Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

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FAO (1957) Protein requirements. FAO Nutritional Stud-

ies. 16. Rome: Food and Agriculture Organization.

FAO (1965) Protein requirements. Report of the FAO/

WHO Expert Committee. FAO Nutrition Report Series

37. Rome: Food and Agriculture Organization.

FAO (1973) Energy and protein requirements. Report of the

Joint FAO/WHO Expert Committee. FAO Nutrition

Report Series 52. Rome: Food and Agriculture Organ-

ization.

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the Joint FAO/WHO/UNU Expert Consultation. WHO

Technical Report Series 724. Geneva: World Health

Organization.

Functional Properties

J K P Weder and H-D Belitz

, Technische Universita

nchen, Garching, Germany

Introduction

0001 Standardization of food properties, in order to meet

nutritional, physiological and toxicological demands,

and requirements of food-processing operations, is a

perennial endeavor. Food production is similar to a

standard industrial fabrication process: on the one

hand is the food commodity with all its required

properties; on the other hand are the components of

the product, each of which supplies a distinct part of

the required properties. Such considerations have

prompted investigations into the relationship in food

between macroscopic physical and chemical proper-

ties and the structure and reactions at the molecular

level. Reliable understanding of such relationships is

a fundamental prerequisite for the design and oper-

ation of a process, either to optimize the process or to

modify the food components to meet the desired

properties of the product.

Functional Properties

0002 In common with other food constituents, proteins

contribute significantly to the physical properties

of foodstuffs, especially through their ability to

build or stabilize gels, foams, doughs, emulsions and

fibrillar structures. Table 1 shows some typical

examples of functional properties of proteins in rela-

tion to important food systems. Foaming, gelling, and

emulsifying properties will be discussed in more

detail.

Foaming Properties

0003Proteins act as foam-forming and foam-stabilizing

agents in various foodstuffs, e.g., in baked products,

sweets, desserts, and beer. The properties of various

proteins are different: serum albumin foams very

well, whereas ovalbumin does not. Mixtures of pro-

teins, e.g., egg white, can be particularly effective. In

that case, the globulins start the formation of foam;

ovomucin is important for its stabilization, and

ovalbumin and conalbumin are responsible for the

heat-setting properties.

0004Foams are dispersions of gases in liquids. Proteins

stabilize such systems by forming flexible, cohesive

films around the surface of the gas bubbles. During

whipping, the protein is adsorbed at the interface via

hydrophobic areas, followed by partial unfolding

(surface denaturation). The decrease of the surface

tension, caused by protein adsorption, facilitates the

formation of new interfaces and further gas bubbles.

The partially unfolded proteins associate under film

formation.

0005The foamability of a protein molecule depends on

its diffusion rate and the ease with which it is de-

natured. These parameters in turn depend on the

molecular mass, the surface hydrophobicity, and the

stability of the conformation.

0006Foams collapse because large gas bubbles grow at

the expense of smaller ones (‘disproportionation‘).

Therefore, the stability of a foam depends on the

stability of the protein film and its permeability for

gases. The stability of the film in turn depends on the

amount of adsorbed protein and on the ability of the

adsorbed protein to associate. Generally, surface de-

naturation exposes additional amino acid side-chains

that can participate in intermolecular interactions.

The stronger the cross-linking, the more stable is the

film. The pH of the system should be near the iso-

electric points of the involved proteins because the

association is promoted by a small net charge.

0007In summary, the ideal foam-forming and foam-

stabilizing protein is characterized by a low molecular

mass, high surface hydrophobicity, good solubility, a

small net charge at the pH of the food, and easy

denaturability.

0008Foams are destroyed by lipids and organic solvents

such as higher alcohols, which, because of their

hydrophobicity, displace proteins from the surface

of the gas bubbles without being able to form stable

films. Egg yolk, for example, prevents the whipping

of egg white, even at low concentrations. The disturb-

ance of protein association by the lecithins is respon-

sible for that effect.

0009The foaming properties of proteins can be im-

proved by chemical and physical modification. Partial

Deceased.

PROTEIN/Functional Properties 4835

enzymatic hydrolysis produces smaller molecules of a

higher diffusion rate, better solubility, and higher

surface hydrophobicity. The disadvantages are the

lower film stability and the loss of heat coagulability.

The introduction of charged or neutral groups or a

partial thermal denaturation (e.g., of whey proteins)

can also improve the desired properties. Recently, the

addition of strongly basic proteins (e.g., clupeines)

has been tested, which obviously increases the protein

association within the films and allows the foaming of

lipid-containing systems.

Emulsifying Properties

0010 Emulsions are disperse systems of two or more im-

miscible liquids. They are stabilized by emulsifiers,

compounds that form interfacial films and prevent

the dispersed phase from coalescing. Proteins are

able to stabilize emulsions (e.g., milk) due to their

amphipathic nature. The emulsifying properties of a

protein depend on the rate at which it diffuses into the

interface, on its adsorbability there, and on the

deformability of its conformation through interfacial

tension (surface denaturation). The diffusion rate

depends on the temperature and the molecular mass

of the protein, which in turn can be influenced by the

pH and the ionic strength. The adsorbability depends

on the exposure of hydrophilic and hydrophobic

groups, and thus on the amino acid composition, as

well as on the pH, ionic strength, and temperature.

The stability of the conformation depends on the

molecular mass, amino acid profile and number of

intramolecular disulfide bonds of the protein.

0011 Thus, a protein with ideal qualities as an emulsifier

for oil-in-water emulsions would have a relatively

low molecular mass, a balanced amino acid compos-

ition in terms of charged, polar and apolar side-

chains, good solubility in water, marked surface

hydrophobicity, and a relatively stable conformation.

