The Study On Control Of Key Pathogenic Bacteria In Cheese By Biocontrol

According to Food and Agriculture Organization of the United Nations(FAO) and world health organization(WHO): Cheese is fresh or ripe dairy products, made from milk, butter, semi-skimmed milk, buttermilk or the mixture of these products, by coagulating and draining whey. Cheese contains abundant nutritional ingredient, including protein, calcium, fat, amino acid, phosphor and vitamin, its nutritive value is high, people all of the world love it deeply. Consumption of cheese is very huge in western countries. In recent years, cheese industry is more and more prosperous in China. But with several large outbreaks of listeriosis have been associated with consumption of contaminated cheese and consumers'interest in health food is increasing, people pay attention to whether the cheese is safty and sanitary. Bacause Listeria monocytogenes is one of key pathogenic bacterium in cheese, the development of a safty and innoxious method to control L. monocytogenes in cheese becoming the focus of how to resolve safty and sanitary problem of cheese.As all known, organic acids, hydrogen peroxide, diacetyl and other metabolic end products produced by Lactis acid bacteria(LAB) act as bio-preservatives by altering the intrinsie properties of the food to such an extent toactually inhibits poilage microorganisms, and the role of these metabolic end products has long been appreeiated, but the precise nature of this role has been the subject of intensive research in recent times. Bacterioein production could be considered as advantageous to the producer as, in sufficient amounts, these peptides can kill or inhibit bacteria competing for the same ecological niche or the sarne nutrient pool. Although bacteriocins are produced by many Gram-positive and Gram-negative species, those produced by the LAB are of particular interest to the food industry. To date, the only commercially produeed bacteriocins are nisin and Pedioein PA-1. While nisin has been found to be extremely effective as an additive to prevent spoilage and increase shelf-life in a number of food. But some varieties of L. monocytogenes have developed resistance to nisin, so we should exploit anti-Listeria bacteriocins.Two bacteriocin-produeing strains were screened from 51 strains of enterococci from different sources. They were Enterococcus faecalis KLDS6.0611 and E. faecalis KLDS6.0611. The cell-free supernatants (CFSs) of two strains can inhibit indieator strairis strongly excluding inhibitive effect of hydrogen peroxide and organic acid, but the inhibitive activity decreased sharply after treatment with proteinase K. The results confirmed that these inhibitory materials were proteins, could be classed as bacteriocin.No reaction was observed around the colonies of two strains on Blood Agar Base Plates, verdict that both of the strains lack in hemolytic activity. Paper disk method was utilized to examine susceptibility of the Enterococcus strains to the vancomycin, demonstrate that both of the strains have no resistance to vancomycin. It is elementary proved that two strains are safe.Bacteriocins were produced by the strains grown in MRS media at 37℃, maximal amounts of bacteriocins produced by KLDS6.0610 were reached after 20h of incubation, amounts of bacteriocins produced by KLDS6.0611 were reached after 16h of incubation. Both of the bacteriocins were not sensitive to heat, the activities remained stable after treatment at 121℃for 30min. The bacteriocins had antimicrobial activity between pH2.5 and 6.5, but lost activaty when pH exceed 6.5. Part inactivation of antimicrobial activity was observed after treatment of bacteriocins produced by KLDS6.0610 with pepsin, proteinase andα-chymotrypsin, no change in activity was recored when bacteriocins was treated withα-amylase. Part inactivation of antimicrobial activity was observed after treatment of bacteriocins produced by KLDS6.0611 with pepsin, proteinase,α-chymotrypsin, trypsin andα-amylase. Both of the bacterions were insensitivity to papain. The action mode of bacteriocins produced by KLDS6.0610 was bacteriostasis, the mechanism of action of bacteriocins produced by KLDS6.0611 was bactericidal. The bacterioeins did not adhere to the surface of the producer bacteria. Two bacteria produced bacteriocins in skimmed milk, but the production was lower than in MRS media.The preliminary purification of bacteriocins were undertook by ammonium sulfate precipitation, dissolved in MilliQ water of 1/40 initiate volume. Tricine-SDS-PAGE was used to determine mass of bacteriocins, results showed mass of bacteriocins produced by KLDS6.0610 and KLDS6.0611was below 3.3k Da.Humidity of each cheese sample during storage time was evaluated, known that humidity of each cheese sample was about 67%, correspond to the requirement to soft cheese in GB/T 21375-2008. The protein level of each cheese sample during storage time was between 25.5%~28%, have no marked change. Compared to the control, the counts of L. monocytogenes in cheese samples added bacteriocins and bacteriocins producers reduced during storage at 4°C for 5 days, the counts of L. monocytogenes in cheese samples added bacteriocins were fewer than that in cheese samples added bacteriocins producers, and the counts of L. monocytogenes in cheese samples added two bacteriocins were fewer than that in cheese samples added one bacteriocin.