Agricultural Engineering College & Research Institute

AGRICULTURAL ENGINEERING COLLEGE & RESEARCH INSTITUTE TAMIL NADU AGRICULTURAL UNIVERSITY, CBE-3

ASSIGNMENT ON RENNET PRODUCTION AGM C51 INDUSTRIAL MICROBIOLOGY (2+1) BY S.SHANMUGA PRIYA BTF-07-023

COURSE TEACHER: DR. N.O. GOPAL EXAMINER:

EXTERNAL

RENNET: Cheese production using rennet is the oldest use of milk. The production of countless different types of cheese use proteolytic enzymes, of which rennin is the most widely used. Rennin is extracted from the gastric mucous membrane of bovines. Milk coagulation is irreversible and has two phases. In the first phase or enzymatic phase, the colloid that protects the casein (K-casein) dissociates the glycomacropeptides (part of K-casein, integrated by non-proteinic nitrogen), thereby making the protective effect disappear. The second stage or coagulation phase involves the formation of saline bridges at favorable temperatures between the calcium-sensitive casein micelles, thereby quickly producing coagulation. DIFFERENT TYPES OF CHEESE: 

Cottage cheese

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Roquefort

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Stilton

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Blue cheese

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Gorgonzola

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Camembert

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Brie

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Swiss

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Baby Swiss

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Gouda

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Edam

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Limburger

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Traditional brick cheese

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Cheddar

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Colby

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Sweet Brick

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Muenster

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Havarti

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Mozzarella

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Provolone

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Parmesan

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Romano

MICROBIAL RENNET: The increasing global cheese production in association with demand for meat made more calves reared to maturity and shortage of calf rennet let to high cost of natural calf rennet and finds alternate proteolytic enzymes that would duplicate the action of calf rennet in cheese vat. MICROBIAL RENNET: Many microorganisms are known to produce rennet-like proteinases which can substitute the calf rennet. Microorganisms like Rhizomucor pusillus, R. miehei, Endothia parasitica, Aspergillus oryzae, and Irpex lactis are used extensively for rennet production in cheese manufacture. Extensive research that has been carried out so far on rennet substitutes has been reviewed by several authors. Different strains of species of Mucor are often used for the production of microbial rennets. Microbial rennets from various microorganisms (marketed under the trade names such as Rennilase, Fromase, Marzyme, Hanilase, etc.) being marketed since the 1970s have proved satisfactory for the production of different kinds of cheese. The molecular and enzymatic properties of chymosins have been studied extensively. Although the proteolytic specificities of the three commonly used fungal rennets are considerably different from those of calf chymosin, these rennets have been used to produce acceptable cheeses. Recently Novo Nordisk has succeeded in expressing just one proteolytic enzyme from the fungus R. miehei in the wellknown organism A. oryzae. This host organism is able to produce the single protease that cleaves the · -casein into a glycomacropeptide and para · -casein by hydrolysing only at the phe105–met106 peptide bond between phenyl alanine and methionine. This monocomponent enzyme product has the trade name Novoren. One major drawback of microbial rennet use in cheese manufacture, is the development of off flavour and bitter taste in the nonripened as well as in the ripened cheeses. The rennets from microbial sources are more proteolytic in nature in comparison to rennet from animal sources, resulting in production of some bitter peptides during the process of cheese ripening. Hence, attempts have been made to clone the gene for calf chymosin, and to express it in selected bacteria, yeasts, and molds. RECOMBINANT RENNET: Gene for calf chymosin was one of the first genes for mammalian enzymes that was cloned and expressed in microorganisms. The use of Chymosin (genetically-engineered rennin or rennet) for cheese-making was the first commercial application of food biotech (1988). In the 1960s the Food and Agriculture Organization of the United Nations predicted a severe shortage of calf rennet. It was anticipated that an increased demand for meat would lead to more calves being reared to maturity, and hence less rennet would be

