| Tremella aurantialba,a kind of precious medicinal and edible fungus,which has the rich nutritional and wide range of pharmacological effects.The polysaccharide from T.aurantialba(TAP)is one of the main active ingredient,which has kinds of pharmacological activities,including anti-tumor,hypoglycemic activity,prevention and treatment of hyperlipidemia and antioxidant,and so on.At present,T.aurantialba fruiting bodies are utilized still in a low stage,mainly in the traditional direct edible sub-entities.Although many domestic and international scholars carried on a great deal of research to T.aurantialba polysaccharide,it is not in a deep-going way compared with other fungus polysaccharide.This study was basaed on the wild T.aurantialba fruiting bodies.Firstly,four methods,including hot water extraction,enzymatic extraction,ultrasonic-assisted extraction and microwave extraction,were used to extract polysaccharides from T.aurantialba fruiting bodies,and four kind of polysaccharide(named H-TAP、U-TAP、E-TAP and M-TAP,respectively)were obtained.Based on the yield of TAP and its antioxidant activity,the optimal extraction method were optimized.The cellulase has greatest impact by single-factor tests and orthogonal experimental design.The extraction conditions were optimized by orthogonal experimental design and box-behnken design,and we not only obtained the optimum cellulase-extraction conditions,but also preliminarily analyzed the polysaccharide structure from four different methods.Finally,the method stepwise alcohol precipitation was selecteded to separate and purify the polysaccharide TAP,and the structure features and antioxidant activities were analyzed.The main methods and results in this study are as follows:The sort of four polysaccharide yield from H-TAP,U-TAP,E-TAP and M-TAP and DPPH free radical removing ability both are H-TAP > U-TAP > E-TAP > M-TAP.Considering the polysaccharide yield and antioxidant activity,enzyme assisted extraction are optimum.Thus,enzyme extraction is the optimal method.Single-factor tests and orthogonal experimental design were used to optimized the multiplex-enzyme extraction conditions.Results showed that the enzyme sequence effecton polysaccharide yield is cellulase > pectinase > papain.Cellulase has the most significant effect on polysaccharide yield.Orthogonal experimental design were used to optimized the multiplex-enzyme extraction conditions.The results showed the order of enzyme influence on polysaccharide yield is temperature > reaction pH > reaction time > water to raw material ratio.On the basis of the above test results,the Box-Behnken response surface design was employed to optimize cellulase extraction conditions.The results showed the optimum conditions were enzymolysis temperature 50.93℃,enzymolysis time 60 min,water to raw material ratio 1:80(g/mL),enzyme dosage 6.7%(w/w),and pH 3.51.Under the optimized conditions,the yield of TAP was 25.96%.Chemical composition and structural features of four polysaccharides were analyzed by HPAEC-PAD and FT-IR spectra.They contain similar monosaccharide components(the order is: D-mannose > D-xylose > D-glucose),and all contain acidic polysaccharides containing D-glucuronic acid,but its mole ratio is obviously different.Three novel polysaccharide fractions,TAP30,TAP60 and TAP80,were isolated and purified by stepwise alcohol precipitation,and the their structural features and antioxidant activities were also analyzed by HPAEC-PAD and FT-IR spectra.The order of DPPH radical scavenging ability was TAP30 > TAP > TAP60 > TAP80,and the order of Hydroxyl radical scavenging ability was TAP30 > TAP60 > TAP > TAP80.Therefore,TAP30,TAP60 and TAP80,especially TAP30,might be a promosing and hopeful ingredient. |
