Food Structure

Volume 11 Number 3 Article 2

1992

Importance of Enzymes to Value-Added Quality of Foods

J. R. Whitaker

Follow this and additional works at: https://digitalcommons.usu.edu/foodmicrostructure

Part of the Food Science Commons

Recommended Citation Whitaker, J. R. (1992) "Importance of Enzymes to Value-Added Quality of Foods," Food Structure: Vol. 11 : No. 3 , Article 2. Available at: https://digitalcommons.usu.edu/foodmicrostructure/vol11/iss3/2

This Article is brought to you for free and open access by the Western Dairy Center at DigitalCommons@USU. It has been accepted for inclusion in Food Structure by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. FOOD STRUCTURE, Vol. II (1992), pp. 201-208 I 046-705XI92$3.00+ .00 Scanning Microscopy International, Chicago (AMF O'Hare), IL 60666 USA

IMPORTANCE OF ENZYMES TO VALUE-ADDED QUALITY OF FOODS

J.R. Whitaker

Department of Food Science and Technology University of California, Davis, CA 95616-8598

Abstract Int roduction

New uses of enzymes are of major importance for Future major increases in jobs in the United value-added quality of foods. At the production and States will be primarily in the service sector, economists

postharvest handling leveiS1 enzymes associated with and planners agree. Much of the emphasis will be on maturation , color, texture, flavor and nutritional changes the high tech industries, i.e. computers, electronics, fine are important. Recent examples are discussed which chemicals, etc. But we must not overlook the important include longer storage prior to initiation of ripening , opportunities that food materials produced by the United

deletion or control of genes that result in softening 1 States will play in helping feed the world. Our past and, browning and other quality defects and the enhancement to some extent, our current role is based primarily on of genes that improve the nutritional quality of foods. the qual ity and quantity of raw food materials produced. At the processing level, new uses of enzymes are de­ However, several other countries now produce and ex­ scribed to change the properties of proteins and lipids, port raw food materials of equivalent quality to products to eliminate off-flavor in beers and sterilized milk, and from the United states, at highly competitive, or in some to monitor adequate blanching of fruits and vegetables. cases lower, prices. This competitive environment has led to emphasis from the United States on "value added products" for export. Value added products usually im­ ply post harvest modifications (formulations for exam­ ple) to increase the net value of the product. In pursu­ Key Words: Enzymes, value-added quality, food qual­ ing this approach, we must remember that it is difficult , ity , sensory properties, nutritional quality, gene ma­ if not impossible, to make a high quality food from a nipulation, protein modification, lipid modification, off­ low quality raw material. flavor elimination, beer, milk, vegetables, blanching. This article explores several applications of en­ zymology that significantly enhance the quality of foods or raw materials. The discussion emphasizes several practical applications to be considered in applying en­ zymes to: a) improve the quality of raw food materials; b) control and/or monitor food quality altributes; c) stabilize foods; d) enhance nutritional, safety, and functional qualities of processed foods ; and e) produce food ingredients via cell cultures. Improve the Quality of Raw Food Materials Initial paper received March 16, 1992 Enzymes are very important in the growth, matu­ Manuscript received June 25 , 1992 ration and post harvest stability of raw food materials. Direct inquiries to J.R. Whitaker They are also very important in the quality (color, fla­ Telephone number: (916) 752-1479 vor, aroma, texture and nutritional quality) of foods. Fax number: (916) 752-4759 For many years, plant and animal breeders have diligent­ ly developed crops and animals which provide greater yield. More recently, attention has been given to devel­ oping plants and animals with improved disease and drought resistance and animals with improved yields based on feed consumption and improved lean to fat ratios. Less attention has been given to improving the

