| Probiotics have good prebiotic characteristics and can effectively improve intestinal diseases caused by pathogenic bacteria by competing for the adhesion and colonization sites of pathogenic bacteria.Probiotics can give full play to their prebiotic function only by ensuring a certain number of viable bacteria in the intestine.Therefore,Bifidobacterium adolescens FS2-3 and Bacillus subtilis SN15-2 probiotic dual-bacteria microcapsules were prepared by endogenous emulsification and extrusion.The two probiotics are encapsulated in different compartments and wrapped in the same microcapsule to avoid the interaction between bacteria before taking.B.subtilis SN15-2 is released first and consumes free oxygen in the intestine.B.adolescentis FS2-3 is released later to play a probiotic role together.The stability of microcapsules was determined by in vitro simulation experiment.Then,the mouse bacterial enteritis model was induced by E.coli O157: H7 to explore the alleviating effect of probiotic dual-bacteria microcapsule on bacterial enteritis.The main results are as follows:(1)According to the bacteriostatic effect,two strains were selected,B.adolescentis FS2-3 and B.subtilis SN15-2.The antibacterial activity test in vitro showed that the direct mixing of B.adolescentis FS2-3 and B.subtilis SN15-2 had no significant effect on the antibacterial activity.The results showed that B.adolescentis FS2-3 had a certain acid producing ability.When the pH of LB medium reached 2,B.subtilis SN15-2 lost its activity.The two strains were co-cultured and the viable number of B.adolescentis FS2-3 was calculated.It was found that B.subtilis SN15-2 could promote the reproduction of B.adolescentis FS2-3 in a certain time.(2)Sodium sulfhydryl carboxymethyl cellulose(CMC-SH)was obtained by chemical modification to increase the adhesion of probiotics in the intestine.The content of sulfhydryl group was 139.02 ± 3.14 μmol/g,the measured CMC-SH potential is-44.2 m V.The bacteriostatic effect showed that the addition of CMC-SH had no effect on the bacteriostatic activity of probiotics.Through in vitro intestinal mucus and intestinal adhesion test,it was verified that CMC-SH could improve the adhesion of B.adolescentis FS2-3 in the intestine.At the same time,rheological test and classical tensile test showed that the mixture of CMC-SH group and intestinal mucus had viscoelasticity,and there was a significant interaction between CMC-SH and intestinal tissue.(3)Probiotic dual-bacteria microcapsules were prepared by endogenous emulsification and extrusion,which avoided the direct contact between the two strains,prevented the effect of acidic environment on the activity of B.subtilis SN15-2,and provided a good anaerobic environment for B.adolescentis FS2-3.The optimum technological conditions were determined as 1.82 % sodium alginate with inner core;The concentration of CMC-SH in bacterial solution was 0.9 %;Water oil volume ratio 1:2.9;The concentration of sodium alginate in the outer layer is 2.9 %.It was observed by microscope that the microcapsule was successfully encapsulated,and the particle size of probiotic dual-bacteria microcapsules was about 304.29 ± 15.24 μm.After continuous simulated artificial gastrointestinal fluid treatment,it was found that probiotic dual-bacteria microcapsules could significantly improve the survival rate of probiotics,the survival rate reached 63.19 ± 1.48 %,and had good release.The stability of probiotic dual-bacteria microcapsules was studied under different storage conditions.The results showed that the survival rate of probiotic dual-bacteria microcapsules stored at 4 ℃ for 42 days was the highest and had good storage performance.(4)The mice model of bacterial enteritis induced by E.coli O157: H7 was established to evaluate the effect of probiotics dual-bacteria microcapsules on alleviating bacterial enteritis in mice.After 5 days of administration,the weight and food intake of mice recovered.The effect of probiotic dual-bacteria microcapsule on intestinal flora of mice was detected by 16S rDNA sequencing.It was found that probiotic dual-bacteria microcapsules could change the diversity and richness of intestinal flora and regulate the relative abundance of harmful and beneficial bacteria in intestinal flora.The levels of pathological sections,immunohistochemistry and inflammatory factors were detected.It was found that probiotic dual-bacteria microcapsule could improve intestinal inflammation,regulate TLR-4overexpression and significantly reduce TNF-α,IL-1β and IL-6 cytokines and CRP content,so as to play a role in alleviating bacterial enteritis. |
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