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UNDER THE GUIDANCE OF EXTERNAL GUIDE : PRIYA RAY (Royal Life Sciences Pvt Ltd) INTERNAL GUIDE : Dr. SANJEEB KUMAR MANDAL ( Assistant Professor B iotechnology CBIT).

INTRODUCTION. In the stomachs of ruminant mammals, a complex collection of enzymes called rennet is created. The protease enzyme chymosin, which is a crucial part of it, causes milk's casein to curdle. Rennet also includes additional enzymes such a lipase and pepsin in addition to chymosin (Silveira et al., 2005) ( wikipedia ).

MECHANISM OF RENNET ENZYME. 3. app Primary Phase Negatively charged end Chymosin enzyrrw Secondary Phase.

MECHANISM OF RENNET ENZYME. Casein is the main protein found in milk. Milk is full of protein particles; most of these (>80%) are formed into the casein micelle. The slightly negatively charged glycomacropeptide (GMP) on the surface of the casein micelle is eliminated by cleavage. Due to the fact that molecules with opposing charges repel one another, the GMP prevents casein micelles from adhering to one another. When the GMP is removed, the casein micelles may begin to cluster and lose their polar charge, rising out of the polar water molecules and joining non-polar milk fat as a component of the cheese curd. This process is made easier by strong ions, including those created by calcium and phosphate. Rennet contains an enzyme ( chymosin ) which cuts this negatively charged kappa casein protein so that the negative end of the chain dissolves into the liquid (it will leave with the whey).This is the primary phase of the coagulation. The secondary phase can commence after the protein micelle has been neutralised ; the proteins will now start to bind to one another and stop repelling one another. This occurs as a result of the newly chopped kappa casein chain's (now known as para-kappa- casein's) sensitivity to mineral uptake. Its cut end forms a bridge that connects the casein micelles with phosphate and then calcium minerals found in milk (effectively creating a protein web)..

Milk that is poor in calcium and phosphate (such as goats’ and cows’ milk – particularly if it is not fresh) may have calcium phosphate added to help improve this stage: this is why it can sometimes be seen on the ingredients list of cheese). As the proteins join together (coagulate) to form the curd/junket, the web also traps in the fats and minerals. This junket will form the cheese (after other stages of manufacture: cutting, draining, pressing, ageing). Most of the rennet will leave with the whey: approximately 0.0000005g per kg is left in the finished cheese..

WHY SOLID STATE FERMENTATION IS USED FOR ENZYME PRODUCTION?.

WHY MICROBIAL RENNET ENZYME IS EFFECTIVE IN CHEESE PRODUCTION?.

AIM: This project aim’s to identify, characterize, and purify microbial rennet enzyme from fungi(mucor) isolated from dairy effluent soil and extract bromelain from pineapple and use these enzymes for making cheese ..

NOVELTY. Analysis of combination of two enzymes (microbial rennet and plant protease(bromelain) in the cheese production. There have been no publications published to date that compare the activity of two enzymes in cheese manufacturing..

LITERATURE SURVEY. 10. The production of extracellular milk-clotting protease by locally isolated fungal specie, Aspergillus niger FFB1 under solid-state fermentation (SSF) using cheep agro-industrial byproduct (wheat bran) was optimized. The effects of several physicochemical and environmental factors were investigated to select the optimal conditions that ensure the best milk-clotting activity by application of "One-factor-at-a-time" method. A trial of cheese production using the crude extract was also carried out ..

A crude enzyme produced by the potential isolate was characterized after partial purification by acetone and ammonium sulfate precipitation. A total of 9 fungal isolates showed protease production in primary and secondary screening; however, one potential isolate (Z1BL1) was selected for further study based on its protease activity. 'e isolate was identified to the genus Aspergillus based on their morphological features. 'e maximum acid protease from the isolate Z1BL1 was obtained using fermentation media containing wheat bran as a solid substrate, 1 mL of 3.2 × 10 6 inoculum size, 50% moisture content, and pH 4.5 upon 120-h incubation at 30°C..

Comparative study of clotting actiity of crude gastric emmes atracted from camels' abomasum at different ages and commercial (rennet and bovine and tamel Inuk Salihl, l.aleyC, Inis:•, MoghMiti Farid, Mahboub and Mdi Mürnhnjne' Eminln d de 9 tvyn• iGEC) d«løg GEC Nh' i« nn 'knit/ K" Cod •iL.

MATERIALS AND METHODS. Glassware Sabouraud dextrose agar(1.5g) Lactophenol cotton blue stain Pineapple Distilled water Ice pack Ammonium sulphate (8.8g) Multigrain bran(44g) Milk Dialysis bag Mortar and pestle Deae -cellulose Chromatography column milk powder(4.0g) cheese cloth(1meter) 25mMtris HCL(5ml) filter paper 0.001M tris HCL buffer Burette stand Centrifuge Alkaline cupric sulphate reagents :mix solA + solB in ratio(50:1) (solA:0.1N Sodium Hydroxide +2%Sodium Hydroxide) ( solB:0.5%Cupric(2)Sulphate+1%Potassium Sodium Hydroxide) 22.Folin ciocalteu phenol reagent :dilute 1 part of phenol reagent to 4 part distilled water.