201 J. R. Whitaker

Table t. Problems and solutions~

Problems Solutions I. Deficit of casein clotting enzymes I. Produce chymosin in microorganisms 2. Disposal of lactose containing whey 2. Produce lactase in yeast 3. Enzyme instability/stability 3. Change critical amino acids via recombinant DNA 4. Low yields of needed enzymes and proteins 4. Amplify number of copies of genes 5. Frost damage to plants 5. Delete ice nucleation protein from Pseudomonas syringae 6. Chilling damage to plants 6. Insert gene for antifreeze protein 7. Softening of fruits and vegetables 7. Regulate expression or level of pectic enzymes 8. Lipoxygenase caused off flavors 8. Limit expression of gene 9. Polyphenol oxidase caused browning 9. Limit expression of gene I 0 . Low nutritional value of proteins 10. Increase digesibility by enhancing instability; amplifying genes for proteins with higher level of essential amino acids II. Control ripening ll . Repress gene for ethylene producing enzyme 12. Insect resistance 12. Incorporate genes for insect inhibitors 13. Selective resistance to herbicides 13. Insert gene for enzyme to catalyze herbicide •Adapted in part from Wasserman er al., 1988 . nutritional quality, safety and sensory properties of raw fruits never seems to be the same as a fruit ripened on food materials. Post harvest storage under controlled the vine or tree. Very recently , Oeller er a/. (1991) atmospheric con ditions has significantly increased th e demonstrated that tomato fruit senescence can be revers­ storage life of products by maintaining the color, flavor, ibly inhibited by antisense RNA. They engineered into and texture of some foods. Particularly important has the tomato the expression of antisense RNA to the been controlled atmosphere storage of apples, and more biosynthesis of l-aminocyclopropane- 1-carboxylate recently fi sh and salads. Results with apples now allow synthase (ACC synthase), the rate-limiting enzyme step delivery of high quality apples year-round. in the production of ethylene required for ripening (Eqn. What opportunities exist for further improve­ I ; Yang and Hoffman , 1984). The inhibition of ethylene ments? Recent research has suggested several intriguing production allows storage of tomato in a suspended mat­ and potentially major opportunities which are worth con­ uration state, until exogenous ethylene or propylene is sidering (Table 1) . These opportunities include: a) con­ administered to initiate continuation of the ripening trol , reduction or elimination of enzymes that adversely steps. The authors discuss the prospect that the life~span affect food quality; b) elimination of toxic and/or anti­ of plant tissues can be extended, th ereby increasing and nutritional components; and c) quantitative increases in controlling the storage time. An example of a current enzymes that lead to more desirable qualities. Let us practice is the maintenance of bananas at the green ma­ now consider some specific examples of enzyme control ture stage in storage, and ripening by ethylene adminis­ or application that can significantly impact food quality. tration ; this enhanced storage time has been practiced Color for several years. Changes from green to red or yellow during the ripening of tomatoes, strawberries, red delicious apples, bananas, etc., are essential, important positive changes , while the browning of bruised or sliced potatoes, apples , peaches , and leafy salads, and the bleaching of the green color of green beans, English peas, and leafy vegetables OH OH 0 are usually considered undesirable changes. A PPO /yOH ,,o, No Color develops during ripening. It results from v+11202-y ~y ___,.____,..____,..Melanins(2) the maturation (senescence) process that leads to rapid CH 3 CH3 CH3 increase in cell size, flavor enhancement, texture de­ crease, and other changes, all catalyzed by various en­ Enzymatic browning of fruits and vegetables is zymes. Senescence also signals the near completion of usually considered an undesirable change. This brown­ the life cycle of the fruit at which point continued ing of fruits and vegetables is primarily due to the action changes lead to deterioration. of the enzyme polyphenol oxidase (PPO). PPO catalyzes Ideally, we want to stop or delay the process at the oxidation of phenols to Q-benzoquinones which then this point through processing. Currently, delivery of polymerize nonenzymatically to melanins (Eqn. 2) of quality products such as for climacteric fruits requires various colors. Up to 50% of some tropical fruit crops harvesting at the mature but green stage to allow for are lost commercially due to browning. pH adjustment, storage and transportation time. The quality of such or addition of sodium bisulfite, sulfur dioxide, ascorbic

