Food Enzymic and Microbial Methods

Enzymic and Microbial Methods In vitro enzymic methods are sometimes used to measure the digestibility and release of essential amino acids. In one method, test proteins are first digested with pepsin and then with pancreatin (freeze-dried powder of pancreatic extract) [70]. In another method, proteins are digested with three enzymes, namely, pancreatic trypsin, chymotrypsin, and porcine intestinal peptidase, under standard assay conditions [31]. These methods, in addition to providing information on innate digestibility of proteins, are useful for detecting processing-induced changes in protein quality. Growth of several microorganisms, such as Streptococcus zymogenes, Streptococcus faecalis, Leuconostoc mesenteroides, Clostridium perfringens, and Tetrahymena pyriformis (a protozoan) also have been used to determine the nutritional value of proteins [34]. Of these Pag e 402 microorganisms, Tetrahymena pyriformis is particularly useful, because its amino acid requirements are similar to those of rats and humans. 6.7 Processing-Induced Physical, Chemical, and Nutritional Changes in Proteins Commercial processing of foods can involve heating, cooling, drying, application of chemicals, fermentation, irradiation, or various other treatments. Of these, heating is most common. This is commonly done to inactivate microorganisms, to inactivate endogenous enzymes that cause oxidative and hydrolytic changes in foods during storage, and to transform an unappealing blend of raw food ingredients into a wholesome and organoleptically appealing food. In addition, proteins such as bovine blactoglobulin, a-lactalbumin, and soy protein, which sometimes cause allergenic or hypersensitive responses, can sometimes be rendered innocuous in this regard. Unfortunately, the beneficial effects achieved by heating proteinaceous foods are generally accompanied by changes that can adversely affect the nutritive value and functional properties of proteins. In this section, both desirable and undesirable effects of food processing on proteins will be discussed. 6.7.1 Changes in Nutritional Quality and Formation of Toxic Compounds 6.7.1.1 Effect of Moderate Heat Treatments Most food proteins are denatured when exposed to moderate heat treatments (60–90°C, 1 h or less). Extensive denaturation of proteins often results in insolublization, which can impair those functional properties that are dependent on solubility. From a nutritional standpoint, partial denaturation of proteins often improves the digestibility and biological availability of essential amino acids. Several purified plant proteins and egg protein preparations, even though free of protease inhibitors, exhibit poor in vitro and in vivo digestibility. Moderate heating improves their digestibility without developing toxic derivatives. In addition to improving digestibility, moderate heat treatment also inactivates several enzymes, such as proteases, lipases, lipoxygenases, amylases, polyphenoloxidase, and other oxidative and hydrolytic enzymes. Failure to inactivate these enzymes can result in development of off-flavors, rancidity, textural changes, and discoloration of foods during storage. For instance, oilseeds and legumes are rich in lipoxygenase. During crushing or cracking of these beans for extraction of oil or protein isolates, this enzyme, in the presence of molecular oxygen, catalyzes oxidation of polyunsaturated fatty acids to initially yield hydroperoxides. The hydroperoxides subsequently decompose and liberate aldehydes and ketones, which impart off-flavor to soy flour and soy protein isolates and concentrates. To avoid off-flavor formation, it is necessary to thermally inactivate lipoxygenase prior to crushing. Moderate heat treatment is particularly beneficial for plant proteins, because they usually contain proteinaceous antinutritional factors. Legume and oilseed proteins contain several trypsin and chymotrypsin inhibitors. These inhibitors impair efficient digestion of proteins, and thus reduce their biological availability. Furthermore, inactivation and complexation of trypsin and chymotrypsin by these inhibitors induce overproduction and secretion of these enzymes by the pancreas, which can lead to pancreatic hypertropy (enlargement of the pancreas) and pancreatic adenoma. Legume and oilseed proteins also contain lectins, which are glycoproteins. These are also known as phytohemagglutinins because they cause agglutination of red blood Pag e 403 cells. Lectins exhibit a high binding affinity for carbohydrates. When consumed by humans, lectins impair protein digestion [90] and cause intestinal malabsorption of other nutrients. The latter consequence results from binding of lectins to membrane glycoproteins of intestinal mucosa cells, which alters their morphology and