Screening Of High Yield Nisin-Producing Strains And Optimizating Of Its Fermentation Conditions

Nisin is a natural antimicrobial peptide produced by microorganism. It is a 34 amino acid polypeptide that effectively inhibits Gram-positive bacteria and also the outgrowth of its spores. Significantly, Nisin was assessed to be food preservatives by the Joint Food and Agriculture Organization/World Heald Organization (FAO/WHO) Expert Committee. It has been accepted as a safe and natural preservative in more than 50 countries. Nisin is innocuous, sensitive to proteases, that can be hydrolyzed into amino acids in the alimentary tract by a-chyomotrypsin. Applied as food preservative forty years, Nisin does not induce a wide range of resisitance, which makes their biosynthesis of widespread concern.The bacteriocin Nisin was first marked in Aplin&Barrett company in England in 1953. After fifty years of development, there has been reported that Nisin production level has reached 8000 IU/mL in England. Our country is relatively backward in this field, the titre of Nisin-producing bateria has only reached 4000 IU/mL for the moment. It is a large gap in Nisin production level between foreign and domestic. At present, the research on Nisin are focused on optimizating of its medium and fermentation conditions. The low titer of Nisin-producing strains is main reason of its application restrictions.In this study, Lactococcus lactis subsp.lactis ATCC11454 by mutanted by UV, MW, NaNO2 and NTG. The mutants were screened by Nisin as antibiotic agent combined with agar diffusion method, and a high-yield mutant E-15 was got. The strain has a titer of 6200 IU/mL, which is as 5 times compared with Lactococcus lactis subsp.lactis ATCC11454. Pass-generation test showed that the heredity character of mutant strain E-15 was stable.The culture condition of E-15 in flask was investigated, then the optimal fermention medium and conditions were confirmed. The optimal medium were got as:peptone 20 g/L, casein peptone 15 g/L, Lactoalbumin hydrolysate 15 g/L, sucrose 30 g/L, MgSO4-7H2O 0.2 g/L, KH2PO410 g/L, NaCl 2 g/L, Tween-801 mL/L, pH 6.8, autoclaved at 121℃/20 min. The effects of inoculum size and culture time were studied in this work. The result suggests the optimal culture condition as:inoculum size 5%, culture time 28 h.The pilot-scale fermentation was carried in 10 L fermenter. The pH feed-back controlled anaerobic fed-batch fermentation was carried out in this work. To make use of the principle of the pH feed-back controlled, the mixed solution of sucrose and sodium hydroxide added to the fermenter by a certain speed, and maintain pH value remained at about 6.5, the sucrose concentration to maintain a stable of 10-15 g/L, The titer of Nisin reaches 9600 IU/mL after 28 h fermentation.Then the downstream processing was studied, plate and frame filter was used to remove bacteria from the fermentation broth. With a pore size of 3000 Da ultrafiltration membrane by ultrafiltration, and then spray drying, the coefficient of recovery is up to 96.1%.