Enzymes in Animal Products Processing

1. Enzymes in Animal Products Processing

2. Meat tenderizing enzymes • Plant source: papain, bromelain, ficin, • Microbial source: Bacillus subtilis, Aspergillus oryzae. • Pancreatic: trypsin, chimotrypsin, carboxypeptidase.

3. Characteristics of papain • Papain is a sulfhydryl protease from Carica papaya latex. • Papain degradates myofibrillar and collagen proteins, yielding protein fragments of several sizes. • The majority of the tenderizing activity occurs during the cooking process.

4. Characteristics of papain • Papain is a single peptide chain of 211 residues folded into two parts that form a cleft. • The molecule has one free SH group which is functional. • Molecular weight: 23,000 • Temperature optimum for activity: 65oC • pH optimum for activity: 6.0 - 7.0 • Isoelectric point: pH 9.6

5. Characteristics of papain • Activator: papain is activated by cysteine, sulfide, sulfite, etc. • Inhibitor: substances which react with sulfhydryl groups including heavy metals, carbonyl reagents, aldehydes, benzoylamidoacetonitrile, H2O2 • Stability: papain as a crystalline suspension is stable at 5°C for 6-12 months. Stabilizing agents are EDTA, cysteine and dimercaptopropanol.

6. Characteristics of bromelain • Bromelain is a digestive enzyme found in the stem and fruit of the pineapple plant. • It enhances the absorption of nutrients from foods, supplements and probiotics. • Bromelain works specifically on protein and, unlike other protein digestive enzymes, it works effectively in all the acid and alkaline conditions found throughout the gastrointestinal tract.

7. Characteristics of bromelain • Bromelain first degrades 40% of the collagen in the sarcolemma followed by degradation of myosin in the myofibrillar component. • There is a significant increase in tenderness when an enzyme solution is injected into muscle versus dipping or tumbling in brine. • Temperature optimum for activity: 50-60oC • pH optomum for activity: 4.5-5.5

8. What are the benefits? • Bromelain has been used successfully to treat abnormal heart functions, arthritis, upper respiratory tract infection. • Bromelain has been used to heal wounds caused by burns. • Bromelain aids in gastrointestinal imbalances and bloating caused by poor digestion;

9. What are the benefits? • Bromelain aids in the healing of gastric ulcers; used as a digestive enzyme for pancreatic insufficiency; • Bromelain gives relief from the symptoms of angina, thrombosis, thrombophlebitis, varicose veins and atherosclerosis. • Bromelain reduces joint inflammation in rheumatoid arthritis, osteoarthritis, sciatica, bursitis, tendonitis and scleroderma;.

10. Characteristics of ficin • Ficin, a vegetable-based enzyme, is derived from figs. • This thiol protease, although capable of minimal degradation of collagen and elastin, will preferentially degrade myofibrillar protein. • Ficin is able to degrade elastin at temperatures as low as 20oC whereas it has little activity against collagen and myofibrillar proteins below 40oC.

11. Characteristics of ficin • Optimal activity occurs between 60-70oC. • The optimal pH for activity is near 7 for collagen and myofibrillar proteins and approximately 5.0-5.5 for elastin degradation. • Sensory evaluations tend to show only a marginal increase in tenderness when compared to untreated samples.

12. Protease from Aspergillus oryzae • Aspergillus oryzae produces an aspartic protease that shows a self-limiting proteolytic activity in meat systems. • Myofibrillar proteins are the main substrate for activity while little, if any, collagen breakdown occurs. • This aspartic protease shows minimal activity during refrigerated storage through 14 days of storage but increases to an optimum at 55oC before dropping dramatically at 60oC.

13. Protease from Aspergillus oryzae • The enzyme from Aspergillus is active in acidic conditions and activity is maintained up to pH 7.0 before rapidly declining. • As a self-limiting enzyme, some limited degradation can improve tenderness without the risk of a mushy or mealy texture.

14. Protease from Bacillus subtilis • Bacillus subtilis proteasesshow alkaline and neutral elastase specifically degrades collagen and elastin with little degradation of myofibrillar protein resulting in less tenderizing effect. but also decreasing the possibility of over-tenderizing. • Bacillus subtilis proteases’ optimal and activity ranges vary depending upon the organism from which they are produced. • Neutral proteases activity rapidly increase at 50oC with a dramatic drop above 65oC.

15. Protease from Bacillus subtilis • pH activity from 5.0-9.0 pH with an optimal pH found at 7.0. • Enzymatic activity occurs during storage for one, two and three days at 4oC • This bacterial protease was developed to be an inexpensive alternative for ficin.

19. Advantage of using enzymes • Papain is able to withstand until 170-185oF before it becomes completely inactivated. A piece of meat cooked to medium rare will not reach a temperature high enough to inactivate the papain. That means the subsequent storage of the meat will allow the enzyme to continue to tenderize. • Bromelain has a lower temperature of inactivation, which is around 160oF. This will not be high enough for inactivation in a medium rare piece of beef. • The rate of action for both papain and bromelain are similar.

