Chapter 2: Proteins PDF

Chapter 2: Proteins |1 Proteins in Food Proteins are essential for human Chapter 2...

Chapter 2: Proteins |1 Proteins in Food Proteins are essential for human Chapter 2 health. They build your muscles, Proteins Proteins bones, skin and hair structure. They defend you against infections and as enzymes they catalyse many reactions. In food, proteins for example provide structure (gluten in bread), hold water (in meat), stabilise foams (cappuccino foam) or emulsions (mayonnaise). The main sources of proteins are meat, fish, eggs, milk, legumes (beans) and grains. These raw materials are processed into products such as cheese and yoghurt (from milk) and tofu (from soybeans), or into ingredient such as whey proteins (from milk), soy proteins and gelatin (from bones). The properties of proteins can be changed by chemical reactions that occur during processing. Examples are denaturation, Copyright © 2024 Laboratory of Food Chemistry, Wageningen University. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by Maillard reactions, or formation any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. of lysino-alanyl derivates. Proteins in Food 2 2.1 Introduction and learning outcomes Table of contents Proteins in Food..................................................................................................................................................... 1 Proteins in Food..................................................................................................................................................... 1 2.1 Introduction and learning outcomes................................................................................................................. 3 2.2 Occurrence and structural properties........................................................................................................... 6 2.2.1 Amino acids......................................................................................................................................... 6 2.2.2 Peptides............................................................................................................................................... 9 Peptide bond.................................................................................................................................................. 9 Nomenclature.............................................................................................................................................. 10 Examples of naturally occurring peptides in foods..................................................................................... 11 2.2.3 Proteins.............................................................................................................................................. 13 Structural forces in protein folding and stability......................................................................................... 14 2.3 Protein classification.................................................................................................................................. 17 2.3.1 Classification based on conformation................................................................................................ 17 2.3.2 Classification based on solubility...................................................................................................... 18 2.4 Effect of pH on the charge of amino acids.................................................................................................. 19 2.5 Chemical changes of amino acids and proteins.......................................................................................... 21 2.5.1 Chemical reactivity of free amino acids............................................................................................ 21 Decarboxylation and deamination of the -carboxylic acid and -amino group........................................ 22 Strecker degradation at the -amino and the  -carboxyl group................................................................. 22 Maillard reactions at the -amino group..................................................................................................... 23 2.5.2 Reactions at the functional group of the side chain........................................................................... 23 Formation of S-S bridges and reshuffling of S-S bridges........................................................................... 24 Oxidation of Methionine............................................................................................................................. 24 Formation of isopeptide bonds.................................................................................................................... 25 2.5.3 Protein hydrolysis.............................................................................................................................. 28 2.6 Physical changes – Protein denaturation....................................................................................................... 28 2.7 Roles of proteins, peptides and amino acids in food................................................................................... 30 2.7.1 Nutrional value of proteins................................................................................................................ 30 2.7.2 Taste of amino acids and peptides..................................................................................................... 32 Taste of amino acids................................................................................................................................... 32 Taste of peptides......................................................................................................................................... 33 2.7.3 Solubility of proteins......................................................................................................................... 34 2.7.4 Water holding capacity...................................................................................................................... 36 2.8 Analysis of proteins..................................................................................................................................... 38 2.8.1 Analysis of content............................................................................................................................ 38 Nitrogen content.......................................................................................................................................... 38 UV absorption amino acids......................................................................................................................... 39 2.8.2 Analysis of composition.................................................................................................................... 40 SDS gel Electrophoresis.............................................................................................................................. 40 2.9 Literature.................................................................................................................................................... 41  Chapter 2: Proteins |3 2.1 Introduction and learning outcomes Amino acids, peptides and proteins are important elements of foods. They contribute to the nutritional value of foods, since they are the source of the building blocks (amino acids) required for the biosynthesis of endogenous proteins in humans. See for their schematic representation Figure 1. Proteins can also contribute to the physical properties of foods by their ability to form and stabilize emulsions and foams, or gels and fibrillar structures upon processing. Moreover, amino acids and peptides can contribute to the flavour of foods. This contribution to taste can be direct (since they have a taste already) or indirect (since they are precursors of aroma compounds formed during the processing of foods). Proteins Figure 1. Schematic representations of an amino acid, peptide and protein Traditionally milk, meats, eggs, cereals, legumes and oilseeds are major sources of food proteins (see Table 1). However, because of growing world population, demand for good quality food (and animal feed) protein increased and therefore also novel sources of food proteins are explored such as protein from potato and algae. Each source has a different protein content (g protein / 100g dry matter), but also the types of proteins that are present will be different. While most of these proteins can be used as food proteins, the types of applications where they can be used depends on the protein properties. For practical purposes food proteins are usually evaluated for their nutritional quality, their digestibility and their techno-functional (e.g. foaming) properties in food products, and their availability (how much is available) and the sustainability to grow these proteins sources. We will first discuss the protein structure and amino acid composition and how these are affected by chemical reactions or physical changes in the structure, so we can then understand how these factors influence the nutritional quality and (techno)-functional properties of proteins (§2.7). Table 1 Protein and non-protein content (in g/100 g dry matter) in different agricultural products %(w/w) Egg-white Meat Fish Milk Soy Pea Nuts Potato Algae Moisture 90 70-75 68-80 88 ~10 ~10 ~5 75 ~10 Protein 85 74-94 78-90 27 41 28 13-27 10 36 % Non-protein 0.6 3-6 9-18 3 3 8 n.d. 24 25 Nitrogen/ Total N 4 2.1 Introduction and learning outcomes Learning outcomes for the chapter proteins §2.2  Know and recognize the structure of an amino acid, (oligo)peptide and protein. §2.2.1  Know and recognize the general structure of amino acids.  