- 學位論文
金針菇熱水萃取物中beta-D-葡萄聚糖 與多酚氧化酶抑制劑之分離
Separation of beta-D-glucans and polyphenol oxidase inhibitor from hot-water extracts of golden needle mushroom, Flammulina velutipes
摘要
金針菇含有許多生理活性物質，包括具免疫調節活性之β-D-葡萄聚醣、真菌免疫調節蛋白質與多酚氧化抑制劑。由於β-D-葡萄聚醣、真菌免疫調節蛋白質與多酚氧化抑制劑之分子量分布依序由大至小，因此本論文利用中空纖維膜超過濾技術，連續序列劃分上述三類物質，所使用之超過濾膜之分子量截切範圍，由大至小分別為100kDa，10kDa與3kDa。結果顯示熱水萃取物中不能通過100kDa超過濾膜之滯留物主要均為β-D-葡萄聚醣，蛋白質主要分布於10kDa-100kDa之分子量截切範圍，多酚氧化抑制劑主要分布於分子量小於3kDa之區分。為暸解各劃分步驟之效率，並使用實驗室常用之乙醇沉澱劃分方式收集(1,3)-β-D-葡萄聚醣做為對照。本試驗以含6.8% (1,3)-β-D-葡萄聚醣之金針菇熱水萃取物為試驗溶液，結果顯示40%乙醇濃度與76%乙醇濃度操作時的(1,3)-β-D-葡萄聚醣回收率分別為85.0%及94.6%。以超過濾法10kDa中空纖維膜分離操作，產率為熱水萃取物之28.7%，其中(1,3)-β-D-葡萄聚醣的回收率可達95.1%，含量佔碳水化合物組成的96.6%，並進一步利用β-D-葡萄聚醣具「高分子量」及「分子間易相互聚集」的特性，組合10kDa超過濾搭配冷凍-解凍操作將β-D-葡萄聚醣形成一凝膠，凝膠產率是熱水萃取物的3.8％，凝膠中(1,3)-β-D-葡萄聚醣回收率為17.0％，純度提升為4.5倍。金針菇之萃取物及小分子量區分可顯著抑制蘋果泥之酵素褐變，有效抑制褐變濃度為熱水萃取物最終濃度在18.75mg/mL以上或3kDa以下區分物最終濃度12.5 mg/mL以上。
並列摘要
Flammulina velutipes, a popular edible mushroom contain many biological active substances including immuno-modulating β-D-glucans, fungal immuno-modulating proteins (FIP) and polyphenol oxidase (PPO) inhibitor. The molecular-weight distribution of the glucans, FIP and the PPO inhibitor are following the order from large to small. In this study we successfully used hollow-fiber ultra-filtration technique to enrich these three substances into fractions in succession. The sequence of molecular-weight cut-off (MWCO) values of ultra-filtration membrane was 100, 10 and 3kDa following the separation order. The results indicated that retentate of 100kDa MWCO was mainly β-D-glucans, proteins were dispersed in 10-100kDa MWCO range and the polyphenol oxidase inhibitor was in fraction of molecular weight smaller than 3kDa. To understand the efficiency of each separation steps, we used ethanol precipitate fractionation to collect (1, 3)-β-D-glucans as control. The hot-water extracts containing 6.8% of (1, 3)- β-D- glucans was used in this study. The results indicated that (1, 3)-β-D-glucans recovery of using 40% ethanol precipitation and 76% ethanol precipitation were 85.0% and 95.1%, respectively. The ultra-filtration separation using 10kDa MWCO could recover 28.7% of solid and 95.1% (1, 3)-β-D-glucans in hot-water extracts, respectively. A method combining freeze-thaw cycle has been developed to recover (1, 3)-β-D-glucans which is large in molecular weight and having highly tendency to aggregate. The combination method could recover 3.8% of solid and 17.0% of (1, 3)-β-D-glucans in hot-water extracts, respectively. The content of (1, 3)-β-D- glucans was enriched 4.5 folds. The polyphenol oxidase inhibitors were concentrated in the fraction which permeated the MWCO of 3kDa membrane. The fraction could significant inhibited enzymatic browning of apple puree. The effective concentration was 12.5 mg/mL comparing to the effective concentration 18.75mg/mL for hot-water extract.