Food Modification By Enzymes

Food Modification By Enzymes

Enzymes have a very important inpact on the quality of our foods. In fact, without enzymes there would be no food. But then there would be no need for food, since no organism could live without enzymes. They are the catalysts that make life possible, as we know it. For any organism, life begins with enzyme action in the gestation and fertilization processes. The growth and maturation of our foods depend on enzyme actions. While we have known for some time that environmental conditions during growing affect the composition, including enzymes, of our plant foods, a recent review [16] has detailed just how much effect moisture deficiency has on the expression of genes during growth and maturation of plants. Following maturation, the harvesting, storage, and processing conditions can markedly affect the rate of food deterioration. Enzymes can also be added to foods during processing to change their characteristics, and some of these changes will be discussed. Microbial enzymes, left after destruction of the microorganisms, continue to affect the quality of processed and reformulated foods. For example, starch-based sauces can undergo undesirable changes in consistency because of heat-stable microbial a-amylases that survive a heat treatment sufficient to destroy the microorganisms. Because of their high specificity, enzymes are also the ideal catalysts for the biosynthesis of highly complex chemicals. 7.8.1 Role of Endogenous Enzymes in Food Quality 7.8.1.1 Color (See also Chap. 10.) Color is probably the first attribute the consumer associates with quality and acceptability of foods. A steak must be red, not purple or brown. Redness is due only to oxymyoglobin, the main pigment in meat. Deoxymyoglobin is responsible for the purple color Pag e 493 of meat. Oxidation of the Fe(II) present in oxymyoglobin and deoxymyoglobin, to Fe(III) producing metmyoglobin, is responsible for the brown color of meat. Enzyme-catalyzed reactions in meat can compete for oxygen, can produce compounds that alter the oxidation-reduction state and water content, and can thereby influence the color of meat. The quality of many fresh vegetables and fruits is judged on the basis of their “greenness.” On ripening, the green color of many of our fruits decreases and is replaced with red, orange, yellow, and black colors. In green beans and English green peas, maturity leads to a decrease in chlorophyll level. All of these changes are a result of enzyme action. Three key enzymes responsible for chemical alterations of pigments in fruits and vegetables are lipoxygenase, chlorophyllase, and polyphenol oxidase. Lipoxygenase Lipoxygenase (lineoleate:oxygen oxidoreductase; EC 1.13.11.12) has six important effects on foods, some desirable and others undesirable. The two desirable functions are (a) bleaching of wheat and soybean flours and (b) formation of disulfide bonds in gluten during dough formation (eliminates the need to add chemical oxidizers, such as potassium bromate). The four undesirable actions of lipoxygenase in food are (a) destruction of chlorophyll and carotenes, (b) development of oxidative off flavors and aromas, often characterized as haylike, (c) oxidative damage to compounds such as vitamins and proteins, and (d) oxidation of the essential fatty acids, lineoleic, linolenic, and arachidonic acids. All six of these reactions result from the direct action of lipoxygenase in oxidation of polyunsaturated fatty acids (free and lipidbound) to form free radical intermediates (Fig. 36). In steps 2, 3, and 4 (Fig. 36) free radicals are formed, while in step 5 a hydroperoxide is formed. Further nonenzymatic reactions lead to formation of aldehydes (including malondialdehyde) and other components that contribute to off flavors and off aromas. The free radicals and hydroperoxide are responsible for loss of color (chlorophyll; the orange and red colors of the carotenoids), disulfide bond formation in gluten of doughs, and damage to vitamins and proteins. The most oxidation-sensitive amino acid residues in proteins are cysteine, tyrosine, histidine, and tryptophan. Antioxidants, such as vitamin E, propyl gallate, benzoylated hydroxytoluene, and nordihydroguaiacetic acid, protect foods from damage from free radicals and hydroperoxides. Chlorophyllase Chlorophyllase (chlorophyll chlorophyllido-hydrolase, EC 3.1.1.14) is found in plants and chlorophyll-containing microorganisms. It hydrolyzes the phytyl group from chlorophyll to give phytol and chlorophyllide (see Chap. 10). Although this reaction has been attributed to a loss of green color, there is no evidence to support this as chlorophyllide is green. Furthermore, there is no evidence that the chlorophyllide is any less stable to color loss (loss of Mg2+) than is chlorophyll. The role of chlorophyllase in vivo in plants is not known. Very few studies have been done on chlorophyllase-catalyzed hydrolysis of chlorophyll during storage of raw plant foods (Chap. 10). Polyphenol Oxidase Polyphenol oxidase (1,2-benzenediol:oxygen oxidoreductase; EC 1.10.3.1) is frequently called tyrosinase, polyphenolase, phenolase, catechol oxidase, cresolase, or catecholase, depending on the substrate used in its assay or found in the greatest concentration in the plant that serves as a source of the enzyme. Polyphenol oxidase is found in plants, animals and some microorganisms, especially the fungi. It catalyzes two quite different reactions with a large number of phenols, as shown in Equations 58 and 59. Pag e 494 (58) (59) The 4-methyl-o-benzoquinone (Eq. 59) is unstable and undergoes further non-enzyme-catalyzed oxidation by O2, and polymerization, to give melanins. The latter is responsible for the undesirable brown discoloration of bananas, apples, peaches, potatoes, mushrooms, shrimp, and humans (freckles), and the desirable brown and black colors of tea, coffee, raisins, prunes, and human skin pigmentation. The o-benzoquinone reacts with the e-amino group of lysyl residues of proteins, leading to loss of nutritional quality and insolubilization of proteins. Changes in texture and taste also result from the browning reactions. It is estimated that up to 50% of some tropical fruits are lost due to enzymatic browning. This reaction is also responsible for deterioration of color in juices and fresh vegetables such as lettuce, and in taste and nutritional quality. Therefore, much effort has gone into developing methods for control of polyphenol oxidase activity. As discussed earlier in Section 7.7.5, elimination of O2 and the phenols will prevent browning. Ascorbic acid, sodium bisulfite, and thiol compounds prevent browning due to reduction of the initial product, o-benzoquinone, back to the substrate, thereby preventing melanin formation. When all of the reducing compound is consumed, browning still may occur, since the enzyme may still be active. Ascorbic acid, sodium sulfite, and thiol compounds also have a direct effect in inactivating polyphenol oxidase due to destruction of the active-site histidines (ascorbic acid) or in removal by (sodium bisulfite and thiols) of the essential Cu2+ in the active site [84]. 4-Hexylresorcinol, benzoic acid, and some other nonsubstrate phenols are effective inhibitors of some polyphenol oxidases, with Ki values around 1–10mM. Most likely, inhibition results from competitive binding of this type of inhibitor into the reducing compound (BH2) binding site (Eq. 58). 7.8.1.2 Texture Texture is a very important quality attribute in foods. In fruits and vegetables, texture is due primarily to the complex

