 0.01), long-lasting mouth coating ( p < 0.01), and denseness ( p < 0.05), and significantly decreased the score for melting speed in the mouth ( p < 0.05). The addition of TUG-891 did not significantly change the scores for the initial fresh milk flavor, middle and last fresh milk flavor, middle and last heated milk flavor, initial sweetness, or middle and last sweetness. These results indicated that the addition of 10 ppm TUG-891 to normal-fat whipped cream significantly increased the intensities of similar sensory characteristics, such as the middle and last thickness of taste, mouth coating (initial, middle and last, and long-lasting) and denseness, and decreased that of melting speed in the mouth. The results obtained also demonstrated that the scores for attributes such as mouth coating (initial, middle and last, and long-lasting) were similar to those for full-fat whipped cream, which was used as a reference sample (Table 16.2). Therefore, TUG-891, a potent GPR120 agonist, may be used to enhance some of the functions of fat in dairy cream,

16

Involvement of GPR120 in Perception of Fatty Oral Sensations in Humans

283

Table 16.3 Results of descriptive sensory analysis on the effect of TUG-891 on the normal-fat whipped cream

Sensory attributes Initial raw milk flavor Middle and last raw milk flavor Middle and last heated milk flavor Initial sweetness Middle and last sweetness Long-lasting sweetness Middle and last thickness of taste Initial dairy-fat like mouth coating Middle and last dairyfat like mouth coating Long lasting dairy-fat like mouth coating Denseness Speed of melting in the mouth

Control whipped cream 2.99 ± 0.21 3.02 ± 0.23

Whipped cream with TUG-891 3.01 ± 0.04 3.16 ± 0.19

Significance for difference N.S. N.S.

Full-fat whipped cream (reference)a 4.0 3.5

3.08 ± 0.27

3.27 ± 0.19

N.S.

3.3

3.12 ± 0.30 3.21 ± 0.48

3.12 ± 0.15 3.38 ± 0.17

N.S. N.S.

3.0 3.5

3.26 ± 0.53 3.17 ± 0.42

3.40 ± 0.17 3.58 ± 0.30

N.S. *

3.5 4.0

3.13 ± 0.27

3.57 ± 0.33

*

3.5

3.12 ± 0.37

3.71 ± 0.36

**

3.6

3.12 ± 0.37

3.76 ± 0.39

**

3.6

3.14 ± 0.36 2.94 ± 0.16

3.48 ± 0.22 2.72 ± 0.27

* *

4.0 2.5

N.S. Not significant, * p < 0.05, ** p < 0.01, paired t-test Full-fat whipped cream was used as a reference sample

a

particularly mouth coating orosensations. Among GPR120 agonists, free fatty acids, such as oleic acid and linoleic acid, have been shown to enhance the fatty after orosensation (Iwasaki et al. 2021). However, effective concentrations of oleic acid and linoleic acid were 9093 μM (0.275 wt%) and 3780 μM (0.1 wt%), respectively, which were markedly higher than those of TUG-891 (13.0 and 27.4 μM in previous studies). Furthermore, these free fatty acids may be easily oxidized and generate aroma compounds, such as aldehydes and alcohols (Chang et al. 2019), which have been shown to cause off-flavors in foods (Cheng 2010; Zhang et al. 2012; Li et al. 2020). To the best of our knowledge, TUG-891 and other potent GPR120 agonists are chemically stable. Therefore, these agonists may be utilized to enhance the function of fat in dairy cream, particularly mouth coating orosensations. Previous studies revealed differences in mouth coating between low- and full-fat foods. Low-fat ice cream was found to have a weaker mouth coating than full-fat ice cream (Liou and Grun 2007). Furthermore, low-fat peanut butter has less oiliness, defined as an oily mouth coating, than full-fat peanut butter (Miyamura et al. 2015). Collectively, these findings indicate that the fatty orosensation is one of the functions of fat in several fat-containing foods.

