Study On The Adsorption, In Vitro Digestion And Intestinal Flora Fermentation Characteristics Of Four Kinds Of Poria Cocos Polysaccharide

【Objective】Polysaccharide is one of the main effective components of traditional Chinese medicine（TCM）Poria cocos.According to its extraction method or its solubility difference,Poria polysaccharides can be divided into water-soluble polysaccharide（WSP）,which only accounts for 0.29%-3.36%of content in Poria cocos,and alkali-soluble polysaccharide（ASP）,which accounts for 70%-90%of content in Poria cocos.Today,the pharmacologic and healthcare functions of Poria cocos mainly focus on WSP.ASP of Poria cocos was discarded in the preparation of TCM decoction for insolubility.With the clinical application and promotion of TCM formula granules,Poria cocos,as a TCM variety with huge clinical usage amount,had an amount of Poria residue（mainly ASP）after the production of TCM formula granules.In this study,WSP,ASP and carboxymethyl poria polysaccharide（CMP）were prepared,and the chemical structure of different polysaccharides were characterized and identified.On the basis of these polysaccharide preparation,the functional characteristics including adsorption properties,antioxidant activities,simulated digestion and fermentation characteristics of intestinal flora in vitro were studied.It was intended to provide theoretical basis for the high value utilization of ASP of Poria Cocos.【Methods】1.Extraction,modification and characterization of Poria polysaccharides.With Poria cocos as raw material,WSP was extracted by water extraction and alcohol precipitation.The total ASP was extracted by alkali extraction and acid precipitation,and the sedimentation products ASP-9and ASP-11 were obtained by gradient p H value method.Using ASP-11 as raw material,CMP was synthesized by water media process.The structures of WSP,ASP-9,ASP-11 and CMP were characterized by ultraviolet（UV）,infrared spectroscopy（IR）and nuclear magnetic resonance spectroscopy（NMR）.2.Study on adsorption characteristics and antioxidant activity of Poria polysaccharides.The oil holding capacity,cholesterol adsorption capacity,polyphenol adsorption capacity and metal ion adsorption capacity of Poria polysaccharides were determined by adsorption experiment.The antioxidant activity of Poria polysaccharides were evaluated in vitro by total reducing capacity,DPPH·scavenging capacity and·OH scavenging capacity.This experiment was mainly intended to assess the application value of Poria polysaccharides from the perspective of dietary fiber.3.Study on simulated digestion and intestinal flora fermentation of Poria polysaccharides in vitro.The simulated digestion model of stomach and small intestine fluid was established to observe the release of monosaccharides and the changes of antioxidant capacity of digestive fluid.The residue of simulated digestive fluid after dialysis was mixed with healthy human feces for fermentation,and the effect of digestive residues of polysaccharides on intestinal flora were observed by 16S r RNA three-generation gene sequencing technology.The effect of polysaccharides on short-chain fatty acids（SCFAs）production in intestinal flora fermentation were observed by gas chromatography.【Results】1.Extraction,modification and characterization of Poria polysaccharides.The functional group region of IR spectra showed that WSP,ASP-9,ASP-11 and CMP had two characteristic absorption peaks of polysaccharide,namely O-H stretching vibration peak between 3300-3500cm^-1and C-H asymmetric stretching vibration peak near 2900 cm^-1,which determined preliminarily that the four samples prepared were polysaccharides.The fingerprint region of IR spectra showed that there was almost no difference between ASP-9 and ASP-11,indicating that ASP-9 and ASP-11 may be the same polysaccharide with different molecular weight,but the difference of WSP,ASP and CMP fingerprint region indicated that the molecular structure of different polysaccharides was different.¹³C NMR spectra showed that the anomeric carbon chemical shift of the four kinds of Poria polysaccharide was 100-106 ppm,which was determined to containβanomeric carbon.ASP-9,ASP-11 and CMP all had only one signal value between 95 and 110 ppm,which was determined to be homopolysaccharide,which was speculated to beβ-glucan,while WSP had multiple signal values,which was determined to be heteropolysaccharide.The peak number of CMP increased compared with the modified preform ASP-11.Carboxymethyl carbon signal appeared at 178 ppm and-CH₂signal appeared at 57.34ppm,indicating that carboxymethylation was successfully modified,and the degree of substitution was 1.3.