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3.1 Introduction Proteins are complex macromolecules made up of carbon, hydrogen, oxygen, nitrogen and usually sulphur. They are constructed from amino acids and they are present in all living tissue, where they function as enzymes e.g. pepsin and amylase; structural materials e.g collagen, keratin and membrane proteins; in transport e.g. haemoglobin and in storage e.g. ferritin and casein and as hormones e.g. insulin. Many of the structural or biologically active proteins are food proteins. Food proteins are those that are palatable, digestible, nontoxic, and available economically for humans. The deficit in nutritional proteins is very large for some segments of the world population. Food proteins are more expensive to produce than carbohydrate or lipids and in order to satisfy the steadily growing demand for proteins, new protein sources must be found and methods developed for their technological utilization. Conventional proteins must be used more efficiently. It is therefore, very important to have available as much data as possible on the physical, chemical and biological properties of food proteins. Knowing the effects of technological treatments on these proteins is also important, as it allows their properties, particularly nutritional quality and functional characteristics to be improved.

3.2 Physicochemical properties of amino acids 3.2.1 Structure and classification of amino acids Amino acids are the basic structural units of proteins. The basic structure of amino acids consists of an carbon atom covalently attached to a hydrogen atom, an amino group, a carboxyl group and a side -chain R group:




There are 20 different types of amino acids, differing in the R group. Every amino acid has a characteristic R side chain which influences its physicochemical properties and the properties of

the protein to which it belongs. Amino acids vary in size, shape, charge, hydrogen bonding capacity and chemical reactivity and are grouped into 4 families depending on the polarities of their side chains: 1. Amino acids with positively charged side chains lysine (lys), arginine (arg) and histidine (his) basic side chains with a positive charge at neutral pH

2. Amino acids with negatively charged side chains aspartate and glutamate acidic side chain with a negative charge at neutral pH hydrophilic side chain

3. Amino acids with uncharged polar side chains no formal charge but have polar properties serine (ser), cysteine (cys), asparagines (asn), glutamine (gln), threonine (thr), tyrosine (tyr) 4. Amino acids with hydrophobic side chains very little affinity for water glycine (gly), alanine (ala), valine (val), leucine (leu), isoleucine (ile), tryptophan (trp), phenylalanine (phe), methionine (met), proline (pro)

3.2.2 Stereochemistry of amino acids Except for glycine, the C atom of amino acids is asymmetric, so amino acids can exist as L- or D- amino acids:







Only the L- amino acids are used as building blocks for proteins.

3.2.3 Acid- Base properties of amino acids Amino acids contain carboxyl (acidic) and amino (basic) groups and can thus behave as both acids and bases (ampholytes). The ionization state of an amino acid depends on the pH e.g. glycine exists in 3 ionized states depending on pH:












In acid solution, the carboxyl group is unionized (COOH) and the amino group is ionized (+NH3). In alkaline solution, the carboxyl group is ionized (COO-) and the amino group unionized (NH2). The pH with at which the dipolar ion is electrically neutral (zero charge) is termed the isoelectric point (pI) of that amino acid and the amino acid exists as a dipolar ion or zwitterion.

3.2.4 Chemical reactions of amino acids Amino acids have various reactive groups e.g. amino, carboxyl, sulfhydryl, phenolic, imidazole and guanyl groups. Reactions of these groups alter the hydrophilic, hydrophobic and functional properties of proteins and peptides. Some of the reactions are used in the quantification of amino acids in proteins e.g: 1. Amino acid reaction with ninhydrin The ninhydrin reaction is used to detect and estimate amino acids quantitatively in small amounts. Heating with excess ninhydrin yields a purple color with all amino acids having a free -amino group, whereas a yellow product is formed from proline, in which the amino group is substituted. Under appropriate conditions, the intensity of the color produced can be used to measure the amino acid concentration colorimetrically.

2. Amino acid reaction with 1-fluoro-2,4-dinitrobenzene (FDNB) In mildly alkaline solution, FDNB reacts with a-amino acids to yield 2,4-dinitrophenyl derivatives, which are useful in the identification of individual amino acids. The reaction is useful in the determination of the amino acid sequence of peptides. 3. Amino acid reaction with flourescamine Flourescamine reacts with primary amines, forming highly fluorescent derivatives. The reaction allows the rapid and sensitive quantitative determination of amino acids, peptides and proteins.