202 fmportance of Enzymes to Value-Added Quality of Foods

acid or thiol compounds are effective methods to control cloud, due to suspended pectic substances, is highly de­ enzymatic browning in juices, purees and slices but can­ si rable in citrus juices. When PME removes the me­ not be used in intact fruits. Conventional plant breeding thoxy group by hydrolysis, forming pectic acid (Eqn. 4) , has reduced the amounts of PPO in some fruits, such as Ca2 + cross-links pectic acids, leading to precipitation. peaches. Further reduction should be possible by re­ Possible solutions, all at the juice level, include heat in ­ combinant DNA technology through repression of the activation of PME (leading to some loss of fresh flavorL expression of the PPO gene (antisense RNA) or by re­ removal of Ca2+ (not very practi cal) or addition of ex ­ moval of the genes that express PPO. The only known ogenous polygalacturonase which hydrolyzes the pectic physiological function of PPO is in wound healing of acid formed to smaller fragments that still give a cloud, plant ti ss ue. but do not precipitate with Ca2+ (Baker and Bruemmer, 1972). Two possible solutions at the production side H H H H H H H would be to reduce the PME level or enhance the poly­ I I I I I I 1 CH (CH ) C=CCH C=C(CH )XCOO- + 0 -+ CH3(C H ) 9HC=rC=C{CH 2JxCOO- (3) 3 2 3 2 2 2 2 3 galactu ronase level by recombinant DNA techniques. 00. H The author is not aware of any research along these lines. Another undesirable color change is the loss of Flavor green color in certain vegetables. Bleaching of the Citrus juices, especially grapefruit, can be quite green color, due to chlorophyll, of green beans, English bitter due to naringin and limonin. Naringinase has been peas and some leafy vegetables is due primarily to hy ­ used in batch and immobilized column formats , to hy­ droperoxy-free radicals produced by lipoxygenase acting drol;ze the bitter compounds to non-bitter compounds on polyunsaturated lipids and/or polyunsaturated fatty (Maier eta/., 1973). There has also been substantial re­ acids (Eqn. 3). The lipoxygenase level of soybean seeds search on inhibition of the biosynthesis of naringin and has been substantially reduced without adverse physio­ Jimonin, since the pathway of their biosynthesis via the logical effects (Kitamura, 1984; Wang eta/., 1990). mevalonic pathway is well known (Hasegama and There is no apparent reason why lipoxygenase levels of Herman, 1986; Ou et al., 1988) and interv enti on can other plants cann ot be reduced by recombinan t DNA take place at several po ints to effectively inhibit techniques, to minimize the effect of this enzyme on formation of naringin or limonin . quality of raw foods. Nutritional Quality Texture Relatively lirtle attention has been given to im ­ Ripening of fruits and some vegetables leads to proving the nutritional quality of raw food materials. softening. Some softening is essential, but too much Nutritional quality of products might be enhanced in one softening is undesirable. Softening is primarily there· of two ways, either by decreasing the levels of toxic or suit of pectic enzymes acting on the pectic substances in antinutritional factors naturally present or by increasing the middle lamella between cells. In some cases, cellu­ the nutritional quality of the product. Some antinutri­ lases and hemicellulases may also play a role. In toma­ tional compounds in foods include erucic acid , raffinose, toes, control of softening has been attempted through phytic acid, cyanogenic glycosides, thioglycosides, two approaches which have been used to try to decrease solanine, protease inhibitors, and a-amylase inhibitors. the level of one or more of the polygalacturonases. There is considerable opponunity to reduce the levels of These approaches included manipulation of the gene ex­ these antinutritional compounds in foods by traditional pressing one of the polygalacturonases (Bennett et a/., plant breeding or by recombinan t DNA techniques. 1989; DellaPenna eta/., 1990); the other method used They can also be reduced or removed via added enzymes antisense RNA to inhibit expression of polygalacturonase during processing (Whitaker, 1990). (Smith eta!. , 1988). As demonstrated by Oeller eta/. Many plants, especially th e cereals and legumes, (1991), expression of the polygalacturonase gene during produce high levels of protein in their seeds. However, ripening appears to be ethylene independent, raising the these proteins are frequently deficient in one or more provocative question of what is the control mechanism essential am ino acids such as methionine (legumes) and for polygalacturonase production in the senescencing lysine (cereals). Recently, de Lumen and his co-work­ tomato. ers, and others (Gepts and Bliss, 1984), have begun to address this. As shown by de Lumen and coworkers, the I o 0 " soybean contains a small amount of a protein high in ~ o, methionine (- 12% ) . The protein has been isolated, sequenced and there is research designed to insert the HO~ O~ (4) isolated gene for this protein into common beans ~o, (Phaseolus vulgaris) genome and amplify its expression H O~ 0 in the seed (A.A. George and 8 .0 . de Lumen, personal I communication, 1992). Two other methods are important in enhancing the Pectin methylesterase (PME), a pectic enzyme, is nutritional levels of methionine available from beans. responsible for cloud separation in citrus juice. The

