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Enzymes - Principle of food chemistry
INTRODUCTION Enzymes, although minor constituents of many foods, play a major and manifold role in foods. Enzymes that are naturally present in foods may change the composition of those foods; in some cases, such changes are desirable but in most instances are undesir- able, so the enzymes must be deactivated. The blanching of vegetables is an example of an undesirable change that is deactivated. Some enzymes are used as indicators in ana- lytical methods; phosphatase, for instance, is used in the phosphatase test of pasteurization of milk. Enzymes are also used as processing aids in food manufacturing. For example, rennin, contained in extract of calves' stom- achs, is used as a coagulant for milk in the production of cheese. Food science's emphasis in the study of enzymes differs from that in biochemistry. The former deals mostly with decomposition reactions, hydrolysis, and oxidation; the lat- ter is more concerned with synthetic mecha- nisms. Whitaker (1972) has prepared an extensive listing of the uses of enzymes in food processing (Table 10-1) and this gives a good summary of the many and varied possi- ble applications of enzymes. NATURE AND FUNCTION Enzymes are proteins with catalytic prop- erties. The catalytic properties are quite spe- cific, which makes enzymes useful in ana- lytical studies. Some enzymes consist only of protein, but most enzymes contain addi- tional nonprotein components such as carbo- hydrates, lipids, metals, phosphates, or some other organic moiety. The complete enzyme is called holoenzyme; the protein part, apoenzyme\ and the nonprotein part, coj'ac- tor. The compound that is being converted in an enzymic reaction is called substrate. In an enzyme reaction, the substrate combines with the holoenzyme and is released in a modified form, as indicated in Figure 10-1. An enzyme reaction, therefore, involves the following equations: *i Enzyme + substrate ^ ^* complex *2 *3 ^- enzyme + products The equilibrium for the formation of the complex is given by K = [E][S] m [ES] Enzymes CHAPTER 10 Enzyme Amylases Cellulase Dextran-sucrase Invertase Lactase Tannase Pentosanase Naringinase Pectic enzymes (use- ful) Food Baked goods Brewing Cereals Chocolate-cocoa Confectionery Fruit juices Jellies Pectin Syrups and sugars Vegetables Brewing Coffee Fruits Sugar syrups Ice cream Artificial honey Candy Ice Cream Feeds Milk Brewing Milling Citrus Chocolate-cocoa Coffee Fruits Fruit juices Olives Wines Purpose or Action Increase sugar content for yeast fermentation Conversion of starch to maltose for fermentation; removal of starch turbidities Conversion of starch to dextrins, sugar; increase water absorption Liquidification of starches for free flow Recovery of sugar from candy scraps Remove starches to increase sparkling properties Remove starches to increase sparkling properties An aid in preparation of pectin from apple pomace Conversion of starches to low molecular weight dex- trins (corn syrup) Hydrolysis of starch as in tenderization of peas Hydrolysis of complex carbohydrate cell walls Hydrolysis of cellulose during drying of beans Removal of graininess of pears; peeling of apricots, tomatoes Thickening of syrup Thickening agent, body Conversion of sucrose to glucose and fructose Manufacture of chocolate-coated, soft, cream can- dies Prevent crystallization of lactose, which results in grainy, sandy texture Conversion of lactose to galactose and glucose Stabilization of milk proteins in frozen milk by removal of lactose Removal of polyphenolic compounds Recovery of starch from wheat flour Debittering citrus pectin juice by hydrolysis of the glucoside, naringin Hydrolytic activity during fermentation of cocoa Hydrolysis of gelatinous coating during fermentation of beans Softening Improve yield of press juices, prevent cloudiness, improve concentration processes Extraction of oil Clarification continues Table 10-1 Uses and Suggested Uses of Enzymes in Food Processing Enzyme Pectic enzymes (deteriorative) Proteases (useful) Proteases (deteriorative) Lipase (useful) Lipase (deteriorative) Phosphatases Nucleases Peroxidases (useful) Food Citrus juice Fruits Baked goods Brewing Cereals Cheese Chocolate-cocoa Eggs, egg products Feeds Meats and fish Milk Protein hydrolysates Wines Eggs Crab, lobster Flour Cheese Oils Milk Cereals Milk and dairy products Oils Baby foods Brewing Milk Flavor enhancers Vegetables Glucose determinations Purpose or Action Destruction and separation of pectic substances of juices Excessive softening action Softening action in doughs; cut mixing time, increase extensibility of doughs; improvement in grain, tex- ture, loaf volume; liberate p-amylase Body, flavor and nutrients development during fer- mentation; aid