Extraction,isolation Of Grifola Frondosa Polysaccharide And Its Effect On Intestinal Flora And Application

Grifola frondosa,as a medicinal and edible fungus,has attracted much attention due to its good taste and rich nutrition.Grifola frondosa contains a variety of bioactive substances,thus possessing a good development prospect as a raw material for functional foods.Polysaccharide is one of the main components and has been proved to have a series of biological activities.In this thesis,the extraction rates of crude polysaccharide from Grifola frondosa by hot water extraction and Vc/H₂O₂ assisted method were compared,and the method with higher polysaccharide yield was selected for optimization.Grifola frondosa polysaccharide（GFP）was obtained from crude polysaccharide by Vc/H₂O₂ assisted extraction after protein removal;two fractions of GFP（GFP-H,GFP-L）were obtained by separation with ultrafiltration.The physicochemical properties of GFP、GFP-H、GFP-L were measured and their structures were characterized,their effects on human intestinal flora were studied.Finally,a healthy food product was developed using Grifola frondosa water extract as a raw material.The main research contents and corresponding results are as follows:1.The extraction rates of crude polysaccharide from Grifola frondosa were compared between hot water extraction method and Vc/H₂O₂ assisted extraction method.The yield of the two methods was 10.53%and 14.77%respectively.Compared with hot water method,Vc/H₂O₂assisted method had advantages of higher polysaccharide yield and lower molecular weight of the polysaccharides.Single factor experiments were carried out to optimize processing parameter of Vc/H₂O₂ assisted method.The optimal extraction conditions are as follows:temperature 95℃,Vc/H₂O₂ concentration 20 m M.Under these optimal conditions,the yield of crude polysaccharide was 15.83%,total sugar content was 61.26%.2.The molecular weight distribution of GFP was 433.7,15.5 and 3.9 k Da measured by HPGPC.The larger molecular weight fraction GFP-H and smaller molecular weight fraction GFP-L were separated by ultrafiltration（Mw cut-off 100 k Da）.Molecular weight of the major component of GFP-H was 396.4 k Da,and the molecular weight distribution of GFP-L was38.6 k Da and 12.5 k Da.The total sugar content of GFP,GFP-H and GFP-L were 72.25%,62.35%and 64.61%respectively.Three polysaccharides contained small amounts of protein,sulfate group and uronic acid.The monosaccharide composition assays showed that GFP,GFP-H and GFP-L were all mainly composed of glucose,galactose,mannose and fucose.3.IR spectra analysis showed that three polysaccharides contained pyranose rings.NMR spectra analysis indicated that both GFP-H and GFP-L hadα-andβ-type glycosidic bonds.Based on NMR,monosaccharide composition and methylation analysis,it is inferred that the main chain of GFP-H might be composed of（1→4）-β-Glcp and（1→6）-β-Glcp,and the branch chain may contain（1→3,6）-α-Glcp andα-T-Glcp sugar units;The main chain of GFP-L includes（1→4）-β-Glcp,the branch chain might be connected byα-T-Glcp and（1→3,6）-α-Manp.The rheological property analysis showed that GFP,GFP-H and GFP-L had good viscoelasticity.Thermogravimetric analysis and differential thermal analysis showed that the thermal stability of GFP,GFP-H and GFP-L were different,but they all had good thermal stability.4.To study the effects of GFP and its fractions on the metabolites of intestinal flora,in vitro human intestinal fermentation model was constructed.The results illustrated that compared with blank group,the GFP,GFP-H and GFP-L groups all down-regulated the production of CH₄,NH₃ and H₂S and up-regulated for CO₂.The degradation rates of GFP,GFP-H and GFP-L were 63.43%,40.18%and 28.32%respectively.The content of short-chain fatty acids（SCFAs）after fermentation was determined by gas chromatography.Compared with the blank group,the total SCFAs content increased in the experimental group.The acetic acid content increased significantly in the GFP group compared with the blank group（p<0.05）,and the propionic acid content increased significantly in the GFP-L group compared with the FOS group（p<0.05）5.The intestinal flora composition after fermentation was analyzed by Illumina system.Species richness and diversity were reduced in the experimental groups compared with blank group.Analysis of microbial composition revealed that GFP,GFP-H and GFP-L had different abilities to regulate the intestinal flora.GFP and GFP-H increased the relative abundance of Firmicutes and Actinobacteriota,while GFP-L increased the relative abundance of Bacteroidota.GFP,GFP-H and GFP-L could reduce the relative abundance of Escherichia coli.GFP and GFP-H could promote the proliferation of Bifidobacterium;GFP-L could promote the proliferation of Bacteroides.GFP,GFP-H and GFP-L had significant enrichment effect on Lactobacillus and Lactococcus.GFP and FOS had similar enrichment ability to Lactobacillus.Therefore,GFP and its fractions could promote the growth of probiotics during in vitro fermentation,and were beneficial to the health of intestinal flora.6.A novel Grifola frondosa solid beverage was developed.To improve the flavor of Grifola frondosa complex beverage,the extract of Grifola frondosa was mixed with kiwi juice to make complex solution,and the ingredients such as sugar,citric acid and maltodextrin were added.The solid beverage was prepared using microencapsulation technology by spray drying.Through single factor and response surface experiments,the optimal formulation was ascertained as:white granulated sugar addition,55.8%of solid content;citric acid addition,0.8%of solid content;and maltodextrin addition,24.8%of solid content.The best sensory evaluation was obtained for the solid beverage based on this formulation.