203 J.R. Whitaker

Sgarbieri and his colleagues in Brazil have developed a 1980b; Motoki and Nio, 1983). The enzyme, transglu­ new cultivar of Carioca (Carioca 80) in which greater taminase, catalyzes the nucleophilic addition of an amine than 80% of the methionine is biologically available, group (on a protein , amino acid or other compound) to whereas less than 50% of the methionine is biologically the side chain of a glutaminyl group of a protein (Eqn. available in Carioca and other common beans (Tezoto 5). Therefore, one can: a) cross-link molecules of the and Sgarbieri, 1990). George era/. (1992) have sug­ same protein, doubling, tripling, etc., the molecular gested explanations for this difference in biological weight and changing the functional properties; b) cross­ availability of methionine in Carioca 80 seeds versus link molecules of two or more proteins, increasing the other common bean seeds, based primarily on differ­ molecular weight and substantially changing the func­ ences in digestibility of the proteins. In a second meth ­ tional properties; c) covalently attach amino acids (such od, substantial improvement in nutritional quality could as essential amino acids) ; or d) covalently attach homo­ be achieved by preventing the rapid loss of methionine, geneous or heterogeneous synthetic polymers of amino the limiting essential amino acid , during storage. The acids. The covalent attachment of essential amino acid­ loss is probably a result of lipid peroxidation, which containing polymers can alter sub stantially the nutrition­ might be eliminated by preventing lipid peroxide forma­ al quality of a protein , as well as its functional proper­ tion by enhanced antioxidant levels in the bean , changing ties. Based on research of Puigserver eta/. (1979), the lipid composition of the bean or removing lipoxygenase peptide bonds of the polymer are hydrolyzed by intes­ from the bean, all by recombinant DNA biotechnology. tinal proteases. Other Space does not permit discussion of other oppor­ tunities to improve the quality and quantity of raw food materials by controlling: a) chilling damage by incorpo­ ration of genes for antifreeze proteins (Feeney et al. , 1986); b) deleting ice nucleation protein from Pseudo ­ monas syrir~gae (Lindow and Connell, 1984; Orser et Change in Lipid Properties a/. , 1985); c) decrease insect damage by in corporation The fatty acid composition and location of a fatty of genes for inhibitors of insects; and d) selective acid on glycerol markedly affect the physical states of resistance to herbicides by amplification of key enzymes triglycerides. The higher the molecular weight and the (Stein rG cken and Amrhein, 1984; see Table 1). more saturated the fatty acid, the higher the melting point of a triglyceride. Tributyrin is a liquid at room lmprove the Quality or Processed Foods temperature (melting point, m.p. -75 °C) while tri­ myristin is a solid (m.p. 56-57 °C). Tristearin melts at Whitaker (1990) has recently described many new 55 °C, while triolein melts at -4 to -5 oc. Homogene­ and future uses of enzymes in food processing (Table 2) . ous triglycerides have sharp melting points, while heter­ Discussed are enzy mes used to: a) report on the quality ogeneous triglycerides or mixtures of several homogene­ of selected foods ; b) produce wanted compounds; c) re­ ous triglycerides melt at a lower temperature and over a move unwanted compounds; d) control mi croorganisms; broader range. Synthetic lipids can now be tailored to e) modify/change pectic substances; f) improve specifi­ have properties approximating those of high value natu­ city by purification; g) modify lipids; and h) modify se­ ral lipids such as found in cocoa butter. Required is lected food products. He made a special case for use of knowledge not only of the fatty acid composition but more highly purified enzymes, so that the specificity and also percentage of each fatty acid in the cr and {3 posi­ selectivity of enzymes would be paramount. A few se­ tions. Then, by the use of I ,3 - and 2- specific lipases lected examples are detailed here to illustrate the the fatty acids can be correctly attached to glycerol to diversity of use of enzymes in food processing. give a mixture of triglycerides with properties similar to Change in Protein Properties that found in nature, such as cocoa butter (Sawamura, 1988). The reactions involved with specific lipases are Proteins are responsible for many functional prop­ shown in Equations 6 and 7, in contrast to that with a erties of foods. Sometimes, the source of the protein, as non-specific lipase (Eqn. 8). well as the proteases, pentosanases and other enzymes, can make a difference in propenies, as in bread, crack­ ers and cakes. These properties can be further modified o{x+L ~ t{'•o (6) by limited (2-5%) specific hydrolysis of proteins, with ' ' resulting changes in solubility, emulsifying, foaming and whipping properties (Chobert eta/., 1987; F. Vojdani 20{x+JL~ o{'• o{L+JX and J.R. Whitaker, 1992, unpublished data). Major (7) ' ' l changes in functionality can also be achieved by cross­ linking of proteins, either molecules of the same protein or different proteins (Neidle et a/. , 1958; Cooke and ' l {' {' {' {' Holbrook, 1974 ; Soria eta/., 1975; Jkura eta/., 1980a, 5 o{X + 9 L ~ o{X + 0 l + L X+ L X + L L + 6 X + 3 0 (8)

