| Probiotics can maintain the balance of gut microbiota,enhance the organic metabolism and immune response,thus resulting in an improvement of healthy status of animals.Lactobacillus,Bifidobacterium,Bacillus and Saccharomyces are actually the widely used probiotics in feed,among which Bacillus subtilis is preferred due to the high resistance to harsh environment and high production ability of metabolic enzymes,and bio-resistence of intestinal pathogenic bacteria.However,probiotics are viable bacterias,their security should be evaluates strictly.Nowadays,the safety assessments are mainly concentrated on potential pathogenicity and antibiotic resistance.Bacillus subtilis,the situation is similar,which has been assessed these aspects,also there are some evaluation criterions about the safety of Bacillus cereus-like enterotoxin.While studies on security issues for metabolites of Bacillus subtilis itself or which may cause metabolic differences in terms of the body still lag behind.So,we isolated and identified Bacillus subtilis J1220.Then detected the changes of serum biochemical and immune indices and investigated the effect of Bacillus subtilis on intestinal flora by establishing a real time-PCR method.Also,we established methods of extraction and detection for metabolites from this strain cultured in vitro and the different metabolites from intestinal fluid,plasm and edible tissues after pigs feeding the Bacillus subtilis J1220 by LC/MS-IT-TOF.Finally we get the different metabolites information in vitro and in vivo.In the fulture we will develop a qualitative and quantitative analysis for the different metabolites and a food safety assessment of probiotics Bacillus subtilis for animal use by microbial metabolomics.1 Isolation and identification of Bacillus subtilis J1220 and biological activity determinationA Bacillus subtilis was isolated from a related probiotic product for animal feeding and identified by traditional physiological and biochemical tests and PCR,then it was named as Bacillus subtilis J1220.After that we determined its growth curve by turbidimetric method,and we found that the fermentation supernatant of Bacillus subtilis J1220 at the end of logarithmic growth phase can inhibit Staphylococcus aureus ATCC29213 and Escherichia coli ATCC25922.The antimicrobial susceptibility test of Bacillus subtilis J1220 was determined by broth dilution method which indicated that this strain has a resistance to penicillin G and sulfonamides.2 Effects of Bacillus subtilis J1220 on piglets’ biochemical and immune indices as well as intestinal microfloraTwelve healthy piglets weighing about 20kg were divided into two groups randomly.Then we established the T fistula model in the ileum of piglets.After postoperative nursing and withdrawal period,the control group fed with basal diet,while the experimental group fed basal diet with the Bacillus subtilis J1220 preparation.The serum biochemical and immune indeces were detected at the 0th,7th,14th,28th day.The results showed that the concentrations of serum total protein,albumin,globulin and ceratinine increased significantly(P<0.05)in the PRO group,while the ratio of albumin to globulin decreased(P<0.05)on day 14 and day 28.Moreover,piglets in the PRO group had a higher level(P<0.05)of IgG,IgM and IgA on day 28.At the 0th,3th,7th,10th,14th,28th day after feeding,the ileum contents and animal feces of each group were collected and stored in-70℃.And we established a method of RT-PCR to detect the populations of Bacillus subtilis,Lactobacillus,Bifidobacterium,Enterobacteriaceae,Enterococci in intestinal contents and faeces.The results showed that the number of Bacillus subtilis in intestinal and feces increased over time after experimental group was fed the probiotic Bacillus subtilis J1220 preparation(P<0.01),and it reached steady state at the 14th day compared with the control group.For Lactobacillus and Bifidobacterium,a significant increase in the experimental group of intestinal contents after the 7th day(P<0.05),and after the 10th day in faeces(P<0.05).However,compared with control group,the number of Enterobacteriaceae and Enterococci in intestinal contents and faeces had a significant decrease in experimental group after the 7th day(P<0.05).3 Metabolism studies of Bacillus subtilis J1220 in vitroFirst,we make sure the condition of liquid phase and mass spectrometry of LC/MS-IT-TOF by observing ion peak integrity and resolution.And then the method of sample preparation was determined by single factor test and orthogonal test,also the stability of the method has been investigated by intra-and inter-coefficient of variation.After that we extracted and measured the metabolites of Bacillus subtilis J1220 cultured at the end of logarithmic growth phase,and compared the differences of ion information between the test strain and blank broth.Also we set an EX-vivo model to compare the interaction of Bacillus subtilis J1220 and intestinal flora.The result shows that the samples were extracted with dichloromethane and ethyl acetate(V/V,1:1)by liquid-liquid extraction 5 min and purified by acetonitrile and n-hexane.The analytes were eluted with 0.1%formic acid in water(A)and acetonitrile(B)gradiently.The intra-and inter-coefficient of variation of the method were all below 15%.At last,there were 15 different ion informations including m/z 470.2441,474.9189,221.1185,277.1827,316.6631,340.1951,472.8264,453.2828,431.2678,409.2591,443.3213,501.8521,479.8179,460.2983 and 438.2803.We also found that some ion informations were disappeared when Bacillus subtilis J1220 co-cultured with intestinal flora.4 Effects of Bacillus subtilis J1220 on the piglets’ metabolismAt first,the methods of sample preparation for intestinal liquid,plasm and edible tissues were established and their repeatabilities were investigated by intra-and inter-coefficient of variation.After that we sampled the intestinal liquid and plasm at the 0th,3th,7th,10th,14th,28th day after feeding by T fistula model in the ileum of piglets.At the 28th day,we slaughtered these animals and collected edible tissues.These animal experimental samples were test by the methods which had been established before and targeted the 15 different metabolites information by extracting the TIC.The results showed that the repeatability of each method was well with intra-and inter-coefficient of variation all below 15%.And we found the main different metabolites informations betwween experimental group and control group were m/z 470.2441 and m/z 474.9189,which appeared in intestinal liquid after the 3th day and the 7th day in plasm of experimental group respectively.As for the edible tissues,there existed obvious differences in liver,kidney,small intestine and large intestine at the 28th day of experimental group,while the differences in muscle and stomach were unconspicuous.In summary,Bacillus subtilis J1220 was isolated and identified,then the antimicrobial susceptibility and bacteriostasis ability was determined.The beneficial gut microbiota was improved after piglets feeding J1220,while the potential harmful microbiota was decreased.And it also found that J1220 could enhance metabolism and immunity.There were 15 different ion informations in vitro by metabolome,and two main ion informations including m/z 470.2441 and m/z 474.9189 were found in EX-vivo and in vivo. |
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