284

N. Iwasaki et al.

*

** ** *

Normal fat-cream

*

+TUG891 10ppm (27.4μM)

Full-fat cream

Fig. 16.9 Results of descriptive sensory analysis of normal-fat whipped cream with or without TUG-891. Blue solid line, control normal-fat whipped cream; red solid line, normal-fat whipped cream with 10 ppm (27.4 μM) TUG-891; green dotted line, full-fat whipped cream evaluated as reference. I, initial (sensed at 0–2 s after tasting). M, middle (sensed at 2–5 s after tasting); L, last (sensed at 5–10 s after tasting); Last, lasting (sensed after 20 s after tasting); Fl, Flavor.. *: p < 0.05, **: p < 0.01.

The present study examined changes in the sensory characteristics of normal-fat whipped cream following the addition of TUG-891, a potent GPR120 agonist. We did not investigate the effects of TUG-891 on these characteristics in low-fat whipped cream because of the difficulties associated with controlling its texture. The effects of TUG-891 on low-fat foods are now being investigated in our laboratory and the results obtained will be published in the future.

16.2.4 Conclusion The present results indicated that TUG-891, a potent GPR120 agonist, enhanced some of the functions of fat in dairy cream, particularly mouth coating orosensations, when added to normal-fat whipped cream. However, since TUG-891 is a chemically synthetic compound and not currently a food additive, the development of a potent GPR120 agonist with consumer acceptance is needed. The development of potent food grade GPR120 agonists is now in progress in our laboratory. Further studies on changes in the sensory characteristics of low-fat foods by the addition of potent

16

Involvement of GPR120 in Perception of Fatty Oral Sensations in Humans

285

GPR120 agonists using a descriptive analysis as well as consumer preference tests are also now being conducted in our laboratory. Acknowledgments The authors are grateful to J-Oilmills Corporation for providing the canola oil sample. We sincerely thank Mr. Naohiro Miyamura of Ajinomoto Co., Inc. for his continuous encouragement and valuable discussions. We also appreciate all of the participants in the sensory evaluations.

References Adachi S, Eguchi A, Sakamoto K, Asano H, Manabe Y, Matsumura S, Tsuzuki S, Inoue K, Fushiki T (2014) Behavioral palatability of dietary fatty acids correlates with the intracellular calcium ion levels induced by the fatty acids in GPR120-expressing cells. Biomed Res 35:357–367 Amelia I, Drake MA, Nelson B, Barbano DM (2013) A new method for the production of low-fat Cheddar cheese. J Dairy Res 96:4870–4884 An T, Zhang X, Li H, Dou L, Huang X, Man Y, Zhang X et al (2016) GPR120 facilitates cholesterol efflux in macrophages through activation of AMPK signaling pathway. FEBS J 287:5080–5095 Cartoni C, Yasumatsu K, Ohkuni T, Shigemura N, Yoshida R, Godinot N, le Coutre J et al (2010) Taste preference for fatty acids is mediated by GPR40 and GPR120. J Neurosci 30:8376–8382 Chale-Rush A, Burgess JR, Mattes RD (2007a) Evidence for human orosensory (taste?) sensitivity to free fatty acids. Chem Senses 32:423–431. https://doi.org/10.1093/chemse/bjm007 Chale-Rush A, Burgess JR, Mattes RD (2007b) Multiple routes of chemosensitivity to free fatty acids in humans. Am J Physiol Gastrointest Liver Physiol 292:G1206–G1212 Chandrashekar J, Hoon MA, Ryba NJP, Zuker CS (2006) The receptors and cells for mammalian taste. Nature 444:288–294 Chang C, Wu G, Zhang H, Jin Q, Wang X (2019) Deep-fried flavor: characteristics, formation mechanisms, and influencing factors. Crit Rev Food Sci Nutr 60:1496–1514 Cheng H (2010) Volatile flavor compounds in yogurt: a review. Crit Rev Food Sci Nutr 50:938–950 Christiansen E, Watterson KR, Stocker CJ, Sokol E, Jenkins L, Simon K, Grundmann M et al (2015) Activity of dietary fatty acids on FFA1 and FFA4 and characterization of picolenic acid as a dual FFA1/FFA4 agonist with potential effect against metabolic diseases. Brit J Nutr 113: 1677–1688 Drewnowski A (1992) Sensory properties of fats and fat replacements. Nutr Rev 50:17–20 Fang SD, Jiango S, Jingyuan MA, Aaron N, Phuongly P, Imad N, Christopehr R Jr, Xin C. (2010) GPR120 receptor agonists and use thereof. Patent Application, WO2010/080537 Fukuwatari T, Kawada T, Tsuruta M, Hiraoka T, Iwanaga T, Sugimoto E, Fushiki T (1997) Expression of the putative membrane fatty acid transporter (FAT) in taste buds of the circumvallate papillae in rats. FEBS Lett 414:461–467 Fukuwatari T, Shibata K, Iguchi K, Saeki T, Iwata A, Tani K, Sugimoto E, Fushiki T (2003) Role of gustation in the recognition of oleate and triolein in anosmic rats. Physiol Behav 78:579–583 Galindo MM, Voigt N, Stein J, van Lengerich J, Rafuse J-D, Hofmann T, Meyerhof W, Behrens M (2012) G protein-coupled receptors in human fat taste perception. Chem Sense 37:123–139 Godinot N, Yasumatsu K, Barcos ME, Pineau N, Ledda M, Viton F, Ninomiya Y et al (2013) Activation of tongue-expressed GPR40 and GPR120 by noncaloric agonists is not sufficient to drive preference in mice. Neurosci 250:20–30 Goff HD, Verespej E, Smith AK (1999) A study of fat and air structures in ice cream. Int Dairy J 9: 817–829 Greenberg D, Smith GP (1996) The controls of fat intake. Psychosom Med 58:559–569