Recognize the carboxylic acid group/ amino group/ side chain/ α-C-atom/ D or L enantiomer.  Be familiar with the amino acid classification based on side chain properties.  Be able to classify amino acids based on the side chain. §2.2.2  Recognize N- and C-terminal.  Know how a peptide bond is formed.  Understand why a peptide naturally occurs in its trans-configuration.  Understand the words di-, tri-, tetra-, oligo- and polypeptides, and protein.  Recognize the α/β/γ-C-atom.  Know what an iso-peptide is. §2.2.3  Know what the primary, secondary, tertiary and quaternary structure of proteins comprises.  Know how different molecular forces work in protein interactions: - Covalent bond (disulphide bonds) - Electrostatic interactions - Hydrogen bonds - Hydrophobic interactions - Van der Waals interactions §2.3.1  Know how globular, fibrillar and random-coil-like proteins avoid exposure of hydrophobic parts to water. §2.3.2  Know the nomenclature of different classes of proteins. §2.4  Recognize which parts of an amino acid can be charged.  Know and understand the definition of the iso-electric point.  Know how net charge is influenced by pH.  Be able to draw a titration curve. §2.5  Understand how protein properties are affected by chemical reactions. §2.5.1  Know the decarboxylation and deamination reactions.  Know the Strecker degradation reaction.  Know where amino groups can be located that can react in the Maillard reaction.  Understand what effect the Maillard reaction has on the net charge of (parts of) protein.  Understand how isopeptide bonds are formed.  Understand how hydroalanine residues are formed. §2.5.2 + 2.5.3  Know how biological value of proteins can be affected.  Know in what reactions disulphide bonds can be involved.  Understand the mechanism of isopeptide formation.  Chapter 2: Proteins |5  Understand the mechanism of crosslinking of protein via formation of formaldehyde.  Know the hydrolysis reaction of protein. §2.6  Know what denaturation is and under which conditions is occurs.  Know what the effects of denaturation of protein are. §2.7.1  Understand definitions of protein content, protein quality, and amino acid availability. §2.7.2  Know what amino acid properties influence the taste in what manner (D/L, polar/non-polar).  Know what peptide properties influence the taste in what manner. §2.7.3 Proteins  Know which factors influence the solubility of protein: - Hydrophobic/hydrophilic surface - Charge on surface - Solubility minimal at pH=pI - Salt concentration (ionic strength) §2.7.4  Understand how water holding capacity is influenced by pH and salt concentration. §2.8  Understand definitions of protein content and composition  Know the analysis techniques to determine protein content or protein composition. 6 Occurrence and structural properties 2.2 Occurrence and structural properties 2.2.1 AMINO ACIDS In nature, more than 300 different amino acids have been identified. Of these amino acids, the majority is only present as free amino acids, and only ± 20 different amino acids are commonly present in proteins. By definition, amino acids contain at least one amino group (-NH2) and one carboxyl group (-COOH). In proteins these groups are attached to the α-carbon atom next to a side chain and hydrogen atom (see Figure 2). With the exception of glycine, this α-carbon atom is a chiral atom. In glycine the α-carbon atom is not chiral, since for glycine the R group is a hydrogen (H). Due to the presence of this chiral atom, the amino acids can occur as stereoisomers: the L- and D-enantiomers (see Figure 3). All proteins found in nature contain only L- amino acids. Figure 2. Basic structural units of an amino acid In the projection formula of amino acids, for the L-configuration the amino group is shown on the left side, with the carboxylic group on the top, and the side chain (R) on the bottom. Figure 3. L- and D-stereoisomers of tyrosine (Tyr) Each type of amino acids has a unique side chain (Table 3). The amino acids are encoded by either a single- letter code or three-letter code (Table 3). Based on the properties of the side chain, amino acids are classified into three groups (see Table 2): 1. amino acids with non-polar (hydrophobic) non-charged side chains: aliphatic (linear or branched), or aromatic (cyclic) hydrocarbon chains 2. amino acids with polar (hydrophilic) non-charged side chains: amide (-CO-NH2), thiol (-SH), or hydroxyl (-OH) groups 3. amino acids with polar (hydrophilic) charged side chains: carboxyl (-COOH) or amine (-NH2) groups  Chapter 2: Proteins |7 Based on the ability of the body to synthesize the amino acids, the ± 20 amino acids present in proteins are divided into three categories: indispensable, conditionally dispensable and dispensable amino acids. See also § 2.7.1 for more information on the nutritional value of amino acids Table 2: Classification of amino acids occurs according to their side chain Class Functional group of Amino acids side chain non-polar, non-charged side chains aliphatic group glycine, alanine, leucine, - low solubility (hydrophobic) isoleucine, valine - mainly present on the inside of the protein aromatic group phenylalanine, tryptophan miscellaneous proline, methionine polar, non-charged side chains hydroxyl group serine, threonine, tyrosine Proteins - reactive (-OH) - hydrophilic (good solubility) sulfhydryl group cysteine - mainly present on the outside of the protein (-SH) amide group asparagine, glutamine (-CO-NH2) polar, charged side chains carboxyl group glutamic acid, aspartic acid - charge depends on pH (-COOH and -COO-) - reactive amino group lysine, arginine, histidine - hydrophilic (good solubility) (-NH2 and -NH3+) - mainly present on the outside of the protein Table 3. The structure of amino acids (protein building blocks), three and one letter symbols. Glycine (Gly. G) L-Alanine (Ala. A) L-Leucine (Leu. L) Non-polar, non-charged side chains L-Isoleucine (Ile. L) L-Valine (Val. V) L-Phenylalanine (Phe. F) L-Tryptophan (Trp. W) L-Proline (Pro. P) L-Methionine (Met. M) 8 Occurrence and structural properties Polar, non-charged side chains L-Serine (Ser. S) L-Threonine (Thr. T) L-Tyrosine (Tyr. Y) L-Cysteine (Cys. C) L-Asparagine (Asn. N) L-Glutamine (Gln. Q) Polar, charged side chains L-Glutamic acid (Glu. E) L-Aspartic acid (Asp. D) L-Lysine (Lys. K) L-Arginine (Arg. R) L-Histidine (His. H)  Chapter 2: Proteins |9 2.2.2 PEPTIDES Peptide bond Peptides (and proteins) are sequences of amino acids that are linked to each other by a peptide bond. A peptide bond results from condensation of the -COOH group of one amino acid with the -NH2 group of another amino acid with removal of a water molecule (Figure 4). Proteins Figure 4. Formation of a dipeptide from two amino acids with loss of one water molecule. By convention the structure of a peptide, as well as a protein, is always written with the N-terminal amino acid on the left (see Figure 5). This is the amino acid where the -NH2 group is still free. The other terminal amino acid, with a free -carboxyl group, is written on the right and called the C-terminal amino acid. Figure 5. Peptide with N-terminal on the left and C-terminal on the right. The peptide bond has a planar (flat) structure, and consequently it has the possibility of cis-trans isomerization. In the cis-form both side chains are on the same side of the plane of the peptide bond, which results in steric hindrance, especially for larger side chains (Figure 6). For this reason, the peptide bonds normally are in the trans-form, where the side chains are on opposite sides of the plane of the peptide bond. R2 O O H O CH H 2N C N C OH H 2N C N C OH CH CH CH H O R1 R2 R1 Cis Trans Figure 6. Spatial configuration of the peptide bond. 10 Occurrence and structural properties Nomenclature Peptides are denoted by the number of amino acids they contain, such as dipeptides, tripeptides, and tetrapeptides. This terminology is used until about 10 amino acids. The expression oligopeptides is used for chains with less than 100 amino acids. The term protein is used for larger peptides (polypeptides) in the state that they are made by the organism (plant / animal). Most proteins have a molecular mass larger than 10,000 Da (1 Da = 1 g/mol), which is equal to approximately 100 amino acids. When a protein is hydrolysed (by breaking several peptide bonds) the products are also referred to as peptides. The name of the peptide is formed by first mentioning the N-terminal amino acid, then the next amino acids are mentioned until the C-terminal amino acid. The amino acids of which the –COOH group has reacted to form the peptide bond receives a different name. Usually the extension –yl is used. For example, glutamic acid is named glutamyl when present in a peptide, as is shown in Figure 7. The C-terminal amino acid (of which the –COOH group has not reacted) keeps its normal name. Figure 7. Nomenclature free amino acids and of peptides. In case of a tripeptide consisting of cysteine, glutamic acid and glycine the name cysteinyl-glutamyl-lysine is used (Figure 8A). To make it more easy, usually the three-letter code, or even the one-letter code for amino acids is used. In this particular case it is: cys-glu-lys, or CEK. Peptide bonds are not only formed through the α-amino and α-carboxyl groups. The amino or carboxyl groups in the side chain (R-chain) of amino acids can also form peptide bonds. For example, glutamic acid has a -carboxyl group and (lysine) has an -amino group (in Figure 8). In the example given Figure 8B the glutamyl has formed a peptide bond with its -carboxyl group and not with its -carboxyl group. The name of this tripeptide is cysteinyl-- glutamyl-lysine. Such peptide bonds, that are not on the backbone of the peptide (i.e. not on the -carboxyl or -amino group) are called iso-peptide bonds. The peptides that contain iso-peptide bonds are called iso- peptides.  