Isolation And Identification Of Bacteriocin-Producing Lactic Acid Bacteria And Study On Their Culture Optimization And Induction Of Bacteriocin Synthesis

Lactic acid bacteria are well-loved by the general public because of their probiotic,safe,efficient antibacterial and wide source properties.Bacteriocins produced by them have protein properties which can be degraded by the human intestinal tract,and play an inhibitory or bactericidal role against many pathogenic or spoilage microorganisms at low concentrations,so it has wide application prospect in food preservation and medical fields.However,up to now,bacteriocins that can inhibit Gram-negative bacteria are still in the minority and most of them are still in laboratory research instead of produced industrially on a large scale,one of the most critical factors is the low yield.Consequently,Aiming at the specific predominant spoilage bacteria Pseudomonas.koreensis PS1 that caused the spoilage of chilled pork,this study isolated and screened bacteriocin-producing lactic acid bacteria that have obvious inhibitory effects from soil and cow dungs,and performed strain identification and growth kinetics research.Then the liquid fermentation process was optimized,and the effects of exogenous substances and auto-induced effects on bacteriocin synthesis were further investigated with a view to promoting the production of bacteriocin of L.plantarum C010.And the main results of the study were as follows:（1）Screening and identification of bacteriocin-producing lactic acid bacteria and growth kinetics study.The test used the specific spoilage bacteria P.koreensis PS1 as an indicator for chilled pork,and 182 strains of lactic acid bacteria were isolated and screened from 25 soil and cow dung samples.Among them,6 strains of lactic acid bacteria have antibacterial activity after removing organic acid and hydrogen peroxide and has a good antibacterial effect on bacteria and molds,especially the active substance produced by strain C010 inhibition is the most significant,in addition,the substance in multiple generations can still maintain the stability of antibacterial activity and sensitive to protease,the preliminary determination of the active substance is bacteriocins.The strain was identified as Lactobacillus plantarum.The physicochemical properties of the bacteriocins produced by the bacteriocins and its growth law were studied.It was found that the bacteriocins produced by the bacteria were stable under heat treatment,ultraviolet irradiation and acidic pH（pH 2.0-6.0）,and the bacteriocins synthesized in the The growth phase reaches the highest in the stable phase,which is in line with the characteristics of primary metabolites.（2）Optimization of the liquid fermentation process of L.plantarum C010.The experiments of single factor,response surface and orthogonal were used to optimize the liquid fermentation process of L.plantarum C010,and the optimal medium for bacteriocin synthesis was determined as:10 g/L peptone,28.0 g/L beef extract,0.3 g/L MgSO₄·7H₂O,0.1 g/L Mn SO₄·H₂O,1.5 ml/L Tween 80,20.0 g/L sucrose,5.0 g/L CH₃COONa,2.0 g/L K₂HPO₄·3H₂O,2.0 g/L C₆H₁₇N₃O₇;and optimal culture conditions were:inoculum concentration of 10⁹ CFU/mL,incubation temperature of 37°C,loading volume of 90 mL,pH 5.5,and incubation time of 20 h.Under the optimized conditions of the fermentation process,the antibacterial effect of the bacteriocin produced by L.plantarum C010 against P.koreensis PS1 was further improved,and the diameter of the inhibition zone could reach 16.93 mm,which was 17.49%higher than that before the optimization（14.41 mm）.（3）Study on the auto-induction effect of L.plantarum C010 liquid fermentation.The liquid fermentation supernatant concentrate of L.plantarum C010 was added to the culture medium to study its effect on the synthesis of bacteriocins.It was found that the bacteriostatic effect of the bacteriocin produced by L.plantarum C010 decreased when the initial inoculation concentration was 10⁴ CFU/mL,and the diameter of inhibition zone was only 10.08 mm at that time.Then found that the addition of 2%supernatant concentrate on this basis could induce the synthesis of bacteriocin 4 h earlier than CK,and the diameter of inhibition zone could reach 14.89 mm at 36 h of fermentation,which increased the antibacterial activity by 9.32%compared with the CK control（13.62 mm）.However,after proteinase K degradation,the antibacterial activity was not significantly different from CK treatment,indicating that the substance that induced bacteriocin synthesis had protein properties,which might be the bacteriocin itself.（4）The effect of exogenous addition of intermediate metabolites on bacteriocin production of L.plantarum C010.Different intermediate metabolites were selected to be added to the liquid medium to investigate their effects on the bacteriocin synthesis of L.plantarum C010 in this experiment.The results showed that:glycerol,sodium pyruvate andα-ketoglutarate all showed a good inducing effect on the synthesis of bacteriocins,while sodium succinate played an inhibitory role.When added in combination,it was found that the antibacterial effect of each combination was less significant than added alone.When glycerol orα-ketoglutarate was added at different time points,it was found that the best bacteriocin synthesis was achieved when both were added at 4 h.（5）The effect of amino acid exogenous additions on bacteriocin production of L.plantarum C010.Different amino acid exogenous additions to liquid medium were selected to investigate their effects on the bacteriocin synthesis of L.plantarum C010 in this experiment.The results showed six amino acids,including Ala,Val,Thr,His,Trp and Tyr,had positive induction effects on bacteriocin synthesis,among which Ala,Thr,His,Trp and Tyr were more effective in bacteriocin synthesis at low concentrations（0.5g/L）.Further investigation of the five amino acids added in combination showed that the antibacterial effect of each combination was not significantly different from that of the individual addition on bacteriocin synthesis,and only AE（Ala+Trp）was slightly better.And when the AE ratio was 1:4 and added at 0 h,the antibacterial activity of bacteriocin were improved better.Further comparison with the initial medium,optimized medium and glycerol-added optimized medium showed that the combination of AE（Ala+Trp）could induce further improvement of bacteriocin,and the diameter of inhibition zone was20.78 mm,an increase of 36.24%compared to the initial medium CK.The above research shows that the bacteriocins produced by L.plantarum C010were better enhanced when alanine and tryptophan were added exogenously to the optimized MRS medium,and the bacteriocins had a broad antibacterial effect,stable at pH 2.0-6.0,heat resistance,UV irradiation,and sensitivity to some proteases.The results of the study provide theoretical basis and data for the enhancement of bacteriocin production by L.plantarum C010 and its application in the preservation of chilled pork and its products.