Effect of heat treament on food protein

Effect of heat treatment 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 triplestranded 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, 4hydroxyproline 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: -, -, -,-,
and -caseins, constituting 34, 8, 9, 25, and 4% of total protein, respectively. In cheesemaking, the specific cleavage of -casein by chymosin into para--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 -lactoglobulins, -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 -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:
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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.
-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 to . Such a pleated structure is stabilized when more chains
interact along the 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, -turns, or -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 -bends on the basis of energy


considerations. Different types of -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 -helix, chain segments
with antiparallel -structures (-meander structure) and combinations of -helix and
-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 (-helix), silk fibroin (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) -helices occur
only; (2) -structures occur only; (3) -helical and -structural portions occur in separate
segments on the peptide chain; (4) -helices and -structures alternate along the peptide
chain; and (5) -helices and -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 -carboxyl and -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 -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 - or -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.
-Halogen carboxylic acids, -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-3di(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 -iodosobenzoic acid and 3-bromo-3-methyl2-(2-nitrophenylmercapto)-3H-indole. Selective modification of histidine is possible with 2hydroxy-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
-arsanilic acid reacts with tyrosine ( substitution) and with histidine, lysine, tryptophan, and
arginine. Tetranitromethane introduces a nitro group into the 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 sidechains, 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, -aminoalanine, lysinoalanine, ornithinoalanine, lanthionine,
methyllanthionine and --isoleucine, as well as other -amino acids.

Reaction under oxidative conditions
Oxidative changes in proteins primarily involve methionine, which forms methionine
sulfoxide relatively readily. After in reduction to methionine, protein-bound methionine
sulfoxide is apparently biologically available.

Functional properties
Functional property

Mode of action

Food system

Solubility

Water absorption
and binding

Solvation, pH-dependent
Hydrogen bonding, entrapment of water

Beverages
Meat, sausages, bread,
cakes

Viscosity

Thickening, water binding

Soups, gravies

Gelation

Matrix formation and setting

Cohesion-adhesion

Emulsification

Adhesive material
Hydrophobic bonding in gluten, disulfide
bridges in gels (deformable)
Formation and stabilization of fat
emulsions

Meat, curds, cheese
Meat, sausages, baked
goods, pasta products

Fat adsorption

Binding of free fat

Meat, sausages, doughnuts

Flavor binding

Adsorption, entrapment, release

Foaming

Formation of stable films to entrap gas

Simulated meat, bakery,
Whipped toppings, chiffon
desserts, angel cakes

Elasticity

Meat, bakery
Sausages, soup, 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 230C 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
65C. Gluten protein denaturation and gelation occur at higher temperatures than starch
gelatinization, beginning around 74C, 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 100C include
blanching, boiling, steaming, scalding, and poaching. Pressure canning foods can reach
temperatures in excess of 116C. 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 78C, 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 -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 60C
for 3.5 min or 64C 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
50C. In the range of 10–40C, every 10C 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 -casein from the casein micelle and
induce micelle aggregation. The maximum rate of aggregation is achieved at 40C, and no
aggregation occurs below 18C or above 60C. At temperatures above 50C, denaturation of
the enzyme causes it to lose activity. Therefore, incubation at 40C will maximize chymosin
activity, and increasing temperatures above 50–60C 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 -elimination reactions;
however, many of these reactions may occur at temperatures as low as 0C 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 -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 140C, 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 disomers occurs when heating proteins either above 200C 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 40C, most proteins exhibit increasing solubility; however, hydrophobic
proteins such as -casein show the opposite solubility trend in this temperature range and
may be most soluble around 4C. As temperatures rise above 40C 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 50C, 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 eamino 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.

References
B. Caballero, P. Finglas, F. Toldra (2003) Encyclopedia of Food Sciences and Nutrition .2nd ed.
Academic Press.