| Probiotics are a new type of green and pollution-free feed additive that can inhibit the growth of pathogenic bacteria,improve gastrointestinal function,and promote the healthy growth of livestock and poultry.In this experiment,the probiotics in the cecum of White Leghorn laying hens were isolated and identified,and their in vitro and in vivo probiotic functions were studied,providing a basis for the development and application of chicken-derived probiotics.Experiment one:The experiment used the caecum of White Leghorn laying hens as samples.And 5 different culture media were used to anaerobically culture the cecal microbiota at 37℃and 25℃for 1 and 7 days,respectively.The isolated strains were identified by 16S r RNA technology.The results show that:A total of 345 bacterial strains were isolated and screened from eight healthy chickens.Fifteen strains were screened according to Gram staining,tactile enzyme reaction,nitrate reduction reaction and gelatin liquefaction test for 16S r RNA identification and glycolysis test.Among them,11 strains were identified as Enterococcus faecalis(F1-F11)and four strains were identified as Enterococcus faecium(S1-S4).Experiment two:The 15 strains of Enterococcus isolated in Experiment 1 were analyzed for their acid tolerance,bile salt tolerance,adhesion ability,in vitro antioxidant capacity,antibacterial activity,and drug resistance.The probiotic characteristics study showed that all 15 strains of isolated bacteria could survive in acidic and high-concentration bile salt solutions and exhibited good adhesion and in vitro antioxidant capabilities.They also showed strong inhibitory effects on Staphylococcus aureus and acid-producing ability.Hemolysis and drug sensitivity tests indicated that the isolated strains did not produce hemolytic zones and were sensitive to multiple antibiotics.Among them,strain F4 showed the most significant probiotic effects and was selected as the strain for animal feeding trials.Experiment three:In this experiment,strain F4 of the screened bacteria from Experiment 2 was used as the test strain for in vivo testing.The animal experiment used a 2×2 two-factor design method,choose 80 healthy SPF White Leghorn chicks hatched by myself and randomly divided into 4 groups:specific-pathogen-free(SPF)group,SPF+10~8CFU/kg Enterococcus faecalis group,commercial cage group,and cage+10~8CFU/kg Enterococcus faecalis group,with 20 chicks in each group.The test period was42 days,with sampling on the 21st and 42nd days.The serum biochemical and immune antioxidant indicators,cecal microbiota diversity,and gene expression of intestinal mucosal immune factors and tight junction proteins among the groups were detected.The animal feeding test showed that adding Enterococcus faecalis to the diet significantly increased the body weight of the chickens.Enterococcus faecalis and the cage environment increased the richness of the Firmicutes and Lactobacillus in the cecum and reduced the richness of the Proteobacteria.Enterococcus faecalis and the cage environment significantly increased the levels of Ig A,Ig M,SOD,T-AOC,GSH,CAT,TP,ALB and TC in the serum(P<0.05),reduced the level of MDA and ALP in the serum(P<0.05),and improved the immune and antioxidant capabilities of the chickens.Enterococcus faecalis improved the height of the villi in the jejunum and ileum,and increased the m RNA expression of IL-1,IL-6,ZO-1,and Claudin-1 in the jejunum mucosa(P<0.05).In conclusion,in SPF and cage environment,the addition of Enterococcus faecalis to feed can improve the immunity and antioxidant capacity of laying hens,regulate intestinal flora,and promote the health of livestock and poultry.In conclusion,the Enterococcus faecalis screened based on culturomics has good probiotic properties such as stress resistance and bacteriostasis,and can improve the immunity of White Leghorn chicks and promote the healthy growth of chickens.This study provides excellent candidate strains for the further development of chicken-derived probiotics,which has certain production practical significance. |
