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The Chemistry of Food

The Chemistry of Food Jan Velı́šek Department of Food Analysis and Nutrition, Faculty of Food and Biochemical Technology, Institute of Chemical Technology Prague, Czech Republic

This edition first published 2014 © 2014 by John Wiley & Sons, Ltd Registered office: John Wiley & Sons, Ltd, The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK Editorial offices: 9600 Garsington Road, Oxford, OX4 2DQ, UK The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK 111 River Street, Hoboken, NJ 07030-5774, USA For details of our global editorial offices, for customer services and for information about how to apply for permission to reuse the copyright material in this book please see our website at www.wiley.com/wiley-blackwell.

The right of the author to be identified as the author of this work has been asserted in accordance with the UK Copyright, Designs and Patents Act 1988.

All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by the UK Copyright, Designs and Patents Act 1988, without the prior permission of the publisher.

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Limit of Liability/Disclaimer of Warranty: While the publisher and author(s) have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose.

It is sold on the understanding that the publisher is not engaged in rendering professional services and neither the publisher nor the author shall be liable for damages arising herefrom. If professional advice or other expert assistance is required, the services of a competent professional should be sought.

Library of Congress Cataloging-in-Publication Data Velisek, Jan, 1946[Chemie potravin. English] The chemistry of food / Jan Velisek. pages cm Includes bibliographical references and index.

ISBN 978-1-118-38384-1 (cloth) –ISBN 978-1-118-38381-0 (paperback) 1. Food–Analysis–Textbooks. 2. Food–Composition–Textbooks. I. Title.

TP372.5.V4513 2013 664 .07–dc23 2013026547 A catalogue record for this book is available from the British Library.

Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic books.

Cover image: istock © merrymoonmary Cover design by Andy Meaden Typeset in 9.5/12 pt Minion by Laserwords Private Limited, Chennai, India

1

2014

Contents

PREFACE

vii

ABOUT THE COMPANION WEBSITE

5.9 5.10 5.11 5.12 5.13 5.14 5.15

ix

CHAPTER 1

INTRODUCTION

CHAPTER 2

AMINO ACIDS, PEPTIDES AND PROTEINS

2.1 2.2 2.3 2.4 2.5

Introduction 4 Amino acids 4 Peptides 27 Proteins 35 Reactions 63

CHAPTER 3

3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8

SACCHARIDES 198

Introduction 198 Monosaccharides 199 Derivatives of monosaccharides Oligosaccharides 217 Polysaccharides 230 Complex saccharides 278 Reactions 280

CHAPTER 5

5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8

FATS, OILS AND OTHER LIPIDS 87

Introduction 87 Classification 87 Fatty acids 88 Homolipids 108 Heterolipids 123 Miscellaneous simple and complex lipids Substances accompanying lipids 132 Reactions 145

CHAPTER 4

4.1 4.2 4.3 4.4 4.5 4.6 4.7

1

VITAMINS 335

Introduction 335 Vitamin A 337 Vitamin D 345 Vitamin E 349 Vitamin K 356 Thiamine 359 Riboflavin 364 Niacin 367

207

4

Pantothenic acid 370 Pyridoxal, pyridoxol and pyridoxamine Biotin 375 Folacin 377 Corrinoids 380 Vitamin C 384 Other active substances 396

CHAPTER 6

6.1 6.2 6.3 6.4 6.5 6.6 6.7

129

477

FLAVOUR-ACTIVE COMPOUNDS

499

Introduction 499 Odour-active substances 500 Taste-active substances 621

CHAPTER 9

9.1 9.2 9.3 9.4 9.5 9.6 9.7 9.8

WATER 459

Introduction 459 Drinking water 459 Water in foods 463 Structure 464 Properties 466 Interactions 466 Phase interfaces 473 Food dispersed systems Water activity 494

CHAPTER 8

8.1 8.2 8.3

MINERALS 402

Introduction 402 Chemistry of minerals 404 Essential elements 416 Non-essential elements 442 Toxic elements 444 Toxic inorganic anions 451 Radionuclides 454

CHAPTER 7

7.1 7.2 7.3 7.4 7.5 7.6 7.7 7.8 7.9

372

PIGMENTS AND OTHER COLORANTS 656

Introduction 656 Tetrapyrroles 657 Other nitrogen pigments Flavonoids 677 Xanthones 699 Curcuminoids 701 Isochromenes 701 Quinoid pigments 703

669

vi

9.9 9.10 9.11 9.12

CONTENTS

Carotenoids 713 Iridoids 730 Other terpenoid pigments 730 Enzymatic browning reactions 732

CHAPTER 10 ANTINUTRITIONAL, TOXIC AND OTHER BIOACTIVE COMPOUNDS 741

10.1 Introduction 741 10.2 Antinutritional compounds 10.3 Toxic compounds 747 CHAPTER 11

11.1 11.2 11.3 11.4 11.5

743

11.6 Substances increasing biological value 11.7 Other food additives 888 CHAPTER 12

12.1 12.2 12.3 12.4 12.5 12.6 12.7 12.8

FOOD CONTAMINANTS

892

Introduction 892 Technological contaminants 894 Microbial toxins 942 Persistent organohalogen contaminants 963 Chlorinated aliphatic hydrocarbons 999 Pesticides 999 Veterinary drugs 1022 Contaminants from packaging materials 1031

FOOD ADDITIVES 847

Introduction 847 Substances prolonging the shelf life of foods 848 Substances regulating odour and taste 863 Substances modifying colour 875 Substances modifying texture 882

888

BIBLIOGRAPHY INDEX

1079

1041

Preface

During the 15 years that have elapsed since the first Czech edition of the textbook The Chemistry of Food was published, many important monographs and scientific articles from various areas of food science have appeared in the literature, thanks to developments in analytical instrumentation and advances in the knowledge in the field of food technology. All these, plus the interest expressed by the readers, were the impetus that led to the third Czech edition of the book in 2009. This first English edition of The Chemistry of Food has remained essentially unchanged in terms of the basic structure of the 12 chapters and the majority of the text, tables, figures and formulae. Certain specific parts, however, have, necessarily, been revised, supplemented and updated.

The Chemistry of Food is the result of many years of experience by workers at the Department of Food Chemistry and Analysis (now Department of Food Analysis and Nutrition), Institute of Chemical Technology in Prague, through the teaching of this subject, plus other related topics taught in the Faculty of Food and Biochemical Technology. The reader will first be introduced to the chemical composition of foods, the important properties of food components and their functions. After these set descriptions, subsequent sections deal with the changes and reactions that occur, or may occur, in foods under certain conditions. It is necessary for the reader to be able to understand the context and complexity of all the processes taking place in foods. As in the previous Czech editions, the authors have tried to reduce to a minimum the amount of text, as is typical for textbooks on biochemistry, microbiology, organic, inorganic and physical chemistry, so that it should not be necessary to consult other specialised textbooks too often, but, at the same time, ensuring the text is clear to both students and professionals.

The textbook contains an introductory chapter and then 11 chapters dealing with the main and accessory nutrients that determine the nutritional and energy value of food raw materials and foods. There is a chapter describing amino acids, peptides and proteins, a chapter dealing with fats, oils and other lipids, as well as chapters on carbohydrates, vitamins, mineral substances and water.

Another chapter considers compounds responsible for the aroma, taste and colour attributes that determine the sensory quality of food raw materials and foods. The remaining chapters discuss substances that affect, or may affect, the hygienic–toxicological quality of food raw materials and foods, including antinutritional, toxic

and other biologically active food components, food additives and food contaminants.

The third Czech edition featured the work of many workers from the Department of Food Chemistry and Analysis, along with colleagues from other departments of the Faculty of Food and Biochemical Technology and also external authors. Jan Pánek acted as a co-author of the chapters dealing with the main nutrients, and Helena Čı́žková and Michal Voldřich participated in these and other chapters. Co-authors of the chapter on amino acids, peptides and proteins were Karel Cejpek and Roman Kubec, of the chapter on fats, oils and other lipids Jana Dostálová and Vladimı́r Filip, of the chapter on carbohydrates Karel Cejpek and Kamila Mı́ková, of the chapter on minerals Richard Koplı́k, of the chapter on flavour-active substances Jan Šavel, of the chapter on pigments and other colourants Jana Dostálová, of the chapter of antinutritional and toxic substances Pavel Kalač and Přemysl Slanina, and of the chapter on contaminants Jaroslav Dobiáš, Marek Doležal and Jana Hajšlová. The co-authors of the sections dealing with food legislation were Vladimı́r Kocourek and Kamila Mı́ková. Most of these co-authors were also involved in the first and second Czech editions of this publication. In addition, co-authors of the first and second Czech editions included Jan Pokorný (the chapter on fats, oils and other lipids), Jiřı́ Davı́dek (the chapter on vitamins and contaminants), Tomáš Davı́dek (the chapter on carbohydrates), Karel Hrnčiřı́k (chapters on vitamins and antinutritional and toxic compounds) and Helena Valentová (the chapter on pigments and other colourants). I would like to take this opportunity to thank all of my co-authors and other colleagues for their thorough work.

Considerable thanks are also due to the reviewers of the first Czech edition, Prof. Ing. Alexander Prı́bela, DSc. (Slovak University of Technology in Bratislava, Slovak Republic) and Ass. Prof. RNDr Jan Staněk, PhD, who also did a great deal of hard work on the project.

The co-authors of the Czech editions were Ass. Prof. Ing. Karel Cejpek, Ing. Helena Čı́žková, PhD, Prof. Ing. Jiřı́ Davı́dek, DSc., Ass. Prof. Ing. Jaroslav Dobiáš, PhD, Ass. Prof. Ing. Marek Doležal, Prof. Ing. Jana Dostálová, PhD, Prof. Ing. Vladimı́r Filip, PhD, Prof. Ing. Jana Hajšlová, PhD, Prof. Ing. Vladimı́r Kocourek, PhD, Prof. Dr Ing. Richard Koplı́k, Ass. Prof. Ing. Kamila Mı́ková, PhD, Ass. Prof. Ing. Jan Panek, PhD, Prof. Ing. Jan Pokorný, DSc., Ass. Prof. Dr Ing. Kateřina Riddellová and Prof. Ing. Michal

viii

PREFACE

Voldřich, PhD, staff of the Faculty of Food and Biochemical Technology, Institute of Chemical Technology in Prague. External co-authors were Ing. Tomáš Davı́dek, PhD (Nestlé, Orbe, Switzerland), Ing. Karel Hrnčiřı́k, PhD (Unilever, Vlaardingen, The Netherlands), Prof. Ing. Pavel Kalač, PhD (University of South Bohemia, České Budějovice), Ing. Roman Kubec, PhD (University of South Bohemia, České Budějovice), Prof. Přemysl Slanina, DVM,

PhD (National Food Administration, Uppsala, Sweden), Ass. Prof.

Ing. Jan Šavel, PhD (Budweiser Budvar n.p., České Budějovice) and Ing. Helena Valentová, PhD (Hügli Food Ltd, Zásmuky).

Jan Velı́šek Prague, February 2013

About the Companion Website

This book is accompanied by a companion website: www.wiley.com/go/velisek/foodchemistry The website includes:

• Powerpoints of all figures from the book for downloading • PDFs of tables from the book • Powerpoints of all formulae from the book

1 Introduction

The term food means material (eaten or drunk) that contains or consists of nutrients (proteins, fats, carbohydrates, vitamins and minerals) and many other chemical substances. Food is usually of plant or animal origin but, to a lesser extent, it may come from other sources, such as algae or microorganisms. Organisms use food to perform one or more of the four essential functions: to supply energy, to build, repair and maintain body tissues, to supply chemical substances that regulate body processes and to supply chemical substances that protect the organism. Traditionally food was provided through agriculture, but today most of the food consumed by the world’s population is either supplied by the food industry or home-grown.

Human food, including such items as meat, poultry, fish, milk, vegetables, fruit and beer for example, is chemically very complex.

According to rough estimates, fresh foods contain about half a million different chemical compounds. A much larger number of compounds are the result of biochemical (enzymatic) and chemical (non-enzymatic) reactions that occur during the storage of raw materials and foods and during their industrial and culinary processing.

Food science – the scientific study of food – is one of the major branches of the life sciences. Every aspect of food is covered, integrating and incorporating concepts and information from many different fields, including natural, technical and social sciences. It draws primarily on the knowledge of chemical science (biochemistry, organic, inorganic, physical and analytical chemistry), some areas of physics (such as the mechanics of solids and liquids), biology (especially microbiology), biotechnology, some of the branches of medical science (human nutrition, human physiology, pharmacology, toxicology) and agricultural science (e.g., crop and livestock production, post-harvest plant physiology and post-mortem muscle physiology). It also uses a range of experiences from technical engineering disciplines, such as agricultural and food engineering, particularly in the form of genetic food engineering. Food science may also call upon some experience of economics, sociology, psychology and other branches of social science.

The most important building blocks of food science are food chemistry and food technology. Food chemistry deals not only with the composition of food raw materials and end food products, but also with the behaviour, interactions and reactions of food components and the changes occurring in these food raw materials and end food products under various conditions during production, storage, processing, preparing and cooking. Food technology covers the entire gamut from procuring food raw materials to processing them into food products to preserving, packaging and delivering the food products to the consumer market. The common interest of both disciplines is examination of the possibilities of enhancing the positive changes and preventing the unwanted ones, elimination of antinutritive and toxic food components, and prevention of possible contamination with substances that can pose health risks and guarantee food safety. Producing safe and nutritious food for human and animal consumption is one of the principal aims of both food chemistry and food technology.

The most important natural constituents of foods are the nutrients that all organisms need in order to live and grow. The main (primary or basic) nutrients are proteins, fats (mainly triacylglycerols and phospholipids, but also numerous other lipids) and carbohydrates (such as some poly-, oligo- and monosaccharides) that determine the nutritional (nutritive) value of food, as they provide structural material (e.g., proteinogenic amino acids used for protein synthesis and lipids from which cell membranes are built) and energy. They may also have other functions. Nutrients are said to be essential if they must be obtained from an external source, either because the organism cannot synthesise them or it cannot produce them in sufficient quantities. Essential main nutrients include not only essential amino acids, but also lipids that are precursors of biologically active substances (such as essential fatty acids and some signalling molecules) and thus have protective functions. Also, some oligosaccharides are biologically active substances (e.g., breast milk oligosaccharides that act as receptor analogues to inhibit the adhesion of pathogens on the epithelial surface).

