Chapter 10

Amino Acids: Carriers of Nutritional and Biological Value Foods

Fanny Ribarova    Medical College Yordanka Filaretova, Medical University Sofia, Sofia, Bulgaria

Abstract

Amino acids are major building elements of protein, determining its biological value, carriers of gene information, and playing a biochemical role, specific for each amino acid.

The aim of this chapter is to present information on the development of knowledge on amino acids, emphasizing the methods for assessment of their content in foods, and the methods for assessment of the biological value of food protein.

The chapter presents general information on amino acids completed with important historical points in the development of the knowledge on them, substantiating the need to proceed further with scientific research. The current status of analytical methodology, concerning particularly amino acid analysis and their participation in biological methods for evaluation of food proteins is characterized. An option for a more correct calculation approach to amino acid content versus total protein in foods is proposed.

The analysis of the current scientific information outlines the need to standardize the analytical methods and calculation approaches implemented in the assessment of amino acid content in food products.

Keywords

amino acids

chemistry

analysis

protein quality

biological value

1. Introduction

The quantitative and qualitative characteristics of protein intake are essential for the proper somatic and neuropsychic development and for the health status and work ability of the population. FAO reports have revealed that some 800 million of the global population suffer from chronic malnutrition resulting from lack or insufficient amount of food, most frequently caused by insufficient protein and energy intake (Moughan, 2012).

The development of contemporary precise indicators and criteria to characterize the protein component in conventional food products, novel protein foods, or protein supplements are currently actively discussed by nutritional scientists (Millward, 2012a; Rutherfurd, 2015; Tome, 2012). The most important criterion revealing the protein quality and physiological importance is its amino acid composition. Amino acids are involved in various numerous biochemical reactions and mechanisms associated with the normal functions of the organism and maintaining its proper health status. The successful implementation of amino acids in establishing healthy, preventive, and/or curative diets requires comprehensive knowledge on amino acids content in foods, their metabolism and usage as food supplements to support healthy human nutritional status, and as therapeutic products administered in the course of treatment of various diseases.

Amino acids realize their biological function in the organism not only in their capacity of essential protein elements, but as compounds with individual activity and engagement in a rich variety of biochemical and physiological processes.

Considering the historical development of the knowledge on amino acids, it could be stated that it contains three sequential stages progressing in line with the development of the protein scientific research, keeping at the same time the individuality of the knowledge on the role of free amino acids and their derivatives.

The first stage started yet in the 19th century when certain amino acids were detected as individual compounds. Further studies, striving to clarify the composition and structure of newly found proteins, resulted in the establishment of new amino acids and provided evidence affirming amino acids as major building elements of protein (Belitz et al., 2009).

The second stage progressed in the early 1800s and was associated with an intense search for new protein raw materials because of the scientific assertions stating the existence of real danger for global protein hunger and the necessity to increase not only the quantity but also the quality of food proteins with an emphasis on the amino acids building them. Thus, this stage marked a certain progress in the methods for amino acids analysis used to control the food proteins quality (Butikofer et  al.,  1991; Gilani et  al.,  2008; Otter,  2012).

The third stage already enhanced the knowledge horizons penetrating in the bioaccessibility, bioavailability, and metabolism of certain amino acids and their derivatives involved in various biochemical reactions and in the composition of bioactive compounds vitally important for the organism (Jonker et al., 2012; Katsanos et al., 2016; Rutherfurd-Markwick, 2012).

Different hypotheses are presented currently referring to the involvement of proteins, respectively, the amino acids building them in the mechanisms outlining the relationship between food, nutrition, and human health. Protein priority is reasonable as they are not only carriers of genetic information but are also active biocomponents, essential for the organism in most cases. Their significance for the life, growth, development, and health of living beings is outlined by their name “proteins,” evolving from the Greek word Πρωτɛύζ; first, most important, fundamental. This chapter presents scientific information in two basic dimensions of the knowledge on amino acids: markers for food proteins quality and approaches for analysis and assessment of amino acid content. The following aspects are discussed in this context: Historical remarks, Chemistry and classification of amino acids, amino acids—markers for the food protein quality, Methods for amino acids analysis.

2. General Information

2.1. Historical Remarks

The isolation and identification of certain amino acids as individual compounds started in the early 1800s. The first one, isolated by the French chemists Vauguelin and Robiquet in 1806, was asparagine, isolated from asparagus. Twenty-six years later it was established again by another researcher as a component of the protein edestin. Asparagic acid was also discovered later, being isolated from legumes by Ritthausen in 1868. Only a few years separated the isolation of the first amino acid from the discovery (1810) of the sulfur-containing amino acid cystine and its monomer, cysteine, was found many years later, in 1884. Many efforts were necessary in those times to achieve the discovery of compounds, though similar in composition and structure, that was clearly manifested by the sequential reports on amino acids. The last one to be detected was the amino acid threonine (1935) by Rose who proposed the term “essential amino acids” and established the minimum daily requirements of amino acids for optimal growth (Belitz et al., 2009).

Following the history of amino acids discoveries it could be evidenced that it was associated with development of the knowledge on proteins. Thus, for example, lysine, methionine, tryptophan, thyrosine, and serine were isolated from milk protein, casein; glutamic acid, from wheat gluten; histidine, from prolamin; isoleucine, from fibrin; leucine, from wool and muscle mass (Belitz et al., 2009). The term amino acid has been used since 1898 and in 1902 Emil Fischer and Franz Hofmeister described the binding of amino acids one to another in linear chains by an amino-group of one and carboxylic group of the other amino acid, respectively. Those compounds are known as peptides and amino acids in those times, regarded only as basic building units of the proteins.