Gel Formation

0012Gels are disperse systems of at least two components

in which a solid phase (dispersed phase) forms a

cohesive network in a liquid phase (continuous

phase). Gels are characterized by their lack of fluidity

and their elastic deformability. They are placed

between solutions with repulsive forces between

molecules of the dispersed phase predominating,

and precipitates with strong intermolecular forces

predominating. A differentiation is made between

two types of gels, the ‘polymeric networks‘ and the

‘aggregated dispersions.‘ Intermediate forms are also

known.

0013Examples of polymeric networks are the gels

formed by gelatin or by polysaccharides such as

agarose or carrageenan. The formation of a three-

dimensional network takes place by aggregation of

unordered fibrous molecules via limited ordered

structures, e.g., double helices. Gels of this type are

characterized by a low concentration of polymer

(c. 1%), transparency and fine texture. Gel formation

is started by adjusting to a suitable pH, by adding

suitable ions, or by heating followed by cooling. Since

the aggregation takes place mainly via hydrogen

bonds, which are easily solved when heated, poly-

meric networks are ‘thermo-reversible,‘ i.e., they are

formed when a solution is cooled, and they melt again

when they are heated.

0014Examples of aggregated dispersions are the gels

formed by globular proteins after denaturation by

heat. The thermal unfolding of the protein exposes

amino acid side-chains, which can take part in inter-

molecular interactions. The subsequent association

leads to small spherical aggregates, which combine

into linear strands whose interaction results in the

three-dimensional gel network. Because of the unor-

dered type of aggregation, gel formation requires a

relatively high protein concentration (5–10%). To

tbl0001 Table 1 Typical functional properties caused by proteins in food systems

Functional property Mode of action Food system

Solubility Protein solvation, pH-dependent Beverages

Water absorption and

binding

Hydrogen bonding of water, entrapment of water

(no drip)

Meat, sausages, bread, cakes

Viscosity Thickening, water binding Soups, gravies

Gelation Protein matrix formation and setting Meat, curds, cheese

Cohesion–adhesion Protein acts as adhesive material Meat, sausages, baked goods, pasta products

Elasticity Hydrophobic bonding in gluten, disulfide bridges

in gels (deformable)

Meat, bakery

Emulsification Formation and stabilization of fat emulsions Sausages (e.g., Bologna), soup, cakes

Fat adsorption Binding of free fat Meat, sausages, doughnuts

Flavor binding Adsorption, entrapment, release Simulated meat, bakery, etc.

Foaming Formation of stable films to entrap gas Whipped toppings, chiffon desserts, angel cakes

Modified from Kinsella and Srinivasan (alias Damodaran S) (1981).

4836 PROTEIN/Functional Properties

avoid the formation of coarse and fairly unstructured

gels, in particular in the area of the isoelectric point,

the aggregation rate should be slower than the

unfolding rate. The degree of unfolding necessary to

start aggregation seems to depend on the particular

protein. Since partial denaturation exposes mainly

hydrophobic groups, the aggregation takes place pre-

dominantly via intermolecular hydrophobic bonds,

and ‘thermoplastic‘ (thermo-irreversible) gels are

formed. This type of gel does not liquefy when

heated, but can soften or shrink. Besides hydrophobic

bonds, disulfide bonds formed from exposed thiol

groups can also contribute to cross-linking, as can

electrostatic bonds between proteins with different

isoelectric points in heterogeneous systems (e.g., egg

white).

0015 Addition of salts improves the gel formation. The

moderate increase in ionic strength promotes inter-

action between charged macromolecules or molecule

aggregates through charge shielding without precipi-

tation. An example is the improvement of thermal

gelling of soya globulins in soya milk by calcium

ions (soya curd, tofu).

Modification of Functional Properties

0016 Modification of proteins is still far from being a

common method in food processing, but it is increas-

ingly being recognized as essential, for two main

reasons. First, proteins fulfill many functions in

food, and some of these can be served better by

modified than by native proteins. Second, persistent

nutritional problems the world over necessitate the

utilization of new raw materials. Modifying reactions

can insure that such new raw materials (e.g., proteins

of plant or microbial origin) meet stringent standards

of food safety, palatability, and acceptable biological

value. This review includes several protein modifica-

tion reactions that are being used or are being con-

sidered for use. They involve chemical or enzymatic

reactions or a combination of both. Examples have

been selected to emphasize existing trends. Modifica-

tion of the properties of proteins is possible by

changing the amino acid composition or the size of

the molecule, or by removing or inserting hetero

constituents. Such changes can be accomplished by

chemical and/or enzymatic reactions.

0017From a food processing point of view, the aims of

protein modification are:

0018blocking the reactions involved in deterioration of

food (e.g., the Maillard reaction);

0019improving some physical properties of proteins

(e.g., texture, foam stability, whippability, solubil-

ity);

0020improving the nutritional value (increasing the

extent of digestibility, inactivation of toxic or

other undesirable constituents, introducing essen-

tial ingredients such as some amino acids).

Chemical Modification

0021A selection of chemical reactions of proteins that are

pertinent to, and of current importance in, food pro-

cessing is shown in Table 2.

