Abhimanyu Thakur What is an enzyme? • A protein catalyst that speeds up a chemical reaction. Lowers the activation energies of a substance • Term enzyme : W. Kuhne • James Sumner(1926): Isolate and crystallized urease He postulated that all enzymes are proteins. • 1930: J. Norhrop, M. Kunitz crystallized pepsin, tripsin and others Racki, 2006 • Pectinases: Juice clarification in the 1930s, and for a short period during World War II • Invertase: Used for the production of invert sugar syrup : pioneered the use of immobilized enzymes in the sugar industry • Large-scale application of enzymes: 1960s, traditional acid hydrolysis of starch was replaced by an approach based in the use of amylases and amyloglucosidases. Racki, 2006 Estimated world food and beverage enzyme demand in million dollars from 2000 to 2020 Source: Adapted from The Freedonia Group Inc., World Enzymes to 2015 AN OVERVIEW OF ENZYMES USED IN FOOD PROCESSING Class Enzyme Glucose oxidase Laccases Oxidoreductases Lipoxygenase Transferases Lyases Isomerases Role Dough strengthening Clarification of juices, flavour enhancer (beer) Dough strengthening, bread whitening Cyclodextrin Cyclodextrin production Transglutaminase Modification of viscoelastic properties, dough processing, meat processing Acetolactate decarboxylase Beer maturation Xylose (Glucose) Glucose isomerization to fructose isomerase Fernandes, 2010 Class Enzyme Role Starch liquefaction Increasing shelf life and improving quality by retaining moist, elastic and soft nature Bread softness and volume Juice treatment, low calorie beer Glucanase Enhanced digestibility in barley and oats used in animal feed. Invertase Sucrose hydrolysis, production of invert sugar syrup Amylases Hydrolases Lactase Lactose hydrolysis, whey hydrolysis Lipase Cheese flavour, dough conditioning, synthesis of aromatic molecules Fernandes, 2010 Class Enzyme Role Proteases Protein hydrolysis (chymosin, Milk clotting, papain) Infant-food formulation with enhanced digestibility and utilization Flavor improvement in milk and cheese Meat tenderizer, prevention of chill haze formation in brewing Hydrolases Pectinase Mash treatment, juice clarification Peptidase Hydrolysis of proteins, cheese ripening Phytases Release of phosphate from phytate, enhanced digestibility Enhanced digestibility, dough conditioning Xylanases Fernandes, 2010 FRUIT, VEGETABLE AND PLANTATION CROP PRODUCTS CLARIFICATION OF FRUIT JUICES Pectolytic enzymes: Increase the yield in juice during crushing and pressing. Example : Clear juices (Apple) Crushing: Pectinases and cellulases for the complete extraction of the juice. Pressing: Pectic enzymes are used to facilitate the pressing and juice extraction. Will et al., 2000 BENEFITS OF ENZYMES FOR FRUIT PROCESSING • Enzymes are the processing aid used for the fruit processing particularly for clear fruit juice and concentrates. Advantages of enzymes are : • Economic: When added to fruit after crushing, amylases and pectinases quickly decrease the viscosity, facilitate juice extraction from pulpy fruit and increase the overall productivity of plant. • Quality: Fast juice processing with enzymes lower the risk of microbial spoilage, reduced oxidation and improved juice and concentrate shelf life. • Sustainability : Use of enzymes has positive effect, they lower energy consumption, reduced waste flow, reduced dependency on chemical used. EFFECT OF TEMPERATURE (LOWER RANGE) AND HOLDING TIME ON APPLE JUICE CLARIFICATION (PECTINOLYTIC ENZYME + GELATIN) Singh and Gupta, 2003 EFFECT OF TEMPERATURE (HIGHER RANGE) AND HOLDING TIME ON APPLE JUICE CLARIFICATION (PECTINOLYTIC ENZYME + GELATIN) Singh and Gupta, 2003 CLARIFICATION OF JUICE (PECTINOLYTIC ENZYME + GELATIN) Clarification of apple juice Change in viscosity of apple juice after clarification Singh and Gupta, 2003 Cloudy juices (i.e., citrus, tomato, nectars) Pectic enzymes: Polygalacturonases Orange juice extraction Pectinases can be added at the end of the pulp wash extraction. Higher yield in juice, Better extraction of soluble solids and to Lower viscosity. Kashyap et al., 2001 EXTRACTION OF VEGETABLE OIL Vegetable oils: Olive, sunflower, coconut, palm or canola are obtained by extraction with organic solvents such as hexane. Pectolytic enzymes: Allows the better extraction of vegetable oils. Enzymes: Cellulases, hemicellulases and pectinases. Improves Stability Increases Polyphenols Increases Vitamin E Content Better Organoleptic Quality Sieiro et al., 2012 EFFECT OF CONCENTRATION OF PECTINEX ULTRA SP ON EXTRACTION YIELD AND SUGAR CONTENT OF JICAMA (YAM BEAN) JUICE Lien and Man, 2010 EFFECT OF TIME OF APPLICATION OF PECTINEX ULTRA SP ON EXTRACTION YIELD AND SUGAR CONTENT OF JICAMA (YAM BEAN) JUICE Lien and Man, 2010 COFFEE, COCOA AND TEA FERMENTATION Fermentation of coffee is made with pectolytic microorganisms in order to remove the layer of mucilage from the