Study On Sulfation Of Hyperbranched β-Glucans From Pleurotus Tuber-regium

Hyperbranchedβ-glucan（HBG）from the sclerotia of Pleurotus tuber-regium has a special highly branched structure with a degree of branching（DB）up to 70%.HBG not only has good water solubility,high dispersion,low viscosity,and many terminal hydroxyl groups,which are convenient for functional modification,but also has a variety of biological activities such as anti-tumor,anti-coagulation and enhancing human immunity,so it has attracted wide attention.The existing research on HBG is mainly concentrated on its isolation and purification,fermentation optimization with the aim of improving yield,but less on its activity development and application,thus limiting further research on its efficacy,so it is especially important to modify the structure and conformation of HBG to enhance its biological activity.To address the above issues,this study used the chlorosulfate-pyridine method（CSA-Pyr）to modify HBG by sulfation.The derived polysaccharides were structurally characterized and then performed on the structure-function relationship analysis.On this basis,the in vitro antioxidant activity,formation of the advanced glycation end-products（AGEs）and hypoglycemic activity of HBG were measured to provide a theoretical basis for expanding the use of fungal polysaccharides in the fields of functional food and medicine.The main findings of the study are as follows:The main findings are as follows:（1）The esterification agent ratios that could successfully sulfate the HBGs were screened by barium sulfate turbidimetry and FT-IR detection methods.The results showed that the effective ratios of esterification agent on S-HBGs were 1:2,1:2.5,1:3 and 1:3.5 to produce different degrees of substitution,molecular weights and microscopic conformations.Among them,S-HBG_1:2 exhibited the greatest molecular weight of 13.5×10⁵ g·mo L^-1,and S-HBG_1:2.5possessed the highest degree of substitution（DS）of 0.75.In addition,the substitution position of the-SO₃H group of S-HBGs was found to be at the C-6 position.（2）The structural and morphological characterization of S-HBGs showed that sulfated modification and polysaccharide degradation occurred simultaneously.The molecular weights（Mw）of S-HBGs were reduced in varying degrees.With the smallest DS,the Mw and particle size of S-HBG_1:3.5 reduced to 9.58×10⁵ g·mo L^-1 and 53.29 nm,respectively.In addition,the water solubility of S-HBGs was greatly improved relative to that of HBG.With the highest DS,water solubility of S-HBG_1:2.5 increased to 83.55%.However,the triple helix structure and branched structure of HBG were not significantly destroyed after sulfation modification.（3）The antioxidant activity of HBG was significantly increased after sulfation.S-HBG_1:2having the highest ABTS⁺radical scavenging ability and reducing power,while S-HBG_1:2.5having the highest DPPH scavenging ability of 78.73%.S-HBG_1:2（0.55）and S-HBG_1:3.5（0.27）with smaller DS showed the best inhibition of non-enzymatic glycosylation end-products（AGEs）.In addition,S-HBGs showed better inhibition ofα-amylase andα-glucosidase than HBG,and the inhibition effect was positively correlated with the degree of substitution,indicating that S-HBGs have a better hypoglycemic effect.（4）The inhibition mechanism of S-HBGs againstα-amylase was further investigated by the Dixon equation and Cornish-Bowden equation.S-HBG_1:2 and S-HBG_1:3.5 exhibited Mixed Type inhibition,and S-HBG_1:2.5 and S-HBG_1:3 exhibited competitive inhibition.The results of Lineweaver-Burk plotting method preliminarily showed that the inhibition type ofα-glucosidase by S-HBGs was relevant to the DS of S-HBGs,and the inhibition type showed a transition trend from competitive inhibition to Mixed Type inhibition with the decrease of DS,and a non-competitive inhibition when DS continued to decrease.The results of fluorescence quenching experiments and circular dichroism showed that the fluorescence of S-HBGs exhibited static quenching for both enzymes,and that S-HBGs had less effect on the secondary structure content of the enzymes and thus less destructive effect on the enzyme structure.