Effects Of Compound Probiotics On Intestinal Microflora And Health Of Rabbits

Probiotics have been widely used in animal production.In the era of "anti-antibiotic",probiotics have become a new research direction of green feed additive industry because of its many advantages.To better evaluate the potential of probiotics in production,this study screened probiotics by testing their enzyme production capacity,ability to tolerate artificial gastric and intestinal fluids,adhesion capacity and the ability to inhibit the adhesion of Caco-2cells by Enterohemorrhagic Escherichia coli（EHEC）.The screened strains with excellent performance were combined to study the role of the complex probiotics in EHEC-infected RK-13 cells and rabbits in vitro and in vivo experiments.In order to verify the probiotic effect produced by the compound probiotics in animals,this study was conducted to evaluate the effects of the compound probiotics on growth performance,immune capacity,antioxidant capacity and intestinal barrier function in rabbits by feeding experiments.We also investigated the effects of compound probiotics on the species and abundance,functional pathways and resistance genes of rabbit cecum flora by metagenomics method.This study is divided into three main sections:1.Screening and combination of probioticsIn this study,the protease production capacity of 11 strains of lactic acid bacteria was firstly evaluated and the results showed that Lactococcus lactis 6242,Lactobacillus acidophilus6075 and Lactobacillus plantarum 21790 had high protease production capacity.By testing the tolerance of lactic acid bacteria to artificial gastric juice and intestinal juice,it was found that Lactococcus lactis 6242,Lactobacillus plantarum 21809 and 21790 and Enterococcus faecalis24203 could maintain a high survival rate when passing through gastric juice,with survival rates exceeding 40%.Lactobacillus rhamnosus 6141 and 6137,Lactobacillus acidophilus 6075,Lactobacillus plantarum 21790 and Enterococcus faecalis 24203 could maintain high viability in the intestinal fluid,with survival rates exceeding 60%.In addition,the adhesion ability of lactic acid bacteria was tested,and it was found that Lactococcus lactis 6242 and Lactobacillus delbrueckii subsp.bulgaricus 6103 had stronger adhesion among all tested strains.Subsequently,the ability of lactic acid bacteria to inhibit the adhesion of EHEC to Caco-2 cells was studied.The results showed that Lactococcus lactis 6242,Lactobacillus plantarum 21790 and Lactobacillus acidophilus 6075 had the highest inhibition rate on EHEC adhesion to Caco-2 cells among all tested strains.The four lactic acid bacteria with better performance were screened as Lactococcus lactis 6242,Lactobacillus plantarum 21709,Lactobacillus acidophilus 6075 and Enterococcus faecalis 24203 by combining the protease production ability,the ability to tolerate artificial gastric and intestinal fluids,the adhesion power,and the ability to inhibit the adhesion of EHEC to Caco-2 cells.The selected lactic acid bacteria were combined with Bacillus coagulans 24625,Bacillus subtilis BYS2,Saccharomyces cerevisiae33298 and Clostridium butyricum 10390,and divided into lactobacillus group（6242,21709,6075,24203）and compound probiotics group（24625,BYS2,21790,33298,10390）for subsequent experiments.2.Effects of compound probiotics on growth performance,antioxidant capacity,intestinal flora and immunity of rabbitsTo test the effects of compound probiotics on growth performance,antioxidant capacity,intestinal barrier function,intestinal flora and immunity of rabbits,a total of 300 New Zealand white rabbits were randomly divided into four groups for feeding experiment: lactobacillus group（group A）,compound probiotics group（group B）,control group（group C）and antibiotic group（group D）.By detecting the serum immunoglobulin content and immune organ index of rabbits,it was found that the immune organ index and serum Ig G and Ig M content in group B were significantly increased（P < 0.05）,which indicated that probiotics could improve the body’s immune ability.The antioxidant capacity of the rabbits was examined and it was found that the content of superoxide dismutase（SOD）was significantly higher in group B compared with other groups（P < 0.05）,the content of catalase（CAT）was significantly higher in group A（P < 0.05）,and the