 carbohydrates: pectic substances, cellulose, hemicelluloses, starch, and lignin. There are one or more enzymes that act on each of the complex carbohydrates that are important in food texture. Proteases are important in the softening of animal tissues and high-protein plant foods. Pectic Enzymes Three types of pectic enzymes that act on pectic substances are well described. Two (pectin methylesterase and polygalacturonase) are found in higher plants and microorganisms and one type (the pectate lyases) is found in microorganisms, especially certain pathogenic microorganisms that infect plants [109]. Pag e 495 FIGURE 36 Reaction catalyzed by lipoxyg enase. See the test for a description of the sequence of reactions. (From Ref. 111, p. 588.) Pectin methylesterase (pectin pectylhydrolase, EC 3.1.1.11) hydrolyzes the methyl ester bond of pectin to give pectic acid and methanol (Eq. 60). (60) Pag e 496 The enzyme is sometimes referred to as pectinesterase, pectase, pectin methoxylase, pectin demethoxylase, and pectolipase. Hydrolysis of pectin to pectic acid in the presence of divalent ions, such as Ca2+, leads to an increase in textural strength, due to the cross bridges formed between Ca2+ and the carboxyl groups of pectic acid. Polygalacturonase (poly-a-1,4-galacturonide glycano-hydrolase, EC 3.2.1.15) hydrolyzes the a-1,4-glycosidic bond between the anhydrogalacturonic acid units (Eq. 61). (61) Both endo- and exo-polygalacturonases exist; the exo type hydrolyzes bonds at the ends of the polymer and the endo type acts in the interior. There are differences in opinion as to whether plants contain both polymethylgalacturonases (act on pectins) and polygalacturonases (act on pectic acid), since pectin methylesterase in the plant rapidly converts pectin to pectic acid. Action of polygalacturonase results in hydrolysis of pectic acid, leading to important decreases in texture of some raw food materials, such as tomatoes. The pectate lyases [poly(1,4-aD-galacturonide) lyase, EC 4.2.2.2] split the glycosidic bond of both pectin and pectic acid, not with water, but by b-elimination (Eq. 62). They are found in microorganisms, but not in higher plants. (62) Splitting of the glycosidic bond gives a product with a reducing group and another product with a double bond. Polygalacturonases and pectate lyases both produce reducing groups when the glycosidic bond is split, resulting in a decrease in texture, so they cannot be distinguished by this method. However, the double bond in the second product (Eq. 62) has an extinction coefficient of 4.80 ×103 M-1 cm-1 at 235 nm. Therefore, a change in absorbance at 235 nm is the method of choice for distinguishing between polygalacturonases and pectate lyases. There are both endo- and exo-splitting pectate lyases, as well as pectate lyases that act on either pectin or on pectic acid. A fourth type of pectin-degrading enzyme, protopectinase, has been reported in a few microorganisms. Protopectinase hydrolyzes protopectin, producing pectin. However, it is not clear yet whether the protopectinase activity in plants is due to the combined action of pectin methylesterase and polygalacturonase or to a true protopectinase. It is unlikely that protopectinase is an “H-bondase” as suggested by some researchers. Pag e 497 Cellulases Cellulose is abundant in trees and cotton. Fruits and vegetables contain small amounts of cellulose, which has a role in the structure of cells. Whether cellulases are important in the softening of green beans and English green pea pods is still a matter of controversy. Abundant information is available on the microbial cellulases because of their potential importance in converting insoluble cellulosic waste to glucose [70]. Pentosanases Hemicelluloses, which are polymers of xylose (xylans), arabinose (arabans), or xylose and arabinose (arabinoxylans), with small amounts of other pentoses or hexoses, are found in higher plants. Pentosanases in microorganisms [109], and in some higher plants, hydrolyze the xylans, arabans, and arabinoxylans to smaller compounds. The microbial pentosanases are better characterized than those in higher plants. Several exo- and endo-hydrolyzing pentosanases also exist in wheat at very low concentrations, but little is known about their properties. It is important that these pentosanases receive more attention from food scientists. Amylases Amylases, the enzymes that hydrolyze starches, are found not only in animals, but also in higher plants and microorganisms. Therefore, it is not surprising that some starch degradation occurs during maturation, storage, and processing of our foods. Since starch contributes in a major way to viscosity and texture of foods, its hydrolysis during storage and processing is a matter of importance. There are three major types of amylases: a-amylases, b-amylases, and glucoamylases. They act primarily on both starch and glycogen. Other starch-splitting enzymes also exist (Table 14). The a-amylases, found in all organisms, hydrolyze the interior a-1,4-glucosidic bonds of starch (both amylose and amylopectin), glycogen, and cyclodextrins with retention of the a-configuration of the anomeric carbon. Since the enzyme is endo-splitting, its action has a major effect on the viscosity of starch-based foods, such as puddings, cream sauces, etc. The salivary and pancreatic a-amylases are very important in digestion of starch in our foods. Some microorganisms contain high levels of aamylases. Some of the microbial a-amylases have high inactivation temperatures, and, if not activated, they can have a drastic undesirable effect on the stability of starch-based foods. b-Amylases, found in higher plants, hydrolyze the a-1,4-glucosidic bonds of starch at the