The interest in amino acids increased particularly in the 1950–60s of the 20th century when multiple surveys and expert statements instigated the opinion of an existing world protein crisis (Webb, 2008). Considering the protein significance for human health and development of the generations, a large-scale research was initiated to search for an outcome of this issue. The solution was suggested to be in finding new protein sources and in formulating food products enriched with proteins and amino acids (single-cell protein, soybean, fishmeal, peanuts, genetically improved plants, synthetic amino acids, etc.). This scientific and industrial progress imposed and required the establishment of the amino acid content of the new products and, respectively, an assessment of their quality compared to that of animal protein foods, thus placing amino acids in the center of new productions control and activating the search for more precise assessment methods. Discussing the issue from the position of current knowledge Webb (2008) described the so-called protein crisis and clarified the causes for its appearance. The authors outlined that most possibly the world protein crisis was the result of massive overestimation of the protein needs of children, as well as that the children could manage on a lower minimum protein concentration in their diets than adults could (Webb, 2008). The current recommendations for children’s protein intake on a weight-for-weight basis are only about twofold, and not fivefold greater than those for adults as provided by past recommendations. The calculations then were based on three main sources: physiological protein needs depending on the age; population number and its distribution in age groups; and the global production of protein foods. The calculations showed the necessity of large-scale increases of protein production. All projects and technologies, developed at that time for the achievement of new protein-rich foods required lots of research, scientific potential, and abundant financing. New scientific results have been obtained though, unfortunately, in spite of the good idea and intentions of the presumable and prospected desired effectiveness was not achieved. Thus, several decades of expensive research efforts did not provide any success. An admitted cause is an eventual error in the extrapolation of the results obtained by experimental studies to human needs. Today it is considered that the more rarely met protein deficiency characteristic for some individual regions and for particular risk groups is due not as much as to the increased amounts and accessibility of protein foods as to modified recommendations and real involvement of total macronutrients building the diet. The substantial differences in the scientific views concerning the recommendations for protein, respectively, amino acids intake in the progress of this knowledge provide particular arguments in favor of further, broader research of this issue. One of the positive aspects of the state addressing protein hunger is the desire to develop more precise methods for assessment of the nutritional and biological value of dietary protein, covering mainly the composition and content of amino acids (Butikofer et  al.,  1991; Gilani et  al.,  2008; Rutherfurd and Dunn,  2011; Rutherfurd and Moughan,  2012).

The historical development of the knowledge on amino acids continues with the search for specific functions of each amino acid not only as a building element of protein but as an individual participant in the vital processes of the organism (Levesque,  2015; Levesque et  al.,  2012). Some scientific publications outlined several hypotheses for the role of amino acids in the food–health relationship. The oldest one was that of Krichevsky in the 1960s of the past century on the role of arginine, explaining the positive effect of vegetable proteins. The homocysteine hypothesis was especially emphasized in the clarification of the mechanisms for the initiation and development of cardiovascular diseases, pointing out that not only the lipid hypothesis could clarify the progress of those diseases. The most modern hypothesis that gained broad popularity is the antioxidant one where various amino acids find their role and significance. Those are the amino acids–building glutathione and the amino acid arginine, which, when modified into citrulline causes the formation of nitric oxide—a powerful relaxing agent.

Nowadays the knowledge on amino acids is focused more on their engagement in genetic mechanisms and on their inclusion in food supplements as bioactive ingredients with wide application in the current global population diet.

3. Chemistry and Classification of Amino Acids

Amino acids are low molecular organic compounds. Their specific physical and chemical properties are determined by the simultaneous binding of the amino and carboxylic group to one and the same carbon atom in their molecule. The number of amino groups differentiates them as mono- and diamino acids and the number of carboxylic groups classifies them as mono- and dicarboxylic ones. The location of the amino group versus the carboxylic group defines the isomeric forms marked with numbers or Greek letters α, ß, γ, and so on. The amino group in α-amino acids is separated from the carboxylic group by only one carbon atom, which is why it is named after the first letter of the Greek alphabet—α. When the carboxylic and amino group are separated by two and more carbon atoms, they are called β-, γ-, and so on, amino acids. Only α-amino acids are found in nature. They the generalized chemical formula shown in Fig. 10.1.

Figure 10.1 General Chemical Formula of Amino Acids.

When R is substituted (represented) by H, this is the amino acid glycine. The location of R could contain aliphatic, aromatic, heterocyclic, or other functional groups. With the exception of glycine all α-amino acids have two enantiomers—l and d forms. Proteinogenic amino acids are only l-forms.

Amino acids are the main elements building proteins. Peptide bonds link them with the nitrogen atom of the amino group of one of the acids and is bound to the carbon atom of the carboxylic group of the other acid, releasing one molecule of water and forming popypeptide chains (Fig. 10.2). The different proteins are built of a different number of amino acids arranged one after the other in a certain sequence that is gene determined, thus specific for each protein.

Figure 10.2 Peptide Chain.

More than 200 amino acids are known but only 20 of them, often called proteinogenic, are genetically determined and take part in protein synthesis. The other amino acids are not engaged in protein synthesis. They are formed in vivo during and after protein translation or in vitro, through various experimental synthesis. They are usually detected as free amino acids or as their derivatives in the biological fluids and tissues of the organism.

Depending on their chemical structure amino acids are classified as aliphatic and nonaliphatic into two main groups as presented in Table 10.1 with the respective subclassification.

Table 10.1

Amino acids classification.

Aliphatic	Nonaliphatic
1. Monoaminocarbonic a. Glycine b. Alanine c. Isoleucine d. Valine e. Leucine 2. Hydroxymonoaminocarbonic a. Serine b. Threonine 3. Monoaminodicarbonic a. Asparagic acid b. Glutamic acid	1. Aromatic a. Phenylalanine b. Thyrosine 2. Monoaminodicarbonic ω-amides a. Asparagine b. Glutamine 3. Diaminocarbonic a. Arginine b. Lysine 4. Sulfur containing a. Cysteine b. Cystine c. Methionine 5. Heterocyclic a. Tryptophan b. Histidine c. Proline d. Hydroxyproline

Having in mind the ability of the organism to synthesize amino acids and their demand, amino acids could be distributed in three groups: essential (irreplaceable), nonessential (replaceable), and conditionally essential. Essential amino acids cannot be synthesized by the animal organism. The only way to be supplied is dietary. Plants and microorganisms can synthesize all necessary amino acids, which is why there are no irreplaceable amino acids for them. Conditionally replaceable amino acids are irreplaceable only in certain cases (e.g., newborns, small children, pregnant women, people with metabolic diseases); for the other people they are replaceable.

The essential dietary amino acids are: lysine, leucine, tryptophan, valine, methionine, isoleucine, threonine, phenylalanine, and histidine.

Cysteine and tyrosine are not included because of the option to obtain them from methionine and phenylalanine, respectively. They can be conditionally essential in premature babies, whose enzyme system, providing the respective translation of amino acids, is not yet developed and in patients with liver diseases when the synthesis of those amino acids is disturbed. In case of hereditary phenylketonuria there is a genetic defect in the enzyme, converting phenylalanine to tyrosine, thus in this case determining tyrosine as an essential amino acid (Webb, 2008). For many years histidine has been accepted as an essential amino acid only in the diet of small children, but due to the sufficient recent new evidence and data about its essential role in the adult organism it was included in the basic inventory of amino acids essential for the human organism.

