Lactic acid bacteria(LAB)is a group of Gram-positive bacteria that could produce much lactic acid by fermenting carbohydrates(glucose or lactose).This bacteria is mainly isolated from natural soil,feces and intestinal tracts.LAB can produce organic acid,hydrogen peroxide,bacteriocin and other substances with antibacterial activity.It is one of the ideal alternatives of antibiotics.Aeromonas veronii,one common harmful aquatic organisms and releases hemolysin,cytotoxin and enterotoxin,causing huge economic losses to aquaculture industry.In this stuy,120 strains were screened out from intestinal contents of cyprinuscarpio,carassiusauratus and megalobramaamblycephala respectively.Observing the colony morphology,HE staining and touch enzyme experiment,we identified 98 strains of suspected Lactobacillus.The aseptic cell culture medium was prepared.Using the A.veronii TH0426 as the indicator bacteria,the bacteriostatic test was carried out.The results of we found a total of 15 strains with strong bacteriostatic effects.Then we extracted the genome of the 15 strains.16 S rDNA gene sequence analysis showed that 11 strains were enterococcus,3 strains were Lactobacillus casei and the last one was Lactobacillus plantarum.Secondly,resistance,tolerance(bile salt,different pH,protease,temperature),antimicrobial spectrum test,adhesion,animal safety,growth curve test and other biological characteristics were detected.The results indicated that only 4 strains(L31,L2,W21,D2)Lactobacillus had resistance to Aeromonas veronii TH0426.L31,L2 and W21 could tolerate 0.1%-0.4% bile salt,while D2 showed bile salt intolerance.Three strains of LAB pH3~ pH7,the numbers of living bacteria increased;trypsin had no effects on the survival rate of LAB;L31 and L2 could survive at 50℃,while living numbers decreased significantly at 60℃ and 70℃.In addition,the three strains of LAB all had strong inhibitory effects and good adhesion ability among which Lactobacillus casei L31 had the strongest adhesionability.Three strains of LAB had no effect on mice and carp in vivo by animal experiment.Finally,preliminary study on bacteriostatic activity of Lactobacillus L31、 L2 and W21 strains showed that the culture liquid and culture supernatant both had significant bacteriostatic effects.When the pH value of the culture supernatant was 6,the three strains of LAB had no inhibition effects;three different protease treatment,and antibacterial activity of the culture supernatanthad no obvious change;the inhibitory activity of the culturesupernatant after treatment had no significant changes;the bacteriostasis activity of the culture supernatant didn’t changed after heat treatment.Electron microscopy observation indicated that after treatment of Lactobacillus plantarum W21,Lactobacillus casei L2 and Lactobacillus casei L31 respectively,Aeromonas veronii TH0426 were damaged to different degrees.In conclusion,culture supernatant of LAB may be dominated by organic acid,which plays a key role in inhibiting harmful bacteria. |