3.3 Physichochemical properties of proteins 3.3.1 Protein structure Four levels of protein structure are distinguished: primary, secondary, tertiary, quaternary Primary structure The primary structure of a protein refers to the linear sequence of constituent amino acids covalently linked through peptide bonds and to the position of disulphide bridges within the protein. The formation of a peptide linkage is shown below:

H C R1

O C OH + H2N

H C R2



H H2N C R1


H C R2


The chain length and sequence of amino acids in proteins determine the physicochemical, structural and biological properties of the proteins. Secondary structure The secondary structure of a protein represents the spatial arrangement of amino acid residues. Two important forms of secondary structure are the -helix structure and the -pleated sheet structure. - helix structure is a rod-like structure in which the amino acid side chain is wound up in a spiral-like shape. There are 3.6 amino acid residues per turn and the structure is stabilized by hydrogen bonds in the same chain:

In the -pleated sheet structures, amino acids are present as extended parallel chains, in which each oxygen atom of one chain is hydrogen bonded to a nitrogen atom in a neighbouring chain. There is fusion of 2 or more polypeptide chains which may be parallel or antiparallel. There is interchain H-bonding: Tertiary structure The overall three dimensional structure of a polypeptide chain is the tertiary structure. The structure is stabilized by hydrogen bonds between the side chains, thus the amino acid sequence of a protein specifies its three dimensional structure. Fibrous proteins are elongated and sparingly soluble while globular proteins are ellipsoidal, water soluble, random coils. Quartenary structure Proteins containing more than one polypeptide chain exhibit an additional level of structural organisation. Quarternary structure refers to the arrangement of polypeptide chains in relation to one another in a multi-chained protein. The polypetide chains are mainly joined together by hydrogen bonds between the side chains of the different peptide chains. Chains are also linked via noncovalent interactions such as van der Waals interactions, electrostatic interactions and hydrophobic interactions.

3.3.2 Protein Denaturation


The primary structure of a protein comprises the covalent bonds: peptide bonds can only be broken by boiling in strong acid and disulphide bonds by treating the protein with mercaptoethanol. The secondary and tertiary structures are kept together by weak hydrogen bonds and can be lost easily, e.g by shortly heating the protein. The native form of a protein is transformed into an arbitrary structure in which only the primary structure is intact. This is called denaturing of a protein. Denaturation is therefore defined as any modification in the conformation of a protein (secondary, tertiary or quartenary structure) , without the disruption of peptide bonds involved in the primary structure. Disadvantages of protein denaturing are that it may lead to insolubilization of proteins and loss of some functional properties of the protein. Water-binding capacity of proteins is altered, and there is increased susceptibility to attack by proteases and inability to crystallize. Advantages of protein denaturing are that there is increased digestibility of some proteins, such as the increased digestibility of legume proteins upon denaturation of trypsin inhibitors, better foaming and emulsifying properties are obtained when proteins are partially denatured and denaturing is necessary for heat-induced gelation of food proteins. Protein denaturing may be reversible or irreversible depending on the denaturing conditions. Physical protein denaturing agents Temperature Heat is the most common physical agent capable of denaturing proteins. For many reactions, the rate of protein denaturation increases about 600 fold with a 10oC increase in temperature. Destabilization of major noncovalent interactions occurs. Heating to a temperature above the critical temperature results in a transition from the native to the denatured state. The susceptibility of proteins to denaturation by heat depends on the nature of the protein, protein concentration, water activity, pH, ionic strength and the kind of ions present. Heat denaturation is often followed by a decrease in solubility due to exposure of hydrophobic groups and to aggregation of unfolded protein molecules. Heat can cause chemical alteration of some amino acid residues e.g. deamidation of glutamine and asparagine. These changes can alter the nutritive and functional properties of proteins. Low temperature can result in denaturation of some proteins. Some enzymes, which are stable at room temperature, may become less stable at 4oC. Reversible denaturation may occur at low

temperatures e.g. glycinin in soya bean loses its activity at 4oC, and regains the activity at ambient temperature.

Hydrostatic pressure Hydrostatic pressure can have a denaturing effect at pressures around 12kbars. Denaturation occurs due to the flexibility and compressibility of proteins. Pressure results in elimination of void spaces in the protein. The denaturation is highly reversible.

Shear Some mechanical treatments such as shaking, kneading or whipping may denature proteins as a result of shearing forces. There is incorporation of air bubbles and adsorption of protein molecules to the air-liquid interface. Repeated stretchings modify the protein network by disruption of helices. Highly flexible proteins readily denatured. Irradiation The effects of electromagnetic radiation on proteins vary depending on the wavelength and energy involved. Ultraviolet radiation is absorbed by aromatic amino acid residues and this can result in modification of conformation. radiation and other ionizing radiations can also produce changes in conformation, together with oxidation of amino acid residues, rupture of covalent bonds, ionization, formation of protein free radicals and recombination and polymerization reactions.