C h a p t e r 2 : P r o t e i n s | 11 Proteins Figure 8. Two different peptide linkages of glutamic acid (glutamyl group). A is α-glutamyl; B is γ-glutamyl. In the short notation of a peptide sequence, the presence of an iso-peptide bond is indicated by a thin angular line (∟) in the three letter notation (Figure 9). Figure 9: Example of the notation for the iso-peptide cysteinyl--glutamyl-lysine Examples of naturally occurring peptides in foods. Peptides are naturally present in non-processed agricultural raw materials such as wheat flour and meat. In plants small peptides are often used as signalling agents, similar to hormones in animals. In animals, including us humans, peptides contribute to the buffering capacity of blood, to maintain the homeostasis in the body. In addition, peptides are commonly present in fermented foods (e.g. cheese, fermented sausage, soy sauce). Such peptides are formed after partial hydrolysis of the original proteins present in the starting material during fermentation. Protein hydrolysates are also made industrially to be used as an ingredient. In infant formula and clinical nutrition hydrolysates are often used to increase digestibility, or to reduce allergic response that the people may have to the intact proteins. In addition, some peptides may have a direct biological activity. Examples of naturally occurring peptides are glutathione, carnosine, anserine, balenine, nd nisin. Glutathione In biological tissues the redox-peptide glutathione (-glutamyl-cysteinyl-glycine, Figure 10) is involved in the active transport of amino acids and in many redox-reactions. In food processing, it influences the rheological properties of wheat flour dough by means of thiol-disulphide exchange with disulphide cross-linked cysteine moieties of wheat gluten proteins. Glutathione reduces the disulphide bonds between the gluten proteins, resulting in a weaker (better) dough. 12 Occurrence and structural properties Figure 10. Structure of the tri-peptide glutathione (γ-glutamyl-cysteinyl-glycine). Carnosine, anserine, and balenine The meat peptides carnosine (β-alanyl-histidine), anserine (β-alanyl-3-methyl-histidine) and balenine (β-alanyl-1- methyl-histidine) are examples of peptides containing a -amino acid (the NH2-group is attached to second C-atom, which is named the β-C atom). The structures of these bioactive dipeptides are given in Figure 11. These peptides are supposed to have a biological function in revitalizing muscles and exert a buffering capacity at neutral pH. Figure 11. Structures of carnosine (β-alanyl-histidine), anserine (β-alanyl-3-methyl-histidine) and balenine (β-alanyl- 1-methyl-histidine). Nisin The antibiotic-peptide nisin is composed of 34 amino acids (Figure 12). It is produced by several strains of lactic acid bacteria. It is active against spores of Gram-positive bacteria. It is permitted in a number of countries as a preservative of cheese to inhibit the spoilage of cheese by butyric acid bacteria. In nisin a number of unusual amino acid residues are present (Figure 12): dehydroalanine (Dha), dehydro--methyl-alanine (Dhb), lanthionine and - methyllanthionine.  C h a p t e r 2 : P r o t e i n s | 13 Proteins Figure 12. The primary structure of nisin and its unusual amino acids. In green: dehydrobutyrine. In blue: dehydroalanine. In light yellow: lanthionine (Ala-S-Ala). In dark yellow: β-methyllanthionine (Abu-S-Ala). 2.2.3 PROTEINS Proteins in food are derived from different sources. Traditional sources are: milk, meat, eggs (animals) and soy, wheat (plants), but currently also other sources (e.g. potatoes, algae) are investigated. The natural functions of proteins in the organism are to provide structure (e.g. collagen), nutrition (e.g. milk), or to actively work as an enzyme (e.g. pepsin in the stomach). In food products, the proteins are used for nutrition (e.g. essential amino acids), to provide structure to a product (gluten in bread), or flavour (source of Maillard reaction products). To understand the differences between different proteins and their properties, it is important to know the amino acid composition and structure of the proteins. Whereas commonly 20 different amino acids are present, not all amino acids are present in the same frequency. In Table 4 the average frequency of amino acids in proteins in general is listed. This is an average of 1021 different kinds of proteins. It can be seen that the aliphatic amino acids alanine and leucine are the most abundant amino acids, followed by glycine. It is interesting to note that cysteine (conditionally essential amino acid) and tryptophan are the least abundant amino acids present in proteins, followed by histidine and methionine. Cysteine and tryptophan rich proteins/peptides have received special attention because of their bio-active functions. In α-lactalbumin e.g. a relatively high concentration of cysteine and tryptophan can be found compared to the average frequency of amino acids in proteins (see Table 4). Table 4. Frequency of average amino acids in 1021 different kinds of proteins and of α-lactalbumin. Amino acid Average α-lactalbumin Amino acid Average α-lactalbumin Alanine 8.3 % 1.6 % Leucine 9.0 % 10.4 % Arginine 5.7 % 1.1 % Lysine 5.7 % 10.7 % Aspartic acid 5.3 % 10.6 % Methionine 2.4 % 0.9 % Asparagine 4.4 % 6.5 % Phenylalanine 3.9 % 4.0 % Cysteine 1.7 % 5.9 % Proline 5.1 % 1.4 % Glutamic acid 6.2 % 6.3 % Serine 6.9 % 4.5 % Glutamine 4.0 % 5.4 % Threonine 5.8 % 5.1 % Glycine 7.2 % 2.7 % Tryptophan 1.3 % 5.0 % Histidine 2.2 % 2.8 % Tyrosine 3.2 % 4.4 % Isoleucine 5.2 % 6.4 % Valine 6.6 % 4.3 % 14 Occurrence and structural properties The structure and the properties of proteins depend on the number of amino acids (length of the peptide chain), the properties of the amino acids (a chain with many hydrophobic amino acid residues does not dissolve well in water) and the order of the amino acid in the chain. The amino acid sequence (the order of amino acids in the chain) is called the primary structure of the protein. Due to different non-covalent interactions, parts of the sequence will fold into repetitive spatial structures, the -helix, or the -sheet. The parts of the sequence that are not structured are called random coil. These three elements describe the secondary structure, and are formed by the formation of hydrogen bonds between the carbonyl oxygen atoms and the amide hydrogen atoms involved in the peptide bonds. The ultimate shape (3D structure) of the folded peptide chain is named the tertiary structure. This tertiary structure defines how the different elements of the secondary structure are oriented relative to each other in the three-dimensional space. Some proteins are not formed by one, but by several polypeptide chains. The way that the different polypeptide chains are associated (linked together) is called the quaternary structure. Proteins that have quaternary structure are also called multimeric proteins. The structure of the proteins determines many of their properties, such as their solubility. The structure can be changed due to for instance heating or pH. primary structure tertiary structure dimer secondary structure quaternary structure Figure 13. Representation of the primary, secondary, tertiary and quaternary structure of a protein. Structural forces in protein folding and stability To understand how certain factors influence the structure of proteins, it is important to know the forces that stabilise the protein structure. The primary structure is formed by the peptide bonds, which are covalent bonds. These are the most stable bonds in the protein structure. Peptide bonds are only broken by proteases (enzymes), or using high temperature at high concentrations of acid or alkali. For example, to determine the amino acids in a protein sample, the proteins are hydrolysed by heating for 23 hours at 100 °C in 6M HCl. The secondary structure of proteins is stabilised by hydrogen bonds between C=O and N-H groups in the peptide backbone (Figure 14). In a polar environment, hydrogen bonds can also be formed with the solvent (water). Therefore the stability of the secondary structure is slightly lower in water than in an apolar environment. It is important to realise that in the inside of the protein normally very few water molecules are present. Consequently in the folded protein, the environment is not very polar and there hydrogen bonds are mostly between the parts of the peptide backbone. The amino acid proline does not have a primary (but only a secondary) α-amino group, so there  C h a p t e r 2 : P r o t e i n s | 15 are no possibilities for H-bonding and for proline to take part in a secondary structure element. The result is that for instance an alpha-helix structure is always interrupted when there is a proline in the sequence. Figure 14. A. hydrogen bond between two peptide groups B. hydrogen bonds between side chain amide groups The tertiary and quaternary structure are stabilised both by the same set of bonds and interactions, namely Proteins disulphide bonds, and electrostatic and hydrophobic interactions. The disulphide bond is a covalent bond between the side-chains of two cysteine residues (Figure 15). Disulphide bonds are commonly present in most proteins. The disulphide bonds stabilise all conformations of a protein equally well, i.e. coincidentally formed sulphur bonds in a denatured structure are as strong as the “correct bonds” in a native structure. Figure 15. Disulphide bridge chemical structure (left) and in a protein structure linking two β-sheets (right) Electrostatic interactions are formed between charged groups on the side chains (Figure 16). If positively and negatively charged residues interact this is called an ionic bond. This can stabilize the structure of proteins. However, if similar charges interact (e.g. positive with positive) they will repel each other. This will destabilize the protein structure. These repulsive or attractive interactions can also occur between protein molecules. This is important for the solubility of proteins. Electrostatic interactions also occur between charged amino acid side chains and ions. For example, in milk calcium bridges are important. These calcium bridges can be formed between dissociated carboxyl groups and/or phosphate groups that are present in casein protein molecules. These positive, attractive interactions result in a tight clustering and stability of the casein micelles in milk. Figure 16. Electrostatic interaction 16 Occurrence and structural properties Hydrophobic interactions (Figure 17) make an important contribution to the stability of the structure of a protein. The hydrophobic interaction originates from the hydrogen bonds between water molecules in the solution. Since nonpolar residues cannot form hydrogen bonds, the water around the nonpolar residue are in a higher energy state. This is unfavourable from a free energy point of view. When two nonpolar residues come together, some of the bound water molecules are released, so they can interact freely with bulk water, thereby lowering the free energy of the system. Hence, nonpolar residues have a tendency to aggregate in water, resulting in the release of water molecules. Figure 17. Hydrophobic interactions Van der Waals interactions work at very short distance and do not require a characteristic chemical structure. The basis of a van der Waals interaction is that the distribution of electrons around an atom changes with time. At any instance, the charge distribution is not perfectly symmetric. This transient asymmetry in the electronic charge around an atom acts through electrostatic interactions to induce a complementary asymmetry in the electron distribution around its neighbouring atoms. The resulting attraction between two atoms increases as they come closer, until they are separated by the van der Waals contact distance. At a shorter distance than this, very strong repulsion becomes dominant because the outer electron clouds overlap. In the nucleus of globular proteins the side chains are ordered in such a way that the number of van der Waals bonds is maximal. In summary, the primary structure is formed by covalent bonds. All higher structures are stabilised mostly by non- covalent bonds. The secondary structure is mostly stabilised by hydrogen bonds between the C=O and N-H groups in the peptide bond backbone. The tertiary and sometimes in the quaternary structure are also mostly stabilised by non-covalent bonds, but here also one type of covalent bond is important. The bonds and interactions that stabilise the protein structure, and their properties are listed in Table 5. Table 5. Molecular forces involved in protein interactions Type Mechanism Energy Group involved Examples (kJ/mol) Covalent bond sharing of electrons 130-500 C-C, C-N, C-O, peptide bond, disulphide C-H, C-N-C, S-S bond and the original bonds in the amino acid Hydrogen Dipole-dipole interaction 8-33 N-H---O=C, amid-carboxyl group, serine, bond involving hydrogen and two OH-- threonine, electro-negative atoms NH-- tyrosine, polar side chains NH2--  C h a p t e r 2 : P r o t e i n s | 17 Electrostatic Coulomb interaction 45- 85 -NH3+ -amino group, lysine, interactions between charged groups =NH+ arginine, histidine, (ionic bond or COOH -carboxyl group, repulsion) aspartic acid and glutamic acid Hydrophobic Low entropy of water 5- 15 non-polar non-polar side chains interaction around hydrophobic molecules compared to bulk solution Van der Waals dipole –induced dipole 1–9 steric hindrance all groups Interactions interaction between nearby between the side situated non-polar groups chains of amino acid Proteins residues 2.3 Protein classification 2.3.1 CLASSIFICATION BASED ON CONFORMATION Proteins consist of polar and apolar (nonpolar) amino acids. Since the apolar amino acids do not interact with water, proteins adopt a certain structure to minimize the contact of hydrophobic amino acids with the aqueous (water) environment. This is done in different ways, and as a consequence proteins can be divided into three groups on the basis of their conformation: Globular proteins Fibrillar or fibrous proteins Proteins that are neither globular nor fibrillar (e.g. caseins; random-coil like) Globular proteins typically occur as soluble, monomeric or multimeric proteins. They have a combination of -helices and β-sheets, alternated by random coils. The proportion of regular structural elements (α-helix + β- sheets) is highly variable. For example: only 45% in lysozyme, but 75% in myoglobin. In globular proteins the hydrophobic amino acids are mostly on the inside of the protein molecule. The surface of globular proteins contain mostly the polar charged or non-charged amino acids. Fibrous proteins are typically associated into larger complexes. In such proteins the entire peptide chain is arranged within a single regular secondary structure. Examples are wool keratin (-helix), silk fibroin (-sheet structure) and collagen (triple helix). Muscle proteins are fibrous proteins, and they form a triple helix. Since most fibrous proteins are used to provide structure to the tissue, they are characterized by a low solubility in aqueous systems. In addition, fibrous proteins have a tendency to cluster together, so that hydrophobic parts of the sequence of one protein are covered by hydrophobic parts of another protein. Random coil proteins do not have any fixed secondary or tertiary structure. To avoid exposure of hydrophobic groups to water they typically associated into micelles. Caseins from milk are a good example of such proteins. Proteins differ significantly in size (Figure 18). Proteins can be as small as a 100 amino acids (Mw  10.000 Da), but can also be present as multimeric proteins, consisting of multiple polypeptide chains, with a molecular mass of several million Da. Also, proteins differ significantly in their shape. 18 Protein classification Figure 18. Relative dimensions of some protein molecules (with molar mass in Da between brackets). 2.3.2 CLASSIFICATION BASED ON SOLUBILITY A classification that is of prime importance to the food technology is the one according to solubility (Figure 19). In general, fibrillar proteins are not water-soluble. This is often related to their biological function, such as keratin (hair), myosin (muscle tissue) and collagen. Figure 19. Fractionation of wheat flour and whey protein to different protein fractions. The solubility of globular proteins is often defined by making use of specific solvents. In 1907 T.B. Osborne introduced a separation of wheat proteins, on the basis of their solubility, into four different fractions. The wheat flour was sequentially extracted with water, a saline solution (e.g. 0.4 M NaCl), an aqueous organic solvent solution (e.g. 70 % v/v ethanol). This resulted in the water-soluble albumins, the salt-soluble globulins and the 70% aqueous ethanol-soluble prolamins leaving a protein fraction in the flour residue, denoted the glutelins. For wheat, the prolamins and glutelins together form the ‘gluten’ fraction. This protein classification has ever since been used widely for food proteins. Conditions for extraction may vary in order to accommodate a better separation. For example, for the extraction of prolamins also t-butanol is used instead of ethanol and the molarity of the salt solution can differ from 0.1-0.5 M. For milk a similar classification in albumins and globulins is made. In this case the proteins are already soluble in the starting material. The classification is then based on the concentration of ammonium sulphate at which the proteins precipitate. The loss of solubility at these high concentrations of salts is also referred to as salting out.  C h a p t e r 2 : P r o t e i n s | 19 Make digital exercises: ‘Protein exercises – Structure and classification' 2.4 Effect of pH on the charge of amino acids Amino acids have both an acidic and a basic character, due to the acidic carboxyl group (-COOH) and the basic amino group (-NH2) (Figure 20). In addition, the side chain (R) of an amino acid may contain a charged (basic or acid) group that will affect the overall charge of the amino acid. Proteins Figure 20. Carboxyl and amino group of an amino acid Net charge of amino acids, peptides and proteins When we consider the amino acid in Figure 20, what is the net charge at any pH and how do we determine this? First, the carboxylic group can only be charged (COO-) or neutral (COOH). However, this state is always changing. There is an equilibrium with COO- + H+  COOH. On average, at any moment in time, a certain percentage of all the molecules in solution will have a carboxylic group in the charged state. This percentage depends on the pH and the dissociation constants (pKa) of the charged group (Table 6). For each group (e.g. COOH), It is important to remember that at pH = pKa the 50% of the acidic group is in the protonated state, and 50% in the deprotonated state. The same is of course true for basic groups. At pH > pKa the acid (or base) is mostly in the deprotonated form (so COO ⎯ / NH2). At any pH we can calculate the % of each group that is either positively (+1), or negatively charge (-1), or neutral. Summing up these values for all charged groups in the amino acid we determine the net charge of the amino acid. When we talk about ‘the charge’ of an amino acid, peptide or protein, we mean ‘the average charge’ of all the molecules of this amino acid in a solution. At a certain pH, the total number of positive and negative charges on the amino acid (or peptide, protein) will be equal. In other words, in that pH the net-charge is zero. This pH is called the iso-electric point (pI). In Table 6 the values for the pI of the different amino acids is also shown. Just like free amino acids, also peptides exhibit acid/base character. It is important to realise that when amino acids are linked to form a peptide, or a protein, the whole (poly-) peptide chain will only contain one -COOH and one -NH2 group. The longer the peptide is, the less the contribution of the N-terminal -NH2-group and the C-terminal α-COOH-group to the total acid/base behaviour of the peptide. For long peptides (>100 amino acids), the charge (and pI) is therefore mostly determined by the amino acid side chains (Table 6). Since proteins typically have both acidic and basic amino-acid side-chains, they are called polyampholytes. 