Fermentation-produced chymosin
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Rennet () is a complex set of enzymes produced in the stomachs of ruminant mammals. Chymosin, its key component, is a protease enzyme that curdles the casein in milk. In addition to chymosin, rennet contains other enzymes, such as pepsin and a
lipase Lipase ( ) is a family of enzymes that catalyzes the hydrolysis of fats. Some lipases display broad substrate scope including esters of cholesterol, phospholipids, and of lipid-soluble vitamins and sphingomyelinases; however, these are usually tr ...
. Rennet has traditionally been used to separate milk into solid curds and liquid whey, used in the production of cheeses. Rennet from calves has become less common for this use, to the point that less than 5% of cheese in the United States is made using animal rennet today. Most cheese is now made using chymosin derived from bacterial sources.


Molecular action of rennet enzymes

One of the main actions of rennet is its protease chymosin cleaving the kappa casein chain. Casein is the main protein of milk. Cleavage causes casein to stick to other cleaved casein
molecules A molecule is a group of two or more atoms held together by attractive forces known as chemical bonds; depending on context, the term may or may not include ions which satisfy this criterion. In quantum physics, organic chemistry, and bioche ...
and form a network. It can cluster better in the presence of calcium and phosphate. This is why those chemicals are occasionally added to supplement pre-existing quantities in the cheese making process, especially in calcium phosphate-poor goat milk. The solid truncated casein protein network traps other components of milk, such as fats and minerals, to create cheese.


Extraction of calf rennet

Calf Calf most often refers to: * Calf (animal), the young of domestic cattle. * Calf (leg), in humans (and other primates), the back portion of the lower leg Calf or calves may also refer to: Biology and animal byproducts * Veal, meat from calves * ...
rennet is extracted from the inner
mucosa A mucous membrane or mucosa is a membrane that lines various cavities in the body of an organism and covers the surface of internal organs. It consists of one or more layers of epithelial cells overlying a layer of loose connective tissue. It is ...
of the fourth stomach chamber (the abomasum) of young, nursing calves as part of livestock butchering. These stomachs are a
byproduct A by-product or byproduct is a secondary product derived from a production process, manufacturing process or chemical reaction; it is not the primary product or service being produced. A by-product can be useful and marketable or it can be consid ...
of
veal Veal is the meat of calves, in contrast to the beef from older cattle. Veal can be produced from a calf of either sex and any breed, however most veal comes from young male calves of dairy breeds which are not used for breeding. Generally, v ...
production. Rennet extracted from older calves (
grass-fed There are different systems of feeding cattle in animal husbandry. For pastured animals, grass is usually the forage that composes the majority of their diet. Cattle reared in feedlots are fed hay supplemented with grain, soy and other ingredi ...
or grain-fed) 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 species, milk-specific rennets are available, such as kid goat rennet for
goat's milk Goat milk is the milk of domestic goats. Goats produce about 2% of the world's total annual milk supply. Some goats are bred specifically for milk. Goat milk naturally has small, well-emulsified fat globules, which means the cream will stay in ...
and lamb rennet for
sheep's milk Sheep's milk (or ewes' milk) is the milk of domestic sheep. It is commonly used to make cultured dairy products such as cheese. Some of the most popular sheep cheeses include feta (Greece), ricotta (Italy), and Roquefort (France). Sheep breeds S ...
.


Traditional method

Dried and cleaned stomachs of young calves are sliced into small pieces and then put into salt water 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 2 to 4 litres of milk.


Modern method

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 form and are activated by the stomach acid. The acid is then neutralized and the rennet extract is filtered in several stages and concentrated until reaching a typical potency of about 1:15,000; meaning 1 g of extract can coagulate 15 kg of milk. One kg of rennet extract has about 0.7 g of active enzymes – the rest is water and salt and sometimes sodium benzoate (
E211 Sodium benzoate is the sodium salt of benzoic acid, widely used as a food preservative (with an E number of E211) and a pickling agent. It appears as a white crystalline chemical with the formula C6H5COONa. Production Sodium benzoate is comm ...
), 0.5%–1.0% for preservation. Typically, 1 kg of cheese contains about 0.0003 g of rennet enzymes.


