Purification,Inhibitory Characterization,and Mechanism Preliminary Study Of Lactocins Produced By Lactobacillus Paracasei

Lactocins,a kind of protein or polypeptide with inhibitory and bactericidal effects,are natural preservatives and can be used in food industry.In this study,nineteen strains of lactic acid bacteria isolated from Luzhou-flavor pit mud were used as the starting strains.The strain SLP16 with high inhibitory activity was selected by agar diffusion method.By excluding the interference of acid and hydrogen peroxide and protease treatment,it was determined that the inhibitory substance produced by strain SLP16 had protein properties and was a lactocin.Strain SLP16 was identified as Lactobacillus paracasei through morphological,physiological,biochemical and 16S rRNA sequence analysis.The lactocin produced by the strain SLP16 was named Lactocins SLP16.Combined with single factor test,Plackett-Burman test,steepest climb test and Box-Behnken test,obtaining the improved MRS medium for producing Lactocins SLP16:20 g glucose,11.63 g soy peptone,5 g yeast extract,7.02 g sodium acetate,10 g beef extract,2 g citric acid diammonium hydrogen,2 g KH₂PO₄,0.25 g MnSO₄,0.78 g MgSO₄,1 mL Tween-80 and 1000 mL water.The fermentation conditions of Lactocins SLP16 were optimized by orthogonal design,showing fermentation time 36 h,initial pH6,inoculum size 6%,and culture temperature 37?.The ratio and fermentation conditions were used for the validation experiment,showing the titer reached 1.8×10⁴IU/mL,3.31 times as much as the original.Liquid fermentation was performed on strain SLP16.The crude lactocins were obtained by two aqueous phase extraction.Orthogonal method was used to optimize extraction conditions of 22%PEG2000,18%ammonium sulfate and pH3.The purified lactocins were obtained by dialysis,freeze drying and Sephadex G-75 gel column chromatography.The final yield was 81.3%,and the purification was 59.2 times,the titer reached 2.3×10⁵ IU/mL.The molecular weight of Lactocins SLP16 was 26 kDa by SDS-PAGE electrophoresis.Lactocins SLP16 had 17 kinds of amino acids,and the content of glycine was the highest.The minimum inhibitory concentration of Lactocins SLP16 against Bacillus subtilis was 12.5?g/mL and the minimum bactericidal concentration was 50?g/mL.Lactocins SLP16 had good thermal stability,after 121?high temperature and high pressure processing 20 min remained 90%of inhibitory activity.It had acid-base tolerance in pH2-10 range,but had inhibitory effect only in acidic and neutral environment.The inhibitory activity was maintained above 80%by ultraviolet irradiation.It had a broad inhibitory spectrum,and had inhibitory effect on Bacillus subtilis?Escherichia coli and Staphylococcus aureus and partial lactic acid bacteria,had no inhibitory effect on partial mold and yeast.The inhibitory effect of Lactocins SLP16 on Bacillus subtilis was to make its intracellular nucleic acid,protein substances and charged substances leak in large quantities,so that the cells cannot synthesize the required energy substances normally.The morphology of Bacillus subtilis was observed by scanning electron microscopy,and autolysis,porosity,deformity and fragmentation occurred,leading to cell death.