Safety And Probiotic Evaluation Of Lactobacillus Paracasei L1 And Its Application In Hybrid Chicken

Lactobacillus paracasei L1 is a predominant lactic acid bacteria isolated from natural fermented sweet potato sour milk.It can rapidly proliferate in high starch content medium,produce acid efficiently,adhere to starch and settle starch rapidly.It is used to make flocculant of sweet potato starch.In Henan,Shandong and Shaanxi provinces where the production of sweet potato starch by acid slurry method is prevalent,the local people have the habit of drinking naturally fermented sweet potato sour slurry.For this reason,this study evaluated the safety and probiotics of L.paracasei L1,and evaluated the effects of L.paracasei L1 on the growth performance,immune function and intestinal flora of hybrid chickens through feeding experiments.It laid a theoretical foundation and provided practical guidance for the production and application of L.paracasei L1 in human and feeding probiotics.The main research methods and results are as follows:1.Safety evaluation of L.paracasei L1.The drug resistance of L1 was determined by K-B?drug-sensitive paper agar diffusion method? and plasmid extraction.By detecting amino acid decarboxylase,nitrate reductase,indole test and hemolysis test,we can judge whether L1 can produce harmful metabolites.Toxicological evaluation of L1 was carried out through mouse experiments.The results showed that L.paracasei L1 was resistant to some antibiotics and no plasmid existed in the bacteria;in the detection of harmful metabolites,the strain showed negative reaction in the process of proliferation and non-toxic?-hemolysis in hemolysis test.During the acute toxicity and subchronic toxicity experiments,mice did not die,their life activities were normal,their diet and drinking water were stable,their hair was white and their excreta was normal,and the mice grew well,there was no significant difference between the two groups.On the 8th and 31st day,there were no pathological changes in the heart,liver,spleen,lung and kidney of mice,and there was no significant difference in organ index between L.paracasei L1 group and saline group.2.Probiotic evaluation of L.paracasei L1.The environmental tolerance of L1 was determined by adding acid,bile salt and high salt to the medium,the self-aggregation and hydrophobicity of L1 were measured to determine whether the bacteria had adhesion ability,L1 and Caco-2 cells were co-cultured to observe and count the adhesion,so as to determine whether L1 had the ability to adhere to intestinal epithelial cells.The results showed that the total viable bacteria count of L.paracasei L1 was always above 10⁷ CFU/mL in acidic and biliary environments,and the total viable bacteria count of L1 was always above 10¹⁰ CFU/mL in high salinity environments,indicating that L1 had good environmental tolerance.The self-aggregation of L1 was 15.8%±1.2 after incubation for 1 hour,20.4%±2.3 after incubation for 2 hours,47.2%±0.8 after incubation for 24 hours,and the hydrophobicity of L1 was 38.1%±2.7.L1 had good adhesion ability of Caco-2 cells,L1 cells adhesion to each Caco-2 About 30±2.5,L1 cell adhesion rate was 22.37%±1.44.3.Effects of L.paracasei L1 on hybrid chicken performance.240 Dagu×Xianju chickens were randomly divided into two groups.The control group was fed with basic diet added sweet potato juice liquid medium,and the experimental group was fed with basic diet added L1 bacterium liquid.After the experiment,the average daily feed intake,average daily gain and feed-weight ratio of broilers were measured,the results showed that there were no significant differences in average daily gain and feed-to-weight ratio between the experimental group and the control group 40 days before the experiment;but in the latter 20 days,average daily gain also increased significantly,especially in the aspect of material weight ratio decreased significantly.4.Effects of L.paracasei L1 on immune performance of hybrid chicken.At the end of the experiment,three chickens in each group were slaughtered,the immune organ index was calculated and the immune factors in serum were analyzed.The results showed that the thymus index of broilers in the experimental group was 25.1% higher than that in the control group,and the spleen index was 17.6% higher than that in the control group,The difference was significant.The bursa index of fabricius tends to increase,but the difference is not significant.Compared with the control group,the level of IL-4 in serum of hybrid chickens in the experimental group increased significantly,the contents of IL-2 and IL-10 increased,but the difference was not significant,L1 had no significant effect on serum IgA and IgG levels in hybrid chickens,the contents of IFN-?and INS increased,but the difference was not significant.5.Effects of L.paracasei L1 on intestinal flora in hybrid chicken.The effects of L1 on intestinal flora and gene function of hybrid chickens were studied by 16S rDNA high throughput sequencing.The results showed that judging by diversity index,L.paracasei L1 improved the community diversity and abundance in small intestine.The richness,but the difference was not significant,while the Shannon values of large and small intestines in the control group were significantly different,which showed that L1 made the distribution of bacteria in large and small intestines more uniform.The distribution of OTUs in intestine was analyzed by Venn diagram,and it was found that feeding L1 increased the number of OTUs in small intestine.Analysis of intergroup differences,in the large intestine,the Bacteroides S24-7 subgroup in the experimental group was significantly higher than that in the control group.L1 significantly increased the number of Bacteroidetes in the small intestine,the number of Synergistetes,and the number of Bacteroides.In gene function analysis,the experimental group improved the functions of propionate metabolism,fatty acid biosynthesis and glycerolipid metabolism in the large intestine,the function of multiple carbohydrates metabolism in small intestine was improved.