nonreducing end to give b-maltose. Since they are exo-splitting enzymes, many bonds must be hydrolyzed before an appreciable effect on viscosity of starch paste is observed. Amylose can be hydrolyzed to 100% maltose by b-amylase, while b-amylase cannot continue beyond the first a1,6-glycosidic bond encountered in amylopectin. Therefore, amylopectin is hydrolyzed only to a limited extent by b-amylase alone. “Maltose” syrups, of about DP 10, are very important in the food industry. b-Amylase, along with a-amylase, is very important in brewing, since the maltose can be rapidly converted to glucose by yeast maltase. b-Amylase is a sulfhydryl enzyme and can be inhibited by a number of sulfhydryl group reagents, unlike a-amylase and glucoamylase. In malt, b-amylase is often covalently linked, via disulfide bonds, to other sulfhydryl groups; therefore, malt should be treated with a sulfhydryl compound, such as cysteine, to increase its activity in malt. Proteases Texture of food products is changed by hydrolysis of proteins by endogenous and exogenous proteases. Gelatin will not gel when raw pineapples is added, because the pineapples contains Pag e 498 TABLE 14 Some Starch- and Glycog en-Deg rading Enzymes Type Config uration of g lucosidic bond Comments Endo-splitting (config uration retained) a-Amylase (EC 3.2.1.1) a-1,4 Initial major products are dextrins; final major products are maltose and maltotriose Isoamylase (EC 3.2.1.68) a-1,6 Products are linear dextrins Isomaltase (EC 3.2.1.10) a-1,6 Acts on products of a-amylase hydrolysis of amylopectin Cyclomaltodextrinase (EC 3.2.1.54) a-1,4 Acts on cyclodextrins and linear dextrins to g ive maltose and maltotriose Pullulanase (EC 3.2.1.41) a-1,6 Acts on pullulan to g ive maltotriose and on starch to g ive linear dextrins Isopullulanase (EC 3.2.1.57) a-1,4 Acts on pullulan to g ive isopanose and on starch to g ive unknown products Neopullulanase a-1,4 Acts on pullulan to g ive panose and on starch to g ive maltose Amylopullulanase a-1,6 Acts on pullulan to g ive maltotriose a-1,4 Acts on starch to g ive DP 2–4 products Amylopectin 6-g lucano hydrolase (EC 3.2.1.41) a-1,6 Acts only on amylopectin to hydrolyze a1,6-g lucosidic linkag es Exo-splitting (nonreducing end) b-Amylase (EC 3.2.1.2) a-1,4 Product is b-maltose a-Amylase a-1,4 Product is a-maltose; there are specific exoa-amylases that produce maltotriose, maltotetraose, maltopentaose, and maltohexaose, with retention of config uration Glucoamylase (EC 3.2.1.3) a-1,6 b-Glucose is produced a-Glucosidase (EC 3.2.1.20) a-1,4 a-Glucose is produced; there are a number of a-g lucosidases Transferase Cyclomaltodextrin g lucanotransferase (EC 2.4.1.19) a-1,4 a- and b-Cyclodextrins formed from starch with 6–12 g lucose units bromelain, a protease. Chymosin causes milk to gel, as a result of its hydrolysis of a single peptide bond between Phe105-Met106 in k-casein. This specific hydrolysis of k-casein destabilizes the casein micelle, causing it to aggregate to form a curd (cottage cheese). Action of intentionally added microbial proteases during aging of brick cheeses assists in development of flavors (flavors in Cheddar cheese vs. blue cheese, for example). Protease activity on the gluten proteins of wheat bread doughs during rising is important not only in the mixing characteristics and energy requirements but also in the quality of the baked breads. Pag e 499 The effect of proteases in the tenderization of meat is perhaps best known and is economically most important. After death, muscle becomes rigid due to rigor mortis (caused by extensive interaction of myosin and actin). Through action of endogenous proteases (Ca2+-activated proteases, and perhaps cathepsins) on the myosin-action complex during storage (7–21 days) the muscle becomes more tender and juicy. Exogenous enzymes, such as papain and ficin, are added to some less choice meats to tenderize them, primarily due to partial hydrolysis of elastin and collagen. 7.8.1.3 Flavor and Aroma Changes in Foods Chemical compounds contributing to the flavor and aroma of foods are numerous, and the critical combinations of compounds are not easy to determine. It is equally difficult to identify the enzymes instrumental in the biosynthesis of flavors typical of food flavors and in the development of undesirable flavors. Enzymes cause off flavors and off aromas in foods, particularly during storage. Improperly blanched foods, such as green beans, English green peas, corn, broccoli, and cauliflower, develop very noticeable off flavors and off aromas during frozen storage. Peroxidase, a relatively heat-resistant enzyme not usually associated with development of defects in food, is generally used as the indicator for adequate heat treatment of these foods. It is clear now that a higher quality product can be produced by using the primary enzyme involved in off flavor and off aroma development as the indicator enzyme. With this in mind, Whitaker and Pangborn [74, 75, 101] and their students determined that lipoxygenase is responsible for off flavor and off aroma development in English green peas, green beans, and corn, and that cystine lyase is the primary enzyme responsible for off flavor and off aroma development in broccoli and cauliflower. Evidence to support the important role of lipoxygenase as a catalyst of off-flavor development in green beans is shown in Figure 37. Additional work indicated that the flavor stability of frozen food (of types mentioned above), blanched to the endpoint of the responsible enzyme, is better than that of comparable samples blanched to the peroxidase endpoint. Naringin is responsible for the bitter taste of grapefruit and grapefruit juice. Naringin can be destroyed by treating the juice with naraginase. Some research is underway to eliminate naringin biosynthesis by recombinant DNA techniques. 7.8.1.4 Nutritional Quality There is relatively little data available with respect to the effects of enzymes on nutritional quality of foods. Lipoxygenase oxidation of linoleic, linolenic, and arachidonic acids certainly decreases the amounts of these essential fatty acids in foods. The free radicals produced by lipoxygenase-catalyzed oxidation of polyunsaturated fatty acids decrease the carotenoid (vitamin A precursors), tocopherols (vitamin E), vitamin C, and folate content of foods. The free radicals also are damaging to cysteine, tyrosine, tryptophan, and histidine residues of proteins. Ascorbic acid is destroyed by ascorbic acid oxidase found in some vegetables such as squash. Thiaminase destroys thiamine, an essential cofactor involved in amino acid metabolism. Ribo-flavin hydrolase, found in some microorganisms, can degrade riboflavin. Polyphenol oxidase-caused browning decreases the available lysine content of proteins. 