286

N. Iwasaki et al.

Hiraoka T, Fukuwatari T, Imaizumi M, Fushiki T (2003) Effects of oral stimulation with fats on the cephalic phase of pancreatic enzyme secretion in esophagostomized rats. Physiol Behav 79: 713–717 Iwasaki N, Sakamoto K, Kitajima S, Maruyama Y, Kuroda M (2021) GPR120 agonists enhance the fatty orosensation when added to fat-containing system, but not evoke it by themselves. Physiol Behav 234:113387 Kawai T, Fushiki T (2003) Importance of lipolysis in oral cavity for orosensory detection of fat. Am J Physiol Regul Integr Comp Physiol 285:R447–R454 Kurihara K (1987) Recent progress in the taste receptor mechanism. In: Kawamura Y, Kare MR (eds) Umami: a basic taste. Marcel Dekker, New York, pp 3–39 Laugerette F, Passilly-Degrace P, Patris B, Niot I, Febbralo M, Montmayeur JP, Besnard P (2005) CD36 involvement in orosensory detection of dietary lipids, spontaneous fat preference, and digestive secretions. J Clin Invest 115:3177–3184 Li X, Wang K, Yang R, Dong Y, Lin S (2020) Mechanism of aroma compounds changes from sea cucumber peptide powders (SCPPs) under different storage conditions. Food Res Int 128: 108757 Lin C, Colquitt L, Wise P, Breslin PA, Rawson NE, Genovese F, Maina I et al (2020) Studies of human twins reveal genetic variation that affects dietary fat perception. Chem Sense 45:467– 481 Liou BK, Grun IU (2007) Effect of fat level on the perception of five flavor chemicals in ice cream with or without fat mimetics by using a descriptive. J Food Sci 72:S595–S604 Mattes RD, Lawless HT (1985) An adjustment error in optimization of taste intensity. Appetite 6: 103–114. https://doi.org/10.1016/s0195-6663(85)80031-3 Méndez-Velasco C, Goff HD (2012) Fat structure in ice cream: a study on the types of fat interactions. Food Hydrocoll 29:152–159 Miyamura N, Jo S, Kuroda M, Kouda T (2015) Flavour improvement of reduced-fat peanut butter by addition of a kokumi peptide, γ-glutamyl-valyl-glycine. Flavour 4:16 Moore K, Zhang Q, Murgolo N, Hosted T, Duffy R (2009) Cloning, expression, pharmacological characterization of the GPR120 free fatty acid receptor from cynomolgus monkey: comparison with human GPR120 splice variants. Comp Biochem Physiol B 154:419–426 Munro IC, Renwick AG, Danielewska-Nikiela B (2008) The threshold of toxicological concern (TTC) in risk assessment. Toxic Lett 180:151–156 Murtaza B, Hichami A, Khan AS, Shimpukade B, Ulven T, Ozdener MH, Khan NA (2020) Novel GPR120 agonist TUG891 modulates fat taste perception and preference and activates tonguebrain-gut axis in mice. J Lipid Res 61:133–141 Newman L, Haryono R, Keast R (2013) Functionality of fatty acids chemoreception: a potential factor in the development of obesity? Nutrients 5:1287–1300. https://doi.org/10.3390/ nu5041287 Pangborn RM, Braddock KS (1989) Ad libitum preference for salt in chicken broth. Food Qual Pref 1:47–52 Pimentel TC, Cruz AG, Prudencio SH (2013) Influence of long-chain inulin and Lactobacillus paracasei subspecies paracasei on the sensory profile and acceptance of a traditional yogurt. J Dairy Sci 96:6233–6241 Ramirez I (1993) Role of olfaction in starch and oil preference. Am J Phys 265:R1404–R1409 Roland AM, Phillips LG, Boor KJ (1999) Effects of fat content on the sensory properties, melting, color, and hardness of ice cream. J Dairy Sci 82:33–38 Roper SD, Chaudhari N (2017) Taste buds: cells, signals and synapses. Nat Rev Neurosci 18:485– 497 Running CA, Mattes RD (2015) Humans are more sensitive to the taste of linoleic and α-linoleic than oleic acid. Am J Physiol Gastrointest Liver Physiol 308:G442–G449 Running CA, Craig BA, Mattes RD (2015) Oleogustus: the unique taste of fat. Chem Sense 40:507– 516

