Molecular Mechanism Of Competitive Regulation Of Bacteroides In Two Chinese Herbal Compound Polysaccharides Based On PULs

Objective:Traditional Chinese medicine polysaccharide is one of the main components of traditional Chinese medicine compound.Traditional Chinese medicine polysaccharide can regulate the structure of intestinal flora.Based on PULs of the molecular mechanism of Shenling baizhu power and Lizhong decoction in regulating intestinal bacillus is regulated,which is adjusted for different Chinese medicine compound.Different ways of gut microbiota provide modern evidence.Methods:1.Two strains of Bacteroides(Bacteroides thetaiotaomicron ATCC 29148,Bacteroides fragile ATCC 25285)and two strains of Firmicutes(Clostridium sp.YT2,Bacillus sp.T30)were selected to mix four strains of Bacteroides and Firmicutes respectively to form four combination(Bacteroides thetaiotaomicron ATCC 29148 and Clostridium sp.YT2,Bacteroides thetaiotaomicron ATCC 29148 and Bacillus sp.T30,Bacteroides fragile ATCC 25285 and Clostridium sp.YT2,Bacteroides fragile ATCC 25285 and Bacillus sp.T30),respectively in BHI medium,Shenling baizhu power and Lizhong decoction were used as the sole carbon source for in vitro mixed culture,and samples were taken continuously at different time periods.Under different Chinese herbal compound polysaccharides as bacterial carbon sources,Bacteroides and Firmicutes were tested in different combinations.The growth curve was used to obtain the effect of different polysaccharide carbon sources on the carbon source competition of the mixed bacteria.2.The two strains of Firmicutes(Clostridium sp.YT2 and Bacillus sp.T30)isolated by self were sequenced using the third-generation PacBio RS II high-throughput sequencing platform.The whole genome sequences of two strains of Bacteroides and two strains of Firmicutes were analyzed by PULs.The polysaccharides from the perspective of PULs were used to explain the micro-ecological structure of the intestinal flora by Shenlingbaizhu powder and Lizhong decoction.At the same time,the transcriptome data of two strains of Bacteroides with strong ability to decompose polysaccharides were analyzed.Results:1.The mixed bacteria of four different combinations were cultured in vitro under different carbon sources(Shenling baizhu power and Lizhong decoction).For the combination of Bacteroides thetaiotaomicron ATCC 29148 and Clostridium sp.YT2,cultured in different mediums(BHI,Shenling baizhu power,Lizhong decoction),the total peak concentration of mixed bacteria at 32 h,44 h,24 h(31.97±0.67)×10~8 CFU/ml?(11.17±0.13)×10~8 CFU/ml?(8.64±0.26)×10~8 CFU/ml;The combination of Bacteroides thetaiotaomicron ATCC 29148 and Bacillus sp.T30,respectively(BHI,Shenling baizhu power,Lizhong decoction)cultured in different mediums,the total peak concentration of mixed bacteria at 36 h,32 h,36 h was(24.90±0.88)×10~8 CFU/ml?(14.74±0.36)×10~8 CFU/ml?(18.37±1.00)×10~8 CFU/ml;for the combination of Bacteroides fragile ATCC 25285 and Clostridium sp.YT2,different media(BHI,Shenling baizhu power,Lizhong decoction)under the environment,the total peak concentration of mixed bacteria at32 h,44 h,and 32 h was(62.07±1.26)×10~8 CFU/ml?(32.66±1.63)×10~8 CFU/ml?(27.46±1.53)×10~8 CFU/ml;Combination of Bacteroides fragile ATCC 25285 and Bacillus sp.T30,respectively(BHI,Shenling baizhu power,Lizhong decoction)The total peak concentration of mixed bacteria at 36 h,32 h,and 36 h was(62.84±0.74)×10~8 CFU/ml?(37.22±1.39)×10~8 CFU/ml?(64.90±1.45)×10~8 CFU/ml.Under the environmental culture of Shenling baizhu power and Lizhong decoction:Bacteroides has been growing faster than Firmicutes;in the BHI composite carbon source environment culture:the growth of Firmicutes is faster than that of Bacteroides,and the growth of Bacteroides is better for Firmicutes.2.The whole genome sequence of Bacillus sp.T30 was 5288332 bp,the GC content was35.35%,and the coding gene was 5427.Bacillus sp.T30 has two gene clusters and recognizes a total of 128 carbohydrate enzyme gene expression.The whole genome sequence of Clostridium sp.YT2 was 3264034 bp,the GC content was 28.76%,and the coding gene was3098.Clostridium sp.YT2 has one gene cluster and recognizes 79 carbohydrate enzyme gene expressions.Comparing the two genomes of Bacteroides with two strains of thick-walled bacteria,it was found that Bacteroides thetaiotaomicron ATCC 29148 has a large number of carbohydrate genes,and Bacteroides thetaiotaomicron ATCC 29148 has 88PULs,and 163kinds of starch proteins can be used.15 kinds of lyase and 226 kinds of glycoside hydrolase;Bacteroides fragile ATCC 25285 can encode 5 kinds of carbohydrate lipase,3 kinds of polysaccharide lyase,141 kinds of glycoside hydrolase and 83 kinds of glycosyltransferase,among which polysaccharide utilization in whole genome is predicted 31 sites.Bacillus sp.T30 has been predicted to have 71 PULs gene clusters,of which 47 PULs are clearly included in the carbohydrate gene.The whole genome of the thick-walled fungus T30 can encode 6 kinds of carbohydrate lipase,20 kinds of glycoside hydrolase and 8 kinds of glycosyltransferase.Clostridium sp.YT2 has been predicted to have 30 PULs gene clusters.Among them,there are 23 PULs with carbohydrate genes,including 7 kinds of carbohydrate lipases,14 glycoside hydrolases,and glycosides.11 kinds of transferases.Bacteroides has a variety of different PULs,and has a strong ability to decompose polysaccharides;thick-walled bacteria have few carbohydrate genes relative to Bacteroides,and lack of PULs recognition gene pairs,the ability to decompose polysaccharides is very weak,just like the previous carbon according to the conclusion of the source competition experiment,in the culture of polysaccharides,the growth of Bacteroides is better than that of thick-walled bacteria.For the analysis of two Bacteroides transcriptome data,it was found that there were 10PULs in the 12 PULs that were significantly expressed in the environmental culture of Shenling baizhu power and Lizhong decoction.The Lizhong decoction was better expressed than that of Shenling baizhu power.Two PULs were better expressed in Lizhong decoction than that of Shenling baizhu power.In the environmental culture of Shenling baizhu power and Lizhong decoction,11 PULs were significantly expressed in Lizhong decoction.It is better expressed;there are two PULs which are better expressed in lizhongtang decoction than in the environmental culture of Shenling baizhu power.Conclusion:The mixed bacteria of Bacteroides and Firmicutes are cultured in the medium of Shenlingbaizhu powder and lizhong decoction as the sole carbon source.The growth of Bacteroides is better than that of Firmicutes.Bacteroides has a competitive advantage of carbon source.By comparison of whole genome carbohydrates,Bacteroides has more PULs than Firmicutes,and the ability of Bacteroides to decompose polysaccharides is stronger than that of Firmicutes.Compared with carbon source competition,Bacteroides has a carbon source competitive advantage.In accordance with PULs,it may be one of the mechanisms for the regulation of intestinal flora by Shenling baizhu power and Lizhong decoction.Through the analysis of Bacteroides transcriptome data,which proved that different Chinese medicine polysaccharides can induce the differential expression of the typical Bacteroides PULs.This may be the molecular mechanism by which different Chinese herbal compounds regulate Bacteroides in different ways.