Interfaces Protein molecules that adsorb at interfaces of water and air, or water and a nonaqueous liquid or solid phase, usually become irreversibly denatured. Protein denaturation begins with diffusion of the macromolecule toward the interface. At this stage, the protein would interact with highenergy, interfacial water molecules, many protein-protein hydrogen bonds would be simultaneously disrupted and microunfoldings of the structure would take place. The partially unfolded protein is hydrated and activated, and also unstable, since many hydrophobic groups are exposed to the aqueous phase. It is by further unfolding and spreading of the protein at the interface that hydrophilic and hydrophobic residues can attempt to reorient themselves,

respectively, in the aqueous and nonaqueous phases. The protein thus adsorbed at the interface is denatured. Chemical denaturation agents pH The pH of the medium in which the protein is placed has considerable influence on the denaturation process. Proteins are stable at their isoelectric points when they are electrically neutral. Extreme pH values result in protein swelling and unfolding due to strong electrostatic repulsions of ionized groups inside the molecule. In most cases, the protein may recover its native structure when pH is brought back to the initial stability range.

Organic solvents Most organic solvents alter the dielectric constant of the medium and therefore the electrostatic forces that contribute to the stability of proteins. The extent of denaturation depends on the effect on polar and nonpolar interactions. Nonpolar organic solvents are able to penetrate into hydrophobic regions, disrupting hydrophobic interactions and promoting denaturation of the protein. Certain solvents e.g. chloro-2-ethanol, increase the prevalence of helices. Ovalbumin has 31% helices in an aqueous medium and 85% in chloro-2-ethanol.

Detergents Surface-active agents such as sodium dodecylsulfate (SDS) preferentially bind to protein, contributing to the unfolding of the protein. The denaturation is irreversible as there is strong binding.

Salts Nonspecific electrostatic interactions occur at low salt concentrations, while at high salt concentrations, greater than 1M, ion specific effects influence the structural stability of proteins. The denaturation mechanism is not well understood.

Metals Alkaline metals, such as sodium and potassium ions react only to a limited extent with proteins, whereas alkaline earth metals, such as calcium and magnesium, are more reactive. Transition metals , e.g. copper react readily with proteins, many forming stable complexes with thiol groups.

3.4 Functional properties of proteins The functional properties of proteins influence the sensory properties of foods e.g. in bakery, meat and dairy products. The functionality of a food protein is defined as physical and chemical properties which affect the behavior of the protein in food systems. Examples of functional properties of proteins required in various foods are presented in the table below:

Food Beverages

Functionality Solubility at different pH, heat stability, viscosity

Soups, sauces Dough formation, baked products

Viscosity, emulsification, water retention Formation of a matrix and film with viscoelastic properties, cohesion, gelation, water absorption, emulsification, foaming, browning

Dairy products

Emulsification, fat retention, viscosity, foaming, gelation, coagulation

Meat products

Emulsification, gelation, cohesion, water and fat absorption and retention

3.4.1 Protein hydration The conformation of individual proteins in solution is largely dependent on interactions with water. Swelling occurs due to movement of water into clefts and crevices of proteins. Water-

protein interactions affect the rheological and textural properties of foods such as solubility, thickening, gelation,coagulation, emulsification and foaming. Protein hydration is dependent on the number of charged amino acid residues. There is least hydration at isoelectric pH when the protein is neutral. The water binding capacity of denatured protein is about 10 times greater than of native protein due to the increase in surface area to mass ratio. However, in denatured, aggregated protein, water binding capacity may decrease The high affinity ionic groups are solvated first at low water activity, followed by polar and nonpolar groups. Protein hydration is important in the juiciness and tenderness of meat products and the textural properties of bakery products.

3.4.2 Protein solubility The degree of solubility of a protein is an important attribute of proteins as it affects thickening, foaming, emulsification and gelling of foods. Solubility is dependent on the hydrophilicity and hydrophobicity of the protein surface. Most denatured food protein exhibits low solubility. Solubility is influenced by pH, ionic strength, temperature and organic solvents. Influence of pH on solubility Proteins exhibit minimum solubility at isoelectric point when they are not charged. The lack of electrostatic repulsion promotes agrregation/ precipitation. The pH-solubility profile of proteins is altered by heat as there is an increase in hydrophobicity of the protein surface due to unfolding Influence of ionic strength on solubility At low inonic strength (<0.5), ions neutralize charges at the surface of proteins. The charge screening affects solubility in two different ways, depending on the characteristics of the protein surface. Solubility decreases for those proteins that contain a high incidence of nonpolar patches, and it increases for those that dont. While the decrease in solubility is caused by enhanced hydrophobic interactions, the increase in solubility is caused by a decrease in the ionic activity of the protein macroion. At ionic strength >1.0, salts have ion specific effects on protein solubility. Sulphate and fluoride ions progressively decrease solubility (salting out), whereas thiocyanate and perchlorate salts increase solubility (salting in).

