Effect of heat treament on food protein

Nguyen Le Huy 20190624

Through their provision of amino acids, proteins are essential to human growth, but they
also have a range of structural and functional properties which have a profound impact on
food quality. Proteins play a fundamental role not only in sustaining life, but also in foods
derived from plants and animals. Foods vary in their protein content and even more so in
the properties of those proteins. In addition to their contribution to the nutritional
properties of foods through provision of amino acids that are essential to human growth and
maintenance, proteins impart the structural basis for various functional properties of foods.

Definition of protein
The word “protein” is defined as any of a group of complex organic compounds, consisting
essentially of combinations of amino acids in peptide linkages, that contain carbon,
hydrogen, oxygen, nitrogen, and usually, sulfur. Widely distributed in plants and animals,
proteins are the principal constituent of the protoplasm of all cells and are essential to life.
(“Protein” is derived from a Greek word meaning “first” or “primary” because of the
fundamental role of proteins in sustaining life.)

Protein in food
Amino acids, peptides, and proteins are important constituents of food. They supply the
required building blocks for protein biosynthesis. In addition, they directly contribute to the
flavor of food and are precursors for aroma compounds and colors formed during thermal
or enzymatic reactions in production, processing, and storage of food. Other food
constituents, e.g., carbohydrates, take part in such reactions. Proteins also contribute
significantly to the physical properties of food through their ability to stabilize gels, foams,
doughs, emulsions, and fibrillar structures.
The most important protein sources and their contribution to world-wide production of
protein. Cereals contribute to protein production by more than half, followed by oil seeds
and meat. Besides plants and animals, algae, yeasts and bacteria may be used for protein
production (single-cell protein (SCP)). The protein content varies as follows: > 20% (cheeses,
meat, legumes, oil seeds); 10–20% (fish, eggs); 5–10% (cereals); and < 5% (milk, roots, tubers,
vegetables, fruits, mushrooms).
Cereals and cereal products
Cereals and cereal products are amongst the most important staple foods of mankind.
Proteins provided by bread consumption in industrial countries meet about one-third of the
daily requirement. The major cereals are wheat, maize, rice, barley, sorghum, oats, millet,
and rye. Wheat and rye have a special role since only they are suitable for bread-making.
With the example of wheat, the cereal proteins have been separated by the basis of their
solubility, into four fractions: the water-soluble albumins, the salt-soluble globulins, the 70%
aqueous ethanol-soluble prolamins, and the remaining glutelins. The levels of fractions differ
amongst cereals with albumins amounting to 4–44%, globulins 3–12%, prolamins 2–48%,
and glutelins 24– 77% of the whole protein fraction. Each of fractions consists of a larger
number of proteins. Albumins and globulins contain the enzymes, whereas prolamins and
glutelins are storage proteins.
Meat and meat products
Meat and meat products are other important staple foods, in particular in industrial
countries. The main meat-producing warm-blooded animals are pig, cattle, poultry, sheep,
goats, and buffalo. Meat proteins, i.e., the proteins of the muscle, are divided into three
groups: proteins of the contractile apparatus (myofibrillar proteins), soluble proteins
(sarcoplasma proteins), and insoluble proteins (connective tissue and membrane proteins).
The myofibrillar proteins of a typical mammalian muscle amount to about 60% of total
muscle protein, with myosin (29%) and actin (13%) as their predominating components and
about 20 minor components including connectin, tropomyosins, troponins, and actinins. The
sarcoplasma proteins form about 30% of total protein. They contain most of the enzymes, in
particular those of glycolysis and the pentosephosphate cycle, but also considerable
amounts of creatine kinase (2.7% of total protein), myoglobin, and some hemoglobin. The
insoluble proteins contain collagen as the main component, besides elastin, insoluble
enzymes, and cytochrome c. In connective tissue, collagen forms a triple-stranded helix
composed of α-helices. Covalent cross-links are formed between the fibers of collagen
during maturation and aging, thus improving its mechanical strength. When heated, collagen
fibers shrink or are converted into gelatine, depending on the temperature. The structure of
the gelatin obtained after cooling depends on the gelatine concentration and temperature
gradient. Collagen contains two unusual amino acids, 4-hydroxyproline and 5-hydroxylysine.
Since the occurrence of the former is confined to connective tissue, its determination
provides data on the extent of connective tissue content of a meat product.
Milk and dairy products
Milk and dairy products form a further important group of staple foods. Milk generally means
cow’s milk, but milk from buffalo, goats, and sheep is of importance in some regions. Milk
proteins, in particular the caseins, play an important role in processing to yield dairy products
such as cheese and sour milk products. The caseins make up about 80% of total milk proteins.
They have been separated later into different fractions: 𝛼 -, 𝛼 -, 𝜅-,𝛽-, and 𝛾-caseins,
constituting 34, 8, 9, 25, and 4% of total protein, respectively. In cheese-making, the specific
cleavage of 𝜅 -casein by chymosin into para- 𝜅 -casein and a glycopeptide reduces the
solubility of the casein complexes and casein micelles, thus leading to their aggregation
followed by gel formation (curd formation). The whey proteins (about 20% of total protein)
consist of 𝛽 -lactoglobulins, 𝑎 -lactalbumins (both in different genetic variants), serum
albumin, immunoglobulins, and proteose-peptone. Also, more than 40 enzymes occur in the
whey protein fraction, but in much lower quantities than the other components. Whey
proteins can be incorporated into the curd using several new processing methods of cheese-
making in order to increase the yield and also to reduce waste water or whey treatment
costs.
Legumes
Legumes (pulses) are very important staple foods in some parts of the world, e.g., soya beans
in South-east Asia and common beans in Latin America. Other legumes, some of greater
regional importance, include peas, peanuts, chick peas, broad beans, and lentils. Legume
proteins, when fractionated in a similar way to cereal proteins, yield three fractions:
albumins, globulins, and glutelins. The portion of the fractions varies, depending on the
legume species, but globulins always predominate. The globulins are subdivided, initially
according to sedimentation during ultracentrifugation, into 11S and 7S globulins (legumins
and vicilins, respectively). Again, the subfractions derived from different legumes are
sometimes designated by special names, e.g., glycinin and arachin for soya bean and peanut
legumin, as well as 𝛽-conglycinin and phaseolin for soya bean and common bean vicilin,
respectively. Soya protein isolates, produced by diluted alkali extraction of defatted soya
bean flakes followed by acid precipitation, are texturized and flavored for use as meat
substitutes or are added to foods to raise their protein level and improve their processing
qualities such as the water-binding capacity or emulsion stability. The isolates contain about
95% protein and consist of 11S and 7S globulins. The similarity between the caseins from
bovine milk and the globulins from soya beans is reflected by the production of some typical
Asian foods such as soy milk, soy curd (tofu), and soy cheese (sufu).
Eggs
Eggs are used as a food not only because of their 0excellent nutritional quality but also
because of their functional properties. Eggs generally means chicken eggs; those of other
birds (geese, ducks, plovers, seagulls, quail) are less important. Egg proteins are divided into
those of egg white and those of egg yolk. Egg white proteins (about 10% of total egg white)
are ovalbumin, conalbumin (ovotransferrin), ovomucoid, ovomucin, lysozyme, ovoglobulin
G2, ovoglobulin G3, and some minor components (54, 12, 11, 3.5, 3.4, 4, 4, and 2.5% of total
egg white protein, respectively). Ovalbumin, conalbumin, ovomucin, and the ovoglobulins G
contribute to foam formation and foam stability. Yolk proteins (about 17% of total yolk) are
phosvitin, the livetins, and the protein moieties of high-density lipoproteins (HDL) and low-
density (LDL) lipoproteins (13, 31, 36, and 20% of total yolk protein, respectively). Apart from
phospholipids, LDL and proteins are responsible for the emulsifying effect of whole eggs or
egg yolk alone. Owing to the ability of all egg proteins, except ovomucoid and phosvitin, to
coagulate when heated, egg products are important food binding agents.
The nutritional quality of a food protein depends on the absolute content of essential amino
acids, the relative proportions of essential amino acids, and their ratios to nonessential
amino acids. In addition, the digestibility of the food protein, the influence by other food
components such as dietary fibers, polyphenols, or proteinase inhibitors, and also the total
food energy intake are of importance. During pregnancy and lactation, the first 6 months,
and after 6 months, the daily requirement increases by 13, 24, and 18%, respectively. The
biological value of a protein is generally limited by the following amino acids:
 Lysine: deficient in proteins of cereals and other plants;
 Methionine: deficient in proteins of bovine milk and meat;
 Threonine: deficient in wheat and rye;
 Tryptophan: deficient in casein, corn and rice.

