Lysozyme (EC is a glycoside hydrolase. It is known also as muramidase or N- acetylmuramide glycanhydrolase. It damages bacterial cell walls by catalyzing hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in a peptidoglycan and between N-acetyl-D-glucosamine residues in chitodex- trins. Lysozyme is widely distributed in nature (birds, mammals, insects, bacteria, plants), and it is abundant in egg white, tears, and human milk, while it is almost absent in cow milk. It is quite a stable protein, active between 1° and 100°C. It is not denatured by solvents, and its maximum activity is between pH 5.3 and 6.4. In 1992, JEFCA (Joint FAO/WHO Expert Committee on Food) declared it safe for food application.

It was characterized by Alexander Fleming in 1922, who studied lysozyme extensively, discovering during his research also Micrococcus lysodeikticus, a bacterium, especially susceptible to lysozyme, which is still used for lysozyme activity essays.

The enzyme works by attacking the peptidoglycans present in the cell walls of Gram-positive bacteria (Gram-negative are less subject to its attack due to the complexity of their cell wall). The antimicrobial effect is not due only to the catalytic effect, because chemically or thermally denatured lysozyme is amazingly more active against Gram-negative bacteria (Cegielska-Radziejewska et al., 2008). This specific activity justifies its use in cheese production in order to prevent late blowing of hard cheese due to Clostridia development. Clostridium tyrobutiricum mainly, Clostridum butiricum, and Clostridum sporogenes can cause relevant damages in some long-ripened cheeses as Grana Padano, Emmentaler, Gouda and Edam type, Manchego type, Prato, Pategras, Barra, Regianito, Sbrinz type. Their spores, if present in cheese, start germinating when the content of oxygen inside the wheel of cheese is low enough. Normally, it happens two or three months after the cheese manufacture, during ripening. The active Clostridia metabolize lactic acid and produce butyric acid and relevant quantity of gas (hydrogen and methane) that can cause blowing of the wheel of cheese or at least cracks. The spores survive at pasteurization. 99% of the Italian Grana Padano and 85% of France's hard cheese market uses lysozyme.

Classified as a food additive by Joint FAO/WHO Expert Committee on Food Additives (JEFCA), affirmed as GRAS by the FDA and approved for use in organic products by the USDA, products containing lysozyme should label the presence of “egg white lysozyme” on the ingredient list. In the European Union, lysozyme is considered an additive E 1105 without any given limit of dosage.

Lysozyme is sold as almost pure enzyme in powder or granular form and as an aqueous solution at the enzyme concentration of 22%. Usually 25 grams of lysozyme are added to 1000 liters of milk. The powder is dissolved in water by gentle stirring, avoiding foam formation that inactivates the enzyme, and then it is added to the milk before adding the starters and the rennet. The lysozyme associates strongly with aS-casein, less with p-casein, and not with к-casein. The effect of temperature and pH on association is small. Since after association with casein, lysozyme is still active, it is suggested that the active site of the molecule is not involved in the association (de Roos, et al., 1998). Most of the lysozyme stays inside the curdle and only a small part is lost in the whey. The enzyme is still detectable in the cheese after many months.

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