The Development Of The Compound Of Enzyme And Probiotics And Its Effect On The Growth And Immune Function Of Mice

With the rapid development of China’s breeding industry,antibiotics are often used as feed additives in livestock production to prevent and treat diseases.However,the abuse of antibiotics will lead to a large number of drug-resistant bacteria,and a large number of residues in livestock and poultry products,which will affect the safety of animal derived food and human health.Probiotics and enzymes can regulate the intestinal health of animals,promote growth and enhance immunity.Therefore,it is urgent to develop enzyme and new microecological agent compound for aquaculture and reduce the use of antibiotics.In this experiment,probiotics and enzyme preparation were compounded and vacuum freeze-drying technology was used to make a new enzyme and microecological preparation compound.The safety test,growth performance and immune function of probiotics and microecological preparation were tested in mice.The research contents are as follows:Experiment 1,preparation and detection of the compound of enzyme and probiotics.The neutral protease and cellulase were added to the culture media of Bacillus subtilis,Saccharomyces cerevisiae and Lactobacillus acidophilus for fermentation.The protective effect,gastric acid tolerance,enteric release and storage time at different temperatures were studied by vacuum freeze-drying technology.The results showed that the addition of neutral protease and cellulase in Bacillus subtilis was 1g/L and 3g/L.The addition amount of neutral protease and cellulase were 2 g/L and 3g/L respectively.When Lactobacillus acidophilus was cultured,the addition amount of neutral protease and cellulase were 2 g/L and 4 g/L.The best protectants were 6% gelatin,25% skimmed milk powder and 8% glycerol.The survival rate and enteric release rate of Lactobacillus acidophilus,Bacillus subtilis and Saccharomyces cerevisiae in gastric acid were high;It could be stored at-20℃ and 4℃ for 120 days and at room temperature for about 30 days.Experiment 2,the safety test of the compound of enzyme and probiotics.The BALB/c mice were given different doses(0 g,2.5 g,5 g,10 g/kg body weight)of enzyme and probiotics,The growth status of mice was observed,blood routine,blood biochemistry and bacterial translocation were detected,and the liver and kidney of experimental mice were taken to make tissue sections.The results showed that no toxic symptoms,no death and no bacterial translocation were found in the mice treated with the compound of enzyme and probiotics.There were no significant difference in body weight and blood biochemical indexes between the two groups.The number of leukocytes and lymphocytes in the middle dose group and the high dose group increased significantly(P < 0.05),but they were in the normal range.The structure of liver and kidney were normal,and no abnormality was found.Experiment 3,the effects of the compound of enzyme and probiotics on growth performance and immune function of mice.The BALB/c mice were given different doses of the compound of enzyme and probiotics The blood samples and intestinal tissues were taken,and immune organs were taken for determination.of immune organ index.The content of lysozyme in blood was detected by turbidimetric method.The content of Cytokine,SIg A and MUC2 in jejunum mucosa of mice were detected by ELISA.Immunohistochemistry(IHC)and q RT-PCR were used to detect the expression of MUC2 protein and m RNA in intestinal mucosa.The results showed that the weight,immune organ index and lysozyme content of mice in each experimental group were higher than those in the control group(P<0.05 or P<0.01).The contents of IL-4,IL-10 and s Ig A in jejunal mucosa of each experimental group were significantly higher than those of the control group(P<0.05 or P<0.01).The contents of TNF-γ in each groups were higher than that of the control group(P<0.05 or P<0.01).The results of immunohistochemistry showed that MUC2 was mainly expressed on the mucosal surface of jejunum and ileum,and the expression of MUC2 in jejunum and ileum of each experimental group were higher than that of the control group(P<0.05 or P<0.01).The results of fluorescence quantitative analysis showed that the expression of MUC2 m RNA in jejunum and ileum of each experimental group were higher than that of the control group(P<0.05 or P<0.01).Conclusion: The compound of enzyme and probiotics was successfully prepared by vacuum freezing.The compound has high number of the live bacteria,gastric acid resistance,high enteric coated release rate,no miscellaneous bacteria,and was not easy to be affected by moisture at room temperature.It could be preserved for about 120 days at-20℃ and 4℃,and the best preservation period was about 30 days at room temperature(25-30℃).It could promote the growth of mice,increase the index of immune organs,increase the lysozyme content of mice,and enhance the intestinal mucosal immune function of mice.