| Kefir is a traditional fermented dairy,with special nutritional and probiotic function.There are many kinds of microbes in Kefir grains,including lactic acid bacteria(LAB),acetic acid bacteria and yeasts.LAB have quite a lot probiotic properties.The metabolites,such as organic acid,hydrogen peroxide,bacteriocin and extracellular polysaccharide,can effectively inhibit the growth of spoilage and pathogenic bacteria in food.It is natural,safe,and doesn't cause resistance to pathogenic bacteria.It is of great potential to develop substitutes for disinfection,sterilization products and new food preservatives.In this study,the characteristics of13 strains of lactic acid bacteria isolated from Tibet Kefir grains and the standard strain ATCC8014 were analyzed,and the content of metabolites and the antimicrobial activities were explored.In addition,the antibacterial and antibiofilm effects of L10strain(Lactobacillus casei)and the metabolites on three common foodborne pathogens were further studied.The gene expression difference of biofilm cells which were treated with Lactobacillus casei metabolites was analyzed by transcriptomic technique.This study will provide theoretical basis and data for developing lactic acid bacteria related products and novel antibiofilm preparations.The main contents and results of the study are as follows:The characteristics of LAB,such as agglutination,biofilm formation and antibiotic resistance,were investigated.14 strains of LAB showed strong auto-aggregation and co-aggregation with three kinds of foodborne pathogenic bacteria(Vibrio parahaeomolyticus,Staphylococcus aureus,and Listeria monocytogenes).The biofilm formation abilities of different strains were different.Carbon source,metal ion and adhesion material influenced the formation of biofilm.LAB was easy to form biofilm on stainless steel sheet.The signal molecule autoinducers-2(AI-2)of different strains were detected by bioluminescence method.In a certain period of time,the cell density of LAB was positively correlated with the yield of signal molecule AI-2,and was closely related to biofilm formation.The drug resistences of LAB were evaluated using Kirby Bauer agar diffusion method,the results showed that all the tested strains were multi-resistant strains,displaying resistence to three major kinds of antibiotics that inhibit the synthesis of cell wall,protein and nucleic acid.The main metabolites and antibacterial activity of LAB were analyzed.In the metabolites of LAB,the contents of extracellular polysaccharide were 69.9 ~ 182.9mg/L,hydrogen peroxide content 15.17 ~ 24.5 g/mL,lactic acid content 1.994 ~2.774 mg/mL,acetic acid content 0.878 ~ 2.163 mg/mL,propionic acid content 0.91 ~1.93 mg/mL.The metabolites of LAB had a certain antibacterial effect on the three pathogens.The inhibition effect on V.parahaemolyticus was the strongest.The optimum fermentation conditions for the bacteriostasis of L10 strain were obtained by the response surface methods.The fermentation time was 48 h,the inoculation amount was 6.2%,the temperature was 38?,and the amount of glucose was 3.87%.It was found that L10 broth could inhibit gram positive and negative pathogens,and had thermal stability.The antibacterial substances were mainly organic acids.The inhibition and eradication effects of L10 strain and its fermented supernatant on biofilm formation and mature biofilm of three food borne pathogens were determined.The results showed that the number of culturable bacteria in biofilm was significantly reduced.,When L10 strain was co-cultured with Vibrio parahaemolyticus and Staphylococcus aureus.The inhibitory effects gradually inhenced with the increase of dosage.The inhibition rates on biomass and metabolic activity of biofilms were about 50% at MIC.When the amount of L10 fermented supernatant was higher than MIC,it had a certain clearance effect on mature biofilms of V.parahaemolyticus and S.aureus,and the inhibitory effect was concentration dependent.The inhibition rate on mature biofilms was about 50% at 4MIC.Moreover,L10 fermentation supernatant has obvious permeability to the outer membrane and plasma membrane of the pathogen cells.Biofilms were observed by fluorescence microscope and laser scanning confocal microscopy(CLSM),and the inhibition and eradication effects were verified.Transcriptomic method was used to analyze the gene expression difference of V.parahaemolyticus biofilm cells after L10 fermented supernatant treatment.The study found that the number of differentially expressed genes was 4290,the up-regulated genes were 2047,and the down regulated genes were 2245.There were 14 significant differentially expressed genes.Differential expressed genes were related to metabolic process,establishment of localization,biological regulation,and located in cell,membrane part.Molecular functions focused on binding,transporter activity,catalytic activity.KEGG pathway analysis revealed that the significant differentially expressed genes were related to glyoxylate and dicarboxylate metabolism,glycine,serine and threonine metabolism,2-oxocarboxylic acid metabolism and so on.In conclusion,LAB isolated from Kefir grains has a variety of features,showing antimicrobial effects,inhibitory and eradicative functions on biofilms of food borne pathogens.It can influence the gene expression of V.parahaemolyticus biofilm cells.Therefore,it has important research significance and development potential. |
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