Protein properties
Conformation
Primary structure
The primary structure gives the sequence of amino acids in a protein chain with peptide
linkage. The peptide bonds have partial (40%) double-bond character with p-electrons
shared between the C–O and C–N bonds. Normally the bond has a trans configuration, i.e.,
the oxygen of the carbonyl group and the hydrogen of the NH group are in the trans position;
a cis configuration only occurs in exceptional cases.
Secondary structure
The secondary structure reveals the arrangement of the chain in space. The peptide chains
are not in an extended or unfolded form.
𝜷-sheet The peptide chain is always lightly folded on the C atom, thus the R side chains
extend perpendicularly to the extension axis of the chain, i.e., the side chains change their
projections alternately from +𝑧 to −𝑧. Such a pleated structure is stabilized when more
chains interact along the 𝑥 axis by hydrogen bonding, thus providing the crosslinking
required for stability. When adjacent chains run in the same direction, the peptide chains
are parallel. This provides a stabilized, planar, parallel sheet structure. When the chains run
in opposite directions, a planar, antiparallel sheet structure is stabilized.
Helical structures The peptide chains are coiled like a threaded screw. These structures are
stabilized by intrachain hydrogen bridges which extend almost parallel to the helix axis,
cross-linking the CO and NH groups.
Reverse turns An important conformational feature of globular proteins is the reverse turns,
𝛽 -turns, or 𝛽 -bends. They occur at “hairpin” corners, where the peptide chain changes
direction abruptly. Such corners involve four amino acids residues, among them frequently
proline. Glycine is favored in the third position of 𝛽 -bends on the basis of energy
considerations. Different types of 𝛽-turns are known, for which different amino acids are
allowed.
Super secondary structures Analysis of known structures has demonstrated that regular
elements can exist in combined forms. Examples are the coiled-coil 𝛼-helix, chain segments
with antiparallel 𝛽-structures (𝛽-meander structure) and combinations of 𝛽-helix and 𝛽-
structure.
Tertiary and Quaternary Structure
Proteins can be divided into two large groups on the basis of conformation: (1) fibrillar
(fibrous) or scleroproteins, and (2) folded or globular proteins.
Fibrous proteins The entire peptide chain is packed or arranged within a single regular
structure for a variety of fibrous proteins. Examples are wool keratin (𝛼-helix), silk fibroin (𝛽-
sheet), and collagen (a triple helix). Stabilization of these structures is achieved by
intermolecular binding (electrostatic interaction and disulfide linkages, but primarily
hydrogen bonds and hydrophobic interactions).
Globular proteins Regular structural elements are mixed with randomly extended chain
segments (random-coiled structures) in globular proteins. The proportion of regular
structural elements is highly variable: 20–30% in casein, 45% in lysozyme, and 75% in
myoglobin. Five structural subgroups are known in this group of proteins: (1) 𝛼-helices occur
only; (2) 𝛽-structures occur only; (3) 𝛼-helical and 𝛽-structural portions occur in separate
segments on the peptide chain; (4) 𝛼-helices and 𝛽-structures alternate along the peptide
chain; and (5) 𝛼-helices and 𝛽-structures do not exist. The process of peptide chain folding
occurs spontaneously, probably arising from one center or from several centers of high
stability in larger proteins. Folding of the peptide chain packs it densely, by formation of a
large number of intramolecular noncovalent bonds.
Quaternary structures In addition to the free energy gain by folding of a single peptide chain,
association of more than one peptide chain (subunit) can provide further gains in free energy.
In principle, such associations correspond to the folding of a larger peptide chain around
several structural domains without covalently binding the subunits.
Denaturation
It is a reversible or irreversible change of native conformation (tertiary structure) without
cleavage of covalent bonds (except for disulfide bridges). Denaturation is possible with any
treatment that cleaves hydrogen bridges, or ionic or hydrophobic bonds. This can be
accomplished by changing the temperature, adjusting the pH, increasing the interfacial area,
or adding organic solvents, salts, urea, guanidine hydrochloride, or detergents such as
sodium dodecyl sulfate. Denaturation is generally reversible when the peptide chain is
stabilized in its unfolded state by the denaturing agent and the native conformation can be
reestablished after removal of the agent. Irreversible denaturation occurs when the
unfolded peptide chain is stabilized by interaction with other chains (as occurs for instance
with egg proteins during boiling). During unfolding, reactive groups, such as thiol groups,
that were buried or blocked may be exposed. Their participation in the formation of disulfide
bonds may also cause an irreversible denaturation. Denaturation of biologically active
proteins is usually associated with loss of activity. The fact that denatured proteins are more
readily digested by proteolytic enzymes is also of interest.

