| In the prevention and control for Aquatic Animals diseases, many problems, such as multipleantibiotic resistance, residues and pollutions, has been caused, due to the abuse of antibiotics. This madepeople dedicate to seek medicines and products which are harmless to human and environment, so thatwe can take the place of antibiotics completely or partially. However the occurrence and application ofProbiotic Bacteria can solve these problems. On account of ideas mentioned above, we isolate Probioticstrains of Lactobacillus which is of Anti-Resistance, Antagonistic to Pathogen, and relatively safe,from Intestinal mucosa of Cyprinus carpio firstly in China。This research sets a good base for theapplication of it in aquaculture.This research is mainly divided into two experiments as follows:The first experiment is mainly the screening of Probiotic strains of Lactobacillus. In the first place,isolate 8 strains of Lactic Acid Bacteria from Intestinal mucosal flora of Cyprinus carpio using bile saltsculture. Then, screen two strains of Bacteria of strong Anti-Resistance using the tests of tolerance of bilesalts, tolerance of pH, tolerance of protease and tolerance of heat. In the test of tolerance of bile salts,over 30％of YL-1 and YL-7 can survive after 4-hour culture in the bile salts of 4％concentration, in thetolerance of pH, the 2 strains of Lactic Acid Bacteria can survive both in acid environment and alkalineenvironment; Trypsin didn't affect the survival of these two strains; and the bacteria show a certaintolerance in the heat condition of 70℃. In bacteriostastic test in vitro, we find that YL-1 is better thanYL-7 in the inhibition to Aeromonas hydrophila, thus YL-1 is selected as the candidate bacteria ofprobiotics. Through the analysis to the cultured supernatant of YL-1, we can find that the bacteriostasticsubstance is mainly Organic Acid and substance sensitive to Trypsin. In sensitivity test of drugs forYL-1, we find that this strain has certain drug resistance. Through the inoculation on mice and Cyprinuscarpio, we identify the safety of YL-1. Finally, we identify YL-1 as Enterococcus faecalis by a series ofphysiological and biochemical tests.The second experiment is the optimization of culture medium for YL-1. The first half ofexperiment is mainly the screening of the best Carbon source, the best Nitrogen source, the bestInorganic Salts Ion, and the best stimulating factors. After the screening of various favorable factors forYL-1, with the help of SAS software, through Plackett-Burman design, we can screen the importantfactors favorable to the growth of YL-1, then with Steepest Ascent experiments, approach the maximum response region. Finally, with the help of Box-Behnken design, we can identify the maximum responsevalue. Through verification, it shows good fitting between the estimated value and the real value, andthe model is reliable. At last, the optimization of cultured medium of YL-1 is as follows: 1.365％peptone, 1.25％extractum carnis, 0.625％yeast powder, 2.5％maltose, 0.1％Tween-80, 0.2 KH2PO4,0.16％ammonium citrate, 0.25％MgSO4, incipient pH 6.4. |
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