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they are also more specific and can be used in low concentrations under mild reaction conditions The products formulated with enzymes tend to be more uniform, and their action in Food Biochemistry and Food Processing, Second Edition Edited by Benjamin K Simpson, Leo M.L Nollet, Fidel Toldr´a, Soottawat Benjakul, Gopinadhan Paliyath and Y.H Hui C 2012 John Wiley & Sons, Inc Published 2012 by John Wiley & Sons, Inc 181 P1: SFK/UKS BLBS102-c09 P2: SFK BLBS102-Simpson March 21, 2012 11:15 182 Trim: 276mm X 219mm Printer Name: Yet to Come Part 2: Biotechnology and Enzymology foods can be stopped relatively easily after the desired transformation is attained The chapter is organized in six sections and covers the major groups and sources of food enzymes, their properties and modes of action, the rationale for their use in food, their use in the manufacture of various foods, and the different strategies employed to control their undesirable effects in foods The chapter also provides information on the future prospects of enzymes INTRODUCTION Enzymes are biological molecules that enable biological reactions proceed at perceptible rates in living organisms (plants, animals, and microorganisms) and the products derived from these sources Living organisms produce basically the same functional classes of enzymes to enable them carry out similar metabolic processes in their cells and tissues Thus, it is to be expected that foodstuffs would also possess endogenous enzymes to catalyze biological reactions that occur in them both pre- and postharvest The molecules that enzymes act upon are called substrates, while the resulting compounds from the enzymatic conversions are called products Enzymes are able to speed up biological reactions by lowering the free energy of activation ( G*) of the reaction (van Oort 2010) Unlike chemical catalysts, enzymes are much more specific and active under mild reaction conditions of pH, temperature, and ionic strength Humans have intentionally or unintentionally used enzymes to modify foodstuffs to alter their functional properties, extend their storage life, improve flavors, and provide variety and delight, and the use of enzymes in food processing has been increasing for reasons such as consumer preferences for their use in food modification instead of chemical treatments, their capacity to retain high nutritive value of foods, and the advances in biotechnology that are enabling the discoveries of new enzymes that are much more efficient in transforming foods The recent advances in molecular biotechnology that are permitting the discoveries of new and better food enzymes augur well both for the need and capacity for food technologists and food manufacturers to produce food products in highly nutritious, safe, and stable forms as part of the overall strategy to achieve food security However, not all reactions catalyzed by enzymes in foods are useful; naturally present enzymes in fresh foods and their counterparts that survive food-processing operations can induce undesirable changes in foods, some of which may even be toxic Thus, there is the need to also develop novel and more effective strategies to control the deleterious effects of enzymes in foods to reduce postharvest food losses and/or spoilage Some Terms and Definitions Several useful terms are encountered in the study of enzymes The active enzyme molecule may comprise a protein part exclusively, or the enzyme protein may require as essential nonprotein part for its functional activity The essential nonprotein part that some enzymes require for functional activity is known as a cofactor or prosthetic group For those enzymes requiring cofactors for activity, the enzyme–cofactor complex is known as the holoenzyme, and the protein part that has no functional activity without the cofactor is known as apoenzyme Thus, cofactors or prosthetic groups may be regarded as “helper molecules” for the apoenzymes Cofactors may be inorganic (e.g., metal ions like Zn, Mg, Mn, and Cu), or organic (e.g., riboflavin, thiamine, or folic acid) materials Examples of food enzymes that not require cofactors or prosthetic groups for activity include lysozyme, pepsin, trypsin, and chymotrypsin; examples of those enzymes that require cofactors or prosthetic groups for functional activity include carboxypeptidase, carbonic anhydrase and alcohol dehydrogenase (all have Zn2+ as cofactor), cytochrome oxidase (has Cu2+ as cofactor), glutathione peroxidase (which has Se cofactor), catalase (with either Fe2+ or Fe3+ as cofactor), and pyruvate carboxylase (that has thiamine in the form of thiamin pyrophosphate or TPP as cofactor) Enzymes have a catalytic region known as the active site where substrate molecules (S) bind prior to their transformation into products (P) The active site is a very small region of the very large enzyme molecule, and the transformation of substrates to products may be measured by either the rate of disappearance of the substrate (−δS/δt) or the rate of appearance of the products (δP/δt) in the reaction mixture The activity of the enzyme is usually expressed in units, where a unit of activity may be defined as the amount of enzyme required to convert µmol of the substrate per unit time under specified conditions (e.g., temperature, pH, ionic strength, and/or substrate concentration) Enzyme activity may also be expressed in terms of specific activity, which may be defined as the number of enzyme units per unit amount (e.g., milligram) of enzyme; or it may be denoted by the molecular activity or turnover number, defined as the number of enzyme units per mol of the enzyme at optimal substrate concentration The efficiency of an enzyme (catalytic efficiency) may be derived from knowledge of the binding capacity of the enzyme for