Physical properties
Dissociation
Proteins, like amino acids, are amphoteric. Depending on pH, they can exist as polyvalent
cations, anions, or zwitterions. Since 𝛼-carboxyl and 𝛼-amino groups are linked together by
peptide bonds, the uptake or release of protons is limited to free terminal groups, and mostly
to side chains. In contrast to free amino acids, the pKa values fluctuate greatly for proteins
since the dissociation is influenced by neighboring groups in the macromolecule. In the
presence of salts, e.g., when binding of anions is stronger than that of cations, the isoelectric
point is lower than the isoionic point. In most cases the shift in pH is consistently positive,
i.e., the protein binds more anions than cations.
Solubility, Hydration, and Swelling Power
Protein solubility is variable and is influenced by the number of polar and apolar groups and
their arrangement along the molecule. Generally, proteins are soluble only in strongly polar
solvents such as water, glycerol, formamide, dimethylformamide, or formic acid. In a less
polar solvent such as ethanol, few proteins have appreciable solubility. The solubility in
water is dependent on pH and on salt concentration. Protein solubility is decreased (“salting-
out” effect) at higher salt concentrations due to the ion hydration tendency of the salts.
Since proteins are polar substances, they are hydrated in water. The degree of hydration
(grams of water of hydration per gram protein) is variable. It is 0.22 for ovalbumin (in
ammonium sulfate), 0.06 for edestin (in ammonium sulfate), 0.8 for 𝛽-lactoglobulin, and 0.3
for hemoglobin.
The swelling of insoluble proteins corresponds to the hydration of soluble proteins in that
insertion of water between the peptide chains results in an increase in volume and other
changes in the physical properties of the protein. The amount of water taken up during
swelling can exceed the dry weight of the protein by several times.