7.8.2 Enzymes Used as Processing Aids and Ingredients Enzymes are ideal for producing key changes in the functional properties of food, for removal of toxic constituents, and for producing new ingredients. This is because they are highly specific, act at low temperatures (25–45°C), and do not produce side reactions. Full utilization of the high Pag e 500 FIGURE 37 Effect of added enzymes on development of undesirable aroma descriptors in g reen beans. Averag e aroma differences are shown between sample and a blanched reference sample to which no enzymes were added. An averag e aroma intensity of 50 is the same as the reference. The shaded bars indicate aroma descriptor intensity differences statistically different from the reference. (From Ref. 114, p. 134, with permission of Institute of Food Technolog ists.) specificity of enzymes is compromised currently because relatively crude enzyme systems are used, because of cost. Crude preparations contain other enzymes that may produce unwanted products. Of major concern is that the crude enzyme preparations often differ substantially from batch to batch. With recombinant DNA methods, the time is closer when pure enzymes can be used in food processing. There are some major successes in the use of food-related enzymes. Production of high-fructose corn syrup is one example. This involves a relatively heat-stable a-amylase, glucoamylase, and glucose isomerase: (63) Starch is heated to 105°C, Bacillus licheniformis a-amylase is added, and dextrins of DP 10–12 are produced by the endosplitting enzyme. The soluble digest is passed through giant columns (6–10 ft in diameter and 20 ft high) of immobilized
 glucoamylase where glucose is produced. The glucose-containing stream is then run through giant columns of immobilized glucose isomerase where approximately equimolar concentrations of glucose and fructose are produced. The fructose is separated from glucose by differential crystallization and is used as a major sweetener in the food industry (~100 billion tons/year). The glucose or a mixture of Pag e 501 TABLE 15 Biocatalytic Production of Sweeteners Feedstock Product Enzyme(s) Starch Corn syrups a-Amylases, pullulanases Glucose a-Amylases, g lucoamylase Fructose a-Amylase, g lucoamylase, g lucose isomerase Starch + sucrose Sucrose derivatives Cyclodextring lucosyltransferase and pullulanase (or isoamylase) Sucrose Glucose + fructose Invertase Sucrose Isomaltulose b-Glucosyltransferase and isomaltulose synthetase Sucrose + fructose Leucrose a-1,6-Glycosyltransferase Lactose Glucose + g alactose b-Galactosidase Galactose Galacturonic acid Galactose oxidase Glucose Galactose epimerase Several Aspartame Thermolysin, penicillinacylase Stevioside a-Glycosylated stevioside a-Glucosidase Ribaudioside-A b-Glycosyltransferase Source: Ref. 110, p. 676. glucose and fructose is used as sweetener, or the glucose is recycled to produce more fructose. Other sweeteners can also be produced enzymatically (Table 15). A second example is the use of aminoacylases to separate racemic mixtures of DL-amino acids, in multi-ton lots. A third example is the use of specific lipases to tailor-make lipids with respect to melting point, unsaturation, or specific location of a fatty acid in a triacylglycerol. This requires a beginning lipid, the required concentrations of free fatty acids, appropriate enzymes, and appropriate conditions (Table 16). For example, a 1,3-specific lipase catalyzes transesterification of fatty acids at the 1 – and 3-positions of glycerol, a 2-specific lipase catalyzes transesterification only at the 2-position of glycerol, and a nonspecific lipase catalyzes transesterification at the 1-, 2-, and 3-positions of glycerol. An example using 1,3-specific lipase is: (64) TABLE 16 Enzymes for Modifying Lipids Enzyme Products Lipases Rearrang ed trig lycerides W axes via esterification Monog lycerides Phospholipase A Lysophospholipid Phospholipase D Phosphotidylg lycerol Source: Ref. 110, p. 685. Pag e 502 7.8.2.1 Specialty Products and Ingredients via Enzymology The following text and tables illustrate some of the current or potential uses of enzymes on an industrial scale. Enzymatic Production of Valuable Compounds Listed in Table 17 are 14 enzymes used to make specialty compounds as additives in foods. Enzymatic Removal of Undersirable Compounds Raw food materials often contain toxic or anti-nutrient compounds that are sometimes removed by proper heat treatment, extraction or by enzymatic reactions (Table 18). There are more than 12,000 plants in the world that may have potential as food sources. Many are not used because of undesirable properties, some of which could be overcome by the proper use of enzymes. Enzymes in Milk and Dairy Products Bovine milk contains many enzymes (see Chap. 14), and other enzymes are added during processing (Table 19). Of most importance economically is the use of chymosin (rennet) in production of several kinds of cheese. b-Galactosidase is potentially of great importance in the commercial hydrolysis of lactose in milk and dairy products, so that these products can be consumed by individuals who are deficient in b-galactosidase. b-Galactosidase is also used to convert whey lactose to glucose and galactose, sweeteners with greater commercial demand. Enzymes in Baking Several enzymes are used in breadmaking (Table 20). Additions of amylases and proteases has been common for years. Several enzyme preparations are available for the stated purpose of reducing the rate of staling in bread. These include (a) a debranching enzyme (1,6-splitting; TABLE 17 Enzymatic Production of W anted Compounds or Creation of Desirable Effects Enzymes Purpose Aminoacylases Resolve DL-amino acids Aspartase Produce aspartate Proteases Surfactants Peroxidase Phenol resins 5′-Phosphodiesterases 5′-Nucleotides for flavor enhancers 5′-Adenylic deaminase Produce 5′-inosinic acid for flavor Lipases (preg astric) Cheese/butter flavors Proteases Decrease ripening time of cheeses Tenderization of meat Lipases/esterases Flavor esters Proteases, nucleases Meaty flavors from yeast hydrolysis Fumarase Fumaric acid as acidulant Cyclomaltodextrin g lucanotransferase Cyclodextrins for inclusion complexation Tannase Antioxidants, such as propylg allate a-Galactosidase Modified food g ums Source: Ref. 110, p. 674pullulanase-type) from Bacillus acidopullulyticus along with a-amylase; (b) a genetically engineered a-amylase from Bacillus megaterium that rearranges the branched chains of amylopectin, via chain transfer, to give a linear polymer; (c) a mixture of cellulase, b-glucanase, and pentosanase produced by Trichoderma reesei; and (d) a mixture of cellulase, hemicellulases, and pentosanases used in conjunction with fungal a-amylase. Preparations containing pentosanases are reported to increase the moisture content of rye bread products by decreasing the high pentosan content of rye flour. TABLE 19 Enzymes in Milk and Diary Products Enzymes Function Chymosin Milk coag ulation For rennet pudding s Chymosin, fung al proteases For cottag e cheese For brick cheese Proteases Flavor improvement, decrease ripening time of cheeses Lipases Flavor improvement;