Amino acids, synthesized in the organism by intermediate metabolites of the carbohydrate and lipid metabolism or intermetabolize are called replaceable. The replaceable amino acids present in food protein are: alanine, arginine, proline, asparagic acid, hydroxyproline, asparagine serine, glutamic acid, glycine, and glutamine.

The role of free amino acids found in the cellular cytoplasm, forming the so-called amino acid pool, acting as a stock of the building elements for protein synthesis in the organism is also important (Ribarova et al., 1987).

The properties of amino acids depend on their chemical composition, structure, and form of existence. They, depending on the pH of the milieu, can be cations, zwitterions, and anions. The specific rotation of amino acids is strongly influenced by pH, as well. The amino acids found in proteins have the same α-C-atom configuration, determine their optical activity as l-amino acids. d-forms of amino acids have also been found in nature, most often in proteins of microbial origin.

The chemical properties of amino acids are determined by the presence of carboxylic and amino group. Their solubility in water varies widely—from easily soluble, proline, hydroxyproline, glycine, and alanine to the almost insoluble cystine and tyrosine. The polar characteristics of amino acids explains their poor solubility in organic solvents. Esterification is the typical reaction for carboxylic groups. The esters of free amino acids are able to form cyclic dipeptide or open-chain polypeptide. The reactions defined by the presence of an amino group are: acylation, alkylation, and reactions with carbonyl compounds (very important reaction for spectrophotometric quantitative determination of amino acids).

The quality of food products could be affected by the reactions of amino acids at high temperatures when side products might potentially be formed because of Maillard reaction (Webb, 2008). The moderate thermal processing leads to enhanced protein decomposition via denaturation of native proteins and inactivation of certain protease inhibitors, contained in the foods, but at the same time it triggers changes in the amino acid composition. Thus, the thermal processing of dairy products with high content of reducing sugars causes certain losses of the essential amino acid lysine. The processing at high temperatures causes the formation of internal peptide bridges between the ɛ-amino group of lysine and the carboxylic groups of amino acids in the protein (asparagines, glutamine). Those bridges are decomposed at the protein salt–acid hydrolysis and this type of damage cannot be detected by chromatographic amino acid analysis. Only the deviations in the content of cystine could be accepted as indirect proof for such thermal damage.

The combination of processing at higher temperatures and alkaline media results in the formation of the isopeptide lysinoalanine (LAL) that is a form nonassimilable by the organism. LAL formation has been observed also in nonalkaline conditions only at heating, for example, in condensed milk, acid caseinate, poultry. Soy globulin, ovoalbumin, lysozyme, and casein are natural proteins, naturally contained in foods, able to produce LAL when heated in nonalkaline media. LAL is regarded as a toxic compound as it causes cytomegalic lesions of the renal tubules. The dose, provoking this effect, depends on LAL form—free state or bound with the protein. It has been established that free LAL has a 15–2-fold stronger effect (Finot, 1983). Digestibility and net protein utilization (NPU) decrease with an increase in LAL content. The reduction in digestibility is related to the inability of trypsin to break peptide bond in the LAL crosslink. A beneficial fact is that free LAL is rarely found in the popular foods, and is in minimal amounts. Yet in 1982 the Otava meeting of Codex alimentarius on vegetable proteins discussed LAL content from the viewpoint of reduced biological value of the protein in the food rather than from the aspect of its toxicity (because of the low content).

Cysteine and cystine are particularly sensitive to the different culinary and technological processes. The thermal processing of protein results in the release of significant amounts of SH2 and other volatile sulfur compounds as a result from cystine decomposition. The oxidation processes themselves cause the formation of cysteic acid that is completely not assimilated by the organism (Chang,  1983; Ribarova et  al.,  1987). The sulfur-containing amino acid methionine is stable at high temperatures. It is quickly oxidized to methionine sulfoxide and further to methionine sulfone that also leads to its conversion into a form inassimilable by the organism.

Histidine dipeptides are stable at high temperatures—anserine, carnosine, and balenine that are used as indicators for determination of the meat type in the different technologically processed meat food products.

Many scientific surveys have been dedicated to the development of methods for determination of accessible and inaccessible amino acid forms aiming to precisely define the real biological value of food proteins. This characteristic can be achieved most accurately at combination of the chemical analysis with the biological experiment.

4. Amino Acids and Proteins Biological Value

The capacity of food proteins to satisfy the demands of the organism of nitrogen and essential amino acids determines their quality. Generally, the term presented by the wording biological value of proteins means that the extent of nitrogen retention by the organism consuming protein food products. The effectiveness of nitrogen assimilation in building the young, growing, developing organism or in supporting the nitrogen balance in the adult organism depends on protein amino acid composition and its structural features. The biological role of each individual amino acid depends on its chemical features and on the specifics of the processes it is engaged in. It is doubtless that the content of essential amino acids that are absolutely necessary for building the organism’s own proteins in the process of its development, growth, and functioning, is a criterion for the quality of food protein.

T. Voit was the first researcher to announce that the different foods and food proteins had different effect on the growth and retention of nitrogen in young animals as well as on the nitrogen balance in adult individuals. The first methods for evaluation of the quality of food proteins were proposed almost a century ago. They were indirect methods based on the growth of test animals fed with the protein foods that were in the focus of the study.

In 1946, Block and Mitchell proposed a calculation method for estimation of the biological value of protein based on its amino acid composition and referred to it as method of the amino acid score (Pellet and Young, 1980). It was based on the comparison of the determined amounts of individual amino acids in the studied protein to those in appropriate protein referent materials or amino acid mixtures. In 1973, the Expert Committee at FAO/WHO on protein requirements studied in depth the papers and standpoints of numerous research groups and commissions engaged in protein quality assessment. This work resulted in proposing a standard ideal protein, corresponding most precisely to the amino acid demands of the organism. This FAO document also listed the formula for calculation of amino acid score that was proposed as a temporary criterion. It is still used currently by the laboratories engaged with assessment of food protein. The formula is:

Amino acid score=mg essential amino acid in 1 g of tested proteinmg essential amino acid in 1 g of reference protein

The nutritionist literature uses, besides amino acid score, also the terms chemical score and protein score, all of them bearing the same meaning and content. The use of the term amino acid score reflects more precisely the meaning assigned to it as it involves the content of the individual amino acids.

The amino acid with the smallest score (respectively, smallest amino acid number) sets a limit to the biological value of food proteins and is called first limiting amino acid. The prevailing limiting amino acids in food products are lysine, tryptophan, and sulfur-containing amino acids.

