| At present,the research on sika deer is mainly focused on the medical value,feeding technology and disease prevention,however,there is no report on the isolation of probiotics from Sika deer.In this study,two strains of Bacillus were isolated from the intestinal secretions of sika deer,study of the two strains of Bacillus species,cellulose degrading solution ability,pathogenic effect,tolerance sensitivity,optimization of the optimum growth conditions and explore the influence of intestinal tract and immune ability of mice.It will provide reference for the future research of sika deer's health.Text 1:The strain of sika deer was screened by high temperature water bath to isolate the strain with high temperature.Afterseparation and purification,microscopic examination,physiological and biochemical analysis,16SrRNA sequence analysis and homology comparison,the strains were identified.The bacteriostatic effect of XF-1 and XF-3 was studied by using Oxford cup method.Text two:(1)the determination of strain growth in different time and after 24h cultured in different culture medium pH,different temperature,inoculation amount and different liquid liquid volume on the influence of strain growth,to determine the optimum growth conditions of different strains;(2)The ratio of cellulose degradation ring of the two strains was measured to determine the effect of cellulose degradation.a total of 30 Kunming SPF mice were divided into 3 groups,they are control group,XF-1 group,XF-3 group.15 days later,evaluation of the safety of two strains of Bacillus by scan and organ coefficient.Text three:60 mice were randomly divided into 6 groups,each group of 10.The rats were divided into normal control group(C),Escherichia coli model group(E),XF-1 probiotics group(P1),XF-3 probiotics group(P3),XF-1 group(T1),E.coli XF-3(T3).Continuous irrigation for 30 days.Finally,the effects of body weight index,serum lysozyme concentration and serum IgG concentration on immune function of mice were measured;The effect of Bacillus on intestinal growth was evaluated by measuring the length of small intestinal mucosa,intestinal mucosa thickness and crypt depth.1.The deer intestinal secretions obtained by separation and purification of two Bacillus strains,after examination,physiology and biochemistry,molecular biology identification technology,XF-1 was identified as Bacillus licheniformis XF-3,Bacillus amyloliquefaciens.2.The inhibitory effect of two strains of Bacillus on the four pathogenic bacteria of was studied by using the Oxford cup method.XF-1 had a good inhibitory effect on Escherichia coli,Streptococcus and Salmonella;XF-3 had a good inhibitory effect on Escherichia coli,Staphylococcus aureus,Streptococcus,Salmonella.3.CMC-Na medium was used to detect the Cellulase Producing Ability of two strains of bacillus.The cellulose degradation index of strain XF-1 was D/d=2.18,the cellulose degradation index of strain XF-3 was D/d=3.18,and the cellulose degrading capacity of XF-3 was higher than XF-1.Through the toxicity test,it was confirmed that the isolated Bacillus could not cause the pathological changes of the main organs of the mice.Compared with the blank group,there was no significant difference in the organ coefficient(p>0.05).4.In the experiment,the survival rate of bacteria in different acid environment and bile salt environment was calculated,result were indicated;after 24h,the survival rate of XF-1 in the pH3.0 environmentwas 7.3%,the survival rate was 8.57%in the 0.2%bile salt environment.After 24h,the survival rate of XF-3 in the pH3.0 environmentwas 12.5%,the survival rate was 6.67%in the 0.2%bile salt environment.These two strains have certain resistance to stress.5.The results showed that the lysozyme activity in serum of P3 group was significantly higher than that of control group(p<0.05),and serum lysozyme activity in P1 and P3 group was significantly higher than that in group E(IgG)(p<0.05);Compared with the IgG concentration in each group,the P3 group was significantly higher than that of the control group(p<0.05),and was significantly higher than that of the E group,the difference between the treatment group and the E group was not significant(p>0.05).The results showed that probiotics group can effectively enhance the concentration of IgG in normal mice.6.By observing the jejunum and ileum tissue sections and measuring intestinal villi and other indicators,compared with the control group jejunum,T3,P1,P3 group significantly difference in length of villi(p<0.01);The thickness of T3 and P3 were significantly different(p<0.05),crypt depth of P1 and P3 were significantly lower than that of control group(p<0.01).Compared with the group of E.coli,the villus length of each group was significantly higher than that of group E(p<0.01),and the thickness of PI,P3 and T3 was significantly higher than that of group E(p<0.01),The crypt depth of probiotics group was significantly lower than that of E group(p<0.01),and the treatment group was significantly lower than that of E.coli group(p<0.05).Compared with the control group,the T1 and E groups had significant differences in villus length,which was significantly lower than that of the control group(p<0.01),and the difference of the mucosal thickness was not significant(p>0.05),and the crypt depth was significantly lower in the P3 group(p>0.05).Compared with the Escherichia coli group,T3 group,probiotics group villus length were significantly higher than E group(p<0.05),and P1 was significantly higher than that of group E(p<0.01),P3 group,mucosal thickness was higher than that in group E(p<0.05),probiotics group P3 crypt depth was lower than that of group E(p<0.05).In this study,the Bacillus licheniformis and Bacillus subtilis were isolated and had the effects of probiotics and stress resistance,which could improve the intestinal immunity of mice. |
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