20 Effect of pH on the charge of amino acids Table 6. The dissociation constants and iso-electric points of chargeable groups of free amino acids at 25 C. Amino acid pK-COOH pK-NH2 pKside chain pI Alanine 2.34 9.69 6.0 Arginine 2.18 9.09 12.60 10.8 Asparagine 2.02 8.80 5.4 Aspartic acid 1.88 9.60 3.65 2.8 Cysteine 1.71 10.66 8.35 5.0 Glutamine 2.17 9.13 5.7 Glutamic acid 2.19 9.67 4.35 3.2 Glycine 2.34 9.60 6.0 Histidine 1.80 9.07 5.99 7.5 4-Hydroxyproline 1.82 9.65 5.7 Isoleucine 2.26 9.62 5.9 Leucine 2.36 9.60 6.0 Lysine 2.20 8.90 10.28 9.6 Methionine 2.28 9.21 5.7 Phenylalanine 1.83 9.13 5.5 Proline 1.99 10.60 6.3 Serine 2.21 9.15 5.7 Threonine 2.15 9.12 5.6 Tryptophan 2.38 9.39 5.9 Tyrosine 2.20 9.11 5.7 Valine 2.32 9.62 6.0 To illustrate how the charge of a single amino acid changes with the pH, the titration curve (charge versus the pH) of the amino acid aspartic acid can be calculated using the pKa values of the three charged groups in the amino (Figure 22). To understand this graph, or schematically draw such a titration curve, you can first make an overview of the different charge states of the amino acid at different pH values using these pKa values, (Figure 21). At pH 1 (below pKa1,2 and 3) the amino acid is positively charged, because the all groups are in their protonated state. The protonated amino group is positively charged, and the protonated carboxyl groups are neutral (COOH). This corresponds with the leftmost molecular structure. When increasing the pH, the pKa value (1.88) of the -carboxyl group is reached, yielding an average charge on the COOH group of -0.5, and a total charge on the amino acid of +1- 0.5-0 = +0.5. Above pH 1.88 this group becomes predominantly negatively charged. The same then occurs for the other carboxylic group, so that finally, at pH 12, the net charge is –2, since both carboxyl groups are negatively charged. This corresponds with the most right molecular structure. Figure 21. pH equilibria of aspartic acid (an amino acid)  C h a p t e r 2 : P r o t e i n s | 21 Aspartic acid: 3 pK1=1.88 pK2=3.65 2 pK3=9.60 1 pK1 Charge 0 0 2 4 6 8 10 12 pK2 -1 pK3 -2 Proteins -3 pH Figure 22. Calculated titration curve, i.e. charge versus pH, of the amino acid aspartic acid. 2.5 Chemical changes of amino acids and proteins During the processing of raw materials into food products several chemical and/or physical changes can take place on amino acids and on proteins. These changes are often induced because of heating the proteins. Temperature, time and pH all play a role in the speed of these reactions. Some reactions (decarboxylation, deamination) are mostly due to enzymatic activity, due to microbial spoilage. Chemical reactions can take place with the reactive side chains of amino acids and proteins. These reactive groups are mainly the amino groups (NH2), the carboxyl groups (COOH), the hydroxyl groups (OH) and the sulfhydryl groups (SH). Since most of the essential amino acids contain such side groups, one clear result of these chemical reactions is a loss of essential amino acids. In addition, the reactions can change the charge of the proteins, thereby affecting solubility and other properties. Finally, the more extended reactions that for instance result in crosslinking of peptides will significantly decrease the digestibility of proteins. The following reactions will be discussed in this chapter: Chemical reactivity of amino acids Chemical reactivity of proteins Decarboxylation and deamination Formation of S-S bridges and reshuffling of S-S bridges Strecker degradation Formation of isopeptides (lysinoalanine) Crosslinking of proteins via the production of Maillard reactions formaldehyde Reactions at the functional group of the side Protein hydrolysis chain 2.5.1 CHEMICAL REACTIVITY OF FREE AMINO ACIDS Free amino acids (amino acids not part of a protein or peptide) can react via: - the -carboxyl group and the -amino group - the functional group in the side chain. 22 Chemical changes of amino acids and proteins Decarboxylation and deamination of the -carboxylic acid and -amino group Free amino acids are prone to degradation caused by micro-organisms. Decarboxylation and/or deamination of free amino acids often occurs during decay and spoilage of protein-rich foods, as a result of bacterial enzyme activities (Figure 23). However, since fermentation of foods also involved bacteria or yeasts, the same reactions can occur during fermentation. Figure 23. Decarboxylation of an amino acid (top) and the deamination of an amino acid (bottom). In Table 7 some examples are given of the products that are formed after decarboxylation or deamination of several amino acids. Mostly, the resulting molecules are volatiles that provide a specific odour (e.g. cadaverine, putrescine). However, some can also have biological effects. Especially the decarboxylation of the amino acid histidine is of importance. Histidine is converted to the amine histamine. When histamine is consumed and absorbed in substantial amounts it can cause a pseudo-allergic reaction. The reason is that histamine is also naturally formed in the body and is released in the allergic response once an allergen has been in contact with the immune system. The consumption of histamine in the diet can lead to pseudo-allergic reactions and therefore products that contain relatively large amounts of histamine should be taken notice of. Table 7. Overview of amines and carboxylic acids that can be formed during enzymatic degradation of amino acids. Decarboxylated form  Amino acid → Deaminated form Cadaverine Lysine Putrescine Omithine Isobutyl amine Valine Tyramine Tyrosine Tryptophan Tryptamine Aminobutyric acid Glutamic acid -ketoglutaric acid Aspartic acid Fumaric acid Threonine α-ketobutyric acid Alanine Pyruvic acid Histamine Histidine Strecker degradation at the -amino and the  -carboxyl group The Strecker degradation, is a purely chemical reaction that occurs when free amino acids react with dicarbonyl compounds (Figure 24). This reaction results in a transamination and a decarboxylation. However, it is important that the Strecker degredation is not related to the deamination and decarboxylation discussed above, since those reactions occur as the result of enzymatic (microbial) reactions. The dicarbonyl compounds can be generated during food processing or prolonged storage by both the Maillard reaction as well as by the enzymatic browning reaction (also named the polyphenol oxidation reaction). More details on these reactions is given in the chapters on  C h a p t e r 2 : P r o t e i n s | 23 Carbohydrates (Chapter 1) and Phenolic compounds (Chapter 4). After the Strecker degredation, the amino acid is converted into an aldehyde. Often, these aldehydes have a specific flavour. Figure 24. The Strecker degradation of free amino acids Maillard reactions at the -amino group Proteins During storage and processing of foods various browning reactions can occur. Besides caramelization and enzymatic browning, in particular the Maillard reactions are of the utmost importance. In the first step of the Maillard reaction a free amino group (-NH2) reacts with the reducing end of a reducing sugar. The amino groups can come from the -amino group of free amino acids, the -amino group of lysine, or the free amino group of the N-terminal amino acid in a peptide. The amino groups (basic groups) after the reaction are secondary amines, which cannot be protonated. As a result, there are less positive charges on the protein at any given pH. By different successive reaction steps brown coloured compounds and aromatic substances can be formed. This is of importance in, for instance, cocoa, coffee, nuts, soy sauce and bread (crust). A surprising range of different flavours can be obtained after Maillard reaction, depending on the amino acids and carbohydrates used. The Maillard reaction results in: Brown pigments, called melanoidins. Only little is known about the structure of these compounds. Brown colouring is desired during baking and frying, but not in products such as powdered milk and dried soups. Volatile compounds. These volatiles often are desired aroma compounds that are released during baking and frying, but they can also be off-flavours that are formed during storage of (dried) products or during pasteurisation/sterilization. Loss of essential amino acids (lysine, cysteine and methionine). Compounds with possible mutagenic or carcinogenic properties. Acrylamide, HMF and furan are examples of toxicants formed during Maillard reaction. Compounds that can cause cross-linking of proteins and consequently cause changes in the functionality of the proteins. 