The method of the amino acid score provides many benefits and disadvantages. It is simple and economical. It also enables the determination of limiting amino acids and the amounts of supplementing proteins. Its shortcomings are the lack of information about amino acid accessibility, on the extent of releasing amino acids during digestion (bioavailability), the role of essential nitrogen, and on the balance with replaceable amino acids. This information can be acquired after biological tests that underlie the determination of protein requirements, as well.

The onset of the efforts to establish the physiological requirements of protein and amino acids could be traced back decades. An expert group of FAO/WHO published yet in 1973 the physiological protein and essential amino acids age-dependent demands setting three population age groups: 3–6 months; 10–12 years; 22+ years (FAO/WHO, 1973). The scientific knowledge in this field followed its upward progress and the following years brought new suggestions from various authors to reconsider the reference protein, proposed by the expert group. Those views were supported by the results achieved by the studies of Torun et al. (1981), establishing that the values of the essential amino acids methionine, cystine, threonine, and valine in the reference protein provided by FAO in 1973 were raised. Those data correlated with the results of Pineda et al. (1981) and showed the necessity to modify the content of the ideal protein used for calculation of the amino acid score. New data were also published on the amino acid requirements of various animals and humans of different age and belonging to different risk groups, underlining the specifics of the requirements depending on the status of the individual or particular organism (Deglaire and Moughan, 2012). The demand of one essential amino acid is determined by its minimal amount necessary to maintain the nitrogen balance even in the excessive presence of other essential amino acids. When one essential amino acids is missing, there is a net loss of body nitrogen, that is, a body protein depletion. The concept of limiting amino acids has limited practical relevance in human nutrition, as the different diets have various structure and composition and the unification of one approach, such as the amino acid score requires its upgrading with additional evidence provided by biological measurements of protein quality. NPU is such a method. It is mostly used in the evaluation of animal nutrition but is sometimes implemented currently in assessing human nutrition. It reflects the percentage of protein retained in the organism of growing test animals in conditions of limited protein intake. Using nitrogen balance, NPU can be assessed directly for humans, using the following formula:

NPU=Amount of retained nitrogenNitrogen intake×100

Retained nitrogen=Intake−(Loss−Loss on protein-free diet)

NPU is about 70 typically for most diets and it is not affected by the rate of dietary animal protein (Webb, 2008).

The reported comprehensive studies confirm more and more the lack of information on the degree of digestibility of protein as a whole and of the amino acids building it. The chemical analysis of food protein and its bioefficacy in the organism are separated by a need to clarify the relationship between the chemical assessment of the protein and its physiological significance for the organism. Many various studies concentrate their attention in this direction. Batterham (1992) elaborated the formulation for bioavailability of amino acids as the dietary proportion of amino acids absorbed in a chemical form suitable for protein synthesis in the organism. It is clear that the assessment of bioavailability is a multiple-step process depending on various factors but it is also clear that only calculation approaches based on chemical analysis are not sufficient for correct comprehensive assessment of food protein quality. The need to update the information and assessment of protein biological value trigger the development and implementation of test models aiming to acquire evidence on the digestibility of amino acids in humans implementing different criteria to nutritional protein quality estimation. Attempts have been made to upgrading the amino acid score in order to ensure more real compliance with the protein requirements of the organism. The simplest approach using the amino acid score in this aspect is to evaluate comparatively the limiting amino acid in the test protein and the content of the same amino acid in 1 g of egg protein as it is a protein with a high degree of bioavailability. Another similar protein that can be used as reference protein is mother’s milk. The calculation follows the formula:

Amino acid score= mg of limiting amino acid in 1 g of test protein mg of this amino acid in 1 g of egg protein×100

This approach does not always provide correct assessment of the bioavailability of amino acids in the studied food proteins as, with some proteins difficult to digest, this calculation technique can significantly overestimate the biological quality of the tested protein and to provide a value, much different from the real content of essential amino acids. Many scientific discussions have been organized on the selection of reference protein and the development of this knowledge was presented in details in the review of Millward (2012b), analyzing consecutively the expert reports of FAO/WHO/UNU of 1985, 1991, and 2007 on the dietary protein quality. Particularly the report of 2007 was discussed in details and assessed outlining that the proofs are insufficient to enable recommendations for specific health outcomes. Nitrogen balance studies have shown 10% higher protein requirements for adults, and also some uncertainty has remained about the interpretation of results from the majority of studies on amino acid requirements (Millward, 2012b). That is why the studies continue in further search of appropriate assessment methods.

The increased production and, respectively, consumption of food supplements, containing bioactive proteins, peptides, and different combinations of amino acids requires precise analysis, specific criteria, and correct assessment of the protein enabling the establishment of their composition, and argumentation of their respective claims that, on its side demanding standardization of the relevant methodology, recommendations recorded in the scientific papers and analyses of numerous researchers (Rutherfurd-Markwick, 2012; Tome, 2012). In this aspect Gilani et al. (2008) showed the need to standardize the determination of amino acids and bioactive peptides for evaluating protein quality and to assess the protein claims of foods. Protein digestibility-corrected amino acid score (PDCAAS), which requires amino acid composition data, is the official method for assessing protein claims of foods and supplements, recommended by FAO/WHO (Gilani et al., 2008).

In the analysis of the international activities on the issue of protein quality, Gilani et al. (2008) successfully presented the development of the topic in the time. The onset period was in the period 1982–89, when the Codex Committee addressed the subject of vegetable proteins, followed by the activities of FAO/WHO (2001) and FAO/WHO/UNU (2002) expert reviews. All those years evidenced the implementation of continuous changes and amendments to the recommendations referring to the methodology for protein quality assessment. Thus, for example, after the adoption by FAO/WHO (1991) of PDCAAS (that included not only the chemical assessment of amino acid content but also the idea of their bioavailability), this method has been criticized for a number of reasons. The FAO/WHO/UNU (2002) Expert Consultation on Protein and Amino Acid Requirements endorsed the PDCAAS method with minor modifications to the calculation method, including: calculation of scoring patterns, protein digestibility, amino acid digestibility, amino acid digestibility by fecal and ileal methods, bioavailability of lysine in the processed proteins, truncation of the amino acid score, and so on (Gilani, 2012). The author of this analysis clearly showed the significance of the issue, the need to enhance the spectrum of indicators and criteria and the demand for new regulatory activities.

Certain contemporary studies have used direct test methods enabling the measuring of the requirement of indispensable amino acids, and the extent of amino acids availability. Traditionally, the methods to establish amino acids availability are based on intestinal absorption or digestibility. The dietary intake of amino acids increases at taking into account the nitrogen balance and/or growth of the test animals until constant levels (plateaus) were reached. Other methods utilized tagged atoms (Elango et al., 2012), and another group of methods emphasized on the oxidation of particular amino acid differing from test indispensable amino acids—they are called indicator amino acids (Elango et  al.,  2012; Humayun et  al.,  2006). The oxidation of indicator amino acids was accepted as a marker of protein synthesis that decreased with the increasing of the level of oxidized amino acids (Elango et  al.,  2008; Levesque et  al.,  2012).