Alternative sources

Because of the limited availability of mammalian stomachs for rennet production, cheese makers have sought other ways to coagulate milk since at least Roman times. The many sources of enzymes that can be a substitute for animal rennet range from plants and fungi to microbial sources. Cheeses produced from any of these varieties of rennet are suitable for lactovegetarians. Fermentation-produced chymosin is used more often in industrial cheesemaking in North America and Europe today because it is less expensive than animal rennet.


Vegetable

Many plants have coagulating properties. Homer suggests in the '' Iliad'' that the Greeks used an extract of
fig The fig is the edible fruit of ''Ficus carica'', a species of small tree in the flowering plant family Moraceae. Native to the Mediterranean and western Asia, it has been cultivated since ancient times and is now widely grown throughout the world ...
juice to coagulate milk. Other examples include several species of Galium, dried caper leaves, nettles, thistles, mallow, '' Withania coagulans'' (also known as Paneer Booti, Ashwagandh and the Indian Cheesemaker), and
ground ivy ''Glechoma hederacea'' is an aromatic, perennial, evergreen creeper of the mint family Lamiaceae. It is commonly known as ground-ivy, gill-over-the-ground, creeping charlie, alehoof, tunhoof, catsfoot, field balm, and run-away-robin. It is als ...
. Some traditional cheese production in the Mediterranean uses enzymes from thistle or ''
Cynara ''Cynara'' is a genus of thistle-like perennial plants in the family Asteraceae. They are native to the Mediterranean region, the Middle East, northwestern Africa, and the Canary Islands. The genus name comes from the Greek ''kynara'', which mea ...
'' (artichokes and cardoons). Phytic acid, derived from unfermented soybeans, or fermentation-produced chymosin (FPC) may also be used. Vegetable rennet might be used in the production of
kosher (also or , ) is a set of dietary laws dealing with the foods that Jewish people are permitted to eat and how those foods must be prepared according to Jewish law. Food that may be consumed is deemed kosher ( in English, yi, כּשר), fro ...
and halal cheeses, but nearly all kosher cheeses are produced with either microbial rennet or FPC. Commercial so-called vegetable rennets usually contain an extract from the mold ''
Rhizomucor miehei ''Rhizomucor miehei'' (also: ''Mucor miehei'' ) is a species of fungus A fungus ( : fungi or funguses) is any member of the group of eukaryotic organisms that includes microorganisms such as yeasts and molds, as well as the more familiar ...
'' described below.


Microbial

Some molds such as ''
Rhizomucor miehei ''Rhizomucor miehei'' (also: ''Mucor miehei'' ) is a species of fungus A fungus ( : fungi or funguses) is any member of the group of eukaryotic organisms that includes microorganisms such as yeasts and molds, as well as the more familiar ...
'' are able to produce proteolytic enzymes. These molds are produced in a fermenter and then specially concentrated and purified to avoid contamination with unpleasant byproducts of the mold growth. The traditional view is that these coagulants result in bitterness and low yield in cheese, especially when aged for a long time. Over the years, microbial coagulants have improved a lot, largely due to the characterization and purification of secondary enzymes responsible for bitter peptide formation/non-specific proteolytic breakdown in cheese aged for long periods. Consequently, it has become possible to produce several high-quality cheeses with microbial rennet. It is also suitable for the elaboration of vegan cheese, provided no animal-based ingredients are used in its production.


