Study On Preparation Of Astragalus Polysaccharides And Oligosaccharides And Their Probiotic Effects

Astragalus polysaccharide is one of the more abundant components in Astragalus.It has various effects such as antioxidant,anti-inflammatory,and immunity enhancement.However,there are few studies on its degradation product oligosaccharides.Polysaccharides and oligosaccharides can regulate the composition of intestinal microorganisms and promoting human health as prebiotic substances.Studies on the the prebiotic activity of Astragalus Polysaccharides are mostly concentrated on animal experiments,but animal experiments have limitations of long experimental period,high cost and complicated operation.At the same time,there are few studies on the probiotic effects of Astragalus Oligosaccharides.In response to this problem,Astragalus Polysaccharides(APS)and Astragalus Oligosaccharides(AOS)were obtained from Astragalus membranaceus and the structure and degrees of polymerization were characterized.And then APS and AOS were used to fermente with fecal microorganisms in vitro.The changes in pH,gas production,total sugar and short-chain fatty acids were measured to evaluate their prebiotic effects.Finally,the intestinal reactor was used to further evaluate the prebiotic effects of APS and AOS.The main contents and results are as follows:(1)Through single factor optimization and orthogonal optimization experiments,the best extraction conditions of APS were determined as follows:extraction temperature 80?,material-liquid ratio 1:50,extraction time 2.5 h.The extraction temperature had the greatest effect on the extraction of APS,and the final yield was 26.23%.After deproteinization with papain and Sevage reagent,the two components APS-1 and APS-2 were purified by DEAE Fast Flow.Then,trifluoroacetic acid was used to degrade APS to obtain AOS.(2)The molecular mass,monosaccharide composition,the type and way of glycosidic bond of APS-1 and APS-2 were analyzed by High Performance Gel Filtration Chromatography(HPGFC),Ion Chromatography(IC),Fourier Transform Infrared Spectrometry(FT-IR),~1H-Nuclear Magnetic Resonance(~1H-NMR)and methylation analysis.The results showed that the molecular masses of APS-1 and APS-2 were 38.4 kDa and 5.2 kDa,respectively.APS-1 was mainly composed of galactose and glucose with a molar ratio of 1:49.76.APS-2 was mainly composed of rhamnose,galactose and glucose,and the molar ratio was 1:2.99:16.26.APS-1mainly consisted of?-type glycosidic bonds,while APS-2 contained both?-type glycosidic bonds and?-type glycosidic bonds.The main ways of glycoside bond connection of APS-1were:T-Glcp,1,4-Glcp,1,6-Glcp and 1,4,6-Glcp,the molar ratio was 1.16:1.27:12.32:1.The main ways of glycoside bond connection of APS-2 were:T-Galp,1,6-Galp,1,4-Galp,1,4,6-Galp,T-Glcp,1,4-Glcp,1,3-Glcp,1,3,6-Glcp and 1,2,3,6-Glcp,the molar ratio was 4.66:4.42:7.7:2.75:2.77:30.09:2.34:1:5.12.Using MALDI-TOF MS to determine the degree of polymerization of AOS was 2?20.(3)In vitro simulated the digestion process of human saliva,gastric juice and intestinal fluidd of APS and AOS.The determination of total sugar and reducing sugar,molecular weight distribution and degree of polymerization indicated that APS and AOS were not be digested and degraded by saliva,gastric juice and intestinal fluid.Then,in vitro fermentation of APS and AOS was performed using mixed stools from 3 healthy volunteers and individual stools from 7healthy volunteers,respectively,and the changes in the total sugar content,pH,gas production and SCFAs content showed that APS and AOS could significantly reduce the pH and promote the production of SCFAs,especially butyric acid.The results of microbial composition showed that APS and AOS could promote the growth of beneficial bacteria such as Bacteroides,Faecalibbacterium,and Bifidobacterium,reduced the abundance of harmful bacteria such as Holdemanella,Sutterella,Dorea,Streptococcus,etc.,and changed the structure of intestinal flora.In short,APS and AOS had a good prebiotic effects.(4)GSR simulates the fermentation of APS and AOS in the colon in vitro.The real-time detection of the changes in bacterial concentration,total sugar content,reducing sugar content,pH,NaOH supplementation and SCFAs during the fermentation process further shows that APS and AOS had a good prebiotic activity,which could significantly reduce pH in intestinal and promote the production of SCFAs,especially in the ability to produce butyric acid and valeric acid significantly superior to inulin.