20. Application methods of enzymes • Sprinkling enzyme powder on the meat. • Dipping the meat in the enzyme solution. • Spraying the enzyme solution on meat cuts. • Injecting the enzyme solution into meat cuts. • Injecting the enzyme solution into circulatory system on animal shortly before slaughter.

21. Disadvantages of enzyme applications • Ununiform distribution of enzymes in meat cuts. • Discolorization due to high level of salt and water. • Need frozen storage after enzyme application. • Microbial spoilage problems and loss in enzyme action.

22. Injection into circulatory system • Vascular system is excellent distribution system. • Heart is an efficient pump. • Bloodstream acts as diluent for enzyme, providing a uniform method of obtaining distribution. • Enzymes have ability to tenderize meat.

23. Other applications of enzymes • Preparation of corned beef for dry oven roasting. • Process for removal of meat from bones. • The retort process in canning industry. • Hydrolysis of renderer`s meat scrap. • Degreasing bones for gelatin production.

24. Enzyme in milk processing • Rennet is an extract from the fourth stomach of young ruminants, such as cows, goats, and sheep. • Rennet contains many enzymes, including a proteolytic enzyme (protease) that coagulates the milk, causing it to separate into solids (curds) and liquid (whey). • The active enzyme in rennet is called chymosin or rennin but there are also other important enzymes in it, e.g., pepsin or lipase. • Natural calf rennet is extracted from the inner mucosa of the fourth stomach chamber (the abomasum) of young calves.

25. Rennin • Chymosin, known also as rennin, is a proteolytic enzyme synthesized by chief cells in the stomach. • Its role in digestion is to curdle or coagulate milk in the stomach, a process of considerable importance in the very young animal. • Chymosin is secreted as an inactive proenzyme called prochymosin that, like pepsin, is activated on exposure to acid. • Chymosin is also similar to pepsin in being most active in acidic environments, which makes sense considering its mission.

26. How chymosin coagulates milk? • The majority of milk protein is casein and there are four major types of casein molecules: alpha-s1, alpha-s2, beta and kappa. • The alpha and beta caseins are hydrophobic proteins that are readily precipitated by calcium. • Kappa casein is a distinctly different molecule - it is not calcium-precipitable. • Kappa casein normally keeps the majority of milk protein soluble and prevents it from spontaneously coagulating.

27. How chymosin coagulates milk ? • Chymosin proteolytically cuts and inactivates kappa casein, converting it into para-kappa-casein and a smaller protein called macropeptide. • Para-kappa-casein does not have the ability to stabilize the micellar structure and the calcium-insoluble caseins precipitate, forming a curd. • Whey is the liquid portion of the milk after the rennet curd is removed. • Whey contains some of the rennin ferment, a small amount of soluble protein (lactalbumin), a slight amount of fat, about 4% of milk-sugar, and the salts of the milk with the exception of the calcium phosphate.

28. Chimosin in cheese industry • Chymosin is a very important industrial enzyme because it is widely used in cheesemaking. • Approximately 6% of the rennin used to coagulate milk is retained in active form in cheese curd. • During cheese ripening, rennin modifies the curd protein structure through its proteolytic action on α-casein, leading to textural changes described as a loss of curdiness. • Casein peptides resulting from rennin action become precursors for flavor compounds in some cheeses such as Cheddar. • "Rennet" is the name given to any enzymatic preparation that clots milk.

29. Preparation of rennet • Obtaining abomasa: Abomasa should be obtained from preferably unweaned calves • Preparation of abosama: Abosama should be washed and the fat and veins removed, should be sliced into thin strips, 5 mm wide. • Soaking: Abosamsa strips should be soaked in a 10 percent sodium chloride (salt) and 1 percent sodium benzoate solution. • Treatment of the liquid: The solution is reacidified with hydrochloric acid to a pH of 4.8, and allowed to settle for two hours to eliminate the mucilage in suspension in the extract, • Determination of strength: Rennet strength is the number of volumes of coagulated milk clotted by one volume of rennet in 40 minutes at 35oC. Liquid rennet strength should be 1/10 000 (1 litre of rennet clots 10 000 litres of milk at 35°C in 40 minutes).

30. Enzymes in egg processing • Traditionally, eggs were supplied in the form of whole eggs. • Food processors use egg as liquid, frozen, dried, whole eggs, whites or yolks. • Fresh shell eggs and liquid products had the best functionality. • Liquid and dried egg products can be treated with enzymes to improve functionality for specific applications.

31. Enzymes in egg processing • Dried egg products have the advantages: • They can be easily pasteurized, • They have excellent shelf life and stability, • They are easier and cheaper to ship due to reduced volume, and • They can be tailored with specific functionality.