transport properties [84]. Both protease inhibitors and lectins found in plant proteins are thermolabile. Toasting of legumes and oilseeds or moist heat treatment of soy flour inactivates both lectins and protease inhibitors, improves the digestibility and PER of these proteins (Fig. 26), and prevents pancreatic hypertropy [43]. These antinutritional factors do not pose problems in home-cooked or industrially processed legumes and flour-based products when heating conditions are adequate to inactivate them. Milk and egg proteins also contain several protease inhibitors. Ovomucoid, which possesses antitryptic activity, constitutes about 11% of egg albumen. Ovoinhibitor, which inhibits trypsin, chymotrypsin, and some fungal proteases, is present at a 0.1% level in egg albumen. Milk contains several protease inhibitors, such as plasminogen activator inhibitor (PAI) and plasmin inhibitor (PI), derived from blood. All of these inhibitors lose their activity when subjected to moderate heat treatment in the presence of water. The beneficial effects of heat treatment also include inactivation of proteinaceous toxins, such as botulinum toxin from Clostridium botulinum (inactivated by heating at 100°C) and enterotoxin from Staphylococcus aureaus. 6.7.1.2 Compositional Changes During Extraction and Fractionation Preparation of protein isolates from biological sources involves several unit operations, such as extraction, isoelectric precipitation, salt precipitation, thermocoagulation, and ultrafiltration/diafiltration. It is very likely that some of the proteins in the crude extract might be lost during these operations. For example, during isoelectric precipitation, some sulfur-rich albumin-type FIGURE 26 Effect of toasting on trypsin inhibitory activity ( ) and PER ( ) of soy flour. Adapted from Ref. 36. Pag e 404 proteins, which are usually soluble at isoelectric pH, might be lost in the supernatant fluid. Such losses can alter the amino acid composition and nutritional value of protein isolates compared to those of crude extracts. For example, the chemical scores of Met and Trp in crude coconut meal are about 100 and 89, respectively, whereas they are almost zero in coconut protein isolate obtained by isoelectric precipitation [42,61]. Similarly, whey protein concentrate (WPC) prepared by ultrafiltration/diafiltration and ion-exchange methods undergoes marked changes in their proteose-peptone contents. This markedly affects their foaming properties. 6.7.1.3 Chemical Alteration of Amino Acids Proteins undergo several chemical changes when processed at high temperatures. These changes include racemization, hydrolysis, desulfuration, and deamidation. Most of these chemical changes are irreversible, and some of these reactions result in formation of amino acid types that are potentially toxic. Racemization Thermal processing of proteins at alkaline pH, as is done to prepare texturized foods, invariably leads to partial racemization of L-amino acid residues to D-amino acids [66]. Acid hydrolysis of proteins also causes some racemization of amino acids [32], as does roasting of proteins or protein containing foods above 200°C[45]. The mechanism at alkaline pH involves initial abstraction of the proton from the a-carbon atom by a hydroxyl ion. The resulting carbanion loses its tetrahedral asymmetry. Subsequent addition of a proton from solution can occur either from the top or bottom of the carbanion. This equal probability results in racemization of the amino acid residue [66]. The rate of racemization of a residue is affected by the electron-withdrawing power of the side chain. Thus, residues such as Asp, Ser, Cys, Glu, Phe, Asn, and Thr are racemized at a faster rate than are other amino acid residues [65]. The rate of racemization is also dependent on hydroxyl ion concentration, but is independent of protein concentration. Interestingly, the rate of racemization is about 10 times faster in proteins than in free amino acids [65], suggesting that intramolecular forces in a protein reduce the activation energy of racemization. In addition to racemization, the carbanion formed under alkaline pH also can undergo b-elimination reaction to yeild dehydroalanine. Cysteine and (76) phosphoserine residues display greater propensity for this route than do other amino acid residues. This is one of the reasons why a significant amount of D-cysteine is not found in alkali-treated proteins. Racemization of amino acid residues causes a reduction in protein digestibility because Pag e 405 the peptide bonds involving D-amino acid residues are less efficiently hydrolyzed by gastric and pancreatic proteases. This leads to loss of essential amino acids that have racemized, and impairs the nutritional value of the protein. D-Amino acids are also less efficiently absorbed through intestinal