Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/3657
標題: 藥用菇菌—豬苓液態醱酵培養菌絲體與胞外多醣生產之探討
Studies on mycelia growth and exopolysaccharide production in submerged culture of medicinal mushroom-Grifola umbellata
作者: 黃宏彰
Huang, Hung-Chang
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: [1] 水野卓和川和正允, 菇類的化學、生化學. 國立編譯館. (1997) 38. [2] 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. [3] 賴進此, 菇類機能性成分的分離與純化. 食品工業, 5. (2002) 36-48. [4] 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. [5] 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. [6] 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. [7] Ukai, S., T. Kiho, C. Hara, I. Kuruma, and T. Y, Polysaccharide in fungi. Anti-inflammatory effect of the polysaccharides from the fruit bodues of several fungi. J Pharmacobiodyn, 6. (1983) 983-990. [8] Hetland, G., N. Ohno, I.S. Aaberge, and M. Lovik, Protective effect of ?glucan against systemic Streptococcus pneumoniae infection in mice. FEMS Immunol Med Mic, 27. (2000) 111-116. [9] Kim, S.W., H.J. Hwang, J.P. Park, Y.J. Cho, C.H. Song, and J.W. Yun, Mycelial growth and exo-biopolymer production by submerged culture of various edible mushrooms under different media. Lett Appl Microbiol, 34. (2002)1 56-61. [10] Zhang, G.L., Y.H. Wang, W. Ni, H.L. Teng, and Z.B. Lin, Hepatoprotective role of Ganoderma lucidum polysaccharide against BCG-induced immune Liver injury in mice. World J Gastroentero, 8. (2002) 728-733. [11] 王伯徹和陳啟楨, 常見食藥用菇類介紹. 食品工業. (1994) 25-26. [12] 王林麗、吳寒寅和羅桂芳, 豬苓的藥理作用及臨床應用. 中國藥業, 9. (2000) 58-59. [13] 王培銘和羅國仁, 豬苓液態培養技術之開發與應用. 食品工業, 35. (2003) 22-28. [14] 吳琇卿, 豬苓多糖及併用WR-2721對輻射與化學性遺傳損傷的防護作用研究. 中國醫藥學院, 博士論文. (2003). [15] Miyazaki, T., N. Oikawa, H. Yamada, and T. Yadomae, Structural examination of antitumour, water-soluble glucans from Grifora umbellata by use of four type of glucanase. Carbohydr. Res., 65. (1978) 235-241. [16] Ueno, Y., Y. Okamoto, R. Yamauchi, and K. Kato, An antitumor avtivity of the alkali-soluble polysaccharide ( and its derivatives) obtained from the sclerotia of Grifora umbellata (Fr.) Plite. Carbohydr. Res., 101. (1982) 160-167. [17] You, J.S., D.M. Hau, K.T. Chen, and H.F. Huang, Combined effects of chuling (Polyporus umbellatus) extract and mitomycin C on experimental liver cancer. Am. J. Chin. Med., 22. (1994)1 19-28. [18] Ohsawa, T., M. Yukawa, C. Takao, M. Murayama, and H. Bando, Studies on constituents of fruit body of Polyporus umbellatus and their cytotoxic activity. Chem. Pharm. Bull., 40. (1992)1 143-7. [19] Inaoka, Y., A. Shakuyo, H. Fukazawa, H. Ishida, H. Nukaya, K. Tsuji, H. Kuroda, M. Okada, M. Fukushima, and T. Kosuge, Studies on active substance in herbs udes for hair treatment. I. effect of herb extract on hair growth and isolation of an active substance from Polyporus umbellatus F. Biol. Pharm. Bull., 42. (1994) 530-533. [20] Ishida, H., Y. Inaoka, J. Shibatani, M. Fukushima, and K. Tsuji, Studies of the active substances in herbs used for hair