Isolation,Screening And Probiotic Effects Of Probiotics From The Vagina Of Healthy Dairy Cows

Lactic acid bacteria are regarded as members of probiotics,whose living bacteria and metabolites not only have the effect of bacteriostasis,but also can enhance the immunity.Meanwhile,they are hardly to develop drug resistance and have no toxic or side effects on human beings and animals.Lactic acid bacteria are recognized as the dominant species in healthy vaginal microbial flora.Lack of lactic acid bacteria is believed to destroy the balance of vaginal microbial flora,reduce the immune barrier function,and then lead to various reproductive tract inflammation,parasitic diseases,fungal and chlamydial infections,etc.In this study,the dominant lactic acid bacteria with probiotic characteristics from the vagina of healthy dairy cows have been isolated,purified and screened,which are none-spore forming,atrichous,morphologically normal,gram-positive and genetically stable in terms of colony morphology,bacterial characteristics and growth characteristics.On this basis,the antibacterial effect and antibacterial components were studied.In addition,effects of SQ0048 on TLRs-MyD88/NF-KB signaling pathway were discussed in order to provide experimental basis and lay theoretical foundation for the preparation of microecological preparations for the treatment of cow vaginal inflammation.Specific research contents are as follows:1.In order to obtain probiotic lactic acid bacteria strains capable of preventing and treating cow reproductive tract inflammation,vaginal flora of 50 healthy cows were preliminarily isolated by microbiological methods.The subculture stability,dyeing characteristics,classification and acid production capacity of the preliminarily isolated lactic acid bacteria were determined by fermentation engineering technology,microscopic examination,Gram staining method and acidity determination method respectively.The morphological identification,physiological and biochemical characteristics and 16SrDNA identification of 8 strains of probiotic lactic acid bacteria were carried out by microscopy,biochemical identification and molecular biology methods respectively;Acidity determination method,catalase test and oxford cup were used respectively to further determine the acid production,hydrogen peroxide production and antibacterial activity of the strain identified by 16SrDNA.The results showed that 55 strains of lactic acid bacteria were isolated from vaginal samples of healthy dairy cows.28 strains were screened out,which arc none-spore forming,atrichous,morphologically normal,gram-positive and genetically stable in terms of colony morphology,bacterial characteristics and growth characteristics.Among them,8 strains had strong acid-producing ability and faster acid-producing speed.According to the identification of this 8 strains,SQ0012 strain was Lactobacillus plantarum;SQ0015 strain was Lactobacillus brevis;SQ0030 strain was Enterococcus faecalis;SQ0041 strain was Enterococcus faecalis;SQ0045 strain was Lactobacillus kitasatonis;SQ0048 strain was Lactobacillus johnsonii;SQ0049 strain was Lactobacillus amylovorus;SQ0054 strain was Lactococcus garvieae.As SQ0030 and SQ0041 strains belonged to the same strain,with SQ0030 retained,finally 7 strains were identified as probiotic lactobacillus strains.Among the 7 strains identified,SQ0048 and SQ0054 strains had stronger acid-producing ability and the fastest acid-producing speed,while SQ0048 also had the characteristic of hydrogen peroxide production.The diameter of inhibition zone of SQ0048 on Staphylococcus aureus was 11±0.5 mm,which showed strong inhibitory effect.The diameter of inhibition zone of SQ0054 strain was 8±0.89 mm,which had slight inhibition effect.The diameter of inhibition zone of SQ0054 strain was significantly smaller than penicillin（P<0.05）,while the diameter of inhibition zone of SQ0048 strain had no significant difference compared with penicillin group（P>0.05）.The diameters of inhibition zone against Escherichia coli was 13±0.45 mm of SQ0048 strain which showed strong inhibition effect,and 10±0.2 mm for SQ0054 strain,which had slight inhibition effect.The inhibition diameters of SQ0048 and SQ0054 strains were significantly larger than streptomycin（P<0.05）.To sum up,SQ0048 and SQ0054 strains had better probiotic characteristics than other strains.2.In order to deeply analyze the main components of SQ0048 and SQ0054 that play an antibacterial role,antibacterial peptide genes of SQ0048 and SQ0054 were cloned and expressed by molecular biological methods,and the antibacterial properties of antibacterial peptide gene expressions were also studied.The results showed that the antimicrobial peptide genes of SQ0048 and SQ0054 strains were successfully cloned and expressed.The homology between SQ0048 positive clone strain and Lactobacillus johnsonii NCC 533（NC₀05362.1）was 100%.The expressed recombinant plasmid had 100%homology