in filtration and clarification, chill- proofing Modify proteins to increase drying rate, improve product handling characteristics; manufacture of miso and tofu Casein coagulation; characteristic flavors during aging Action on beans during fermentation Improve drying properties Use in treatment of waste products for conversion to feeds Tenderization; recovery of protein from bones, trash fish; liberation of oils In preparation of soybean milk Condiments such as soy sauce and tamar sauce; specific diets; bouillon, dehydrated soups, gravy powders, processed meats Clarification Shelf life of fresh and dried whole eggs Overtenderization if not inactivated rapidly Influence on loaf volume, texture if too active Aging, ripening, and general flavor characteristics Conversion of lipids to glycerol and fatty acids Production of milk with slightly cured flavor for use in milk chocolate Overbrowning of oat cakes; brown discoloration of wheat bran Hydrolytic rancidity Hydrolytic rancidity Increase available phosphate Hydrolysis of phosphate compounds Detection of effectiveness of pasteurization Production of nucleotides and nucleosides Detection of effectiveness of blanching In combination with glucose oxidase continues Table 10-1 continued Enzyme Peroxidases (deteriorative) Catalase Glucose oxidase Polyphenol oxidase (useful) Polyphenol oxidase (deteriorative) Lipoxygenase Ascorbic acid oxidase Thiaminase Food Vegetables Fruits Milk Variety of products Variety of products Glucose determination Tea, coffee, tobacco Fruits, vegetables Vegetables Vegetables, fruits Meats, fish Purpose or Action Off-flavors Contribution to browning action Destruction of H 2 O 2 in cold pasteurization To remove glucose and/or oxygen to prevent brown- ing and/or oxidation; used in conjunction with glu- cose oxidase Removal of oxygen and/or glucose from products such as beer, cheese, carbonated beverages, dried eggs, fruit juices, meat and fish, milk powder, wine to prevent oxidation and/or browning; used in conjunction with catalase Specific determination of glucose; used in conjunc- tion with peroxidase Development of browning during ripening, fermenta- tion, and/or aging process Browning, off-flavor development, loss of vitamins Destruction of essential fatty acids and vitamin A; development of off-flavors Destruction of vitamin C (ascorbic acid) Destruction of thiamine Source: Reprinted with permission from J. R. Whitaker, Principles of Enzymology for the Food Sciences, 1 972, by courtesy of Marcel Dekker, Inc. Table 10-1 continued where E, S, and ES are the enzyme, substrate, and complex, respectively K m is the equilibrium constant This can be expressed in the form of the Michaelis-Menten equation, as follows: v = v is] [S] + K n where v is the initial short-time velocity of the reaction at substrate concentration [S] V is the maximum velocity that can be attained at a high concentration of the substrate where all of the enzyme is in the form of the complex This equation indicates that when v is equal to one-half of K the equilibrium constant K m is numerically equal to S. A plot of the reaction rates at different substrate concentrations can be used to determine K m . Because it is not always possible to attain the maximum reac- tion rate at varying substrate concentrations, the Michaelis-Menten equation has been modified by using reciprocals and in this form is known as the Lineweaver-Burke equation, i - I K ™ v " V + V[S] Plots of 1/v as a function of 1/[S] result in straight lines; the intercept on the Y-axis rep- resents 1/V; the slope equals K n JV', and from the latter, K m can be calculated. Enzyme reactions follow either zero-order or first-order kinetics. When the substrate concentration is relatively high, the concen- tration of the enzyme-substrate complex will be maintained at a constant level and the amount of product formed is a linear func- tion of the time interval. Zero-order reaction kinetics are characteristic of catalyzed reac- tions and can be described as follows: d[S]_ k . dt where S is substrate and k° is the zero-order reac- tion constant First-order reaction kinetics are character- ized by a graduated slowdown of the forma- tion of product. This is because the rate of its formation is a function of the concentration Products Figure 10-1 The Nature of Enzymes—Substrate Reactions Holoenzyme - product complex Holoenzyme-substrote complex Holoenzyme Apoenzyme - substrote complex Substrote Cofoctor Apoenzyme of unreacted substrate, which decreases as the concentration of product increases. First- order reaction kinetics follow the equation, ^ = ft 1 ([Sl-[Fl) where P is product and k { is the first-order reac- tion constant For relatively short reaction times, the amount of substrate converted is proportional to the enzyme