The metabolic availability method, based on the indicator amino acid oxidation technique was first tested, for example, on amino acids lysine availability in growing pigs, fed with peas as a protein source (Moehn et al., 2005). Some other studies have shown the effect of heat treatment on the bioavailability of lysine, using also the indicator amino acid oxidation technique and others have adapted the metabolic availability method for use in humans (Levesque,  2015; Prolla et  al.,  2013). Many studies are dedicated to the selection of test animals, adequate for the task, as it is necessary that they match as much as possible to the human physiological specifics. Thus, besides the routinely used laboratory rats, models with pigs whose upper digestive tract is anatomically and physiologically more similar to that of humans, and, besides that, they are fed with foods included in the human diet, are promoted (Deglaire and Moughan, 2012). In clinical tests the knowledge on the behavior of amino acids along their route in the organism is particularly important, that is, assessment of their bioefficacy. In this aspect, special scientific attention has been paid to anabolic amino acids—leucine, arginine, and citrulline (Jonker et al., 2012), and other authors have focused on distinction between nutritional dispensability and clinical efficacy of amino acids as homocysteine and cardiovascular disease (Wang et al., 2015); homocysteine and cancer and other diseases (Deng and Zheng,  2012; Lin et  al.,  2010; Miller et  al.,  2013; Wang et  al.,  2012; Zhang et  al.,  2012;  2015); glycine as an immunonutrient (D’Mello, 2012); tryptophan—medical aspects (Palego et al., 2016); amino acids and kidney diseases (Ribarova et  al.,  2003; Vazelov et  al.,  1999; Vazelov and Ribarova,  2013); excitatory amino acids and neurodegenerative disorders (Flores et al., 2012), and many others (Jonker et  al.,  2012; Katsanos et  al.,  2016).

Ghosh et al. (2012) presented a very interesting study, examining the effect of adjusting total dietary protein for quality and digestibility (PDCAAS), including data from 116 countries, from FAO/FBS (food supply), from USDA nutrient tables, and from other data sources, by using modern statistical analyses. They have shown that protein and utilizable protein availability were independently and negatively associated with stunting (P = 0.017). The utilizable protein, according to the authors, was a better index of population impact of risk/prevalence of protein inadequacy (Ghosh et al., 2012).

Those facts imperatively challenge the nutritionists with the important task to unify the criteria for assessment and standardization of the used analytical methods and approaches for calculation and processing of the results revealing the amino acid content of food products.

5. Amino Acid Analysis of Food Proteins

The methods for determination of amino acids are among the most difficult ones for implementation in food analytical chemistry. They progress through several stages. Besides the stages, relevant for almost all methods for food analysis (sampling, homogenization, quantitative, and qualitative determination), there is a substantial difference in the processing of laboratory samples including determination of total protein, protein hydrolysis, and derivatization of the obtained amino acids. Hydrolysis and derivatization are most critical for the quality of the analyses (Finley,  1985; Rutherfurd and Moughan,  2012).

Sampling is a very important stage of the analytical procedure. The reproducible sampling is associated with the heterogenicity of the products, the content of high amounts of fat in animal products and fibers in plant foods. Fats impede the proper homogenization of the sampled aliquot parts of the food product while with the greater amount of fibers the homogenization could hardly be realized only by grinding.

Special attention is paid to sampling in the basic European regulations on the content of various chemical substances in foods. The fundamental international standard EN ISO 17025 explicitly postulates that such methods must have a developed optimal sampling plan. The number of collected samples most generally depends on the type of analyzed foodstuff, the size of the lot and, last but not least, on the repeatability (coefficient of variation) of the method itself.

Sample preparation for amino acid analysis covers the following stages: homogenization; determination of total protein; hydrolysis and derivatization of the obtained amino acids.

The homogenization depends on the type and structure of the particular samples.

A mandatory step for all samples is to convert them into air-dry state, recording the weight of eliminated water. Similarly, the greater fat amount is to be removed, particularly concerning foodstuffs of animal origin (Finley,  1985; Ribarova et  al.,  1987; Rutherfurd and Moughan,  2012). After this preliminary processing the homogenization implements two main approaches: grinding for most sample types and cutting into pieces with further grinding of the samples with higher fiber content.

5.1. Determination of Total Protein

In order to establish the amino acid composition of the foods knowledge on their total protein content, including all protein types, found in the product composition is essential in the first place. Thus, cereals contain albumins, globulins, prolamins, glutelins, with different amino acid composition and content, respectively. The use of the total protein content in the calculation of amino acids in a product enables the elaboration of an assessment of its biological value based on essential amino acids. If the aim is to determine the composition of a particular protein, it has to be preliminarily separated from the other protein components, building the particular product. The total protein content is an important indicator both for the foods and for the assessment of the dietary intake. The protein content in foods is determined routinely by Kjedahl’s method, one of the oldest and standardized methods in food analytical chemistry, which enables the simultaneous determination of protein and nonprotein nitrogen. Although it is not possible to distinguish quantitatively the nitrogen, obtained from the proteins and from the nonprotein compounds in the food, this method is a preferred analytical approach because of the comparatively good analytical parameters, reasonable cost, and the option for automation (Greenfield and Southgate, 1992). It is based on the determination of total nitrogen in a sample, which, multiplied by a coefficient, relevant for the sample, produces the total protein amount. And, although at nitrogen determination the errors can be minimized, the values of calculated protein bear always some uncertainty. This is due to the fact that one part of the nitrogen is not protein, that is, foodstuffs contain a significant number of substances, often in great amounts that increase the nitrogen content of nonprotein origin. Those are purines, pyrimidines, urea, amino sugars, creatine, creatinine, methylamino compounds, and so on. Only the nitrogen from the nitrate and nitrite salts contained in the samples is not included in the total nitrogen content as it is released into the environment during the analysis. Nonprotein nitrogen can often represent one half and even more of the total determined amount. Fish and seafood are rich in nonprotein nitrogen. The ratio between protein and nonprotein nitrogen to the greatest extent depends on the origin (nature) of the sample. The effect of interspecies differences is not to be neglected, as well as the implemented food processing technology. There is evidence for efforts to achieve greater precision when calculating total nitrogen, protein nitrogen, and amino acid nitrogen as well as separate determination of nucleic acids, but Kjedahl’s method is still relevant in routine protein analysis. The main points in this analysis are the selection of a catalyst and digestion methods. The temperature during digestion is a critical point and it must be kept between 370 and 410°C. The catalyst CuSO₄/TiO₂ is preferred to HgO because of environmental concerns. There are also other methods, such as Dumas’s method, determining total nitrogen by combustion with oxygen, followed by reduction to liberate nitrogen gas. There is an automated version of the method with nitrogen evolved being measured by thermal conductivity. Comparative studies of the two methods have shown good compliance with very small increase of the values obtained by Dumas’s method. To make things easier at application of Kjeldahl’s method, numerous companies have developed and offered semiautomatic and automatic apparatuses. The international experience has enabled the calculation of conversion coefficients for nitrogen-to-protein conversion. For most basic food products with high protein content the recalculation with coefficients reduces to a certain degree the deviations in the determined total protein amounts. Those coefficients have been recommended by FAO/WHO (FAO/WHO, 1973). Some of them are presented in Table 10.2. It is recommended to list always the used recalculation coefficients when presenting data for food protein content.