The Chemistry of Food, First Edition. Jan Velı́šek. © 2014 John Wiley & Sons, Ltd. Published 2014 by John Wiley & Sons, Ltd. Companion Website: www.wiley.com/go/velisek/foodchemistry.

2

CH 01 INTRODUCTION

The energy value of consumed food depends on the content of nutrients and on such factors as their digestibility, utility, content of some other substances, dietary regime, mental health status and other factors. Energy value is mainly related to proteins, fats and carbohydrates.

Organic nutrients also include vitamins. Inorganic chemical compounds, such as dietary minerals, water (and oxygen), may also be considered nutrients. Vitamins and dietary minerals are collectively known as accessory (additive) nutrients and are often referred to as essential nutritional factors. With the exception of some vitamins that humans cannot synthesise, these accessory nutrients must be obtained from foods. They are therefore known as exogenous factors. Another essential nutrient is water, which is obtained in small amounts by oxidation of primary nutrients, but in much larger quantities from foods, and especially from beverages.

Most foods contain a mixture of some or all of the nutrient classes, together with many other substances. Some nutrients can be stored internally (such as the fat soluble vitamins), while others are required more or less continuously. Poor health may be caused by a lack of required nutrients or, in extreme cases, too much of a required nutrient.

Some nutrients, such as proteins, peptides and amino acids, lipids, carbohydrates, vitamins and minerals, are also sensorially active substances (e.g., sweet or bitter). The main nutrients along with other substances (certain organic acids, such as acetic and citric acids, and sugar alcohols, such as glucitol, known as sorbitol) can simultaneously also be a source of energy, for example, ethanol has a relatively high energy value.

In human nutrition, nutrients are often inaccurately classified in a way that reflects the amount that the body requires. These nutrient classes can be categorised as either macronutrients (required in relatively large amounts) or micronutrients (required in smaller amounts). The macronutrients include proteins, fats, carbohydrates and water; the micronutrients are minerals and vitamins. A third class of dietary material, known as fibre (such as non-digestible polysaccharide cellulose), is also necessary, for both mechanical and biochemical reasons. Other micronutrients include antioxidants and various phytochemicals, which are said to influence or protect some body systems. Their necessity is not well established.

Besides nutrients, foods contain many substances that influence the food sensory impression and its organoleptic properties. These food constituents are known as sensorially active compounds.

They determine the sensory value (quality) of foods, inducing an olfactory sensation (perception), which is described as the aroma, odour and smell, gustative perception, which is the taste, visual perception, which is the colour, haptic (tactile) perception, which is the touch and feel, and auditorial perception, which is the sound. The olfactory sensation is derived from odouractive compounds and the gustative perception from taste-active compounds. Flavour is the sensory impression determined by the chemical senses of both taste and smell and is caused by flavouractive food components. Haptic sensation is the texture, which is affected mainly by high molecular weight compounds, such as proteins and polysaccharides, often referred to collectively as hydrocolloids. Geometric aspects of texture that evoke both haptic and visual sensations symbolise the terms appearance and shape.

Aspects of texture related to mechanical properties of food are called consistency. Auditorial sensations are associated with a range of textural characteristics (such as crispiness).

Food also contains a range of substances with beneficial or, vice versa, with a negative impact on human health. There is now increasing evidence to support the hypothesis that some foods and food components have beneficial physiological and psychological effects over and above the provision of the basic nutrients.

Food that does not have a significant history of consumption or is produced by a method that has not previously been used for food is called novel food. This was first termed by the European Union (EU) in Regulation (EC) No. 258/97 of the European Parliament and of the Council of 27 January 1997 concerning novel foods and novel food ingredients. An example of a novel food is margarine containing phytosterols that help to reduce the blood cholesterol level. Other examples include canola oil produced from rapeseed with low levels of both erucic acid and glucosinolates and exotic fruits and vegetables, which have a long history of safe use. Functional foods are defined as foods (beverages) having disease preventing and/or health promoting benefits in addition to their nutritive or processing value. A food can be regarded as functional if it is satisfactorily demonstrated to affect beneficially one or more target functions in a body, beyond adequate nutritional effects. Functional food can be a natural food, a food to which an existing ingredient has been added, or a food from which a harmful component has been removed by technological or biotechnological processes. The term functional food (officially not used by the EU) was first used in Japan in the 1980s.

(FOSHU, Ministry of Health, Labor and Welfare, Japan. Available at: http://www.mhlw.go.jp/english/topics/foodsafety/fhc/02.html.)

Examples of functional foods with a long history are table salt fortified with iodine or margarine fortified with vitamin D. Another example is yoghurts with live microorganisms beneficial to the host organism (bacteria of the genus Bifidobacterium), which have advantageous effects on health and are called probiotics. Prebiotics are non-digestible food ingredients (usually oligosaccharides and other substances classified as soluble dietary fibre) that stimulate the growth and/or activity of beneficial bacteria in the small intestine.

Symbiotics contain both prebiotics and probiotics. Nutraceuticals are foods and food products, in some countries physiologically active compounds isolated from foods, which reportedly have a physiological benefit or provide protection against chronic diseases. Such products may range from nutrients (essential amino acids, essential fatty acids, vitamins and minerals), to dietary supplements (also known as food supplements or nutritional supplements) and specific diets, to genetically modified foods and other products. Dietary supplements provide nutrients, fibre and various biologically active substances from foods, usually classified according to their effects as antioxidants and anticarcinogens, which may be missing or may not be consumed in sufficient amounts in food. Some countries define dietary supplements as foods, while in others they are defined as drugs or natural health products.

Supplements containing vitamins or dietary minerals are included as a category of food in the ‘Codex Alimentarius’, a collection of internationally recognised standards, codes of practice, guidelines and other recommendations relating to foods, food production and

INTRODUCTION

food safety. Organic food is food that is produced using methods that do not involve modern synthetic inputs, such as synthetic pesticides and chemical fertilisers, and does not contain genetically modified organisms or food additives and is not processed using irradiation.

The food components that impair utilisation of nutrients (proteins, lipids, carbohydrates, vitamins and minerals) by biochemical mechanisms are called antinutritional substances. An example of such an antinutritional substance is oxalic acid, found in spinach and rhubarb, and which binds up calcium to insoluble calcium oxalate and prevents its absorption. Many foods (especially foods of plant origin) contain various natural toxic substances. These may be toxic only to certain individuals causing an allergy, which is related to the immune system response of the organism, or a food intolerance, which occurs when food irritates a person’s digestive system or when a person is unable to properly digest or breakdown the food. The most common food allergies are peanuts, tree nuts (such as walnuts, pecans and almonds), fish and shellfish, milk, eggs, soy products and wheat. Intolerance to lactose in milk and other dairy products is the most common food intolerance.

Substances toxic to all individuals are called toxic substances or toxins. Once a toxic substance has contacted the body it may have either acute (immediate) or chronic (long term) effects. Most of food-born toxins are substances with low acute toxicity (such as the pungent alkaloid piperine in black pepper), although some may present chronic effects, such as hepatotoxic pyrrolizidine alkaloids in plants (such as comfrey and coltsfoot species) or cause pathological changes to the respiratory system (e.g., tobacco smoke).

Many chemical compounds in foods (as well as in feeds for livestock) are added intentionally as food additives. The purpose is to protect foods against spoilage, oxidation and increase some aspects of the food quality (e.g., nutritional value, aroma, taste, colour or texture). Preservatives, colours and flavours are the bestknown additives, but there are in fact many categories of additives, each tailored to a specific purpose.

Contaminants are harmful chemical substances (chemical contamination) and toxic microorganisms (microbiological contamination) that have not been intentionally added to food but may be present there as a result of human activities in agriculture or industry. They may enter food at various stages of its production, packaging, transport or storage. A separate issue is genetically modified food or the presence in food of ingredients from genetically modified organisms, which is sometimes referred to as a form of food contamination. In relation to food, contaminants are sometimes differentiated into primary or exogenous contaminants that come from the external environment (e.g., residues of agrochemicals such as pesticides) and secondary or endogenous contaminants, which are, under certain circumstances, generated during food processing (e.g., by heating and fermentation) from

3

natural food components (nutrients and other constituents). These contaminants are often called technological, processing or processinduced contaminants. They are absent in the raw materials, and are formed by chemical reactions between natural and/or added food constituents during processing. Typical examples are acrylamide formed in potato chips from asparagine, methanol produced in fruit alcoholic beverages by hydrolysis of pectin and chloropropanols formed from fats and hydrochloric acid in hydrolysed vegetable proteins.

Food contaminants and some additives used inappropriately, and sometimes even natural toxic substances, are collectively termed xenobiotics. The term is primarily understood to describe artificial substances that do not exist in nature and also covers substances that are present in much higher concentrations than are usual. Their presence in foods is related to the hygienic–toxicological quality.

As food chemistry is a dynamic discipline, it also deals with the biochemical and chemical reactions, interactions and physical processes that occur during food processing and storage. Knowledge of these processes is a prerequisite for their eventual regulation and also allows the optimisation of production processes, so that it is possible to produce high value foods in all aspects of quality, foods with high nutritional, sensory and hygienic–toxicological value (healthy foods), while satisfying all the requirements and demands of the consumers. In addition, knowledge of these processes can be applied in ordinary culinary experiences. Finally, food chemistry examines the ways in which to enhance positive changes and to prevent the unwanted ones, eliminate the antinutritive and toxic substances, and prevent possible contamination with substances that can pose health risks to guarantee food safety. Producing safe and nutritious food for human and animal consumption is one of the principal aims of both food chemistry and food technology.

This book takes you on a tour of food chemistry, an incredibly fascinating field of study that will provide you with a constant sense of discovery. For better or for worse, food chemistry is all around us every day and everything in food is chemistry, as chemistry is essential to meet our basic needs of food, energy, health, water and air. Food plays a role in everyone’s lives and touches almost every aspect of our existence in some way. Therefore, the magic of food composition and its changes has attracted inquisitive minds for centuries. The understanding of the nature of chemicals and pivotal chemical processes occurring in foods, which the reader will gain, has been divided into 11 chapters, each of which provides insights into different classes of food constituents and related reactions, including proteins, peptides and amino acids, fats and other lipids, saccharides and their derivatives, vitamins, mineral compounds, water, odour- and taste-active substances, colours, beneficial, antinutritive and toxic components, food additives and contaminants and covering all aspects of the subject that are necessary for a comprehensive understanding about how things work. Let us wade into this information soup.

2 Amino Acids, Peptides and Proteins

2.1 Introduction Aminocarboxylic acids (see p. 5) occurring in nature have vital functions in living organisms. Many of these amino acids are found as free substances or higher molecular weight compounds, where the amino acid building units are connected to each other by amide bonds, –CO–NH–, termed peptide bonds. Depending on the size of the molecule (the number of bound amino acids), these compounds are divided into two large main groups, which are:

• peptides, usually composed of 2–100 monomers • proteins, which contain more than 100 monomers, but also hundreds or even thousands of amino acids.

Proteins and peptides may even contain some further compounds in addition to amino acids. Proteins are undoubtedly the most important amino acid derivatives: they are the basic chemical components of all living cells and therefore are also part of almost all food raw materials and foods of plant, animal and microbial origin. In organisms, proteins perform a number of unique and extraordinary functions. Along with ribonucleic acids (RNA) and deoxyribonucleic acids (DNA), polysaccharides, some lipids and other macromolecules (such as polyisoprene, the major component of rubber tree latex), peptides and proteins are often known as biological polymers or biopolymers. Nucleic acids (RNA and DNA) have almost no significance as food components in human and animal nutrition, although they play fundamental roles in living systems. Plants and some microorganisms are capable of synthesising proteins from basic substrates such as carbon dioxide, water and inorganic nitrogen compounds, but animals rely on getting their necessary vegetable or animal protein from their food. In the process of digestion, the food proteins are enzymatically broken down (hydrolysed) to their building blocks, from which animals may synthesise their own proteins

or use them (along with carbohydrates and lipids) as a source of energy. Therefore, proteins, together with carbohydrates and lipids, belong to the category of main (primary) nutrients. In human and animal nutrition, proteins are irreplaceable because they cannot be substituted by other nutrients over a long period of time.

The following sections deal with important amino acids, peptides and proteins, their structure, occurrence, properties, fate in the human organism, nutritional aspects and important interactions and reactions that fundamentally affect the nutritional value, organoleptic properties (odour, taste, colour and texture) and the hygienic–toxicological quality of food commodities.

2.2 Amino acids Amino acids are found in foods – they are the building units of proteins, peptides and many other compounds – but they also occur as free compounds. Plants, animals and other organisms have been shown to contain more than 700 different amino acids.

Some of these are spread quite generally throughout nature, while others occur only in certain species of plants, animals or in other organisms. According to their origins, therefore, the following two groups of amino acids are recognised:

• amino acids found in all living organisms (bound in proteins, peptides or occurring as free amino acids)

• amino acids found only in some organisms (bound in peptides

or present as free compounds) that are not protein constituents.

The amino acids bound in proteins (22 compounds) are called proteinogenic, encoded, basic, standard or primary amino acids; 21 of them are constituents of proteins, food raw materials and foods. Of the 22 proteinogenic amino acids, 20 are encoded by the

The Chemistry of Food, First Edition. Jan Velı́šek. © 2014 John Wiley & Sons, Ltd. Published 2014 by John Wiley & Sons, Ltd. Companion Website: www.wiley.com/go/velisek/foodchemistry.