204 Importance of Enzymes to Value-Added Quality of Foods

Table 2. Improve quality of processed foods. 0 COOH 0 H H H II I (A) II I (B) I I CH3- c-9-CH3 ~ CH3- C- 9 - CH3 ~ CH3-9 - 9-CH3 {10) Problems Solutions OH C02 ~ ~ ~ I. Resolve DL-amino I. Aminoacylases Acetolactate Acetoin 2,3-Butylene glycol acids 2. 5 '-Nucleotides for 2. 5' Phosphodiesterases; During fermentation, acetolactate is produced by flavor 5' Adenylic deaminase the yeast as an intermediate in the biosynthesis of 3. Cheese/butter flavors 3. Lipases (pregastic) isoleucine and valine. The acetolactate left at the end of 4. Decrease cheese 4. Proteases, Jipases the primary fermentation is oxidized nonenzymatically ripening times to diacetyl (Eqn. 9). The level of diacetyl at the end of Cyclod~xtrim; fur 5. 5. Cyclomaltodextrin primary fermentation is around 0. 10-0.15 mg/liter, while separations gl ucanotransferase the taste threshold is 0.05 rug/liter. Therefore, beer is 6. Modified food gums 6. a-Galactosidase 7. a. Remove raffinose 7. a. a-Galactosidases subjected to a secondary fermentation, lasting several b. Remove phytic acid b. phytase weeks, during which the acetolactate diffuses back into c. Remove oxidized c. sulfhydryl oxidase the yeast cells where it is reduced to acetoin. By adding flavor of milk acetolactate decarboxylase, the acetolactate is decar­ d. Remove carbamates d. urease boxylated to acetoin and C02 (Step A, Eqn. 10) and !he e. Remove bitter e. naringinase acetoin is then reduced by a dehydrogenase to 2,3- compounds in citrus butylene glycol (Step B, Eqn. 10), which contributes no f. Remove cyanogenic f. linamarinase flavor. glycosides The gene for acetolactate decarboxylase has been 8. Specialty triglycerides, 8. Specific lipases inserted into brewing yeast, so that the acetolactate is re­ others moved as rapidly as formed during the primary fermen­ 9. Soybean milk 9. Microbial proteases tation step (Sone eta/., 1987). coagulation 10. Increase dough 10. Pentosanases Elimination of Cooked Flavor in Milk moistness Ultra high temperature (UHT) treatment of milk, II. Light beer II. Amyloglucosidases sterilization, permits storage of milk for several weeks 12. Avoid diacetyl in beer 12. Acetolactate at room temperature. This process is widely used in decarboxylase some countries, such as Mexico and Brazil, because of Waxes and specific di - and monoglycerides can lack of refrigerators in many homes. The process is not also be produced by transesterification reactions (Heldt­ used in the United States because of the cooked off-fla­ Hansen e1 a/., 1989). vor. It has been shown that the off-flavor results from Brewing the thermal reduction of protein disulfides, forming thiol compounds (Swaisgood and Horton, 1989; Swaisgood et There are at least three major changes in uses of a/., 1982). Sulfhydryl oxidase-treated UHT milk does enzymes in brewing during the last five to six years. not develop the typical oxidized-type flavors during long These include the addition of a-amyloglucosidases in the storage, since the sulfhydryl groups are oxidized to di­ mashing phase to hydrolyze the a - 1 ,6 glucose linkages sulfides (Eqn. II; Swaisgood eta/., 1982; Swaisgood of amylopectin, thereby permitting complete conversion and Horton, 1989). Sulfhydryl oxidase has also been of starch to glucose and its fermentation to C02 and eth­ used to strengthen weak wheat doughs by catalyzing anol (light beer; Scott, 1989). ,6-Glucanases are also disulfide formation (Scott, 1989). added during the mashing phase to hydrolyze the glucans present from plant cell walls, thereby reducing the vis­ cosity of the mash, and facilitating the filtration step (1 1) (Enari et al., 1987). The most remarkable enzyme advance in brewing Blanching of Fruits and Vegetables has been the use of acetolactate decarboxylase (Olsen Fruits and vegetables hear treated to kill microor­ and Aunstrup, 1986; Rostgaard eta/. , 1987; Soneeta/. , ganisms and inactivate enzymes can be stored for several 1987). Addition of acetolactate decarboxylase is design­ months in the frozen state, as long as they are protected ed to prevent formation of diacetyl, in order to avoid an from recontamination with microorganisms as shown off-taste and to cut the fermentation time. The reaction first by Kohman (1928). In order to preserve as much involved in diacetyl formation is shown in Equation 9. of the fresh qualities (flavor, aroma, color, texture and nutrition) as possible, at the same time killing microor­ 0 COOH 0 0 ganisms and inactivating enzymes, the minimum heat II I II II CH3- C- 9 - CH 3 + 0.5 0 2 __.. CH 3-C - C - CH3 + C~ + H20 (9) possible should be applied. Almost from the beginning OH of the frozen food industry in the 1930' s, there was dis­ Acetolactate Diacetyl agreement as to the indicator enzyme to use to determine adequate heat treatment. Catalase was used in some

