| China is the major producer and consumer of Auricularia auricula-judae(Bull.).The research on Auricularia auricula-judae(Bull.)is mainly concentrated in China,While the research is relatively rare in foreign countries rare.Most of the domestic studies have been conducted focused on chemical composition,structure,and physiological function,but the digestion of Auricularia auricula-judae(Bull.)have not been reported yet.In recent years,a large number of studies have shown that some polysaccharides can be degraded after digestion,and the physiological activities of the degraded polysaccharides may be changed accordingly.Therefore,this article simulates the digestive system of the gastrointestinal tract to digest the Auricularia auricula polysaccharide(AAP)in vitro.The composition,characterization structure and in vitro antioxidant activity of the product before and after digestion are explored by chemical analysis,liquid chromatography,infrared spectroscopy and gel chromatography.Also,phagocytic ability,NO content and the expression level of inflammation factor in RAW 264.7 are detected to estimate the changes in the immune activity of the AAP and d-AAP.Finally,real-time quantitative PCR and western blotting were applied to explore the effect of d-AAP on the mRNA and protein expression levels of RAW264.7 cells,and the following conclusions were obtained:(1)AAP is more sensitive to lower pH,so it can be quickly digested in the gastric juice without further degradation in the intestine juice.And in the process of digestion,the three branched sugars of glucuronic acid,fucose and rhamnose are separated from the main chain to form free monosaccharides;after digestion in the intestinal juice,the mannose is degraded to form mannose monosaccharide.(2)Compared with the undigested AAP,the composition of the d-AAP is changed.The reducing sugar increases,the uronic acid decreases,the protein increases and the fucose disappears in d-AAP.Also,the AAP with an average molecular weight of 1210 kD is degraded into two parts(825 kDa and 735 Da)after digestion.Finally,the three-stranded helix conformation in the AAP disappeared after digestion in vitro.(3)The d-AAP maintains good ABTS and DPPH free radical scavenging ability and total reducing power,but the antioxidant activity is significantly reduced compared with AAP.The same results also appears in the viability of superoxide dismutase and cell viability of RAW264.7 cells.(4)However,after examining the phagocytic ability,NO content and the expression level of IL-6 and TNF-?,it is found that the digestion product of d-AAP can stimulate the secretion of the above inflammatory factors and activate the RAW264.7 cells,which is significantly higher than undigested AAP.(5)The RAW264.7 cells was used to detect the mRNA and protein expression of inflammatory factors(IL-6,TNF-?)in signal pathways.It is indicated that by activating TLR4 receptor,the d-AAP can increase the phosphorylated expression of JNK and ERK proteins in RAW264.7 cells,and further activate the phosphorylated expression of NF-?B,which can stimulate the mRNA expression of inflammatory factors(IL-6,TNF-?)in cells,and finally increase the expression level of inflammatory factors.The expression of these inflammatory factors and the increase of macrophage activity will improve immune activity.In conclusion,after the digestion of AAP in the gastrointestinal juice,the composition and structural characterization of the AAP change,which lead to a decrease in antioxidant activity and an increase in immune activity. |
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