P1: SFK/UKS BLBS102-c09 P2: SFK BLBS102-Simpson March 21, 2012 11:15 200 Trim: 276mm X 219mm Printer Name: Yet to Come Part 2: Biotechnology and Enzymology sterilization, blanching, UHT, high temperature short term treatment, pasteurization, and related thermal treatments to curtail undesirable enzymatic reactions in food and protect foods from enzyme-induced postharvest and post-processing spoilage that could arise from enzymes naturally occurring in food materials, or from enzymes deliberately added to foods to bring about particular transformations Nevertheless, thermal processing of foods has the disadvantage of destroying heat-labile essential components in foods such as some vitamins and essential oils, thus the need for effective nonthermal techniques Table 9.3 pH Stabilities of Selected Enzymes Enzyme Pepsin Malt amylase Invertase Gastric lipase Pancreatic lipase Maltase Catalase Trypsin pH Stability Optimum 1.5 4.5–5.0 4.5 4.0–5.0 8.0 6.5 7.0 8.0 Low Temperature Treatments A decrease in temperature slows down the average kinetic energy of biomolecules such as enzymes and their substrates The molecules are sluggish at low temperatures and collide less frequently and effectively with one another, thus reaction rates are relatively slow Hitherto, advantage is taken of lower thermal energy in approaches such as refrigeration, iced storage, refrigerated sea water storage, and frozen storage to slow down the undesirable effects of enzymes in foods after harvest Low temperature treatments are generally used to slow down the deleterious effects of enzymes in fresh foods (vegetables, eggs, seafood, and meats); however, other processed food products such as high-fat food spreads (margarine and butter), vacuum-packaged meats and seafood products, pasteurized milk, cheeses, and yoghurt also benefit from the desirable effects derived from low temperature treatments It must be noted here that there are some enzymes that are quite stable and active in extreme temperatures (high- or low-temperature-adapted enzymes—known as extremophiles), which may survive the traditional temperature treatments and induce autolysis and spoilage in foods For these enzymes, other techniques are needed to stop their undesirable effects Low-temperature treatments (refrigeration, chilling, and freezing) all slow down enzyme activity but not completely inactivate the enzymes Enzymatic activity still takes place in foods thus treated, albeit at much reduced rates Once the food material is out of the source of the low temperature, once the frozen food material is thawed, enzyme activity may restart and cause undesirable autolytic changes in foods Refrigerated and chilled storage in particular may not be considered as effective methods for long-term storage of fresh foods in high quality In household food preparations, vinegar or lemon juice is often sprinkled on fresh foods (e.g., vegetable salads) to prevent dark discolorations Lowering pH of citrus juice with HCl has been used to achieve irreversible inactivation of pectin esterase (PE; Owusu-Yaw et al 1988), and LOX activity in soy flour is drastically reduced at pH ≤ 5.0 (Thakur and Nelson 1997) Effect of Inhibitors Enzyme inhibitors are substances that slow down or prevent catalytic activities of enzymes They this by either binding directly to the enzyme or by removing co-substrates (e.g., O2 in enzymatic browning) in the reaction catalyzed by particular enzymes Enzyme inhibitors fall into several categories, such as reducing agents that remove co-substrates such as O2 (e.g., sulfites and cysteine), metal chelators that bind or remove essential metal cofactors from enzymes (e.g., EDTA, polycarboxylic acids, and phosphates), acidulants that reduce pH and cause enzyme inactivation (e.g., phosphoric acids, citric acid, sorbates, benzoates), and enzyme inhibitors that bind directly to the enzymes and prevent their activity (e.g., polypeptides, organic acids, and resorcinols) Food-grade protein inhibitors from eggs, bovine/porcine plasma, and potato flour are all used to control undesirable proteolytic activity in foods Egg white contains ovomucoid, a serine protease inhibitor; bovine and porcine plasmas have the broad spectrum protease inhibitor α -macroglobulin; and potato flour has a serine protease inhibitor (potato serine protease inhibitor or PSPI) Fractions containing these inhibitors have been used to prevent surimi texture softening due to proteolysis (Weerasinghe et al 1996) Effect of pH Enzyme activity in foods is pH dependent (Table 9.3) In general, enzymes tend to be destabilized and irreversibly inactivated at extreme pH values, and advantage is taken of this property of pH to control enzymatic activity in foods For example, the enzyme PPO is known to cause undesirable browning