with Lactobacillus johnsonii NCC 533（NC₀05362.1）,the SQ0054 positive clone strain had 95%homology with Lactococcus garvieae Lg2（12479992）,and the expressed recombinant plasmid had 100%homology with Lactococcus garvieae Lg2（12479992）.The expressed products of SQ0048 antibacterial peptide against Staphylococcus aureus was 15±0.45 mm,which had very strong inhibitory effect.The expressed products of SQ0054 antibacterial peptide against Staphylococcus aureu was 11±0.8 mm,which had strong inhibitory effect.The diameter of inhibition zone of SQ0048 antimicrobial peptide expression product on Staphylococcus aureus was significantly larger than SQ0054 antimicrobial peptide expressed products（P<0.05）,and was significantly larger than PBS（P<0.01）.Compared with penicillin,there was no significant difference（P>0.05）.The diameters of inhibition circle of expressed products of SQ0048 antibacterial peptide was 18±0.60mm against Escherichia coli,which had very strong inhibition effect,while it was 13±0.5 mm of SQ0054,which had strong inhibitory effect.The diameter of inhibition zone of SQ0048 antibacterial peptide expressed products to Escherichia coli was significantly larger than antibacterial peptide expressed products of SQ0054,PBS and streptomycin（P<0.01）.To sum up,it could be seen that the antibacterial ability of the antibacterial peptide expression of SQ0048 strain was better than that of SQ0054 strain,which had strong antibacterial effect.3.In order to further explore the molecular mechanism of probiotic effects of SQ0048 and SQ0054 strains,the following studies were carried out:（1）Cow vaginal epithelial cells were isolated,cultured and purified by primary cell separation and culture technology.Morphological,immunofluorescence and PCR methods were used to identify them.Cell purity was determined by flow cytometry.The growth characteristics of cells were monitored by RTCA system.The optimal conditions for cell cryopreservation were determined by single factor method.The results showed that the isolated,cultured and purified cow vaginal epithelial cells were mostly flat polygonal or short spindle-shaped（paving stone-like）,with clear boundaries,full cytoplasm,clear and visible nucleus,round shape,in line with the morphological characteristics of epithelial cells,the cells of vaginal tissue samples were identified by immunofluorescence,with blue nucleus,rich cytoplasm and red color,showing that cytokeratin CK-18 was positive,and the expression of cytokeratin CK13 gene was significantly different from that of the control group（P<0.01）,proving that the isolated and purified cells were cow vaginal epithelial cells.The purity of purified cells was 94%.When the cell inoculation density was 1.63×104 cfu/mL,the growth curve was in the best state and showed a steady upward trend.When the ratio of fetal bovine serum to DMSO in cell cryopreservation solution was 9:1,the number of activated living cells after cryopreservation was more,and the activated cells were in a better state and could be subcultured.This laid a good experimental foundation for further research on the effect of SQ0048 strain on TLRs-MyD88/NF-κB signaling pathway in cow vaginal epithelial cells.（2）The RT-qPCR method was used to analyze the regulatory effect of SQ0048 strain on TLRs-MyD88/NF-KB signaling pathway in cow vaginal epithelial cells.The optimal concentration and time of TLR4,TLR2 and TLR4,MEKK1,IKK,NF-κB inhibitors were determined.At the same time,the CCK-8 method was used to detect the inhibitor at the optimal concentration and time of action and the toxic effect of SQ0048 strain on cow vaginal epithelial cells at medium,high and low concentrations.It was confirmed that the inhibitor and SQ0048 strain had no significant toxic effect on cow vaginal epithelial cells.（3）RT-qPCR,ELISA and western blot methods were used to determine the effect of SQ0048 on TLRs-MyD88/NF-KB signaling pathway after acting on cow vaginal epithelial cells.The results showed that SQ0048 strain at the medium concentration could up-regulate the expression of MyD88,MEKK1,IKK,NF-κB factors by exciting TLR2 and TLR4,which had an effect on TLR2/TLR4 mediated NF-κB signaling pathway,suggesting that SQ0048 lactobacillus strain could exert its biological function through TLR2/TLR4 mediated NF-κB signaling pathway.After the excitement of NF-κB induced by strain SQ0048,it could up-regulate the expression of IL-1β,IL-6,IL-8 and TNF-a,while down-regulate the expression of IL-10.Conclusion:SQ0048 strain has strong acid-producing ability,fast acid-producing speed and probiotic effect of hydrogen peroxide production.SQ0048 strain and its expressed antimicrobial peptide have significant inhibitory effects on Staphylococcus aureus and Escherichia coli.The probiotic effect of SQ0048 is related to its activation of MyD88/NF-κB signaling pathway mediated by TLR2 and TLR4,up-regulating the expression of IL-1β,IL-6,IL-8,TNF-a and down-regulating the expression of IL-10.