 decrease ripening time of cheeses Sulfhydryl oxidase Remove cooked flavor b-Galactosidase Lactose removal Microbial proteases Soybean milk coag ulation Source: Ref. 110, p. 687. Pag e 504 TABLE 20 Enzymes in Baking Enzymes Purpose Amylase To maximize fermentation process; prevent staling Proteases Improve handling and rheolog ical properties a-Glutamyl transferase, g lutathione oxidase, cysteinyl g lycine dipeptidase Improve doug h elasticity, loaf volume, crumb structure, and retention of bread softness on storag e Pentosanases In rye bread products to decrease doug h development time and power requirements, increase moistness Sulfhydryl oxidase Streng then weak doug hs by -S-S- formation Source: Ref. 110, p. 689. Enzymes in Brewing Several recent advances have been made with regard to the use of enzymes in brewing (Table 21). There is increasing interest in the possible use of blended a-amylase and protease preparations as replacements for malt in brewing. This is attributable to the expense and limited supplies of malt and the possibility that better quality control might be achieved. The industry has also used amyloglucosidases recently to make “light” beer. Amyloglucosidases hydrolyze the a-1,6-glucosidic bonds of the amylopectin fraction, permitting the complete fermentation of starch. Use of b-glucanases may solve the high viscosity/slow filtration rate problems caused by mannans from cell walls. The most exciting advance, however, is the use of acetolactate decarboxylase, now cloned into brewers yeast, to shorten the fermentation time by avoiding diacetyl formation (Eq. 65) [83a]. (65) Enzymes for Control of Microorganisms Enzymes have potential for destroying microorganisms by several means (Table 22). The means range from hydrolysis of cellwall compounds, such as b-glucans, chitin, and peptidoglycans, to production of H2O2 and
 which oxidize the essential -SH group of key sulfhydryl enzymes or polyunsaturated fatty acids in cell walls. These are interesting possibilities, worthy of being tested at the commercial level. 7.9 Immobilized Enzymes in Food Processing 7.9.1 Advantages and Disadvantages Why immobilized enzymes? The objectives of immobilizing enzymes are to permit their repeated use to make products, and at the end, to have a product free of enzyme. In Section 7.8.2, Pag e 505 TABLE 21 Enzymes in Brewing Enzymes Purpose Amylases (a and b) Convert nonmalt starch to maltose and dextrins; fermented by yeast to alcohol and CO2 Proteases (endo and exo) Hydrolyze proteins to amino acids; used by yeast to g row Papain Chillproofing beer Amylog lucosidase Hydrolyze 1,6-linkag es in amylopectin permitting complete fermentation of starch (lig ht beer) b-Glucanases Hydrolyze g lucans to reduce viscosity and aid filitration Acetolactate decarboxylase Decrease fermentation time, by avoiding diacetyl formation Source: Ref. 110, p. 690. the discussion focused on the many applications and potential applications of enzymes in food processing, their use in developing food ingredients and in the removing of unwanted compounds. It was mentioned that crude enzyme preparations produce numerous side reactions because of contaminating enzymes. An example of this problem can occur during fruit juice production. Pectic enzyme preparations from Aspergillus niger are often used to increase yield and clarity. However, there are several polymer-hydrolyzing enzymes in the A. niger enzyme preparation (Table 23). The net effect observed after enzyme treatment of the juice is a composite of the activities of many of the enzymes present. For example, in apple juice an arabinosidase removes the side chain arabinose units from the linear araban chain, which then aggregates to TABLE 22 Enzymes for Control of Microorg anisms Enzyme Function Oxidases Removal of O2, NADH or NADPH; produce H2O2 and which oxidize -SH g roups and polyunsaturated lipids Xylitol phosphorlase Conversion of xylitol to xylitol 5-phosphate, which kills microorg anism Lipases Liberation of free fatty acids, which are toxic to protozoa Giardia lamblia Lactoperoxidase Uses & H2O2, produced by oxidase, to convert SCN-to SCNO – and -SH g roups to -S-S-, -S-SCN, or -S-OH Myeloperoxidase W ith added H2O2 and CI- , produces HOCI and cholroamines Lysozyme Effective ag anist a number of g ram-positive org anisms via hydrolysis of cell-wall peptidog lycans; hydrolysis of protein-mannan outer layer of yeast when added with and endo- b-1,3-g lucanase Mannanase Lysis of b-g lucans-protein cell wall of yeast Chitinase Effective ag anist chitin in cell wall of several fung i Antienzymes Proteases, sulfhydryl oxidase, dehydrog enases, lipoxyg enases Source: Adapted from Ref. 92. Pag e 506 TABLE 23 Enzymes Produced by Aspergillus niger That Hydrolyze Ploymers Substrate Enzymes Arabinans a-L-Arabinofuranosidase Cellulose Cellulases a Dextran Dextranase DNA, RNA Deoxyribonuclease, ribonuclease b-Glucans b-Glucanase Hemicellulose Hemicellulases (pentosanases) Inulin Inulinase Mannans b-Mannanase Pectic substances Pectin methylesterase, pectate lyase, polyg alacturonase Proteins Proteases Starch a-Amylase, g lucoamylase Xylans Xylanase aAll types of cellulases (see Section 7.11.2). Source: Ref. 110, p. 684. give turbidity. Also, the next batch of A. niger pectic enzymes may perform differently unless the growth conditions are controlled exactly. At the present time, the cost of a more purified enzyme preparation would be prohibitive if used only once. The advantage of immobilized enzymes is that they can be used repeatedly, greatly decreasing the overall cost. The cost of fructose production from corn starch would be prohibitive if the glucoamylase and glucose isomerase where used for only one batch. Contrast that with the current system using immobilized glucoamylase and glucose isomerase where the cost of producing fructose is about 65% of that of producing sucrose from sugar cane and sugar beets. Why are immobilized enzyme systems not used more in food processing? The answer is that most food systems are too complex, physically. Chemical complexity is not a problem. Because of the high specificity of enzymes, a single compound such as glucose, for example, can be selected from a complex mixture, bound by glucose oxidase, and converted to product. The thousands of other compounds are not changed. In an immobilized enzyme system, special arrangements are necessary to bring the enzyme and substrate into contact. This can be done by adding an immobilized enzyme preparation and providing thorough circulation, or by flowing the food past a stationary immobilized enzyme (column, coated tube wall, etc). The immobilized enzyme can be used for several months. Immobilized enzymes are especially valuable for analytical processes. This topic will be discussed in Section 7.12.4. 