205 J. R. Whitaker cases, peroxidase in others. Eventually , peroxidase was Food Ingredients By Cell Culture almost universally the enzyme of choice, because its in­ One can clearly envision the time, not too distant, activation led to the best keeping quality of the frozen when many food ingredients will be produced in facto­ foods. This is not surprising since peroxidase is usually ries using plant and animal cell culture, just as many of the most heat-stable enzyme found in vegetables and our ingredients for food and medicine are made by bac­ fruits, so by the time it is inactivated no other enzymes terial fermentations. Plant and animal cells are more or microorganisms remain. But there is no evidence that fragile than microbial cells, so that must be researched peroxidase is involved in deteriorative reactions in the and solved. Removal of heat and transport of nutrients food. in and products out through the cell wall and how to turn Williams eta/. (1986) discussed the question of on the desired genes and turn off others, including those which indicator enzyme to use. It would appear obvious for proteases, neetl to be sol vet!. Minimization or elimi­ the best indicator would be the enzyme causing the prob­ nation of toxic substances is needed. But colors and fla­ lem. Enzymatic browning is caused by polyphenol oxi­ vor compounds, vitamins and enzymes have been pro­ dase; bleaching of carotenoids and chlorophyll is gener­ duced in the laboratory. Pilot scale-up and commerciali­ ally caused by lipoxygenase. Softening is caused by the zation is not too far away, especially where the pectic enzymes and flavor loss or off-flavor is caused by economics are favorable. a number of enzymes. Williams eta/. (1986) developed a protocol, combining protein chemistry, enzymology Summary and sensory testing, to determine the key enzyme(s) in­ Several enzymatic methods have been described volved in quality deterioration. Their research has led for value added enhancement of raw and processed to the conclusion that lipoxygenase is responsible for foods. These methods include strategies for color, off-flavor development in green beans, English peas and flavor, texture and nutritional quality improvement of corn (Lim eta/., 1989; Velasco eta/., 1989) while cys~ the products. In some cases, the strategy calls for tine lyase is the key enzyme in development of off-flavor elimination of one or more deteriorative enzymes by use in broccoli and caulinower (Velasco eta/., 1989). Off~ of selective heat treatment or recombinant DNA technol­ flavor development by lipoxygenase is due to polyunsat­ ogy. Tn other cases, the genes for an undesirable en­ urated fatty acid peroxidation, regardless of whether the zyme may be removed by recombinant DN~ technolo~y fatty acids are free or in triglyceride form. Cystine or a desired enzyme may be inserted or mcreased 1n lyase converts cystine to pyruvate, hydrogen sulfide and level, by recombinant DNA technology. Exogenous ammonia. The off-flavor and off-odor results from the enzymes can be added during processing to change the last two compounds. Pilot plant studies, including stor­ properties of proteins, lipids and carbohydrates in foods, age, show that a superior product results when ~hese thereby improving the water-holding properties, the enzymes are used as indicators of adequate blanching. foaming properties, the emulsifying properties, and/or Tailor Enzyme Preparations for Value-Added Foods the solubilities of key food ingredients. Crude en zyme preparations used in food proces­ To date, most enzyme preparations used in food sing will soon be replaced by more highly purified processing are crude extracts. For example, pectic en­ enzyme preparations, leading to more specificity and zyme preparations produced by Aspergillus niger have control of the desired changes. Extraction of food been used for a long time to increase juice yield and to ingredients, especially flavors and colors will shortly be clarify juices. But, as shown in Table 3, there are replaced with use of plant, animal and microbial cell numerous other hydrolytic enzymes present that also cultures that synthesize predominately one ingredient; break down polymers in plants. The overall results - this will greatly improve the efficiency of production, juice yield, clarity, color, flavor, taste- occur from the and the economy and quality of product by minimizing composite effect of these enzymes. Would better food extraction and purification steps required. products be achieved if only the pectic enzymes were used? Perhaps only one of the three groups of pectic en­ References zymes should be used? Could industry afford to use Baker RA, Bruemmer JH (1972). Pectinase stabi~ more highly purified enzymes? No one probably knows lization of orange juice cloud. J. Agric. Food Chern . 20, the answers to these questions at the moment. But if we 1169~ 1173. are to be competitive with the European Common Mar­ Bennett AB, DellaPenna D, Fischer RL, ket, Japan and other developed, and developing coun­ Giovannoni J, Lincoln JE (1989). Tomato fruit poly­ tries, we need answers to these questions. New, large galacturonase: gene regulation and enzyme function. In: scale purification techniques, production of desired Biotechnology and Food Quality, Kung S, Bills DB, enzymes by recombinant DNA techniques and increasing Quatraro R (eds.). Butterworths, Boston, MA. 167 ~ 180. knowledge of enzymology permit us to make better food Chobert JM, Sitohy MZ, Whitaker JR (1987). products, if we are willing to ask and answer the right Specific limited hydrolysis and phosphorylation of food questions of what needs to be done and how we can do proteins for improvement of functional and nutritional it. properties J. Amer. Oil Chern. Soc. 64, 1704~1711. Cooke RD , Holbrook JJ (1974). Calcium and the

