Screening Of Lactic Acid Bacteria For Feeding And Their Microencapsulation Preparation

Enterococcus faecalis is a kind of parthenogenic anaerobic Gram-positive bacteria widely used in lactic acid bacteria microecological preparations,which exists widely in the environment and belongs to intestinal symbiotic bacteria,which can adhere and colonize in animal intestine and form a dominant flora.Its metabolites are important for the suppression of intestinal pathogenic bacteria,intestinal flora stability and livestock growth.In this study,an acid-producing strain of Enterococcus faecalis,which can inhibit intestinal pathogenic bacteria,was purposefully screened from the manure of healthy weaned calves,and its bacterial inhibitory properties and main metabolites were studied,and the preparation technology of highly active bacterial agent was carried out.The main results of the study are as follows:(1)Three acid-producing strains were screened from weaned calf manure through acid production and gas production and bacterial inhibition experiments,among which the best bacterial inhibition was strain SL-2901.SL-2901 was finally identified as Enterococcus faecalis through morphology,sugar fermentation and other physiological,biochemical and molecular biological identification.(2)The inhibition experiments of Enterococcus faecalis SL-2901 fermentation solution against five common pathogenic bacteria in the intestinal tract revealed that SL-2901 has broad-spectrum inhibition and is more effective in inhibiting Gram-negative bacteria than Gram-positive.(3)The inhibition effect of the fermentation supernatant of Enterococcus faecalis SL-2901 was greatly influenced by the p H value.p H=2.00～5.00,when decreasing the p H value could significantly improve its inhibition effect.There was no significant change in its inhibition after high temperature treatment,indicating that the inhibition product of Enterococcus faecalis SL-2901 was more heat resistant.The effect of pepsin,trypsin,proteinase K and papain treatment on the bacterial inhibition effect of Enterococcus faecalis SL-2901 fermentation broth was weak.(4)The metabolites of Enterococcus faecalis SL-2901 were quantified by LC-MS untargeted metabolomics combined with multivariate statistical methods,and a total of 443 differential metabolites were screened by matching the differential metabolites with the HMDB database,among which benzenes and their derivatives accounted for 9.93%,lipids and lipid-like molecules for 34.09%,organic acids and their derivatives for 15.12%,organic nitrogen compounds for 14.31%,organic oxygen compounds for 8.58%,and organic heterocyclic compounds for 22.12%.15.12%,organic nitrogen compounds 14.31%,organic oxygen compounds 8.58%,and organic heterocyclic compounds 22.12%.Among them,nucleotides,organic acids and their derivatives and amino acids were the most varied substance groups between groups.Metabolites such as histamine,DL-4 hydroxyphenyl lactate,phenylpyruvate,acetyl phosphate,indole-3-lactic acid and dehydro complexine were the effective inhibition products produced by Enterococcus faecalis SL-2901.(5)In order to enhance the resistance of Enterococcus faecalis SL-2901 and improve its survival rate after gastrointestinal passage,the preparation process of Enterococcus faecalis microcapsules was optimized using response surface testing.The results showed that the optimal process conditions for the preparation of E.faecalis microcapsules were 3.0% SA mass fraction,2.5% PVA mass fraction and 2 h immobilization time,and the microcapsule embedding rate reached 85.7% under these conditions;the survival rate in simulated gastric juice reached 70.1%,which was 43.9% higher than the survival rate of the strain without microcapsule embedding.