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Thông qua việc cung cấp các axit amin, protein cần thiết cho sự phát triển của con người, nhưng chúng cũng có một loạt các đặc tính cấu trúc và chức năng có ảnh hưởng sâu sắc đến chất lượng thực phẩm. Protein đóng một vai trò cơ bản không chỉ trong việc duy trì sự sống mà còn trong các loại thực phẩm có nguồn gốc từ thực vật và động vật. Thực phẩm khác nhau về hàm lượng protein của chúng và thậm chí nhiều hơn về đặc tính của những protein đó. Ngoài việc chúng đóng góp vào các đặc tính dinh dưỡng của thực phẩm thông qua việc cung cấp các axit amin cần thiết cho sự tăng trưởng và duy trì của con người, protein còn truyền cơ sở cấu trúc cho các đặc tính chức năng khác nhau của thực phẩm. Định nghĩa về protein Từ “protein” được định nghĩa là bất kỳ nhóm hợp chất hữu cơ phức tạp nào, bao gồm cơ bản là sự kết hợp của các axit amin trong các liên kết peptit, chứa cacbon, hydro, oxy, nitơ và thông thường, lưu huỳnh. Phân bố rộng rãi trong thực vật và động vật, protein là thành phần chính của nguyên sinh chất của tất cả các tế bào và rất cần thiết cho sự sống. (“Protein” có nguồn gốc từ một từ tiếng Hy Lạp có nghĩa là “đầu tiên” hoặc “chính” vì vai trò cơ bản của protein trong việc duy trì sự sống.) Protein trong thực phẩm Axit amin, peptit và protein là những thành phần quan trọng của thực phẩm. Chúng cung cấp các chất xây dựng cần thiết cho quá trình sinh tổng hợp protein. Ngoài ra, chúng trực tiếp góp phần tạo nên hương vị của thực phẩm và là tiền chất cho các hợp chất tạo mùi thơm và màu sắc được hình thành trong quá trình phản ứng nhiệt hoặc enzym trong sản xuất , chế biến và bảo quản thực phẩm. Các thành phần khác của thực phẩm, ví dụ như carbohydrate, tham gia vào các phản ứng như vậy. Protein cũng đóng góp đáng kể vào các đặc tính vật lý của thực phẩm thông qua khả năng ổn định gel, bọt, bột nhào, nhũ tương và cấu trúc sợi. Các nguồn protein quan trọng nhất và sự đóng góp của chúng trong việc sản xuất protein trên toàn thế giới. Ngũ cốc đóng góp hơn một nửa vào sản xuất protein, tiếp theo là hạt có dầu và thịt. Bên cạnh động thực vật, tảo, nấm men và vi khuẩn có thể được sử dụng để sản xuất protein ( protein đơn bào (SCP)). Hàm lượng protein thay đổi như sau:> 20% (pho mát, thịt, các loại đậu, hạt có dầu); 10–20% (cá, trứng); 5–10% (ngũ cốc); và < 5% (sữa, rễ, củ, rau, quả, nấm).
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 months, and after 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 -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 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 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 other heating methods However, foods cooked in a microwave 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 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 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 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 ... 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,... 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;... 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