Screening And Physiological Function Of Forage Probiotic Bacteria

Lactobacillus reuteri is one of the important probiotics in animal microecological preparation group. And its metabolites and bacteria itself can inhibit the piglets intestinal pathogen adhesion and growth. So it can improve the immunity of the piglets and reduce piglet mortality, improving the quality of the aquaculture industry. In this study, lactobacillus were isolated from the feces of livestock and poultry. The results of our work are as follow:(1)The screening of feeding probiotics. There are three acid producing strain are selected after initial screening of acid resistance and bile salts. NCU801 determined as Lactobacillus reuteri by physiological and biochemical characteristics, sugar fermentation and molecular biology identification.(2)The growth curve drawing of NCU801. Lactobacillus reuteri NCU801 belongs to different fermentation, so it can be able to use glucose metabolism to produce lactic acid, acetic acid, ethanol and carbon dioxide. 0-4 h was lag phase of the bacterial growth, 4-16 h belongs to logarithmic phase, while 16-24 h was stationary phase and after 24 h, the bacteria entered the decline phase.(3)The animal gastrointestinal tolerance of NCU801. The D value of L. reuteri NCU801 was 60 min in artificial gastric fluid with pH 2.5. There are still 38.6% survival rate through the simulated intestinal fluid resistance experiments on animals after 4 hours. It cultivated in the improved MRS culture medium with concentration 0.3% bile salt, but there were still a number of living bacterium 9.7× 105 CFU/m L after 24 h. So L. reuteri NCU801 has strong resistance of animal gastrointestinal adverse environment.(4)The antibacterial performance of NCU801. It found L. reuteri NCU801 bacterial itself, fermentation liquor and cell-free solution that were studied to detect the antibacterial activity in vitro based on oxford plate assay system, which were based on the common nine kinds of pathogenic bacteria in the gut bacteriostatic experiment. Fermentation liquor and cell-free solution of NCU801 had the stronger inhibiting effect than bacteria itself. The fermentation liquor and cell-free solution for e. coli had strongest inhibitory effect, bacteriostatic ring reach more than 18 mm in diameter. It could be concluded that the antibacterial substance contains L. reuteri NCU801 bacterial metabolic product of its fermentation liquor.(5)The intestinal adhesion performance of NCU801. NCU801 adhere to Caco- 2 cells that related with concentration of bacteria, bacteria fermentation time, incubation time and pH. The adhesion(5.08 ± 0.15)CFU/cell of L. reuteri NCU801 was very excellent. Results showed that both the living and dead cells could inhibit E.coli and S.aureus adhesion to Caco-2 cells on the competition, exclusion, displacement experiments. In the exclusion experiment, the living cell was able to significantly inhibit the adhesion of E. Coli, and the levels was 86.8 ± 4.5(%).(6)The high density culture conditions of NCU801.The best fermentation for L. reuteri NCU801 carbon source, nitrogen source, inorganic salt, buffer salt ions were glucose, beef extract and yeast extract, NaAc, Mg2+ through single factor experiment. The orthogonal test optimization of culture medium formula for: 1.5% beef extract, 1.5% yeast extract, 5% glucose, 0.06% MgSO4, 0.01% MnSO4, 0.001% NaCl, 0.2% K2HPO4, 0.2% ammonium citrate tribasic, 0.3% CH3 COONa. And the microbial concentration is 1.3×109 CFU/mL.