Study On The Mechanism Of Antibacterial Activity Of Probiotic Bacillus Subtilis Ma139in Vitro And Its Label With Green Fluorescent Protein Gene

Probiotics are living microorganisms which act to modulate the population ofmicrobiota in the intestine. Probiotics have an antimicrobial effect through modifyingthe microflora, secreting antibacterial substances, competing with pathogens toprevent their adhesion to the intestine, competing for nutrients necessary for bacterialsurvival. However, the mechanism of Bacillus probiotics on modifying the balance ofmicroflora remains largely unknown.In this study, the strain Bacillus subtilis MA139with probiotic potential wasstudied to explore the mechanism on modifying microflora in simulated intestinalenvironment. The co-culture of B. subtilis MA139, Escherichia coli K88andlactobacilli was carried out under microaerobic condition either in MRS medium withinitial pH6.5or in intestinal fluid. The results of co-culture were as follows:(1) E.coli K88was not significantly inhibited in the presence of B. subtilis MA139compared with pure culture;(2) Addition of B. subtilis MA139resulted in ansignificant increase of L. salivarius G1-1and an decrease in the pH of the medium (P<0.05). Viable cells of the L. reuteri G8-5had higher improvement than those of L.salivarius G1-1.0.42log at6h and0.20log at12were enhanced. At6h, the mediumpH was reduced from5.2to5.0in the mixed cultures. Therefore, under microaerobiccondition, B. subtilis MA139exhibited slightly inhibition against E coli K88, andenhanced the growth of lactobacilli and subsequently decreased medium pH in theMRS cultures. The results of the co-culture experiment in the small intestine in vitrowere as follows:(1) B. subtilis MA139reduced the viable cell of E. coli K88from9.47log to8.51log in the small intestinal fluid of the forepart at the end of theco-culture (P <0.05), and no notable reduction in the small intestinal fluid of thehindgut (P>0.05).(2) B. subtilis MA139significantly enhanced the growth of both L.salivarius G1-1and L. reuteri G8-5at48h and60h in the small intestinal fluid of theforepart (P <0.05).In order to investigate the distribution, colonization and antibacterial activity ofB. subtilis MA139in gastrointestinal tract, B. subtilis MA139and E. coli K88werespecifically labeled by gfp gene. The promoter4412of plasmid pGFP4412wasreplaced by the promoter of xylR gene of B. subtilis168and rpsD promoter of B.subtilis MA139to construct new vectors of pGFP-xylR and pGFP-rpsD．The shuttlevectors were transformed into both B. subtilis MA139and E. coli K88and thepositive colonies was selected for the analyses of GFP quantity and genetic stability.The labeled strains of B. subtilis MA139-pGFP-rpsD and E. coli K88-pGFP-rpsD were finally obtained. The green fluorescence of the strains detected by thefluorescence mmicroscope was easily observed. The labeled strains provided the basisof study on the colonization and inhibition of B. subtilis MA139against E. coli K88in vivo.