the substrate (Km , which is the apparent Michaelis–Menten constant) and the subsequent transformation of the substrate into products (Kcat or Vmax ) The catalytic efficiency of an enzyme is defined as the ratio of the maximum velocity (Vmax ) of the transformation of the substrate(s) into product(s) to the binding capacity of the enzyme (or the apparent Michaelis–Menten constant, Km , i.e., Vmax /Km or Kcat /Km ) The catalytic efficiency is a useful parameter in selecting more efficient enzymes from a group of homologous enzymes for carrying out particular operations Rationale for Interest in Food Enzymes Beneficial Effects Enzymes have several advantages for food use compared to conventional chemical catalysts They are relatively more selective and specific in their choice and action on substrates, thus obviating side reactions that could lead to the formation of undesirable coproducts in the finished products They have higher efficiency and can conduct reactions several times faster than other catalysts They are active in low concentrations and perform well under relatively mild reaction conditions (e.g., temperature and pH); thus, their use in food processing helps to preserve the integrity of heat-labile essential nutrients They can also be P1: SFK/UKS BLBS102-c09 P2: SFK BLBS102-Simpson March 21, 2012 11:15 Trim: 276mm X 219mm Printer Name: Yet to Come Enzymes in Food Processing immobilized onto stationary support materials to permit their reuse and thereby reduce processing costs Most of them are quite heat labile; thus, they can be readily inactivated by mild heat treatments after they have been used to achieve the desired transformation in foods, and they are natural and relatively innocuous components of agricultural materials that are considered “safe” for food and other nonfood uses (e.g., drugs and cosmetics) Their action on food components other than their substrates are negligible, and more gentle, thus resulting in the formation of purer products with more consistent properties; and they are also more environmentally friendly and produce less residuals (or processing waste that must be disposed of at high costs) compared to traditional chemical catalysts (van Oort 2010) Because of these beneficial effects, enzymes are used in the food industry for a plethora of applications including: baking and milling, production of (both alcoholic and nonalcoholic beverages), cheese and other dairy products manufacture, as well as the manufacture of eggs and egg products, fish and fish products, meats and meat products, cereal and cereal products, and in confectionaries Undesirable Effects Certain food enzymes cause undesirable autolytic changes in food products, such as excessive proteolysis to produce bitterness in cheeses and protein hydrolysates, or excessive texture softening in meats and fish products (e.g., canned tuna) Enzymes like proteases, lipases, and carbohydrases break down biological molecules (proteins, fats, and carbohydrates, respectively) to adversely impact flavor, texture, and keeping qualities of the products Decarboxylases and deaminases degrade biomolecules (e.g., free amino acids, peptides, and proteins) to form undesirable and/or toxic components, e.g., biogenic amines, in foods Some others, for example, polyphenol oxidases (PPO) and lipoxygenases (LOX), promote oxidations and undesirable discolorations and/or color loss in fresh vegetables, fruits, crustacea, and salmonids, and others like thiaminase and ascorbic acid oxidase cause destruction of essential components (vitamins) in foods Thus, more effective treatments must be developed and implemented to safeguard against such undesirable effects of enzymes in foods SOURCES OF FOOD ENZYMES (PLANT, ANIMAL, MICROBIAL, AND RECOMBINANT) Enzymes have been used inadvertently or deliberately in food processing since ancient times to make a variety of food products, such as breads, fermented alcoholic beverages, fish sauces, and cheeses, and for the production of several food ingredients Enzymes have been traditionally produced by extraction and fermentation processes from plant and animal sources, as well as from a few cultivatable microorganisms Industrial enzymes have traditionally been derived from plant, animal, and microbial sources (Table 9.1) Examples of plant enzymes include α-amylase, β-amylase, bromelain, β-glucanase, 183 ficin, papain, chymopapain, and LOX; examples of animal enzymes are trypsins, pepsins, chymotrypsins, catalase, pancreatic amylase, pancreatic lipase, and rennet; and examples of microbial enzymes are α-amylase, β-amylase, glucose isomerase, pullulanase, cellulase, catalase, lactase, pectinases, pectin lyase, invertase, raffinose, microbial lipases, and proteases Microorganisms constitute the foremost enzyme source because they are easier and faster to grow and take lesser space to cultivate, and their use as enzyme source is not affected by seasonal changes and inclement climatic conditions and are thus more consistent Their use as sources of enzymes is also not affected by various political and agricultural policies or decisions that regulate the slaughter of animals or felling of trees or plants Industrial microbial enzymes are obtained from bacteria (e.g., α- and β-amylases, glucose isomerase, pullulanase, and asparaginase), yeasts (e.g., invertase, lactase, and raffinose), and fungi (e.g., glucose oxidase (GOX), catalase, cellulase, dextranase, glucoamylase, pectinases, pectin lyase, and fungal rennet) Even though all classes of enzymes are expected to occur in all or most microorganisms, in practice, the great majority of industrial microbial enzymes are derived from only a very few GRAS (generally recognized as safe) species, the predominant ones being types like Aspergillus species, Bacillus species, and Kluyveromyces species This is because