Table 10.2

Coefficients for conversion of total nitrogen into total protein.

Food Products	Factors
Wheat	5.83
Wheat flour	5.70
Pasta	5.70
Rice	5.95
Rye, barley, etc.	5.83
Peanuts	5.46
Soy	5.71
Walnuts	5.18
Various seeds	5.30
Milk	6.38
Cheese	6.38
Other products	6.25

5.2. Protein Hydrolysis

The procedure that is most critical for the analytical quality and getting satisfactory results is the one concerning the breaking of the peptide bonds, which, as a principle, are very strong from a chemical point of view. Another important issue is that the different amino acids are significantly variably stable to the possible chemical mixes applicable in the hydrolysis (strong acids or bases). The presence of oxygen in the mix during the hydrolysis is also a risk for reduced analytical recovery of the analysis of certain amino acids. On the other hand, protein type, its molecular mass, and qualitative composition are important as they are responsible for the moment when an acid will be released from the protein molecule, respectively, how long it will be subject to the decomposing effect of the aggressive hydrolysis media. The most unstable amino acids are methionine, cystine, threonine, and tryptophan. Branched-chain amino acids need a longer hydrolysis time in order to be separated from the peptide chain (Benson et  al.,  1981; Ribarova et  al.,  1987). The protein composition in food products varies in a broad range. Thus, the achievement of perfect hydrolysis conditions, specific for the respective products is a difficult task addressed by numerous scientific studies and literature reviews (Liu and Chang,  1971; Pellet and Young,  1980; Williams,  1986). Different versions of hydrolysis are applied for protein analysis: acid—using various acids; alkaline—with different bases; enzyme—involving different peptidases. There are many published methods with various hydrolysis conditions concerning hydrolyzing chemical mixes and hydrolysis time, as well as the used heating equipment (muffle furnaces and microwave ovens) (Kabaha et al., 2011). The book Amino Acids in Higher Plants, J.P.F. D’Mello, ed., 2015, presents rich information concerning the diversity of conditions used in protein hydrolysis though the majority of them are not implemented in everyday routine practice. This fact is explained to a certain degree by the nonlinear relationship between the degree of protein decomposition and the duration of the hydrolysis process in similar other conditions (chemical agent and temperature). This relationship is close to exponential and is different for the particular amino acids, and, particularly, for more unstable ones. When more accurate amino acid composition data are required, least squares nonlinear regression can be used to estimate the amino acid content of a protein source. This method corrects for losses of amino acids during hydrolysis based on the amino acid yield determined using a range of hydrolysis times. In routine analyses those differences can be compensated to a great extent by adequate method validation.

Some scientists yet in the past century, aiming at most effective hydrolysis, have proposed to perform five individual hydrolyses: three with 6 N HCL, duration respectively 24, 48, and 74 h; one acid after oxidation with performic acid and one hydrolysis with 6 N NaOH (Pellet and Young, 1980). The extrapolation of the obtained results from the first three acid hydrolyses enables the more precise determination of the amino acid composition of the protein. The oxidation with performic acid causes the transformation of cystine and cysteine into cysteic acid and of methionine—into methionine sulfate. Those compounds are resistant to acid hydrolysis, and, after it has finished, they can be determined quantitatively together with the other amino acids. In some cases cysteine oxidation to cystine is practiced through alkalization of the hydrolysis after it was finalized down to pH 6.8 and leaving it to the effect of the ambient air for 4 h. The essential amino acid tryptophan is almost fully decomposed by acid hydrolysis. That is why its determination is made after alkaline or enzyme hydrolysis (Andrews and Baldar,  1985; Lin et  al.,  1985; Vries et  al.,  1980). Contradicting evidence has been published on the methods for determination of sulfur-containing amino acids. According to Pienjacek et al. (1975), the application of preoxidation of sulfur-containing amino acids with performic acid provides certain possibilities for errors as the analyzed food product itself can contain oxidized forms of sulfur-containing amino acids that are not assimilated by the organism. The assimilation itself depends on the oxidation degree. While methionine sulfoxide is assimilated, methionine sulfone cannot be assimilated by the organism. Many researchers, striving to save time, looked for more rational routes to apply such hydrolysis that provided maximum real amounts for all amino acids, with minimum losses. Liu and Chang (1971) were successful in this effort, hydrolyzing the protein with 3 N solution of p-toluenesulfonic acid. Penke et al. (1974) have performed hydrolysis with mercaptoethanolsulfonic acid, which preserved tryptophan up to 95%. Simpson et al. (1976) achieved the same results using hydrolysis with methanesulfonic acid. All approaches implemented for hydrolysis have advantages and disadvantages. The major disadvantages are the low analytical recovery and poor reproducibility that significantly reduce the informative value of the overall analysis and can even lead to false conclusions in some cases. The hydrolysis conditions (temperature, duration, sample/acid, sample/base ratio) must be maximally optimized with a view to improving the reproducibility of this stage of amino acid analysis.

5.3. Qualitative and Quantitative Determination of Amino Acids

After the protein was decomposed to amino acids their qualitative and quantitative determination is performed (derivatization, chromatographic separation of the individual amino acids with further detection). The literature provides many various descriptions of approaches and techniques for derivatization, separation, and detection. The derivatization of the produced by hydrolysis amino acids also poses certain risks for worse results (analytical recovery), although, with the time certain techniques have established themselves and the optimization is significantly easier to perform compared to protein hydrolysis.