Chemical Reactions
In contrast to free amino acids, except for the relatively small number of functional groups
of the terminal amino acids, only the functional groups in protein side chains are available
for chemical reactions.
Lysine Residue
Reactions involving the lysine residue can be divided into several groups: (1) reactions
leading to a positively charged derivative; (2) reactions eliminating the positive charge; (3)
derivatizations introducing a negative charge; and (4) reversible reactions. The last are of
particular importance.
Arginine Residue
The arginine residue of proteins reacts with 𝛼- or 𝛽-dicarbonyl compounds. Reaction of the
arginine residue with 1,2-cyclohexanedione is highly selective and proceeds under mild
conditions. Regeneration of the arginine residue is again possible with hydroxylamine.
Glutamic and Aspartic Acid Residues
These amino acid residues are usually esterified with methanolic hydrochloric acid. There
can be side reactions, such as methanolysis of amide derivatives or N,O-acyl migration in
serine or threonine residues. Diazoacetamide reacts with a carboxyl group and also with the
cysteine residue to carboxamidomethyl derivatives.
Amino acid esters or other similar nucleophilic compounds can be attached to a carboxyl
group of a protein with the help of a carbodiimide. Amidation is also possible by activating
the carboxyl group with an isoxazolium salt to an enolester and its conversion with an amine.
Cystine Residue
Reductive cleavage of cystine occurs with sodium borohydride and with thiols. odification.
Cleavage of cystine is also possible by a nucleophilic attack.
Electrophilic cleavage occurs with Ag+ and Hg+ or Hg2+. The sulfenium cation which is formed
can catalyze a disulfide exchange reaction. In neutral and alkaline solutions a disulfide
exchange reaction is catalyzed by the thiolate anion.
Cysteine Residue
A number of alkylating agents yield derivatives which are stable under the conditions for acid
hydrolysis of protein. The reaction with ethylenimine gives an S-(2-aminoethyl) derivative
and, hence, an additional linkage position in the protein for hydrolysis by trypsin. lodoacetic
acid, depending on the pH, can react with cysteine, methionine, lysine, and histidine residues.
Cysteine is readily converted to the corresponding disulfide, cystine, even under mild
oxidative conditions, such as treatment with iodine or potassium hexacyanoferrate(III).
Stronger oxidation of cysteine, and also of cystine, e.g., with performic acid, yields the
corresponding sulfonic acid, cysteic acid.
Methionine Residue
Methionine residues are oxidized to methionine sulfoxide with hydrogen peroxide. The
sulfoxide can be reduced, regenerating methionine, using an excess of thiol reagent. With
performic acid, methionine sulfone is formed.
𝛼 -Halogen carboxylic acids, 𝛽 -propiolactone, and alkyl halides convert methionine into
sulfonium derivatives, from which methionine can be regenerated in an alkaline medium
with an excess of thiol reagent. Reaction with cyanogen bromide, which splits the peptide
bond on the carboxyl side of the methionine molecule, is used for selective cleavage of
proteins.
Histidine Residue
Diethyl pyrocarbonate reacts with histidine to form N-(ethoxycarbonyl)histidine. With
iodoacetamide, N-1-(carboxamidomethyl)-, N-3-(carboxamidomethyl)-, or N-1,N-3-
di(carboxamidomethyl) histidine are formed.
Selective modification of histidine residues present on active sites of serine proteinases is
possible. Substrate analogs such as halogenated methyl ketones inactivate such enzymes by
N-alkylation of the histidine residue.
Tryptophan Residue
N-Bromosuccinimide oxidizes the tryptophan side chain and also tyrosine, histidine, and
cysteine. Other oxidative cleaving reagents are 𝜊-iodosobenzoic acid and 3-bromo-3-methyl-
2-(2-nitrophenylmercapto)-3H-indole. Selective modification of histidine is possible with 2-
hydroxy-5-nitrobenzyl bromide (Koshland reagent I) and 2-nitrophenylsulfenyl chloride.
Tyrosine Residue
Selective acylation of tyrosine can occur with 1- acetylimidazole as a reagent. Diazotized 𝜌-
arsanilic acid reacts with tyrosine (𝑜𝑟𝑡ℎ𝑜 substitution) and with histidine, lysine, tryptophan,
and arginine. Tetranitromethane introduces a nitro group into the 𝑜𝑟𝑡ℎ𝑜 position.
Bifunctional Reagents
Bifunctional reagents enable intra-and intermolecular cross-linking of proteins. Examples are
bifunctional imidoester, maleimides, fluoronitrobenzene, and isocyanate derivatives.

Interactions and Reactions Involved in Food Processing

Reaction with carbohydrates
Many 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 ‘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, primary
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:

 Brown pigments (known as ‘melanoidins‘): baking and roasting,

 Volatile compounds: contribute aroma of cooking, frying, roasting, baking besides
the generation of off-flavors in food storage and processing.
 Bitter substances: desired to coffee, but can cause off-flavor
  Reductones: highly reductive properties and contribute to the stabilization of foods
against oxidative deterioration.
 Losses of essential amino acids
 Mutagenic compounds.
 Cross-linking of proteins.

Reaction with lipid oxidation products

Products Formed from Hydroperoxides
Fatty acid hydroperoxides formed thermally or enzymatically 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 trihydroxy 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
Studies 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 several isomers. However, in the presence of oxygen, covalently
bound amino acid–fatty acid adduct formation is significantly suppressed; instead, oxidized
fatty acids are formed.
Protein Changes
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).
Decomposition of Amino Acids
Studies 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. The strong dependence of this loss on the
nature of the protein and on reaction conditions is obvious.