16

Involvement of GPR120 in Perception of Fatty Oral Sensations in Humans

287

Scrafani A, Ackroff K, Abumrad NA (2007) CD36 gene deletion reduced fat preference and intake but not post-oral fat conditioning in mice. Am J Physiol Regul Integr Comp Physiol 293: R1823–R1832 Shimpukade B, Hudson BD, Hovgaard CK, Milligan G, Ulven T (2012) Discovery of a potent and selective GPR120 agonists. J Med Chem 55:4511–4515 Simons PJ, Kummer JA, Luiken JJ, Boon L (2010) Apical CD36 immunolocalization in human and porcine taste buds from circumvallate and foliate papillae. Acta Histochem 113:839–843 Stewart JE, Feinie-Bisset C, Gloding M, Delahunty C, Clifton PM, Keast RS (2010) Oral sensitivity to fatty acids, food consumption and BMI in human subjects. Br J Nutr 104:145–152 Stewart JE, Newman L, Keast RS (2011) Sensitivity to oleic acid is associated with fat intake and body mass index. Clin Nutr 6:838–844 Takeda M, Sawano S, Imaizumi M, Fushiki T (2001) Preference for corn oil in olfactory-blocked mice in the conditioned place preference test and the two-bottle choice test. Life Sci 69:847–854 Tomaschunas M, Zorb R, Fischer J, Kohn E, Hinrichs J, Busch-Stockfisch M (2013) Changes in sensory properties and consumer acceptance of reduced fat pork Lyon-style and liver sausages containing inulin and citrus fiber as fat replacers. Meat Sci 95:629–640 Xiahui XE, Xnefeng Z, Kunyong Y, Roberts E, Hong L (2010) Compounds and methods for modulating G protein-coupled receptors. Patent Application, WO2010/008831 Zhang Y, Guo S, Liu Z, Chang SK (2012) Off-flavor related volatiles in soymilk as affected by soybean variety, grinding, and heat-processing methods. J Agric Food Chem 60:7457–7462 Zheng LI, Marshall R, Heymann H, Fernando L (1997) Effect of milk fat content on flavor perception of vanilla ice cream. J Dairy Sci 80:3133–3140