mucosa cells, and even if absorbed, they cannot be utilized in in vivo protein synthesis. Moreover, some D-amino acids, such as D-proline, have been found to be neurotoxic in chickens [16]. In addition to racemization and b-elimination reactions, heating of proteins at alkaline pH destroys several amino acid residues, such as Arg, Ser, Thr, and Lys. Arg decomposes to ornithine. When proteins are heated above 200°C, as is commonly encountered on food surfaces during broiling, baking, and grilling, amino acid residues undergo decomposition and pyrolysis. Several of the pyrolysis products have been isolated and identified from broiled and grilled meat, and they are highly mutagenic as determined by the Ames test. The most carcinogenic/mutagenic products are formed from pyrolysis of Trp and Glu residues [14]. Pyrolysis of Trp residues gives rise to formation of carbolines and their derivatives. Mutagenic compounds are also produced in meats at moderate temperatures (190–200°C). These are known as aminoimidazoazaarenes (AIAs). One of the classes of compounds is imidazo quinolines (IQ compounds), which are condensation products of creatinine, sugars, and certain amino acids, such as Gly, Thr, Ala, and Lys [51]. The three most potent mutagens formed in broiled fish are shown here. (77) Following heating of foods according to recommended procedures, IQ compounds are generally present only at very low concentrations (microgram amounts). 6.7.1.4 Protein Cross-Linking Several food proteins contain both intra- and intermolecular cross-links, such as disulfide bonds in globular proteins, desmosine, and isodesmosine; and di- and trityrosine-type cross-links in fibrous proteins such as keratin, elastin, resilin, and collagen. Collagen also contains e-N-(g-glutamyl)lysyl and/or e-N-(g-aspartyl)lysyl cross-links. One of the functions of these cross-links in native proteins is to minimize metabolic proteolysis. Processing of food proteins, especially at alkaline pH, also includes crosslink formation. Such unnatural covalent bonds between polypeptide chains reduce digestibility and biological availability of essential amino acids that are involved in, or near, the cross-link. As discussed in the previous section, heating of protein at alkaline pH, or heating above 200°C at neutral pH, results in abstraction of the proton from the a-carbon atom, resulting in formation of a carbanion. The carbanion derivative of Cys, cystine, and phosphoserine undergoes b-elimination reaction, leading to formation of a highly reactive dehydroalanine residue (DHA). DHA formation can also occur via a one-step mechanism without formation of the carbanion. Pag e 406 (78) Once formed, the highly reactive DHA residues react with nucleophilic groups, such as the e-amino group of a lysyl residue, the thiol group of Cys residue, the d-amino group of ornithine (formed by decomposition of arginine), or a histidyl residue, resulting in formation of lysinoalanine, lanthionine, ornithoalanine, and histidinylalanine cross-links, respectively, in proteins. Lysinoalanine is the major cross-link commonly found in alkali treated proteins because of the abundance of readily accessible lysyl residues. (79) Pag e 407 The formation of protein-protein cross-links in alkali-treated proteins decreases their digestibility and biological value. Both digestibility (and PER) and net protein utilization (NPU) decrease with an increase in lysinoalanine content. The decrease in digestibility is related to the inability of trypsin to cleave the peptide bond in the lysinoalanine cross-link. Moreover, the steric constraints imposed by the cross-links also prevent hydrolysis of other peptide bonds in the neighborhood of the lysinoalanine and similar cross-links. Evidence suggests that free lysinoalanine is absorbed in the intestine, but it is not utilized by the body and most of it is excreted in the urine. Some lysinoalanine is metabolized in the kidney. Rats fed 100 ppm pure lysinoalanine or 3000 ppm protein-bound lysinoalanine exhibit nephrocytomegaly (i.e., kidney disorder). However, such nephrotoxic effects have not been observed in other animal species, such as quails, mice, hamsters, and monkeys. This has been attributed to differences in the types of metabolites formed in rats versus other animals. At levels encountered in foods, protein-bound lysinoalanine apparently does not cause nephrotoxicity in humans. Nevertheless, minimization of lysinoalanine formation during alkali processing of proteins is a desirable goal. The lysinoalanine contents of several commercial foods are listed in Table 21. The extent of formation of lysinoalanine is dependent on pH and temperature. The higher the pH, the greater is the extent of lysinoalanine formation. High-temperature heat treatment of foods, such as milk, causes a