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Studies on mycelia growth and exopolysaccharide production in submerged culture of medicinal mushroom-Grifola umbellata
|關鍵字:||Grifola umbellata;豬苓;submerged culture;polysaccharide;polyethylene glycol;whey;液態培養;多醣體;聚乙二醇;乳漿蛋白||出版社:||化學工程學系所||引用:|| 水野卓和川和正允, 菇類的化學、生化學. 國立編譯館. (1997) 38.  杜巍、李元瑞和袁靜, 食藥用菌多糖生物活性與結構關係. 中國藥學, 21. (2002) 32-34.  賴進此, 菇類機能性成分的分離與純化. 食品工業, 5. (2002) 36-48.  Hikino, H., C. Kanno, Y. Mirin, and T. Hayashi, Isolation and hypoglycemic activity of ganoderans A and B, glycans of Ganodderma lucidum fruit bodies. Planta. Med., 4. (1985) 39-40.  Kim, D.H., B.K. Yang, S.C. Jeong, J.B. Park, S.P. Cho, S. Das, J.W. Yun, and C.H. Song, Production of hypoglycemic, extra cellular polysaccharide from the submerged culture of the mushroom, Phellinus linteus. Biotechnol. Lett., 23. (2001) 513-517.  Sone, Y., M. Kakuta, and A. Misaki, Isolation and characterization of polysaccharide of "Kikurage" fruit body of Auricularia auricula-judae. Agr. Biol. Chem. Tokyo, 42. (1978) 417-425.  Ukai, S., T. Kiho, C. Hara, I. Kuruma, and T. Y, Polysaccharide in fungi. 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豬苓Grifola umbellata 是中藥上常用的一種藥用真菌，在文獻上亦有別稱Polyporus umbellatus或Chuling。一般中藥使用的豬苓其實是菌核部分，有利尿滲濕、解熱止渴、消炎止瀉之功效。從野生豬苓分離出的一種水溶性多醣，具有良好的抗癌作用，這引起醫學界對豬苓研究的重視。目前豬苓的人工栽培還存在一定的困難，主要靠採挖野生資源供藥用，在豬苓的自然資源不斷減少下，其供求矛盾日趨突出。相較於液態培養方式則有培養週期較短、產品品質較穩定、易於商業化大規模生產等優點。因此，利用液態培養方式進行藥用真菌豬苓之生物活性物質的開發與應用是極值得進行的研究題材。
第二部分為培養條件對豬苓菌絲體生長與多醣體生產之探討。實驗結果顯示，葡萄糖為較適合豬苓菌絲體生長與多醣體生產之碳源。yeast extract與skim milk有利於豬苓菌絲體生長；然而，觀察胞外多醣生產發現，skim milk可更有效提升胞外多醣生產。培養液之初始pH值，以pH 6較有利於菌絲體生長，而pH 5則有利於胞外多醣之生產。最適培養豬苓生產胞外多醣體之培養基組成為glucose 3%, skim milk 0.2%, 0.1% KH2PO4, 0.1% MgSO4 7H2O, 0.005% Vitamin B1。使用Skim milk為氮源可得較高多醣體產率，比產率為112.35 mg/g,約為複合氮源的4.36 倍。
第三部分於豬苓液態醱酵培養過程中，分別使用不同添加劑（如：有機酸、醇類、植物油與高分子等）加入培養基中，探討添加劑與豬苓液態醱酵培養菌絲體生長與多醣體生產之影響，以期促進豬苓菌絲體生長與多醣體生產之效率。結果顯示，添加聚乙二醇(PEG)，可有效提升豬苓菌絲體生長與多醣體生產。PEG添加組，培養至第十四天可達最高菌絲體量，菌體量從5.236 g/l提升至至6.316 g/l，約為為未添加組的1.21倍。培養至第十二天可得最高多醣體生成量，產量從0.484 g/l增加至0.769 g/l，約為為未添加組的1.59倍。
Grifola umbellata is one of the most famous traditional Chinese medicinal mushrooms and has also been termed as Polyporus umbellatus or Chuling in scientific reports. The fruiting bodies of G. umbellata are commonly used for treating edema and promoting diuretic process. It has been reported that G. umbellata possesses outstanding anti-tumor effects. In light of its health benefits, G. umbellata of various compositions could be used as medicinal mushroom in the global market. Owing to inefficient protection, the wild sources of Grifola umbellata have been seriously depleted. To meet the mass demand of global market, it is necessary to cultivate the strain of Grifola umbellata under artificial conditions. However, the production of fruiting bodies of Grifola umbellata from field cultivation is a time-consuming and labor-intensive process. Meanwhile, the quality of G. umbellata under soil cultivation is difficult to control. Therefore, submerged fermentation for mycelia culture has recently received great interest as a promising alternative for efficient production. The study contains four parts.
In the first part, a brownish Grifola umbellata broth concentrate (GBC) harvested at the beginning of the growth phase was collected and used as an additive in the medium to investigate its effect on facilitating mycelia germination. The results showed that the addition of GBC in solid-state culture promoted mycelia growth. Moreover, the GBC addition in submerged culture significantly shortened the lag phase and the maximum mycelia production was enhanced by 18 %, from 10.8 g/l to 12.78 g/l. These results demonstrate that GBC is capable of stimulating Grifola umbellata growth in all respects.
In the second part, the effects of cultural condition on the mycelial growth and exopolysaccharide (EPS) production were studied. The results showed that glucose was the best carbon source for mycelia growth and EPS production. Yeast extract was the best nitrogen source for mycelia growth, whereas skim milk as nitrogen source can remarkably improve EPS production. The optimal medium constituents for EPS production were as follows: glucose 3%, skim milk 0.2%, 0.1% KH2PO4, 0.1% MgSO4、7H2O, 0.005% Vitamin B1. The initial pH value of 5 was the most efficient to EPS production. The Grifola umbellata culture with skim milk as nitrogen source displayed a much higher specific EPS yield of 112.35 mg/g, accounting for a 4.36 times increase compared to that with combined nitrogen source medium.
In the third part, several different additives were supplied in the cultural medium to find the one with the most stimulatory effect on G. umbellata mycelia growth and exopolysaccharide production. The results show that polyethylene glycol PEG (Mw 8000Da) is the most effective stimulator. With its addition, the maximum mycelia production at day 14 was enhanced from 5.236 g/l to 6.316 g/l, accounting for a 21% increase. Meanwhile, the exopolysaccharide production was enhanced from 0.484 g/l to 0.769 g/l at day 12, accounting for a 59 % increase.
Finally, a novel approach to utilizing whey, the cultivation of mycelial growth and exopolysaccharide production by medicinal mushroom Grifola umbellata is introduced. The result shows that whey could be an alternative substrate for mycelia growth and polysaccharide production. Therefore, cultivation of Grifola umbellata mycelia and exopolysaccharide production using whey can provide a unique solution to solve the dual problems of an alternative utilization of the whey and waste management.
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