5

2.2 AMINO ACIDS

universal genetic code. The remaining two amino acids (selenocysteine and pyrrolysine) are incorporated into proteins by unique synthetic mechanisms. For each of the encoded amino acids, specific transfer RNA molecules exist, and proteins of each organism arise as products of protein synthesis controlled by the genetic code. Amino acids bound in peptides or free amino acids have the same nutritional value as proteins, but their importance in nutrition is usually negligible due to the small amounts in which free amino acids and peptides commonly occur in foods.

An example of an amino acid occurring only in some organisms is pyrrolysine, which occurs as a component of enzymes involved in the production of methane in some methanogens, members of a group of single-celled microorganisms of a relatively new domain, Archaea, in the classification of life.

The process of protein biosynthesis is called translation. Posttranslational oxidation, alkylation and esterification of some amino acids that are bound in proteins yield modified proteinogenic amino acids (see Section 2.2.1.1.2). Non-proteinogenic (non-encoded, non-standard or secondary) amino acids do not function as building blocks of proteins, as they have other roles in organisms. Amino acids also have an influence on the organoleptic properties of food, especially on their taste. Products of reactions of amino acids are often important compounds influencing odour, taste and colour of foods.

2.2.1 Structure, terminology, classification and occurrence Amino acids are organic compounds, containing at least one primary amino group, –NH2 , together with at least one carboxyl group, –COOH, and various side groups in the molecule. They can be also defined as amino group substituted carboxylic acids.

According to the distance of the amino group from the carboxylic group, amino acids (2-1) are generally divided into:

• 2-aminocarboxylic acids (α-aminocarboxylic acids) that have

the amino and carboxyl groups attached to the same carbon atom (such as α-alanine);

• 3-aminocarboxylic acids (β-aminocarboxylic acids) that have the amino and carboxylic groups attached to adjacent carbon atoms (such as β-alanine);

• 4-aminocarboxylic acids (γ-aminocarboxylic acids) that have

one carbon atom (group) between the amino and carboxylic groups (such as γ-aminobutyric acid);

• 5-aminocarboxylic acids (δ-aminocarboxylic acids) that have

two carbon atoms (groups) between the amino and carboxylic groups (such as 5-aminolaevulinic acid, the precursor of the biosynthetic pathway that leads to haem in mammals and chlorophyll in plants);

• 6-aminocarboxylic acids (ε-aminocarboxylic acids) that have three carbon atoms (groups) between the amino and carboxylic groups (such as lysine).

NH2 R

COOH n

2-1, 2-amino acid (n = 0) 3-amino acid (n = 1) 4-amino acid (n = 2) 5-amino acid (n = 3) 6-amino acid (n = 4)

The amino acids also include carboxylic acids that contain a secondary amino group –NH– in the molecule that is a part of three-, four-, five- or six-membered rings. These amino acids are actually derivatives of the saturated nitrogen heterocycles aziridine, azetidine, azolidine (pyrrolidine) and azinane (piperidine) or of other more complex heterocyclic compounds. For example, the only proteinogenic amino acid with a pyrrolidine ring is proline, which in biochemical literature is sometimes incorrectly classified as an imino acid, but imino acids contain imino –C(=NH)– functional groups instead of secondary amino groups.

In most foods, about 99% of amino acids are bound in proteins and peptides. The rest (about 1%) are free amino acids. There is a larger amount of free amino acids in foods in which proteolytic enzymes or chemical agents have hydrolysed proteins during manufacture or storage. Larger amounts of free amino acids can be found in some cheeses, beer and wine. The enzymatic hydrolysates of proteins (such as soy sauce) or acidic protein hydrolysates (used as soup condiments) contain only free amino acids with small amounts of peptides, but no protein.

2.2.1.1 Amino acids of proteins 2.2.1.1.1 Proteinogenic amino acids Proteinogenic amino acids bound in all proteins are exclusively 2-amino (α-amino) acids that have the primary or secondary amino and carboxyl groups attached to the same carbon atom in position 2 (α) to the carboxyl group. In other words, the amino groups are attached to the carbon adjacent to the carboxyl groups.

The carbon atom in position 2 (α) is commonly referred to as Cα carbon in biochemical literature.

The proteins in most organisms contain only 21 basic amino acids, of which 20 amino acids have the primary amino group (2-2), while one amino acid is an alicyclic amino acid with a secondary amino group (2-3). All amino acids except glycine (which is achiral) are optically active (chiral) compounds that contain an asymmetric centre (chiral atom) and thus can occur in two non-superimposable mirror-image forms, d- and l-forms (optical isomers or enantiomers) that are the mirror images of each other. Proteinogenous amino acids almost exclusively belong to the l-series, thus have the l-configuration (see Section 2.2.3.2).

R

COOH NH2

COO−

R +

NH3

2-2, aliphatic α-amino acid

6

CH 02 AMINO ACIDS, PEPTIDES AND PROTEINS

n

N H

n

COOH

+

H

N

COO− H

2-3, alicyclic α-amino acid

One or more hydrogen atoms of the substituent R of l-α-amino acids, known as a side chain, specific to each amino acid (2-2), may be substituted by a carboxyl group (called a distal carboxyl group), an amino group (or distal amino group) or other functional groups, such as a hydroxyl group –OH, a sulfhydryl (mercapto) group –SH, a sulfide group –S–CH3 , a guanidino group –NH–C(=NH)–NH2 or a phenyl group –C6 H5 . The methylene group or nitrogen atom of the secondary amino group of α-amino acids can also be substituted (2-3).

The proteinogenic amino acids are often known by their trivial names, which are derived from their properties or the source from which they were first isolated. Systematic names of the

proteinogenic amino acids are rarely used, but are more frequent in the non-protein amino acids. Each amino acid has both a threeletter code (mostly the first three letters of the trivial name) and a single-letter code that is used for the registration of long sequences of amino acids in proteins (Table 2.1).

Each classification of amino acids is somewhat imprecise and conforms to certain purposes. The most common way of sorting the proteinogenic amino acids in food chemistry is classification according to the structure of their side chain and functional groups present therein. This will distinguish the following groups of amino acids:

• aliphatic amino acids (monoaminomonocarboxylic acids) with

an unsubstituted side chain, which include the simplest amino acid glycine (sometimes also classified as the only amino acid without a side chain), its higher homologue alanine (known as α-alanine) and branched chain amino acids valine, leucine and isoleucine;

Table 2.1 Trivial and systematic names of proteinogenic amino acids and their codes.

Trivial name

Systematic name

Three-letter code

One-letter code

Glycine

Aminoacetic acid

Gly

G

L-Alanine

L-2-Aminopropionic acid

Ala

A

L-Valine

L-2-Amino-3-methylbutanoic acid

Val

V

L-Leucinea

L-2-Amino-4-methylpentanoic acid

Leu

L

L-Isoleucinea

L-2-Amino-3-methylpentanoic acid

Ile

I

L-Serine

L-2-Amino-3-hydroxypropanoic acid

Ser

S

L-Threonine

L-2-Amino-3-hydroxybutanoic acid

Thr

T

L-Cysteine

L-2-Amino-3-mercaptopropanoic acid

Cys

C

L-Selenocysteine

L-2-Amino-3-selanylpropanoic acid

Sec

U

L-Methionine

L-2-Amino-4-methylthiobutanoic acid

Met

M

L-Aspartic acida

L-Aminosuccinic acid

Asp

D

L-Glutamic acida

L-2-Aminoglutaric acid

Glu

E

L-Asparaginea

L-2-Amino-4-carbamoylbutanoic acid

Asn

N

L-Glutaminea

L-2-Amino-5-carbamoylpentanoic acid

Gln

Q

L-Lysine

L-2,6-Aiaminohexanoic acid

Lys

K

L-Pyrrolysine

(2R,3R)-N6-[3-Methyl-3,4-dihydro-2H-pyrrole-2-ylcarbonyl]-L-lysine

Pyl

O

L-Arginine

L-2-Amino-5-guanidylpentanoic acid

Arg

R

L-Histidine

L-2-Amino-3-(4-imidazolyl)propionic acid

His

H

L-Phenylalanine

L-2-Amino-3-phenylpropionic acid

Phe

F

L-Tyrosine

L-2-Amino-3-(4-hydroxyphenyl)propionic acid

Tyr

Y

L-Tryptophan

L-2-Amino-3-(3-indolyl)propionic acid

Trp

W

L-Proline

L-Pyrrolidine-2-carboxylic acid

Pro

P

a In addition to the specific amino acid codes, three-letter code and one letter-code placeholders are used for ambiguous amino acids, i.e. Asx and B for aspartic acid or asparagine, Glx and Z for glutamic acid and glutamine, Xle and J for leucine or isoleucine and Xaa (Unk) and X for unspecified (unknown) amino acid.

2.2 AMINO ACIDS

• aliphatic hydroxyamino acids, which include serine and threonine;

• aliphatic sulfur-containing amino acids cysteine and

7

• hydrophilic amino acids with polar side chains, which include

serine, threonine, cysteine, selenocysteine, aspartic and glutamic acids and their amides asparagine and glutamine, as well as lysine, pyrrolysine, arginine and histidine.

methionine;

• • amino acids with carboxyl groups in the side chain (monoaminselenoanalogue of cysteine called selenocysteine;

odicarboxylic acids), such as aspartic acid and glutamic acid;

Hydrophilic amino acids are classified according to the ionic form in which they occur in living organisms into:

• neutral (polar side chain has no electric charge in neutral solutions), which includes most amino acids;

• their monoamides (amino acids with carboxamide groups in the

• acidic (polar side chain has a negative charge in neutral solu-

• amino acids with basic functional groups in the side chain, that is,

• basic (polar side chain has a positive charge in neutral solutions),

side chains): asparagine and glutamine;

tions), which includes aspartic acid and glutamic acid;

diaminomonocarboxylic acid lysine, lysine derivative with a 1pyrroline ring known as pyrrolysine, arginine with a guanidino group in the side chain and the derivative of 1H-isomer of imidazole called histidine;

According to the significance in human nutrition, proteinogenic amino acids are divided into:

• amino acids with aromatic and heterocyclic side chains, which

• essential (or indispensible: valine, leucine, isoleucine, threonine,

• proline is the amino acid, in which the functional group is

• semi-essential (arginine and histidine); • non-essential (other amino acids; tyrosine forms by hydrox-

include phenylalanine, its hydroxyl derivative tyrosine and tryptophan with an indole ring;

involved in the ring structure.

For clarity, formulae of amino acids are shown in the nonionised form. Non-ionised molecules are, however, virtually absent in aqueous solutions and in animal and plant tissues. Amino acids are ionised and form inner salts (2-2 and 2-3), and carboxyl groups exist as –COO– anions and amino groups as –NH3 + cations. The amino acid molecule simultaneously carries positive and negative charges. Because the resulting amino acid contains one positive and one negative charge, it is a neutral molecule called a zwitterion.

These forms are the predominant ionic forms of amino acids under neutral conditions (about pH 7 in solution). Under these conditions, the distal carboxyl groups of aspartic and glutamic acids, distal amino group of lysine, guanidine group of arginine and imidazole cycle of histidine are also more or less ionised. The nitrogen atoms of the imidazole ring of histidine are denoted by pros (‘near’, abbreviated π) and tele (‘far’, abbreviated τ ) to show their position relative to the side chain.

In biochemistry, the most common and perhaps most practical classification of proteinogenic amino acids is according to the side chain polarity and its ionic forms occurring in neutral solutions, which is related to non-bonding interactions in proteins (see Section 7.6.2.2). The following groups of amino acids are recognised:

• hydrophobic amino acids with non-polar side chains, which include valine, leucine, isoleucine, methionine, phenylalanine, tyrosine and proline; sometimes the hydrophobic amino acids also include glycine, alanine and tryptophan, even though these amino acids are rather amphiphilic amino acids, which forms a transition between the hydrophobic amino acids and the following group;

such as lysine, pyrrolysine, arginine and histidine.

methionine, lysine, phenylalanine and tryptophan) that cannot be synthesised by the human body;

ylation of the essential amino acid phenylalanine) that are synthesised by the human body de novo.

Amino acids that the body cannot synthesise must be obtained entirely from food. These amino acids are called essential amino acids. Essential amino acids are routine constituents of most protein-based foods or dietary proteins and are fairly readily available in a reasonably well-balanced diet. However, there are some amino acids that are present in lower concentrations than the same amino acid in a high quality protein (e.g. lysine in wheat and eggs).

These amino acids are called limiting amino acids, because if a person’s diet is deficient in one of them, this amino acid will limit the usefulness of the others (and will limit the extent of protein synthesis in the body), even if the others are present in what would otherwise be large enough quantities. Limiting amino acids are distinct from non-essential amino acids that the body can synthesise and are therefore sometimes used for food and animal feed enrichment.

In rapidly growing organisms (such as infants, for instance), some non-essential amino acids (arginine and histidine), which the young organism cannot synthesise in sufficient quantities, become essential amino acids. These amino acids are sometimes called semi-essential amino acids. Arginine is synthesised at a rate that is insufficient to meet the growth needs of the body, and the majority of it that is synthesised is cleaved to form urea. Histidine was initially thought to only be essential for infants, but longer-term studies established that it is also essential for adult humans. The amino acids methionine and phenylalanine are also sometimes considered semi-essential for reasons not directly related to lack

8

CH 02 AMINO ACIDS, PEPTIDES AND PROTEINS

of synthesis. Methionine is required in large amounts to produce cysteine if the latter amino acid is not adequately supplied in the diet. Similarly, phenyalanine is needed in large amounts to form tyrosine if the latter is not adequately supplied in the diet.

The amino acids that the body can synthesise are called nonessential amino acids. They are biosynthesised from intermediates of the citric acid cycle used by all aerobic organisms to generate energy and through other metabolic pathways (glycolysis and the pentose cycle). All living organisms, including humans, synthesise glutamic acid from 2-oxoglutaric acid (derived from the citric acid cycle) and ammonium ions. This reaction is known as reductive amination. Glutamic acid then becomes the precursor of glutamine, proline, ornithine and arginine. Transamination of oxaloacetic acid (from the citric acid cycle) yields aspartic acid that is a precursor of asparagine. Transamination of pyruvic acid (a product of glycolysis) yields alanine. Another intermediate of glycolysis, 3-phospho-dglyceric acid (and d-ribulose 1,5-bisphosphate in plants), yields serine, which is a precursor of glycine, cysteine and selenocysteine. d-Ribose 5-phosphate from the pentose cycle (the product of photosynthesis in plants) is a precursor of histidine.