7.9.2 Methods of Immobilizing Enzymes Enzymes can be immobilized by several methods: (a) covalent attachment to insoluble support materials such as metals, glass, ceramic, nylon, cellulose, Sepharose, or Sephadex; (b) entrapment in a gel matrix made from, for example, polyacrylamide, Sephadex or agar; (c) adsorption on an insoluble matrix by hydrophobic, electrostatic or other noncovalent affinity methods; (d) adsorption on an insoluble matrix and then covalent cross-linking to the matrix; (e) intermolecular cross-linking of enzymes to form an insoluble matrix that is used in granular form; (f) attachment to semipermeable membranes; and (g) suspension of insoluble enzyme particles in immiscible organic solvents. Pag e 507 Some of the criteria used in deciding on the method of immobilization include: (a) stability of the enzyme during repeated use; (b) retention of activity of the enzyme following immobilization; (c) stability of the matrix to other enzymes, flow, pressure, temperature and other conditions encountered; (d) accessibility of the substrate to the enzyme (diffusion of substrate to the enzyme and products away); (e) susceptibility of the enzyme system to fouling by the substrate feed; (f) efficiency of converting substrate to product; (g) compatibility of the system with subsequent steps in conversion and recovery of product; (h) need for cofactors; (i) compatibility of pure enzyme, crude enzyme, or cells to be immobilized; (j) susceptibility to microbial contamination; (k) renewal efficiency; and (1) cost. Ideally the immobilized enzyme system should be reusable hundreds of times (3–6 months) before there is 50% loss of enzyme activity. Do such systems exist for food applications? Yes, they do. Three will be described. One of the best types of systems might involve countercurrent flow of two immiscible liquids, one containing an enzyme able to
 function at an interface and the other containing a substrate that can concentrate at the interface. This type of system could potentially be used to transesterify triacylglycerols. The substrates (triacylglycerol and fatty acids) are soluble in organic phase, and the lipase is soluble in the aqueous phase and functions at the interface of micellular substrates. Such systems are now in use. Another system potentially could involve enzymes immobilized to an insoluble magneticsensitive matrix. After reaction with the desired food components in a stirred reactor, the insoluble magnetic matrix could easily be removed and reused. A third method might involve use of a semipermeable membrane through which the enzyme is circulated while immersed in the food. The substrate must diffuse into the semipermeable membrane and the product must diffuse out. 7.9.3 Applications The starch to fructose conversion described above is almost an ideal system. The starch granules are ruptured by heating at 105°C to achieve solubility; however, the resulting solution is too viscous to manage. Hydrolysis of starch with the relatively heat-stable Bacillus lichenformis a-amylase to DP 10 solves that problem. Also, heating at 105°C destroys any microorganisms in the starch and solvents. Therefore, the remainder of the hydrolysis and isomerization can be carried out in giant columns of glucoamylase and glucose isomerase, which are also relatively stable. Fouling and regeneration of the columns are not major problems. Other major commercial applications of immobilized enzymes also have similar features (Table 24). How many other applications of enzymes are awaiting commercial application? Immobilized b-galactosidase for hydrolysis of lactose in milk is being considered in the United States. Column-based systems would be hampered by fouling, but it is surprising that some of the other ways in which immobilized enzymes can be used (discussed earlier) are not yet commercially acceptable. Only Italy commercially processes milk to hydrolyze lactose enzymatically. 7.9.4 Immobilized Cells In some cases, immobilized microbial cells can be employed to produce useful compounds (Table 25) or to remove toxic compounds from waste products (Table 26). Japanese scientists have contributed especially to developments in this area. In theory, this should be a very important method, since the desired enzyme is already immobilized inside the cell, cofactors can be regenerated by appropriate metabolic enzymes, the cell is often living and can replenish enzyme losses, and complex reactions requiring several steps can be carried out. It has also been suggested that other enzymes required for hydrolysis of feedstock, cell metabolism, or cofactor Pag e 508 TABLE 24 Commercial Applications of Immobilized Enzymes in the Food and Related Industries Immobilized enzymes or cells Substrates Products Aminoacylase Synthetic acyl-DL-amino acid + H2O L-Amino acid + acyl-Damino acid Aspartase, Escherichia coli Fumarate + NH3 L-Aspartate Fumarase, Brevibacterium ammoniagenes Fumarate + H2O L-Malate Glucose isomerase D-Glucose D-Fructose Sterptomyces sp. Bacillus coagulans Actinoplanes missouriensis Arthrobacter sp. a-Galactosidase (used to hydrolyze raffinose in sug ar beet juice, which hinders crystallization of sucrose) Raffinose D-Galactose and sucrose Source: Ref. 88, p. 436. production might be immobilized on the surface of the microbial cell [45]. For example, if endocellulases were immobilized on microbial cells they would convert insoluble cellulose to soluble oligosaccharides; these would diffuse into the cell, be further hydrolyzed by exocellulases and cellobiase to glucose, and glucose would then be metabolized to ethanol and CO2, both useful products. However, there are formidable conditions that must be met before immobilized microbial TABLE 25 Production of Useful Compounds by Immobilized Living Microbial Cells Useful compounds Microbial cells Carriers for immobilization Ethanol Saccharomyces carlsbergensis Carrag eenan Saccharomyces cerevisiae