Enzymes used in cheese production
Rennet preparations from the stomachs of calves, lambs and kids have been used in cheese production for thousands of years. These rennets contain the enzyme rennin (chymosin, an aspartic protease), which performs limited proteolysis of milk protein (casein) to form curds at the start of cheese making. Fig sap, containing the proteolytic enzyme ficin, has sometimes been used for the same purpose, but many other proteases carry out too much proteolysis.
Animal rennets, particularly from calves, predominated in cheese manufacture until the 1960s when an increase in cheese production coincided with a shortage of available animal rennet. Specific fungal proteases, which have very similar properties to calf chymosin, were then developed as microbial rennets, such as proteases from Rhizomucor miehei and R. pusillus. This overcame the shortfall and facilitated the production of so-called ‘vegetarian’ cheese. Some microbial enzyme preparations are also more temperature-sensitive (thermolabile) than calf rennet, which is useful in certain cheese-making processes.
More recently, the calf chymosin gene has been introduced into several microorganisms, including E. coli, K. lactis, Aspergillus nidulans and A. niger var. awamori. These genetically engineered microorganisms are capable of expressing and secreting the enzyme. Recombinant chymosin preparations now have approval for use as food additives in over 20 countries. Animal and microbial chymosins (constituting about one-third of the market) have combined world sales worth in excess of $75 million. Microbial lipases are also used in dairy products, especially cheeses, for the hydrolysis of fatty acid esters to accelerate flavour development.
Enzymes used in plant juice production
Several microbial enzymes are employed in fruit juice processing, but probably the most important are pectinases. These processing aids are often produced by solid-substrate fermentation of A. niger or Penicillium species. Fruits and berries contain varying amounts of pectin, which acts as a binding layer between plant cells to hold adjacent cell walls together. Pectin is a heteropolymer of galacturonic acid, methyl esters of galacturonic acid and other sugar residues.
In plant juice production, some of this pectin is extracted during pressing. It causes an increase in juice viscosity, leading to difficulties in obtaining optimal juice yields, and in juice clarification and filtration. These problems can be overcome by adding pectinase preparations to the fruit pulp before pressing. Similar enzyme treatment is used to increase the yield of oils from olive pulp, palm fruit and coconut flesh.
The commercial ‘pectinase’ is not one enzyme, but is usually a complex cocktail of enzymes (pectin methyl esterases, polygalacturonases and pectin lyases) capable of attacking a variety of bonds in correspondingly diverse pectin molecules. Compositions of commercial pectinase preparations vary considerably in the proportions of these different enzymes. Fungal pectinases are also available that remain active in very acidic juices from citrus fruits, where the pH can be as low as 2.2–2.8. They may be used in the enzymic peeling of citrus fruit for canning.
The polysaccharide araban, a polymer of the pentose arabinose, is also an important component of fruit cell walls. Like pectin, it is often extracted during pressing of some fruits, especially pears. This may lead to haze formation, but it can now be eliminated by using commercial arabanases. Also, some extracts, such as apple juice, contain starch, which must be degraded to produce clear juices and concentrates. This is achieved by adding amylases, along with the pectinase, during depectinization of the juice.
In wine making, commercial enzymes are used for several purposes, as well as for juice extraction. For red wines, colour extraction from the grape skins during pressing can be promoted by the addition of commercial cellulases, e.g. from the fungus Trichoderma reesei. In addition, glycosidases can be employed to hydrolyse terpenyl glycosides, releasing the terpenes that are important constituents of the wine bouquet. Wines prepared from grapes allowed to undergo attack by the fungus Botrytis cinerea before harvesting (‘noble rot’) are often difficult to clarify and filter, due to the presence of fungal β-glucans. These polymers can be degraded by adding a specific microbial β-glucanase to the wine.
Citrus fruits, such as grapefruit and bitter oranges, contain the bitter-tasting fruit flavonoid called naringin. The bitterness of products derived from these fruits can be adjusted using naringinase (α-rhamnosidase + β-glucosidase) from A. niger. It first converts naringin to the less bitter compound prunin, then on to non-bitter naringenin. The level of naringin hydrolysis may be controlled by regulating the flow rate of fruit juice through a column of immobilized naringinase.
Glucose oxidase from A. niger or Penicillium species, often coupled with catalase, can be employed to remove molecular oxygen from wine, beer, fruit juices and soft drinks. This prevents potentially damaging oxidation that otherwise affects product quality.
2 glucose + O2 —-glucoseoxidase+catalase—-> 2 gluconic acid
Reference and Sources:
- https://www.nature.com/articles/s41529-022-00277-7
- https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7449042/
- https://www.sciencedirect.com/science/article/pii/B9780128132807000049
- https://www.nature.com/articles/s43705-023-00275-z
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