2.5.2 REACTIONS AT THE FUNCTIONAL GROUP OF THE SIDE CHAIN When the amino acids are part of a peptide or protein only the side chains can be reactive, since most of the α- amino group and the α-carboxyl group have already formed peptide bonds. In addition, in most foods the research has focussed on proteins, rather than the (small amount of) free amino acids in the materials. Therefore, the following reactions have been mostly studied in proteins. At elevated temperatures proteins (and also amino acids and peptides) can take part in several reactions. The nature and extent of the chemical changes in proteins by food processing depends on a number of parameters: processing conditions (pH, temperature, ionic strength, presence of oxygen) and the composition of the food (presence of other reactive compounds). As a consequence of these reactions the biological value as well as the techno-functional properties (e.g. solubility, foaming, gel forming, etc.) may be decreased. The biological value can be decreased because of: Degradation of essential amino acids Conversion of essential amino acids into derivatives that cannot be metabolised Decrease in the digestibility of protein as a result of intra- or interchain cross linking (decreased accessibility for the proteases) 24 Chemical changes of amino acids and proteins Formation of S-S bridges and reshuffling of S-S bridges Cysteine, containing a sulfhydryl group (-SH), can take part in redox reactions. These reactions lead to formation or breaking of disulphide bonds. Disulphide bridges within one peptide chain (or protein) will stabilise the structure of the peptide. In addition, disulphide bridges can be formed between different peptides (or proteins) to form larger complexes. For example, it is generally accepted that the rheological properties of bread dough is related to its gluten network. This three-dimensional gluten network seems to be dependent on the arrangement and number of disulphide bonds and sulfhydryl groups on and between the gluten proteins. The disulphide bonds can be involved in the following reactions: 1) Oxidation of sulfhydryl groups or reduction of disulphide bonds: Under influence of an oxidizing agent (oxidant, e.g. O2), two cysteine residues can react to form a cystine residue, forming a disulphide bond. For analysis, sometimes reducing agents are added to proteins, to reduce (break) the disulphide bonds. 2) Reshuffling of disulphide bonds (S-S bridges) for which a minimum concentration of ionized sulphide groups (R-S-) is needed: This reaction seems to yield a similar result as the combination of oxidation and reduction, but it follows a different reaction mechanism. Oxidation of Methionine Methionine, containing a sulphide (-C-S-C-), can be oxidized to methionine sulfoxide, which can then be further oxidized to methionine sulfone. (Figure 25). This reaction can take place during heating, and in the presence of peroxides. It is also one of the processes leading to off-flavour in milk when it is exposed to too much light. Figure 25. Oxidation of methionine upon heating  C h a p t e r 2 : P r o t e i n s | 25 Formation of isopeptide bonds There are three main reactions by which isopeptide bonds can be formed within or between proteins. 1- Glutamic acid and aspartic acid esters can undergo a reshuffling (Figure 26). In this example, the -amino group that was linked to the -carboxylic group of the aspartic acid becomes linked to the -carboxylic group. Proteins Figure 26: Isopeptide bond formation 2- Reaction between the -amino group of lysine residues and the - or -carboxamide groups of asparagine and glutamine residues, respectively. These isopeptide bonds can be formed when heating proteins under dry conditions at neutral pH results. As a result of the reaction between these groups, ammonia (NH3) is released and a cross link (peptide bond, denoted isopeptide bond) is formed between the two residues. These isopeptide bonds are cleaved during acidic hydrolysis of protein and, therefore, do not contribute to the occurrence of unusual amino acids as is the case in the formation of lysinoalanine like compounds and crosslinking with formaldehyde. 3- Reaction between -amino group of lysine or sulfhydryl group of cysteine or guanidino group of arginine with dehydro-alanine, resulting in the isopeptides lysinoalanine (LAL), lanthionyl (LAN) and ornithionalanine (OAL), respectively.. The first step in the formation of LAL, LAN and OAL is the formation of dehydroalanine residues from cysteine and methionine (Figure 27), which occurs at elevated temperatures and pH. This reaction occurs in alkaline conditions and results in a double bond between the -carbon atom and the -carbon atom. If the reaction occurs in a protein, the new residue is called a dehydro-alanyl residue, the free compound is dehydroalanine. Figure 27. Formation of dehydrolalanine residue from cysteine in alkaline conditions In the second step, the newly formed dehydroalanyl residues reacts to form an isopeptide bond with the side chain of other amino acids. Only side chains with a nucleophilic character can take part in this reaction, for instance, lysine (-NH2), cysteine (-SH), and arginine (-NH-C(NH)-NH2). In Figure 28 the formation of formation of lysinoalanine (LAL), as a result of reaction of lysine with dehydroalanine is described. 26 Chemical changes of amino acids and proteins Figure 28. Formation of lysinoalanine (LAL) Similar reactions can occur with cysteine (formation of lanthionine; LAN) and with arginine, forming orntihinoalanine; OAL). In the latter case first a conversion of arginine to ornithine takes place. These reactions are demonstrated in Figure 29.  C h a p t e r 2 : P r o t e i n s | 27 Proteins Figure 29. Formation of LAN and OAL The isopeptides (LAL, etc), also called crosslinked amino acids, have been used to determine or quantify the extent of heat damage that certain products had been subjected to. Of these compounds, lysinoalanyl (LAL) has most often been studied, for instance in hard-boiled eggs (eggs have a pH between 7 and 9). The formation of LAL (and the other compounds) is faster at alkaline pH. However, since these compounds are found in most common food products, it is apparently a combination of time, temperature, concentrations and pH that influences their formation. Table 8 gives examples of processed food products with various concentrations of LAL. These newly formed amino acids cannot be metabolised by humans or animals. Hence, the nutritional value of the protein is lowered. Moreover, their formation involves essential amino acids, another reason that nutritional value is decreased. Lastly, the crosslinking of proteins through the formation of these crosslinked amino acids may also decrease their accessibility to digestive enzymes, leading to decreased digestibility and therefore lower nutritional value. Table 8. Variation in LAL content for several food products. Food source LAL (μg/g) References Cereal products 200 - 390 Antilla et al., 1987; Hasegawa et al., 1987; Sternberg and Kim, 1977; Struthers et al., 1980 Chicken meat (processed) 370 Sternberg and Kim, 1977 Eggs (processed) 160 - 1820 Fritsch and Klostermeyer, 1981a,b; Hasegawa et al., 1987; Murase and Goto, 1977; Raymond, 1980; Sternberg et al., 1975a,b; Sternberg and Kim, 1977 Infant formulas, dry 150 - 920 Antilla et al., 1987; Bellemonte et al., 1987 Infant formulas, liquid 160 - 2120 Bellemonte et al., 1987; Langhendries et al., 1992; Pfaff, 1984; Pompei et al., 1987 Milk powders 150 - 1620 Aymard et al., 1978; Fritsch and Klostermeyer, 1981a,b; Hasegawa et al., 1987; Sternberg and Kim, 1977 28 2.6 Physical changes – Protein denaturation 2.5.3 PROTEIN HYDROLYSIS Hydrolysis of proteins can occur as a result of enzymatic reactions, but can also result from non-enzymatic reactions. In both cases, the peptide bonds are hydrolysed (Figure 30), releasing the carboxylic and the amino group that were linked in the original peptide bond. Partial hydrolysis results in peptides, complete hydrolysis results into free amino acids. Chemical hydrolysis (with acids or alkaline) can be used to completely hydrolyse a protein (sample) to the constituent amino acids to determine the amino acid composition. For this typically 23 h heating at 100 °C in 6M HCl is used. Under milder conditions, or longer incubation times of course some partial hydrolysis can occur. However, most hydrolysis observed in food processing will be the result of enzymatic hydrolysis (with proteolytic enzymes). Figure 30: Hydrolysis of a peptide bond. Enzymatic protein hydrolysis (proteolysis) can be used in clinical or infant food production to for instance decrease allergenicity. It also occurs in food due to the presence of microbial proteases, for instance in Parma ham, or cheese. The effects of hydrolysis are several. Hydrolysis leads to changes in taste, solubility, gelling, foaming and emulsifying properties, water holding capacity. In addition, the study of enzymatic hydrolysis is used to increase our understanding of the digestive system. Food product example: Production of gelatin from collagen Gelatine is produced from collagen. Collagen is a protein in skins and bones, the by-products of the meat industry. These materials are boiled in water or dilute acid solutions, during which the hydrolysis of collagen takes place, after which the product is obtained that we call gelatin. Gelatin is used to make a gel structure in candies (jelly gums, marshmallows) and puddings. Make digital exercises: ‘Protein exercises – Chemical properties' 2.6 Physical changes – Protein denaturation The most important physical change of proteins that can occur during food processing (i.e. heating) is unfolding. Unfolding, also called denaturation, is the transformation from a well-defined, folded structure of a protein (formed under physiological conditions) to an unfolded state. It is important to realise that the protein can retain part of its secondary structure, and anyway will keep some ‘shape’. In other words, a globular protein will still be more or less globular in the unfolded state. Still, the more open structure of the unfolded state results in exposure of hydrophobic amino acids that were previously hidden on the inside of the protein structure. This results in an increase of the exposed hydrophobicity and can lead to aggregation of proteins and consequent loss of solubility.  