Screening Of Chicken Probiotics And Cloning And Construction Of Shuttle Vector

With the shortcomings exposed by the abuse of antibiotics,the country is paying more and more attention to breeding without resistance.Therefore,it is very urgent to screen probiotics with good properties to replace antibiotics.Probiotics as feed additives have become a research hotspot.In this study,chicken manure samples from chickens within 1 week of birth were collected from chicken flocks of self-raised and self-propagating farmers in Guangxi,from which candidate strains with inhibitory effects on pathogenic microorganisms and characteristics as feed additives were screened.The inhibitory ability,antibiotic sensitivity and tolerance to simulated gastrointestinal fluid,high osmotic pressure and strong acidity of candidate strains were evaluated.The types of inhibitory substances of each strain were preliminarily analyzed,and the shuttle plasmid of E.coli-lactic acid bacteria was constructed.In this study,four strains(numbered J13,J26,J30 and J31)with strong inhibitory effects on pathogenic E.coli,Salmonella and S.aureus were obtained through primary screening and re-screening.After preliminary identification by morphology and 16 S r DNA sequence analysis,all four strains belonged to lactic acid bacteria,and the strain J13 belonged to Enterococcus faecium,strains of J26 and J31 belong to Lactobacillus plantarum,and the strain J30 belongs to Lactococcus lactis.The results of growth curve analysis showed that the growth trends of J26 and J31 were similar,they all entered logarithmic phase after 2-4 h of culture,stable phase after 16-20 h,slightly decreased after 20 h of cultivation,and became stable after 24 h of cultivation.The logarithm growth period of j13 and J30 was shorter,J13 entered stable period after 12 hours of culture,J30 entered stable period after 16 hours of culture.Four strains of lactic acid bacteria showed stronger inhibitory ability to S.aureus than pathogenic E.coli and Salmonella.The diameter of inhibition zone reached 12.75-23.22 mm.Two strains of Lactobacillus plantarum showed stronger inhibitory ability to pathogenic microorganisms than Lactococcus lactis and Enterococcus faecium.The effects of temperature,p H and proteinase K on the stability of the extracellular antibacterial substances of the strains were studied.The results showed that temperature and proteinase K had no effect on the stability of the extracellular inhibitory substances of the strains.But,p H had a greater effect on the extracellular substances of the strains,the higher the p H,the worse the antibacterial ability,indicating that the major inhibitory substances were greatly affected by p H.According to the reported bacteriocin gene sequences on NCBI,bacteriocin genes were successfully cloned from strains J26 and J31,indicating that thw extracellular bacteriostatic substances include bacteriocins.Disk diffusion method was used to identify the drug susceptibility of four strains.All strains were sensitive to ampicillin and neomycin.Strain J30 showed tolerance only to rifampicin,while strain J13 was more tolerant to most antibiotics including enrofloxacin,streptomycin,kanamycin and erythromycin.The tolerance of strains J26 and J31 to simulated gastric juice,intestinal juice,low p H,high concentration of bile salts and high osmotic pressure were analyzed by in vitro experiments.The results showed that the log survival rate of the J26 and J31 remained above 80% after 3 h of treatment in the simulated gastric juice experiment in vitro.In the simulated intestinal juice experiment in vitro,they also showed good tolerance ability after 3 h of treatment that the logarithmic survival rate of the J26 was maintained above 75% and the J31 was maintained above 85%.They could survive normally in the environment of p H 2.0 and p H 3.0 and tolerate 0.25%-1% bile salt concentration.In the environment of 10% concentration of Na Cl,they could not survive normally.However,in the environment of 2%-6% concentration of Na Cl,the logarithmic survival of strain J26 remained above 7.66 Log cfu/m L and the J31 remained above 7.32 Log cfu/m L.It meets the requirements of intestinal colonization ability of animals.Based on the E.coli-lactic acid bacteria shuttle plasmid p MG36 e and E.coliBacillus subtilis shuttle plasmid p BE-S,the E.coli-lactic acid bacteria shuttle expression vector was successfully constructed,named p EL.The recombinant expression plasmid p EL-egfp was constructed by connecting egfp as a reporter gene to p EL,and the recombinant vector was transformed into strain J30 to obtain a single colony of the recombinant on a resistant plate.Validation of colony with target by PCR,but it can’t detect in solution of colony.It indicates that the recombinant plasmid cannot exist stably in the J30 strain.