in fresh fruits and vegetables By adding acids such as citrate, lactate, or ascorbate, acidic conditions are created (around pH 4) where the PPO enzyme is inactive A similar effect is achieved when glucose/catalase/GOX cocktail is used on raw crustacea (e.g., shrimp); the GOX oxidizes the glucose to gluconic acid and this is accompanied by a drop in pH, which inactivates the PPO Effect of Water Activity Enzymatic activity depends to the availability of water Water availability, and hence water activity (Aw ), in foods may be modified by using procedures that remove moisture, such as drying, freezing, addition of water-binding agents or humectants (such as salt, sugar, honey, glycerol, and other polyols), and by lyophilization (Tejada et al 2008) Most enzyme-catalyzed reactions require moisture to progress effectively for several reasons: (i) a thin film of moisture (bound water) is required to maintain proper enzyme conformational integrity for functional activity, (ii) to solubilize substrates and also serve as the reaction medium, 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 and (iii) to participate in the reaction as co-reactant When moisture content is reduced by dehydration, there is conformational destabilization and loss of catalytic activity For example, most enzymes such as proteases, carbohydrases, PPO, GOX, and peroxidases require Aw ≥ 0.85 to have functional activity, the wellknown exceptions being lipases that may actually gain in activity and remain active at Aw of 0.3, perhaps as low as 0.1 (Loncin et al 1968) The unusual behavior of lipases is observed in lipase-catalyzed reactions with their water-insoluble substrates, lipids, and is due to the interfacial phenomenon that proceeds better in a reduced moisture milieu Foodstuffs prepared based on Aw reduction to control the undesirable effects of enzymes (and microorganisms) include the use of sugar in food spreads (jams and jellies), salt in pickled vegetables, glycerol in cookies and liqueurs, and gelatin in candies and confectioneries Effect of Irradiation Irradiation of foods (also known as cold pasteurization) is a process during which foods are subjected to ionizing radiations to preserve them Food irradiation methods entail the use of gamma rays, X-rays, and accelerated electron beams, and can preserve food by curtailing enzymatic (and microbial) activities However, the irradiation dosage needed to achieve complete and irreversible inactivation of enzymes may be too high and could elicit undesirable effects of their own (e.g., nutrient loss) in food materials The technique is used to some extent in meats, seafood, fruits, and vegetables (especially, cereal grains) for long-term preservation; however, the procedure has low appeal and acceptability to consumers and, therefore, not extensively used in food processing Effects of Pressure High-pressure treatment, also known as high-pressure processing (HPP) or ultra high-pressure processing, is a nonthermal procedure based on the use of elevated pressures (400–700 MPa) for processing foods At the elevated pressures, there is inactivation of both enzymes and microorganisms, the two foremost causative agents of food spoilage, and the inactivation arises from conformational changes in the 3D structure of the enzyme protein molecules (Cheftel 1992) Enzymes in foodstuffs display different sensitivities to pressure; while some may be inactivated at relatively low pressures (few hundred MPa), others can tolerate pressures up to a thousand MPa The pressure effects may also be reversible in some enzymes and irreversible in others, depending on the pressure intensity and the duration of the treatment Those enzymes that can tolerate extreme pressures may be deactivated using appropriate pressure treatments in combination with other barriers to enzyme activity such as such as temperature, pH, and/or inhibitors (Ashie and Simpson 1995, Ashie et al 1996, Sareevoravitkul et al 1996, Katsaros et al 2010) HPP has minimal effects on the attributes of food, such as flavor, appearance, and nutritive value, compared with other procedures like thermal processing, dehydration, or irradiation In contrast to those other processing methods, high-pressure treat- 201 ments keep foods fresher and highly nutritious, improve food texture, make foods look better and taste better The HPP approach also retains the native flavors associated with foods, protects heat-labile essential food components, and extends product shelf life with minimum need for chemical preservatives The technology has been used mostly by companies in Japan and the United States to process foods such as ready-to-eat meats and meat products, food spreads (jams), fresh juices and beverages (sake), processed fruits and vegetables, fresh salads, and