PROCEDURE 1.MICROBIAL R ENNET. 14. Isolation, identification of mucor strain for enzyme production.

Filtration and centrifugation of enzyme and Obtaining crude enzyme Then performing enzymatic assay.

Quantification of protein using Lowry’s method. 16.

2. ENZYMATIC ASSAY. 10mM calcium chloride solution was prepared (pH 4.5). 10.4% of milk solution was added to above 10mM calcium chloride solution. Above 2ml substrate solution was incubated at 30 0 c for 45 minutes. After incubation period,0.2ml of crude enzyme was added to substrate solution at 30 0 c and observed for the clot formation in milk. Time taken for the formation of clot in milk was recorded. (Milk-clotting activity(MCA) was defined by ( Berridge method )as one unit of enzymatic activity or rennet unit (RU)corresponds to the number of units of volume of milk that can be coagulated with 1ml of coagulant preparation in 100 seconds and at optimal pH and temperature ) Unit of milk-clotting activity (RU) formula: (RU)=(10*V)\(T C *Q) T c =time taken for clot formation (s) V=Volume of substrate in (ml) Q=volume of coagulant (ml).

3.BROMELAIN ENZYME. 18. The pineapple fruit and peel were separated and cut into small pieces..

19. Then juice was filtered with the help of whatmann filter paper.

4.CHEESE PRODUCTION. 20. Preparation of 2 sets of mozzarella cheese.

21. 250 mL fresh, raw Usually, cow milk that has been heated to roughly 32 °C is utilized.

22. The milk was heated until it coagulated and there was a clear separation of curds and whey, at which point the pot was taken off the heat and the curd and whey were poured into cheesecloth that was positioned over a container for collecting the whey. The milk was heated slowly with intermittent stirring clockwise and anticlockwise until it reached boiling..

Schematic representation of overall work. SERIAL DILUTION OF SOIL SAMPLE ENZY'Æ FROM PURIFICATION BROMELAIN ENZYME STAINItØ COTTCN sos POINT INEULAT'ION QUANTITATIVE ESTIMATION OF PROTEIN BY LOWRYS METHOD ENZYMATIC ASSAY OF BROMELAIN ENZYME CL.RTURE PREPARATION EXCHANGE CHEESE • SOUD STATE FERMENTATION AMMONIUM SULPHATE CRUDE PRECIPITATION BROMELAIN AND RENNET.

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26. abstract. abstract. abstract. Fig6: Mucor Seed Culture Obtained After 7 Days Of Incubation.

27. Fig9:Filtration Of Crude Enzyme. Fig10:Filtered Crude Enzyme.

28. Fig11: Substrate For Enzymatic Assay. Fig12:Milk-clotting Activity Of Crude Rennet.

29. abstract. abstract. Fig13:Crude Enzyme Obtained After Centrifugation.

30. abstract. abstract. Fig15: Enzyme Obtained After Salt Precipitation.

31. abstract. Fig17: Pellet Dissolved In 25mMtris Hcl.

32. Fig 19:Dialysis Of Rennet Enzyme. Fig20: Milk-clotting Activity Of Dialysed Sample.

33. Fig21: Stationary Phase. Fig22:Enzyme Loaded Into Stationary Phase.

34. taxm LOL. Fig 23 : 6 Eluates With 25mm Tris-hcl,( 25mm,50mm,75mm,100mm,125mm,150mm )Nacl Were Prepared In 6 Different Test Tubes..

35. Fig24:Enzymatic Assay Of Eluates.

36. abstract. Fig25:SDS PAGE. Fig26: Bands obtained after SDS PAGE lane A: enzyme purified by dialysis, lane B: enzyme purified by ammonium sulphate precipitation, lane C: ion-exchange chromatography..

37. Fig27:Peel Of Pineapple Crushed. Fig28:Fruit Part Of Pineapple Crushed.

38. Fig29:Filtered Crude Enzyme. Fig30:Crude Enzyme Obtained After Centrifugation.

39. Fig31:Left Side(precipitated Bromelain Enzyme) Right Side(precipitated Rennet Enzyme).

40. BOROSILG„. Fig33:Milk-clotting Activity Of Crude Bromelain Enzyme.

41. Fig36:250ml Amul Full Fat Cow Milk. Fig37:Clotting Of Milk.

42. Fig38: Cheese( Rennet+Bromelain ). Fig39: Cheese (Rennet).

ENZYMATIC ASSAY RESULTS. 43. Enzyme used Time required for clotting of 2ml of substrate Milk-clotting activity units (RU/ml) Crude rennet(0.2ml) 25min 0.067 Precipitated rennet (0.2ml) 15min 0.093 Dialysis(0.2ml) 10min 0.167 Elute2(0.2ml) 10min 0.167 Elute3 (0.2ml) 5min 0.334 Elute4 (0.2ml) 7min 0.238 Elute5 (0.2ml) 27min 0.062 Elute6 (0.2ml) 37min 0.046 Precipitated bromelain (0.2ml) 18min 0.112 Bromelain+rennet (0.1ml+0.1ml) 4min 0.416.

