Isolation, Characterization Of A Lactic Acid Bacteria With High Bile Salt Tolerance And Its Application In Soyabean Meal Fermentation

Soybean meal is the most wildly used vegetable protein, however the anti-nutritional factors seriously restricted the application of soybean meal, how to remove the anti-nutritional factors in soybean meal has become the primary problem of feed industry. In decades, many researches proved that microbial fermentation can effectively remove anti-nutritional factors. Large soybean protein molecules can be broken down into peptides and amino acids by microbial fermentation. Lactic acid bacteria is the earliest and the most widely applied probiotics. This research isolated a Lactic acid bacteria with high protease and high bile salt resistant from the intestinal tract of healthy piglet. After physiological and biochemical identification of the bacteria and study of the biological characteristics, the researcher moves on to optimize the culture medium composition and freeze-dried protective agent. Finally the researcher using the strain fermenting soybean meal, and compared with Bacillus subtilis and Lactobacillus plantarum, find the optimal conditions for soybean fermentation. This research can provide basic data for remove the anti-nutritional factors in soybean meal. The main research content and summary are as the follows:(1)The high-yield strain was obtained through primary screening and re-screening with protaese screening plate. The bacteria is gram-positive bacteria, the colonies is raised, white, neat and moist, the diameter is 1.0~1.5 mm. The strains under the microscope is cossus and the diameter is about 0.8 μm without capsule and spore. The strain WLEF-01 was identified as Enterococcus faecium based on physiological and biochemical characteristics and 16 S rDNA sequences analysis.(2)The biological characteristics research showed WLEF-01 grows fast, the logarithmic phase stars around 0.5 h and end in 5 h, it has a long stability. The strain has a strong ability to produce acid, the medium pH drops rapidly during the logarithmic phase, and finally reached 4.5. WLEF-01 has acid-resistant ability, the survival rate is above 50% when the medium pH in 4~6.8. The bacteria shows outstanding bile salt resistant ability, the survival rate reaches 90% at 0.3% bile salted after 2 hours and 83% at 1% bile salted after 2 hours. WLEF-01 revealed antagonism against pathogenic E. coli and S. aureus and have tolerance to some antibiotics.(3)The optimum composition of culture medium is 1% lactose, 4% yeast powder, 0.25% NaAc, 15% tomato juice, pH 6.5 and the value of OD600 reached 1.617. The optimum composition of protective agent is 10% skimmed milk powder, 5% trehalose, 5% lactose, 7.5% maltodextrin, 5% peptone, and the survival rate of WLEF-01 is 41.85%.(4)The fermentation experiment of soybean meal showed WLEF-01 have a better ability to break down protein, and compound strains fermentation is better than single strain fermentation to remove the anti-nutritional. The compound strains fermentation orthogonal experiment shows that the optimum inoculation is 5% Bacillus subtilis, 1% Lactobacillus plantarum and 5% WLEF-01. In this condition, the anti-nutritional factors decomposed into peptides and amino acids, no obvious protein bands. The pH of fermented soybean meal is 4.8 and the inhibition zone diameter of E.coli reached 13.75 mm. The WLEF- 01 count up to 3.92 x 1010 cfu/g after vacuum freeze-drying.