Essential and semi-essential amino acids

• Valine Valine occurs in animal and plant proteins (meat, cereals) in amounts of 5–7% (average content is 6.9%). Egg and milk proteins contain 7–8% valine. The structural protein elastin has the highest amount of valine (16%).

• Leucine Leucine occurs in all common proteins, usually in

amounts of 7–10% (average content is 7.5%). Cereals contain a variable amount of leucine, wheat proteins contain about 7% and maize proteins about 13%. Free leucine forms in larger amounts during cheese ripening due to bacterial activity.

• Isoleucine The largest amounts of isoleucine are found in milk and egg proteins (6–7%), while meat and grains contain 4–5% of leucine (average content is 4.6%).

• Threonine A rich source of threonine is meat and brewer’s yeast.

Its content in animal proteins (meat, eggs and milk) is around 5%. There is a relatively high amount of threonine in wheat, but its content in other cereals is lower (often around 3%), meaning that threonine sometimes becomes the limiting amino acid.

• Methionine Animal proteins contain 2–4% of methionine, whereas plant proteins contain only 1–2% (average content is 1.7%). Methionine is the limiting amino acid in legumes.

Methionine (and cysteine) is present only in small amounts in histones and is completely lacking in protamines.

•

Lysine The average content of lysine in proteins is 7%. High amounts of lysine are found in most animal proteins; meat, eggs and milk proteins commonly contain 7–9% of lysine, fish and shellfish proteins contain 10–11% of lysine. In contrast, vegetable proteins, such as proteins of cereals (especially gliadins, but not

glutelins) and cereal products contain only 2–4% of lysine, which is the limiting amino acid.

• Phenylalanine Good sources of phenylalanine are meat, fish and

most protein containing foods, where this amino acid occurs in sufficient amounts (4–5%, average content is 3.5%). In some individuals, its presence in the diet causes phenylketonuria, an inherited defect in which phenylalanine is incompletely and abnormally metabolised (see Section 10.3.1.2).

• Tryptophan The average tryptophan content in proteins is 1.1%.

Animal proteins contain 1–2% of tryptophan, except for histones and collagen, which do not contain tryptophan at all. Tryptophan is also not present in gelatine or in acid protein hydrolysates used as soup seasonings. Therefore, the tryptophan content in meat products can serve as an indicator of the quality of the meat used. (The contents of 3-methylhistidine or creatinine have been measured for the same purpose.) Cereals contain <1% of tryptophan; the glutenine fraction of gluten has a somewhat higher content of tryptophan. In fruits and fruit juices N1 (β-d-glucopyranosyl)-l-tryptophan is also found. Its amount in juices ranges from 0.1 to >10 mg/l. The highest amount of this glycoconjugate was found in pear juices (13.5 mg/l).

Humans can partly use tryptophan for biosynthesis of nicotinic acid (see Section 5.8.3). In ruminants, if present in excessive amounts in the diet, tryptophan causes pulmonary oedema (fluid accumulation in the lungs) that may cause respiratory failure. The cause is the heterocyclic compound 3-methylindole (skatole), which is a fermentation product of tryptophan in the rumen.

• Arginine Arginine occurs in all proteins in amounts of 3–6%

(average content is 4.7%). Basic proteins protamines from fish roes have particularly high levels of arginine. Peanuts and other oilseeds are also rich sources (arginine content of up to 11%).

• Histidine Common proteins contain 2–3% histidine (average

content is 2.1%), and blood plasma proteins contain up to 6% histidine. The flesh of some fish (especially mackerel and tuna) contains from 0.6 to 1.3% (and sometimes more than 2%) of free histidine. The free histidine content in the flesh of other fish is only 0.005–0.05%.

Non-essential amino acids

• Glycine Glycine is contained in a significant amount (25–30%) mainly in the structural protein collagen and in gelatine; in the majority of albumins it is not present at all (average content is 7.5%).

• Alanine Alanine occurs in almost all proteins in amounts of

2–12% (average content is 9.0%). Maize prolamine protein zein and animal protein gelatine contain about 9% of alanine.

• Serine Serine is found in many proteins; generally in an amount of 4–8% (average content is 7.1%).

9

2.2 AMINO ACIDS

H3C

COOH

HOOC

NH2

NH2

alanine

glutamic acid

NH H2N

proline

arginine

H3C

NH

asparagine

NH2 pyrrolysine

NH2

selenocysteine

CH3 H3C

isoleucine

COOH NH2

NH2

serine

H3C

threonine

COOH

COOH CH3

cysteine

NH2

N H

leucine

COOH

COOH

H3C

NH2

NH2

aspartic acid

OH

COOH

HO

COOH

NH2

COOH NH2

histidine

COOH

HS

O

HSe

NH2

HOOC

COOH

COOH N

O

H N

glycine

COOH

H2N

N

NH2

NH2

NH

NH

COOH

COOH

N H

COOH

COOH

COOH

H2N

NH2 tryptophan

COOH

COOH

NH2

NH2

phenylalanine

lysine

NH2

HO

tyrosine

CH3

O COOH

H2N

H3C

S

NH2 glutamine

COOH

COOH

H3C

NH2

NH2

methionine

valine

2-4, basic amino acids

• Cysteine The highest amount of this amino acid and its oxidation

product cystine (2-5) is present in keratin (up to 17%); it occurs in many other proteins in smaller amounts (1–2%). The average content is 2.8%. In the organism, cysteine can partially replace the essential amino acid methionine.

• Aspartic acid and asparagine The average content of aspartic acid in proteins is 5.5%; the average content of asparagine is 4.4%. Aspartic acid is the major amino acid of animal proteins known as globulins and albumins (6–10%). Vegetable proteins contain 3–13% aspartic acid, mainly in the form of asparagine (e.g. wheat proteins contain about 4% and maize proteins about 12%).

• Glutamic acid and glutamine The average content of glutamic

acid and glutamine in proteins is 6.2 and 3.9%, respectively.

Glutamic acid is the most abundant amino acid in the nervous tissue. In conventional proteins, both amino acids are usually found in larger quantities (especially in globulins) in cereal and legume proteins (18–40%). Wheat gluten (in its component gliadin) contains about 40%, soy protein contains about 18% and milk proteins contain about 22% of glutamic acid.

• Selenocysteine In most foods of both vegetable and animal

origin, selenocysteine is the main form of selenium bound in proteins. The content of this amino acid, as well as the contents of other amino acids and peptides containing selenium (l-selenocystine, Se-methyl-l-selenocysteine, l,lselenocystathionine, l-selenomethionine and γ-glutamylSe-methyl-l-selenocysteine), is unknown. Selenocysteine is typically located in a small number of active centres of proteins of Archaea, bacteria and eukaryotes (in glutathione

10

CH 02 AMINO ACIDS, PEPTIDES AND PROTEINS

peroxidase, thioredoxin reductase, formiate dehydrogenase, glycine reductase and in some hydrogenases). Usually only one molecule of selenocysteine is bound in the peptide chain; however proteins containing multiple molecules of this amino acid are also known. For example, the isoenzyme of mammalian glutathione peroxidase, known under the abbreviation GPx6, is a selenoprotein in humans with cysteine-containing homologues in rodents.

therefore correlates with the lower quality of raw material used, for instance where the products contain skin, wherein collagen is the major protein. 4-Hydroxyproline also occurs in smaller amounts in plants. For example, concentrations ranging from 1.0 to 4.2 mg/kg have been found in the edible part of bergamot fruits (Citrus bergamia, Rutaceae).

HO

• Tyrosine Tyrosine accompanies phenylalanine in most proteins

N H

in an amount of 2–6% (average content is 3.5%). Gelatine contains only traces of tyrosine.

• Proline Proline is present in most proteins in amounts of 4–7%;

the average content is 4.6%. Its content in the component of wheat gluten, gliadin, is about 10%, and about 12% of proline contains casein. Proline content in gelatine can be up to 13%. In bacteria, plants and animals it also plays a role as an osmoprotectant that helps stabilise proteins and cell membranes from the damaging effect of high osmotic pressure.

2.2.1.1.2 Modified proteinogenic amino acids Post-translational modification, which extends the range of functions of the protein, is one of the later steps in the biosynthesis of many proteins. Post-translational modification of proteins occurs by methylation, hydroxylation, acetylation and phosphorylation of the protein functional groups, by attaching various lipids and carbohydrates to the protein molecule and by making structural changes such as the formation of disulfide bonds from cysteine residues by oxidation.

Post-translational oxidation of the thiol groups of cysteine residues in proteins yields l-cystine (Cys-Cys, 2-5). The disulfide bond (or bridge) plays an important role in the structure of many proteins, as it combines two different polypeptide chains or two molecules of cysteine in the same peptide chain. For example, the major globular protein of milk β-lactoglobulin contains two disulfide bridges, and the glycoprotein of egg white ovomucoid has three domains connected by disulfide bridges. The protein keratin has a high cystine content: for example, human hairs contain about 5% cystine.

HOOC

2-6, 4-hydroxyproline

4-Hydroxyproline in collagen is accompanied by a small amount of its isomer, l-3-hydroxyproline (l-3-hydroxypyrrolidin2-carboxylic acid) and by a hydroxy derivative of lysine, also known as l-5-hydroxylysine, (2S,5R)-5-hydroxylysine, l-5hydroxy-2,6-diaminohexanoic acid (abbreviated Hyl, 2-7). In glycoproteins, hydroxylysine is bound as O-glycoside. Small amounts of hydroxylysine occur in plant materials (as a free amino acid in alfalfa forage, Medicago sativa, Fabaceae).

OH H2N

COOH NH2

2-7, 5-hydroxylysine

The minor amino acid typically present in the meat myofibrillar protein actin (also in some myosin isoforms and in dipeptide anserine, see Section 2.3.3.1.3) is l-3-methylhistidine (2-8), which is formed by methylation of histidine bound at the 73rd position of the protein chain. The functional significance of this modification of actin is not known; it is probably related to the metabolism of phosphates, with which the side chain of methylhistidine interacts.

Methylhistidine does not occur in other protein-rich foods such as milk, eggs and soybeans. Its contents might therefore serve as a criterion for determining the quality ingredients in meat products.

COOH

N

NH2 S

S

COOH NH2

2-5, cystine

Another common post-translationally modified amino acid is the proline derivative l-4-hydroxyproline, (2S,4R)-4-hydroxyproline, also known as l-4-hydroxypyrrolidine-2-carboxylic acid (abbreviated Hyp, 2-6), which is an important structural component of collagen, gelatine (about 12% of content) and the polypeptide (glycopeptide) of plant cell walls known by the trivial name extensin (see Section 4.5.1.3.1). Its content is low in most other proteins. The amount of 4-hydroxyproline in meat products

COOH

N

NH2

CH3 2-8, 3-methylhistidine

The other amino acid that is usually modified is l-serine. It can be esterified with phosphoric acid. This ester, O-phosphoserine (2-9), occurs in many proteins, such as glycophosphoprotein phosvitin (also known as phosphovitin) from egg yolk (see Section 2.4.5.3.2).

Phosvitins are one of the most phosphorylated (10%) proteins in nature, and are important for sequestering cations of calcium, iron and other metals for the developing embryo. Serine in phosvitin represents nearly 50% of the amino acids and there

11

2.2 AMINO ACIDS

are about 90% of the molecules of serine phosphorylated. Phosphoserine also occurs in the glycoprotein αS1 -casein (eight phosphoserine residues) and β-casein (five phosphoserine residues).

Phosphoserine (as phospatidyl-l-serine) is also a component of phospholipids distributed widely among animals, plants and microorganisms. Phosphoserine usually constitutes less than 10% of the total phospholipids, the greatest concentration being in myelin from brain tissue and in wheat germ. Threonine residues in proteins are also esterified fairly frequently with phosphoric acid yielding O-phosphothreonine. Phosphatidyl-l-threonine was first detected in animal brains and tuna muscle, before it was characterised definitively as a minor component of neurons, macrophages, viruses and some bacterial species.

O HO

OH P

COOH

O

NH2

in living organisms and not by function (these functions often are not well known).

2.2.1.2.1 Neutral aliphatic and alicyclic amino acids 3-Amino acids and 4-amino acids In addition to α-amino acids, β- and γ-amino acids can also be found in food. Naturally occurring β-alanine (3-aminopropionic acid, 2-10) acts in the biosynthesis of pantothenic acid, acetyl coenzyme A, other acyl coenzymes A (see Section 5.9.1) and some histidine dipeptides. β-Alanine is synthesised in several different ways, by aspartic acid decarboxylation (in bacteria), but also by the transformation of propionic acid (in bacteria and some plants), by degradation of the polyamines spermine and spermidine (in yeasts and many plants, such as tomato, soybean and maize), or arising from the pyrimidine derivative uracil that occurs in all animals and in some plants, such as wheat.

2-9, O-phosphoserine

COOH H2N

2.2.1.2 Other amino acids Plants and microorganisms are able to generate all 21 amino acids necessary for protein synthesis, and they can additionally synthesise many more. Foods contain numerous other less common amino acids in addition to those that are constituents of proteins (proteinogenic) and modified proteinogenic amino acids.

