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.
In 1 kg of rennet extract there are about 0.7 grams of active enzymes – 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.
As the proper coagulation is done by enzymatic activity, the task was to find enzymes for cleaving the casein that would result in a taste and texture similar to animal-based rennet.
Many plants have coagulating properties. Some examples include fig tree bark, nettles, thistles, mallow, and Creeping Charlie. Enzymes from thistle or cynara are 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 specially 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.
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, fungi or yeasts 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. One example of a commercially available genetically engineered rennet is Chymax, created by Pfizer.
Today the most widely used genetically engineered 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 genetically engineered 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 genetically engineered 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.
Cream cheese, paneer, and rubing are traditionally made this way (see Acid-set cheeses for others), and this form of coagulation is sometimes used in cheap mozzarella production without maturation of the cheese.
The acidification can also come from bacterial fermentation such as in cultured milk.