206 Importance of Enzymes to Value-Added Quality of Foods

assays of human plasma clotting factor XIIJ. Biochem . Table 3. Enzy mes produced by Aspergillus nige r J. 141 , 71-78. which hydrolyze polymers~ DellaPenna D, Lashbrook CC, Toenjes K Giovannoni JJ : Fischer RL, Bennett AB (1990). Poly: Enzyme Substrate galactu ronase 1sozymes and pec tin depolymerization in a-L-A rabi nofu ranosidase Arabinans ~r;~~~enic rin tomato fruit. Plant Physiol. 94 , 1882- Cellulases, cell obiohydrolases, ll- Ce\lulose Enari TM, Knowles A, Lehtinen U Nikkola M gl ucosidases Penttila M: Suihko ML , Home S, Vilpo,la A ( 1987)'. Glucano lytJc brewer's yeast. Proc. 2 1st Congress Eur. Dextranase Dextran Brewt:ry Convention , Madrid , Spain; as cited in Deoxyribonuclease, ribonuclease DNA, RNA Brewers' Guardian 116, \0- 11 , Ap ri l 1987. Feeney RE, Burcham TS, Yeh Y (1986). Anti ­ /l-G \u canase /l-Glu cans freeze g lycoproteins fro m polar fis h blood. Ann . Rev. Biophys. Chern. 15, 59-78. Hemicellulases Hemicellulose George AA , de Lumen BO , Whitaker J R Inulinase Inulin Sgarbieri VC (1992). Methionine-containing proteins i~ two Phaseolus vulgaris cultivars with different (3- Mannanase Mannans methionine bioavailabilities. Plant Foods Human Nutr. Pectin methylesterase, pectate Pectic In press. lyase, polygalacturonase substances Gepts P, Bliss FA (1984). Enhanced available methionine concentration associated with higher phase­ Pro teases Proteins olin levels in common bean seeds. Theor. Appl. Genet. a-Amylase, glucoamylase Starch 69 , 47-53. . Hasegama S, Herman Z (\986). Biosynthesis of Xy lanase Xylans hmon01ds: conversion of deacetylnomilinate to nomilin in Cilrus limon. Phytochemistry 25, 2523-2524. *Enzymes referenced in Chemical Abstracts 1977-1982 alone. Heldt-Hansen HP, Ishii M, Patkar SA, Hansen 48, 561 -566. TT, Eigtved P (1989). A new immobilized positional Neidle A, Mycek MJ, Clarke DD, Waelsch H non ~ p ecific .lipase fo r fat modification and ester syn­ (1958). Enzymic exchange of protein amide groups. the ~ I S. In: B10catalysis in Agricultural Biotechnology, Arch . Biochem. Biophys. 77, 227-229. Whttaker JR, Sonnet P (eds.). ACS Symp. Ser. 389, Oe\ler PW, Min-Wong L, Taylor LP, Pike DA , American Chemical Society, Washington, DC , 158-172. Theologis A (199 1). Reve rsible inhibition of tomato fruit Ikura K, Kometani T , Yoshikawa M, Sasaki R, senescence by antisense RNA. Science 254, 437-439. Chiba H ( \ 980a). Cross lin king of casein components by Olsen F, Aunstrup K (\986). Alpha-acetolactate transglutaminase. Agric. Bioi. Chern. 44, \567- 1573. decarboxylase enzyme and preparation thereof. U.S. lkura K, Kometani T , Sasaki R, Chiba H ( 1980b). Patent 4,617,273. Cross linking of soybean ?Sand liS proteins by trans­ Orser C, Staskawicz BJ , Panopoulos NJ , glutaminase. Agric. Bioi. Chern. 44, 2979-2984. Dahlbeck D, Li ndow SE (1985). Cloning and expression Kitamura K (1984). Biochemical characterization of bacterial ice nucleation genes in Escherichia coli. 1. of lipoxygenase lacking mutants, L-1-less, L-2-less and Bacterio\. 164, 359-366. L-3-less soybeans. Agric. Bioi. Chern, 48, 2339-2346. Ou P, Hasegawa S, Herman Z, Fong CH (1988). Kohman EF (1928). As referred to in Kohman EF Limonoid biosynthesis in th e stem of Citrus limon . (\936) Enzymes and the storage of perishables. Food Phytochemistry 27, 115 -\18. Ind. 8 , 287-288. Puigserver AJ , Sen LC , Clifford AJ, Feeney RE, Lim MH, Velasco PJ, Pangborn RM , Whitaker JR Whitaker JR ( 1979). Covalent attachment of amino acids (1989). Enzymes involved in off-aroma formation in to casein. 2. Bioavailability of methionine and N-acetyl broccoli. In: Quality Factors of Fruit and Vegetables, methionine covalentl y linked to casein . J. Agric. Food len JJ (ed.). ACS Symp. Ser. 405, American Chemical Chern. 27, 1286-1 293. Society, Washington, DC , 72-83. Rostgaard Jensen B, Svendsen I, Ottesen M Lindow SE, Conne\1 JH (1984). Reduction of (1987). Isolation and characterization of an fl-acetolac­ frost injury to almond by control of ice nucleation. 1. tate decarboxylase useful for accelerated beer matura­ Amer. Soc. Hort. Sci. 109, 48-53 . tion. Proc. 21st Congress Eur. Brewery Convention, Maier VP, Brewster LC, Hsu AC ( 1973). Ethyl­ Madrid, Spain; as cited in Brewers' Guardian 116, lO­ ene-accelerated limonoid metabolism in citru s fru its. ll , April 1987. Process for reduci ng juice bitterness. J. Agric. Food Sawamura N (1988). Transesterification of fa ts Chern. 21 , 490-495. and oils. In : Enzyme Engineering, Blanc HW, Klibanov Motoki M, Nio N (1983). Cross linking between MA (eds.). Ann. N.Y. Acad. Sci. 542, The New York different food protein s by transglutaminase. J. Food Sci. Academy of Sciences, New York, NY, 266-269.

