Effects Of Aqueous Extract From Eleutherococcus Senticosus On EXOPOLYSACCHARIDES Fermentation Of Auricularia Auricular And The Possible Mechanism

Recent studies found that the addition of Eleutherococcus senticosus in the cultivation substrate of Auricularia auricular had an influence on the yield and the function of its fruit body polysaccharide. However, until now there has been no report about the effect of Eleutherococcus senticosus on the liquid fermentation of Auricularia auricular. Here, the effect of aqueous extract from Eleutherococcus senticosus on the fermentation yield, as well as the composition and bioactivity of the exopolysaccharide of Auricularia auricular HA-1 was studied. Moreover, the metabolic regulation mechanism of the extract on the biosynthesis of exopolysaccharide was explored. The main research contents were as follows:1. The effect of aqueous extract from Eleutherococcus senticosus and fermentation time on the production and properties of the exopolysaccharide of Auricularia auricular was studied. It was found that by the addition of aqueous extract from Eleutherococcus at 2.5 g/L,the highest yield of exopolysaccharide was reached as 7.60 g/L, the highest mole ratio of galactose and mannose was also reached as 15.34% and 15.21% in the monosaccharide composition, respectively. Furthermore, the obtained exopolysaccharide had the highest ABTS radical scavenging capacity as 27.79?mol Trolox/g. With the extended fermented time (0?9 d),the molecular weight distribution of the exopolysaccharide became homogeneous, and the ratio of galactose and mannose in the monosaccharide composition of the exopolysaccharide was also increased.2. The crude exopolysaccharides, produced from the fermentation with the addition of 2.5 g/L aqueous extract, were purified and characterized. After purification with DEAE-650 ion chromatography and Sephacryl 300 gel chromatography,two exopolysaccharides named CEPSN-land CEPSN-2 were obtained with the molecular weight of 4.6 and 6.7 kD.Besides the high content of glucose (mole ratio >97%), CEPSN-1had 0.4% galactose while CEPSN-2 had 1% galactose and 0.4% fucose in their monosaccharide composition. FT-IR spectra, Methylation reaction and GC-MS analysis showed that in the two exopolysaccharides glucose units existed as the galactopyranose type and their main chains were both consisted of 1,4,6-Glcp.3. The cytotoxicity and immune activity of CEPSN-1and CEPSN-2 were investigated.The two polysaccharides at 50-250 ?g/mL not only showed no toxicity to mice macrophage Raw 264.7 cells, but also could promoting the release of NO and cytokines (IL-6, IL-10 and TNF-a), and their promoting effect on the release of IL-6 and IL-10 was similar. The release of IL-6 and IL-10 reached the highest values under the polysaccharide concentration of 200 and 50 ?g/mL, respectively. (IL6: CEPSN-1 : 192.97 pg/mL, CEPSN-2: 226.96 pg/mL; IL10: CEPSN-1:192.51 pg/mL, CEPSN-2: 172.14pg/mL)?4. The metabolic regulation mechanism of the aqueous extract of Eleutherococcus senticosus on the biosynthesis of exopolysaccharide was explored. Using real-time PCR method, the expression level of three key genes (UDP-glucose-4-epimerase, hexokinase and phosphoglucomutase) in the biosynthesis of exopolysaccharide at the fermentation by the addition of 2.5 g/L extract was investigated. The result showed that under the stimulation of the aqueous extract, the expression of UDP-glucose-4-epimerase and hexokinase was up-regulated with the extended fermented time of 5?7 d,and reached the highest value at 7d, which was 1.95-fold and 1.77-fold of the control, respectively. While the expression of phosphoglucomutase gene was just up-regulated at 8 d. The result suggested that the aqueous extract of Eleutherococcus senticosus could accumulate the content of galactose and mannose in the exopolysaccharide synthesis.