Screening Of Lactobacillin Producing Lactic Acid Bacteria And Preliminary Study On Preserve Effect On Prawn

The purpose of the study was to isolate lactic acid bacteria from intestinal tract of10species of marine animals collected from the South China Sea, and the isolated srtainswere identified by using the polyphasic taxonomy. Strains with antibacterial activity werescreened by Oxford plate double-layer agar experiment, and strains with antibacterialactivity were studied for further to exclude inhibitive effect of organic acid, H2O2andobserve four proteases’ sensitivity. The biological features of lactobacillin-producingstrains and its lactobacillin were analyzed. The crud lactobacillin were used forpreservation of penaeus vanmamei, and the preservation effect was evaluated. The mainresults were as follows:One hundred and fifty-nine suspicious lactic acid bacteria were screened fromintestinal tract of marine animals, which produce soluble calcium circle. Among of43strains with antibacterial activity. The43strains were identified as Lactobacillu、Lactococcus、Enterococcu、Leuconostoc and Pediococcus, respectively, according to theirmorphological and physiological-biochemical characteristics.13of them, which had broadantibacterial spectrum, strong antibacterial activity and representative, were identified with16S rRNA sequence analysis.The results showed that there were2strains Enterococcusdurans、2strains Lactococcus lactis subsp.lactis、1strain Weissella. confusa、2strainsLactobacillus pentosus、3strains Lactococcus garvieae、2strains Leuconostoc citreum and1strain Pediococcus pentosaceus.Thirty-nine strais, which can inhibit two or more kinds of indicator bacteria, werestudied for further to exclude inhibitive effect of organic acid and H2O2. Strains ZH-54andLY-87were screened as suspected lactobacillin-producing strains, then proteases’sensitivity were observed. The cell-free supernatant of strain ZH-54, was treated by trypsinand proteinase K, showed inhibitory activity against Staphylococcus aureus reducing todifferent degrees. While the cell-free supernatant of strain LY-87showed sensitive topapain and proteinase K. These results showed that the inhibitory substance produced bystrain ZH-54and LY-87were proteins or polypeptide sassumed lactobacillin-likesubstance. Based on morphological and physiological-biochemical characteristics and16S rRNAsequence analysis, strain ZH-54was identified as Enterococcus durans, while strain LY-87was identified as Lactococcus lactis subsp. Lactis，and the accession number of16S rRNAin GenBank was JX258806and JX258807,respectively.Research on biological characteristics of Lactobacillin Bact.54and Bact.87showedthat the Bact.54produced by strain ZH-54have good stability against temperature, theantimicrobial activity only have12.14%loss suffered under the temperature of100℃for15min and18.50%loss suffered under the temperature of121℃; However, theBact.87produced by strain LY-87antimicrobial activity have39.70%loss suffered underthe temperature of100℃, and61.76%loss suffered under the temperature of121℃. pHhad significant effect on Bact.54and Bact.87, the antimicrobial activity decreased whilethe pH value increase, the antimicrobial activity lost while the pH value surpass7.0.Bact.54could inhibit some gram positive bacteria and some gram negative bacteria, whileBact.87produce only antimicrobial activity to some gram positive bacteria.The study on the effect of the crud Bact.54and Bact.87as the preservation on thepenaeus vanmamei showed that they both have clear preservative effect, but the Bact.54ismuch better. The Bact.54could extend the shelf-life of penaeus vanmamei to2-3days,while Bact.87extend to1-2days. Bact.54and Bact.87can effectively inhibit the growth ofartificial inoculated Staphylococcus aureus. Therefore, the lactobacillin produced byZH-54and LY-87could enhance the security of penaeus vanmamei and has broadprospects as biological preservative for aquatic product.