0022Acylation Treatment with succinic anhydride gener-

ally improves the solubility of a protein. For example,

succinylated wheat gluten is quite soluble at

pH 5. This effect is related to disaggregation of high-

molecular-weight gluten fractions. In the case of

succinylated casein, the modification shifts the isoelec-

tric point of the protein (and thereby the solubility

minimum) to a lower pH. Succinylation of leaf pro-

teins improves the solubility as well as the flavor and

the emulsifying properties. Succinylated yeast protein

not only has an increased solubility in the pH range of

4–6 but is also more heat-stable above pH 5. It has

better emulsifying properties, surpassing many other

proteins, and has an increased whippability.

tbl0002 Table 2 Chemical reactions of proteins significant in food

Reactive group Reaction Product

—NH

Acylation —NH—CO—R

—NH

Reductive alkylation with HCHO — N(CH

)

—NH

Reductive glycosylation —NH—CH

—(CHOH)

—CH

—CONH

Hydrolysis —COOH

—COOH Esterification —COOR

—COOH Amidation (‘glycosylation‘) —CO—NH—CH(CHO)—(CHOH)

—OH Esterification —O—CO—R

—SH Oxidation —S—S—

—SH Alkylation —S—R

—S—S— Reduction —SH

—CO—NH— Hydrolysis —COOH þH

N—

PROTEIN/Functional Properties 4837

0023 Introduction of aminoacyl groups into protein

can be achieved by reactions involving amino acid

carboxy anhydrides, amino acids and carbodiimides,

or by t-butyloxycarbonyl (BOC)-amino acid N-

hydroxysuccinimide esters with subsequent removal

of the amino-protecting group (BOC). Feeding tests

with casein with attached methionine, as produced by

this method, have demonstrated a satisfactory avail-

ability of methionine. Such covalent attachment of

essential amino acids to a protein may avoid the

problems associated with food supplementation with

free amino acids: losses during processing, develop-

ment of undesired aroma due to methional, etc. With

b-casein as an example, it was shown that the associ-

ation of a protein is significantly affected by its acyla-

tion with fatty acids of various chain lengths.

0024 Alkylation Modification of proteins by reductive

methylation of amino groups with formaldehyde

and sodium borohydride (NaBH

) retards Maillard

reactions. The resultant methyl derivative, depending

on the degree of methylation, is less accessible to

proteolysis. Hence, its value from a nutritional and

physiological point of view is still an open question.

0025 Sugars such as glucose, fructose, maltose, or lactose

can be bound to the amino groups of proteins (casein,

legumin, b-lactoglobulin) by reductive glycosylation

with sodium cyanoborohydride. Whereas neutral

sugars favor foaming capacity and foam stability,

charged carbohydrates improve emulsifying proper-

ties. Amino sugars can also be bound to the carboxyl

group of proteins through an amide bond by the

carbodiimide method.

0026 Reactions involving cysteine and cystine Disulfide

bonds exert a strong influence on the properties of

proteins. Wheat gluten can be modified by reduction

of its disulfide bonds to sulfhydryl groups and subse-

quent reoxidation of these groups under various con-

ditions. Reoxidation of a diluted suspension in the

presence of urea results in a weak, soluble, adhesive

product (gluten A), whereas reoxidation of a concen-

trated suspension in the presence of a higher concen-

tration of urea yields an insoluble, stiff, cohesive

product (gluten C). Additional viscosity data have

shown that the disulfide bridges in gluten A are

mostly intramolecular, whereas those in gluten C are

predominantly intermolecular.

0027 Disulfide cleavage in soya protein by 0.1 M sodium

sulfite produces an adhesive with an acceptable vis-

cosity and improved adhesive strength and hydropho-

bicity compared with unmodified soya protein when

spray-dried, but a very low viscosity when freeze-

dried.

Enzymatic Modification

0028Of the great number of possible enzymatic reactions

with protein as a substrate, only a small number have

been found so far to be suitable for use in food

processing.

0029Dephosphorylation With b-casein as an example, it

was shown that the solubility of a phosphoprotein in

the presence of calcium ions is greatly improved by

enzymatic dephosphorylation.

0030Plastein reaction The enzyme-catalyzed formation

of larger polypeptides (about 3 kDa) from peptide

fragments of a hydrolysate, i.e., the formation of

peptide bonds, is called the ‘plastein reaction.‘

Amino acid esters that are added to the hydrolysate

are incorporated into the resulting products, which

are called ‘plasteins.‘ Incorporation of amino acid

esters into the polypeptide is affected by the nature

of the amino acid side-chain and by the length of the

alkyl chain of the ester. Amino acids with hydro-

phobic side-chains and long-chain alkyl esters are

preferred. Thus, an amino acid with a short side-

chain should be applied as an ester of a long-chain

alcohol. The plastein reaction can help to improve the

biological value of a protein. An example is the plas-

tein enrichment of zein with tryptophan, threonine,

and lysine.

0031Enrichment of a protein with selected amino acids

can be achieved with the corresponding amino acid

esters or, equally well, by using suitable partial hydro-

lysates of another protein. For example, the enrich-

ment of soya protein with sulfur-containing amino

acids is possible through ‘adulteration‘ with the

partial hydrolysate of wool keratin. The protein effi-

ciency ratio (PER) value of such a plastein is signifi-

cantly improved. In this way, the production of

plasteins with an amino acid profile very close to

that recommended by the Food and Agriculture

Organization and the World Health Organization

(FAO/WHO) can be achieved from very diverse

proteins, e.g., from leaf, bacterial and algal proteins.

