Research On The Micro-ecological Mechanism Of The Jianpi Recipe Shenlingbaizhusan Based On "Traditional Chinese Medicine Polysaccharides-Butyric Acid Producing Bacteria"

Objective:As a commonly used prescription,Shenlingbaizhusan has a significant clinical effect in the treatment of diarrhea of spleen deficiency and dampness,and is widely used.Experimental studies have shown that these traditional Chinese medicine compounds can regulate the intestinal flora and achieve the purpose of treating diarrhea.However,there are few studies on which components of Shenlingbaizhusan can regulate intestinal flora.Polysaccharide,as one of the main components of traditional Chinese medicine,can be used as a unique carbon source for specific intestinal flora in the fermentation process,which may be the functional component of regulating intestinal flora.Butyric acid producing bacteria as a kind of functional flora,its metabolite butyric acid has been proved to have therapeutic effect on diarrhea.At present,the research on"traditional Chinese medicine polysaccharide butyrate producing bacteria"is less and needs to be strengthened.Based on the above background,heat shock method,gas chromatography,16S r RNA gene sequencing,second-generation sequencing,third-generation sequencing,in vitro carbon source experiment with Shenlingbaizhusan as the sole carbon source,and other technologies were studied around"traditional Chinese medicine polysaccharide butyrate producing bacteria",aiming to study the microecological mechanism of Shenlingbaizhusan and its polysaccharide components regulating intestinal butyrate producing bacteria through animal experiment and in vitro carbon source experiment.Methods:(1)Bacillus was screened by water bath heat shock,and then isolated by coating and scribing.Butyric acid producing bacteria were identified by acid production experiment,Gram staining,gas chromatography,16S r RNA gene and other physiological experiments and molecular analysis methods.(2)Sequencing the whole genome of the isolated butyric acid-producing b acteria through the second and third generation sequencing methods,namely Ill umina and Pac Bio,and an overview of the genome information,including the analysis of genome size,t RNA,r RNA,and CDS number.Use Glimmer(http://ccb.jhu.edu/software/glimmer/index.shtm L),Gene Mark S,Prodigal software to pre dict the coding sequence(CDS)in the genome.The nucleic acid sequence and amino acid sequence of the functional gene are obtained by prediction,which can be used for subsequent function and systematic evolution analysis.By def ault,glimmer is used to predict the assembly results of scanning maps,glimme r is used to predict chromosome genome by default,and Gene Mark S is used t o predict plasmid genome by default.The characteristics of raz1 gene were an alyzed by whole genome analysis.(3)A model of AAD(Antibiotic-associated diarrhea)rat model was constructed by gavage of antibiotics and Clostridium difficile to induce intestinal flora imbalance.After the model was established,the rats appeared to eat less food,feces were not formed,and the reaction was sensitive.Reduce other symptoms.After treatment with Shenlingbaizhusan decoction,AAD rats gradually resumed diet,stool formation,and sensitivity improved.Collect rat feces at each stage and extract DNA,use butyryl-Co A-Co A transferase gene primers BCo Atscr F and BCo ATcr R to PCR amplify fecal DNA genes,and then use TA cloning sequencing platform to detect rat in normal stage,AAD model The diversity of the intestinal butyric acid-producing bacteria and the changes of the flora structure in the stage and treatment stage,the research is based on the results of the effect of Shenlingbaizhusan compound on the intestinal butyric acid-producing bacteria.(4)Polysaccharide culture medium was constructed by using Shenlingbaizhusan polysaccharide as the sole carbon source The growth curve and butyric acid concentration curve of butyric acid producing bacteria in PM,PM and PM degradation solution(respectively by Bacteroides fragile,Bacteroides thetaiotaomicron and Clostridium sp.YT2)were determined by continuous sampling method.The effects of polysaccharide components of Shenlingbaizhusan on the growth and butyric acid concentration of butyric acid producing bacteria were further studied,and whether the polysaccharide components of Shenlingbaizhusan can regulate the growth of butyric acid producing bacteria in intestinal tract was completed in vitro study.Results:(1)A butyric acid-producing bacterium RAZ1 was isolated from the feces of healthy adults.The colony morphology was smooth,milky white,round convex,and Gram stain was positive.Gas chromatographic analysis results show that RAZ1 has the ability to produce butyric acid.The 16S r RNA gene phylogenetic tree results show that RAZ1 belongs to Clostridium.(2)The number of chromosomal genomes of the strain RAZ1 sample is 1,without the plasmid genes of the genome,the genome size is 4056467 bp,the number of genes is 3902,the total gene length is 3427287 bp,the genome GC content is28.05%,and the sequencing depth is 378.42.There are 85 t RNAs and 30 r RNAs.The comparison result of the NR database is a total of 3899 encoded proteins;the comparison result of the Swiss-Prot database is a total of 2592 annotated proteins;the comparison result of the Pfam database is a total of 3039 annotated proteins;the comparison result of the COG database is a total of 3196 Annotated proteins;the comparison result of GO database is a total of 2783 annotated proteins;the comparison result of KEGG database is a total of 1811 annotated proteins.