Screening Of Probiotic Enterococcus Faecalis And Preliminary Study On Its Antibibacterial Substances

Bacteriocin is a kind of substance with antibacterial activity secreted by bacteria in the process of growth and reproduction,and has the property of protein.Many bacteriocins have been isolated and purified as food preservatives,therapeutic agents or biological control agents.Therefore,the research and development of broad-spectrum,high-efficiency,stable performance of new bacteriocin has a positive role in food preservation,the development and utilization of antibiotic substitutes.The strain CG-9 isolated from human saliva in our laboratory has strong antibacterial properties.The strain CG-9 was identified as Enterococcus faecalis by 16s r DNA gene.The probiotics of Enterococcus faecalis CG-9 showed that the strain could tolerate the environment of p H 3,had certain tolerance to bile salt,maintained a high survival rate in artificial simulated gastrointestinal fluid,and had good free radical scavenging ability.The results showed that the antibacterial substances were proteins.The culture conditions of Enterococcus faecalis CG-9 producing bacteria were optimized by single factor experiment,orthogonal test and box Behnken experiment.The optimal medium was obtained as follows:peptone 10 g/L,yeast extract 15 g/L,sucrose 20 g/L,KH₂PO₄2.5 g/L,Mg SO₄0.4 g/L,anhydrous sodium acetate 5 g/L,manganese sulfate 0.2 g/L,diammonium hydrogen citrate2 g/L,Tween-80 1 ml/L.The optimum fermentation conditions were as follows:culture temperature 36℃,medium p H 6.2 and culture time 20 h.Under these conditions,the maximum diameter of inhibition zone can reach 19.67 mm,which is 19.53%higher than that before optimization,and the antibacterial characteristics are significantly improved.In order to further study Bacteriocin Produced by Enterococcus faecalis CG-9,the bacteriocin produced by Enterococcus faecalis CG-9 was purified by ammonium sulfate precipitation,cation exchange chromatography and gel filtration chromatography.After purification,the specific activity of bacteriocin reached 17239.06 AU/mg,and the final yield was 16%,purified 34.37 times;Tricine The results of SDS-PAGE showed that the molecular weight of the bacteriocin was about 6.5kda;the study of its characteristics showed that the bacteriocin produced by Enterococcus faecalis CG-9 had heat resistance;the effect of p H value on the bacteriocin activity showed that the bacteriocin produced by Enterococcus faecalis CG-9 still maintained high activity in the p H range of 2-10;at the same time,the bacteriocin could tolerate 6%sodium chloride and still maintain at 8%sodium chloride concentration It has a wide range of antibacterial activities,and has a certain inhibitory effect on Gram-positive bacteria and Gram-negative bacteria.During the freeze-drying process of Bacteriocin Produced by strain CG-9,the bacteriocin activity（diameter of bacteriostatic zone）after freeze-drying was optimized.When the freezing temperature was-80℃,the concentration of trehalose was 1.5%,and the thickness of enzyme solution was 7.0 mm,the diameter of bacteriocin freeze-dried powder was 17.73 mm,which maintained high activity.During the freeze-drying process of bacteriocin produced by Enterococcus faecalis CG-9,the bacteriocin activity（diameter of bacteriostatic zone）after freeze-drying was used as the index to optimize the freeze-drying conditions.The optimal conditions were obtained as follows:the pre freezing temperature was-80℃,the concentration of trehalose was 1.5%,and the thickness of enzyme solution was 7.0 mm.The results showed that the diameter of bacteriostatic zone of freeze-dried powder was 17.73 mm,and the activity was still high,which laid a theoretical foundation for the industrial production of bacteriocin produced by Enterococcus faecalis CG-9.