0032The plastein reaction also makes it possible to im-

prove the solubility of a protein, e.g., by increasing

the content of glutamic acid. Soya protein (24% glu-

tamic acid) yields a plastein with 25% glutamic acid

and a Glu-plastein with 42% glutamic acid. Soya

protein has a pronounced solubility minimum in the

pH range of 3–6. The minimum is much less pro-

nounced in the case of the plastein, and the glutamic

acid-enriched Glu-plastein has a satisfactory solubil-

ity over the whole pH range and is also resistant

to thermal coagulation. Proteins with an increased

content of glutamic acid show an interesting sensory

4838 PROTEIN/Functional Properties

effect: partial hydrolysates of such plasteins do not

taste bitter, but exhibit a pronounced ‘meat broth‘

flavor.

0033 Elimination of the bitter taste from a protein

hydrolysate is also possible without incorporation of

hydrophilic amino acids. Bitter-tasting peptides, such

as leucyl-phenylalanine, which are released by partial

hydrolysis of proteins, react preferentially in the

subsequent plastein reaction and are incorporated

into higher-molecular-weight peptides with a neutral

taste.

0034 The versatility of the plastein reaction is also

demonstrated by examples wherein undesired amino

acids are removed from a protein. A phenylalanine-

free diet, which can be prepared by mixing amino

acids, is recommended for certain metabolic defects.

However, the use of higher-molecular-weight phenyl-

alanine-free peptides is more advantageous from a

sensory and osmotic point of view. Such peptides

can be prepared from proteins by the plastein reac-

tion. First, the protein is partially hydrolysed with

pepsin (pepsin A, EC 3.4.23.1). Then, treatment

with pronase (mycolysin, EC 3.4.24.31) under suit-

able conditions preferentially releases amino acids

with long hydrophobic side-chains such as phenyl-

alanine. The remaining peptides are separated by gel

chromatography and subjected to the plastein reac-

tion in the presence of added tyrosine and tryptophan

ethyl esters. This yields a plastein that is practically

phenylalanine-free and has a predetermined ratio of

other amino acids, including tyrosine. The plastein

reaction can also be carried out as a one-step process,

thus putting these reactions to economic, industrial-

scale use.

0035 Associations involving cross-linking Cross-linking

between protein molecules is achieved with peroxid-

ase (EC 1.11.1.7). The cross-linking occurs between

tyrosine residues when a protein is incubated with

a mixture of peroxidase and hydrogen peroxide

). Incubation of a protein with a mixture of

peroxidase, H

, and catechol also results in cross-

linking. In this case, oxidative deamination of lysine

residues takes place, followed by aldol and aldimine

condensations, i.e., reactions analogous to those cata-

lyzed by lysyl oxidase in connective tissue.

Texturized Proteins

0036 The world protein production for nutrition is cur-

rently about 22% from animal sources and 78%

from plant sources. The plant proteins are primarily

from cereals (53%) and oilseed meal (18%). Some

nonconventional sources of protein (single cell pro-

teins, leaves) have also acquired some importance.

Proteins are responsible for the distinct physical struc-

ture of a number of foods, e.g., the fibrous structure

of muscle tissue (meat, fish), the porous structure of

bread, and the gel structure of some dairy and soya

products.

0037Many plant proteins have a globular structure and,

although available in large amounts, are used to only

a limited extent in food processing. In an attempt to

broaden the use of such proteins, a number of pro-

cesses were developed in the mid-1950s that confer a

fiber-like structure to globular proteins. Suitable pro-

cesses yield products with cooking strength and a

meat-like structure. They are marketed as meat

extenders and meat analogs and can be used

whenever a lumpy structure is desired.

0038Starting material The following protein sources are

suitable for the production of texturized products:

soya; casein; wheat gluten; oilseed meals such as

from cottonseed, groundnut, sesame, sunflower, saf-

flower or rapeseed; zein (corn protein); yeast; whey;

blood plasma; or packing plant offal such as lungs or

stomach tissue. The required protein content of the

starting material varies and depends on the process

used for texturization. The starting material is often a

mixture such as soya protein with lactalbumin, or

protein plus acidic polysaccharide (alginate, carra-

geenan, or pectin).

0039The suitability of proteins for texturization varies,

but the molecular weight should be in the range of

10–50 kDa. Proteins of less than 10 kDa are weak

fiber builders, whereas those higher than 50 kDa are

disadvantageous due to their high viscosity and their

tendency to gel in the alkaline pH range. The propor-

tion of amino acid residues with polar side-chains

should be high in order to enhance intermolecular

chain binding. Bulky side-chains hinder such inter-

actions, so that the proportions of amino acids with

these structures should be low.

0040Texturization The globular proteins are unfolded

during texturization by solving the intramolecular

bonds. The resultant extended protein chains are sta-

bilized through interactions with neighboring chains.

In practice, texturization is achieved in one of two

ways:

0041The starting protein is dissolved, and the resultant

viscous solution is pressed through a spinning

nozzle into a coagulating bath (‘spin process‘).

0042The starting protein is moistened slightly, and then,

at high temperature and pressure, the molten pro-

tein is extruded with shear force through the orifice

of a die (‘extrusion process‘).

PROTEIN/Functional Properties 4839

In the spin process, the starting material (protein

content > 90%, e.g., soya protein isolate) is sus-

pended in water and dissolved by the addition of

alkali. The 20% solution is then aged at pH 11

through constant stirring. The viscosity increases

during this step as the protein unfolds. The solution

is then pressed through the orifices of a nozzle

(5000–15 000 orifices, each with a diameter of

0.01–0.08 mm) into a coagulating bath of pH 2–3.

The bath contains an acid (citric, acetic, phosphoric,

lactic, or hydrochloric) and, usually, 10% sodium

chloride. Spinning solutions of protein–acidic poly-

saccharide mixtures also contain earth alkali salts.