(3)In the experiment of treating aad rats with Shenlingbaizhusan,the diversity of butyric acid producing bacteria in the faeces of AAD rats was as follows:in the normal feeding stage,Roseburia was the main genus,and there were 8 genera,including Clostridium,Eucterium,Lacrimispora,Psychrosphaera,agathobacter,Geobacillus and Oscillibacter;In the modeling stage,there are only three genera,with Clostridium being the most,followed by Roseburia and Anaerofusis;In the treatment stage,the number of genera gradually recovered to 6,with Eubacterium being the most,Clostridium and Roseburia being the second,Lacrimispora,Hydrogeniciclostidium and Bacteroides being the second.The phylogenetic tree showed that all the sequences could be classified into 16 clusters.Based on 16S analysis,the dominant flora of butyrate producing bacteria was Firmicutes,and some butyrate producing bacteria were also found in Bacteroidetes and Proteobacteria.Percentage analysis of the clusters of each stage of the treatment of AAD rats with Shenlingbaizhusan decoction showed that:SPD1,butyric acid-producing bacteria is the most widely distributed,distributed in 11 clusters;The diversity of SPD2 butyric acid-producing bacteria was significantly reduced,only distributed in 5 Clusters,of which Cluster11 accounted for the main part;SPD3 butyric acid-producing bacteria are distributed in 7 Clusters.Compared with SPD1,the diversity is reduced,but compared with SPD2,the diversity has recovered and the structure of butyric acid-producing bacteria has also changed.The proportion of Cluster11 is reduced,and the proportion of Cluster4 is significantly increased.(4)The results showed that the combination of butyric acid producing bacteria and polysaccharide degrading bacteria in Shenlingbaizhusan polysaccharide medium had the highest growth:The combination of butyrate producing bacteria and Bacteroides thetaiotaomicron was better than that of butyrate producing bacteria and Clostridium sp.YT2.The combination of Clostridium sp.YT2 was better than that of butyrate producing bacteria and Bacteroides fragile,and better than that of butyrate producing bacteria cultured alone.The maximum growth results were as follows:(0.959±0.09)×10~9CFU/m L、(0.929±0.09)×10~9CFU/m L、(0.686±0.09)×10~9CFU/m L、(0.129±0.09)×10~9CFU/m L.The results of the highest butyric acid-producing concentration of the combination of butyric acid-producing bacteria and polysaccharide-degrading bacteria in the polysaccharide medium of Shenlingbaizhusan were:3540.67 mg/L、3614.67 mg/L、2725 mg/L、342 mg/L。Compared with the control group BHI composite carbon source,the combination of butyric acid-producing bacteria and polysaccharide degrading bacteria,the maximum growth result comparison is:butyric acid-producing bacteria alone is better than the combination of butyric acid-producing bacteria and Bacteroides thetaiotaomicron better than butyric acid-producing bacteria.The combination of acid bacteria and Clostridium sp.YT2 is better than the combination of butyric acid-producing bacteria and Bacteroides fragile.The maximum growth results are:(0.916±0.09)×10~9CFU/m L、(0.474±0.09)×10~9CFU/m L、(0.263±0.09)×10~9CFU/m L、(0.220±0.09)×10~9CFU/m L.The concentrations of butyric acid produced are:2835.67 mg/L、1625mg/L、1126.67 mg/L、1147.5 mg/L.The maximum growth of butyric acid-producing bacteria in the PM degradation liquid of Shenlingbaizhusan degraded by polysaccharide degrading bacteria was greater than that in the BHI degradation liquid degraded by polysaccharide degrading bacteria.Conclusion:According to the results of physiological and molecular experiments,the experiment successfully isolated a strain of RAZ1 from healthy adult feces,which has the function of producing butyric acid,which meets the definition of butyric acid-producing bacteria.The whole genome results showed that the genome size of the bacteria was 4056467 bp,the number of genes was 3902,the total length of genes was 3427287 bp,the GC content of the genome was 28.05%,and the sequencing depth was 378.42.There are 85 t RNAs and 30 r RNAs.There are a large number of carbohydrate active enzymes in raz1,including glycoside hydrolase,glycosyltransferase,carbohydrate esterase and auxiliary enzymes.It may include the butyric acid production pathway of raz1,and analyze the metabolic pathway of butyric acid production from the perspective of genome.In the experiment,a purchased Clostridium butyricum strain was selected and compared with the butyricum strain isolated in the laboratory for subsequent carbon source experiment in vitro.The results showed that Shenlingbaizhusan could regulate the diversity of intestinal butyric acid producing bacteria in AAD model rats,and could restore the diversity of intestinal butyric acid producing bacteria destroyed by antibiotics.According to in vitro experiments with Shenlingbaizhusan polysaccharide as the sole carbon source,the results suggest that:The mechanism of Shenlingbaizhusan regulating intestinal butyric acid producing bacteria may be that the polysaccharide components of Shenlingbaizhusan are first degraded into monosaccharide/pyruvate by intestinal polysaccharide degrading bacteria,and then the monosaccharide/pyruvate is decomposed and metabolized into butyric acid by butyric acid producing bacteria to regulate intestinal flora and intestinal butyric acid concentration,so as to achieve therapeutic effect.However,there are differences in the growth of different combinations of polysaccharide degrading bacteria and butyric acid producing bacteria and the regulation effect of different carbon sources on the combination of different polysaccharide degrading bacteria and butyric acid producing bacteria.This may be the reason for the different therapeutic effects of different Chinese herbal compounds,which needs further study.