¹H NMR spectra showed that the spectrograms of the four samples were characteristic spectrogram of typical polysaccharide characteristics.2.Comparison of adsorption characteristics and antioxidant activity of Poria polysaccharides in vitro.The adsorption results showed that the oil holding capacity was in the order of ASP-9>ASP-11>WSP>CMP;The cholesterol adsorption capacity was in the order of ASP-9>ASP-11>CMP>WSP;The adsorption capacity on gallic acid and chlorogenic acid was in the order of ASP-9>ASP-11>CMP>WSP,and that on EGCG and tea polyphenols was in the order of ASP-11>ASP-9>WSP>CMP;The Cd²⁺and Zn²⁺adsorption capacity was in the order of ASP-9>ASP-11>CMP>WSP,and Mg²⁺adsorption capacity was in the order of WSP>CMP>ASP-11>ASP-9.In conclusion,the adsorption capacity of water-insoluble ASPs on vegetable oil,cholesterol,polyphenols and heavy metal ions(Cd²⁺/Zn²⁺)was superior to that of the water-soluble polysaccharides WSP and CMP.The experimental results of antioxidant capacity showed that the total reducing capacity,DPPH·scavenging capacity and·OH scavenging capacity of WSP were superior to those of the other three polysaccharides samples,followed by CMP,and ASPs was weak.The antioxidant capacity of all Poria polysaccharides increased with concentration,showing an obvious dose-effect relationship,and WSP showed the strongest dose-effect relationship of antioxidant capacity,followed by CMP,ASP-9 and ASP-11,which exhibited the same rule as the antioxidant capacity of polysaccharides.3.Simulated digestion and intestinal flora fermentation of Poria polysaccharides in vitro.The results showed that WSP had a large amount of reduced sugar release during the simulated digestion of stomach and small intestine fluid,while the amount of reduced sugar release of ASP-9,ASP-11 and CMP was very low,indicating that WSP was partially digested,while ASP and CMP were difficult to digest.In the whole digestion stage,the antioxidant capacity of WSP digestive fluid was the highest,followed by CMP,and ASP-9 and ASP-11 were both very low,which was consistent with the rule of in vitro antioxidant capacity of polysaccharides.The results of intestinal flora fermentation experiment showed that ASP-11 and ASP-9 could significantly increase the richness and diversity of intestinal microorganisms,while WSP and CMP had little effect on it.From phylum level,all Poria polysaccharides could promote the growth of Firmicutes,and CMP was also beneficial to the growth of Bacteroidetes.ASP-9,ASP-11 and CMP all inhibited the growth of Proteobacteria.From genus level,ASP-9,ASP-11and CMP all promoted the growth of Clostridium,and the effect of the two kinds of ASP was more obvious.CMP could promote the growth of beneficial bacteria Parabacteroides.All Poria polysaccharides could inhibit the growth of Klebsiella,and the inhibitory effects of WSP and CMP were better than those ASPs.ASP-9,ASP-11 and CMP could inhibit the growth of Escherichia,but ASP had better inhibitory effect.From species level,ASP-9,ASP-11 and CMP all promoted the growth of beneficial bacteria Enterococcus faecium,and ASP-11 had a better effect.CMP could promote the growth of beneficial bacteria Parabacteroides distasonis.WSP can promote the growth of beneficial bacteria Weissella confusa.All Poria polysaccharides could inhibit the growth of opportunistic pathogen Klebsiella pneumoniae,and the inhibitory effects of WSP and CMP were better than those of the two kinds of ASP.ASP-9,ASP-11 and CMP could inhibit the growth of opportunistic pathogen Escherichia coli,but ASP had better inhibitory effect.The results of SCFAs content detection of intestinal flora fermentation showed that,compared with blank control group,all Poria polysaccharides could promote the production of SCFAs by intestinal microorganisms,but there were differences in the of SCFAs among all groups.The production content of acetic acid and propionic acid was in the order of WSP>CMP>ASP-9>ASP-11.The production content of butyric acid was in the order of CMP>ASP-11>ASP-9>WSP.The different strain Megasphaera elsdenii was significantly positively correlated with propionic acid.Parabacteroides distasonis,Bacteroides thetaiotaomicron and Eubacterium eligens were positively correlated with butyric acid.Klebsiella pneumoniae was negatively correlated with both acetic acid and propionic acid.【Discussion】WSP,ASP-9,ASP-11 and CMP were successfully prepared by IR,¹³C NMR and¹H NMR characterization.The adsorption capacity of the two water-insoluble ASPs for vegetable oil,cholesterol,polyphenols and heavy metal ions(Cd²⁺/Zn²⁺)was better than that of the water-soluble WSP and CMP.The antioxidant capacity of water-soluble WSP and CMP was better than that of water-insoluble ASP in vitro.All Poria polysaccharides could affect the structure of intestinal flora,and improve the richness and diversity of intestinal flora in order of ASP>WSP>CMP.ASP and CMP were significantly superior to WSP in promoting probiotics and inhibiting harmful bacteria.All poria polysaccharides could promote the production of SCFAs,suggesting that ASP and CMP could be used as potential prebiotics to regulate the homeostasis of intestinal flora.