The resulting protein filaments are extended further

(to about two to four times the original length) in a

‘winding up‘ step, and are bundled into thicker fibers

with diameters of 10–20 mm. The molecular inter-

actions are enhanced during stretching of the fibers,

thus increasing the mechanical strength of the fiber

bundles.

0043 The adherent coagulation solvent is then removed

by pressing the fibers between rollers, followed by

passing a neutralizing bath (sodium bicarbonate plus

sodium chloride) of pH 5.5–6 and, occasionally, also

a hardening bath (concentrated sodium chloride).

The fiber bundles may be combined into thicker

cords with diameters of 7–10 cm. Additional treat-

ment involves passage of the bundles through a bath

containing a binder (a protein that coagulates when

heated, such as egg protein) and other additives

such as modified starch or other polysaccharides,

aroma compounds or lipids. This treatment produces

bundles with improved thermal stability and aroma.

A typical bath for fibers that are processed to produce

a meat analog might consist of 51% water, 15%

ovalbumin, 10% wheat gluten, 8% soya flour, 7%

onion powder, 2% protein hydrolysate, 1% sodium

chloride, 0.15% monosodium glutamate, and 0.5%

pigments. Finally, the soaked fiber bundles are heated

and chopped.

0044 In the extrusion process, the moisture content

of the starting material (protein content about 50%,

e.g., soya concentrate) is adjusted to 30–40%,

and additives (sodium chloride, buffers, aroma com-

pounds, pigments) are incorporated. Aroma com-

pounds are added in fat as a carrier, when necessary,

after the extrusion step to compensate for aroma

losses. The protein mixture is fed into the extruder

(a thermostatically controlled cylinder or conical

body that contains a polished, rotating screw with

a gradually decreasing pitch) that is heated to

120–180



C and develops a pressure of 3–4 10

Pa.

Under these conditions, the mixture is transformed

into a plastic, viscous state in which solids are dis-

persed in the molten protein. Hydration of the protein

takes place after partial unfolding of the globular

molecules and stretching and rearrangement of the

protein strands along the direction of mass transfer.

The process is affected by the rotation rate and shape

of the screw, and by the heat transfer and viscosity of

the extruded material and its residence time in the

extruder. As the molten material exits from the extru-

der through a die of 4 mm diameter, the water vapor-

izes, leaving behind vacuoles in the ramified protein

strands.

0045The extrusion process is more economical than the

spin process. However, it yields fiber-like particles

rather than well-defined fibers. A great number and

variety of extruders are now in operation. As with

other food processes, there is a trend toward develop-

ing and utilizing high-temperature/short-time (HTST)

extrusion cooking.

See also: Aerated Foods; Casein and Caseinates: Uses

in the Food Industry; Colloids and Emulsions;

Emulsifiers: Organic Emulsifiers; Enzymes: Uses in

Food Processing; Functional Foods; Protein: Chemistry;

Quality; Sources of Food-grade Protein; Rheological

Properties of Food Materials; Single-cell Protein:

Yeasts and Bacteria; Soy (Soya) Beans: Processing for

the Food Industry; Soy (Soya) Cheeses; Stabilizers:

Types and Function; Wheat: Grain Structure of Wheat and

Wheat-based Products

Further Reading

Damodaran S and Paraf A (eds) (1997) Food Proteins and

their Applications. New York: Marcel Dekker.

Froning GW (1988) Nutritional and functional properties

of egg proteins. In: Hudson BJF (ed.) Developments

in Food Proteins, vol. 6, pp. 1–34. London: Elsevier

Applied Science.

Hettiarachchy NS and Ziegler GR (eds) (1994) Protein

Functionality in Food Systems. New York: Marcel

Dekker.

Kinsella JE (1978) Texturized proteins: fabrication,

flavoring, and nutrition. Critical Reviews in Food

Science and Nutrition 10: 147–207.

Kinsella JE and Shetty KJ (1978) Yeast proteins: recovery,

nutritional and functional properties. Advances in

Experimental Medicine and Biology 105: 797–825.

Kinsella JE and Srinivasan D (1981) Nutritional, chemical

and physical criteria affecting the use and acceptability

of proteins in foods. In: Solms J and Hall RL (eds)

Criteria of Food Acceptance, pp. 296–332. Zurich:

Forster Verlag.

Mitchell JR (1986) Foaming and emulsifying properties of

proteins. In: Hudson BJF (ed.) Developments in Food

Proteins, vol. 4, pp. 291–338. London: Elsevier Applied

Science.

Morrissey PA, Mulvihill DM and O’Neil EM (1987) Func-

tional properties of muscle proteins. In: Hudson BJF

4840 PROTEIN/Functional Properties

(ed.) Developments in Food Proteins, vol. 5, pp. 195–

256. London: Elsevier Applied Science.

Morrissey PA, Mulvihill DM and O’Riordan D (1991)

Functional properties of blood proteins. In: Hudson

BJF (ed.) Developments in Food Proteins, vol. 7, pp.

125–166. London: Elsevier Applied Science.

Phillips LG, Whitehead DM and Kinsella J (1994)

Structure–Function Properties of Food Proteins. San

Diego, CA: Academic Press.

Pomeranz Y (1991) Functional Properties of Food Com-

ponents, 2nd edn. San Diego, CA: Academic Press.

Poole S and Fry JC (1987) High-performance protein

foaming and gelation systems. In: Hudson BJF (ed.)

Developments in Food Proteins, vol. 5, pp. 257–298.

London: Elsevier Applied Science.