significant amount of lysinoalanine to form even at neutral pH. Lysinoalanine formation in proteins can be minimized or inhibited by adding low-molecular-weight nucleophilic compounds, such as cysteine, ammonia, or sulfites. The effectiveness of cysteine results because the Table 21 Lysinoalanine (LAL) Content of Processed Foods Food LAL (mg /g protein) Corn chips 390 Pretzels 500 Hominy 560 Tortillas 200 Taco shells 170 Milk, infant formula 150–640 Milk, evaporated 590–860 Milk, UHT 160–370 Milk, HTST 260–1030 Milk, spray-dried powder 0 Skim milk, evaporated 520 Simulated cheese 1070 Eg g -white solids, dried 160–1820 Calcium caseinate 370–1000 Sodium caseinate 430–6900 Acid casein 70–190 Hydrolyzed veg etable protein 40–500 W hipping ag ent 6500–50,000 Soy protein isolate 0–370 Yeast extract 120 Source: Ref. 103. Pag e 408 nucleophilic SH group reacts more than 1000 times faster than the e-amino group of lysine. Sodium sulfite and ammonia exert their inhibitory effect by competing with the e-amino group of lysine for DHA. Blocking of e-amino groups of lysine residues by reaction with acid anhydrides prior to alkali treatment also decreases the formation of lysinoalanine. However, this approach results in loss of lysine activity and may be unsuitable for food applications. Under normal conditions used for processing of several foods, only small amounts of lysinoalanine are formed. Thus, toxicity of lysinoalanine in alkali-treated foods is not believed to be a major concern. However, reduction in digestibility, loss of bioavailability of lysine, and racemization of amino acids (some of which are toxic) all are undesirable. Excessive heating of pure protein solutions or proteinaceous foods low in carbohydrate content also results in formation of e-N- (g-glutamyl)lysyl and e-N-(g-aspartyl)lysyl cross-links. These involve a transamidation reaction between Lys and Gln or Asn residues. The resulting cross-links are termed isopeptide bonds because they are foreign to native proteins. Isopeptides resist enzymatic hydrolysis in the gut, and these cross-linkages therefore impair digestibility of proteins and bioavailability of lysine. (80) Ionizing radiation of foods results in the formation of hydrogen peroxide through radiolysis of water in the presence of oxygen, and this, in turn, causes oxidative changes in, and polymerization of, proteins. Ionizing radiation also may directly produce free radicals via ionization of water. (81) (82) The hydroxyl free radical can induce formation of protein free radicals, which in turn may cause polymerization of proteins. (83) (84) Heating of protein solutions at 70–90°C and at neutral pH generally leads to sulfhydryl-disulfide interchange reactions (if these groups are present), resulting in polymerization of Pag e 409 proteins. However, this type of heat-induced cross-link generally does not have an adverse effect on the digestibility and bioavailability of proteins and essential amino acids because these bonds can be broken in vivo. 6.7.1.5 Effects of Oxidizing Agents Oxidizing agents such as hydrogen peroxide and benzoyl peroxide are used as bactericidal agents in milk, as bleaching agents in cereal flours, protein isolates, and fish protein concentrate, and for detoxification of oilseed meals. Sodium hypochlorite is also commonly used as a bactericidal and detoxifying agent in flours and meals. In addition to oxidizing agents that are sometimes added to foods, several oxidative compounds are endogenously produced in foods during processing. These include free radicals formed during irradiation of foods, during peroxidation of lipids, during photooxidation of compounds such as riboflavin and chlorophyll, and during nonenzymatic browning of foods. In addition, polyphenols present in several plant proteins can be oxidized by molecular oxygen to quinones at neutral to alkaline pH, and this will lead ultimately to peroxides. These highly reactive oxidizing agents cause oxidation of several amino acid residues and polymerization of proteins. The amino acid residues most susceptible to oxidation are Met, Cys/cystine, Trp, and His, and to a lesser extent Tyr. Oxidation of Methionine Methionine is easily oxidized to methionine sulfoxide by various peroxides. Incubation of protein bound mehtionine or free methionine with hydrogen peroxide (0.1 M) at elevated temperature for 30 min results in complete conversion of methionine to methionine sulfoxide [18]. Under strong oxidizing conditions, methionine sulfoxide is further oxidized to methionine sulfone, and in some cases to homocysteic acid. (85) Methionine becomes biologically unavailable once it is oxidized to methionine sulfone or homocysteic acid. Methionine sulfoxide, on the other hand, is reconverted to Met under acidic conditions in the stomach. Further, evidence suggests