Reaction under alkaline condition

Losses of available lysine, cystine, serine, threonine, arginine, and some other amino acids
occur at high pH values. Hydrolysates of alkali-treated proteins often contain some unusual
compounds such as ornithine, 𝛽-aminoalanine, lysinoalanine, ornithinoalanine, lanthionine,
methyllanthionine and D-𝑎𝑙𝑙𝑜-isoleucine, as well as other D-amino acids.
Reaction under oxidative conditions
Oxidative changes in proteins primarily involve methionine, which forms methionine
sulfoxide relatively readily. After in 𝑣𝑖𝑣𝑜 reduction to methionine, protein-bound
methionine sulfoxide is apparently biologically available.

Functional properties
Functional property Mode of action Food system

Solubility Solvation, pH-dependent Beverages

Water absorption Hydrogen bonding, entrapment of water Meat, sausages, bread,

and binding cakes
Viscosity Thickening, water binding Soups, gravies

Gelation Matrix formation and setting Meat, curds, cheese

Cohesion-adhesion Adhesive material Meat, sausages, baked

goods, pasta products
Elasticity Hydrophobic bonding in gluten, disulfide Meat, bakery
bridges in gels (deformable)
Emulsification Formation and stabilization of fat Sausages, soup, cakes
emulsions
Fat adsorption Binding of free fat Meat, sausages, doughnuts

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

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

desserts, angel cakes

Heat treatment for food protein

Amino acid composition and sequence determine the native structure, functionality, and
nutritional quality of a protein in a set environment. During food processing, heat is often
added to the protein’s environment, and this addition of energy can change any or all of the
structural, functional, and nutritional characteristics of the native protein. Foods are
complex systems, and it is important to recognize that pH, water activity, food composition,
and interactions of these with temperature also affect protein properties to varying extents.