207 J. R. Whitaker

Scott D (1989). Specialty enzymes and products Discussion with Reviewers for the food industry. In : Biocatalysis in Agricultural Biotechnology, Whitaker JR, Sonnet P (eds.). ACS R . F. McFeeters: Has it been demonstrated that reduc­ Symp. Ser. 389, American Chemical Society, tion of polygalacturonase activity by manipulation of Washington , DC, 176- 192. gene expression in the tomato has actually affected Smith CJS, Watson CF, Ray J, Bird CR, Morris softening of the tomato tissue during ripening? PC, Schuch W, Grierson D (1988). Antisense RNA Author: This is an important question since different inhibition of polygalacturonase gene expression in results are reported in the literature. The Flavr Savr transgenic tomatoes. Nature 334, 724-726. tomato, developed by Calgene using an antisense RNA Sane H, Kondo K, Fujii T, Shimuzu F, Tanaka J, method against a polygalacturonase, does have a sub­ Inoue T (1987). Fermentation properties of brewer's stantially reduced rate of p~ctin breakdown, effectivdy yeast having ,8-acetolactate decarboxylase gene. Proc., extending the storage time. Other workers (Bennett et 21st Congress Eur. Brewery Convent. , Madrid, Spain; al., 1989; DellaPenna eta/., 1990) do not find similar as cited in Brewers' Guardian 116, 10-11, April 1987. results in transgenic rin tomato fruits. The rin tomatoes Soria A , Soria C, Boulard C (1975). Fibrin are mutants with other properties also different from stabilizing factor (F XIII) and collagen polymerization. wild type tomatoes. Experientia 31, 1355-1357. Steinrucken HN, Amrhein N (1984). 5-Enol­ J .E. Spradlin: Do you think it feasible to expand the pyruvylshikimate-3-phosphate synthase of Klebsiella delayed ripening of tomatoes via antisense RNA concept pneumoniae. I. Inhibition by glyphosate [N-(phospho­ to a wide variety of other fruits and vegetables such that nomethyl)glycine]. Eur. J. Biochem. 143, 351-357. in the future produce can be ripened on demand either Swaisgood HE, Horton HR (1989). Immobilized by the grocer or in the home by the consumer? enzymes as processing aid or analytical tools. In : Author: The antisense method of inhibiting expression Bic.catalysis in Agricultural Biotechnology, Whitaker of a gene is a general approach. The method requires JR, Sonnet P (eds.). ACS Symp. Ser. 389, American isolating the gene for a particular enzyme or protein, se­ Chemical Society, Washington, DC, 242-261. quencing a portion of the gene and synthesizing a com­ Swaisgood HE, Sliwkowski MX, Skudder PJ, plimentary antisense (reverse) RNA segment. All of Janolino VG ( 1982). Sulfhydryl oxidase: characterization these requirements are met usi ng present technologies. and application for navor modification of UHT milk. In: Therefore, there is every reason to expect this method Use of Enzymes in Food Technology, Dupuy P (ed.). can be extended to other fruits and vegetables, and to Technique et Documentation, Lavoisier, Paris, 229-235. animals. Tezoto SS, Sgarbieri VC (1990). Protein nutritive value of a new cultivar of bean (Phaseolus vulgaris, L.). J .E. Spradlin: You referred to antisense RNA and re­ J. Agric. Food Chern. 38, 1152-1156. combinant DNA technologies several times. Do you Velasco PJ, Lim MH, Pangborn RM, Whitaker JR think that commercial quantities of produce will be pro­ (1989). Enzymes responsible for off-flavor and off­ duced from plants resulting from these technologies aroma in blanched and frozen-stored vegetables. Bio­ within the next five to ten years? technol. Appl. Biochem. II, 118-127. Author: Commercial quantities of produce from plants Wang J , Fujimoto K, Miyazawa T , Endo Y, produced by these technologies definitely will be availa­ Kitamura K (1990). Sensitivities oflipoxygenase-lacking ble within five to ten years, and likely as early as 1993. soybean seeds to accelerated aging and their chemilumi­ This results from the decision of the U.S. Department of nescence levels. Phytochemistry 29, 3739-3742. Agriculture in May 1992 to not subject transgenic plants Wasserman BP, Montville TJ, Korwek EL (1988). to more stringent review than plants produced by stand­ Food biotechnology: scientific status report. Food ard breeding practices. Calgene has received approval Techno!. 42, 133- 146. of the U.S. Department of Agriculture to proceed with Whitaker, JR (1990). New and future uses of en­ commercialization of its Flavr Savr tomato, which it ex­ zymes in food processing. Food Biotechnol. 4 , 669-697. pects to do in 1993. Some 70 other transgenic plants are Williams DC, Lim MH , Chen AO , Pangborn RM, in various stages of development. Whitaker JR (1986). Blanching of vegetables for freez­ ing - which indicator enzyme to choose. Food Techno!. 40, 130-140. Yang SF, Hoffman NE (1984). Ethylene biosyn­ thesis and its regulation in higher plants. Annu. Rev. Plant Physiol. 35, 155-189.

208