32. Enzymes in egg processing • Eggs are usually processed in a semi-continuous process. • Eggs are extremely useful food ingredients and have a variety of functional properties including foaming, gelation, emulsification and texturization. • Eggs provide foaming properties in cakes and meringues; gelation in cakes and quiches; emulsifying components in batters and mayonnaise and improve the texture of baked goods.

33. Enzymes in egg processing • The main components of egg are proteins and lipids and these are responsible for the functional attributes. • Other components are present in small quantities. • Egg yolk is a complex oil water emulsion composed of 50% water, 32% lipids and 16% protein. • Approximately 28% of the lipids are phospholipids, of which approximately 80% is phoshatidylcholine, 12% is phosphatidylethanolamine with other phospholipids such as sphingomyline and lyso-phosphatidylcholine. • The surface active properties of these phospholipids can act a little like soap in stabilising oil water emulsions.

34. Enzymes in egg processing • Enzymatic conversion of the phospholipids into lyso-phospholipids increases the emulsion stability produced with these egg yolks. • Egg yolks have extremely useful emulsifying and gelation properties due to the presence of various lipid and protein types and have been extensively used in recipes for products such as mayonnaise.

35. Preventing microbial spoilage using peroxidase and catalase • Processed eggs should be pasteurized to eliminate the presence of possibly harmful bacteria and prevent spoilage. • Micro-organisms in the egg can be killed by exposure to heat or sterilizing chemicals. • Longer heat exposure and higher temperature or higher concentration of the sterilant, the more micro-organisms are killed. • Pasteurization should achieve the same reduction in micro-organisms as heating to 64.4oC for at least 2 minutes and 30 seconds.

36. Preventing microbial spoilage using peroxidase and catalase • Eggs Pasteurization can damage egg proteins changing their functional characteristics. • Hydrogen peroxide can be used to chemically sterilize the solution egg before thermal pasteurization allowing shorter time and / or lower temperature combinations to achieve the desired reduction in micro-organisms. • If hydrogen peroxide has been utilized to assist pasteurization of egg ingredients, Catalase should then be used to remove residual peroxide, breaking it into harmless water and oxygen.

37. Preventing microbial spoilage using peroxidase and catalase • The peroxide should be added slowly to avoid damage to the egg proteins by high peroxide concentrations. • The mixture is held for at least 20 minutes to allow the peroxide to kill vegetative micro-organisms. • Once sterilization is complete, residual peroxide must be eliminated with Catalase. • The combination of peroxide and heat pasteurization achieves a much greater reduction in microbial numbers than either technique can independently.

38. PREVENT BROWNING BY REMOVINGSUGAR WITH GLUCOSE OXIDASE • Another problem occurring during the heat treatment of eggs is browning caused by the Maillard reaction. • This occurs as a result of small amounts of glucose in the egg white reacting with amino acids. • This can be problematic for dried egg whites if the product is traditionally pasteurized after drying in a hot room, for an extended period of time. • Glucose oxidase is able to break down the glucose to products which do not cause browning.

39. Prevent browning by renoving sugar with glucose oxidase • Glucose oxidase requires the presence of dissolved oxygen to function so it is important to ensure the liquid egg is well aerated. • Glucose oxidase can be used to remove sugar from whole liquid eggs, egg yolk or liquid egg white. • Ensure the peroxide is added slowly to avoid denaturing the egg and take care as the solution may bubble and foam as oxygen is released. • Because the glucose oxidase has eliminated the glucose, there will be much less Maillard reaction products formed during pasteurization resulting in a brighter looking product with no browning.

40. Improve foaming properties of egg white by removing contaminating yolk lipids • The main functional property of egg white is its high foaming capacity. • Cross contamination of egg white with egg yolk lipids greatly reduces foaming capacity. • Lipase is needed to breaks down the lipid complexes and ensures the egg white maintains full foaming capacity. • The enzyme may be added to the liquid egg white.

41. Improve foaming properties of egg white by modifying protein • Foaming ability can be improved by a minor modification of the egg white proteins. • Some processors incubate dried egg white in a hot box for several days to produce high whip egg white powders. • The heat treatment partially denatures the egg proteins, improving their whipping ability and resulting in greater foam height. • Over hydrolysis decreases in foam stability will be seen. • There is an optimum hydrolysis point whereby foam volume is increased without adversely affecting the foam stability.

42. New flavor enzyme developed for the production of egg white hydrolysate • Egg white protein is a valuable product with important nutritional and functional properties. • The most abundant protein in egg white is ovalbumin. • The proteins present in egg white are high quality proteins, are relatively easy to digest and are efficiently absorbed into the body. • Protease, designed to efficiently hydrolyse egg white proteins to produce a bland, non bitter tasting egg white hydrolysate.

43. New flavor enzyme developed for the production of egg white hydrolysate • The enzyme results in a reduction in the size of the egg white proteins thus allowing them to be more efficiently digested and absorbed into the body. • This is very useful for protein fortification of foods such as nutritional bars or powdered mixes, used by athletes, where clarity of the egg white hydrolysate in the final product is not important.