| One strain of Bacillus subtilis JB286 with relatively high protease content and the largest number of viable bacteria in fermentation broth was screened from 12 strains of Bacillus subtilis preserved in the enterprise.Through the combination of single factor experiment and orthogonal experiment,a suitable strain for JB286 was established.The optimal shake flask culture conditions and fermentation system of the strain:initial p H 7.2,rotating speed 160rpm,culture temperature 37°C,inoculum volume 6%-8%,filling volume 20%;the optimal medium ratio is:peptone 4%,Glucose 2%,sodium chloride 5%,corn flour 5%,soybean meal4%,yeast powder 5%,manganese sulfate 1%;shake flask fermentation for 24 h,the bacterial concentration can be increased from 8×10~8CFU/m L to 3.4×10~9CFU/m L,an increase of325%.Increasing the concentration of viable bacteria in the fermentation broth lays a theoretical foundation for the efficient fermentation technology for the production of Bacillus subtilis.2.Evaluation of strain probiotic potentialThrough in vitro acid and alkali resistance test,the concentration of viable bacteria that can enter the body and its probiotic potential of JB286 strain were determined;then through animal experiments,its safety and probiotic ability were tested.The 60 mice were divided into three groups,A,B,and C,with 20 mice in each group.Group A was the blank control group,group B was fed 2×10~6CFU/m L bacterial solution,and group C was fed 2×10~8CFU/m L bacterial solution,sampling every week for 3 weeks.Using serological detection,q RT-PCR,16S r RNA sequencing and other technologies to detect the effects of different feeding concentrations on immunoglobulins and cells at different feeding stages Effects of factor content,intestinal tight junction protein gene expression,and cecal flora microflora abundance.The results show:（1）Compared with the blank control group（group A）,the plasma immunoglobulin（Ig A,Ig M,Ig G）and cytokine（IL-2,IL-6）contents of mice in groups B and C were significantly increased（P<0.05）;（2）The daily gain of mice in groups B and C was higher than that in group A,but the difference was not significant（P>0.05）.The expression of intestinal tight junction proteins（ZO-1,Occludin,Claudin-1）in the control group was significantly higher than that in the two treatment groups in the first week of the experiment,but significantly lower than that in the two treatment groups in the third week,indicating that JB286 can enhance immunity.and intestinal barrier effects.（3）The diversity of intestinal flora of mice inoculated with JB286 increased,the relative abundance of beneficial bacteria such as Firmicutes in the cecum increased,and the relative abundance of harmful bacteria decreased.In summary,the suitable shake flask fermentation conditions for strain JB286 are initial p H 7.2,rotation speed 160 rpm,culture temperature 37°C,inoculum volume 6%-8%,and liquid filling volume 20%;the optimal medium ratio is:peptone 4%,glucose 2%,sodium chloride 5%,corn flour 5%,soybean meal 4%,yeast powder 5%,manganese sulfate 1%.After 24 h of shake flask fermentation under optimized conditions,the concentration of bacterial liquid can be increased from 8×10~8CFU/m L to 3.4×10~9CFU/m L.Feeding the optimized Bacillus subtilis JB286 strain can improve the humoral immunity of animals,regulate the composition of intestinal flora,improve the diversity and species abundance of intestinal flora,and enhance immunity and anti-infection ability.The most beneficial concentration of JB286 strain was 2×10~6CFU/m L.This study provides data support and theoretical reference for the application of Bacillus subtilis in production. |
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