coffee beans. With the same purpose, commercial enzyme preparations containing pectinase is sprayed onto the beans to ferment. (Reduces the fermentation time) Cocoa fermentation: Develop the chocolate flavour. This fermentation is carried out by a succession of different microorganisms, some of them pectolytic. Pectic enzymes allow the degradation of the cocoa pulp and are indispensable for the fermentation process and the good quality of fermented beans Tea leaves with pectic enzymes of fungal origin (at a dose adjusted to avoid damaging the leaf), facilitates and accelerates the fermentation. Kashyap et al., 2001 WINE INDUSTRY Pectinases alongwith hemicellulases, glucanases and glycosidases. Microbial pectolytic enzymes especially of fungal origin are resistant to the conditions of fermentation and can be used to facilitate processes, improve quality and diversify products. Pectic enzymes: Support the extraction process Maximize juice yield Facilitate filtration Intensify the flavour and colour Sieiro et al., 2012 MEAT AND MEAT PRODUCTS CONSUMER DEMANDS Add value to lower quality meat High quality Improving meat value Moderate Price Maximize efficiency of carcass utilization Tenderization of too tough meat parts APPLICATION Boost manufacturing process Production of fresh low value meat pieces. OF ENZYMES Upgrade meats of poorer quality Trimming to higher quality steaks. Myofibrillar proteins denature. HEATING Collagen in connective tissue denaturizes & shrink. Muscle protein becomes more susceptible to exogenous proteolytic enzymes. Water binding is due to myofibrillar proteins WATER BINDING carrying high net charge Water binding increases with increasing salt content up to 5 % NaCl. Mincing & chopping affect the properties of meat MECHANICAL TREATMENTS Mincing : Keep fibre bundles and fibres intact but cut them across longitudinal axis. Chopping : Swelling & disintegration of myofilaments. PROTEASE RAW MATERIAL MEAT PROCESSING CONDITIONS EFFECT ON MEAT TEXTURE AND OTHER QUALITY FACTORS Turkey drumsticks Hens and roasters Injection of papain More tenderness Injection of papain And treating with high pressure Significant increased tenderness up to 100 MPa Ground beef In sausage manufacturing More protein solubilization and improved water holding capacity Actidin Beef muscle Immersion in crude actidin Tenderness Ginger Extract Sheep and meat chunks Marinating Increased cooking yield and collagen solubility Papain Papain Ficin Beef meat Whitehurst and Oort, 2009 INFLUENCE OF MARINATION WITH PROTEOLYTIC ENZYMES ON pH VALUES Increasing the pH Improvement of water retention Taste and tenderness of meat have improved. TENDERNESS OF MEAT INCREASES PROPORTIONATELY Istrati et al., 2013 WITH INCREASING pH. INFLUENCE OF MARINATION WITH PROTEOLYTIC ENZYMES ON RIGIDITY INDEX OF BEEF CUTS Istrati et al., 2013 During ripening proteolysis takes which is involved in taste and flavour development of meat products. Meat protein hydrolysis is mainly catalyzed by endogenous enzymes, such as cathepsins and trypsin-like peptidases as well as proteases. Lipolysis constitutes another important group of enzymatic reactions which are related to aroma formation of fermented sausages. Dry-cured meat products : long period of ripening : transformation of free amino acids and fatty acids through microbial and chemical ways to yield aromatic compounds. Because long ripening time involves a high cost of storage until a suitable matured state is reached, many attempts have been made to shorten this period. Proteases and lipases have been used for this purpose. The most promising method to shorten ripening time of fermented sausages is by the incorporation of cellfree extracts from lactic acid bacteria and moulds. The addition of cell-free extract from Lactobacillus paracasei sbsp. Paracasei Accelerate the ripening Improve the sensory quality of sausages. The addition of cell-free extracts from moulds such as Mucor racemosus and Penicillium aurantiogriseum Improved sensory properties of fermented sausages Whitehurst and Oort, 2009 Enzyme APPLICATION EFFECT ON MEAT FLAVOUR Papain Spanish dry fermented sausages Slight softening of product Increase in concentrations of some esters. Protease Dry fermented sausages Lactobacillus lactis ssp. cremoris Dry fermented sausages Increase in concentrations of volatile compounds. Cell free extract from Lactobacillus sakei Dry fermented sausages Increase in concentrations of volatile compounds derived from lipid and carbohydrate oxidation Whitehurst and Oort, 2009 ENZYMES IN DAIRY The dairy foods sector of the food manufacturing industry is a traditional user of enzymes. The best known dairy enzyme preparation is, rennet, a collective name for commercial preparations. In addition to the use of milk clotting enzymes to make cheese, the dairy industry also makes use of enzymes such as lipases, non-coagulant