Polyacrylamide Saccharomyces carlsbergensis Polyvinyl chloride Saccharomyces cerevisiae Calcium alg inate L-Isoleucine Serratia marcescens Carrag eenan Acetic acid Acetobacter sp. Hydrous titanium oxide Lactic acid Mixed culture of lactobacilli and yeasts Gelatin Arthrobacter oxidans Ployacrylamide a-Ketog luconic acid Serratia marcescens Collag en Bacitracin Bacillus sp. Polyacrylamide Amylase Bacillus subtilis Polyacrylamide Hydrog en g as Clostridium butyricum Polyacrylamide Rhodospirillium rubrum Ag ar Source: Ref. 21, p. 88, courtesy of International Union of Biochemistry and Elsevier/North-Holland. Pag e 509 TABLE 26 Decomposition of Poisonous Compounds Using Immobilized Living Microbial Cells Applications Poisonous compounds Microbial cells Carriers for immobilization W aste treatment Phenol Candida tropicalis Polyacrylamide Benzene Pseudomonas putida Polyacrylamide W aste water Mixed culture of various microorg anisms Bentonite and Mg 2+Polyurethane spong e Denitrification Nitrate, nitrite Micrococcus denitrificans Liquid membrane Pseudomonas sp. Active carbon Source: Ref. 21, p. 89, courtesy of International Union of Biochemistry and Elsevier/North Holland. cells can be used commercially: (a) the microorganisms must be approved for use in foods and/or in food ingredient manufacture; (b) the substrate must diffuse readily into the cell and the product out (while cell walls can be made more permeable by chemical treatments, there is a limit to how much can be done before the cell is killed and/or becomes too fragile); (c) the “living” cell must not duplicate, but must remain viable in terms of enzyme and cofactor generation; (d) the cell must be rugged enough to permit repeated use; (e) the cell must not make toxic or significant amounts of unwanted products; and (f) the desired product must not be a substrate for other enzymes. It has been proposed repeatedly that plant cells might be immobilized and packed into columns to make flavor constituents, colorants, vitamins, and other valuable products, but this has not been done except in the laboratory. The advantage would be a relatively pure compound that needs little further purification to be of commercial use. Other advantages would be energy efficiency, small space requirement, and independence from climate. Problems with cell fragility, getting feed stock in and product out of the cells, long-term utilization, and efficient heat removal have not been solved. But one day, this approach will be successful and, hopefully, food scientists and the food industry will contribute greatly to this occurrence. 7.10 Solvent-Partitioned Enzyme Systems Continuous-flow systems for mass manufacturing of all kinds of products have important advantages over batch systems. Thus, immobilized enzyme systems used in continuous systems have advantages over batch-wise use of enzymes. In solventpartitioned enzyme systems, there are two phases, but unlike immobilized enzyme columns, both phases can be in motion simultaneously, by counterflow or in stirred reactors. For success, substrates, products, and enzyme(s) must be easily separated when
 appropriate. Two systems appear to meet these expectations: counterflow systems of two immiscible solutions, and systems of “dry” enzyme suspended in an inert organic solvent. 7.10.1 Activity and Stability of Enzymes in Organic Solvents Can hydrolases synthesize bonds? Do hydrolases have activity in water immiscible organic solvents? Are enzymes stable in water-immiscible organic solvents? Some years ago, it became clear to researchers that hydrolases can catalyze the biosynthesis of esters, amides, peptides, and carbohydrates under appropriate experimental conditions. Papain, for example, can form peptide Pag e 510 bonds during hydrolysis of proteins, provided the pH is about 6 and the peptide concentration is 30–35%. Later, it was shown that this biosynthesis is consistent with the intermediate acylenzyme mechanism where an acyl group is transferred to a nucleophilic acceptor molecule (usually water). However, if the concentration of another nucleophile, such as an amine group (amino acid), is high enough, the acyl group of the acylenzyme is partitioned between water and the amino group in accord with their nucleophilicity and relative concentrations. If some of the water is replaced by immiscible organic solvents, biosynthesis occurs even more rapidly (Eqs. 66 and 67), since the concentration of water is decreased, relative to that of H2NR’. (66) (67) It also helps to have the acid protonated. These competitive hydrolysis/biosynthesis reactions have been used successfully in the plastein reaction [6] to alter characteristics of soy protein. This is accomplished by adding methionine methyl ester to the reaction mixture and causing its incorporation into the soy protein. The result is a product that is firmer, chewier, and has improved nutritional quality. Similarly, superior surfactant products have been created by using a protease to form lauryl derivatives of gelatin. Again, replacement of some of the water with immiscible organic solvents can facilitate the biosynthetic reaction. The same considerations apply to reformation of triacylglycerols to yield better melting, greater solubility and unsaturation, and better nutritional properties than those of the original triacylglycerol. An example of reformation of a triacylglycerol by addition of a fatty acid (L) and a 1,3-specific lipase is shown in Eq. 64. If a non-specific lipase is used, the triacylglycerol can be reformulated to several products (Eq. 68), where E is a nonspecific lipase. (68) By using two or more fatty acids in controlled amounts, many types of intermediate products with interesting functionalities can be obtained. Therefore, hydrolases can perform biosynthetic reactions under appropriate conditions (Section 7.5.5) [4, 91, 118], with biosynthesis favored by a low water activity in conjunction with an organic solvent. Rates of lipase-catalyzed lipid transesterification reactions can be increased more than sixfold with a simultaneous 16-fold decrease in the rate of hydrolysis. If it is important not to use organic solvents in the biosynthesis, similar biosynthesis-favored reactions can be accomplished with alkylated lipases [91, 118]. Apparently, the increased hydrophobicity of the lipase surface is responsible for this shift in the ratio of biosynthesis to hydrolysis. Alkylated trypsin is a more effective catalyst for esterification of sucrose by oleic acid than is trypsin [4]. Are the enzymes stable enough to permit biosysthesis in organic solvents? This question was addressed in Section 7.5.6. Ribonuclease and lysozyme are more stable when suspended in immiscible organic solvents than when dissolved in water (Table 10). Therefore, performing Pag e 511 transesterification reactions with enzymes suspended in immiscible organic solvents is entirely feasible. An additional advantage is the avoidance of microbial problems. 