available. Over the last 30 years several substitutes for calf rennet have been developed, allowing the supply of enzymes to keep pace with cheese production. The first Chymosins in the early 1980s were derived from genetically-modified microbes (Escherichia coli, Kluyveromyces lactis and Aspergillus niger). Numerous groups have since followed their lead, using other microbes, so that chymosin has now been obtained from food yeasts. Today about 90% of the hard cheese is made using chymosin from geneticallymodified microbes and 60% of U.S. hard cheese was made with genetically engineered chymosin it has up to 80% of the global market share for rennet.. Chymosin is identical to the enzyme obtained from animals. This can be used to produce better quality cheese than the fungal or other animal (non-calf) rennets. These bioengineered enzymes behave in exactly the same way as calf rennin, but their activity is more predictable and they have fewer impurities. Such enzymes have gained the support of vegetarian organizations and of some religious authorities. Chymosin obtained from recombinant organisms has been subjected to rigorous tests to ensure its purity. Highly recommended by some of the best known cheese makers this is high quality rennet, originating from animal sources, but containing no animal products itself a vegetable product with animal origins. the most widely used genetically engineered rennet is produced by the fungus Aspergillus niger. Other micro organisms are Escherichia coli, Kluyveromyces lactis. Escherichia coli . It is not regarded as GRAS (Generally Regarded As Safe) because recombinant proteins are frequently synthesized as intracellular inclusion bodies process cost increases. Cheese production with chymosin is similar to production with natural calf rennet. Preparation of calf rennet has two forms of chymosin A in proportion 40% & chymosin B 60 % Genetic rennet contains only one of the known main chymosin types, either type A or type B and Other do not exist. Genetically engineered chymosin and natural pepsin is mixed to imitate the complexity of natural rennet and to get the same results in coagulation and in development of flavor and taste. Such rennets are suitable for vegetarians if there was no animal based alimentation used during the production in the fermenter. Aspergillus niger Aspergillus niger is a fungus and one of the most common species of the genus Aspergillus. It is a common contaminant of food. A. niger is cultured for the industrial production of many substances. Various strains of A. niger are used in the industrial preparation of citric acid (E330) and gluconoic acid (E574). A. niger fermentation is "generally regarded as safe“ by the FDA. The industrial production of enzymes requires: Suitable producer organisms (selection, safety evaluation, optimisation of yield, culture media, fermentors, scaling-up. Isolation (purification) of the products (down stream-processing) and formulation. The volume of the fermenting mass increases from few ml to 100-200 m3.

Medium & Cultivation Parameter: The medium used is complex medium maltose as C source. It is a fed batch process and scales approx 50 qm. Production under “contained use” conditions. CONTAINED USE: EU Contained Use Directive (Art. 2c): ‘contained use’ shall mean any activity in which micro-organisms are genetically modified or in which such GMMs are cultured, stored, transported, destroyed, disposed of or used in any other way, and for which specific containment measures are used to limit their contact with, and to provide a high level of safety for, the general population and the environment; FERMENTATION: The production organism is grown in several stages to build up the inoculum for large-scale production. After inoculation to the fermenter, the cells are grown aerobically under the proper conditions of pH, temperature, nutrient composition, etc. After chymosin reaches a desired level in the fermentation broth, the fermentation is stopped and the fungal cells are inactivated and separated from the liquid.

DOWN STREAM PROCESSING:

Chymosin is then recovered from the broth by one of two methods: (1) the broth is filtered, followed by chromatographic purification and concentration of chymosin; or (2) the chromatographic step is preceded by extraction of chymosin from the fermentation broth. Chymosin eluted from the chromatographic column is formulated to the commercial strength (Chr. Hansen's,1989).

THE OVER ALL PROCESS:

Rennet producing industries: • Gist brocades – maxiren – K.lactis • Genecor / Chr. Hansen – chymogen – A.niger • Pfizer – chymax- E.coli • Iranian industrial enzymes company – rennilase