Part V

Future Prospect

Chapter 17

Overview and Future Prospects of Studies on Kokumi Substances Motonaka Kuroda

Abstract This chapter provides an overview of studies on kokumi substances. A kokumi substance is defined as a substance that modifies basic tastes and flavors, such as complexity, mouthfulness, and continuity (lingeringness), when added to foods, although it is tasteless itself at the dose tested. The first kokumi substance to be reported was glutathione (γ-Glu-Cys-Gly) in garlic. Other γ-glutamyl peptides were isolated from various foods, such as edible beans and cheeses, and identified as kokumi substances. Kokumi γ-glutamyl peptides are perceived by the calciumsensing receptor (CaSR) in humans. Screening using CaSR enabled the detection of the potent kokumi peptide, γ-Glu-Val-Gly. The effects of glutathione and γ-Glu-Val-Gly on the sensory characteristics of foods and satiety have also been discussed. Rodents exhibited a preference for kokumi peptides, such as glutathione and γ-Glu-Val-Gly, and these peptides enhanced nerve responses when added to umami solutions. The findings of functional magnetic resonance imaging revealed that the addition of glutathione to a salty/umami solution significantly activated the left ventral insula, which plays a role in processing the presence of a taste stimulus as well as its corresponding pleasantness in humans. Other kokumi substances, such as α-peptides, which are lipid-related compounds, are summarized in this chapter. Although some of the molecular mechanisms underlying the perception of kokumi substances have been elucidated, further studies in the field of physiology, neurobiology and molecular biology, and food science are needed to obtain a more detailed understanding of kokumi substances.

17.1

Definition of a Kokumi Substance

As described in Chaps. 1 and 2, a kokumi substance is a taste-related koku enhancer. Koku is defined as sensations such as complexity, mouthfulness, and lastingness and is mainly attributed to stimulations by taste compounds, aroma compounds, and M. Kuroda (✉) Institute of Food Research and Technologies, Ajinomoto Co., Inc., Kawasaki, Kanagawa, Japan e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2024 M. Kuroda (ed.), Kokumi Substance as an Enhancer of Koku, https://doi.org/10.1007/978-981-99-8303-2_17

291

292

M. Kuroda

texture. A kokumi substance is defined as a koku enhancer that is tasteless at the concentration tested. In the other words, a kokumi substance is a substance that modifies basic tastes and flavors, such as complexity, mouthfulness, and continuity (lingeringness), when added to foods, although it is itself tasteless at the concentrations tested. This book introduces substances that meet this condition.

17.2 17.2.1

Kokumi γ-Glutamyl Peptides Biochemical Studies

The first kokumi substance to be reported was glutathione (γ-Glu-Cys-Gly), which was identified as a kokumi substance of garlic (Chaps. 3 and 4). Glutathione was separated using several types of column chromatography and was identified by nuclear magnetic resonance and mass spectrometry. By utilizing a similar methodology, the research group led by Professor Thomas Hofmann at the Technical University of Munich investigated kokumi substances in various foods. The sensory effects of various compounds isolated from foods were examined by panelists trained to assess changes in mouthfulness and complexity using 5 mM GSH in model chicken broth. γ-Glu-Leu, γ-Glu-Val, and γ-Glu-β-Ala were identified as kokumi γ-glutamyl peptides in edible beans (Phaseolus vulgaris L.), γ-Glu-Glu, γ-Glu-Gly, γ-Glu-Gln, γ-Glu-Met, γ-Glu-Leu, γ-Glu-Val, and γ-Glu-His in cheese, and γ-Glu-Glu, γ-Glu-Gly, γ-Glu-Gln, γ-Glu-Met, γ-Glu-Leu, γ-Glu-Val, and γ-Glu-Val-Gly in soy sauce. Shibata et al. identified γ-Glu-Tyr and γ-Glu-Phe as kokumi substances in soybean seeds using a similar methodology (Chap. 5). These γ-glutamyl peptides enhanced complexity and mouthfulness when added to chicken broth or reconstituted soy sauce solution at concentrations below the threshold in water (intrinsic threshold). Furthermore, the content of the potent kokumi γ-glutamyl peptide, γ-Glu-Val-Gly, was quantified in various foods, and it was also detected in scallop foods and various fermented foods, such as fish sauces, soy sauces, fermented shrimp paste, beer, and cheese (Chap. 6).