content of malondialdehyde（MDA）was significantly lower in group B（P < 0.05）,which indicated that probiotic feeding could enhance the antioxidant capacity of the organism to some extent.At week 5,the cecum contents of rabbits were for metagenome sequencing and found that feeding probiotics increased the relative abundance of Ruminococcus and Akkermansia and decreased the relative abundance of Alistipes and Bacteroides in the cecum flora.According to the linear discriminant analysis effect size（LEf Se）,it was found that the key OTUs in group C were mainly Methanobacteria under Euryarchaeota classification.In groups A and B,the key OTUs were mainly bacteria under Firmicutes and fungi,and in group D,the key OTUs were mainly Proteobacteria,indicating that feeding probiotics significantly affected the microbial composition in the cecum.Comparing the sequencing results with the database of Kyoto Encyclopedia of Genes and Genomes（KEGG）,it was found that the main KEGG pathways in group B were amino acid metabolism pathway,lipid metabolism pathway,xenobiotics biodegradation and metabolism and carbohydrate metabolism pathway.The KEGG pathways of group A mainly focused on metabolism of cofactors and vitamins,glycan biosynthesis and metabolism,metabolism pathways of terpenoids and polyketides and lipid metabolism pathway.The KEGG pathways of group D mainly focused on xenobiotics biodegradation and metabolism and metabolism pathways of terpenoids and polyketides.In addition,this study found that antibiotic resistance ontology（ARO）genes in the rabbit cecum were mainly β-lactamases（OXA-257,CTX-M-106,LEN-12,LRA-12）,extended-spectrum β-lactamases（TEM-147,TEM-16）,macrolide 2’-phosphotransferase II（MPHB）,plasmidmediated quinolone resistance protein（QNRB33）,aminoglycoside phosphoryl transferase（APH（3’）-VIIIa）,and glycopeptide antibiotic（VANTG）.Among them,there were 1964 types of ARO in group B,while the maximum number of types reached 2105 in group D and 1982 in groups A and C,responding that probiotics could reduce the production of antibiotic resistance genes in the intestine of rabbits to some extent.This study tested the growth performance of rabbits,and the results showed that compared with the control group,the average weight of group A and B increased by 14.88% and 12.33%,respectively,indicating that the addition of probiotics to the diet could significantly improve the growth performance of rabbits（P < 0.05）.3.Effects of compound probiotics on RK-13 cells and rabbits infected with EHECThe expression of cytokines in RK-13 cells infected with EHEC was examined.Compound probiotics could significantly decreased the expression of inflammatory cytokine m RNA in RK-13 cells infected with EHEC（P < 0.05）.Compared with the EHEC group,the expression level of DEFB124 in group B was significantly increased（P < 0.05）,suggesting that probiotics could inhibit the inflammatory response induced by EHEC,and enhance the expression of defensins.In addition,this study counted the survival of rabbits infected with EHEC,and found that compared with group C,the survival rate of rabbits in group A increased by 23.51% and that of rabbits in group B increased by 34%.The expression of cytokines in the spleen of rabbits was examined after 3 days of attack,and it was found that the expression of IL-6 was significantly lower（P < 0.05）,while the expression of IL-4 and defensin was significantly higher（P < 0.05）in groups A and B,which was also associated with the increased survival rate of rabbits in the probiotic group after the attack.In vitro and in vivo experiments have demonstrated that probiotics can inhibit the production of inflammatory cytokines induced by EHEC,increase the expression of defensins,and help rabbits exert anti-infective effects.In summary,probiotics,especially in combination with different species,can improve the growth performance,antioxidant capacity,immune ability and disease resistance of rabbits,improve intestinal barrier function,regulate the species,abundance and functional pathways of intestinal flora to produce better probiotic effects on organisms,and to a certain extent,reduce the generation of antibiotic resistance genes in the intestinal tract of rabbits.This study is expected to provide data support and theoretical reference for the use of compound probiotics as green and healthy feed additives.