7.10.2 Advantages of Solvent-Partitioned Enzyme Systems By now, several of the advantages of performing hydrolase-catalyzed reactions in solvent-parti-tioned systems rather than aqueous systems should be obvious: (a) equilibrium is shifted toward biosynthesis; (b) the enzyme is more stable; (c) enzyme is readily removed from the system; (d) product of high purity is more easily obtainable, particularly if it partitions into the organic phase; (e) product is not likely to be contaminated by the enzyme; and (f) microbial problems are minimized. In some situations there is another advantage to enzyme-catalyzed reactions in immiscible organic solvents; that is, the system can favor one stereoisomeric form over another. Table 27 illustrates the enantioselectivity of transesterification between vinyl butyrate and sec-phenethyl alcohol as catalyzed by porcine pancreatic lipase in different immiscible solvents. The enantioselectivity, uR /uS , (where uR and uS are chiralities) decreases from 75 in nitromethane to in decane. Therefore, the enantioselectivity is greater with greater polarity (dielectric constant) of the solvent. 7.10.3 Applications Currently, the most important application of solvent-partitioned enzyme systems is in the restructuring of triacylglycerols for food use. Restructuring of triacylglycerols is important not only for changing physical properties but also for improving nutritional attributes. This area is important enough that genetic engineering companies have invested millions of dollars into tailoring the genes in higher plants to make specialty triacylglycerols. Another application is in the
 manufacture of surfactants and detergents. Long-chain fatty acids can be covalently linked to amides, such as amino acids, or to glucose to give highly functional surfactants for use in soaps and shampoos, as well as in foods. This can result in improved foaming and emulsifying properties, and in easier incorporation of water, insoluble TABLE 27 Enantioselectivity of Porcine Pancreatic Lipase in Transesterification Between Vinyl Butyrate and sec-Phenethyl Alcohol a Solvent n Rb(mM//h) n Sb(mM/h) Enantioselect, (n R/n S Nitromethane 9.7 0.13 75 Dimethylformamide 2.3 0.038 61 Triethylamine 4.2 0.099 42 tert-Amyl alcohol 6.9 0.20 35 Butanone 8.6 0.32 27 Acetonitrile 14 0.64 22 Benzene 10 0.67 15 Cyclohexane 43 3.3 13 Decane 5.5 0.85 6 a 100 mM sec-phenethyl alcohol and 100 mg /ml lipase bChirality of product. Source: Reprinted with permission from Ref. 40, p. 3170, copyrig ht 1991 American ChemicalSociety. Pag e 512 flavors, colors and antioxidants in systems containing incompatible components. Similar advantages can accrue from using enzymes to make totally biodegradable soaps, shampoos, and detergents from naturally occurring compounds, such as glucose. The discovery that enzymes can efficiently catalyze reactions in immiscible solvents, including two-phase solvent systems, surely is one of the major advances in application of enzymology in the last twenty years. 7.11 Enzymes in Waste Management It is estimated that the annual production of major carbohydrate feedstock for use as potential fuels or for manufacturing chemicals in the United States is about 1160 million tons, versus 50 million tons of organic chemical feedstocks [42]. This number includes 160 million tons of municipal solid waste, 400 million tons of agricultural residue, 400 million tons of forest residue, and 200 million tons of corn and grains. These residues pose major environmental problems, since a large amount of the agricultural residues are burned (where permitted), the forest residues result in major fires each year, and the massive municipal landfills are often sources of groundwater pollution. Therefore, if some of these materials could be converted to other forms of fuel (ethanol and methanol) or to fermentation feedstock (glucose) to produce proteins, ethanol, and CO2, disposal problems would be alleviated and nonrenewable fuel supplies would be conserved. The U.S. government is spending considerable money on research on how to efficiently tap these potential sources of fuel and chemicals. What are the compounds that need to be converted? They are largely starch, cellulose, lignin, lipids, nucleic acids and proteins. What enzymes are needed? Primarily the amylases, cellulases, lignin peroxidases, lipases, nucleases, and proteases. These are enzymes we know quite a lot about. So, what is the problem? The major problems are the insolubility of the potential substrates just listed, and the poor stability and catalytic inefficiency of the enzymes required. Some methods exist for increasing the solubility of the substrates and thereby the rate of hydrolysis. But all methods require considerable energy expenditure that makes the process uneconomical at the moment, when petroleum and coal are relatively inexpensive. Do commercially feasible solutions exist for converting biomass to valuable chemicals? Nature does this efficiently, given the huge 1160 million tons to be converted annually. The natural conversion of these compounds occurs by action of the usual classes of enzymes, but the process is slow. Improvements in rate are possible through redesign of
 enzymes with greater efficiency and synergistic properties. Rapid, economical biomass conversion is a difficult task so the approach taken must be multifaceted. More efficient enzymes are needed, and this requires knowledge of why these enzymes are so inefficient in attacking insoluble substrates. Once this is better understood, better enzymes can be searched for or developed by recombinant DNA techniques. One needed property is greater stability, especially at temperatures sufficiently high microbial growth will not be a problem. The pentose-containing polymers are especially difficult to hydrolyze and even more difficult to ferment to ethanol and CO2. Lignin severely limits hydrolysis and fermentation of cellulose, because it is so inert. Enzymes that efficiently attack these substrates are needed. Solution of the biomass conversion problem is a must. Conversion processes must be environmentally acceptable, and all components, even minor ones, must be economically converted to usable or innocuous products to meet waste minimization goals pursued by the U.S. Environmental Protection Agency

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