17.2.2

Physiological Studies

Experiments using agonists and antagonists showed that kokumi γ-glutamyl peptides were perceived by the calcium-sensing receptor (CaSR) in humans (Chap. 8). The molecular mechanisms underlying enhancements in umami and sweetness by kokumi substances were investigated using isolated mouse taste buds and monitoring ATP, a signal molecule of type-II taste cells. γ-Glu-Val-Gly enhanced ATP secretion from mouse taste buds triggered by a sweetener. Based on the finding showing that this enhancement was abolished by atropine, an antagonist of muscarinic acetylcholine receptors, CaSR agonists were suggested to enhance

17

Overview and Future Prospects of Studies on Kokumi Substances

293

sweet-induced ATP release from sweet receptor cells via cholinergic cell-tocell signaling within a taste bud (Chap. 9). In Chap. 10, to elucidate the neurological mechanisms of action of kokumi substances, preference and taste nerve recording tests were conducted using rodents. In the two-bottle preference test, glutathione and γ-Glu-Val-Gly significantly increased intake when added to an umami solution. This increase was abolished by the addition of NPS-2143, an antagonist of CaSR. The recording of chorda tympani indicated that glutathione and γ-Glu-Val-Gly significantly enhanced nerve responses when added to an IMP solution. In Chap. 11, the effects of glutathione on brain activity associated with taste sensations on the tongue were investigated using functional magnetic resonance imaging (fMRI) under standardized taste delivery conditions. The findings of fMRI revealed that the addition of glutathione to a salty/umami solution elicited significant activation of the left ventral insula, which plays a role in processing the presence of a taste stimulus as well as its corresponding pleasantness. From these physiological studies, enhancements in umami and sweetness by kokumi substances were suggested to be due to the increase of ATP, a signal molecule of type-II cells, followed by the increase in the taste-nerve responses to umami and sweetness (Fig. 17.1).

17.2.3

Food Science and Sensory Science

The additive effects of glutathione (Chap. 4) and γ-Glu-Val-Gly (Chap. 12) on the sensory characteristics of foods were investigated using a descriptive sensory analysis. The addition of glutathione significantly enhanced the continuity of flavor, mouthfulness, and the thickness of taste when added to reconstituted model beef extract. It also significantly enhanced sweetness, umami, the thickness of taste, mouthfulness, and the continuity of flavor when added to reconstituted scallop extract (Chap. 4). The addition of γ-Glu-Val-Gly significantly enhanced the umami, mouthfulness, and mouth coating of chicken consommé, the oiliness, umami, thickness of taste of gravy, and the middle and last meaty flavor and pepper flavor of hamburger steak. Moreover, it significantly enhanced the sweetness, thickness of taste, and juiciness of an orange-flavored drink. It also significantly enhanced the thickness of taste, continuity, aftertaste, and oiliness of reduced-fat foods. Collectively, these findings suggest that the kokumi peptides glutathione and γ-Glu-Val-Gly modify several sensory characteristics in various foods. In Chap. 13, the perceptional and nutritional impacts of kokumi materials were investigated by a consumer panel. The panel evaluated the expected calorie content and satiating properties of beef broth with various savory and koku intensities. Although calorie contents were the same in the broth tested, beef broth with high savory and koku intensities was more likely to have both a high expected calorie content and satiating properties, suggesting that koku enhancement by kokumi substances reduced energy intake while maintaining palatability.