10 Influence of temperature on solubility Between 0 and 40oC, solubility of a protein increases with temperature but above 40 oC, aggregation and precipitation of the protein occurs resulting in decreased solubility. Influence of organic solvents on solubility Organic solvents such as ethanol or acetone result in increased intra- and intermolecular electrostatic forces, both repulsive and attractive. Repulsive intramolecular electrostatic interactions cause unfolding of the protein molecule, promoting intermolecular hydrogen bonding between exposed peptide groups and attractive intermolecular electrostatic interactions between oppositely charged groups. The interactions lead to precipitation of the protein in organic solvents or reduced solubility in an aqueous medium.

3.4.3 Flavor binding property of proteins Proteins bind to carbonyl compounds generated from lipid oxidation through noncovalent interactions resulting in off-flavors being imparted e.g to whey protein concentrates. Proteins are used as used as flavor carriers or flavor modifiers, with denatured proteins having greater flavor binding capacity.

3.4.4. Protein gelation A gel is an intermediate phase between a solid and a liquid. Polymers are cross-linked to form a network capable of entrapping water. Heat, enzymes and divalent cations induce formation of a network structure. Most food protein gels are prepared by heating a protein solution. The protein unfolds, losing its native structure to form a progel. A protein network is then formed when interactions occur between the exposed protein groups. Network interactions include H-bonds, hydrophobic interactions and electrostatic interactions. Gel networks may be reversible when mainly H-bonds are involved, as is the case with gelatin gels, which melt upon heating and the setting-melting cycle can be repeated many times. Irreversible gel networks are formed when interactions consist mainly of disulphide bonds and hydrophobic interactions, as is the case with ovalbumin. Two types of protein gels can be formed, depending on the amino acid residues involved in the protein structure. Coagulum gels are formed when there is a large amount of nonpolar amino

acid residues while translucent gels are formed when there are small amounts of nonpolar amino acid residues:

Coagulum type gel


Native protein

Denatured protein

Translucent Gel

A translucent gel holds more water and is less prone to syneresis than a coagulum type gel. Coagulum type gels consist of coarsely aggregated protein particles, are opaque, lack elasticity and are susceptible to syneresis. Gelation plays a major role in the preparation of many foods, including various dairy products, coagulated egg white, gelatin gels and bread doughs. Protein gelation is not only used for the formation of solid viscoelastic gels , but also for improved water absorption, thickening, adhesion and emulsion or foam stabilizing effects. Gelation is affected by pH and salts. Calcium bridges improve the firmness and stability of many gels.

3.4.5 Dough formation The gluten proteins of wheat grain endosperm are able to form a strongly cohesive and viscoelastic paste or dough when mixed and kneaded in the presence of water at ambient temperatures. Gluten is a heterogenous mixture of proteins such as gliadins and glutenins. Due to their low content of ionizable amino acids, the gluten proteins are poorly soluble in neutral


aqueous solutions. Being rich in glutamine and hydroxy amino acids, the proteins are prone to hydrogen bonding. Progress has been made in explaining how gluten proteins of wheat behave during the formation of dough and during bread making. When hydrated bread flour is mixed and kneaded, the shear forces generated cause the gluten proteins orient, align and partially unfold. This enhances hydrophobic interactions and the formation of disulphide cross-links through disulphide interchange reactions. A viscoelastic protein network is established as gluten particles form thread-like polymers, forming a sheet-like film capable of entrapping gas. Strong flours from certain wheat varieties require long mixing times and give very cohesive doughs. Weak flours are less effective, and the gluten network breaks down when the energy or duration of mixing exceeds a certain level, probably because disulphide bonds are ruptured. Dough strength is apparently related to a large content of high molecular weight glutenins. Glutenins are responsible for the elasticity, cohesiveness and mixing tolerance of the dough, and gliadins facilitate fluidity, extensibility and expansion of the dough, contributing to a large bread-loaf volume. Excessive glutenins result in high cohesion, inhibiting expansion of trapped CO2 bubbles during fermentation and dough rising. Excessive gliadins result in high extensibility, causing gluten films that are weak and permeable, poor CO 2 retention and dough collapse. Baking apparently does not induce extensive additional denaturation of gluten proteins. Soluble wheat proteins such as albumins and globulins are denatured and aggregated by baking, and the partial gelation contributes to the setting of the breadcrumbs.