It is estimated that there are around 700 amino acids known in nature, of which at least 300 are found in plants. They are often bound in peptides (see Section 2.3) or are present as free amino acids. In biochemistry, these non-protein amino acids are often classified as the so-called secondary metabolites, as they are the products of three major routes: modification of an existing (often proteinogenic) amino acid, modification of an existing pathway and by novel pathways. These amino acids become the biosynthetic precursors of many biologically active nitrogenous compounds, such as signalling molecules, vitamins, alkaloids (such as valine, leucine, isoleucine, threonine, arginine, lysine, phenylalanine and tryptophan), bile acids and pigments. Some of these amino acids also have specific functions in organisms; they act as neurotransmitters, stimulants and hormones. For example, phenylalanine (or 3,4-dihydroxyphenylalanine) is a precursor of adrenal hormones catecholamines (such as epinephrine, which is also known as adrenaline; see Section 10.3.2.10.1) and of the thyroid hormone thyroxine (see Section 2.2.1.2.5). Other amino acids increase plant tolerance to abiotic stress factors, act as toxic substances which protect plants against invading viruses, microorganisms, other plants and predators (e.g. the arginine analogue canavanine found in some legumes) and also serve as a storage and transport form of nitrogen and sulfur. Some of the unusual amino acids form secondarily during storage of raw materials and food processing via the activities of microorganisms and by chemical transformation of proteins (e.g. lysinoalanine, see 2-121), peptides or free amino acids.

The following sections of this chapter present some of the most important non-protein amino acids in foods and feeds. For clarity, they are sorted according to the structure in which they are present

2-10, β-alanine

The higher homologue of β-alanine is γ-aminobutyric acid (4-aminobutanoic acid), known under the acronym of GABA (2-11), which is formed almost exclusively by enzymatic decarboxylation of glutamic acid. It is mainly present in the brain tissue of animals, where it acts as an inhibitor of nerve impulse transmissions. It is also found in plants and microbial cells, where it has a role as a signal molecule, and is also a constituent of some peptides such as nisine (see Section 11.2.1.2.1).

H2N

COOH

2-11, γ-aminobutyric acid

N-Substituted amino acids N-Alkylsubstituted amino acids are commonly found in foods.

The simplest of these amino acids is N-methylglycine (sarcosine, 2-12), which is a product of the catabolism of choline (see Section 3.5.1.1.1), creatine (see Section 2.2.1.2.4) or forms by Nmethylation of glycine. Sarcosine probably has a regulatory function in methylation reactions. The subsequent N-methylation of sarcosine gives N,N-dimethylglycine (2-13), which is a component of pangamic acid ranked first among the vitamins (see Section 5.15).

N,N,N-Trimethylglycine (N,N,N-trimethylammonioacetate, 2-14) was named glycinebetaine or just betaine after its discovery in sugar beet (Beta vulgaris, Amaranthaceae), where it occurs in the amount of about 2.5 g/kg in the root. Glycinebetaine is a very effective osmolyte of microorganisms and higher plants, which results from choline or by N-methylation of glycine and sarcosine, respectively, and acts in various transmethylation reactions. It is present in higher amounts in sugar beets and molasses. Glycinebetaine was found to accumulate at high levels in some higher fungi. For example, its content in horse mushroom (Agaricus arvensis) is 7.8% dry matter.

12

CH 02 AMINO ACIDS, PEPTIDES AND PROTEINS

H3C

H N

CH3 COOH

H3C

2-12, N-methylglycine (sarcosine)

H3C H3C

N

R COOH

N

R

+

R P R

R R ammonium ion phosphonium ion

2-13, N,N-dimethylglycine

R +

R As R R arsonium ion

+ R

R S

R sulfonium ion

2-15, general structures of some onium ions

CH3 +

+

R N R

COO

−

2-14, N,N,N-trimethylglycine

The terms betaines include glycinebetaine and similar zwitterionic ammonium compounds derived from other amino acids.

By extension, betaines are also neutral molecules having chargeseparated forms with an onium atom which bears no hydrogen atoms (such as phosphonium, sulfonium and arsonium ions) that is not adjacent to the anionic atom. Examples of the most common onium ions (ammonium, phosphonium, sulfonium and arsonium ions) are given by the general formulae 2-15. The attached atoms are typically organic substituents, for example methyl groups. The most common betaines are quaternary ammonium compounds that occur in foods of animal and plant origin. In animals, betaines participate (along with S-adenosyl-l-methionine, pyridoxine, folic acid and vitamin B12 ) in the protection of the cardiovascular system by lowering the potentially toxic concentrations of l-homocysteine. l-Carnitine plays an important role in the metabolism of fatty acids in biological systems plays (see Section 5.15). An arsenic analogue of betaine, arsenobetaine, accumulates in the body of some fish (see Section 6.2.3.1). Betaines are particularly ubiquitous in plants.

They are generally referred to as osmolytes as they, like their amino acid precursors, tend to accumulate in the cytoplasm and intercellular fluids, where they exert protective functions for proteins, nucleic acids and cell membranes in response to abiotic stresses, such as cold, freezing, high temperature, the presence of toxic metals, reduced availability of water and high salinity. Some of the major betaines are listed in Table 2.2. Their structures are given by formulae 2-16.

An important group of N-substituted amino acids are N-acylamino acids (2-17). An important metabolite is hippuric acid, also known as N-benzoylglycine (2-17). Hippuric acid arises from glycine as a result of detoxification of benzoic acid and other aromatic acids in humans and animals. In higher concentrations it occurs in the urine of herbivores and reportedly inhibits some pathogenic bacteria in the urinary tract. Benzoic acid is found (usually in small amounts) in many plant materials, including fruit, vegetables and forage crops (see Section 8.2.6.1.6), and is also used as a food preservative (see Section 11.2.1.1.1). Cows partially excrete hippuric acid into their milk, up to a concentration of 60 mg/kg. Microorganisms used in the production of fermented dairy products hydrolyse hippuric acid to glycine and benzoic acid.

For example, the content of benzoic acid in yoghurts can reach, on average, 15 mg/kg.

N-phenylpropenoyl amino acids (2-17) have been identified as the key contributors to the astringent taste of non-fermented cocoa beans and cocoa products. Besides the already known (E)-N-[3 ,4 -dihydroxycinnamoyl-3-hydroxy-l-tyrosine (known as clovamide), (E)-N-(4 -hydroxycinnamoyl)-l-tyrosine (deoxyclovamide) and (E)-N-(3 ,4 -dihydroxycinnamoyl)-l-tyrosine, seven additional amides derived from cinnamic, 4-coumaric, caffeic and ferulic acids, namely, (+)-(E)-N-(cinnamoyl)-laspartic acid, (+)-(E)-N-(4 -hydroxycinnamoyl)-l-aspartic acid, (+)-(E)-N-(3 ,4 -dihydroxycinnamoyl)-l-aspartic acid, (+)-(E)N-(4 -hydroxy-3 methoxycinnamoyl)-l-aspartic acid, (–)-(E)N-(4 -hydroxycinnamoyl)-l-glutamic acid, (–)-(E)-N-(3 ,4 -dihydroxycinnamoyl)-l-glutamic acid and (–)-(E)-N-(4 -hydroxycinnamoyl)-3-hydroxy-l-tyrosine, were recently identified.

Table 2.2 Some important betaines.

Betaine

Initial amino acid

Occurrence

β-Alaninebetaine (homobetaine)

β-Alanine

Plants of the Plumbaginaceae family, citrus species, meat

Gababetaine

γ-Aminobutyric acid

Molluscs, citrus species

L-Carnitine

Lysine and methionine

Meat, dairy products, legumes, vegetables, higher fungi

Laminine (lysinebetaine)

Lysine

Some higher plants

Stachydrine (cadabine, prolinebetaine)

Proline

Higher plants (e.g. citrus species), fungi

Betonicine (4-hydroxystachydrine, 4-hydroxyprolinebetaine)

4-Hydroxyproline

Plants of the Lamiaceae family

Hercynine (histidinebetaine)

Histidine

Higher fungi, citrus species

L-Ergothioneine (2-thioimidazolebetaine)

2-Mercaptohistidine

Higher fungi, plants

Homarine

Pyridine-2-carboxylic acid

Some higher plants

(Dimethylsulfonium)propanoate

Methionine

Marine phytoplankton, seaweeds

13

2.2 AMINO ACIDS

H3C H3C

COO−

+

N

n

+

H3C

COO−

N

H3C

CH3

H3C

CH3 OH

homobetaine, n = 1 gababetaine, n = 2

COO−

N CH3

carnithine

+

N CH 3 CH3

NH2 laminine

stachydrine

COO−

COO− HO

H3C

COO−

+

N

+

N CH 3 CH3

R

4-hydroxystachydrine

+

+

NH N CH3 H3C CH3

hercynine, R = H ergothioneine, R = OH

H3C

COO−

N

COO−

+

S

CH3

CH3

homarine

(dimethylsulfonium) propanoate

2-16, structures of important betaines O H N

COOH

O

R

COOH N H

COOH

HO

hippuric acid

(E )-N-(4′-hydroxycinnamoyl)-L-glutamic acid, R = H (E )-N-(3′,4′-dihydroxycinnamoyl)-L-glutamic acid, R = OH

2-17, structures of important N-acylamino acids

Exposure to light rapidly converted (E)-isomers of Nphenylpropenoyl amino acids into the corresponding (Z)-isomers.

Alicyclic amino acids A common neutral amino acid derived from cyclopropane is 1-aminocyclopropane-1-carboxylic acid. The precursor of this carboxylic acid is methionine, or strictly S-adenosyl-lmethionine which is derived from methionine (Figure 2.1).

1-Aminocyclopropane-1-carboxylic acid is also present in apples, pears and other fruits. 1-Aminocyclopropane-1-carboxylic acid serves as a precursor of ethylene (ethene) in essentially all tissues of

vascular plants and also in fungi and algae. Ethylene is a signalling molecule in plants (a hormone) that regulates seed germination, plant growth, flowering, ripening of fruits, leaf abscission and aging. Plants also synthesise ethylene in various stressful situations (mechanical damage, frost damage, flooding, pathogen attack and in the presence of heavy metals). Production of ethylene is connected with the formation of hydrogen cyanide, a process known as cyanogenesis (see Section 10.3.2.3).

The seeds of lychee fruit (Litchi chinensis) from the soapberry family (Sapindaceae), native to southern China and Southeast Asia, contains an unusual amino acid l-α-(methylenecyclopropyl)glycine, that is, (2S,3S)-2-(methylenecyclopropyl)

NH2 N H3C

S

N

oxidation

2

+

O

N

1

N 3

H2N COOH

OH

OH

COOH

COOH

NH2

NH

1-aminopropane1-carboxylic acid

CH2 + H2O

OH N-hydroxy-1-aminopropane1-carboxylic acid

S-adenosyl-L-methionine

HOOC C N cyanoformic acid

H C N + CO2 hydrogen cyanide Figure 2.1 Formation of ethylene and hydrogen cyanide in plants.

H2C

ethylene

14

CH 02 AMINO ACIDS, PEPTIDES AND PROTEINS

glycine (2-18), which lowers blood glucose levels. Symptoms of poisoning, known as toxic hypoglycaemic syndrome, occur 6–48 hours after ingestion of seminal immature follicles. The poisoning is manifested by vomiting, drowsiness, fatigue and hypoglycaemia.

The toxin (the active metabolite is methylencyclopropylacetylCoA) interferes with the metabolism of branched-chain amino acids, irreversibly binds to FAD and inhibits acyldehydrogenases acting in β-oxidation of fatty acids.

An important alicyclic amino acid in some genera of tropical plants of the Flacourtiaceae, Passifloraceae and Turneraceae families is cyclopentenylglycine occurring as a mixture of two stereoisomers, (2S,1 R)-2-(cyclopent-2 -en-1 -yl)glycine (2-22) and (2S,1 S)-2-(cyclopent-2 -en-1 -yl)glycine (2-23), in which the (2S,1 R)-isomer predominates. Cyclopentenylglycine is a precursor of unusual fatty acids (see Section 3.3.1.4.3) and also cyanogenic glycosides (see Section 10.3.2.3.1).

H2C H

COOH

H

COOH

NH2

2-22, (2S,1′R)-2-(cyclopent-2′-en-1′-yl)glycine

2-18, 2-(methylenecyclopropyl) glycine

The same amino acid, together with derived γ-glutamyl dipeptide and (2S,1 S,2 S)-2-(2 -carboxycyclopropyl)glycine (2-19) occurs in the seeds of ackee fruit (Blighia sapida, Sapindaceae), native to tropical West Africa. A higher homologue of 2-(methylenecyclopropyl)glycine, that is, l-3-(methylenecyclopropyl)alanine, also known as hypoglycin A (2-19), is present in this fruit in much higher concentrations. Hypoglycin A (now known as hypoglycin) occurs as a mixture of (2S,4R)- and (2S,4S)-diastereoisomers in fruits (in the edible arilli portion) and seeds. The former is the dominant isomer in the arilli. Formula (2-20) shows the (2S,4S)isomer, which also has a hypoglycaemic effect, but the other primary toxic effect is known as Jamaican Vomiting Sickness (see Section 10.3.2.7). Ackee fruit also contains the γ-glutamyl dipeptide of hypoglycin A, known as hypoglycin B (2-21). The amount of hypoglycin A in totally green and yellow–green or orange–green arilli and seeds is 6.7–7.9 and 8.2–8.4 g/kg, respectively. Dull and shrivelled arilli (mainly from red fruits) and seeds contain 0.27–0.55 and 1.5–2.6 g/kg of hypoglycin A, respectively. The content of hypoglycin B in the seeds of unripe fruit is 1.6–3.1 g/kg, while in seeds of ripe fruit the content ranges from 11.7 to 12.6 mg/kg.

Higher concentrations of hypoglycins A and B also occur in seeds of the common sycamore (Acer pseudoplatanus, Aceraceae). For example, The Food and Drug Administration and Health Canada sets the maximum permissible level of hypoglycin A to 100 mg/kg.

COOH H2N

Hydroxyamino acids Various legumes (seeds of plants of the Fabaceae family) are rich sources of free aliphatic hydroxyamino acids. Common amino acids such as 4-hydroxyleucine, 4-hydroxynorvaline and 5-hydroxynorleucine are hydroxyderivatives of branched chain amino acids, l-leucine (2-4), l-isoleucine (2-4), l-norvaline (2-24) and l-norleucine (2-25). Another common amino acid in legumes is l-homoserine (2-26), which is derived from 2-aminobutyric acid, the intermediate in the biosynthesis of threonine from aspartic acid. Frequently O-acyl- and O-amino derivatives of homoserine are seen. An example of an O-aminoderivative of homoserine is a toxic amino acid known as canaline (see Section 2.2.1.2.4). The most common source of this amino acid is the jack bean (Canavalia ensiformis).