that any methionine sulfoxide passing through the intestine is absorbed and reduced in vivo to methionine. However, in vivo reduction of methionine sulfoxide to methionine is slow. The PER or NPU of casein oxidized with 0.1 M hydrogen peroxide (which completely transforms methionine to methionine sulfoxide) is about 10% less than that of control casein. Oxidation of Cysteine and Cystine Under alkaline conditions, cysteine and cystine follow the b-elimination reaction pathway to produce dehydroalanine residues. However, at acidic pH, oxidation of cysteine and cystine in simple systems results in formation of several intermediate oxidation products. Some of these derivatives are unstable. Pag e 410 (86) Mono- and disulfoxides of L-cystine are biologically available, presumably because they are reduced back to L-cystine in the body. However, mono- and disulfone derivatives of L-cystine are biologically unavailable. Similarly, while cysteine sulfenic acid is biologically available, cysteine sulfenic acid and cysteic acid are not. The rate and extent of formation of these oxidation products in acidic foods are not well documented. Oxidation of Tryptophan Among the essential amino acids, Trp is exceptional because of its role in several biological functions. Therefore, its stability in processed foods is of major concern. Under acidic, mild, oxidizing conditions, such as in the presence of performic acid, dimethylsulfoxide, or N-bromosuccinimide (NBS), Trp is oxidized mainly to b-oxyindolylalanine. Under acidic, severe, oxidizing conditions, such as in the presence of ozone, hydrogen peroxide, or peroxidizing lipids, Trp is oxidized to N-formylkynurenine, kynurenine, and other unidentified products. (87) Exposure of Trp to light in the presence of oxygen and a photosensitizer, such as riboflavin, leads to formation of Nformylkynurenine and kynurenine as major products and several other minor ones. Depending upon the pH of the solution, other derivatives, such as 5-hydroxyformylkynurenine (pH > 7.0) and a tricyclic hydroperoxide (pH 3.6–7.1), are also formed [73]. In addition to the photoxidative products, Trp forms a photoadduct with riboflavin. Pag e 411 Both protein-bound and free tryptophan are capable of forming this adduct. The extent of formation of this photoadduct is dependent on availability of oxygen, being greater under anaerobic conditions [97]. The oxidation products of Trp are biologically active. In addition, kynurenines are carcinogenic in animals, and all other Trp photooxidative products as well as the carbolines formed during broiling/grilling of meat products exhibit mutagenic activities and inhibit growth of mammalian cells in tissue cultures. The tryptophan-riboflavin photoadduct shows cytotoxic effects on mammalian cells, and exerts hepatic dysfunctions during parenteral nutrition. (88) These undesirable products are normally present in extremely low concentration in foods unless an oxidation environment is purposely created. Among the amino acid side chains, only those of Cys, His, Met, Trp, and Tyr are susceptible to sensitized photooxidation. In the case of Cys, cysteic acid is the end product. Met is photooxidized first to methionine sulfoxide, and finally to methionine sulfone and homocysteic acid. Photooxidation of histidine leads to the formation of aspartate and urea. The photooxidation products of tyrosine are not known. Since foods contain endogenous as well as supplemented riboflavin (vitamin B2), and usually are exposed to light and air, some degree of sensitized photooxidation of the preceding amino acid residues would be expected to occur. At equimolar concentrations, the rates of oxidation of the sulfur amino acids and Trp are likely to follow the order Met>Cys> Trp. Pag e 412 Oxidation of Tyrosine Exposure of tyrosine solutions to peroxidase and hydrogen peroxide results in oxidation of tyrosine to dityrosine. Occurrence of this type of cross-link has been found in natural proteins, such as resilin, elastin, keratin, and collagen. (89) 6.7.1.6 Carbonyl-Amine Reactions Among the various processing-induced chemical changes in proteins, the Maillard reaction (nonenzymatic browning) has the greatest impact on its sensory and nutritional properties. The Maillard reaction refers to a complex set of reactions initiated by reaction between amines and carbonyl compounds, which, at elevated temperature, decompose and eventually condense into insoluble brown products known as melanoidins (see Chap. 4). This reaction occurs not only in foods during processing, but also in biological systems. In both instances, proteins and amino acids typically provide the amino component, and reducing sugars (aldoses and ketoses), ascorbic acid, and carbonyl compounds generated from lipid oxidation provide the carbonyl component. Some of the carbonyl