Lysozyme (l,4-/WV-acetylmuramidase) is a 14,600 Da enzyme present in avian eggs, mammalian milk, tears and other secretions, insects, and fish. It is most stable under acidic conditions. The enzyme catalyzes hydrolysis of the [1-1,4 glycosidic bonds between iV-acetylmuramic acid and iV-acetylglucosamine of the peptidoglycan of bacterial cell walls. This causes cell wall degradation and eventual lysis.
Lysozyme is most active against Gram-positive bacteria, most likely because of the exposed peptidoglycan in the cell wall. The enzyme inhibits Bacillus stearothermophi-lus, B. cereus, Clostridium botulinum, C. thermosaccharo-lyticum, C. tyrobutyricum, Listeria monocytogenes, and Micrococcus lysodeikticus (18). Variation in susceptibility of Gram-positive bacteria is likely due to the presence of teichoic acids or other materials that bind the enzyme and that certain species have greater proportions of 1,6 or 1,3 glycosidic linkages in the peptidoglycan that are more resistant than the 1,4 linkage (75). For example, certain strains of L. monocytogenes are not inhibited by lysozyme unless EDTA is present (76). Lysozyme is less effective against Gram-negative bacteria due to a reduced peptidoglycan content and presence of outer membrane of lipo-polysaccharide and lipoprotein. Gram-negative cell susceptibility can be increased by pretreatment with chelators (eg, EDTA) or if cells are subjected to shock (pH, heat, osmotic), drying or freeze-thaw cycling (75).
The most common use of lysozyme in foods is in hard cheeses such as Edam and Gouda to prevent a defect known as late blowing. This defect is characterized by formation of holes in the cheese and the production of unacceptable flavors caused by C. tyrobutyricum. In foods other than cheese, lysozyme has been evaluated as an antimicrobial in seafoods, sake, potato salad, sushi, Chinese noodles, creamed custard, and sausage. Lysozyme and lysozyme combined with EDTA may have potential for reducing Salmonella on poultry and spoilage microflora from shrimp. Lysozyme was bactericidal to L. monocytogenes in shredded cabbage and lettuce, fresh green beans, corn, and carrots, and this activity was enhanced by EDTA (76). The compound was less effective in refrigerated meats and soft cheese. In some studies, no inhibition was demonstrated with EDTA and lysozyme against Salmonella typhimurium or Pseudomonas fluorescens (77).
Lysozyme is one of the few naturally occurring antimicrobials approved by regulatory agencies for use in foods. In the United States and Europe, lysozyme is allowed to prevent gas formation in cheeses such as Edam and Gouda by C. tyrobutyricum. Lysozyme is used to a great extent in Japan to preserve seafood, vegetables, pasta, and salads.
Lactoperoxidase (MW = 78,000 Da) is an enzyme that occurs in raw milk, colostrum, saliva and other biological secretions. Bovine milk naturally contains 10 to 60 mg/L of lactoperoxidase (78). This enzyme reacts with thiocyanate (SCN~) in the presence of hydrogen peroxide and forms antimicrobial compound(s). The components are called the lactoperoxidase system (LPS). Fresh milk contains 1 to 10 mg/L of thiocyanate, which is not always sufficient to activate the LPS (79). Hydrogen peroxide, the third component of the LPS, is not present in fresh milk due to the action of natural catalase, peroxidase, or superoxide dis-
mutase. Approximately 8 to 10 mg/L hydrogen peroxide are required to activate the LPS, which can be added directly, through the action of lactic acid bacteria or through the enzymatic action of xanthine oxidase, glucose oxidase, or sulfhydryl oxidase. In LPS reaction, thiocyanate is oxidized to the antimicrobial hypothiocyanate (OSCN~), which also exists in equilibrium with hypothiocyanous acid (78).
The LPS is more effective against active against Gram-negative bacteria, including Pseudomonas, than Gram-positive bacteria. However, it does inhibit both Gram-positive and Gram-negative foodborne pathogens, including Salmonella, Staphylococcus aureus, Listeria monocytogenes, and Campylobacter jejuni (78). There is variable activity against catalase-negative microorganisms, including the lactic acid bacteria such as Lactococcus, Lactobacillus, and Streptococcus.
The LPS system can increase the shelf life of raw milk. This could be useful in countries that have poorly developed refrigerated storage systems (78). LPS has also been used as a preservation process in cream, cheese, liquid whole eggs, ice cream, and infant formula.
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