3

H3C

HOOC

COOH H

NH2

H

2-19, 2-(2′-carboxycyclopropyl)glycine

COOH NH2 COOH O

2-21, hypoglycin B

NH2

2-20, hypoglycin A

H2C H HN

5

COOH

H3C

3

2-24, norvaline

COOH

HO

4

NH2

H2C

COOH

H

2-23, (2S,1′S)-2-(cyclopent-2′-en-1′-yl)glycine

4

H

NH2

NH2

NH2 2-25, norleucine

COOH

2-26, homoserine

These hydroxyamino acids are frequent components of peptides occurring in toxic mushrooms. For example, peptides of some Amanita species (see Section 10.3.2.9.2) contain (2S,4R)- and (2S,4S)-isomers of 4-hydroxyleucine and some other hydroxysubstituted amino acids, including amino acids with unsaturated side chains.

2.2.1.2.2 Sulfur and selenium amino acids Foods of plant origin contain numerous sulfur amino acids, mostly derived from cysteine, methionine and their higher homologues,

15

2.2 AMINO ACIDS

such as l-homocysteine (2-27) and l-homomethionine, also known as l-5-methylthionorvaline (2-28).

HS

COOH

H3C

COOH

S

NH2

NH2

2-27, homocysteine

2-28, homomethionine

Cysteine derivatives Plants synthesise a number of S-cysteine (and S-glutathione) conjugates that are released by the action of l-cysteine-S-conjugate thiol-lyases (deaminating) to produce various volatile flavouractive thiols in fruits and vegetables (see Section 8.2.9.1.2).

Other important derivatives of cysteine are S-alk(en)yl-lcysteines (2-29), which accompany, in small amounts, the predominant S-alk(en)yl-l-cysteine S-oxides, also called S-alk(en) yl-l-cysteine sulfoxides (2-30). S-Alk(en)yl-l-cysteine sulfoxides may occur in two optical isomers (thanks to the free electron pair on the sulfur atom). Only (+)-S-isomers, also known as (SS )-isomers, occur in vegetables. However, isoalliin is an exception. It occurs as an (RS )-isomer because of the priority of the other substituent. The bond between the sulfur and oxygen atoms in S-alk(en)yl-l-cysteine sulfoxides differs from a conventional double bond between carbon and oxygen. As a result, the molecule has a dipolar character, with the negative charge centred on the oxygen and the sulfur atom being a chiral centre. These amino acids originate from γ-glutamyl S-alk(en)yl-l-cysteine storage compounds via S-alk(en)yl-l-cysteines (see Section 2.3.2.1.2) and are the precursors of many biologically and flavour active R

+

COOH

S O−

R

R

2-29, S-alk(en)ylcysteines

The basic member of the homologous series, S-methylcysteine (2-29, R = CH3 ), and its sulfoxide, (RC ,SS )-S-methylcysteine sulfoxide, also known as (+)-S-methyl-l-cysteine sulfoxide or methiin (2-30, R = CH3 ), occurs in cabbage, garlic, onion and other Brassica and Allium vegetables and also in beans. There is roughly 0.2–0.7 g/kg in fresh Brassica vegetables. The methiin levels in Allium vegetables are given in Table 2.3. Note that methiin is a toxic amino acid to ruminants (see Section 10.3.2.7.1). (RC ,SS )-SCarboxymethylcysteine sulfoxide (2-30, R = CH2 –COOH), occurs, for example, in radish (genus Raphanus, Brassicaceae). Some species of Brassica plants and some members of the genus Allium (e.g. elephant garlic, onion, shallot, leek and chive) contain traces of ethiin, that is, (RC ,SS )-S-ethylcysteine sulfoxide (2-30, R = CH2 –CH3 ) (Table 2.3).

O

S

COOH

S

NH2

O marasmin

HN

COOH

S

H3C

NH2

alk(en)ylcysteine sulfoxides

N

COOH

S

NH2

COOH

S O

NH2

compounds (see Section 8.2.9.1.4). Particularly rich sources of these amino acids are some genera of the plant families Brassicaceae (the genus Brassica), Amaryllidaceae (subfamily Allioideae, genera Allium and Tulbaghia) and Fabaceae (Phaseolus, Vigna and Acacia).

S-Alk(en)ylcysteine derivatives also occur in plants belonging to the families Olacaceae (Scorodocarpus) and Phytolaccaceae (Petiveria), in some higher fungi, such as mushrooms in the genera Marasmius, Collybia and Lentinula (known as shiitake), and in brown (Undaria) and red (Chondria) seaweeds.

COOH

S O

NH2

S-(pyridin-2-yl)cysteine sulfoxide

NH2

S-(pyrrol-3-yl)cysteine sulfoxide

2-30, S-substituted L-cysteine sulfoxides Table 2.3 Concentrations of alke(en)yl-L-cysteine sulfoxides in Allium vegetables.

Concentration in g/kg fresh weight Garlic (Allium sativum)

Onion (Allium cepa)

Leek (Allium porum)

Chive (Allium schoenoprasum)

Methyl

0.05–4.24

0.22

0.04

0.32

Ethyl

0–traces

0.05

traces

0.01

Propyl

0–traces

0.06

traces

0.07

Prop-2-en-1-yl (allyl)

1.10-10.5

traces–0.05

traces

0.02

Prop-1-en-1-yl

traces–1.11

0.50–1.31

0.18

0.31

Substituent

16

CH 02 AMINO ACIDS, PEPTIDES AND PROTEINS

Another related amino acids are (RC ,SS )-S-allylcysteine (deoxyalliin), and (RC ,SS )-S-(prop-2-en-1-yl)cysteine sulfoxide, which is known as alliin (2-30, R=CH2 –CH=CH2 ). Alliin is the major sulfur containing amino acid of garlic (Table 2.3).

Its isomer (RC ,RS ,1E)-S-(prop-1-en-1-yl)cysteine sulfoxide, or isoalliin (2-30, R=CH=CH–CH3 ), is the major free amino acid of onions. It is also found in smaller amounts in garlic (Table 2.3).

(RC ,SS )-S-Propylcysteine sulfoxide, or propiin, R = [CH2 ]2 –CH3 (2-30) occurs in onions, shallots and chives and in trace amounts in some garlic species, for example in bulbs of A. vineale (0.01 mg/kg fresh weight), A. ursinum (0.02 mg/kg fresh weight) and A. triquetrum (0.09 mg/kg fresh weight). Its higher homologue butiin, that is, (RC ,SS )-S-butylcysteine sulfoxide, R=[CH2 ]3 –CH3 (2-30), occurs in some species of the genus Allium, in bulbs of A. siculum (0.3 g/kg fresh weight) and A. tripedale (5.7 g/kg fresh weight) together with (RC ,SS ,1E)-S-(but-1-en-1-yl)cysteine sulfoxide, R=CH=CH–CH2 –CH3 (2-30), known as homoisoalliin. The concentrations found in fresh bulbs of the ornamental plants A. siculum and A. tripedale were 3.5 and 1.2 g/kg, respectively. The important cysteine sulfoxide (RS ,RC )-S-(methylthiomethyl)cysteine-4-oxide, known as marasmin (2-30), carrying a methylthiomethyl moiety as the aliphatic residue, was found in South African society garlic (Tulbaghia violacea) and in wild (woodland) garlic (T. alliacea), in A. stipitatum and A. suworowii (Allium subgenus Melanocrommyum) and as a γ-glutamyl derivative, γ-glutamyl-(SS ,RC )-marasmin, in various mushroom species belonging to the genus Marasmius (such as M. alliaceus). Marasmin is the precursor of the thiosulfinate marasmicin, which shows antifungal and tuberculostatic activities.

(RC ,SS )-S-(Pyridin-2-yl)cysteine sulfoxide (2-30) and several pyridyl compounds, which were formed from this amino acid by the action of alliinase, were identified in bulbs of the drumstick onion Allium stipitatum (Allium subgenus Melanocrommyum), which is used as a crop plant and in folk medicine in Central Asia. (RC ,SS )-S-(Pyrrol-3-yl)cysteine sulfoxide is found in another member of the subgenus Melanocrommyum, A. giganteum.

To date, six S-alk(en)ylcysteine sulfoxides, namely S-allyl-, (E)-S(prop-1-en-1-yl)-, S-methyl-, S-propyl, S-ethyl and S-butylcysteine sulfoxides have been found in common vegetables of the genus Allium; the last two compounds are only present in trace amounts.

It has been well documented that the total content and relative proportions of the individual S-alk(en)ylcysteine sulfoxides in alliaceous plants are significantly affected by a number of genetic and environmental factors (e.g. plant species and variety, climatic conditions, sulfur content in the soil, irrigation, fertilisation, harvest date, amongst other things) and is usually around 0.1–0.8%, which represents more than 10% of the crude protein content.

The contents also vary considerably over the growth period. For example, in garlic, the highest amounts are found in spring, in the green parts of the plant, which are the most important sites of cysteine sulfoxide biosynthesis, although some biogenesis may also occur in the bulbs. The cysteine sulfoxide levels in the bulbs start to increase dramatically approximately 5 weeks before harvest. This accumulation in bulbs may be associated with the possible role of cysteine sulfoxides as storage compounds for sulfur and nitrogen during dormancy.

Some vegetables also contain alk(en)ylthio substituted l-cysteine sulfoxides in small amounts. For example, (RC ,SS )-S-methylthiocysteine sulfoxide (R=S–CH3 , 2-30), (RC ,SS )-S-propylthiocysteine sulfoxide (2-30, R=S–CH2 –CH2 –CH3 ) and (RC ,SS ,1E)-S(prop-1-en-1-ylthiocysteine sulfoxide (2-30, R=S–CH=CH–CH3 ) occur in onions at concentrations of 0.19, 0.01 and 0.56 g/kg fresh weight, respectively. These amino acids and the corresponding peptides (see Section 2.3.3.1.2) are formed by reaction of thiosulfinates (see Section 8.2.9.1.4) with cysteine residues.

An interesting sulfur containing amino acid derived from cysteine is 2-amino-4,6,8,10,10-pentaoxo-4,6,8,10-tetrathiaundecanoic acid (2-30, R=CH2 –CH2 -SO–SO–CH2 –SO2 –CH3 ), or deglutamyllentinic acid), which forms from the corresponding γ-glutamyl peptide known as lentinic acid (see Section 2.3.2.1.2) through the action of γ-glutamyl transpeptidase and becomes the precursor of the characteristic sulfur compound (1,2,4,5,6pentathiepane, commonly known as lenthionine; see Section 8.2.12.9.7) in dried edible shiitake mushrooms (Lentinula edodes), one of the most popular edible mushrooms in Japan and other parts of the Far East.

The unusual cysteine derivative l,l-cystathionine (2-31) is an intermediate of the biosynthesis of methionine and of other amino acids. The toxic amino acid l,l-djenkolic acid, also known as 3,3 (methylenedithio)di-l-alanine, consists of two cysteine residues joined by a methylene group between the sulfur atoms (2-32). It was first identified in the urine of Indonesians consuming tropical legumes known as jengkol or jering (Archidendron pauciflorum, syn. Pithecellobium lobatum, Fabaceae). The amino acid l-felinine, 3-hydroxy-1,1-dimethylpropyl-l-cysteine (2-33), occurs in the urine of cats and other felines, and is a precursor of the putative cat pheromone 3-mercapto-3-methylbutan-1-ol, which has a practical use in marking territory. Cysteine (or cystine) is also the precursor of dehydroalanine (2-aminoacrylic acid), from which the amino acid lanthionine is formed (see Section 2.5.1.3.4), which is also a component of the microbial peptide nisin (see Sections 2.3 and 11.2.1.2.1).

Dehydroalanine is a precursor of other unusual amino acids (such as lysinoalanine). Lysinoalanine and its analogues can be generated as cross-links on heating of proteins and, especially, in proteins after treatment in alkaline solutions (as occurs, for example, in soybean processing). Digestive enzymes do not hydrolyse these cross-links. Other sulfur-containing amino acids include N-acetylS-substituted cysteines (or mercapturates, 2-34), which form in the body as products of detoxification in reactions of xenobiotics with glutathione and by hydrolysis of the resulting products.

NH2 S

HOOC

COOH NH2

2-31, cystathionine

HOOC

S NH2

2-32, djenkolic acid

S

COOH NH2

17

2.2 AMINO ACIDS

R

H3 C

CH3

HO

H3C

COOH

S

COOH

S NH

O 2-34, mercapturic acids

NH2 2-33, felinine

Plants also contain other stereoisomers of these amino acids and many other related compounds that exhibit a range of biological effects.

HOOC

COOH CH3

Methionine derivatives

S

COOH NH2

2-35, dihomomethionine

H3C

NH2

S

COOH

COOH CH2

2-37, 4-methylglutamic acid

A common amino acid in Brassica vegetables, S-methyl-lmethionine, was formerly classified as vitamin U (see Section 5.15). Higher homologues of methionine – l-homomethionine, l-dihomomethionine (2-35), l-trihomomethionine up to lhexahomomethionine (2-36) – are starting compounds for the biosynthesis of many important glucosinolates (see Section 10.3.2.4.1). For example, homomethionine is the precursor of sinigrin and glucoibervirin, while dihomomethionine is the precursor of progoitrin and gluconapin.

H3C

HOOC

NH2

2-38, 4-methyleneglutamic acid OH

HOOC

COOH NH2

2-39, 3-hydroxyglutamic acid

N-Ethyl-l-glutamine, also known as l-theanine or l-ethanine (2-40), is a unique major free amino acid in tea leaves. Depending on the variety of Chinese tea (Camellia sinensis, Theaceae), its content in the leaves is normally around 1.3% of dry matter (0.7–2.0%) and is higher in green tea and tea of higher quality (0.8–2.7% in green tea, 0.6–2.1% in black tea, 2.0–9.2% in oolong tea, 0.7–1.8% in decaffeinated black tea). The tea extracts contain about 3% of theanine. The theanine content is used to detect tea extracts in products based on tea (instant and iced tea). Theanine has neuromuscular sedative effects with no side effects and is used in some special food supplements.