3.4.6 Interfacial properties of proteins Several foods are foam- or emulsion- type products, that is, there is some dipersion. A dipersion is a system of discrete particles in a continuous phase. Examples of dispersions are shown in the table below:

Dipersed phase Gas Liquid Solid Solid

Continuous phase Liquid Liquid Gas Liquid


Dispersion type Foam Emulsion Powder Suspension




The stability of dispersions is influenced by the presence of an amphiphilic substance at the interface. Proteins are often surfactants of choice for foams and oil-water emulsions. Proteins form highly viscoelastic films at interfaces with the ability to withstand mechanical shock. Protein surface activity is influenced by the stability and flexibility of the protein, its ease of adaptability to environmental changes and the distribution of hydrophobic and hydrophilic groups of the protein. Desirable surface-active proteins are able to rapidly adsorb at the interface, rapidly unfold and reorient at the interface, interact with other molecules and form a viscoelastic film. Hydrophobic patches influence the probability of protein adsorption. Highly flexible molecules undergo rapid conformational changes at the interface. Configurations at the interface may be tail, loop or train type , with the train configuration having stonger binding:

Intermolecular forces at the interface, such as attractive electrostatic attractions, hydrogen bonding and hydrophobic interactions influence the strength of the protein film. A balance of forces is therefore necessary for the formation of a stable viscoelastic film. Emulsifying properties of proteins Many food products are emulsions e.g. milk, ice cream, butter, cheese and salad dressings. Protein constituents play a major role in stabilizing the colloidal systems. Fundamentals of emulsion formation and stabilization have been described in section 3.4.6. In oil/water emulsions such as fresh milk, proteins adsorb at the interface between dispersed oil droplets and the continuous aqueous phase and contribute physical and rheological properties such as thickness, viscosity and elasticity or rigidity. Proteins are generally poor stabilizers of water/oil emulsions, probably attributable to the predominantly hydrophilic nature of most proteins, causing the bulk of an adsorbed protein molecule to reside on the water side of the interface. Many factors influence the characteristics of emulsions, and these include pH, type of protein, temperature and the presence of low molecular weight surfactants. Most proteins are sparingly soluble at their pI values and at certain ionic strengths, adopting compact structures with high

viscoelasticity. This may either prevent unfolding and adsorption at the interface or stabilize an already adsorbed protein film against surface deformation or desorption, favoring emulsion stability. Some proteins have have optimal emulsifying properties at the pI (gelatin and egg white proteins) and others perform better at pH values away from the pI (soy proteins, peanut proteins, casein). Flexible proteins, able to unfold and spread when in contact with the lipid surface, readily establish hydrophobic interactions with lipid droplets, produce adsorbed films with desirable viscoelasticity properties and successfully stabilize emulsions. Globular proteins, with a stable structure and great surface hydrophilicity such as whey proteins and ovalbumin, are poor emulsifying agents unless they can be unfolded by prior treatment without loss of solubility. Moderate heat treatments can be carried out to partially unfold the proteins. Heating usually decreases the viscosity and rigidity of the protein film adsorbed at the interface, decreasing emulsion stability. However, gelation of the highly hydrated interfacial protein film increases its surface viscosity and rigidity and stabilizes the emulsion. Gealation of myofibrillar proteins contributes to heat stability of meat emulsions such as sausages. The addition of surfactants of small molecular size is usually detrimental to the stability of protein-stabilized emulsions because they decrease the firmness of protein films and lessen the forces causing proteins to remain at the interface. Foaming properties of proteins Food foams are usually dispersions of gas bubbles in a continuous liquid or semisolid phase. A continuous phase of thin liquid layers separates the gas bubbles and mechanical energy is required for creation of this interface. Foams are usually produced by whipping or shaking an aqueous protein solution in the presence of a bulk gas phase or bubbling a gas through a sparger into an aqueous solution of low protein concentration. A major difference between emulsions and foams is that, in foams, the volume fraction occupied by the dispersed phase (gas) varies over a much broader range than in emulsions. Foams exist with widely differing textures such as meringue, cakes, whipped cream and beer froth, and because many foams have very large interfacial areas, they are often unstable. Maintaining the interface against coalescence of gas bubbles necessitates the presence of surfaceactive agents. Proteins are able to form a thin film at the gas-liquid interface, lowering the