Puigserver AJ, Sen LC, Clifford AJ, Feeney RE and

Whitaker JR (1978) A method for improving the nutri-

tional value of food proteins: covalent attachment of

amino acids. Advances in Experimental Medicine and

Biology 105: 587–612.

Watanabe M and Arai S (1988) The plastein reaction and its

applications. In: Hudson BJF (ed.) Developments in

Food Proteins, vol. 6, pp. 179–217. London: Elsevier

Applied Science.

Whitaker JR, Shahidi F, Munguia AL, Yada RY and Fuller G

(eds) (1998) Functional Properties of Proteins and

Lipids, ACS Symposium Series 708. Washington, DC:

American Chemical Society.

Interactions and Reactions

Involved in Food Processing

J K P Weder and H-D Belitz

, Technische Universita

nchen, Garching, Germany

Introduction

0001 The nature and extent of the chemical changes

induced in proteins by food processing depend on a

number of parameters, for example, food compos-

ition and processing conditions, such as temperature,

pH, or the presence of oxygen. The consequences of

such reactions may be desirable or undesirable. For

example, the biological value of proteins may be

decreased by destruction of essential amino acids,

conversion of essential amino acids into derivatives

that are not metabolized, or reduced digestibility of

protein as a result of intra- or interchain cross-

linking. The formation of toxic degradation products

is also possible. The nutritional/physiological and

toxicological assessment of changes induced by pro-

cessing of foods is a subject of some controversy and

opposing opinions.

Reactions with Carbohydrates

0002Many foodstuffs contain reducing sugars and amino

compounds such as proteins, peptides, amino acids,

and amines. Reactions between these components are

usually classed under the term ‘Maillard reaction‘ or

‘nonenzymatic browning.‘ They occur especially at a

higher temperature, low water activity and during

longer storage. Reactive sugars are glucose, fructose,

maltose, lactose, and, to a smaller extent, reducing

pentoses. On the side of the amino components, pri-

mary amines are more important than secondary

amines because their concentration in foods is usually

higher. Exceptions are, for example, malt and corn

products, which have a high proline content. In the

case of proteins, the e-amino groups of their lysine

residues react predominantly, but guanidino groups

of arginine residues can also react. These reactions

result in:

0003Brown pigments, known as ‘melanoidins,‘ which

contain nitrogen in variable amounts, and have

varying molecular masses and solubilities in water.

Little is known about their structure. Browning is

desired in baking and roasting, but not in foods

which are only slightly colored or have a distinct

color (condensed milk, faintly colored dried soups,

tomato soup).

0004Volatile compounds, which often contribute to the

aroma. Aroma formation through the Maillard re-

action is desired in cooking, baking, roasting, and

frying. However, the generation of off-flavors in

food during storage, especially in the dehydrated

state, or during pasteurization, sterilization, or

roasting is not desired.

0005Bitter substances, which are partially desired

(coffee), but can also cause an off-flavor, e.g., in

grilled meat or fish.

0006Reductones, which have highly reductive proper-

ties and contribute to the stabilization of foods

against oxidative deterioration.

0007Losses of essential amino acids (e.g., lysine,

methionine).

0008Mutagenic compounds.

0009Cross-linking of proteins.

The Maillard reaction of the e-amino group of lysine

prevails in the presence of reducing sugars, for

example, lactose or glucose, which yield the protein-

bound Amadori compounds, e-N-deoxylactulosyl-1-

lysine or e-N-deoxyfructosyl-1-lysine, respectively.

Lysine is not biologically available in these forms.

Deceased.

PROTEIN/Interactions and Reactions Involved in Food Processing 4841

Acid hydrolysis of such primary reaction products

yields lysine as well as the degradation products fur-

osine and pyridosine in a constant ratio. A nonredu-

cing sugar (e.g., sucrose) can also cause a loss of lysine

when conditions for sugar hydrolysis are favorable,

leading to the formation of the reactive sugars glucose

and fructose.

0010 Very recently, two intensely red-colored compounds

were isolated from brown–orange melanoidins with

mass > 10 000 Da obtained by thermal treatment of

an aqueous solution of casein and furan-2-carbox-

aldehyde (furfural), a well-known product of the

Maillard reaction. These compounds were identified

as the so-far unknown chromophoric amino acids

(S)-2-amino-6-{4-[ (E)-1-formyl-2-(2-furyl)ethenyl]-

5-(2-furyl)-2-[ (E)-(2-furyl)methylidene]-2,3-dihy-

dro-3-oxo-1H-pyrrol-1-yl}hexanoic acid and its

2-[ (Z)-(2-furyl)methylidene] isomer.

0011 In the late 1970s, it was shown that charred surface

portions of grilled fish and meat, as well as the smoke

condensates, isolated during grilling, exhibit highly

mutagenic effects in microbial tests (Ames test with

Salmonella typhimurium mutant strain TA 98). Model

experiments showed that pyrolysates of amino acids

and proteins are responsible for those effects. Table 1

lists several mutagenic compounds (pyridoindoles,

pyridoimidazoles, pyridocarbazoles, and tetraaza-

fluoranthenes) that were isolated from such pyro-

lysates.

0012 Mutagenic compounds can also be formed at lower

temperatures from amino acids and proteins. The

compounds listed in Table 2 were isolated from

meat extract, deep-fried meat, grilled fish, and heated

model mixtures of creatinine, an amino acid

(glycine, alanine, or threonine), and glucose. The

compounds, mainly imidazoquinolines and imidazo-

quinoxalines, are formed from creatinine, subsequent

products of the Maillard reaction (pyridines, pyra-

zines), and amino acids. Their toxicity is based on

the heteroaromatic amino function.