NH2

2-36, hexahomomethionine

Selenium amino acids Allium vegetables and various other plants contain small amounts of amino acids derived from cysteine and methionine, in which sulfur is replaced by selenium. These amino acids are the main organic form of selenium and, together with Se-containing proteins, represent the main selenium source in foods (see Section 6.2.3.1).

Examples of these amino acids are Se-alk(en)ylcysteines and their γ-glutamyl derivatives.

2.2.1.2.3 Acidic amino acids and their amides Most non-protein acidic amino acids and their amides are structurally related to l-glutamic acid and l-glutamine. For example, sea pea seeds (Lathyrus maritimus, Fabaceae) contain l-4-methylglutamic acid, that is, (2S,4R)-2-amino-4-methylpentanedioic acid (2-37), and the seeds of peanuts (Arachis hypogaea, Fabaceae) contain l-4-methyleneglutamic acid (2-38) together with l-4-methyleneglutamine. (2S,3R)-3-Hydroxyglutamic (threo3-hydroxy-l-glutamic) acid (2-39) is a precursor of ibotenic acid and other toxic compounds in fly agaric (Amanita muscaria) and panther cap (A. pantherina). It is also a precursor of the tricholomic acid found in some agarics (Tricholoma muscarium) (see Section 10.3.2.9.3). The so-called false rhubarb (Rheum rhaponticum), red currant (Ribes silvestre, Grossulariaceae) and garden cress (Lepidium sativum, Brassicaceae) contain l-3,4-dihydroxyglutamic acid, that is, (2S,3S,4R)-2-amino-3,4-dihydroxypentanedioic acid.

O H3C

N H

COOH NH2

2-40, theanine

The fruiting bodies of common mushrooms Agaricus bisporus (also known as button mushrooms, white mushrooms, table mushrooms and champignon mushrooms) contain a mutagenic glutamic acid derived hydrazine β-N-(γ-l-glutamyl)-4(hydroxymethyl)phenylhydrazine known as l-agaritine (10-226) at concentrations of 100–1 700 mg/kg. The metabolic fate of agaritine has been linked with the carcinogenity of this mushroom (see Section 10.3.2.11.4).

2.2.1.2.4 Basic amino acids and related compounds Plants contain various basic diaminomonocarboxylic acids that are homologues of lysine or derivatives of these homologues.

Other arginine derivatives and their homologues are also relatively common. The lowest member of the homologous series of compounds is l-2,3-diaminopropionic acid (2-41), occurring in plants of the genus Mimosa of the legume family Fabaceae. The toxic N3 -methyl derivative occurs in the cycads (plants of the cycas family Cycadaceae) and the N2 -oxalyl derivative is found in vetchlings (Lathyrus spp.) and vetches (Vicia spp.) that belong to the legume family Fabaceae. Together with l-2,4-diaminobutyric acid (2-41), this compound is the originator of neurodegenerative disease neurolathyrism (see Section 10.3.2.7.1).

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si el gobierno hubiera atropellado las leyes para castigar los atropellos de otros, debería haber empezado por embarcarse él para Canarias, y decir: Marchemos todos francamente, y yo el primero, por la senda de presidio? Vaya, Andrés, que eso ni suponerse puede, y si te cuentan que tal caso ha sucedido, puedes decir que el que lo cuente es un malévolo de ésos que traen la anarquía en el bolsillo. Diría el gobierno y diría bien: «Yo no hice tal cosa, y si la hiciera, ¿qué diferencia habría entre los atentados del pueblo y los míos? Porque en fin, mientras que la ley no le ha declarado reo, el condenado es asesinado: en ese caso no habría entre mi atentado y el del pueblo más que una diferencia, á saber: que el pueblo asesinó malamente carlistas y yo asesino malamente liberales».

Asesinatos por asesinatos, ya que los ha de haber, estoy por los del pueblo.

Puedes estar seguro de que hay causa; y si no se les ha formado, es porque andamos de prisa, ó por mejor decir, lo que ha ido á Canarias no ha sido una cadena de culpables, sino una comisión artística compuesta de liberales, que van á costa del gobierno á acabar de descubrir aquellas islas, y escribir una memoria de las alturas del globo, y á dar testimonio al mundo sobre todo de la altura á que estamos, tomando el meridiano del pico de Tenerife.

También te habrán contado posteriormente otra pequeña arbitrariedad ejecutada oficialmente en una vieja, en virtud de un cúmplase de un héroe. ¡Dios nos libre de caer en manos de héroes!

Sólo te diré que á lo menos en Barcelona tuvieron que acometer una fortaleza y exponerse á ser rechazados. Bueno es remontarse á las causas de las cosas, al tronco, y no á las ramas. Es así que la primera causa de que existen facciosos fueron las madres que los parieron; ergo quitando de en medio á las madres; lo que queda.

Los teólogos dicen: sublata causa tollitur efectus. Es lástima que no haya vivido el abuelo, porque mientras más arriba más seguro es el golpe. Pero hemos tenido que contentarnos con la madre. Está probado que así como Sansón tenía la fuerza en el pelo, los facciosos tienen el veneno en la madre, que viene á ser la hiel de ellos; en quitándosela se vuelven como malvas: así lo ha probado la

experiencia, porque de resultas el otro no ha fusilado más que á treinta. ¿Quién sabe los que hubiera fusilado si hubiera tenido madre todavía? Luego, las mujeres son las que están impidiendo la felicidad de España, y hasta que no acabemos con ellas no hay que pensar en tener tranquilidad. En cuanto á las hermanas, como estaban casadas con guardias nacionales, les tocaba fusilar la mitad á los de allá, y la otra mitad á los de acá; pero nosotros, más desprendidos, no quisimos perdonar ni la mitad que nos tocaba, y lo fusilamos todo. ¡Bienaventurados en tiempos de héroes los incluseros, porque ellos no tienen padre ni madre que les fusilen!

Pasadas estas etiquetas de recíproca cortesía, dieron en correr voces de que el ejército estaba descontento, y que la guerra de Navarra no iba lo ligera que debía. Felizmente para todos, algunos amigos tuyos y míos, que así saben mover la pluma como esgrimir la espada, enderezaron la opinión en artículos luminosos, probando lo que ninguno debía tener olvidado, que las guerras civiles son largas, á pesar de todos los programas del mundo; que éstos son por el contrario los que tienen corta vida; que así las civiles como las demás se sostienen con dinero y con soldados; que un gobierno en lucha con una facción pierde más cuando pierde una batalla, que adelanta cuando la gana, y que una derrota nuestra nos quita más honra que gloria da á la facción; que por lo tanto es fuerza no aventurarse sino á ciencia cierta; que la guerra no se hace en el ministerio, sino en Vizcaya; que de real orden se llevan y se traen jueces, se envían buques á Canarias, y se conquistan votos, pero de real orden no se ganan batallas; que algunos descalabros nuestros han sido debidos á reales órdenes; que para hacer la guerra se necesita un plan; que para tener plan es preciso que el general sólo sea responsable; y que Córdoba, en fin, sin que haya necesidad de llamarle héroe, tiene un plan, el cual es forzoso dejarle llevar á cabo, siquiera porque no ha habido hasta ahora otro mejor que el suyo.

Tales razones no convencieron, fué bien acogida la representación del ejército, y si bien ninguno de los que hablaban fué á dar su brazo en vez de su voto, al fin no se admitió la dimisión, y sigue el general, y su plan, y la guerra de Navarra, en el mejor estado posible.

Mientras todo esto pasaba echáronse encima las próximas elecciones, hoy ya pasadas, y porque digo se echaron encima, no vayas á pensar alguna tontería. Dijeron muchos si habría amaños ó si no habría amaños; que se escribió largo y se intrigó más. Lo primero solo prueba cultura en el país, lo segundo arguye talento. ¡Vaya usted á impedir que hablen las gentes! Para que no fuesen las elecciones muy populares bastante amaño era ya la propia ley electoral, en virtud de la cual debían elegir los electores nombrados por los ayuntamientos y los mayores contribuyentes. No hay cosa para elegir como las muchas talegas: una talega difícilmente se equivoca; dos talegas siempre aciertan, y muchas talegas juntas hacen maravillas. Ellas han podido decir á su procurador por boca de los mayores contribuyentes la famosa fórmula aragonesa: «Nos, que cada una de nos valemos tanto como vos, y todas juntas mucho más que vos, os hacemos procurador».

Luego, los elegidos habían de tener doce mil reales de renta: gran garantía de acierto: por poco que valga un real en estos tiempos, no hay real que no valga una idea, sin contar con las muchas que hasta ahora hemos visto que no valían un real, y con los varios casos en que por menos de real daría uno todas sus ideas: bueno es siempre que haya reales en el Estamento por si acaso no hubiese ideas.

Tanto mejor si hay lo uno y lo otro.

No es menos importante lo de los treinta años; no es menos simbólico ni cabalístico el número de treinta que el de tres tan citado, y de que es décuplo; treinta días tiene el mes, treinta minutos cada media hora, por treinta dineros vendió Judas á un Dios, treinta años representa la vida de un jugador, y treinta años, en fin, la capacidad de un procurador. Muchos filósofos han creído que cuando el hombre nace, el Ser Supremo, que esta atisbando, le sopla dentro el alma por medio del mismo procedimiento que usa un operario en una fábrica de cristales para dar forma á una vasija; pero eso es el alma, mas no la capacidad y la facultad de procurar: esta tal otra quisicosa se la infunde el Criador el día que cumple treinta años, por la mañanita temprano, así como la aptitud legal y la mayoría se la comunica á los veinte y cinco. Oh tú, Andrés, que no los has cumplido, está con cuidado el día que los hayas de cumplir,

y escríbeme para mi gobierno lo que sientas en ese día: dime por dónde entra la capacidad, y hacia dónde se coloca en tu persona: prevenido de esa suerte de los síntomas que la anuncian podré yo hacer á la mía, el día que me baje, el recibimiento que se debe á tan ilustre huésped. ¿Cuándo tendremos treinta años? Aquel día seremos ya unos hombrecitos.

Bien ha habido hombres que han discurrido antes de los treinta años, pero ésos son fenómenos portentosos, raros ejemplos de no vista precocidad; y en cuanto á Pitt y otros de su especie, ministros ya mucho antes, ni siquiera es posible considerarlos como monstruos de naturaleza; es fuerza inferir error de cálculo y mala fe en la de bautismo.

El haber nacido en la provincia, ó tener en ella arraigo, no es de menos importancia, si recordamos que las primeras impresiones se graban para siempre en la cabeza del niño, y deciden de lo que ha de ser después cuando grande: ni es posible que un hombre conozca su provincia, y se interese por ella, si no ha nacido por allí cerca. Puede suceder que una provincia tenga más confianza en la reputación, en el saber de un forastero; pero páselo en paciencia la buena de la provincia, que más pasó Cristo por ella.

Dicen sin embargo que todos los electores no han tenido presentes todas esas verdades; así que, unos procuradores no han nacido, otros no tienen la renta, ¡qué sé yo! Esto tiene compostura habiendo comisión de poderes, y en todo caso se aplica la renta de unos á otros, como hacen los buenos cristianos con los méritos de nuestro Señor Jesucristo, que valen mucho más que las rentas; y así poniendo de aquí y quitando de allí tengo para mí que se ha de remediar. Y aun yo diría más. Don Juan Álvarez Mendizábal fué elegido por ejemplo por Barcelona, siendo natural de Cádiz, y no habiendo residido en Cataluña. Decían: «Pero no tiene nada suyo en Cataluña, sino los electores:» ¿pues eso no es tener?, ¿no valen tanto por lo menos los electores como una casa, ó una tapia, ó unas cuantas fanegas de pan llevar? ¡Sino que poniéndose á hablar las gentes!...

Por lo demás es sabido que el gobierno no ha influido absolutamente nada en las elecciones, y desde luego se dijo que eran á pedir de boca. Para que formes una idea, han salido elegidos los sugestos siguientes: Por Barcelona, como llevo dicho, don Juan Álvarez Mendizábal.

Por Cádiz, don Juan Álvarez Mendizábal.

Por Gerona, don Juan Álvarez Mendizábal.

Por Granada, don Juan Álvarez Mendizábal.

Por Madrid, don Juan Álvarez Mendizábal.

Por Málaga, don Juan Álvarez Mendizábal.

Por Pontevedra, don Juan Álvarez Mendizábal, etc., etc., etc.

Que es el cuento de pasó una cabra, y volvió y pasó otra, y volvió á tornar y á pasar otra cabra, y así sucesivamente.

Si oyes decir que se abre el Estamento, di que es broma, que quien se abre es don Juan Álvarez Mendizábal.

No habrás olvidado que los ministros de estado y de hacienda, y el presidente del consejo, son don Juan Álvarez Mendizábal, y que los otros ministros no son sino una manera de ser, distinta sólo en la apariencia del don Juan Álvarez Mendizábal. Ahora figúrate el día que el Estamento don Juan Álvarez Mendizábal pida cuentas al ministro don Juan Álvarez Mendizábal...; aquí llaman esto un gobierno representativo: sin que sea murmuración, confieso que yo llamo esto un hombre representativo.

Una vez conocida la buena índole de las elecciones y la idoneidad de esos diversos señores procuradores, ocurrió la duda de si estas Cortes que iban á reunirse vendrían sólo para hacer una ley electoral mejor que la que les confiere su derecho; ó si podrían constituirse revisoras. Quiénes se agarraron á la legalidad, diciendo que esto último sería ilegal; quiénes intentaron probar que lo de menos era la legalidad, y que lo que importaba era la conveniencia.