derivatives from the nonenzymatic browning sequence react readily with free amino acids. This results in degradation of the amino acids to aldehydes, ammonia, and carbon dioxide, and the reaction is known as Strecker degradation. The aldehydes contribute to aroma development during the browning reaction. Strecker degradation of each amino acid produces a specific aldehyde with a distinctive aroma (see Chap. 11). (90) The Maillard reaction impairs protein nutritional value, and some of the products may be toxic, but probably are not hazardous at concentrations encountered in foods. Because the e-amino group of lysine is the major source of primary amines in proteins, it is frequently involved in the carbonyl-amine reaction, and it typically suffers a major loss in bioavailability when this reaction occurs. The extent of Lys loss depends on the stage of the browning reaction. Lysine involved in the early stages of browning, including the Schiff’s base, is biologically Pag e 413 available. These early derivatives are hydrolyzed to lysine and sugar in the acidic conditions of the stomach. However, beyond the stage of ketosamine (Amadori product) or aldosamine (Heyns product), lysine is no longer biologically available. This is primarily because of poor absorption of these derivatives in the intestine [29]. It is important to note that no color has developed at this stage. Although sulfite inhibits formation of brown pigments [113], it cannot prevent loss of lysine availability, because it cannot prevent formation of Amadori or Heyns products. Biological activity of lysine at various stages of the Maillard reaction can be determined chemically by addition of 1-fluoro-2,4- dinitrobenzene (FDNB), followed by acid hydrolysis of the derivatized protein. FDNB reacts with available e-amino groups of lysyl residues. The hydrolysate is then extracted with ethyl ether to remove unreacted FDNB, and the concentration of edinitrophenyl-lysyl (e-DNP-lysine) in the aqueous phase is determined by measuring absorbance at 435 nm. Available lysine also can be determined by reacting 2,4,6-trinitrobenzene sufonic acid (TNBS) with the e-amino group. In this case, the concentration of e-trinitrophenyl-lysine (e-TNP-lysine) derivative is determined from absorbance at 346 nm. Nonenzymatic browning not only causes major losses of lysine, but reactive unsaturated carbonyls and free radicals formed during the browning reaction cause oxidation of several other essential amino acids, especially Met, Tyr, His, and Trp. Crosslinking of proteins by dicarbonyl compounds produced during browning decreases protein solubility, and impairs digestibility of proteins. Some Maillard browning products are suspected mutagens. Although mutagenic compounds are not necessarily carcinogenic, all known carcinogens are mutants. Therefore, the formation of mutagenic Maillard compounds in foods is of concern. Studies with mixtures of glucose and amino acids have shown that the Maillard products of Lys and Cys are mutagenic, whereas those of Trp, Tyr, Asp, Asn, and Glu are not, as determined by the Ames test. It should be pointed out that pyrolysis products of Trp and Glu (in grilled and broiled meat) also are mutagenic (Ames test). As discussed earlier, heating of sugar and amino acids in the presence of creatine produces the most potent IQ-type mutagens (see Eq. 77). Although results based on model systems cannot be reliably applied to foods, it is possible that interaction of Maillard products with other low-molecular-weight constituents in foods may produce mutagenic and/or carcinogenic substances. On a positive note, some Maillard reaction products, especially the reductones, do have antioxidative activity [75]. This is due to their reducing power, and their ability to chelate metals, such as Cu and Fe, which are pro-oxidants. The amino reductones formed from the reaction of triose reductones with amino acids such as Gly, Met, and Val show excellent antioxidative activity. Besides reducing sugars, other aldehydes and ketones present in foods can also take part in the carbonyl-amine reaction. Notably, gossypol (in cotton seed), glutaraldehyde (added to protein meals to control deamination in the rumen of ruminants), and aldehydes (especially malonaldehyde) generated from the oxidation of lipids may react with amino groups of proteins. Bifunctional aldehydes, such as malonaldehyde, can cross-link and polymerize proteins. (91) This may result in insolublization, loss of digestibility and bioavailability of lysine, and loss of functional properties of proteins. Formaldehyde also reacts with the e-amino group of lysyl residues; the toughening of fish muscle during frozen storage is believed to be due to reactions of formaldehyde with fish proteins.

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