Study On The In Vitro Degradation Mechanism Of "Astragalus Polysaccharides-Butyric Acid” Based On GpPULs Bacteria/Butyric Acid-Producing Bacteria

Objective:To investigate the degradation mechanism of"Astragalus polysaccharide-butyric acid",based on the analysis of the composition of APS,and to investigate the in vitro degradation mechanism of APS to butyric acid by typical Firmicutes（gpPULs bacteria）and butyric acid-producing bacteria in combination with in vitro carbon source experiments,in order to provide a modern basis for APS to regulate the intestinal microecology.provide a modern basis for pharmacological studies on the regulation of intestinal microecology by APS.Methods:1.Extraction,separation and purification of APS:Astragalus crude polysaccharides were extracted by aqueous extraction and alcoholic precipitation method,and then separated and purified by ion exchange column and gel filtration column chromatography after sevage method to remove protein,D101 macroporous resin to remove pigment,and dialysis bag to remove small molecules,and then their molecular weight and monosaccharide composition were determined.2.Two strains of Clostridium sp.YT2 and Bacillus sp.T30 were cultured in vitro with different configurations of polysaccharides（20%APS,60%S,60%L and 60%FOS）as the sole carbon source medium.The growth of the bacteria under different carbon sources was measured by continuous sampling and spectrophotometric methods,while the content of pyruvate and monosaccharides in the intermediate degradation solution was determined by liquid chromatography analysis to obtain whether astragalus polysaccharides could affect the growth of the bacteria.The intermediate degradation solution of the APS group was then filtered through mixed membranes of 0.88μm,0.48μm and 0.22μm pore size in turn,and then two strains of butyric acid producing bacteria（Butyric acid-producing bacteria RAZ1and Clostridium tyrobutyricum ATCC25755）were cultured separately in vitro,and the growth curves were measured by OD₆₀₀and the end product butyric acid content was determined by gas chromatography analysis to further observe the effect of APS.3.Transcriptome analysis of Clostridium sp.YT2 combination of butyric acid-producing bacteria RAZ1 grown to log phase in vitro under different carbon sources was performed using a second generation Illumina high throughput sequencing platform.The gene,pathway and protein expression of the two thick-walled strains on different carbon sources were analysed by GO database,KEGG database and COG database,and the differences between APS and other sugar media in terms of gpPULs and butyric acid gene expression,leading to a degradation mechanism for"Astragalus polysaccharide-butyric acid".4.Transcriptome analysis of two butyric acid producing strains of Bacillus sp.T30combination（Butyric acid-producing bacterium RAZ1,Clostridium tyrobutyricum ATCC25755）grown to log phase in vitro under different carbon sources was performed using a second generation Illumina high throughput sequencing platform.The gene,pathway and protein expression of the two strains were analysed by GO database,KEGG database and COG database on different carbon sources,and the differences between Astragalus polysaccharide and other sugar media in terms of gpPULs and butyric acid gene expression were analysed to the degradation mechanism of"Astragalus polysaccharide-butyric acid".Results:1.The two Astragalus polysaccharides,HQE1F1 and