The Research On Montmorillonite Intercalated With Bacillus Subtilis And Its Antibacterial Effect In Vitro And Effect On Barrier Funvtion

Probiotics made by beneficial microorganisms, developed a new type of green feed additives in recent years, with non-toxic, harmless, no residue and other characteristics. B.subtilis can100%be to animal intestines directly because of its resistance, but, B.subtilis have less competitive than intestinal bacterials, it was difficult for B.subtilis to colonized on Gastrointestinal tract(GI). In this study, The MMT-B.subtilis compounds were synthesized with the Na+-montmorillonite (Na+-MMT) to improve the intestinal colonization of B.subtilis. The main experimental results are as follows:(1) The preparation、characterization and optimization of preparation conditions of MMT-B.subtilis compounds:The structure of compounds was characterized by zeta potential (ξ potential), X-ray diffraction (XRD) and Fourier transformed infrared (FTIR). The results showed, compared with the physical mixture of MMT and B.subtilis, the ξ potential of MMT-B.subtilis compounds was decreased from7.95mV to5.61mV, while expanding the basal spacing (d001) of MMT from1.34nm to1.70nm. The FTIR spectra showed the mechanism of intercalation between B. subtil is and MMT may involve hydrogen bond, amino group, carboxy group and hydroxy group of bacterial surface. Orthogonal optimization experiments derived the optimal reaction condition of MMT-B.subtilis:0.15g weight of montmorillonite, reaction pH=6,0.0001mol/L of ionic strength. The maximum intercalation capacity of montmorillonite was5.82×109CFU/g.(2) The inhibitory effect of MMT-B.subtilis:Escherichia coli, Staphylocus aureus, Salmonella pullorum and Shigella flexneri were used as the tested strains in vitro antibacterial activities. The results showed the antagonistic effect of four pathogens had varied with the concentrations of MMT-B.subtilis.In all, the inhibitory effect increasing with the increasing concentration of MMT-B.subtilis. However, different pathogen showed different sensitivity for the concentration of MMT-B.subtilis, as in the culture at24h,10g/L of MMT-B.subtilis could promote growth of S.aureus and S.flexneri. Comparision of three different antimicrobial agents, we founded that:For E.coli and S.aureus, MMT-B.subtilis compounds had stronger antibacterial activity than the physical mixture and B.subtilis; for S.pullorum, there were no significant differences among MMT-B.subtilis, the physical mixture and B.subtilis (P>0.05); for S.flexneri, there was no siginificant difference between MMT-B.subtilis and the physical mixture(P>0.05).(3) The effect on intestinal mucosal barrier function of MMT-B.subtilis:In this experiment, we used the Caco-2cell monolayer as model, with the amount of bacterial translocation (cell number of pathogen infection) and intestinal villus damage (extracellular lactate dehydrogenase release quantity) as indicator, to study the effect of MMT-B.subtilis on intestinal mucosa. The results showed that the most appropriate concentration of MMT-B.subtilis for Caco-2cell growth was0.02mg/L; Compared with the physical mixture, MMT-B.subtilis greatly reduced the number of colonies of pathogen invasion on Caco-2cell and the difference (compared with the control group) was significant (P<0.05); E.coli and S.pullorum seriously undermined the integrity of Caco-2cell membrane, resulted that the LDH escaped from intracellular and released into the extracellular, and also inhibited the LDH activity of Caco-2intracellular. After joining the MMT-B.subtilis, the Caco-2LDH activity of intracellular was not affected, and overall, the protective effect of MMT-B.subtilis on cell membrane is stronger than the physical mixture of MMT and B.subtilis.