microorganisms can coproduce harmful toxins, and therefore need to be stringently evaluated for safety at high cost before they can be put to use for food production As such, there are only very few microorganisms currently used as safe sources of enzymes and most of these strains have either been used by the food industry for several years or been derived from such strains by genetic mutation and selection The traditionally produced enzymes are invariably not well suited for efficient use in food-processing applications for reasons such as sensitivity to processing temperature and pH, inhibitory reaction components naturally present in foods, as well as availability, consistency, and cost Recent developments in industrial biotechnology (including recombinant DNA and fermentation technologies) have permitted molecular biologists and food manufacturers to design and manufacture novel enzymes tailor-made to suit particular food-processing applications (Olempska-Beer et al 2006) These new microbial enzymes are adapted to have specific characteristics that make them better suited to function under extreme environmental conditions, e.g., pH and temperature The production of recombinant enzymes by recombinant DNA technology entails the introduction of the genes that encode for those enzymes from traditional sources into special vectors to produce large amounts of the particular enzyme Recombinant enzymes are useful because they can be produced to a high degree of purity, and they can be produced in high yields and made available on a continuing and consistent basis at much reduced cost As well, their purity and large-scale production reduce extensive quality assurance practices; and the technique itself enables useful (food) enzymes from unsafe organisms to be produced in useful forms as recombinant enzymes in safer microorganisms Examples of recombinant enzymes available for food use include various amylases, lipases, chymosins, and GOX P1: SFK/UKS BLBS102-c09 P2: SFK BLBS102-Simpson March 21, 2012 11:15 184 Trim: 276mm X 219mm Printer Name: Yet to Come Part 2: Biotechnology and Enzymology Table 9.1 Sources of Industrial Food Enzymes—Selected Traditional and Recombinant Forms Source Enzyme Some Food Applications Plant sources Papaya tree Pineapple stem Papain Bromelain Meats, baked goods, brewing Meats, baked goods Animal sources Stomachs of ruminants Mammals (guts) Chymosin (rennet) Lipase Dairy (cheese) Dairy products, oleo products, baked goods Alcalase α-Amylase β-Amylase Pectinase (alkaline) α-d-Galactosidase Protease Pectinase (acidic) Lipase Glucose oxidase Fructosidase Waste utilization, bioactive compounds Baked goods, beverages, and high-glucose syrups Baked goods, beverages, and high-glucose syrups Fruit juices, coffee, and tea Soybean pretreatment Soy sauce Fruit juices Dairy products, oleo products, baked goods Baked goods, beverages, dried food mixes Fructose production Lipase Dairy products, oleo products, baked goods Fructosyl transferase Fructo-oligosaccharides production Transglutaminase (TGase) Laccase Meats, seafood, dairy products Beverages (wine, fruit juice, beer), jams, baked goods Aspartic proteinase Chymosin Glucose oxidase Laccase Lipase Pectin esterase Phytase Phospholipase A1 α-Amylase Pullulanase α-Acetolactate dehydrogenase Chymosin Xylanase Chymosin α-Amylase Pectin lyase Meats (potential as tenderizer) Dairy (cheese) Baked goods, beverages, dried food mixes Beverages (wine, fruit juice, beer), jams, baked goods Dairy products, oleo products, baked goods Beverages (wine, fruit juice, beer) Baked goods, animal feeds Refining of vegetable oils Baked goods, beverages, and high-glucose syrups Baked goods, beverages, and high-glucose syrups Dairy, flavor generation Dairy (cheese) Cereals, baked goods, animal feed Dairy (cheese) Baked goods, beverages, and high-glucose syrups Beverages (wine, fruit juice, beer) Microbial sources Bacillus spp Aspergillus niger Aspergillus niger, Candida guilliermondii, Kluyveromyces marxianus Candida cylindracea, Penicillium spp., Rhizopus spp., Mucor spp Aspergillus spp., Penicillium spp., Aureobasidium spp Streptoverticillium mobaraense Pleurotus ostreatus Recombinant enzymesa Aspergillus oryzae Aspergillus oryzae, Pichia pastoris Bacillus lichenformis Bacillus subtilis Escherichia coli K-12 Fusarium venenatum Kluyveromyces maxianus var lactis Pseudomonas fluorenscens Biovar Trichoderma reesei a Olempska-Beer et al (2006) Table 9.1 also lists other recombinant food enzymes, their source microorganisms, and their food applications, as well as those of their traditional counterparts MAJOR FOOD ENZYMES BY GROUPS The major enzymes used in the food industry include various members from the oxidoreductase, transferase, hydrolase, and isomerase families of enzymes The oxidoreductases catalyze oxidation–reduction reactions in their substrates Examples of the oxidoreductases are catalases, GOXs, LOX, PPO, and peroxidases The transferases catalyze the transfer of groups between molecules to results in new molecules or form inter-/ intramolecular cross-linkages in their substrates An example of transferase used in the food industry is transglutaminase (TGase) The hydrolases catalyze the hydrolytic splitting ... discoveries of new and better food enzymes augur well both for the need and capacity for food technologists and food manufacturers to produce food products in highly nutritious, safe, and stable forms... sections and covers the major groups and sources of food enzymes, their properties and modes of action, the rationale for their use in food, their use in the manufacture of various foods, and the... transformation in foods, and they are natural and relatively innocuous components of agricultural materials that are considered “safe” for food and other nonfood uses (e.g., drugs and cosmetics)