Microbial Coagulants

Many microorganisms are able to produce extracellular proteases that can curdle the milk. Such coagulants can be produced by fermentation and therefore are almost unlimitedly available. These enzymes show, however, higher proteolytic activity than rennet, which may lead to a loss of protein degradation products into the whey, thus, negatively affecting the cheese yield.

At present, only fungi-derived coagulants are used as microbial coagulants. Three species are commonly used: Rhizomucor miehei, Rhizomucor pusillus and Cryphonectria parasitica.

The aspartic proteinase produced by R. miehei (EC C.A.S. Na 9073-79-4) consists of a single polypeptide chain with a high similarity to chymosin in its threedimensional structure (Chitpinityol and Crabbe, 1998). This proteinase (40.5 kDa, optimum mil- clotting activity at pH 5.6, optimum proteolytic activity at pH 4.1, 50% loss of proteolytic activity after 30 minutes at 45°C; Preetha and Boopathy, 1997) is the most commonly used microbial coagulant for cheese production and commercially available at different levels of thermostability and purity. The protein has no active sulphydryl groups, and the amounts of Phe, Thr, and Lys are equivalent to that of calf chymosin (Sternberg, 1970). The other microbial coagulants are less characterized: purified proteinase from Mucor pusillus has a molecular mass of about 49 kDa, its clotting activity is best at 50°C, and sensitivity to pH and CaCl2 are similar to that of calf chymosin (Nouani et al., 2009). Cryphonectria parasitica proteinase is less characterized, but it is generally recognized that its proteolytic activity is higher (Tam and Whitaker 1972; Vanderporten and Weckx 1972) and that mainly p-casein is hydrolyzed (Ustunol and Zeckzer 1996; Awad et al., 1998; Trujillo et al., 2000; Broome et al. 2006).

Rhizomucor miehei aspartic proteinase has also been produced as recombinant by a genetically modified strain of A. oryzae.

Fungal enzymes are produced by fermentation. A mother culture is propagated on a selected growth medium and grown up to produce sufficient cells to inoculate a large fermenter, possibly containing as much as 50,000 L of the same growth medium. At the end of fermentation, biomass is separated from the fermentate, which contains the enzyme. The fermentate is concentrated by filtration,using reverse osmosis technology, then the enzymatic strength is standardized and the product finally filter sterilized prior to packing.

Some early microbial coagulants did exhibit two major drawbacks in that the enzyme was not heat labile and there were very high levels of nonspecific proteases present. The first problem was highlighted by manufacturers of milk infant formula (IMF), which included whey powder in the formulation. When mothers rehydrated the IMF, the residual enzyme activity clotted the milk. The second area of concern was the poor yields and bitterness developed during maturation of long-ripened cheeses varieties as a result of the high levels of nonspecific proteases in the coagulants.

The two drawbacks were overcome by extensive research work. Better purification of the enzyme removed bad tastes in the cheese. After purification, some chemical modifications (oxidation) are usually applied to decrease the thermostability. The thermostability of fungal proteinase is a big disadvantage in comparison to calf rennet, because after scalding of the cheese curd, the enzyme remains intact, resulting in a high cleavage of casein during ripening. The process by which chemical treatments are applied for increasing thermolability is a trade secret.

In order to increase the clotting activity in comparison to the generic proteolytic activity, the proteinases can be deglycosylated by endo-p-N-acetylglucosamidase H, also called Endo H (EC The milk-clotting activity is increased significantly, the pH dependency of the milk-clotting activity is reduced to values closer to that of calf chymosin, and the ratio between milk clotting and general proteolytic activity becomes higher. Moreover, the deglycosylated enzymes become more heat labile.

The highly thermolable microbial coagulant from Cryphonectria parasitica is mainly used in Emmental production because it gives less fast maturation and better stability regarding texture.

Microbial coagulants are cheap products, but in general, the yield from the transformation of milk in cheese is lower than using rennet or chymosin. Sometimes microbial coagulants leave a bitter taste, especially in long-ripened cheeses.

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