Studies On The Fermentation Process Optimization Of Sparassis Crispa And The Neuro-protective Activities Of Its Polysaccharides

Sparassis crispa, an edible mushroom with various medicinal properties, has been studied for years. As one of the main bioactive components, Sparassis crispa polysaccharides are reported to possess anti-tumor effects, antiallergic, etc.In this thesis, Sparassis Crispa was treated by nitrosoguanidine. A mutant, Sparassis Crispa SC031 with high genetic stability was obtained, whose desirability function value was 1.6-fold higher than that of the wild type Sparassis Crispa. The desirability value and chemometrics methods were also applied to optimize the fermentation medium of Sparassis Crispa SC031. Then the fermentation characteristic of Sparassis Crispa SC031 was studied. Optimum fermentation medium consisted of: 30.67 g/L sucrose, 10.88 g/L yeast extract powder, 10 g/L peptone, 2 g/L KH2PO4, 1.59 g/L MgSO4 and 0.10 g/L vitamin B1.The polysaccharide fractions were purified from Sparassis Crispa SC031 by DEAE-52 cellulose and sephadex G100 chromatography. Two main fractions, SCWEA and SCWEB, were obtained and the chemical characteristics were analyzed. The average molecular weights and partical size of the two fractions were 75 kDa, 184 kDa and 88.9 nm, 1012.2 nm respectively. SCWEA consisted primarily of rhamnose, mannose and galactose, and their ratios were 10: 2: 1. SCWEB largely consisted of glucose with a litte arabinose(20:1). Ultraviolet spectrum indicated the absence of protein and nucleic acid in SCWEA and SCWEB. Both FTIR spectrum of SCWEA and SCWEB displayed a polysaccharide characteristic absorption peak, such as hydroxyl groups, C-H groups, C=O groups, C-O-C groups and C-O-H groups. The analysis of periodate oxidation-smith showed that SCWEA was identified with a triple helix with(1â†'3)-linked Rha in the backbone, and(1â†'2) linkages and(1â†'6) linkages in the side bone, and SCWEB may be a(1â†'3)-beta-glucose.To explore the neuro-protection of SCWEA and SCWEB against the cell injury, the glutamate damaged PC12 cell model was used. MTT method, DCFH-DA staining, Fluo-4-AM staining, JC-1 staining and Western Blot were used to determine the changes of cell viability, intracellular ROS concentration, Ca2+ influx, mitochondrial membrane potential and the expression of Bcl-2, Bcl-x L,Akt, GSK and ect.. Morphological analysis was also involved to discover the 5zou’s effects on inducing PC12 cell differentiation, witnessing neuritogenesis and differentiation in PC12 cells. Results showed that both SCWEA and SCWEB enhanced the cell viability, reduced intracellular ROS level and Ca2+ influx, restored the mitochondrial membrane potential, and increased the expression of Bcl-2 and Bcl-x L. The study conformed that SCWEA and SCWEB have neuro-protective effects on neurotoxins-induced PC12 cells injury via mitochondria related pathway.