Screening And Applications Of Bacterioncin-like Producing Lactic Acid Bacteria Kefir

Kefir, a number of small particles lumping together, is a kind of traditional starter cultures by fermentation of kefir grains, in which various microorganisms inhabit. It has many functions, including lowering blood fat and pressure, enhancing human immunity, preventing and resisting cancer, and so on. The way of fermenting and operating the traditional kefir is more open, and some bacterial strains with antibacterial properties must exist in it. Thus, their products perform an important function in antisepsis and food safety of Kefir. This experiment is intended to separate strains with broad-spectrum antibacterial properties from traditional fermented food, used in dairy fermentation,intended to improve the quality and safety of fermented dairy products,and provide the possibility of developing new food preservations.Kefir particles are used as the microbial separation resource in the experiment. After primary and secondary screening, a lactic acid bacterium with a good antibacterial activity is separated. This strain is named as lactococcus lactis via conventional physiological and biochemical identification combining with the molecular biological identification 16SrDNA.With saturated phenol,chloroform,isoamylalcohol(25:24:1) extraction remains after the fermentation broth inhibited Staphylococcus aureus,further confirmed the non-protein nature of the antibacterial factor.Concentrated fermentation broth by dialysis bags determine the molecular weight antibacterial factor<1000Da.Because of the wide of its bacteriostasis spectra,restrain some gram-positive and gram-negative bacteria,in which the inhibitory effect was Staphylococcus aureus best, followed by E.coli,to mildew, yeast, Bacillus subtilis,Lactobacillus bulgaricus and Streptococcus thermophilus no inhibitory effect. Z-4 and ZKJ-1strains to macrolide antibiotic had drug resistance,detection of Lactococcus lactis on the Z4 and ZKJ-1 inhibitory activity,result show that it without inhibition effect for Z4 and ZJK-1.The medium, temperature, pH, and culture method of the strain are studied through single factor experiment. The result indicates that the optimal conditions the growth of bacterium include M17 medium, 7.0 as the initial fermentation pH, 37℃as the optimal culture temperature, the static culture as the optimal culture method. The OD600 value of the strain before the optimization is 1.160 and the OD600 value of the strain after the optimization increases by 16.47% and becomes 1.351.Firstly, the type of lactic acid bacteria fermentation medium, the carbon source and nitrogen source, metal ions, stimulating factor in the medium are studies via quaternary quadratic orthogonal rotation experiment with bacteriocin-like production as the index. The optimal medium formula confirmed finally includes glucose 16.70 g/L, yeast extract 18.70 g/L, MgSO4·7H2O 0.36 g/L, triammonium citrate 2 g/L, sodium acetate 2.5 g/L, the additive volume of tween 80 1 mL/L. The potency value of the strain before the optimization is 38.62 AU/mL and the potency value of the strain after the optimization increases to 126.58 AU/mL. The type of lactic acid bacteria fermentation conditions, the pH and temperature, seed age, inoculum of the fermentation conditions are studies via quaternary quadratic orthogonal rotation experiment with bacteriocin production as the index. The optimal fermentation conditions finally includes pH 6.0, at 36℃, seed age 12 h,inoculum2.5%. The potency value of the strain before the optimization is 39.86 AU/mL and the potency value of the strain after the optimization increases to 146.92 AU/mL.In the single factor experiment and orthogonal rotation experiment, Lactococcus lactis, Lactobacillus bulgaricus and Streptococcus thermophilus are mingled and put in cultured milk as a certain ratio. The optimal allocation ratio Lactococcus lactis: Lactobacillus bulgaricus: Streptococcus thermophiles in the cultured milk obtained from the experiments is 0.5: 1.5: 1.5, and the inoculum concentration, sugar concentration, milk concentration are 5%, 8%, 9% respectively, prepared under the process yogurt organization delicate,taste and aroma are more pure,no bad fermentation smell,taste softer,no syneresis. at 4℃conditions,bacteriocin-like producing Lactococcus lactis can be effectively inhibit the growth of spoilage bacteria and prolong the shelf life of yogurt.