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Rennet is a natural complex of enzymes produced in any mammalian stomach to digest the mother's milk. Rennet contains 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 rennin or chymosin (EC 3.4.23.4) but there are also other important enzymes in it, e. g., pepsin or lipase. There are non-animal sources for rennet substitutes.

Contents 1 Sources 2 Uses 3 Production of natural calf rennet 3.1 Traditional method 3.2 Modern method 4 Alternative coagulants 4.1 Vegetable rennet 4.2 Microbial rennet 4.3 Genetically engineered rennet 4.4 Acid coagulation 5 See also 6 External links 7 References

The most common source for animal-based rennet is the abomasum (fourth stomach) of slaughtered, milk-fed, newborn cow calves. However, stomachs of other young ruminants such as goats (for goat's milk) or lamb (for ewe's milk), and pseudo-ruminants like camels (for camel's milk) are also used.

The chief use of rennet is in the making of cheese and Junket. Chymosin reacts specifically with κ-casein, cleaving the protein between the amino acids phenylalanine(105) and methionine (106), producing two fragments. The soluble fragment (residues 106-169) which becomes part of the whey is known as glyco macro peptide and contains the glycosylation sites for κ-casein. The other component (residues 1-105) is insoluble. This in the presence of calcium ions causes the coagulation of the casein micelles to form a curd.

Natural rennet is produced in the inner mucosa of the fourth stomach of young ruminants. These stomachs are a by-product of veal production.

Today this method is only used by traditional cheese-makers in central Europe, e.g. in Switzerland, Jura in France, or Alp-Sennereien in Austria.

Dried and cleaned stomachs of young calves are sliced into small pieces and then put into saltwater, together with some vinegar or wine to get a lower pH. After some time – overnight or several days - this solution has to be filtered. This crude rennet can then be used for coagulation of the milk. 1 gram of this solution should then coagulate 2000 to 4000 grams of milk.

Deep-frozen stomachs are milled and put into an extracting solution – in this solution the enzymes are extracted. The crude rennet extract is then activated by adding acid – the enzymes in the stomach are produced in an inactive pre-form and are activated by the stomach acid. After neutralisation of the acid, the rennet extract is filtered in several stages and concentrated until reaching the required potency - e.g. 1:15.000 (1 kg of rennet would have the ability to coagulate 15,000 litres of milk).

1 kg of rennet extract contains about 0.7 grams of active enzymes and no other organic material – the rest is water and salt and sometimes some sodium benzoate, E211, 0.5% - 1% for preservation. 1 kg of cheese contains about 0.0003 grams of rennet enzymes.

Because of the limited availability of proper stomachs for rennet production, cheesemakers have always looked for other ways to coagulate the milk. Artificial coagulants are a useful alternative, especially for cheap or lower-quality cheeses.

As the proper coagulation is done by enzymatic activity, the task was to find enzymes for cleaving the casein that would result in taste and texture similar to what animal-based rennet produces.

Many plants have coagulating properties. Some examples include fig tree bark, nettles, thistles, mallow, and Creeping Charlie. Rennet from thistle or cynara is used in some traditional cheese production in the Mediterranean.

These real vegetable rennets are also suitable for vegetarians. Vegetable rennet might be used in the production of kosher cheeses but nearly all kosher cheeses are produced with either microbial rennet or GM rennet. Worldwide there is also no industrial production for vegetable rennet. Commercial so-called vegetable rennets usually contain rennet from the mold Mucor miehei - see microbial rennet below.

There are some moulds like Rhizomucor miehei that have the capability to produce proteolytic enzymes. These moulds are produced in a fermenter and then especially concentrated and purified to avoid contamination with unpleasant side products of the mould growth. At the present state of scientific research institutes like the EFSA deny the QPS-status - Qualified Presumption of Safety - to enzymes produced especially by these moulds.

The flavour and taste of cheeses produced with microbial rennets tend towards some bitterness, especially if longer maturation is wanted. These so-called "microbial rennets" are suitable for vegetarians if there was no animal-based alimentation used during the production in the fermenter.

Because of the above imperfections of microbial rennets, some big companies sought further replacements of natural rennet. With the development of genetic engineering, it suddenly became possible to use calf-genes to modify some bacteria, fungus or yeast to make them produce Chymosin. Chymosin produced by genetically modified organisms was the first artificially produced enzyme to be registered and allowed by the FDA in the USA. In 1999, about 60% of U.S. hard cheese was made with genetically engineered Chymosin.^[1]

Today the most widely-used genetic rennet is produced by the fungus Aspergillus niger. The problems of destroying the aflatoxins or the antibiotic resistant marker genes seem to be solved.

Cheese production with genetic rennet is similar to production with natural calf rennet. Genetic rennet contains only one of the known main chymosin types – either type A or type B. Some of the other chymosin types found in genetic rennet do not exist in natural rennet. This is also the reason why special analysis can determine what kind of coagulant has been used.

Often a mixture of genetic Chymosin and natural pepsin is used to imitate the complexity of natural rennet and to get the same results in coagulation and in development of flavour and taste.

The so-called "GM rennets" are suitable for vegetarians if there was no animal based alimentation used during the production in the fermenter -- but only for vegetarians who are not opposed to GM-derived foods.

Milk can also be coagulated by adding some acid, e.g. citric acid. This form of coagulation is sometimes used in cheap mozzarella production without maturation of the cheese. Paneer is also made this way. The acidification can also come from bacteria as happens in soured milk.

• Pepsin
• Rennet is the name of a character in the 1932 satire Cold Comfort Farm

• EFSA and Qualified Assumption of Safety
• FDA-registration of recombinant chymosin
• Recombinant Chymosin
• Cheese Yield Experiments and Proteolysis by Milk-Clotting Enzymes
• Validation of recombinant and bovine chymosin by mass spectrometry
• Free Article: Rennet 101
• Mass spectrometric characterisation of proteins in rennet and in chymosin-based milk-clotting preparations

Carroll, Ricki. Making Cheese, Butter, & Yogurt. Storey Publishing 2003.

"Biotechnology and Food: Leader and Participant Guide," publication no. 569, produced by North Central Regional Extension. Printed by Cooperative Extension Publications, University of Wisconsin-Extension, Madison, WI, 1994. Publication date: 1994.

Tom Zinnen, biotechnology specialist, University of Wisconsin-Extension and UW Biotechnology Center, UW-Madison; and Jane Voichick, professor, Nutritional Sciences, UW-Madison and UW- Extension.