0013 Pentosidine (N

-[N-(5-amino-5-carboxy-1-pentyl)-

pyrido[4,5-b]imidazol-2-yl]-2,5-diaminopentanoic

acid), a cross-linked amino acid in which one arginine

and one lysine residue are linked together by a pen-

tose, was recently detected in foods. The low levels

found in commercial thermally processed food

samples (up to 4 mg kg

1

protein in milk products,

up to 40 mg kg

1

protein in roasted coffee beans

and bakery products) indicate that pentosidine

does not contribute significantly to food protein

cross-linking.

0014CROSSPY (1,4-bis-(5-amino-5-carboxy-1-pentyl)-

pyrazinium radical cation), a cross-linked amino acid

in which two lysine residues are linked together

through reaction with two molecules of glycolalde-

hyde, a Maillard reaction product, was more recently

isolated from heated aqueous solutions of bovine

serum albumin and glycolaldehyde and detected also

in toasted wheat bread crust and dark brown roasted

cacao as well as coffee beans by ESR (electron spin

resonance) spectroscopy.

Reactions with Lipid Oxidation Products

Products Formed from Hydroperoxides

0015Fatty acid hydroperoxides formed thermally or enzy-

matically in food are usually degraded further. This

degradation can also be of nonenzymatic nature. In

nonspecific reactions involving heavy metal ions,

heme(in) compounds or proteins, hydroperoxides

are transformed into oxo, epoxy, mono-, di-and tri-

hydroxy carboxylic acids. Unlike hydroperoxides,

i.e., the primary products of autoxidation, some of

these derivatives have a bitter taste. Such compounds

are detected in legumes and cereals. They may play a

role in other foods rich in unsaturated fatty acids and

proteins, such as fish and fish products.

Lipid–Protein Complexes

0016Studies related to the interaction of hydroperoxides

with proteins have shown that, in the absence of

oxygen, linoleic acid 13-hydroperoxide reacts with

N-acetylcysteine, yielding an adduct that consists of

tbl0001 Table 1 Mutagenic compounds from pyrolysates of amino acids and proteins

Mutagenic compound Short form Pyrolyzedcompound

3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole Trp-P-1 Tryptophan

3-Amino-1-methyl-5H-pyrido[4,3-b]indole Trp-P-2 Tryptophan

2-Amino-6-methyldipyrido[1,2-a:3

-d]imidazole Glu-P-1 Glutamic acid

2-Aminodipyrido[1,2-a:3

-d]imidazole Glu-P-2 Glutamic acid

3,4-Cyclopentenopyrido[3,2-a]carbazole Lys-P-1 Lysine

4-Amino-6-methyl-1H-2,5,10,10b-tetraazafluoranthene Orn-P-1 Ornithine

2-Amino-5-phenylpyridine Phe-P-1 Phenylalanine

2-Amino-9H-pyrido[2,3-b]indole AaC Soya globulin

2-Amino-3-methyl-9H-pyrido[2,3-b]indole MeAaC Soya globulin

Modified from Chen C, Pearson AM and Gray JI (1990) Meat mutagens. Advances in Food and Nutrition Research 34: 387–449.

4842 PROTEIN/Interactions and Reactions Involved in Food Processing

several isomers. However, in the presence of oxygen,

covalently bound amino acid–fatty acid adduct for-

mation is significantly suppressed; instead, oxidized

fatty acids are formed.

0017 The difference in reaction products is explained by

different reaction pathways. The thiyl radical, de-

rived from cysteine by abstraction of a hydrogen

atom, is added to the epoxyallyl radical only in the

absence of oxygen. In the presence of oxygen, oxida-

tion of cysteine and fatty acids to their oxidized forms

occurs with a higher reaction rate than in the previous

reaction. As a consequence, a large portion of the

oxidized lipid from a protein-containing food stored

in air does not have lipid–protein covalent bonds and,

hence, is readily extracted with a lipid solvent such as

chloroform/methanol (2:1, v/v).

Protein Changes

0018 Some properties of proteins change when they react

with hydroperoxides or their degradation products.

This is reflected by changes in food texture, decreases

in protein solubility (formation of cross-linked

proteins), changes in color (browning), and changes

in nutritive value (loss of essential amino acids).

0019The radicals generated from hydroperoxides

can abstract hydrogen atoms from proteins, preferen-

tially from the amino acids tryptophan, histidine,

lysine, arginine, cysteine and tyrosine, wherein

the nitrogen-, sulfur- or phenolic hydroxyl-containing

group reacts. Protein radicals combine with each

other, resulting in the formation of a protein net-

work.

0020Malonaldehyde is generated under certain condi-

tions during lipid peroxidation. As a bifunctional

reagent, malonaldehyde can cross-link proteins

through a Schiff base reaction with the e-amino

groups of two lysine residues. The Schiff base product

is a conjugated fluorochrome that has distinct spec-

tral properties (l

max

excitation *350 nm; l

max

emis-

sion *450 nm). Hence, it can be used to detect lipid

peroxidation and the reactions derived from it with

the proteins present.

tbl0002 Table 2 Mutagenic compounds from various heated foods and model systems

MutageniccompoundShortformFood/modelsystem

2-Amino-3-methylimidazo[4,5-f]quinoline IQ 1, 2, 3

2-Amino-3,4-dimethylimidazo[4,5-f]quinoline MeIQ 3

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline MelQx 2, 3

2-Amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline 4,8-DiMelQx 2, 3, 5, 6

2-Amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline 7,8-DiMelQx 4

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine PhIP 2

1, meat extract; 2, deep-fried meat; 3, grilled fish; 4, model mixture of creatinine, glycine and glucose; 5, as 4, but with alanine; 6, as 4, but with threonine.