The initial periods of the development of protein analysis evidenced the comprehensive implementation of microbiological and paper chromatographic methods that have now only historical significance. The next step of the progress of the methodology were the thin layer chromatographic methods with good resolution and low reproducibility. Some impetus was given to those methods with the development of densitometry, contributing to better accuracy, improved reproducibility, and their implementation in routine practice.

Amino acid determination by gas–liquid chromatography requires their quantitative transformation into volatile derivatives, such as methyl, trimethylsilyl, and N-butyl-N-trifluoroacetyl esters (Bos et  al.,  1983; Pellet and Young,  1980). The gas chromatographic conditions require preliminary good cleansing of the hydrolyzates. The quantitative derivatization requires no less efforts without guarantees for good reproducibility. Those difficulties place gas chromatographic methods in the rear of available methods for amino acid analysis and currently they are quite rarely used.

For the separation of amino acids from the obtained mixture after the hydrolysis, because the middle of the 20th century, liquid chromatograph columns using low or high pressure have gained wide popularity (Andrews and Baldar,  1985; Deyl,  1986; Sarwar et  al.,  1983; Sarwar,  1984). Chronologically, the first ones were developed much earlier and were based on the method of Spackman et al. (1958). The columns have relatively large diameter filled with ion exchange resin, the elution is realized with different buffer solutions at low pressure. Because amino acids generally do not absorb light at any useful wavelength, although tryptophan and to some degree tyrosine and phenylalanine absorb significantly at 280 nm, they must be derivatized. Initially, the derivatization was performed with ninhydrin after separating the amino acid mixture with ionexchange chromatography (postcolumn derivatization) (S. Moore). The intensity of absorption of the colored compounds produced by derivatization is determined at wavelength 570 nm for amino acids and 440 nm—for imino acids (proline and hydroxyproline). The mechanization of the overall analytical process resulted in the creation of a number of models and versions of mechanical amino acid analyzers by different companies, used in routine practice. Blackburn (1973) and Benson et al. (1981) published reviews containing details about the development of equipment and methods using ion exchange chromatography.

Although the evaluations of the obtained results by this methodological approach to amino acid analysis have been good, the long time needed for the proper separation of the individual amino acids is still a problem. In some cases the sensitivity is unsatisfactory, thus leading to increasing the sample amount and, hence, to deterioration of the analytical parameters.

In the 1980s, high-performance liquid chromatography (HPLC) was nominated as the most effective method for amino acid determination. It helped to achieve maximal speed, sensitivity, and reproducibility. The majority of described similar methods imply amino acid derivatization aiming at their fluorescent detection (pre- or postcolumn). The more prevalently applied technique is precolumn derivatization with o-phthalaldehyde or phenylisothiocyanate; 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate; dansyl chloride, benzyl chloride, fluorescamine (Chang, 1983; Williams, 1986). The implementation of HPLC with reverse-phase column, precolumn derivatization, and fluorescent detection allows the determination of very small amounts of aminoacids (of the order of picograms) (Andrews and Baldar,  1985; Chang,  1983; Deyl,  1986; Turnell and Cooper,  1982). A proper combination of chromatographic conditions can enable the separation of d- from l-isomers of amino acids (Pellet and Young, 1980).

The available scientific publications have reported numerous studies with a substantial number of derivatization agents that have not been widely implemented in practice. Agents for precolumn derivatization are preferred most of all as the obtained compounds are very well separated by reverse phase HPLC and manifest considerable fluorescence thus guaranteeing reliable identification and high sensitivity.

Recently, after liquid chromatography coupled to mass spectrometers were introduced in the chemical analyses of foods and the relative reduction of the equipment costs, more and more numerous researchers have attempted to implement this technique in the analysis of amino acids. The principal advantages are the high selectivity and sensitivity, good speed of the analysis progress and, before all, the possibility to analyze mixtures of amino acids without derivatization. As, in principle, it is better to use reversed phase chromatography columns with this equipment, and amino acids are strongly polar and cannot be separated well with those columns, the researchers have used ion-pairing reagents. Salt-acid extracts obtained by hydrolysis create certain difficulties and this equipment is mainly used for the determination of free amino acids in some food products and biological materials (Thiele et al., 2012). In spite of the beneficial cheaper costs of this equipment, it is still unavailable at most laboratories engaged in protein analysis.

Validation of the analytical procedure and results: Verification program (EN ISO 17025 general requirements for the competence of testing and calibration laboratories).

Method validation is a procedure where several parameters are calculated based on a performed test. Those parameters provide objective information about the possibilities of the particular method and, respectively, for the reliability of the obtained results:

• • Linear range (linearity): The difference between the smallest and the greatest concentrations with linear relationship between concentration/signal (response) in the range between them.
• • Sensitivity: It is defined by two values: limit of detection and limit of quantification.
• • Repeatability: Relative standard deviation (coefficient of variation). It is calculated using the results for multifold analysis of one and the same sample in equivalent laboratory conditions.
• • Analytical recovery: In the case of amino acid analysis, the most appropriate approach is the one that uses referent materials, but, mainly because of financial concerns the routine laboratory practice still implements the so called method of the standard addition.

The introduction of a system for intralaboratory analytical quality control of the performed analyses (AQA) is essential for the verification of the results.

Principally, the system consists of two parts:

• • Periodical checks of the calibration of the analytical equipment;
• • Periodical analyses of samples with known concentration of amino acids.

5.4. Presentation of the Results

Amino acids are usually expressed as mg/g of nitrogen or as g/16 g nitrogen (approximately 100 g protein). The basis of expression should be chosen to fit the specific use of the data. The most common basis of expression for other food ingredients is g/100 g of edible portion of food.

The method implemented for processing and calculation of the data obtained after the amino acid analysis is important for the accuracy of expression of the amounts of amino acids. The application of various calculation approaches leads to substantial differences between the final results from the amino acid analysis of one and the same product.

In most of the published tables the sum of amino acids per 100 g protein (16 g nitrogen) exceeds significantly 100%. This excess remains frequently unnoticed as the losses during the hydrolysis process are in the opposite direction and are of equivalent value. Many authors have tried to compensate this exceeding of the value by the idea to extrapolate the results to a different value: 90%, 95%, and 100% (normalization).

Having in mind that protein hydrolysis is one of the stages of amino acid analysis with poor reproducibility, the additional calculation error is inadmissible.

