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. If milk were not coagulated, it would rapidly flow through the stomach and miss the opportunity for initial digestion of its proteins.
Chymosin efficiently converts liquid milk to a semisolid like cottage cheese, allowing it to be retained for longer periods in the stomach. Chymosin secretion is maximal during the first few days after birth, and declines thereafter, replaced in effect by secretion of pepsin as the major gastric protease.
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.
In order to understand how chymosin coagulates milk, one needs to know something about milk proteins. 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 - the normal calcium concentration in milk is far in excess of that required to precipitate these proteins. However, kappa casein is a distinctly different molecule - it is not calcium-precipitable. As the caseins are secreted, they self-associate into aggregates called micelles in which the alpha and beta caseins are kept from precipitating by their interactions with kappa casein. In essence, kappa casein normally keeps the majority of milk protein soluble and prevents it from spontaneously coagulating.
Enter chymosin. 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.
Aside from its physiologic role, chymosin is also a very important industrial enzyme because it is widely used in cheesemaking. In days gone by, chymosin was extracted from dried calf stomachs for this purpose, but the cheesemaking industry has expanded beyond the supply of available calf stomachs (remember that these have to be from young calves). It turns out that many proteases are able to coagulate milk by converting casein to paracasein and alternatives to chymosin are readily available. "Rennet" is the name given to any enzymatic preparation that clots milk.RENNET.
Rennet (IPA pronunciation: [ˈɹɛnɪt]) 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 18.104.22.168) but there are also other important enzymes in it, e.g., pepsin or lipase. There are non-animal sources for rennet substitutes.
The chief use of rennet is in the making of cheese, curd, 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, and in the presence of calcium ions causes the coagulation of the casein micelles to form a curd.
Production of natural calf rennet
Natural calf rennet is extracted from the inner mucosa of the fourth stomach chamber (the abomasum) of young calves. These stomachs are a by-product of veal production. If rennet is extracted from older calves (grass-fed or grain-fed) the rennet contains less or no chymosin but a high level of pepsin and can only be used for special types of milk and cheeses. As each ruminant produces a special kind of rennet to digest the milk of its own mother, there are milk-specific rennets available, such as kid-goat rennet especially for goat's milk and lamb-rennet for sheep-milk. Rennet or digestion enzymes from other animals, like swine-pepsin, are not used in cheese production. (Swine-pepsin is, however, used in the analysis of disulfide bonds of proteins.)
Dried and cleaned stomachs of young calves are sliced into small pieces and then put into saltwater or whey, together with some vinegar or wine to lower the pH of the solution. After some time, (overnight or several days) the solution is filtered. The crude rennet that remains in the filtered solution can then be used to coagulate milk. About 1 gram of this solution can normally coagulate 2000 to 4000 grams of milk.
Today this method is used only by traditional cheese-makers in central Europe: Switzerland, Jura, France, Romania and Alp-Sennereien in Austria.
Deep-frozen stomachs are milled and put into an enzyme-extracting solution. 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: about 1:15000 (1 kg of rennet would have the ability to coagulate 15000 litres of milk).
In 1 kg of rennet extract there are about 0.7 grams of active enzymes and no other organic material – the rest is water and salt and sometimes sodium benzoate, E211, 0.5% - 1% for preservation. Typically, 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 no industrial production for vegetable rennet. Commercial so-called vegetable rennets usually contain rennet from the mold Mucor miehei - see microbial rennet below.
Some molds, such as Rhizomucor miehei are able to produce proteolytic enzymes. These molds are produced in a fermenter and then especially concentrated and purified to avoid contamination with unpleasant side products of the mold growth. At the present state of scientific research, governmental food safety organizations such as the EFSA deny QPS (Qualified Presumption of Safety) status to enzymes produced especially by these molds.
The flavor and taste of cheeses produced with microbial rennets tend towards some bitterness, especially after longer maturation periods. These so-called "microbial rennets" are suitable for vegetarians, provided no animal-based alimentation was used during the production.
Genetically engineered rennet
Because of the above imperfections of microbial rennets, some producers 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.
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. Other chymosin types found in natural rennet do not exist in genetic rennet. This is also the reason why special analysis can determine what kind of coagulant has been used by analyzing what bonds have and haven't been cleaved.
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 bacterial fermentation such as in cultured milk.