C h a p t e r 2 : P r o t e i n s | 29 Figure 31: Example of a protein in a folded (Left) and unfolded state (right). As stated earlier, the native structure of a protein is the net result of various attractive and repulsive interactions Proteins originating from various intramolecular forces as well as interaction of various protein groups with the surrounding solvent, usually water. The native state is thermodynamically the most stable with the lowest feasible free energy at physiological conditions. Any change in its environment, such as temperature (processing of foods), ionic strength (production of composite foods out of ingredients), pH (isolation of protein ingredients from agricultural raw materials) will force the molecule to assume a new equilibrium structure. Denaturation does not involve the break-up of covalent bonds, although the interchange and reshuffling of SH/S-S groups/bonds is sometimes included. The transition of a protein from its native conformation to an unfolded state has several important effects: 1) Decreased solubility as more hydrophobic groups are exposed to water 2) Aggregation of proteins. As stated above, upon unfolding hydrophobic groups become exposed. Next, via these exposed hydrophobic groups unfolded proteins interact with each other creating aggregates. These aggregates inhibit the refolding and may even prevent the refolding of individual proteins. 3) Increased accessibility of peptide bonds to proteolytic enzymes, hence, increased degradability. 4) Loss of biological activity. Because biological activity depends on a precise conformation of a protein a change in conformation often results in a lower biological activity. Biological activity usually is referred to enzyme activity, but in many plant protein preparations proteinaceous enzyme inhibitors (e.g. protease inhibitors, amylase inhibitors) are present and inactivation of these inhibitors is of paramount importance for the nutritional properties of the food. 5) Increased reactivity. Due to the unfolding reactive groups that had been buried in the inside of the protein become exposed to the solvent and other protein molecules. In this respect the exposure of the thiol group (-SH) of side chain of cysteine is of special importance, because reshuffling of S-S bridges can take place. Unfolded proteins can refold to their native conformation once the conditions for unfolding are removed. However, in practice unfolding is usually accompanied by various reactions preventing the refolding to their native conformation, such as reshuffling of S-S bridges, deamidation, and aggregation (Figure 32). Figure 32. Unfolding and aggregation of unfolded proteins 30 Roles of proteins, peptides and amino acids in food Food product example: Boiling an egg A typical example of the effect of heating a protein is the boiling of an egg. Egg white mainly consists of proteins (10%) and water (88%). Raw egg whites are liquid. During boiling the proteins unfold (denaturation) and the unfolded protein aggregate to form a gel structure. The boiled egg is solid, although it still contains about 88% water, but now the water is retained in the protein gel structure. 2.7 Roles of proteins, peptides and amino acids in food Proteins, peptides and amino acids are used for, or provide specific properties in foods. These can be positive (nutritional value), or negative (allergenicity). The bio- and techno-functional properties of proteins are: - Nutritional value: Proteins are the source for nitrogen and some indispensable (essential) amino acids - allergen: Allergens are always proteins, e.g. peanut proteins, milk proteins. - bio-active: Decrease of blood pressure (ACE inhibition) - Taste: Amino acids and peptides can have a bitter, sulphur, or even sweet taste - Solubility: Decreased solubility of caseins is used to produce cheese - Water holding capacity: Proteins hold water in meat - Structure: Gluten provide the structure of bread - Gelling agent: The gel structure of gelatin pudding is provided by proteins - Foaming agent: Egg foam, milk foam, even beer foam is (partly) stabilized by proteins - Emulsifying agent: Proteins can stabilize emulsions such as salad dressings 2.7.1 NUTRIONAL VALUE OF PROTEINS Most agricultural products contain proteins, as well as peptides and free amino acids. However, on mass basis, proteins normally represent the major part of the proteinaceous material. Soybeans for instance contain around 40% proteins, but around 2-4 % free amino acids or peptides are much lower. In practice, the amounts of free amino acids and peptides in our food do not contribute strongly to the nutritional value of the food. Nevertheless, their occurrence is of prime importance as they are reactive constituents in many reactions occurring in foods and they can have a buffering capacity in foods. In processed food products, the ratio between proteins and peptides (+ free amino acids) can be dramatically shifted. In fermented foods, such as cheese, soy sauce, etc, most of the proteins will have been converted to peptides (and free amino acids). Other peptide rich products are (enzymatic) protein hydrolysates. With respect to nutritional value, it is not taken into account if the material is present as protein or peptides. The nutritional value is determined by comparing the total amino acid content with the nutritional requirements. Amino acids can be divided into indispensable (essential) amino acids, dispensable (non-essential) and conditionally indispensable (semi-essential) amino acids (see Table 9). Table 9. Classification of amino acids according to their nutritional value Indispensable Dispensable Conditionally dispensable Phenylalanine Alanine Cysteine Isoleucine Aspartic acid Glutamine Leucine Asparagine Arginine Lysine Glutamic acid Tyrosine Methionine Glycine Histidine Threonine Proline Tryptophan Serine Valine  C h a p t e r 2 : P r o t e i n s | 31 The human body itself can synthesize 7 amino acids, which are therefore considered dispensable. Despite being dispensable, these amino acids are a valuable source of amino acids and nitrogen. The protein nutritional value of food products corresponds to its ability to meet nitrogen and amino acid requirement of the consumer and to ensure proper growth and maintenance. This ability depends on the following factors: 1) Protein content. Most staple foods have a protein content of about 10%; the exceptions are potato and cassava. The tubers and roots of these plants contain about 2% of protein, on a wet weight basis (Table 10). 2) Protein quality. The nutritional quality of a food protein depends on the kind and amounts of amino acids present. A balanced or high-quality protein contains essential amino acids in ratios equivalent to human needs. Proteins of animal origin are often of a higher quality than those of plant proteins. This can be determined by comparing the amino acid contents of various proteins with the FAO reference patterns. Vegetable proteins usually are not fully suitable as the sole supplier of amino acids. Cereal proteins are often Proteins low in lysine and in some instances they lack threonine and tryptophan as well. Oilseeds and nuts can have low contents of methionine and lysine, whereas legumes often have low levels of methionine. The indispensable (essential) amino acids in greatest deficit with respect to requirement are denoted ‘limiting’ amino acids. 3) Amino acid bio-availability. The digestibility of a protein is calculated as the ratio between the total amount of amino acids that are absorbed (so, not excreted) and the total amount of amino acids that are consumed. The digestibility depends on the ease with which the proteins can be hydrolysed by the digestive proteases, and the absorption of the peptides and amino acids. The digestion of proteins can be hindered by different factors, such as perhaps aggregation, chemical modification of amino acids, or the presence of crosslinks. A large variability in protein digestion has been reported. Quite often it has been suggested that amino acids from animal sources are usually digested and absorbed to an extent of 90%, whereas those from certain plant proteins may be digested or absorbed to an extent of only 60-70%. Table 10. Protein from agricultural raw materials for foods Moisture Protein content Digestibility Biological value % % (% dry matter) (BV) VEGETABLE Potato 78 2.0 9.1 68 Rice 13 7.6 8.7 96 70 Wheat flour 12 13.3 15.1 91 64 Peas 11 23.8 26.7 83 64 Soybean 9 42.8 47.2 85 82 ANIMAL Milk 87 3.5 27.6 96 85 Egg 74 12.8 49.2 97 99 Beef 62 18.5 48.7 97 74 Chicken 66 20.2 59.4 93 75 The lower utilization of certain proteins may be due to several factors: Protein conformation: fibrous, insoluble proteins are less readily accessible by proteolytic enzymes than soluble proteins. Nevertheless, protein denaturation by mild heating enhances the accessibility. Binding of other components of the food matrix to the proteins (e.g. phenolic components, lipids, carbohydrates) may partially impair digestion. The presence of anti-nutritional factors in plant protein sources, e.g. protease inhibitors and tannins (i.e. oligomeric phenolic components), can impair digestion. Modification/degradation of amino acids, due to prolonged processing at high temperatures. 