Screening Of Pig-derived Lactic Acid Bacteria Lactobacillus Animal 20 And Preparation Of Its Compound Preparation

The misuse of antibiotics has great harm to the development of the livestock and poultry industry and is one of the important public health issues.Lactic acid bacteria are the main representatives of probiotics,which can effectively inhibit the invasion of intestinal pathogens.In this study,lactic acid bacteria were isolated from pig manure,and strains with good probiotic effects were screened through antibacterial activity and tolerance tests.In vitro experiments evaluated the antibacterial activity against E.coli and Staphylococcus aureus.The selected lactic acid bacteria were used to prepare a compound preparation,the fermentation process of the compound strain was optimized,and the effect of the compound preparation on E.coli and S.aureus was evaluated.This research provides the basis and guarantee for the green and healthy breeding of livestock and poultry.The research contents and results are as follows:1.Isolation,identification and biological evaluation of porcine lactic acid bacteriaIn order to screen out lactic acid bacteria with excellent biological activity,from the perspective of lactic acid bacteria tolerance,41 strains were isolated from feces of healthy pigs.Through adhesion colonization,antibacterial activity,tolerance and other experiments to compare the physical and chemical characteristics of lactic acid bacteria.And one strain was Lactobacillus rhamnosus(B8)and two strains are Lactobacillus animalis(B2,T20).Tolerance test results showed that the survival rate of strains B2,T20 and B8 after heat treatment at 60? for 15 min was higher than 70%.The survival rate of B2,B8 and T20 cultured in pH=2 medium for 4h exceeds 37%.After culturing at 0.5% bile salt concentration for 4 hours,the survival rate of B2,B8 and T20 exceeded 30%.The results of adhesion experiments showed that the self-aggregation ability,co-aggregation ability of pathogenic bacteria and surface hydrophobicity among different strains were different(P <0.05).Among them,T20 has the highest self-polymerization ability,being 66.14%.The co-aggregation ability of T20 with pathogenic bacteria was 59.36%.The highest surface hydrophobicity of T20 and B8 was 77.63% and 76.62%,respectively.The intestinal colonization experiment results of lactic acid bacteria showed that B2,B8 and T20 could be stably colonized in the intestine,and the number of cecum colonization was significantly higher than that of duodenum,jejunum and colon(P < 0.05),and there was no significant difference from the ileum.The antibacterial results showed that B2,B8,and T20 can inhibit the growth of ETEC 10 and A65,and there is no difference in the antibacterial ability of bacterial culture and cell-free fermentation broth(CFS).The above experiments indicate that T20 strain has better antibacterial activity and tolerance,and can be stably colonized in the intestine,confirming that T20 is a probiotic strain with excellent probiotic characteristics.T20 was named as Lactobacillus animal 20(L.animal 20),which was used in the next in vitro anti-pathogenic bacteria experiment.2.Study on the anti-pathogen effect of lactic acid bacteria L.animal 20 in vitroIn order to explore the role of lactic acid bacteria against enterotoxigenic Escherichia coli(ETEC 10)and Staphylococcus aureus A65.This section selects the probiotic strain L.animal 20 from the perspective of lactic acid bacteria inhibiting the growth of ETEC 10 and A65,that explore the potential effect of lactic acid bacteria against pathogenic bacteria.Bacterial co-culture assays showed that ETEC 10 and A65 showed different growth patterns in pure culture and co-culture.L.animal 20 can inhibit the growth of ETCC 10 and A65,and the number of viable cells in co-culture is significantly lower than that in pure culture(P < 0.05).L.animal 20 CFS reduced the expression of ETEC 10 and A65 motility and biofilm-related genes,and significantly reduced their motility and biofilm formation(P < 0.05).Transmission electron microscopy(TEM)observed the loss of cell contents,contraction,damage and lysis of ETEC 10 and A65 cells after co-culture.SDS-PAGE verified that the intracellular protein was significantly reduced(P < 0.05).The above experimental results prove that the screened L.animal 20 has anti-enterotoxic E.coli(ETEC 10)and Staphylococcus aureus A65.3.Preparation of porcine-derived lactic acid bacteria compound preparation and process optimizationIn order to prepare a lactic acid bacteria compound preparation with good biological activity,this section uses the bacteriostatic activity of the compound preparation as an indicator to determine the optimal ratio of T20:B2:B88 to 2:2:3.Optimized fermentation medium formula: 0.8% glucose and 0.8% sucrose as a composite carbon source with a ratio of 3:1;2% soybean meal and 1% yeast extract as a composite nitrogen source with a ratio of 1:2;0.05% sodium acetate is used as a buffer salt,the ratio is 1:1;5% tomato and5% cucumber are used as the proliferation factor,the ratio is 3:1.On the basis of optimizing the culture medium,the effects of culture time,temperature,inoculation volume and liquid volume on the antibacterial activity of lactic acid bacteria complex preparation were analyzed.The results showed that the fermentation liquid had the highest antibacterial activity after incubation at 37? for 18 h at 2% inoculation volume and 40%liquid volume.The above research showed that L.animal 20 has good tolerance and antibacterial activity,and can be stably colonized in the intestine.L.animal 20 fermentation supernatant(CFS)has a good in vitro bacteriostatic effect on ETEC 10 and A65;The lactic acid bacteria screened out above to prepare a compound preparation,after optimizing the medium composition and fermentation conditions,the bacteriostatic effect of ETEC 10 and A65 is significantly improved.The research results of this paper have certain application value in antibiotic replacement.