Modified from Chen C, Pearson AM and Gray JI (1990) Meat mutagens. Advances in Food and Nutrition Research 34: 387–449.

tbl0003 Table 3 Amino acid losses occurring in proteins exposed to peroxidized lipids

ReactionsystemReactionconditionsAminoacidslost

(% loss)

Protein Lipid Time Temperature (



CaseinLinoleic ac idethylester24h55

Lys(10),Thr(10),Val(10),Ala(9),Tyr(8),Phe(8),Ser(8),

Arg(8),Asp(8)

c,d

CaseinLinoleic ac idethylester4days60

Lys(50),Met(47),Ile(30),Phe(30),Arg(29),Asp(29),Gly(29),

His(28),Thr(27),Ala(27),Tyr(27)

c,d

OvalbuminLinoleic ac idethylester24h55

Met(17),Ser(10),Lys(9),Ala(8),Leu(8)

c,d

OvalbuminLinoleic ac idethylester4days60

Lys(50),Met(42),Leu(22),His(21),Val(21)

c,d

RibonucleaseLinoleicacid40min37

Met(99),Tyr(62),His(54),Lys(51),Cys(40)

TrypsinLinoleic ac id40min37

Met(83),His(12)

LysozymeLinoleic ac id8days37

Trp(56),His(42),Lys(17),Met(14),Arg(9)

Only the most labile amino acids are listed.

Aqueous system.

Trp not analyzed.

Cys not analyzed.

80% relative humidity.

Data from Gardner HW (1979) Lipid hydroperoxide reactivity with proteins and amino acids: a review. Journal of Agricultural and Food Chemistry 27: 220–

229 and references therein.

PROTEIN/Interactions and Reactions Involved in Food Processing 4843

0021 Reactions resulting in the formation of a protein

network like that outlined above have practical

implications; for example, they are responsible for

the decrease in solubility of fish protein during frozen

storage. Also, the monocarbonyl compounds derived

from autoxidation of unsaturated fatty acids readily

condense with free amino groups of the proteins, thus

forming Schiff bases that can provide brown poly-

mers by repeated aldol condensations. The brown

polymers are often nitrogen-free since the amino

compound can be readily eliminated by hydrolysis.

When hydrolysis occurs in the early stages of aldol

condensations (after the first or second condensation)

and the released aldehyde, which has a powerful odor,

does not re-enter the reaction, the condensation pro-

cess results not only in discoloration (browning) but

also in a change in aroma.

Decomposition of Amino Acids

0022Studies of model systems have revealed that protein

cleavage and degradation of side-chains, rather than

the formation of protein networks, are the preferred

reactions when the water content of protein–lipid

mixtures decreases. Examples for the extent of

losses in amino acids in a protein in the presence

of an oxidized lipid are presented in Table 3. The

strong dependence of this loss on the nature of the

protein and on reaction conditions is obvious. Deg-

radation products obtained in model systems of

pure amino acids and oxidizing lipids are listed in

Table 4.

Reactions Under Alkaline Conditions

0023Losses of available lysine, cystine, serine, threonine,

arginine, and some other amino acids occur at

high pH values. Hydrolysates of alkali-treated pro-

teins often contain some unusual compounds, such

as ornithine, b-aminoalanine, lysinoalanine, ornithi-

noalanine, lanthionine, methyllanthionine and

d-allo-isoleucine, as well as other d-amino acids.

The formation of these compounds is based on

the following reactions: 1,2-eliminations in the

case of hydroxy amino acids and thio amino acids

tbl0004 Table 4 Products formed from amino acids exposed to peroxidizing lipids

Reactionsystem Compoundsformed fromaminoacids

Amino acid Lipid

Histidine Methyl linoleate 3-(Imidazol-4-yl)lactic acid, 3-(imidazol-4-yl)acetic acid; histamine, valine, aspartic acid,

ethylamine

Cysteine Ethyl arachidonate Cystine, hydrogen sulfide, alanine

Linoleic acid Cystine, cysteic acid, cystine disulfoxide

Methionine Methyl linoleate Methionine sulfoxide

Lysine Methyl linoleate Diaminopentane, aspartic acid, glycine, alanine, a-aminoadipic acid, pipecolinic acid,

1,10-diamino-1,10-dicarboxydecane

Modified from Gardner HW (1979) Lipid hydroperoxide reactivity with proteins and amino acids: a review. Journal of Agricultural and Food Chemistry

27: 220–229.

tbl0005 Table 5 Formation of unusual amino acids by alkali treatment

of proteins

Name Formula

3-N

-Lysinoalanine (R

—

3-N

-Lysino-3-methylalanine

—

)

COOH

CHNH

COOH

CHNH

(CH

)

CHR NH

3-N

-Ornithinoalanine (R

—

3-N

-Ornithino-3-

methylalanine (R

—

)

COOH

CHNH

COOH

CHNH

(CH

)

CHR NH

Lanthionine (R

—

3-Methyllanthionine (R

—

)

COOH

CHNH

COOH

CHNH

CHR S

3-Aminoalanine (R

—

2,3-Diaminobutyric acid

—

)

COOH

CHNH

CHRNH

3-N

-Histidinoalanine

COOH

CHNH

HC CH

COOH

CHNH

3-N

-Histidinoalanine

COOH

CHNH

COOH

CHNH

4844 PROTEIN/Interactions and Reactions Involved in Food Processing