Por fin salimos del atolladero, y parece que no tratarán de constituirse por varias razones. Porque no han sido convocadas

para eso. Porque siendo su objeto principal hacer una ley electoral, en virtud de la cual puedan convocarse luego las revisoras, es claro que los demás asuntos que á ellas se sometan, por importantes que sean, habrán de ser subalternos al principal. La nación tiene un cimiento, y necesita una casa: en estas Cortes va á decidir cuáles han de ser las circunstancias del arquitecto que se la puede hacer á su gusto. Por consiguiente, todo lo que sea proceder á construir el que sólo está comisionado para designar el constructor, es hacer la casa y dejar para después el arquitecto: equivale á blanquear después de pintar; es dejar al que venga detrás el derecho de poner en duda la validez de la construcción.

En estas disputas andábamos, cuando otro run run más terrible vino á poner nuevo espanto en nuestro corazón. He aquí que una noche corre la voz de que se va á poner la constitución del año 12. ¡Bravo!, dije yo: esto es lo que se llama andar camino. Aquí no se sabe multiplicar, pero restar á las mil maravillas. Vamos á quién puede más. El año 14 vino el rey y dijo: quien de catorce quita seis, queda en ocho. Vuelvan pues las cosas al ser y estado del año 8. El año 20 vienen los otros y dicen: quien de veinte quita seis, queda en catorce: vuelvan las cosas al ser y estado del año 14. El año 23 vuelve el de más arriba y dice: quien de veinte y tres quita tres, queda en veinte; vuelvan las cosas al ser y estado de febrero del año 20. El año 1836 asoman los segundos, y éstos quieren restar más en grande: quien de treinta y seis quita veinte y cuatro, queda en doce; vuelva todo al año 12. Éstos han pujado, si se exceptúa el del Estatuto, que más picado que nadie cogió y lo restó todo, y nos plantó en el siglo xv. ¡Diantre!, ¡si volveremos todavía á la venida de Túbal! Sepamos primero cómo se entiende nuestro progreso. ¿Hacia dónde vamos? ¿Hacia atrás, ó hacia adelante? Tengamos el cuento del cochero, que, montado al revés, arreaba al coche.

Ya te lo he dicho: tejedores; tejer y destejer. Nadie vende su tela, y nadie hace tela nueva.

Decían ellos que el volver atrás no era más que tomar carrera. ¡Dios los bendiga, y qué larga la toman!

Vamos claros. La constitución del año 12 era gran cosa en verdad, pero para el año 12: en el día da la maldita casualidad de que somos más liberales que entonces: si te he de hablar ingenuamente, á mí me parece poco.

Las circunstancias del año 12, la guerra que sosteníamos apoyada en el fanatismo popular, y el mayor atraso de la época, exigieron concesiones en el día no necesarias, ridículas.

En ellas hablan las Cortes en nombre de Dios Todopoderoso, Padre, Hijo y Espíritu Santo: gran principio para una novena: buena es la devoción, pero á su tiempo: eso es adoptar, heredar de la monarquía el derecho divino: la sociedad puede servir á Dios en toda clase de gobiernos. El Supremo Hacedor no delega facultades temporales ningunas, ni en un soberano, ni en un congreso; la sociedad se hace ella misma por derecho propio sus reyes y sus asambleas. Cristo vino al mundo á predicar, no á redactar códigos. Á Dios daremos cuenta de nuestras creencias, no á los hombres: reflexión igualmente aplicable al capítulo II, artículo 12, porque el Salvador quiso convencer, no obligar, porque no quiere más homenajes que los voluntarios. Ítem más: en la constitución del año 12 no está consignada la libertad de imprenta, sino para las ideas políticas, y eso es decirle á un hombre: Ande usted, pero con una sola pierna.

En cambio nos impone como ley fundamental el amor á la patria y la obligación de ser justos y benéficos... en cambio... Andrés mío, callemos, porque, repito, que la venero, y tengo por indigno de un liberal poner en ridículo el paladión de nuestra independencia nacional, y la cuna de nuestra libertad, por fácil que eso sea. Pero la respeto, como Cristo respetó el testamento viejo, fundando el nuevo.

Veneremos el viejo código, y venga no obstante otro nuevo más adecuado á la época.

Parécense los hombres del año 12, amigo Andrés, al cura que no sabía leer más que en su breviario: ó mejor al gastrónomo en VistaAlegre, que viendo su mesa puesta, pugna por sentarse á ella en cuanto le dejan un momento libre, en cuanto ve un resquicio por donde acercarse á la mesa. El caso es el mismo: todos les hacemos

cumplimientos, pero no les dejamos sentarse. Unas veces se lo impidió el poseedor don Pascual de la Rivera, otras los mozos de su fábrica... Convengo en que es una desesperación; pero culpen, no á nosotros, sino á ellos mismos, que tantas veces se dejaron interrumpir antes de llegar el bocado á la boca.

Aténgome á su artículo, que dice: «La nación española es libre é independiente, y no es ni puede ser patrimonio de ninguna familia, ni persona».

Esto digo yo: entre á gobernar, no éste ni aquél, sino todo el que se sienta con fuerzas, todo el que dé pruebas de idoneidad. Basta de ensayos. Á eso nos responden ellos: «¿Y dónde están esos hombres?» ¿Dónde han de estar? En la calle, esperando á que acaben de bailar los señores mayores, para entrar ellos en el baile. «¿Cómo no salen esos hombres?», añaden. ¿Cómo han de salir?

De Calomarde acá, ¿qué protección, qué ley electoral ha llamado á los hombres nuevos para darles entrada en la república? Cuenta sin embargo con ella, y llámelos la ley presto: ¡¡¡déjese entrar legalmente á los hombres del año 1836, ó se entrarán ellos de rondón!!!

En conclusión, hombres nuevos para cosas nuevas: en tiempos turbulentos hombres fuertes sobre todo, en quienes no esté cansada la vida, en quienes haya ilusiones todavía, hombres que se paguen de gloria, y en quien arda una noble ambición y arrojo constante contra el peligro. «¿Qué saben los jóvenes?», exclaman. Lo que ustedes nos han enseñado, les responderemos, más lo que en ustedes hemos escarmentado, más lo que seguimos aprendiendo. ¡Y qué eran ustedes el año 12! Nosotros fundaremos nuestro orgullo en ser sus sucesores, en aprovechar sus lecciones, en coronar la obra que empezaron. Nosotros no rehusamos su mérito; no rehúsen ellos nuestra idoneidad, que el árbol joven es la esperanza del jardinero, si el viejo ya le da sombra.

Según el miedo que tienen de que la juventud entre en los puestos, no parece sino que es posible hacerlo peor que ellos.

Para el año 1836 la única constitución posible es la constitución de 1836.

Una idea te diría, si no la hubieras de contar; y sólo á ti te la diría, porque ellos la tomarán á personalidad, si de ella hiciese un artículo, y sabe Dios que no lo digo por tal. Mucho venero á los hombres de otra época, Andrés mío; mucho saben, sobre todo en no hablándose de gobernar, para lo cual ya nos han manifestado repetidas veces hasta donde rayan: mucho saben, y tanto que no sólo no los lanzaría yo de la república, sino que los guardara muy guardados como guardaban los romanos los libros sibilinos, para consultarlos con el mayor respeto: de ellos armaría una biblioteca viva, donde vueltos de espaldas en muy pulidos estantes, leyese el estudioso encima Fulano, de Economía Política; Mengano, de Reformas Constitucionales; Zutano, de la Guerra de la Independencia; Perengano, de Metáforas y del Espíritu del Siglo, etc., etc.; de suerte que no hubiese más que volverlos y ojearlos en un apuro, cuidando mucho de quitarles antes y después el polvo, y de tornarlos á volver hasta otra duda, como pergaminos preciosos.

Ahí verás tú si los respeto, y si los tengo en estima.

Hasta aquí de la constitución y de los hombres del año 12. Pasó el susto, y la noticia, como habrás visto, no tuvo consecuencia. Sin duda el ruido que metió fué el último cumplimiento de despedida que nos hizo.

No ganamos para sustos. Posteriormente se cruzaron de palabras el pueblo de Valencia y su capitán general. Éste tomó una porción de providencias, entre otras las de Villadiego; con cuyo ingenioso arbitrio no le pudieron haber los valencianos, que es decir que ha podido más que ellos, que se ha burlado de ellos. Tiene mucho talento. Buen chasco se han llevado. Así, así: á los alborotadores hay que jugarles esas pasadas; con eso escarmientan. Á buen seguro que si Basa hubiera hecho otro tanto, no le hubieran deshecho á él, y el pueblo de Barcelona se hubiera llevado el mismo chasco que el de Valencia. ¿No queréis capitán general? Pues tomad capitán general. ¿No te figuras tú al pueblo de Valencia buscando á su capitán general por todas partes, como quien busca

una sanguijuela extraviada, y él trota que trota para Madrid? Á mí me hace morir de risa. Es lo que él dice: «¿Pues qué, querían ustedes que me mataran?» ¿Qué habíamos de querer?

Conque ahora está aquí bueno, gordo y tranquilo; no ha sido poca fortuna el poderlo contar.

En Zaragoza fué por otro estilo: salieron unos carlistas sentenciados á qué sé yo qué bobería: se levantó el pueblo, sitió á los jueces, y dieron en quererlos juzgar. Al maestro cuchillada. Pero no les da el naipe para esos pasajes á los jueces de Zaragoza, como á los capitanes generales de Valencia.

Entre tanto el ministerio de gracia y justicia sigue siempre de mudanza, y hace bien, porque el juez que no da fruto en una tierra, lo da en otra. El juez ha de ser como el zapato, hecho al pie; por eso el que no le viene bien al uno, le viene al otro.

Para eso el de la gobernación no se mete con nadie, ni habla mal de nadie. Es un excelente señor; á su oficina y no más. Da lástima hacerle daño, y sería completo si se le volviese C la H de su apellido; pero llámalo h.

En cuanto al de la guerra nadie sabe una palabra de él.

En mi última te pintaba en globo la confusión que en el Estamento y fuera de él había causado la ley electoral, y te añadía: «Yo por el pronto sólo veo clara una cosa, y es que para el 22 de marzo se reunirán de nuevo en Madrid otras Cortes... que para entonces es probable que empecemos á entendernos... y que seguramente no tendremos facción, porque estarán al caer los seis meses de la promesa, ó no tendremos ministerio, si no la cumple, porque estará caído, etc.».

De todas esas profecías sólo en la primera acerté; porque en cuanto á entendernos da gusto. Unos dicen que Mendizábal es el primer hombre del mundo; otros que no es tal, sino el último; que el primero es Istúriz y Galiano; te advierto que éste son dos: otros que ni Istúriz ni Mendizábal: no sé qué te diga: quién asegura que esto puede durar unos quince días, quién defiende que durará más que un constipado mal curado: éste no ve más que el prestigio que tiene

todavía en las provincias, el cual no se destruye tan fácilmente, sobre todo cuando no deja de tener algún fundamento; aquél no atiende más que al descrédito en que ha caído en sus corros y cafés, y cree que toda la nación puede juzgarle con igual talento, y tan de cerca como él. Éstos disputan que no hay hombres aquí; aquéllos que sí hay hombres; los de la izquierda que hay dinero; los de la derecha que no hay un cuarto; estoy por éstos. Quién opina que la guerra es inacabable; quién la da por acabada; añadiendo que no falta más que tirar una línea: uno dice que el mal de España no tiene remedio; otro que ésa es la mejor señal, que empieza la revolución, y que en Francia sucedía lo mismo, á pesar de que todo era diferente; varios juzgan que el rigor es de justicia, y que el árbol de la libertad se riega con sangre: algunos creen que la humanidad repugna tales horrores: no falta quien piensa que es guerra de empleos, y sobra quien no piensa ni eso ni nada. Pero todos somos liberales y vamos á una: eso sí. Por lo cual esto se acabará pronto de un modo ó de otro: en prueba de ello te puedo decir que se empiezan ya á acabar dos cosas: el dinero y la paciencia.

Pero son tantas las opiniones en fin y los hechos que se acumulan, y tantas las cosas que van á suceder, sin contar las que han sucedido desde la apertura de las Cortes, que me es indispensable reservarlas para otras cartas: me limito en ésta á ponerme al corriente, saliendo del atraso de noticias en que te tenía. En lo sucesivo aprovecharé todas las ocasiones posibles de escribirte, y al siguiente correo para Francia recibirás la inmediata, salvo extravío, golpe de mano airada, ó caso fortuito.

Si en el ínterin, y en medio de este conflicto de opiniones encontradas, me pides la mía, te contaré un caso que juzgo oportuno.

Sitiaban los Franceses al mando del mariscal Moncey esa misma Valencia, que en distintas épocas han mandado el Cid y Carratalá.

Reuniéronse en tan grave apuro el ayuntamiento y las personas más ricas del pueblo, entre las cuales quedóse dormido de confusión y pesadumbre un confitero, que entendía más de ramilletes que de disturbios políticos. Iba diciendo cada uno en la asamblea su opinión como mejor lo entendía. Llegada que le fué su

vez á nuestro hombre,—y usted, le dijo sacudiéndole del brazo el que á su lado tenía, ¿qué piensa?—Sí, ¿cuál es su opinión de usted?, preguntaron todos á un tiempo; á cuya pregunta contestó despertando y todo despavorido el confitero: ¡¡¡mi opinión, sí, mi opinión, señores, es de que Dios nos asista!!! En cuyo voto imitaba el confitero la rara discreción del padre Froilán Díaz, confesor de Carlos II. Eso mismo opino yo, Andrés mío, por ahora, y mientras no vea levantarse en masa á la nación para ahogar de una vez y para siempre el monstruo que en el norte nos devora, en vez de entretenerse en cuestiones secundarias y en rencillas personales, de las cuales debiera el país hacer justicia, como del orgullo mezquino y de la loca vanidad de sus dueños.—Tu amigo,—Fígaro.

FIN DEL TOMO SEGUNDO

PARIS.—ÉDOUARD BLOT, IMPRIMEUR, RUE BLEUE, 7

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