concentration. Each enzyme has one—and some enzymes have more—optimum pH values. For most enzymes this is in the range of 4.5 to 8.0. Examples of pH optima are amylase, 4.8; invertase, 5.0; and pancreatic a-amylase, 6.9. The pH optimum is usually quite narrow, although some enzymes have a broader opti- mum range; for example, pectin methyl- esterase has a range of 6.5 to 8.0. Some enzymes have a pH optimum at very high or very low values, such as pepsin at 1.8 and arginase at 10.0. Temperature has two counteracting effects on the activity of enzymes. At lower temper- atures, there is a g 10 of about 2, but at tem- peratures over 4O 0 C, the activity quickly decreases because of denaturation of the pro- tein part of the enzymes. The result of these factors is a bell-shaped activity curve with a distinct temperature optimum. Enzymes are proteins that are synthesized in the cells of plants, animals, or microorgan- isms. Most enzymes used in industrial appli- cations are now obtained from microor- ganisms. Cofactors or coenzymes are small, heat-stable, organic molecules that may readily dissociate from the protein and can often be removed by dialysis. These coen- zymes frequently contain one of the B vita- mins; examples are tetrahydrofolic acid and thiamine pyrophosphate. Specificity The nature of the enzyme-substrate reac- tion as explained in Figure 10-1 requires that each enzyme reaction is highly specific. The shape and size of the active site of the enzyme, as well as the substrate, are impor- tant. But this complementarity may be even further expanded to cover amino acid resi- dues in the vicinity of the active site, hydro- phobic areas near the active site, or the presence of a positive electrical charge near the active site (Parkin 1993). Types of speci- ficity may include group, bond, stereo, and absolute specificity, or some combination of these. An example of the specificity of en- zymes is given in Figure 10-2, which illus- trates the specificity of proline-specific pep- tidases (Habibi-Najafi and Lee 1996). The amino acid composition of casein is high in proline, and the location of this amino acid in the protein chain is inaccessible to common aminopeptidases and the di- and tripepti- dases with broad specificity. Hydrolysis of the proline bonds requires proline-specific peptidases, including several exopeptidases and an endopeptidase. Figure 10-2 illustrates that this type of specificity is related to the type of amino acid in a protein as well as its location in the chain. Neighboring amino acids also determine the type of peptidase required to hydrolyze a particular peptide bond. Classification Enzymes are classified by the Commission on Enzymes of the International Union of Biochemistry. The basis for the classification is the division of enzymes into groups according to the type of reaction catalyzed. This, together with the name or names of substrate(s), is used to name individual enzymes. Each well-defined enzyme can be described in three ways—by a systematic name, by a trivial name, and by a number of the Enzyme Commission (EC). Thus, the enzyme oc-amylase (trivial name) has the systematic name a-l,4-glucan-4-glucanohy- drolase, and the number EC 3.2.1.1. The sys- tem of nomenclature has been described by Whitaker (1972; 1974) and Parkin (1993). Enzyme Production Some of the traditionally used industrial enzymes (e.g., rennet and papain) are pre- pared from animal and plant sources. Recent developments in industrial enzyme produc- tion have emphasized the microbial enzymes (Frost 1986). Microbial enzymes are very heat stable and have a broader pH optimum. Most of these enzymes are made by sub- merged cultivation of highly developed strains of microorganisms. Developments in biotechnology will make it possible to trans- fer genes for the elaboration of specific enzymes to different organisms. The major industrial enzyme processes are listed in Table 10-2. HYDROLASES The hydrolases as a group include all enzymes that involve water in the formation of their products. For a substrate AB, the reaction can be represented as follows: AB + HOH -4 HA + BOH The hydrolases are classified on the basis of the type of bond hydrolyzed. The most important are those that act on ester bonds, glycosyl bonds, peptide bonds, and C-N bonds other than peptides. Figure 10-2 Mode of Action of Prolme-Specific Peptidases. Source: Reprinted with permission from M.B. Habibi-Najafi and B.H. Lee, Bitterness in Cheese: A Review, Crit. Rev. Food ScL Nutr., Vol. 36, No. 5, p. 408. Copyright CRC Press, Boca Raton, Florida. Proteases Rennet Trypsin Papain Fungal Fungal (rennins) Bacterial Glycosidases Bacterial oc-amylase Fungal cc-amy- lase (3-amylase Amyloglucosi- dase Pectinase Cellulase Yeast lactase Mold lactase Others Glucose isomerase Glucose oxidase