CLOTTING-STRENGTH. 44. Clotting strength(RU/0.00045) PERCENTAGE Crude rennet(0.2ml) 14.89 Precipitated rennet (0.2ml) 20.67 Dialysis(0.2ml) 37.12 Elute2(0.2ml) 37.12 Elute3 (0.2ml) 74.23 Elute4 (0.2ml) 52.89 Elute5 (0.2ml) 13.78 Elute6 (0.2ml) 10.23 Precipitated bromelain (0.2ml) 24.89 Bromelain+rennet (0.1ml+0.1ml) 92.45.

45. We were able to avoid contamination by using a medium bottle for solid-state fermentation. After a 10-day incubation period at room temperature, we were able to extract the rennet enzyme from the mucor strain. Among the other purification methods, microbial rennet purified by ion exchange chromatography had the fastest milk clotting time (5 minutes). When bromelain and rennet were used together, milk clotting time was faster (4 minutes) than with microbial rennet (5 minutes). The clotting power of the combination of enzymes microbial rennet and bromelain was 92.45% higher than that of microbial rennet alone (74.23%). The cheese yield while utilizing microbial rennet was 15.374%. The cheese yield while utilizing a mix of enzymes, microbial rennet, and bromelain was 18.038% Mozarella cheese has a moisture level that ranges between 45 and 52%. When enzyme microbial rennet and bromelain were combined, the moisture level was 50. 195.When clotting strength, milk-clotting time, and cheese quality were assessed, the results obtained by combining microbial rennet with bromelain surpassed microbial rennet alone..

OUTCOME OF THE PROJECT. Microbial Rennet isolated from mucor . Cheese produced using microbial rennet enzyme. Cheese produced using microbial rennet and bromelain enzyme ..

REVIEW PAPER-TITLE AND CONTENTS. TITLE: ALTERNATIVES TO CALF RENNET AND OTHER INGREDIENTS USED IN CHEESE MAKING 1. ABSTRACT 2. INTRODUCTION 3. TYPES OF CHEESE 4. FLAVOURS ENHANCERS AND DEBITTERING 5. INGREDIENTS FOR CHEESE MANUFACTURING 6. DIFFERENT MILK CLOTTING ENZYMES USED IN CHEESE PRODUCTION 7. CLASSIFICATION OF MICROBIAL MILK CLOTTING ENZYMES 8.DISCUSSION 9. CONCLUSION 10.FUTURE PROSPECTS 12. REFERENCES.

48. DETAILSde DETAILS edeDETAILS RESPO RESPONSE NSE Tentative Title of the project Isolation of microbial rennet enzyme from microorganism and its purification, characterization, and comparison with animal rennet enzyme for various cheese production Tentative Objectives To isolate mucor strain from dairy effluent soil and produce microbial rennet enzyme Novelty statements Comparative analysis of the use of animal and microbial rennet enzymes in the manufacturing of cheese. No of papers referred to identify the gaps and frame the objective 13 No of days required for literature survey 21 days Time required to complete the project in 8 th semester 4 months Chemicals and materials required Sabouraud dextrose agar, animal rennet enzyme, glassware, ammonium sulphate.

49. DETAILS RESPONSE Software Required No Microorganisms required No Planning to do project internally or externally External Expertise available in CBIT NA Any ethical approvals required NA.

REFERENCES. 1.Chinmayee, Cirium V., et al. "Production of highly active fungal milk-clotting enzyme by solid-state fermentation." Preparative Biochemistry & Biotechnology 49.9 (2019): 858-867. 2. Sambo , S., et al. "An overview of the solid state fermentation in the production of fungal protease enzymes." World Journal of Advanced Research and Reviews 9.3 (2021): 085-089. 3. Abada , Emad A. "Application of microbial enzymes in the dairy industry." Enzymes in food biotechnology . Academic Press, 2019. 61-72. 4. Hickey, M. (2017). Chapter 30 - Legislation in Relation to Cheese (Fourth edition) (pp. 757-778). San Diego: Academic Press 5. Johnson, M. E., & Lucey, J. A. (2006). Major technological advances and trends in Cheese. Journal of Dairy Science, 89(4), 1174–1178 6 . Mamo , Jermen , et al. "Optimization of media composition and growth conditions for production of milk-clotting protease (MCP) from Aspergillus oryzae DRDFS13 under solid-state fermentation." Brazilian Journal of Microbiology 51 (2020): 571-584. 7. Rashidinejad, A., Bremer, P., Birch, J., & Oey, I. (2017). Chapter 14 - Nutrients in Cheese and Their Effect on Health and Disease. In R. R. Watson, R. J. Collier, & V. R. Preedy (Eds.), Nutrients in Dairy and their Implications on Health and Disease (pp. 177-192): Academic Press..