interfacial tension and forming an elastic protective barrier between entrapped gas bubbles. Proteins therefore play a role in foam stabilization. Protein stabilized foams are affected by factors such as pH, the presence of salts, sugars and lipids and heat. Many studies stress the importance of high protein solubility as a prerequisite to good foaming capacity and stability, although it also appears that insoluble protein particles can play a beneficial role in stabilizing foams, probably by increasing surface viscosity. Salts affect the solubility, viscosity, unfolding and aggregation of proteins and this can alter foaming properties. Calcium ions may improve foam stability by forming bridges between carboxyl groups of the protein. Sucrose and other sugars often depress foam expansion but improve foam stability, as they increase bulk viscosity. Thus, in making meringues and other foams, it is preferable to add sugar at a late stage, when foam expansion has already taken place. Moderate heat treatments prior to foam formation improve certain foaming properties while more severe thermal treatments impair foaming capacity by causing air expansion, decreased viscosity, bubble rupture and foam collapse, unless gelation of the protein contributes sufficient rigidity to the adsorbed film to stabilize the foam. Surface active polar lipids interfere with the most desirable conformation of adsorbed protein films by situating themselves at the air/water interface, thus imparing foaming performances of proteins.

3.5 Changes in the functional properties of food proteins through processing and storage Proteins used as food ingredients usually undergo physical or chemical treatments during preparation or use, and the treatments can influence protein functionality. Isolated proteins may be intentionally modified to improve existing properties or to develop new ones. 3.5.1 Heat treatment Most food proteins display biological or functional properties only within a narrow temperature range. Extensive denaturation results in insolubilization of proteins and thus has an effect on properties dependent on protein solubility. Partial denaturation improves digestibility and bioavailability of essential amino acids. This is attributable to protein unfolding and the exposure of previously buried amino acid residues, enabling proteases specific for these amino acids to act more quickly and extensively. Blanching or cooking leads to the inactivation of several enzymes such as proteases, lipooxygenases and polyphenoloxidases, that could cause the formation of

undesirable colors or flavors or undesirable changes in texture. Most protein toxins naturally present in foods are denatured and inactivated by heat. Extreme heat such as in baking or grilling leads to amino acid racemization and decomposition. Maillard browning decreases protein solubility and impairs digestibility. Thermal treatments, depending on intensity, can lead to desulphuration, deamidation, isomerization and other chemical modifications of amino acids, sometimes followed by formation of toxic compounds. Sterilization at temperatures above 115oC, causes partial destruction of cysteine residues and formation of hydrogen sulphide, dimethylsulphide and cysteic acid. Such reactions are common with meats, fish and milk. Deamidation reactions take place during the heating of proteins at temperatures above 100oC. The ammonia released comes mainly from the amide groups of glutamine and asparagines, modifying the functional properties of the proteins. Severe heat treatments above 200oC, as well as heat treatments at alkaline pH cause isomerization of amino acid residues. Since most D-amino acids have no amino acids, racemization reduces the nutritional value of amino acids. The presence of D-isomers reduces the digestibility of the protein and certain D amino acids exert a toxic action.

3.5.2 Extraction, fractionation and dehydration Techniques used to purify, concentrate or separate individual proteins may lead to differences in amino acid contents of proteins. The procedures, which include isoelectric precipitation or salting out, usually result in reversible aggregation if conducted at low temperatures. An exception is casein, in which there is a collapse of the quartenary micellar structure in isoelectronically precipitated casein due to isoelectric aggregation and precipitation of the protein. This occurs because carboxyl groups become protonated, leading to a weakening or rupture of carboxyl-Ca2+-carboxyl linkage, release of calcium phosphate and an increased electrostatic attraction among casein molecules. Isoelectric precipitation is commonly used in purification and there is loss of some of the protein in the crude extract. This leads to alteration in amino acid composition and nutritional value of the protein. The partial removal of water from protein solutions leads to increased concentrations of all nonaqueous constituents. As a result, increased protein-protein, protein-carbohydrate, and protein-salt interactions may occur and these may alter a proteins functional properties. Marked changes are observed in whey protein concentrate and this affects foaming, emulsification and gelation properties of the protein.

Protein concentrates prepared from low-salt whey display excellent gelling and foaming properties. Protein inhibitors and toxic factors may be either eliminated or concentrated in the final purified protein. Nearly complete removal of water often causes extensive aggregation (protein-protein interactions), especially if high temperatures are used. This may result in severe loss of protein solubility and surface activity.