Mold catalase Animal catalase Lipase Calf stomach Animal pancreas Carica papaya fruit Aspergillus oryzae Mucorspp. Bacillus spp. Bacillus spp. Aspergillus oryzae Barley Aspergillus niger Aspergillus niger Molds Kluyveromyces spp. Aspergillus spp. Various microbial sources Aspergillus niger Aspergillus niger Liver Molds Source: From G.M. Frost, Commercial Production of Enzymes, in Developments in Food Proteins, BJ.F. Hud- son, ed., 1986, Elsevier Applied Science Publishers Ltd. Enzyme Source Table 10-2 Major Industrial Enzymes and the Process Used for Their Production Submerged Fermentation Surface Fermentation lntracellular Extracellular Concentration Precipitation Drying Pelleting Further Purification Solid Product Solution Product Immobilized Product Esterases The esterases are involved in the hydrolysis of ester linkages of various types. The prod- ucts formed are acid and alcohol. These enzymes may hydrolyze triglycerides and include several Upases; for instance, phos- pholipids are hydrolyzed by phospholipases, and cholesterol esters are hydrolyzed by cho- lesterol esterase. The carboxylesterases are enzymes that hydrolyze triglycerides such as tributyrin. They can be distinguished from Upases because they hydrolyze soluble sub- strates, whereas Upases only act at the water- lipid interfaces of emulsions. Therefore, any condition that results in increased surface area of the water-lipid interface will increase the activity of the enzyme. This is the reason that lipase activity is much greater in homog- enized (not pasteurized) milk than in the non- homogenized product. Most of the lipolytic enzymes are specific for either the acid or the alcohol moiety of the substrate, and, in the case of esters of polyhydric alcohols, there may also be a positional specificity. Lipases are produced by microorganisms such as bacteria and molds; are produced by plants; are present in animals, especially in the pancreas; and are present in milk. Li- pases may cause spoilage of food because the free fatty acids formed cause rancidity. In other cases, the action of Upases is desirable and is produced intentionally. The boundary between flavor and off-flavor is often a very narrow range. For instance, hydrolysis of milk fat in milk leads to very unpleasant off- flavors at very low free fatty acid concentra- tion. The hydrolysis of milk fat in cheese contributes to the desirable flavor. These dif- ferences are probably related to the back- ground upon which these fatty acids are superimposed and to the specificity for par- ticular groups of fatty acids of each enzyme. In seeds, Upases may cause fat hydrolysis unless the enzymes are destroyed by heat. Palm oil produced by primitive methods in Africa used to consist of more than 10 per- cent of free fatty acids. Such spoilage prob- lems are also encountered in grains and flour. The activity of lipase in wheat and other grains is highly dependent on water content. In wheat, for example, the activity of lipase is five times higher at 15.1 percent than at 8.8 percent moisture. The lipolytic activity of oats is higher than that of most other grains. Lipases can be divided into those that have a positional specificity and those that do not. The former preferentially hydrolyze the ester bonds of the primary ester positions. This results in the formation of mono- and diglyc- erides, as represented by the following reac- tion: During the progress of the reaction, the con- centration of diglycerides and monoglycer- ides increases, as is shown in Figure 10-3. Lipase Lipase The (3-monoglycerides formed are resistant to further hydrolysis. This pattern is characteris- tic of pancreatic lipase and has been used to study the triglyceride structure of many fats and oils. The hydrolysis of triglycerides in cheese is an example of a desirable flavor-producing process. The extent of free fatty acid forma- tion is much higher in blue cheese than in Cheddar cheese, as is shown in Table 10-3. This is most likely the result of Upases elabo- rated by organisms growing in the blue cheese, such as P. roqueforti, P. camemberti, and others. The extent of lipolysis increases with age, as is demonstrated by the increas- ing content of partial glycerides during the aging of cheese (Table 10-4). In many cases, lipolysis is induced by the addition of lipoly- tic enzymes. In the North American choco- late industry, it is customary to induce some lipolysis in chocolate by means of lipase. In the production of Italian cheeses, lipolysis is Figure 10-3 The Course of Pancreatic Lipase Hydrolysis of Tricaprylin. MG = monoglycerides, DG = diglycerides, TG = triglycerides. Source: From A. Boudreau and J.M. deMan, The Mode of Action of Pancreatic Lipase on Milkfat Glycerides, Can. J. Biochem., Vol. 43, pp. 1799-1805, 1965. X HYDROLYSIS MOLE V. G LY CERIDES [...]