Common Heat Treatments on Food Proteins

At home or on an industrial scale, the purposes of common heat treatments of foods
containing proteins are similar: to change texture or function, improve safety and quality,
and control enzymes by altering physical, chemical, and biological protein properties. Foods
are baked to change texture and improve safety, vegetables are blanched to inactivate
enzymes, canning temperatures used for low acid foods are designed to prevent toxin
production by pathogenic microorganisms, pasteurization and sterilization are designed to
kill pathogens and inactivate enzymes, and extrusion modifies the texture of
proteincontaining foods. Mild heat treatments, such as incubation, generally do not cause
the same extent of change as higher heat treatments and may be used to promote enzyme
activity instead of destroying it. It is worthwhile to note that proteins usually are most stable
to heat at their isoelectric points.
Dry-heat Food Preparation
Dry-heat food preparation methods at temperatures ranging from 160 to 230°C include
baking, roasting, grilling, and frying. In addition to microbial destruction, dry-heat methods
alter the texture of protein foods by heat gelation of proteins, denature enzymes and
pigments, and with extensive heat may form thermally induced mutagens. When meats are
heated, myofibrillar proteins denature, then form a gel matrix, enzymes such as myosin and
actomyosin are inactivated, and oxidation of denatured myoglobin pigments turns cooked
meats brown. On baking, the elastic wheat gluten network in bread dough expands to
contain leavening gases until the temperature is high enough to gelatinize starch, around
65°C. Gluten protein denaturation and gelation occur at higher temperatures than starch
gelatinization, beginning around 74°C, and continuing for the remaining baking time. The
Maillard reaction between proteins and carbohydrates produces the browning of bread
crusts during baking.
Moist-heat Food Preparation
Moist-heat food preparation methods at temperatures ranging from 65 to 100°C include
blanching, boiling, steaming, scalding, and poaching. Pressure canning foods can reach
temperatures in excess of 116°C. Blanching is a process to inactivate enzymes by dipping
foods, usually vegetables, into boiling water for a short time period. This enzyme inactivation
prevents quality loss by color, texture, or flavor changes during frozen storage. Blanching
also decreases initial microbial load on foods as well as wilts products such as spinach for
tighter packing. Boiling occurs at or near 100 C, depending on elevation, and functions to
inactivate enzymes, denature proteins, change texture, and potentially destroy toxins.
Higher temperatures achieved under pressure, such as for pressure canning, inactivate
enzymes, destroy pathogenic and many spoilage microorganisms, and prevent toxin
formation during storage. Poaching eggs leads to denaturation and coagulation of egg white
proteins. Steaming fish causes texture changes as a result of denaturation and gelation of
proteins. Scalding milk may initiate unfolding of whey proteins and improve select functional
properties desirable in preparing bakery foods.
Microwaving
Microwaving combines properties of dry- and moist-heat food preparations with the
advantage of reducing food cooking times. Heat produced by friction of rotating food
molecules, most notably water, in response to magnetron-generated microwaves denatures
and coagulates food proteins as do other heating methods. However, foods cooked in a
microwave do not brown as they would in dry-heating methods. The crust of microwaved
bread dough does not brown, because the air in the microwave does not reach high enough
temperatures, and the steam generated does not allow the surface to dry sufficiently for
nonenzymatic browning to occur.
Pasteurization
The pasteurization process is designed to destroy any pathogenic microorganisms that might
be present in the food product. Temperatures used for pasteurization also reduce the total
microbial load, thereby increasing shelf-life, and may inactivate select enzymes that lead to
quality loss during storage. Pasteurization of milk, designed to destroy the pathogen Coxiella
burnetti, is accomplished by a time/temperature integrated process. Lower temperatures
take longer times whereas higher temperatures require much shorter process times to
accomplish the same result. At temperatures above 78°C, esterase, a lipase that may cause
hydrolytic rancidity of milk products, is inactivated. Pasteurization of milk also may denature
whey proteins, and higher temperatures (ultrahigh temperature processing) or longer
process times (such as vat pasteurization) may cause cooked and heated flavors primarily
from the volatile sulfur compounds released by 𝛽 -elimination of disulfide bonds in
denatured whey proteins. The high temperatures used for sterilization of aseptic products
lead to microbial safety but do not inactivate all enzymes. During extended storage, these
enzymes may cause age gelation of aseptically processed milk products. Pasteurization of
liquid eggs is a low-temperature, long-time process, either 60°C for 3.5 min or 64°C for 2.5
min, designed to destroy pathogenic microorganisms, specifically Salmonella, without
coagulating the egg proteins.
Extrusion
Extrusion is a high-temperature/high-pressure/highshear process used to convert soy
proteins to textured vegetable protein meat analogs. The texture changes during extrusion
cooking result from orientation and denaturation of proteins followed by cross-linking into
a network of fiber-like proteins. The extruded vegetable protein network mimics the fibrous
texture of meat products. Pressure and shear individually can denature proteins, and lower
temperatures may be used in combination with these forces to achieve the same level of
denaturation as higher temperatures alone.
Incubation
Temperature is the most important factor affecting the rate of enzyme-catalyzed reactions
and also influences the stability of the enzymes. As temperature is increased, reaction rates
increase until a temperature at which the enzyme loses activity is passed, often around 50°C.
In the range of 10–40°C, every 10°C rise in temperature is accompanied by a 1.8 increase in
reaction rate for the enzyme chymosin. The enzyme calf rennet (chymosin) is added to milk
in the cheesemaking process to cleave 𝜅-casein from the casein micelle and induce micelle
aggregation. The maximum rate of aggregation is achieved at 40°C, and no aggregation
occurs below 18°C or above 60°C. At temperatures above 50°C, denaturation of the enzyme
causes it to lose activity. Therefore, incubation at 40°C will maximize chymosin activity, and
increasing temperatures above 50–60°C can be used to stop the enzyme. Temperatures used
for cheese ripening also are controlled to maximize desirable enzyme activity for flavor
development.
Heat-induced Changes in Protein Structure
Common changes in protein structure as a result of thermal processing include denaturation,
aggregation, and thermal degradation. Elevated temperatures also increase rates of
deleterious chemical reactions in proteins that lead to oxidation, isomerization, Maillard
browning, deamidation, desulphuration, and other 𝛽-elimination reactions; however, many
of these reactions may occur at temperatures as low as 0°C as well.
Denaturation
Heat denaturation of proteins involves configurational changes in the thermodynamically
stable native structure of the protein via unfolding or alteration of the quaternary, tertiary,
or secondary structure as a response to heat exposure. Denaturation may disrupt hydrogen
and disulfide bonds, hydrophobic interactions, and salt bridges, but peptide bonds remain
intact. The primary structure, or amino acid sequence, of the protein remains unchanged, as
does the molecular weight. A loss of ordered structure generally occurs in the entropydriven
transition from a native to a denatured protein. Temperatures at which denaturation occurs
vary greatly with protein source and type. Some proteins unfold a few degrees above
temperatures at which they function, whereas others, such as wheat gluten and milk 𝛽-
casein, require much higher temperatures for denaturation. Globular dairy whey proteins
denature at much lower temperatures than casein proteins that have more random coil
native structures.
Functional properties affected by heat denaturation include solubility, emulsifying capacity,
gelation capacity, foaming properties, and enzyme or biological activity. After mild (or
insufficient) heat treatments, protein denaturation may be reversible. For enzymes, this