Fermentation-produced chymosin

Because of the above imperfections of microbial and animal rennets, many producers sought other replacements of rennet. With genetic engineering it became possible to isolate rennet genes from animals and introduce them into certain bacteria, fungi, or yeasts to make them produce recombinant chymosin during fermentation. The genetically modified microorganism is killed after fermentation and chymosin isolated from the fermentation broth, so that the fermentation-produced chymosin (FPC) used by cheese producers does not contain a GMO or any GMO DNA. FPC is identical to chymosin made by an animal, but is produced in a more efficient way. FPC products have been on the market since 1990 and, because the quantity needed per unit of milk can be standardized, are commercially viable alternatives to crude animal or plant rennets, as well as generally preferred to them in industrial production. Originally created by biotechnology company Pfizer, FPC was the first artificially-produced enzyme to be registered and allowed by the US Food and Drug Administration.Staff, National Centre for Biotechnology Education, 2006
Case Study: Chymosin
/ref> In 1999, about 60% of US
hard cheese There are many different types of cheese. Cheeses can be grouped or classified according to criteria such as length of fermentation, texture, methods of production, fat content, animal milk, and country or region of origin. The method most comm ...
s were made with FPC, and it has up to 80% of the global market share for rennet. By 2017, FPC takes up 90% of the global market share for rennet. The most widely used FPC is produced either by the fungus '' Aspergillus niger'' and commercialized under the trademark CHY-MAX by the Danish company
Chr. Hansen Chr. Hansen A/S is a bioscience company based in Hørsholm, Denmark. The company is a supplier of bacteria cultures, probiotics, enzymes and human milk oligosaccharides. Its products are used in the production of fresh dairy, cheese, meat, seafoo ...
, or produced by '' Kluyveromyces lactis'' and commercialized under the trademark Maxiren by the Dutch company DSM. FPC is chymosin B, so it is purer than animal rennet, which contains a multitude of proteins. FPC provides several benefits to the cheese producer compared with animal or microbial rennet: higher production yield, better curd texture, and reduced bitterness. Cheeses produced with FPC can be certified kosher and halal, and are suitable for vegetarians if no animal-based alimentation was used during the chymosin production in the fermenter.


Nonrennet coagulation

Many soft cheeses are produced without use of rennet, by coagulating milk with acid, such as citric acid or vinegar, or the lactic acid produced by soured milk. Cream cheese, paneer, rubing, and other acid-set cheeses are traditionally made this way. The acidification can also come from bacterial fermentation such as in
cultured milk Fermented milk products or fermented dairy products, also known as cultured dairy foods, cultured dairy products, or cultured milk products, are dairy foods that have been fermented with lactic acid bacteria such as ''Lactobacillus'', '' Lactococ ...
. Vegan alternatives to cheese are manufactured without using animal milk but instead use soy, wheat, rice or cashew. These can be coagulated with acid using sources such as vinegar or lemon juice.


In mythology

In Yazidism, the Earth is believed to have coagulated and formed when rennet flowed from the White Spring of the celestial
Lalish Lalish ( ku, لالش, translit=Laliş, also known as Lalişa Nûranî) is a mountain valley and temple in Shekhan, Duhok Governorate in Iraq. It is the holiest temple of the Yazidis. It is the location of the tomb of the Sheikh Adi ibn Musafir, ...
in heaven into the Primeval Ocean.


See also

*
Cheese Cheese is a dairy product produced in wide ranges of flavors, textures, and forms by coagulation of the milk protein casein. It comprises proteins and fat from milk, usually the milk of cows, buffalo, goats, or sheep. During production, ...
* Junket (dessert) * Milk * Pepsin


References


Footnotes


Bibliography

* 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 and Jane Voichick


External links


Fankhauser's Page on Rennet history and use

Appendix D - Assessment of filamentous fungi - Qualified Presumption of Safety





Cheese Yield Experiments and Proteolysis by Milk-Clotting Enzymes

Validation of recombinant and bovine chymosin by mass spectrometry

Native and Biotechnologically Engineered Plant Proteases with Industrial Applications
{{Enzymes Animal glandular products Cattle products Cheese Dairy industry EC 3.4.23