Depolymerization And Degradation Of Hyperbranched β-glucan And Its Application In Nano-carriers

Hyperbranched polysaccharides of edible and medicinal fungi not only have good water solubility,high dispersion,low viscosity,and many terminal hydroxyl groups for functional modification,but also have avariety of biological activites,such as hypoglycemia,hypolipidemia,antioxidant activity,and immune enhancement,and thus have received a lot of attention.Pleurotus tuber-regium hyperbranchedβ-glucan（PTR-HBG）is a kind of natural glucan with with a high branching degree（DB）up to 70%.Although the highly branched structure has various excellent properties,the excessive branches restrict the research of its structure-activity relationship,as well als further development and application.To address these issues,this study employed enzymatic hydrolysis method to depolymerize the PTR-HBG.According to the types of glycosidic bonds,relevant enzymes were selected to explore efficient and available depolymerization and degradation methods,and then analyze the optimum enzymatic hydrolysis conditions.The structure and morphology of samples were determined to elucidate and the enzymatic mechanism.On this basis,the in vitro antioxidative activity of PTR-HBG as well as its efficacy as a nanocarrier for bioactive substances were evaluated to set a foundation for an in-depth analysis of the structure-activity relationship between hyperbranched structure and function.The main results are as follows:（1）When the degradation effects of five different commercial enzymes on PTR-HBG were compared,it was found thatβ-glucosidase and cellulase had better degradation effects.The best degradation conditions were obtained as following:theβ-Glucosidase at the enzyme concentration of 2.5 U·m L^-1,the temperature of 50°C,the p H was 5.2,and the reaction time of90 min;Cellulase at the enzyme concentration of 20 U·m L^-1,the temperature of 40°C,the p H of 4.8,and the reaction time of 120 min,respectively.The enzymatic kinetics analysis showed that the K_m and V_max ofβ-glucosidase were 4.009μmol·L^-1 and 0.377 mmol·L^-1·min^-1,respectively,the K_m and V_max of cellulase were 8.054μmol·L^-1 and 0.523 mmol·L^-1·min^-1,respectively.（2）The depolymerized products ofβ-glucosidase（PGs）and cellulase（PCs）were analyzed from aspects of structure and morphology.The molecular weight（Mw）of PG and PC could be largely decreased to 3.98×10⁵ g·mo L^-1 and 2.74×10⁵ g·mo L^-1,respectively.The molar ratios of1,4-D-Glcp and 1,4,6-D-Glcp residues decreased in PCs,but the 1,6-D-Glcp residue slightly increased.In contrast,the molar ratio of 1,4-D-Glcp residues rose significantly in PGs.In addition,triple helical structures could be observed in PTR-HBG and PCs with DBs of 0.68and 0.69（PC2 and PC3）,respectively,whereas the triple helical structure of PGs vanished with DBs of at least 0.62.（3）The manufactured nanosystem was most stable when the mass ratio of glucan and zein was 1:1.The particle size and potential fluctuation range of PC3/zein were determined to be170 nm to 230 nm and-24.4 m V to-32.6 m V,respectively.Because PCs have less steric hindrance and a highly branched structure,they may better preserve zein nanoparticles,making PC3/zein the most p H stable.Curcumin（Cur）encapsulation rate was up to 83.25%when the mass ratio of zein to Cur was 1:0.017,while tocopherol（TOC）encapsulation rate was up to96.83%when the mass ratio of zein to TOC was 1:0.1.As a result,the PC3/zein nanocarrier system has good encapsulation effect on hydrophobic active small molecules.