利用乙醇劃分沈澱、超過濾與冷凍-解凍循環分離靈芝菌絲液態培養物中具分支之(1,3)-beta-D-葡萄聚醣

Abstract

靈芝多醣已證實具有抑制腫瘤、調節免疫及降血糖等生理功能，其中以具(1,6)-β-D-glucosyl分支的(1,3)-β-D-葡萄聚醣為靈芝熱水萃出物中最主要的活性多醣。本實驗欲利用此β-D-葡萄聚醣具「高分子量」及「分子間易互相聚集」的特性，以乙醇劃分沈澱、超過濾及冷凍-解凍循環處理來分離靈芝菌絲液態培養物內之(1,3)-β-D-葡萄聚醣，並探討不同處理的回收及純化效能。實驗中使用含有3.6% β-D-葡萄聚醣(乾基比例)之菌絲發酵物熱水萃取液為試驗溶液，結果顯示，上述三者處理間以乙醇劃分沈澱法之40%乙醇濃度操作組有最佳的效能，(1,3)-β-D-葡萄聚醣的回收率為68.3%，其含量占碳水化合物組成的93.2%。提高乙醇濃度雖可提高多醣及葡聚醣的回收量，但降低了組成中(1,3)-β-D-葡萄聚醣的含量。超過濾(阻隔分子量【molecular weight cutoff；MWCO】10kDa中空纖維管膜)及冷凍-解凍處理的β-D-葡聚醣回收率分別為73.5%及34.3%，含量占碳水化合物組成的35.3%及14.1%。組合超過濾膜與冷凍-解凍兩操作之回收率為65.3%，雖略低於單一超過濾處理，但可進一步提升組成含量至69.4% (以碳水化合物為基底)。

Polysaccharides from Ganoderma lucidum, a popular Traditional Chinese Medicine ingredient, have been associated with the antitumor, immuno-modulating and hypoglycemic activities of this mushroom. (1,3)-β-D-glucans bearing (1,6)-β-D glucosyl branches has been proved to be the major active component in the hot-water extractable polysaccharides. In this study, we employed ethanol fractional precipitation, ultra-filtration and freeze-thaw cycle techniques to separate the (1,3)-β-D-glucans from broth of mycelium liquid culture of G. lucidum. The separation was based on the properties of the (1,3)-β-D-glucans having larger molecular weight than other polysaccharides and having high tendency to aggregate. A freeze-dried powder containing 3.6% of (1,3)-β-D-glucans was used as raw material for this study. The results indicated that ethanol fractional precipitation adjusting ethanol concentration at 40% was the most efficient way, having 68.3% of recovery, among mentioned methods to purify (1,3)-β-D-glucans from a hot-water extract. The purity of the fractioned polysaccharides was 93.2% on carbohydrate basis. Increasing ethanol concentration increased both recovered quantity of the polysaccharides and the glucans but decreased the content of (1,3)-β-D-glucans. The operations of ultra-filtration using 10 kDa molecular weight cutoff (MWCO) hollow fiber membrane and freeze-thaw cycle recovered 73.5% and 34.3% of the (1,3)-β-D-glucans, respectively; the contents of the glucans were 35.3% and 14.1% on carbohydrate basis, respectively. Combination of ultra-filtration and freeze-thaw cycle could significant increase the content of (1,3)-β-D-glucans up to 69.4% with slightly lower recovery, 65.3%.