HQE1F2,with molecular weights of 7575 k Da and 4087 k Da,respectively,were identified by extraction,isolation and purification,and detection of single symmetric wave peaks by HPGPC.HQE1-F1 was selected for further monosaccharide fraction analysis as it had fewer spurious peaks,resulting in HQE1-F1 consisting of Glc,Ara.Gal and Man,with molar percentages of83.011%,15.401%,1.221%and 0.367%,respectively.2.In an in vitro carbon source experiment with a combination of polysaccharide-degrading bacteria and butyric acid-producing bacteria,polysaccharide-degrading bacteria Clostridium sp.YT2,Bacillus sp.T30 and Bacteroides fragilis were grown in APS medium compared to the control medium with maximum bacterial concentrations of（0.556±0.114）×10⁹CFU·m L^-1,（0.386±0.183）×10⁹CFU·m L^-1,（0.352±0.174）×10⁹CFU·m L^-1.The butyric acid content produced by butyric acid-producing bacteria RAZ1 and Clostridium tyrobutyricum ATCC25755 after degradation in APS medium by Firmicutes/Bacteroides was significantly higher than that directly in APS medium,and the butyric acid content of butyric acid-producing bacteria RAZ1 and Clostridium tyrobutyricum ATCC25755 cultured directly under APS were（11.59±2.57）mg/L,（49.86±3.89）mg/L.After degradation by Clostridium sp.YT2,Bacillus sp.T30 and Bacteroides fragilis,the butyric acid-producing bacteria RAZ1produced butyric acid at concentrations of（120.88±3.48）mg/L,（161.58±2.60）mg/L,（68.65±7.71）mg/L.The concentrations of butyric acid produced by Clostridium tyrobutyricum ATCC25755 were（115.34±4.10）mg/L,（150.30±14.72）mg/L and（100.10±4.15）mg/L.And the bacterial growth of butyric acid-producing bacteria after degradation by polysaccharide degrading bacteria was also significantly better than direct culture,with direct culture of butyric acid-producing bacteria RAZ1 and Clostridium tyrobutyricum ATCC25755.The maximum bacterial concentrations for direct culture of butyric acid-producing bacteria RAZ1 and Clostridium tyrobutyricum ATCC25755 were（0.024±0.070）×10⁹CFU·m L^-1and（0.017±0.009）×10⁹CFU·m L^-1,respectively.After degradation by Clostridium sp.YT2,Bacillus sp.T30,and Bacteroides fragilis,the maximum bacterial concentrations of butyric acid-producing bacteria RAZ1 were（0.385±0.051）×10⁹CFUm L^-1,（0.274±0.023）×10⁹CFU·m L^-1,（0.347±0.125）×10⁹CFU·m L^-1.After degradation by Clostridium sp.YT2,Bacillus sp.T30,and Bacteroides fragilis,the maximum bacterial concentrations of Clostridium tyrobutyricum ATCC25755 were（0.298±0.261）×10⁹CFU·m L^-1,（0.592±0.261）×10⁹CFU·m L^-1,（0.671±0.185）×10⁹CFU·m L^-1. At the same time,we also examined the composition of the intermediate products,mainly including pyruvate and monosaccharide components.Clostridium sp.YT2 degraded APS S and FOS,respectively,and obtained pyruvate contents of（22.45±0.05）μg/ml,（5.27±0.07）μg/ml,（5.01±0.68）μg/ml,respectively.Bacillus sp.T30 degraded APS,S and FOS,respectively,yielding pyruvate levels of 0,（10.37±0.37）μg/ml,（4.87±0.03）μg/ml.Bacteroides fragilis degraded APS,S and L,respectively,and obtained pyruvate contents of（24.81±0.72）μg/ml,（6.87±0.24）μg/ml and 0.Clostridium sp.YT2 degraded APS to obtain48h intermediates containing monosaccharides such as Man,GlcN,Rha,Glc-UA,Gal-UA,Glc,Gal and Ara in the amounts of 1.86μg/mg,2.02μg/mg,3.11μg/mg,0.60μg/mg,8.26μg/mg,128.14μg/mg,18.01μg/mg and 50.17μg/mg,respectively.Bacillus sp.T30 degraded APS to obtain 48h intermediates containing Man,GlcN,Rha,Glc-UA,Gal-UA,Glc,Gal and Ara at 