reversible unfolding always occurs prior to irreversible inactivation. If a thermal process does
not irreversibly inactivate target enzymes, the enzymes may be able to refold and potentially
cause quality issues in food products during storage. Residual enzyme activity in aseptically
processed foods can lead to problems such as thinning of puddings due to amylase activity,
age gelation in ultrahigh-temperature processed milks, and separations in orange juice
caused by pectin methylesterase activity.
Aggregation
Denaturation, or at least a partial denaturation, of a protein is usually required prior to its
ability to aggregate and form a gel or a precipitate. Unfolding of a protein exposes reactive
groups that may then form intermolecular cross-links via covalent, hydrogen, ionic, or other
bonding. Gels are formed when the cross-linked protein network is extensive enough to form
a continuous phase and trap water. Precipitates are formed when the aggregated proteins
become insoluble and settle out of a solution.
Gelation The cross-linking of proteins and entrapment of water during gelation may be
either reversible or irreversible. The polymeric networks formed by hydrogen bonding of
gelatin molecules are thermoreversible. On cooling, the gelatin bonds to form a gel, and on
reheating, the hydrogen bonds break, and gelatin returns to the dispersed phase in the
solution. Conversely, denatured globular proteins form thermoirreversible gels. Whey, soy,
and egg white proteins first denature and then interact via disulfide, hydrophobic, and ionic
bonds to form gels when exposed to heat. These gels are heat-set and may stiffen, instead
of liquefy, when exposed to additional heat. The term ‘coagulation‘ is often applied to
irreversible heatsetting of proteins.
Precipitation Precipitation of food proteins often is controlled by pH, enzymes, or salt
concentration adjustments for food ingredient isolation and applications; however, heat also
may destabilize proteins, causing them to precipitate. Solubility and hydrophobicity, which
are affected by temperature, also influence protein precipitation. As temperatures increase,
the likelihood of protein precipitation increases as hydrophobicity increases and solubility
decreases. Denaturation of whey proteins in milk causes them to precipitate, adhere to the
cooking vessel, and possibly scorch with continued heating.
Thermal Degradation
Thermal degradation of proteins occurs at temperatures higher than those for denaturation,
and as with denaturation reactions, temperatures for thermal degradation vary greatly with
protein type. In baddition to the quaternary, tertiary, and secondary structure disruption in
denaturation, thermal degradation also disrupts the primary structure and peptide bonds of
proteins. Effects of thermal degradation on functionality of proteins are more severe than
the effects of denaturation. Types of protein thermal degradation include hydrolysis,
racemization, and pyrolysis.
Hydrolysis Hydrolysis of proteins for use as functional or flavor ingredients often is
accomplished using acid and enzyme techniques; however, high temperatures also may be
used to hydrolyze proteins into peptides, especially at high or low pH. Hydrolysis of caseins
may occur at 140°C, and heat-induced hydrolysis of meat connective tissue proteins may
increase their solubility. Partially hydrolyzed proteins may be more digestible than native
proteins due to the unfolding of the protein structure.
Racemization Racemization, or isomerization, of amino acid residues from l-isomers to d-
isomers occurs when heating proteins either above 200°C or at alkaline pH. This may reduce
protein digestibility, because d-amino acids are less absorbed and hydrolyzed than l-amino
acids in the digestive tract. Isomerase or racemase enzymes also contribute to these
conversions.
Pyrolysis Pyrolysis is a high temperature degradation of organic materials in nonoxidative
conditions. Amino acid pyrolysis products are formed at temperatures above 250–300 C.
Free radicals formed during pyrolysis may attach to other amino acids, thereby forming new
heterocyclic compounds. Some of these compounds are thermally induced mutagens.
Functional Changes in Heat-treated Proteins
Research indicates that protein structure is optimized for function as opposed to stability.
As a result, when addition of heat destabilizes protein structure and stability, functionality
also is affected. Effects of heat on protein functionality can be positive or negative, and the
degree of heat treatment on a specific protein often differentiates between an increase or
decrease in the desired functionality. Addition of some heat may slightly unfold a protein,
thereby emulsifying, gelation, and foaming properties. In designing functional and
nutritional foods, the extent of protein denaturation is controlled to attain desired functional
characteristics, such as whey proteins designed for use as fat replacers. Higher heat
treatments, especially those leading to thermal degradation, may in turn decrease these
functional properties as the protein unfolds more or loses primary structure. Water–protein
interactions dominate protein functionality in food structure; therefore, effects of heat on
hydrophobicity, solubility, emulsifying and gelation capacities, foaming properties, and
enzyme activity are important.
Hydrophobicity
The surface hydrophobicity of proteins may either increase or decrease as a result of heat
treatments. A thermally induced unfolding of protein molecules exposes hydrophobic sites,
thereby increasing hydrophobicity. Conversely, protein aggregation in response to heat
results in decreased exposure of hydrophobic sites, thereby decreasing the surface
hydrophobicity of aggregated proteins.
Solubility
The solubility of proteins depends on the nature of the protein surfaces in contact with the
environment (usually water). To generalize, a protein with a hydrophilic surface will be more
soluble in water than a protein with a more hydrophobic surface. As temperatures rise from
0 to 40°C, most proteins exhibit increasing solubility; however, hydrophobic proteins such
as 𝛽-casein show the opposite solubility trend in this temperature range and may be most
soluble around 4°C. As temperatures rise above 40°C and proteins unfold, more hydrophobic
sites are exposed, and the solubility of the proteins will decrease. These temperature-
dependent changes in solubility will influence emulsifying, gelation, and foaming properties,
since solubility influences the amount of protein available for reactions.
Emulsifying Capacity
The emulsifying capacity of proteins results from amphipathic structure and is measured as
the volume of oil that can be emulsified per gram of protein in an oil-in-water system. The
protein must be somewhat soluble in water in order to act as an emulsifier, and the increase
in surface hydrophobicity that occurs with partial denaturation of most proteins will increase
their emulsifying capacities. When higher temperatures have caused extensive denaturation
and decreased protein solubility, the emulsifying capacity also will decrease.
Gelation Capacity
The gelation capacity of proteins is measured as the amount of water that can be bound or
trapped per gram of protein. The effects of heat on gelation capacity are similar to those on
emulsifying capacity. Partial denaturation may increase gelation capacity, whereas extensive
denaturation will decrease it.
Foaming Properties
The foaming capacity of a protein is measured as the amount of interfacial area that can be
created by whipping the protein. Foam stability is measured as the time required to lose
either 50% of the liquid or 50% of the volume from the foam. Generally, heating a globular
protein to achieve partial denaturation will increase foaming properties. As the structure
unfolds and exposes hydrophobic sites, it may be able to adsorb more quickly to air–water
interfaces and lower interfacial tension, thereby trapping more air and increasing the
foaming capacity. Extensive heat denaturation of proteins will decrease their ability to form
foams.
Enzyme Activity
Temperature controls the rate of enzyme-catalyzed reactions and influences enzyme
stability. As discussed in the incubation section, moderate temperatures can optimize the
rate of enzyme-catalyzed reactions, and higher temperatures may denature (unfold) the
enzymes, thereby causing them to lose activity. The temperatures at which enzymes
denature vary with the type of enzyme. Chymosin, added to milk for cheese-making, loses
activity above 50°C, whereas pectinmethylesterase present in orange juice is stable to much
higher heat treatments.