dips HPP is also used to shuck and retrieve meats from shellfish (oysters, clams, and lobster) CONCLUDING REMARKS: FUTURE PROSPECTS Foodstuffs have naturally present enzymes as well as intentionally added ones that produce significant effects on food-processing operations The actions of these enzymes may improve food quality or promote food quality deterioration The use of enzymes as food-processing aids has increased steadily for several years now, and this trend is expected to continue for the foreseeable future due to the interest and need for more effective strategies and greener technologies to curtail the reliance on existing technologies and protect the environment The factors that augur well for the expanded use of enzymes as food-processing aids include: consumer preferences for their use instead of chemicals, their use as food-processing aids is perceived to be more innocuous and more environmental friendly, their capacity to selectively and specifically remove toxic components in foods (e.g., glucosinolates with sulfatases, acrylamide with asparaginase, or phytates with phytases), and recent advances in enzyme engineering, which is permitting the discovery and design of new and superior enzymes tailored to suit specific applications Recent developments in enzyme engineering are enabling yields of particular enzymes to be improved by increasing the number of gene copies that code for enzyme proteins in safe host organisms, and this feature is particularly significant because they are permitting useful enzymes from plant and animal sources, and even their counterparts from uncertified microorganisms (including hazardous ones) to be produced in high yields much more consistently, rapidly, and safely for food use It is also significant because the cultivation and growth of microorganisms for the purpose of producing recombinant enzymes is not dependent of weather conditions, proceeds much faster, and require much lesser space and regulations compared with plants or animals Enzyme engineering is also facilitating the design of new enzyme structures with superior performance characteristics with respect to catalytic activity, thermal stability, tolerance to pH and inhibitors, and other properties to make them more suited for applications in several fields of endeavor This capacity is also expected to enhance the design and creation of synthetic or artificial enzymes (Chaplin and Burke 1990) with superior properties for both food and nonfood uses Recombinant enzymes with improved stability would facilitate their use in producing P1: SFK/UKS BLBS102-c09 P2: SFK BLBS102-Simpson March 21, 2012 202 11:15 Trim: 276mm X 219mm Printer Name: Yet to Come Part 2: Biotechnology and Enzymology more useful biosensors for food analysis and other analytical work (Chaplin and Burke 1990), stimulate the manufacture of more immobilized enzymes that are also endowed with superior properties for reuse and cost savings in food processing, produce new enzymes that can function well in nonaqueous milieu, and facilitate the synthesis of new molecules with predetermined structures and functions (as was achieved with the synthesis of aspartame by thermolysin) for use in foods Recombinant enzyme technology is also expected to facilitate the discovery of newer applications for enzymes as food-processing aids and also produce new enzymes to assist the incorporation of specific essential molecules in food products to meet specific dietary needs, as use as dietary supplements to manipulate ingested carbohydrates, lipids, and cereal proteins (e.g., gluten) for improved human health and wellness The undesirable effects of enzymes in foods are controlled to some extent by traditional practices such as thermal treatments, cold storage, water activity (Aw ) reduction, pH control, and treatment with chemicals However, there are various limitations with these methods, such as destruction of heat-labile essential components in foods, continued enzymatic activity (albeit at a reduced rate) even under iced, refrigerated, or frozen storage; and the adverse effects of Aw reducing agents such as salt or sugar as well as chemicals on human health Novel approaches based on nonthermal treatments for controlling enzymes (such as HPP and PEF) 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Food Biochemistry and Food Processing Blackwell Publishing, Ames, IA, pp... 201 ments keep foods fresher and highly nutritious, improve food texture, make foods look better and taste better The HPP approach also retains the native flavors associated with foods, protects... ready-to-eat meats and meat products, food spreads (jams), fresh juices and beverages (sake), processed fruits and vegetables, fresh salads, and dips HPP is also used to shuck and retrieve meats