proteases, aminopeptidases, lactase, lysozyme and lactoperoxidase. Some of these applications are traditional (lipase for flavour enhancement) while others are relatively new (lactose hydrolysis, accelerated cheese ripening and control of microbiological spoilage) ENZYMES IN MILK AND CHEESE INDUSTRY Whitehurst and Oort, 2009 RENNET : A BACKBONE TO CHEESE INDUSTRY Enzyme preparation used to clot milk: Historically Calf Rennet. Plays two roles: Primarily – Clot milk curd. Secondary – Ongoing proteolysis in cheese. Rennet Type Advantages Glewis , 2013 Disadvantages Animal (Calf) Traditional use. Can have slight Historical acceptance. variations in Flavour can vary slightly. manufacture. Animal origin. Fermentation Produced Chymosin Very consistent in manufacture. Suitable for vegetarians. Microbial Non-animal origin More Thermoliable – Mozzarella Perception may cause bitterness. RENNET MODE OF ACTION – CLOTTING MILK Critical factors: pH – optimum 6.0 Temp: optimum 30-32oC. Coagulation does not occur <18oC Calcium – 20% reduction in colloidal calcium phosphate prevents coagulation. CHEESE MATURATION PROCESS OCCURRING IN CHEESE Lipolysis Glycolysis Proteolysis Fats breakdown Sugars breakdown Caseins breakdown CHEESE PRODUCTION PROCESS WITH A MICROBIAL ENZYME High coagulant activity and low proteolytic activity High production yield Ensures optimal conditions for the development of the original aroma, flavor and texture of cheese Unrestricted availability of raw material Inexpensive to obtain due to the possibility of using cheap substrates for fermentation Greater acceptance among people that do not eat food containing animal products Greater acceptance among groups that oppose the use of animal enzymes due to religious and ethical principles. ENZYMES IN BAKERY Source: Adapted from The Freedonia Group Inc., World Enzymes to 2015 Baking comprises the use of enzymes from three sources: The endogenous enzymes in flour Enzymes associated with the metabolic activity of the dominant microorganisms Exogenous enzymes which are added in the dough APPLICATIONS OF STARCH MODIFYING ENZYMES IN BAKING Enzyme Applications in baked products α-Amylases Generation of fermentable compounds β-Amylases Increase in bread volume Reduction in fermentation time Glucoamylase Pullulanase Isoamylase Amylomaltases Amylosucrases Improvement in dough viscosity, rheology and bread softness Improvement in bread texture Formation of reducing sugars and subsequent Maillard reaction products intensifying bread flavor and color Anti-staling effects. Miguel et al., 2013 APPLICATIONS OF CELLULASES & HEMICELLULASES IN BAKING Enzyme Applications in baked products Cellulase Removal of insoluble arabinoxylans Hemicellulase Increase in dough viscosity, stability, with better mouldable form Lamarinase Improvements on rheological properties of dough Lichenase Reduction in fermentation time Endo β(1,4)-Dxylanase α-LArabinosidase β-D-Xylosidase Increase of bread volume Synergistic action of glucanase providing more soluble dietary fibre in bread products Production of prebiotic oligosaccharides in bread. Miguel et al., 2013 APPLICATIONS OF PROTEASES, TRANSGLUTAMINASES, LIPASES, ESTERASES AND OXIDOREDUCTASES IN BAKING Enzyme Proteases Applications in baked products Reduction of dough mixing time Control of dough rheology Enhance dough extensibility Increase loaf or bread volumes Formation of amino acids and flavors Crispness feature on bread crust Transglutaminases Increase volume Improve structure of breads Better retention of gas Improve bread crumb strength Improve dough stability Improve properties of gluten-free breads Protect frozen doughs from damage Enzyme Lipases and esterases Applications in baked products Improvement in bread volume and dough stability Formation of emulsifiers Retard staling Development of flavors. Oxidoreductases Glicose oxidase Control on browning for Maillard Reaction Improvements in crumb properties. Lipoxygenase Bleaching of fat-soluble flour pigments Laccase Dough strength, stability and reduced stickiness Increase in volume Improved crumb structure and softness Miguel et al., 2013 EFFECT OF EXTRUDED BRAN AND ENZYMES ON WHEAT DOUGH MIXING PROPERTIES Dietary fibre : Reduction of the cholesterol levels and the risk of colon cancer. * WEAKENS THE STRUCTURE AND BAKING QUALITY OF WHEAT DOUGH AND DECREASES BREAD VOLUME AND ELASTICITY OF THE CRUMB Bakezyme HSP6000: β-xylanase from A.niger Bakezyme P500: α-amylase from A.oryzae Bakezyme AG800: glucoamylase from A.niger Basinskiene et al., 2008 EFFECT OF EXTRUDED BRAN & ENZYMES ON QUALITY PARAMETERS 0 – without enzymes X – βββxylanase X, G, A – multienzyme mix (β-xylanase, glucoamylase α-amylase) Basinskiene et al., 2008 EFFECT OF EXTRUDED BRAN (EB) AND ENZYMES ON BREAD CRUMB TEXTURE Basinskiene et al., 2008 EFFECT OF EXTRUDED BRAN (EB) AND ENZYMES ON OVERALL ACCEPTABILITY OF BREAD Basinskiene et al., 2008
