Selection Of Lactic Acid Bacteria Strains Producing Protease And Application Research

Along with the improvement of human health awareness and understanding to hazards of antibiotic residues, Lactic Acid Bacteria (LAB) has been more and more widely used to forage production as an additive of green environmental protection type, which could not only improve animal immunity,prevent animal from gastrointestinal diseases, also promote the growth of milk production.At present, the average yield of dairy cows in China is only 2800-3000kg/year, while overseas the average yield of dairy cows's 11,000kg/year, the highest if its is 8000kg/year in America. The microecological agents play an important role in promoting lactating of dairy cows, in addition to dairy cattle varieties, keeping the environment and forage.LAB as microecological agents are widely utilized in production. The most application of lactic acid bacteria is for the character of lactic acid producing which could decrease the pH of gastrointestinal, result in growth inhibition of pathogenic bacterium. However, it's not reported that the LAB could secrete protease to enhance the utilization of forage. The aim of this study was to develop a LAB which could produce lactic acid, decrease pH of gastrointestinal as well as secrete protease to improve feed utilization.The LAB strain was selected from 21 kinds of LAB strains preserved in our laboratory. The research was as follows: selecting the strain of LAB producing protease; identifying the strain of LAB on biochemical and molecular level; isolation and purification of the protease; property study of the protease; selection of the strain from the ultra-condensed medium; the strain producing protease domestication; performance effect of dairy cows when the LAB as microecological agents were applied in the cows forage.The research achievements were as follows:The LAB strain producing the protease was selected out by the transparent collar in casein plate medium and Folin's methods. It belonged to Lactococcus lactis subsp AS1.1936, identified by biochemical methods and specificity PCR. Extracellular protease was purified through Sephadex G-75 filler, which also determined that its molecular weigh was 35KD. The protease was a kind of acid protease through studying on enzyme property, the optimal temperature is 40℃, Mn²⁺ had obviously activated on this protease. The prescription of AS1.1936 ultra-concentrated medium was gained, and viable count was 1.05×10⁹cfu/ml. The methods of domesticating the LAB strain to secrete protease were: first acclimation through the casein protein agar in medium, after rejuvenation, re-acclimation the LAB strain with casein protein concentrations from 0.5% to 5.0% (0.5% as gradient). The LAB strain could significantly increase milk production to 4.08%, milk fat to 19.51%, dry matter to 8.07%, when it's applied in the cows forage as microecological agents, discrepancy were extremely predominance (P<0.01). However, it had no conspicuous impact on the rate of milk protein.The LAB strain producing protease was successfully selected in this study, and animal experiment showed that it's feasible to use the LAB strains producing protease to improve the quantity and quality of milk production as microecological agents.