It has been commonly accepted that the content of each amino acid in g/16 g nitrogen should be calculated using the formula:

C=M⋅V⋅16104⋅W

With: C—the molar concentration, μmol/cm³; M—molecular mass; W—sample mass, g; V—final volume, cm³; 16 g—nitrogen content per 100 g protein

In the protein molecule the amino acids participate as amino acid residues (one molecule of water is missing for each peptide bond). It is reasonable to correct the molecular mass of the individual amino acids in the above formula as M—M(H₂O). In this calculation method the sum of amino acids is smaller than the total protein content. The difference corresponds to the actual losses resulting from the hydrolysis process. The calculation is not correct referring to the free amino acids contained in the food products as well as in relation to the final amino acid residues in the protein molecule. This error, though, is negligibly small as the proteins in the food products are with high molecular mass and the free amino acids are in very low concentration.

The great differences in the analytical recoveries of the salt–acid hydrolysis require that the calculation with corrected molecular masses must absolutely be combined with normalization of the results. In this way the coefficient of variation is significantly improved and the amino acid content corresponds more precisely to the value of total protein.

Table 10.3 presents the amino acid content of egg protein analyzed 7 times implementing both calculation approaches.

Table 10.3

Amino acid composition of egg protein.

Molecular Masses	M			M-MH2O
Calculation Methods	Without Normalization			With Normalization
Amino Acids g/16 g N	X ¯	± ∆ X ¯	CV%	X ¯	± ∆ X ¯	CV%
Essential
Valine	7.33	0.28	3.65	6.68	0.06	0.86
Isoleucine	5.65	0.24	3.98	5.25	0.07	1.35
Leucine	9.20	0.43	4.50	8.54	0.08	0.88
Lysine	7.61	0.31	3.90	7.18	0.18	2.40
Methionine	4.04	0.32	7.46	3.82	0.18	4.43
Cystine	2.83	0.25	8.41	2.59	0.20	7.45
Treonine	4.78	0.29	5.79	4.37	0.11	2.43
Tryptophan	1.16	0.18	14.78	1.14	0.20	17.03
Thyrosine	4.15	0.37	8.48	4.02	0.24	5.59
Phenylalanine	6.6	0.24	3.45	6.33	0.12	1.74
Replaceable
Alanine	6.55	0.26	3.82	5.62	0.06	0.94
Arginine	6.31	0.30	4.59	6.09	0.10	1.59
Aspartic acid	10.59	0.46	4.13	9.86	0.25	2.37
Glycine	3.77	0.16	3.92	3.09	0.03	1.07
Glutamic acid	14.14	0.70	4.69	13.35	0.13	0.90
Proline	3.80	0.27	6.77	3.44	0.18	4.99
Serine	6.92	0.38	5.27	6.17	0.10	1.56
Hydroxyproline	—	—	—	—	—	—
Histidine	2.59	0.12	4.28	2.47	0.04	1.63
Total amino acid content	108.3	—	—	100	—	—

±∆X¯=X¯±s⋅tN, confidence interval of the value; S, standard deviation; t, Fisher’s coefficient; N, number of tests; CV = sx 100% , coefficient of variation (relative standard deviation).

In the course of the amino acid analysis, in spite of the great progress in the development of analytical technique, there are still not fully clarified issues and problematic steps that require comprehensive additional studies in order to elaborate and establish standardized procedures enabling the exchange of relevant data and scientific evidence.

6. Conclusions

Considering the rich and continuously enhancing information during the last two centuries on the role of amino acids as a basic building unit of food proteins and as individual bioactive compounds, engaged in numerous biochemical reactions and physiological processes in the organism, it could be stated that this scientific issue continues to pose a challenge to science, in spite of the significant achievements in the field.

The amino acids issue is discussed in three main aspects. The first one refers to the assessment of the quality of protein foods based on the content of essential amino acids. The second aspect concerns the role of amino acids in the absorption of proteins and covers biological analyses and approaches, and the third aspect reveals the development and upgrading of analytical methods used for analysis of the amino acid content. In spite of the introduction of modern analytical equipment, there are still unclear and unresolved problems along the overall chain of the methodological analytical course, presented in details in the relevant parts of this chapter.

We have also presented evidence from our experience in the analysis of amino acids in Bulgarian food products listing the approach we have applied to the calculation of the content of amino acids in relation to the amount of protein they are contained in. In order to enable the exchange of data and information through the available networks it is necessary to unify the analytical methods and calculation approaches. The establishment of a standardized procedure is the only and unique prerequisite for the organization of a database for the amino acid composition of food products. The need for such a database is urgent and its realization requires exchange of experience and knowledge.

The increasing amount of scientific information during the last years, concerning the individual activity and biological role of each particular amino acid, requires comprehensive analysis and further studies aimed at clarifying the mechanisms of action and the effectiveness of amino acid intake in the form of pharmaceutical products, enriched foods, or food supplements. The concentration of more scientific interest and energy in the field of amino acids will contribute to the better substantiated, supported scientific evidence explanation of the claims listed on the product label on products containing amino acids and, respectively, bioactive proteins. This provides a response in the two dimensions of the issue—science and practice—associated with consumers’ demands.

The progressing unbalance between the global production of foods, availability of food sourced, and raw materials on one hand, and the demographic burst and the physiological requirements of healthy, preventive, and curative nutrition on the other, support even stronger the significance of the protein quality issue and the necessity for its optimal development and resolution. Food proteins are at heart of a healthy diet.

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Further Reading

Alarcyn-Flores MI, Romero-Gonzalez R, Frenich AG, Vidal JLM, Reyes RC. Rapid determination of underivatized amino acids in fertilizers by ultra high performance liquid chromatography coupled to tandem mass spectrometry. Anal. Methods. 2010;2:1745–1751.

FAO, 2011. Dietary protein quality evaluation in human nutrition. Report of an FAO Expert Consultation, 31 March–2 April, Auckland, New Zealand.

FAO, 2013. Food and Nutrition Paper 92. Food and Agriculture Organization of the United Nations, Rome.

Inglis AS. Single hydrolysis method for all amino acids, including cysteine and tryptophan. Methods Enzymol.. 1983;91:26–36.

Makedonski L, Peycheva K, Stancheva M. Laboratory Manual for General and Organic Chemistry Students of Medicine. Varna, Bulgaria: STENO; 2011.

Yan LD, Li Ping Z. Analysis of clinical significance for the detection of serum homocysteine for digestive system cancer patients. Mod. Prevent. Med.. 2012;39:1754–1755.

Yang, Y., Chang, L., Lu, Y., Yang, L., 2010. Analysis of amino acids in foodstuff by nuclear magnetic resonance. In: Yu, W., Zhang, M., Wang, L., Song, Y. (eds), Proceedings of the 3rd International Conference on Biomedical Engineering and Informatics (BMEI). Institute of Electrical and Electronics Engineers, New York, NY, pp. 750–754.