294

M. Kuroda

umami substance

Increase in taste-nerve responses to umami and sweetness Enhancement of umami and sweetness Fig. 17.1 Proposed model for the enhancement in umami and sweet taste by kokumi substances (CaSR agonists). As described in Chap. 9, cell-to-cell communication between CaSR- and T1R3expressing taste cells occurs in mouse taste buds. CaSR stimulation induces acetylcholine secretion. Secreted acetylcholine acts on nearby cholinergic taste cell (e.g., T1R3-expressing cells) to enhance ATP release from basic taste stimulation such as sweet and umami. In Chap. 9, the increase in ATP secretion was observed when T1R3-eppressing cells were stimulated by sweetener. Since T1R3expressing cells are also stimulated by umami taste, it is supposed that umami taste can be enhanced by kokumi substances via the same mechanism. The increase in ATP secretion was suggested to cause the enhancement in sweet and umami taste

17.3 Amino Acids, α-Peptides, and Their Related Kokumi Substances Chapter 14 described α-peptides and N-acyl-amino acids with the properties of kokumi substances. Several leucyl dipeptides from yeast extract and α-peptides from fermented cocoa and soy sauce were found to have lower threshold values in foods than in water. Some types of N-lactoyl- and N-succinyl-amino acids from kimchi and N-acyl-tyrosine from yeast were also shown to have lower threshold values in foods than in water. These findings suggest that these α-peptides and

17

Overview and Future Prospects of Studies on Kokumi Substances

295

N-acyl-amino acids are kokumi substances. However, the molecular mechanisms underlying the perception of these kokumi substances currently remain unclear.

17.4

Lipid-Related Kokumi Substances

Chapter 15 indicated that several octadecadien-12-ynoic acids from golden chanterelles and oxylipins from thermally processed avocado had lower threshold values in foods than in water. These findings indicated that these compounds possessed the properties of kokumi substances. GPR120 agonists, including fatty acids, such as oleic acid and linoleic acid, and synthetic potent agonists, enhanced the fatty mouthcoating sensation, which is similar to lastingness, when added to food, but did not evoke this effect themselves. In addition, based on the finding showing that GPR120 activity positively correlated with the intensity of the mouth-coating enhancement, GPR120 was suggested to contribute to the perception of the fatty mouth-coating sensation.

17.5

Future Prospects of Studies on Kokumi Substances

As described above, the receptors for kokumi γ-glutamyl peptides have been identified as CaSR, and the mechanisms responsible for enhancements in sweet and umami tastes by kokumi substances have been proposed, as shown in Chap. 9. However, the mechanisms underlying enhancements in flavor characteristics, such as complexity, mouthfulness, and lingeringness (continuity), remain unclear. One explanation is that enhancements in these flavor characteristics by kokumi substances are due to increases in the intensities of umami and sweet tastes by kokumi substances. However, the mechanisms underlying enhancements in koku attributes (complexity, mouthfulness, continuity, and lingeringness) by umami and sweet substances remain unclear. Moreover, kokumi substances may enhance flavor characteristics, such as complexity, mouthfulness, and continuity (lingeringness), by mechanisms other than enhancements in umami and sweet tastes. This book discussed several sensory characteristics of a number of candidate kokumi substances, such as α-peptides, N-acylated amino acids, and lipid-related compounds (Chap. 14). Among these compounds, GPR120 agonists, including free fatty acids, enhance the fatty orosensation, which is similar to the mouth coating sensation, when added to food systems, but have no effects by themselves at the concentrations tested. The finding showing that GPR120 activity positively correlated with sensory intensity indicated the involvement of GPR120 in the perception of the fatty orosensation (Chap. 15). However, the mechanisms underlying the perception of α-peptides, N-acylated amino acids, and other lipid-related compounds have not yet been elucidated.

296

M. Kuroda

Further studies in the field of food science (sensory science, food chemistry, and food processing), physiology, neurobiology, and molecular biology are needed. The combination of these studies will contribute to a more detailed understanding of kokumi substances.