3.5.3. Reaction with metal ions At appropriate pH, the presence of polyvalent ions or of some polyelectrolytes enhances the formation of ionic cross-links between protein molecules. Calcium ions (Ca2+) can cross-link proteins through their ionized carboxyl groups at neutral or alkaline pH inducing protein aggregation. Insoluble calcium proteinates can be solubilized by the addition of Ca2+-complexing reagents such as citrates or polyphosphates.The resulting dissociation and unfolding frequently improve water absorption, swelling and surface properties.

3.5.4 Effect of pH Acid or alkaline pH enhances the binding of anions or cations, respectively by proteins. This affects functional properties of proteins, especially solubility. As described in section 3.5.1, heating of proteins in alkaline pH results in deamidation of Asn and Gln residues and -elimination of cysteine residues. Alkaline treated proteins are more soluble with improved emulsification and foaming properties

3.5.5 Effect of mechanical treatments Intense shear forces such as those generated in homogenization of milk cause fragmentation of protein aggregates resulting in a general improvement in protein emulsification properties. Partial denaturation of proteins may stabilize foams while intense forces decrease foam capacity e.g. intense egg beating. Extensive dry milling of protein flours or concentrates results in powders with small particle sizes and large surface areas. This generally results in improved properties of water absorption, protein solubility and foaming properties, as compared with unmilled counterparts. Mechanical forces play an important role in protein texturization


processes such as dough or fiber formation. Protein alterations enhanced by shear forces are molecular alignment, disulphide bond interchange and formation of protein networks.

3.6 Protein modification Protein modification involves alteration of the physicochemical properties of the proteins, usually with the intention of improving the functional properties of the proteins or imparting new functional properties to the proteins. However, intentional protein modification has certain drawbacks, which include damage to nutritional value, formation of amino acid derivatives that may be toxic and the incorporation of reagent residues that may also be toxic. Protein modification is usually chemical or enzymatic.

3.6.1 Chemical modification Alkylation

Alkylation of proteins involves the reaction of amino acids with iodoacetate or iodoacetamide. The disulphide groups (SH) and amino groups of the amino acid react: e.g. with iodoacetate



The increased electronegativity of the treated protein alters its solubility. There is blocking of disulphide-induced protein polymerization. Hydrophobicity of the protein is increased if an aliphatic long chain aldehyde or ketone is used. Interfacial properties of the proteins are affected. Acylation Amino acid reaction with acid anhydrides e.g. acetic anhydride:


The presence of additional negative charges results in electrostatic repulsion, unfolding and dissociation, with an improvement in protein solubility and/or dispersibility, even at the isoelectric pH. Acetylated proteins are more soluble than native proteins. The proteins waterabsorption capacity and heat stability are generally improved, as has been observed with fish proteins, soy proteins and gluten.

3.6.2 Enzymatic modification Enzymatic hydrolysis Improvement of food proteins has been attempted through limited or extensive proteolysis using proteases such as pepsin, papain, trypsin


low Mw peptides

Proteolysis results in solubilization of poorly soluble proteins as in preparation of soluble protein hydrolyzates from fish or vegetable proteins. Hydrolysed proteins useful in liquid type products e.g. soups. Papain has been used in meat tenderization. Limited specific proteolysis can cause protein coagulation, as occurs when chymosin and fungal proteases are used in the precipitation of casein in cheese making. Partial proteolysis has been used to improve the emulsifying or foaming properties of heatdenatured proteins, probably due to increased solubility of the hydrolyzate, which facilitates diffusion and spreading at oil/water and air/water interfaces. Extensive protein hydrolysis often results in the release of bitter tasting peptides with leucine or phenylalanine terminal residues. Such hydrolyzates may, however, be useful for patients with impaired digestive functions and for those allergic to milk or gluten proteins. Extensive hydrolysis damages the gelation, foaming and emulsifying properties of proteins because of the reduction of molecular size while partial hydrolysis improves foaming and emulsifying properties. Plastein reaction In plastein formation, proteolysis is followed by resynthesis of peptide bonds. The first step involves proteolysis of a 5 % protein suspension with pepsin or papain at neutral pH to obtain

peptides of 1-20 000 Da. The second step involves concentrating the hydrolyzate to a 30-40 % protein concentration, adding the same or another protease, and adjusting the pH. This allows random rearrangement of peptides into plasteins through transpeptidation reactions. New polypeptides are formed with altered functional properties. The second reaction may be used to covalently attach sulphur-rich peptides to protein hydrolyzates or incorporate essential amino acids such as lysine or methionine to the protein to improve the nutritional quality of methionine or lysine deficient food proteins. Protein cross-linking This involves the formation of unnatural covalent bonds between polypeptide chains. Transglutaminase catalyses the covalent cross-linking of lysyl residues with glutamine residues:

New forms of protein produced might have improved functional properties. At high protein concentration, protein gels are formed at room temperature. Protein cross-linking may be used to improve protein nutritional quality.