1.76μg/mg,1.99μg/mg,3.54μg/mg,0.59μg/mg,10.35μg/mg,0.65μg/mg,133.90μg/mg,18.45μg/mg and 50.17μg/mg.The 48h intermediate products obtained from the degradation of APS by Bacteroides fragilis contained the monosaccharides Man,GlcN,Rha,Glc-UA,Gal-UA,GalN,Glc,Gal and Ara at 1.86μg/mg,1.80μg/mg,4.12μg/mg,0.51μg/mg,7.54μg/mg,0.74μg/mg,136.43μg/mg,20.22μg/mg and 60.42μg/mg.3.Clostridium sp.YT2 gpPULs were differentially expressed,with significant expression of gpPUL in the APS group（5,13,15,17,22,23）,and gpPUL（15,17,20,21,23,24 and 25）were significantly expressed in the Glc group.Related genes significantly expressed in the astragalus polysaccharide group were GH4（gpPUL5）,CE9（gpPUL5）,MAA（gpPUL13）,cys E（gpPUL17）,AA7（gpPUL21）,AA4（gpPUL22）and CE4（gpPUL23）.Related genes significantly expressed in the Glc group were Nag C（gpPUL15,24,25）,cys E（gpPUL17）,GT2（gpPUL20）,AA7（gpPUL21）,CE4（gpPUL23）. During the degradation of APS by intermediates of Clostridium sp.YT2 degradation solution set by butyric acid-producing bacterium RAZ1 to butyric acid,differential expression of genes related to the butyric acid kinase gene buk,butyryl coenzyme A dehydrogenase（bcd）gene,phosphotrans-butyryl coenzyme（ptb）gene,and3-hydroxybutyryl coenzyme A dehydrogenase（paaH）gene were detected in the APS degradation liquid group compared to the Gal group for the butyric acid production process.4.Bacillus sp.T30 gpPULs were differentially expressed,with eight significantly expressed in the APS group,gpPUL（1,9,14,42,45,53,58 and 62）,respectively.These included significantly expressed genes ABC（gpPUL（1,14））CBM40（gpPUL9）,CLpP（gpPUL45）,CLpX（gpPUL53）,PTS（gpPUL58）,and ATPase（gpPUL62）.The FOS group significantly expressed seven,gpPUL（1,24,42,45,53,58 and 68）,including significantly expressed genes ABC（gpPUL1）,AA4（gpPUL24）,ATP（A-I）（gpPUL42）,CLpP（gpPUL45）,CLpX（gpPUL53）,PTS（gpPUL58）and MFS（gpPUL68）.During the degradation of APS by intermediates of Bacillus sp.T30 degradation solution set by butyric acid-producing bacterium RAZ1 to butyric acid,genes related to the butyric acid production process in the astragalus polysaccharide degradation solution group compared to the galactose group were detected in the butyric acid kinase gene buk,butyryl coenzyme A dehydrogenase（bcd）gene,phosphate trans-butyryl coenzyme（ptb）gene,3-hydroxybutyryl coenzyme A dehydrogenase（paaH）gene,and enoyl coenzyme A hydratase（crt）gene Differential expression was observemuud.During the degradation of intermediate products to butyric acid by Clostridium tyrobutyricum ATCC25755,several genes were detected affecting the butyric acid metabolic process,including the genes gab T,K932＿RS02950,K932＿RS04890,K932＿RS04895,K932＿RS04910,K932＿RS05695,K932＿RS07510,K932＿RS09610,K932＿RS09710,K932＿RS10325,K932＿RS13395,K932＿RS13905.Conclusion:We tracked the in vitro growth and transcriptomic gene analysis of the combined degradation of astragalus polysaccharide by Firmicutes and butyric-producing bacteria,and obtained the in vitro degradation mechanism of typical firmicutes（gpPULs）and butyric-producing bacteria to degrade astragalus polysaccharide into butyric acid:（1）Astragalus polysaccharide can degrade into butyric acid;The degradation can be achieved by gpPULs and butyricogenic bacteria.（2）The process of degradation to butyric acid is closely related to the expression of gpPULs and related hydrolases of astragalus polysaccharide,which is composed of Firmicutes（gpPULs）and butyric acid production butyric kinase pathway（buk gene）.