Optimization Of Fermentation Conditions And Application Of Lactococcus Lactis C15 To Produce Bacteriocins

In recent years,people pay more and more attention to health problems,and the phenomenon of overuse of antibiotics in food still exists.Compared with the adverse consequences brought by the use of antibiotics,the research on probiotics with excellent characteristics has attracted wide attention.Lactobacillus bacteriocins have the advantages of no pollution,no residue and high safety.However,most of the commercial bacteriocins have a narrow bacteriostatic spectrum,which limits application in the field of food.The development of Lactobacillus bacteriocins with a wide bacteriostatic spectrum and relative stability has a great prospect.In this study,L.lactis C15 with antibacterial activity screened in the laboratory was taken as to explore the probiotic characteristics of L.lactis C15,optimize the fermentation conditions for bacteriocins produced by L.lactis C15 through single factor test and response surface method,and finally separate and purify the produced bacteriocins by gel chromatography.Moreover,initially study its application in dairy products,which lays a theoretical foundation for its application in the preservation and preservation of food as a biological preservative.The specific content and research results of the study are as follows.(1)Through auto-aggregation,co-aggregation test,hydrophobicity test,simulated gastrointestinal fluid test,adhesion test,whether the strain C15 had the ability to adhere and colonize the intestine of the body was evaluated.Then,through the drug sensitivity test,hemolytic test,and biogenic amine test,the strain C15 was evaluated for safety.The results showed that the hydrophobicity of L.lactis C15 to xylene and hexadecane reached more than70%,and the auto-aggregation ability was 23.56%.Its co-aggregation ability to Escherichia coli and Listeria monocytogenes reached more than 65%,its survival rate in artificial gastrointestinal fluid was more than 60%,and adhesion test showed that its adhesion rate to Caco-2 cells reached 94.07%.Moreover,drug sensitivity test,hemolysis test and biogenic amine test showed that strain C15 was a safe strain.(2)Bacteriocin produced by L.lactis C15 conducted PCR amplification and identification.Eight sets of specific primers were selected,and the target band was obtained by genome extraction,PCR amplification,and agarose gel electrophoresis.After purification,gene sequencing was performed to identify L.lactis C15 containing Lactococcin B.(3)M17 medium was regarded as the basic medium.Carbon source,nitrogen source,inorganic salt,vitamin,etc.that promote production of bacteriocin were screened.Through the single factor and orthogonal experiment,the optimal medium components,namely glucose,tryptone,anhydrous sodium sulfate,vitamin B₃ and?-glycerophosphate were determined.Then,the single factor test combined with response surface method was used to further optimize the optimal addition amount amount of each component of the medium and optimum culture conditions.Finally,the best medium formula was obtained:the addition amount of glucose was 6.37 g/L,the addition amount of tryptone was 17.03 g/L,the addition amount of anhydrous sodium sulfate was 0.25 g/L,and the addition amount of vitamin B₃ was 0.70 g/L,the addition amount of?-glycerophosphate was 19.00 g/L.The optimal culture conditions were temperature of 33?,time of 16 h,inoculation volume of 2%,relative effect value of2520.27 IU/m L.(4)By using a series of methods such as salting out,dialysis,and gel filtration,bacteriocin produced by L.lactis C15 was purified,and the characteristics of bacteriocin were further studied.The results showed that bacteriocin C15 retained some activity after being treated at121?for 15 min.At 80?for 30 min,the activity of bacteriocin retained about 30%;In the p H range of 2 to 8,the activity was stably maintained.After the p H value was greater than 9,the activity was somewhat reduced but still retained some activity;In the enzyme sensitivity test,it was found that bacteriocin was completely lost after treatment with neutral protease,alkaline protease,pepsin and trypsin;After separately acting with EDTA and urea,the activity of bacteriocin was obviously enhanced.(5)The inhibitory effect of bacteriocin on pathogenic bacteria in skim sterilized milk was preliminarily explored.The crude bacteriocin extract was added to skim sterilized milk with indicator bacteria(E.coli and L.monocytogenes),and nisin was regarded as a positive control,it was observed whether bacteriocin would inhibit the growth of E.coli and L.monocytogenes in the milk system by measuring the number of pathogenic bacterium.The results showed that it was placed at 30?for 24 h.Bacteriocin can effectively inhibit the growth of E.coli and L.monocytogenes in sterilized milk,which showed that bacteriocin had great development potential in the field of food preservation.