Study On Submerged Fermentation Of T. Aurantialba Based On Inhibition Of Non-enzymatic Glycosylation And Preliminary Analysis Of The Active Substance

The non-enzymatic glycosylation reaction is one of main factors causing diabetescomplications, therefore inhibition of non-enzymatic glycosylation reaction becomes aneffective way to control the complications of diabetes. In this thesis, the inhibitory rate byTremella aurantialba culture broth was enhanced by the optimization of medium compositionand culture conditions. At the same time, the extraction conditions and structure ofpolysaccharide and other small molecular weight were determined and analysed preliminary.The main factors influencing the rate of non-enzymatic glycosylation reaction werecarbon and nitrogen sources. In the meantime, the corn meal and bran all could obviouslyimprove the inhibition rate of non-enzymatic glycosylation reaction. Howere, inorganic saltsplayed little role of enhancing the inhibition rate. The culture medium optimized byorthogonal experiments was （g/L）: glucose10, peptone5, corn cob powder20, wheat bran15,CoCl₂0.5, MnSO₄0.5. The optimized culture conditions were25oC, initial pH of6.5,150mL medium in500mL flask and10%（v/v） inoculums. After optimization, the inhibitory rateof non-enzymatic glycosylation reaction achieved89.74%.The active substance was relatively stable on temperature and the inhibition rate offermentation broth boiled by water did not change. The T. aurantialba culture broth waspurified and the main components were analyzed. The result indicated that bothpolysaccharide and other polar substances in culture broth were responsible for the inhibition.Crude polysaccharide that inhibited non-enzymatic glycosylation reaction was extractedby alcohol precipitation and purified by DEAE-cellulose column and gelfiltrationchromatography. Its average molecular weight was1.37×10⁶Da determined by HPGPC.Monosaccharide composition analysis indicated that it was composed of fucose, arabinose,xylose, mannose, glucose and galactose in a molar ratio of0.22:0.13:2.42:10.57:1.00:0.07.FTIR and NMR revealed the typical characteristics of pyranose and the typical peak of α andβ-glycosidic bond. The results of Periodate oxidation and Smith degradation indicated thatstructure of Polysaccharide contained1â†'3,1â†'4and1â†'6glycosidic bond or branch endgroup. Enzymolysis experiments further proofed that the β-1,4glycosidic bond existence.The back bond of polysaccharide was mainly constituted by mannose and mainly contained1â†'3glycosidic bond.Finally, the small molecular weight substances were isolated by macroporous resin, C-18column, and preparative liquid, and the structure of separation substances were analysedpreliminarily using LC-MS. The T. aurantialba fermentation broth may produceoligosaccharide, glycoside or alkaloids inhibiting non-enzymatic glycosylation reaction.