The Research Of Biological Properties Of Lactobacillus Isolated From Tibetan Chickens And Its Effects On The Intestinal Flora In AA Broiler

Lactobacillus are one of the most important probiotics in animal organism,the giant application and scientific interest in Lactobacillus has grown out of safe and effective biological characteristics.The Tibetan Chicken is an aboriginal species which resist and extremely adapt to frigid temperature,hypoxia and roughage due to their special physiological and genetic adaptations.However,few studies have been done on the intestinal flora of Tibetan chickens,and there is little research on the probiotics of chicken.Therefore,we isolated and identified Lactobacillus strains from 23 Tibetan chickens based on morphological,physiological,biochemistry characteristics and 16S rDNA sequencing analysis.The antibacterial characteristics of selected Lactobacillus strains were tested through agar diffusion experiment and molecular biological method included OD₆₀₀,OD₂₆₀ and extracellular potassium ion concentration.And outstanding Lactobacillus strains were screened for zoological experiments based on the research results of antibacterial experiment and biological stability studies.The important part of zoological experiments focused on the effects of Lactobacillus strains on intestinal villus of AA chickens.The last part of our research was conducted to preform small intestine microbiota diversity analysis of Tibetan Chicken by employing high-throughput sequencing of 16S rRNA V3-V4.The main research contents and results are as follows:1.Isolation and identification of Lactobacillus from Tibetan chickens42 Lactobacillus strains included Weissella cibaria,Lactobacillus plantarum,Pediococcus pentosaceus,Pediococcus acidilactici,Lactobacillus reuteri and Lactobacillus mucosae,named WT1,LP1,PT2,PA1,LR1 and LM1.The 6 kinds of Lactobacillus strains we isolated were significantly resistant to 7 antibiotics according to the standard proposed by CLSI,and 36 strains were multidrug-resistant strains.The LP1,PT2,PA1 and WT1 showed good bile salts tolerance and excellent resistance to acid,the LP1,PT2 and WT1’s survivial rates were greater than 40%under the condition of 0.3%bile salt concentration or PH=3,especially,the LP1 survival rate was 72.33 under 0.3%bile salt.2.Bacteriostatic characteristics of LactobacillusBacteriocins derived from WT1 and PT2 strains manifested obvious antibacterial activity against the tested pathogen of Salmonella enterica serovar Enteritidis（S.Enteritidis NCTC 13349）,Escherichia coli（E.coli ATCC 25922）and Staphylococcus aureus（S.aureus ATCC 25923）,LP1 also showed excellent bacteriostatic ability against E.coli（ATCC 25922）and S.aureus（ATCC 25923）.Thetreatment with WT1 gave rise to a significant increase in leakage of nucleic acid from the cells of S.Enteritidis,S.aureus and E.coli.The OD₂₆₀ in the supernatants of the co-cultures of WT1 with S.Enteritidis,S.aureus and E.coli reached 4.05,3.62,3.59respectively.And the OD₂₆₀ in the supernatants of the co-cultures of PT2 with S.Enteritidis,S.aureus and E.coli reached 3.67,3.56,3.85 respectively.In addition,The OD₂₆₀ in the supernatants of the co-cultures of LP1 with S.aureus and E.coli reached3.56 and 3.46 respectively.On the contrary,the OD₂₆₀ of control group still remained 1.50.Moreover,the bacteriocins produced by WT1,PT2 and LP1 have good acid resistance and thermal stability.Furthermore,the PCR product of approximately 1500 bp was amplified from the DNA of WT1,PT2 and LP1with the Ent A primer set.And approximately 350 bp was amplified from the DNA of WT1 and PT2 with the Ent L50primer set.3.The effect of lactobacillus strain on the weight and intestinal villus height of AA broilerIn the experimental group,the intestinal mucosa was intact and small intestinal epithelial cells were lined up.The administration of WT1 could significantly promote the villus height of jejunum（P<0.01）,ileum（P<0.05）and cecum（P<0.05）.PT2 showed the positive effect on promoting villus height of jejunum（P<0.01）and duodenum（P<0.05）.And LP1 could obviously promote the villus height of jejunum（P<0.01）,duodenum（P<0.05）and ileum（P<0.05）.Moreover,WT1,LP1 and PT2 of Lactobacillus could promote the weight gain of AA broiler,and the growth of intestinal villi of AA broiler was positively correlated with weight gain.4.Effect of lactobacillus on intestinal flora of AA broilersAdministration WT1、PT2 and LP1 could promote the content of Lactobacillus and resist the growth of pathogens.The Lactobacillus included WT1,PT2 and LP1manifested the positive adjustment role of intestinal flora.The proportion of Lactobacillus in the WT1 group was 53.8%（ileum）,93.1%（jejunum）and 8.5%（cecum）,respectively.The proportion of Lactobacillus in the group was 93.1%（jejunum）.The proportion of Lactobacillus in LP1 group was 78.8%（cecum）,which was significantly higher than that in the control group.At the same time,the Alpha analysis showed that LP1,WT1,and PT2 in chickens were able to improve the community richness and diversity of intestinal tissue.