... the formation of gluconic acid with uptake of one molecule of oxygen The cata- 4-Methylcatechol A-Methyl 0-benzoquinone L-Ascorbic acid L-Dehydroascorbie acid Figure 1 0-1 4 Reaction of L-Ascorbic Acid with o-Quinone in the Prevention of Enzymic Browning lase decomposes the hydrogen peroxide formed into water and one half-molecule of oxygen The net result is the uptake of one half-molecule of oxygen The... points are 1,6-oc glucosidic bonds Source: From JJ Marshall, Starch Degrading Enzymes, Old and New, Starke, Vol 27, pp 37 7-3 83, 1975 Alpha-amylase (a-l,4-Glucan 4Glucanohydrolase) This enzyme is distributed widely in the animal and plant kingdoms The enzyme contains 1 gram-atom of calcium per mole Alpha-amylase (a-1,4-glucan-4-glucanohydrolase) is an endoenzyme that hydrolyzes the oc-l,4-glucosidic bonds... production of food fats that have a higher essential fatty acid content and lower trans levels than is possible with current methods of hydrogenation Amylases The amylases are the most important enzymes of the group of glycoside hydro- lases These starch-degrading enzymes can be divided into two groups, the so-called debranching enzymes that specifically hydrolyze the 1,6-linkages between chains, and the enzymes. .. deactivates the enzyme, and no off-flavor is formed Blanching of peas and beans is essential in preventing the lipoxygenase-catalyzed development of off-flavor In addition to the development of off-flavors, the enzyme may be responsible for destruction of carotene and vitamin A, chlorophyll, bixin, and other pigments In some cases the action of lipoxygenase leads to development of a characteristic aroma... IMMOBILIZED ENZYMES One of the most important recent developments in the use of enzymes for industrial food processing is the fixing of enzymes on water-insoluble inert supports The fixed enzymes retain their activity and can be easily added to or removed from the reaction mixture The use of immobilized enzymes permits continuous processing and greatly increased use of the enzyme Various possible methods of. .. position of the ester bond in the glycerol molecule, or the nature of the fatty acid Examples of enzymes in this group are lipases of Candida cylindracae, Corynebacterium acnes, and Staphylococcus aureus The second group contains lipases with position specificity for the 1- and 3-positions of the glycerides This is common among microbial lipases and is the result of the steri- Table 1 0-7 Application of Microbial... amylases Beta-galactosidase ($-D-Galactoside Galactohydrolase) This enzyme catalyzes the hydrolysis of pD-galactosides and a-L-arabinosides It is best known for its action in hydrolyzing lactose and is, therefore, also known as lactase The enzyme is widely distributed and occurs in higher animals, bacteria, yeasts, and Table 1 0-8 Development of a-Amylase During Malting of Barley at 2O0C Days of Steeping... Proteases These enzymes require a metal for activity and are inhibited by metal-chelating compounds They are exopeptidases and include carboxypeptidase A (peptidyl-L-amino-acid hydrolase) and B (peptidyl-L-lysine hydro- lase), which remove amino acids from the end of peptide chains that carry a free oc-carboxyl group The aminopeptidases remove amino acids from the free oc-amino end of the peptide chain... (3-amylase When these grains germinate, the (3-amylase level hardly changes, but the a-amylase content may increase by a factor of 1,000 The combined action of a- and (3-amylase in the germinated grain greatly increases the production of fermentable sugars The development of aamylase activity during malting of barley is shown in Table 1 0-8 In wheat flour, high a- amylase activity is undesirable, because... Protein-Cu2 + o-quinone + H2O The protein copper-oxygen complex is formed by combining one molecule of oxygen with the protein to which two adjacent cuprous atoms are attached Catecholase activity involves oxidizing two molecules of odiphenols to two molecules of 0-quinones, resulting in the reduction of one molecule of oxygen to two molecules of water The action sequence as presented in Figure 1 0-1 3 . Acid 4:0 6:0 8:0 10: 0 12:0 14:0 16:0 18:1 and 18:2 18:0 Milk Lipase 13.9 2.1 1.8 3.0 2.7 7.7 21.6 29.2 10. 5 Steapsin 10. 7 2.9 1.5 3.7 4.0 10. 7 21.6 24.3 13.4 Pancreatic Lipase 14.4 2.1 1.4 3.3 3.8 10. 1 24.0 25.5 9.7 Calf . listing of the uses of enzymes in food processing (Table 10- 1) and this gives a good summary of the many and varied possi- ble applications of enzymes. NATURE AND FUNCTION Enzymes are proteins. the complex is given by K = [E][S] m [ES] Enzymes CHAPTER 10 Enzyme Amylases Cellulase Dextran-sucrase Invertase Lactase Tannase Pentosanase Naringinase Pectic enzymes (use- ful) Food Baked goods Brewing Cereals Chocolate-cocoa Confectionery Fruit