| Probioties have become the ideal alternative to antibiotics for its non-toxic,no residue,no drug resistance as well as its low cost.In this study,two different strains of microorganisms were isolated from intestine of Anhui local piglets,and the properties of these strains were evaluated in vitro and cell modle.The in vivo effects and probiotic mechanisms of these microbes as probiotics in weaned piglets were conducted.The contents of each experiment are as follows:1:Isolation and indentification of dominant porcine lactobacillus and bacillusWe screened a lactic acid bacteria and a bacillus bacteria from intestine of Anhui local piglets by dilution coating method,whom were found to be resistant to to the artificial gastric juice,artificial intestinal juice and bile,can also inhibit bacteria reproduction of Staphylococcus aureus,Escherichia coli and Salmonella enteritidis.The strains were calassified as Lactobacillus reuteri and Bacillus licheniformis according to the results of some physiology biochemical tests and the analysis of 16S rDNA sequences,respectively named Lactobacillus reuteri R-1 and Bacillus licheniformis D-2.The two strains were sent to China Center for Type Culture Collection with accession numbers CCTCC M2015660 and CCTCC M2015457.2:Study on properties of probiotics in vitroTo study the enzyme production characteristics,we determined lipase by the olive oil floatation method,protease by the Folin method,amylase by the Yoo modification method,and cellulase by the DNS method?The in vitro antioxidant properties of the two strains were studied by DPPH,·OH and O2-scavenging rate,reducing power and antioxidant enzyme content of supernatants and cell-free extracts of D-2 and R-1 by Oxford-cup test.These results have indicated D-2 can produce protease,lipase,amylase and cellulase,the protease and amylase activity,respectively 70.17U/ml and 31.10U/ml,while R-1 was detected aprotease and cellulase with activity 21.17 U/ml and 4.35U/ml only;,and showed that the antibacterial activity of R-1 decreased significantly after treating lactate dehydrogenase or NaOH,while the ability of D-2 maintain stability under various treatments;The antibiotics sensitivity of D-2 and R-1 were studied by antibiotic susceptibility paper method,D-2 and R-1 were found to be highly sensitive to many kinds of antibiotics which means they are not with the resistance plasmid?3:Research on function and mechanism of probiotics on piglet's intestinal epithelial cellsIn order to study function and mechanism of probiotics on pigle' s intestinal epithelial cells,fluorescent labeling was carried out to investigate the adhesive properties of R-1,D-2 and Escherichia coli K88 to IPEC-J2 cell.And the effects of the strains on IPEC-J2 cell viability were detected by analysis of trypan blue and lactate dehydrogenase activity.The results showed that the adhesion rate of the R-1 to IPEC-J2 cell was higher than that of E.coli K88 and R-lhad significant effect on the adhesion of E.coli K88 to IPEC-J2 cells.While D-2 had lower adhesion rate and had no effect on the adhesion of E.coli K88 to IPEC-J2 cells.The cell survival rate and cell membrane integrity of IPEC-J2 cells were significantly decreased by E.coli K8,whereas R-1 and D-2 did not affect cell survival rate and cell membrane integrity.It showed that Lactobacillus reuteri R-1 and Bacillus D-2 are safe as probiotics.Cytokine contents were measured with enzyme-linked immune-osorbent assay(ELISA)method,which indicated probioties could influence IPEC-J2 cell immunity by regulating cytokine.4:Effect of Probiotics on diarrhea index,antioxidant and immune indexes of weaning pigletsOne hundred and tewnty piglets of Large White×Landrace weaned at 28 day of age were selected and divided into four groups,and the piglets from each group were assigned randomly to three pens(replicates)with tens animals each.Each group was fed one of four diets for 4 weeks.Control:a basal diet without antibiotics and probiotics,I:the basal diet supplemented with D-2(making the number of live bacteria in the diet reached 107CFU/g),?:the basal diet supplemented with Lactobacillus reuteri R-1(making the number of live bacteria in the diet reached 107CFU/g),?:the basal diet supplemented with a mixture of s D-2 and R-1(making the live bacteria number of each strain in the diet reached 5×106CFU/g).The diarrhea rate,tissue stained with he and the antioxidant immune indexes were measured,the results showed that diets supplemented with probiotics can significantly reduce diarrhea rate of weaned piglets.Compared with the control group,probiotic may improve intestinal health and alleviate oxidative stress and immunity functiondecline of early weaning piglets.5:Effect of probiotics colonization on intestinal health microbial diversity of weaning pigletsThe experiment design was the same as experiment4,probiotics in fecal samples of piglets on the 28 day and the 42 day were accurately quantitative analysis by real-time PCR.The results showed that two probiotics have been colonized in the gastrointestinal tract of piglets.To analyze intestinal microflora of weaning piglets,traditional microbial culture and high-throughput sequencing technology were used.According to the results we found probiotics can colonize the digestive tract of piglets to regulate the intestinal flora by promoting intestinal probiotics grow and restraining harmful bacteria.Although high-throughput sequencing technology results showed that adding probiotics have no major impact on the core microflora,but that can improve the intestinal microbial diversity. |
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