32 Roles of proteins, peptides and amino acids in food The heating of protein-rich foods often leads to lysine loss, for instance due to the Maillard reaction, where the ε- amino group of lysine then reacts with reducing sugars. This can have two effects. Firstly, the reacted compound is not lysine anymore. Secondly, the modified lysine may hinder degradation of the whole protein by the digestive enzymes. During the further course of the reaction brown colouring occurs, the so-called Maillard reaction (see chapter 1 ‘Carbohydrates’). These secondary reactions may also have involved other reaction of amino acids, and further hinder enzymatic hydrolysis. As a result of the reaction of lactose with lysine from the milk proteins, sterilised milk typically has a lower biological value than pasteurised milk. UHT (ultra high temperature) processing (e.g.1-4 sec. 130-150 °C) has a germ-killing effect that is comparable to sterilisation, whereas the lysine losses are even lower than in the case of pasteurisation. Not only heat can lead of a loss of protein quality: Methionine (an essential amino acid) is converted to methional by sunlight, which causes the milk to get a bad taste: “light taste”. Proteins can sometimes become better accessible for proteolytic enzymes as a result of (moderate) processing of foods. Size reduction (e.g. minced steak) and frying of meat increases the digestibility. Meat with a high proportion of connective tissue is not well digested unless the connective tissue is denatured by means of heating. Specific proteins (avidin and ovomucoid) in raw eggs that inhibit digestive enzymes are undesired and can be destroyed by boiling or frying the eggs. The heating of protein-rich foods can also be attractive for other reasons, for instance the formation of aroma compounds. There are several ways to determine the protein nutritional value. They can be divided into bioassays and chemical assays. The biological value is an often-used representative of the bioassays used. The Biological value reflects the balance of essential amino acids in the absorbed protein digest. 2.7.2 TASTE OF AMINO ACIDS AND PEPTIDES Taste of amino acids Fermented protein-rich foods owe their taste (in part) to amino acids and peptides, e.g. cheese, herring, soy sauce. Table 11 contains the data with respect to the taste of amino acids. The taste is influenced by the molecular configuration of the amino acid: most sweet amino acids have a D-configuration (enantiomer) and bitter amino acids mainly the L-configuration. The strength/intensity of the taste is expressed by the recognition threshold value, i.e. the lowest concentration that is necessary for a taste panel to reliably recognize the taste of the compound. Table 11 shows that the intensity of the taste of amino acids depends on the hydrophobicity of the side chains. The most hydrophobic amino acids are the bitterest amino acids: L-tyrosine is the most bitter amino acid and D-tryptophan is the sweetest amino acid. The bigger, non-polar D-amino acids are somewhat sweeter (e.g. D-tryptophan is 35 x sweeter than saccharose), whereas the polar amino acids (D-glutamic acid, D-aspartic acid, D-arginine) are nearly tasteless. In addition, the bigger, non-polar L-amino acids are bitter, so that the configuration at the α-carbon atom is important with regard to the taste of the amino acid. In food products amino acids are sometimes added to improve the taste. The amounts are normally so low that it does not influence the nutritional value. Most soup flavours are a mixture of salt (NaCl) and amino acid preparations rich in L-glutamic acid. These flavour preparations are made by hydrolysing glutamic acid-rich and/or glutamine-rich proteins with strong HCl or via enzymatic hydrolysis and subsequently neutralising the hydrolysate with strong NaOH. L-glutamic acid is a special case. At high concentrations, L-glutamic acid has a broth-like taste and at low concentrations it has the effect of a flavour enhancer, i.e. the intensification of the characteristic flavour of a certain food. It is sold as Mono Sodium Glutamate (MSG). Cysteine and especially methionine have a distinct “sulphur taste”.  C h a p t e r 2 : P r o t e i n s | 33 Table 11. The taste of amino acids in water at neutral pH Amino acid Taste L-amino acid D-amino acid Quality Intensitya Quality Intensitya Alanine Sw 12-18 Sw 12-18 Arginine Bi Neu Asparagine Neu Sw 3-6 Aspartic acid Neu Neu Cysteine Neu Neu Glutamine Neu Neu 8-12 Glutamic acid Meat broth Neu Proteins Glycine Sw 25-35 Histidine Bi 45-50 Sw 2-4 Isoleucine Bi 10-12 Sw 8-12 Leucine Bi 11-13 Sw 2-5 Lysine Bi 80-90 Sw Methionine Sulphurous Sulphurous Phenylalanine Bi 5-7 Sw 1-7 Proline Sw 25.40 Neu Bi 25.27 Serine Sw 25.35 Sw 30-40 Threonine Sw 35.45 Sw 40-50 Tryptophan Bi 4-6 Sw 0.2-0.4 Tyrosine Bi 4-6 Sw 1-3 Caffein Bi 1-1.2 Saccharose sw 10-12 a Recognition threshold value (mmol/L); sw = sweet; bi = bitter; neu = neutral Taste of peptides Peptides usually taste bitter, but does not depend on the D or L configuration (as is the case with the free amino acids) (Table 12). Moreover, in general peptides are more bitter than the constituting amino acids. This is especially the case for the aromatic amino acids. The intensity of the taste is influenced by the hydrophobicity of the side chains of the constituent amino acids and sometimes by the order of the amino acids in the peptide chain. Bitter peptides can be formed in foods as a result of enzymatic degradation of proteins, for instance during the ripening of cheese or the production of protein hydrolysates (enzymatically hydrolysed proteins) used in infant milk formula for babies who are allergic to (intact) milk proteins. As bitterness is usually perceived as a detrimental quality parameter of foods, the bitterness of peptides is a major drawback for the application of protein hydrolysates in foods. As is clear from the above, sweet peptides do not occur naturally. However, one of the most commonly used low- calorie sweeteners, aspartame, is in fact a dipeptide. Aspartame does not occur in nature, but is synthetically made from aspartic acid and phenylalanine: Aspartame is a methyl ester of the dipeptide L-asp-L-Phe. The official name is -L-aspartyl-L-phenylalanine methylester. Aspartame is about 140 times sweeter than saccharose (beet/cane sugar; see also Chapter 1, Carbohydrates). Systematic research of the structure of sweet dipeptides showed that conditions for sweetness are: 1) the free α-carboxyl group has to be esterified; 2) - the N-terminal amino acid should be L-ASP and 3) the β-carboxyl group of L-Asp must be involved in the peptide bond. Although in practice quite a number of synthetic peptides suffice as low-calorie sweetener, it is aspartame that has been successfully applied. 34 Roles of proteins, peptides and amino acids in food Table 12. The taste threshold values (in mM) of different peptides at neutral pH: The effects of D,L-configuration, order of the amino acids and length of peptides. Peptidea Taste Quality Intensityb Gly-Leu Bi 19-23 Gly-D-Leu Bi 20-23 Gly-Phe Bi 15-17 Gly-D-Phe Bi 15-17 Leu-Leu Bi 4-5 Leu-D-Leu Bi 5-6 D-Leu-D-Leu Bi 5-6 Val-Ala Bi 65-75 Ala-Val Bi 60-80 Gly-Leu Bi 19-23 Ala-Leu Bi 18-22 Leu-Ala Bi 18-21 Leu-Gly Bi 18-21 Phe-Gly Bi 16-18 Gly-Phe Bi 15-17 Phe-Gly-Phe-Gly Bi 1.0-1.5 Phe-Gly-Gly-Phe Bi 1.0-1.5 a L-Configuration if not otherwise designated; bi = bitter b Recognition threshold value in mmol/L 2.7.3 SOLUBILITY OF PROTEINS When milk gets sour, because the milk is spoiled by acid forming bacteria, lumps appear in the milk. The same thing happens if an acid (e.g. lemon juice) is added to milk. The acid causes the proteins in the milk to stick together (=coagulate=aggregate), which results in the lumps. This coagulation takes place because the acid decreases the pH of milk to around the pI, where the net charge is 0 (Figure 33). If the pH is close to the iso- electric point, for many proteins the solubility decreases, causing them aggregate (Figure 35A). The solubility of a protein in water is predominantly related to the surface properties (the outside) of the protein. In general, all charged amino acids, and most of the polar non-charged amino acids are present on the surface of (globular) proteins. Still, a certain amount of hydrophobic amino acids will also be present at the surface of the protein, although most of the hydrophobic amino acids will be buried in the interior of the protein. The Figure 33: Charge on protein depends on pH. solubility of proteins is the result of the balance between repulsive electrostatic interactions (when all proteins have the same charge sign + or - ) and attractive hydrophobic interactions between proteins. At pH far away from the iso-electric point all the proteins in the solution (if they are the same type of protein) will have the same net charge. As a result, there will be an electrostatic repulsion. In that case, the protein will typically be soluble. However, if the protein also has a high proportion of hydrophobic amino acid residues on their surface, their solubility (even at pH away from pI) will be lower than proteins with a high proportion of  C h a p t e r 2 : P r o t e i n s | 35 hydrophilic (polar charged, or polar non-charged) amino acid residues at their surface. Unfolding of the protein will increase the exposure of hydrophobic groups and thereby also decrease the solubility at pH away from pI. The total net charge on the protein will change with the pH, depending on the number of side chains with a possibly negative (-COOH) or positive (-NH2) charge (see Table 6 for pKa's). When the pH reached the pI of the protein, the number of positive and negative charges on a protein is equal (same number of COO- groups as NH3+ groups), and the net charge is zero. This pH is called the iso-electric point (pI). Since there is no electrostatic repulsion when pH=pI, most proteins show a minimal solubility at the pI (Figure 34). The reason is that the small amount of hydrophobic groups on the surface of the protein is than sufficient to promote aggregation and precipitation. The solubility is also affected by salt. Three different regimes can be identified when discussing the effects of Proteins salt. 1- Starting from water, a s