Heat-induced Protein–Carbohydrate Interactions

Perhaps the most notable protein interaction with other food ingredients is the browning
produced in the Maillard reaction between proteins and carbohydrates. At high
temperatures, low water activity, and/or extended storage times, proteins may react with
reducing sugars to form brown pigments in the Maillard reaction (nonenzymatic browning).
Reactive groups in proteins for the Maillard reaction are primary amines, usually the e-amino
groups of lysine residues. Examples of reducing sugars are glucose, fructose, lactose, and
maltose. When the free eamino group of lysine reacts with a reducing sugar, the lysine is no
longer nutritionally available. Lysine is often the limiting amino acid in protein quality of grain
products, and a decrease in the available lysine due to the Maillard reaction decreases the
overall protein quality of the food. Nonenzymatic browning is desirable in bakery products
such as breads, cooked meats, and caramels for which browning contributes to color and
flavor development. However, too much browning produces burnt or off-flavors in these
products. Nonenzymatic browning also is undesirable in dried milk powders, infant formula,
dehydrated potatoes, dried fruits, and white wine.
Heat Effects on Protein–Lipid Interactions
Heat treatments may affect protein–lipid interactions in terms of free-radical formation,
changes in emulsifying capacity, and alteration of conjugated lipoprotein structure. Lipid–
protein free radicals may be formed when free radicals produced by oxidation of
unsaturated lipids react with proteins. High temperatures greatly increase the rate of
oxidation of sulfurcontaining amino acids via reactions with oxidized lipids. Cysteine and
histidine free-radicals may then cross-link and induce aggregation of proteins. As discussed
in the emulsifying capacity section, partial denaturation of globular proteins may expose
hydrophobic sites and increase emulsifying capacity, thereby increasing the ability of
proteins to interact with lipids; however, higher heat treatments will decrease this ability.
Heat also will denature proteins in conjugated lipoprotein structures and affect the
functionality of these, especially in membrane systems.

Nutritional Changes in Heat-treated Proteins

Heat-induced changes in protein structure can exhibit both advantageous and negative
effects from a nutritional standpoint. Heat denaturation of globular proteins, such as dairy
whey proteins, often leads to increased digestibility and sometimes increased nutritional
value as the structure is unfolded and therefore more susceptible to proteolytic attack by
digestive enzymes. Proteinaceous antinutritional factors, commonly found in legume and
oilseed proteins as well as milk and egg proteins, can be inactivated with sufficient heat,
usually moderate heat treatments, thereby increasing the biological availability and
digestibility of select proteins. Heat inactivation of trypsin and chymotrypsin inhibitors
present in legumes and oilseeds also may protect the pancreas. Once heat denatured, the
lectins (phytohemagglutinins) present in legumes and oilseeds no longer bind to intestinal
cells, causing malabsorption of nutrients. Heat inactivation of ovomucoid and ovoinhibitor
in eggs and plasmin and plasminogen activator inhibitors in milk prevent their protease
inhibitory activities. Once denatured by heat, egg avidin no longer inhibits biotin absorption.
Toxic proteins, including Clostridium botulinum neurotoxin and Staphylococcus aureus
enterotoxin, also are inactivated by heat, although toxin destruction usually requires higher
heat treatments than inactivation of antinutritional factors. Although heat improves safety
and some nutritional aspects of food, heating foods also may detract from nutritional quality,
especially during high heat or long time processes such as charcoal grilling. Protein cross-
linking can decrease proteolysis, thereby decreasing digestibility. The d-amino acid residues
formed in racemization are less digestible than the original l-amino acids. As a result of
Maillard browning, there is a loss of available lysine and consequent decrease in protein
quality. Melanoidins and heterocyclic amines produced in this reaction and during pyrolysis
have been shown to be mutagenic or carcinogenic and classified as thermally induced
mutagens or carcinogens. Consumption of small amounts of these compounds over
extended periods of time may lead to serious health problems.