| The emergence of drug-resistant strains caused by the misuse of antibiotics has become more and more widely concerned.The non-toxic and safe alternatives were looking for in order to change this situation.Years of research and practice show that probiotics instead of antibiotics in some extent because it can maintain the host intestinal microecological balance,improve the level of the host immune,promote the body’s growth and development.Bacillus subtilis is an important bacterial resource of probiotics,which has many functions,such as preventing pathogenic bacteria and improving immunity.Generally,the screening of probiotics is a crucial step in the process of probiotic production,because the strain specificity is filtered through a prerequisite of probiotics.Different sources of Bacillus subtilis has different gastrointestinal physiological function in different animal.Therefore,it is generally believed from the corresponding normal chicken intestinal microecological agents of separation strains screening for poultry is more reasonable.We carry out this experiment in order to isolate the Bacillus subtilis that can resist bacterium.We get the chicken in the age of 1,5,10,20,30 days from the farm and take the intestinal contents of the duodenum,jejunum,ileum,cecum and rectum in 80℃water to bath heating.After that we dilute the intestinal content and coated plate,using specific PCR method for the separation of Bacillus subtilis,with flat growth standoff method to screen out best bacteriostasis effect of Bacillus subtilis BS-7.We studied its basic characteristics and beneficial effect on the body,and to have bacteriostasis of 36h bacteria liquid supernatant as experimental group,with no bacteriostasis of 6h bacteria liquid supernatant as a control group for HPLC/ESI-MS/MS.In the end,we analyze the result from proteomics and metabolomics two directions.The test results show that:To explore the time and the main location of the BS-7 play a useful role in normal chicken,15 strains of Bacillus subtilis were obtained from different day age of different intestinal segment in the farms through the specific PCR method.from the time on 1,5,30days of age of Bacillus subtilis quantity is less,and the age of 10 and 20 days of age distribution of bacillus subtilis accounted for more than fifty percent of the total.Speculates that the reason for 1~7 days of age,the relatively small number of intestinal flora,chicken system were in perfect condition,as time goes by Bacillus subtilis as of beneficial bacteria in the intestine,and in the age of 30,reduce the possible reason is the breeding based on the extensive use of some of the Bacillus subtilis sensitive antibiotic drugs to kill bacteria,affecting the number of Bacillus subtilis.From the distribution position,Bacillus subtilis was mainly distributed in ileum and cecum,accounting for 40%and 26.7%.Ileum plays a dominant role in the digestion and absorption of chicken intestines,while the cecum is the main site for storing content,so it contains more Bacillus subtilis than other intestinal segments.Bacteriostatic screening of all Bacillus subtilis was isolated by flat plate growth,and 3strains of Bacillus subtilis showed different levels of bacteriostatic performance.The best effect is BS-7.Further confirmation was made through morphological,physiological and biochemical tests and molecular biological identification.The results showed that the homology of the BS-7 strain and the standard strain Bacillus subtilis strain CICC 10023(GU980947.1)was 99.5%,so the strain BS-7 was identified as Bacillus subtilis.The training conditions were optimized,and the results were as follows:35~37℃,200rpm/min,the initial pH was 7,and the strain grew best.Based on the research of the strains BS-7 tolerance test found that BS-7 in the gastric acid,intestinal juice and bile salts environment has good tolerance,met the probiotic bacteria in the gut of survival and the prerequisite for the normal work.And processing in 90℃high temperature 30 min,there is still more than 70%of the survival and lay a foundation for its development.In order to study the probiotics of BS-7,we first carried out acute toxicity test and translocation test in vivo,ensuring the safety of the strain.After that,the SE were infected with chicken,and the inhibitory effect of BS-7 on it in vivo was investigated.The test group was divided into six groups,and the experimental group had been fed the BS-7 strain1x10~8CFU/mL every two days from the age of 2 days before the gavage of SE(13 days),and the control group was given gastric saline.The patients in each group were:one group(physiologicalsaline);Twogroups(physiologicalsaline+SE);Threegroups(once BS-7+SE);Four groups(BS-7);Group 5(BS-7+SE);Group 6(BS-7 clear+SE).We measured the role of BS-7 from the weight ratio(F/G),the bacterium capacity of liver,spleen,cecum and the liver and spleen lesions.Results show that compared with the control group 2,continuous gavage BS-7 bacteria liquid and supernatant can make material weight ratio drops,decrease of microbial load and viscera disease in ease,and significant difference(P<0.05).Microbial effect is best,but no significant difference with supernatant effect(P>0.05).Only once BS-7 on the 2nd day and the rest were replaced with physiological saline,and the results showed that there was no significant beneficial effect.To explore BS-7 on antimicrobial constituents in microbial supernatant,the analysis of HPLC-ESI-MS/MS were done in experimental group and control group.Applying iTRAQ technology for quantitative analysis of protein group differences in protein,monitoring to1978 different proteins,of which 811 increase protein,1167 cut protein.Differences in protein were analysis by beneficiation of pathways and GO,in which we focus on the GO annotation molecular functions 10 of higher protein and the pathways of the 10 proteins,most have the function of degradation of RNA,inhibit protein expression,especially mRNA interfere with the enzyme,bacteria can cause apoptosis.They are located in the pathway RNA degradation system,Quorum sensing,and two-component system,it plays a certain role in inhibiting the growth of pathogenic bacteria,and the mechanism is worthy of further study and discussion.Protein deposition method with organic reagent to metabolites extracted samples,and the preparation of QC computer after sample test,then the plane mass spectrometry,including the original data preprocessing,through a series of statistical analysis to identify differentially expressed in different processing conditions of metabolites;Finally,the metabolites and differential metabolites were identified and the pathway analysis was conducted.A variety of factors were selected to select the most differentiated pathways and proteins-Dopaquinone and Metabolic pathways.Dopamine quinone intermediates for the synthesis of melanin,studies have shown that melanin on Staphylococcus aureus have inhibition,we suspect that this is associated with the antibacterial properties of BS-7,the specific mechanism remains to be further explored.This study isolated Bacillus subtilis from poultry intestinal,and get the Bacillus subtilis that have bacteriostatic,study the culture conditions,live action,using proteomics method screening differences pathway and protein,for the further development of the poultry probiotic formulation provides a good reference strains of probiotics. |
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