3.6 Interaction of proteins with other food components

3.7.1 Interaction of proteins with carbohydrates Maillard browning, which is described in detail in section, results in major losses of lysine. Crosslinking of proteins decreases their solubility. Some of the carbonyl derivatives of Maillard browning may react with amines and free amino acids, leading to the formation of ammonia, CO2 and new carbonyl compounds such as aldehydes. This reaction is known as Strecker degradation. Volatile products, such as aldehydes, pyrazines and sugar fragmentation products from the Strecker degradation reaction may contribute to aromas and flavor. Commercially, the Strecker degradation is used to produce the distinctive flavors of chocolate, honey, maple syrup and bread.

3.7.2 Interaction of proteins with lipids Lipoproteins, consisting of noncovalent complexes of proteins and lipids occur widely in living tissues and influence the physical and functional properties of foods. In some cases, covalent

bonds occur between oxidation products of lipids and proteins. Unsaturated lipids (RH) usually undergo autoxidation to form peroxy free radicals (ROO.). The lipid free radicals can add to protein (PH) resulting in formation of lipid-protein free radicals: ROO. + PH ROO.+PH ROOP. ROOH +P.

or the lipid free radical can react with a protein to form a protein free radical:

The protein free radicals formed can react resulting in protein cross-linking and polymerization. Lipid-protein reactions can have adverse nutritional effects. Noncovalent binding of carbonyl compounds to proteins imparts off-flavors

3.7.3 Interaction with polyphenols Polyphenols, which are phenolic compounds naturally occurring in many plant tissues, can, in the presence of oxygen, be oxidized to quinones. Quinones can irreversibly react with SH and NH2 groups of amino acids. Tannins bind SH and NH2 groups of amino acids resulting in decreased digestibility and availability of protein bound lysine and cysteine.

3.7.4 Other protein interactions Poteins react with halogenated solvents e.g methyl bromide, decreasing nutritional value. They also react with nitrates forming carcinogenic nitrosamines. Amino acids such as proline, tryptophan and tyrosine constitute reactive substrates for the reaction. Protein disulphide bonds are reduced by sulfites to yield S-sulfonate derivatives. S-sulfonation causes protein unfolding, affecting functional properties of the protein.

3.8 Specific food proteins

3.8.1 Milk proteins Milk is one of the excellent sources of protein in mans diet. The proteins of cows milk have extensively been studied, and those of human milk have received considerable attention. Milk

proteins can be separated by precipitation at definite pH, salt fractionation, and occasionally, heat coagulation. Casein Casein is the major protein in milk. It has been shown to be a mixture of proteins, which have been named , and caseins and differ from one another in their molecular weights, their rate of migration in an electric field and their phosphorus content. Quantities of casein are 3.0-3.5 % in cows milk and 0.3-0.6 % in human milk. Casein is precipitated by acidifying milk to pH 4.7. or by action of the enzyme rennin or fungal proteases. Whey proteins Whey proteins are not precipitated by acidifying milk to pH 4.7 and represent about 20% of the protein contained in skimmed milk. Examples of whey proteins include albumin, which is soluble in saturated magnesium sulphate, and lactalbumin. Colostrum Colostrum is the first secretion of the mammary gland and consists of 17.5 % protein, 5 % being casein. It carries antibodies for protection against diseases.

3.8.2 Egg proteins Whole egg is an excellent food because it is a very rich source, not only of protein and lipid, but also of most of the vitamins and many of the required minerals except calcium. The proteins of egg are rather numerous are considered to be of high biological value. Some of the proteins of egg white have unusual properties, which give them the ability to protect the embryo from bacterial invasion or regulate the nutrition of the embryo. Lysozyme is an antibiotic, ovomucoid is a trypsin inhibitor, ovomucin is an inhibitor of haemagglutination. Avidin binds biotin, while conalbumin binds iron.


3.8.3 Meat proteins o myosin has a structural role and aids muscle contraction o collagen is hydrolysed to gelatin o collagen affects toughness or tenderness of meat o elastin is not hydrolysed on cooking, makes meat rubbery