treatment. II. Isolation of hair regrowth substances, acetosyringone and polyporusterone A and B, from Polyporus umbellatus Fries. Biol. Pharm. Bull., 22. (1999)11 1189-92. [21] Sekiya, N., H. Hikiami, Y. Nakai, I. Sakakibara, K. Nozaki, K. Kouta, Y. Shimada, and K. Terasawa, Inhibitory effects of triterpenes isolated from Chuling (Polyporus umbellatus Fries) on free radical-induced lysis of red blood cells. Biol. Pharm. Bull., 28. (2005)5 817-21. [22] 王秋穎和徐錦堂, 蜜環菌發酵液在豬苓菌發酵過程中的應用. 中國藥學雜誌, 28. (1993) 466-468. [23] 王秋穎和徐錦堂, 假單抱桿菌、靈芝等幾種菌的發酵液對豬苓菌生長的影響. 中國藥學雜誌, 32. (1997) 459-461. [24] 周曉燕、願順明、張文玉和許周善, 發酵法生產豬芥菌絲體及豬苓多醣的研究. 工業微生物, 31. (2001) 1-4. [25] 願芳紅、殷紅和馬勁, 碳、氮源對豬苓菌絲生長與胞外多醣含量的影響. 西北大學學報, 31. (2001) 437-440. [26] 鄧百方和陳文強, 豬苓菌絲液體培養及富硒研究. 中國馴庰? 22. (2003) 33-34. [27] 陳文強和鄧百方, 豬苓菌絲液體培養及富鋅研究. 中國馴庰? 10. (2003) 25-27. [28] 陳文強、鄧百方和張娟, 豬苓液體培養基篩選研究. 胺基酸和生物資源, 24. (2002) 23-24. [29] Brome, C.A., Physical and chemical methods of sugar analysis. (1948) 895. [30] Reese, E.T. and M. Mandels, Methods in carbohydreate chemistry. New York: Academic. (1963) 139. [31] Nelson, N.A., Photometric adaptation of the somogyi method for the determination of glucose. J. Biol. Chem., 153. (1944) 375. [32] Dubois, M., K.A. Gilles, J.K. Hamilton, P.A. Rebers, and F. Smith, Colorimetric method for determination of sugars and related substances. Anal. Chem., 28. (1956) 350-356. [33] Marshall, R.D., A. Neuberger, and Gottschalk, Amsterdam. Elsevier Publishing Company. (1972) 283-290. [34] Papadopoulas, N.M. and W.C. Hess, Determination of neuraminic (sialic) acid, glucose and fructose is spinal fluid. Archives of Biochemistry and Biophysics, 88. (1960) 167. [35] Fang, Q.H., Y.J. Tang, and J.J. Zhong, Significance of inoculation density control in production of polysaccharide and ganoderic acid by submerged culture of Ganoderma lucidum. Process Biochem, 37. (2002) 1375-1379. [36] Wang, Q.Y., S.X. Guo, J.Y. Fan, and M. Xue, Characterization of Sclerotial formation from hyphae of Grifola umbellata. Acta. Bot. Sin., 46. (2004) 328-331. [37] Fang, Q.H. and J.J. Zhong, Submerged fermentation of higher fungus Ganoderma lucidum for production of valuable bioactive metabolites-ganideric acid and polysaccharide. Biochem Eng J, 10. (2002) 61-65. [38] Xiaoke, X. and G. Shunxing, Morphological characteristics of sclerotia formed from hyphae of Grifola umbellata under artificial conditions. Mycopathologia, 159. (2005)4 583-90. [39] Yao, H. and J.J. Zhong, Improved production of ginseng pokysaccharide by adding conditioned medium to Panax notoginseng cell cultures. Biotechnol. Lett., 13. (1999) 347-349. [40] 許慶波、李太元、李豔茹、梁運江和金慶日, 液態淺層培養對豬苓菌絲體生長及其多醣含量之影響. 吉林農業大學學報, 26. (2004) 287-290. [41] Zhong, J.J. and Y.J. Tang, Submerged cultivation of medicinal mushrooms for production of valuable bioactive metabolites. Adv. Biochem. Eng. Biotechnol., 87. (2004) 25-59. [42] Huang, H.C. and Y.C. Liu, Acceleration of mycelia growth by adding grifola umbellata broth concentrate in soild-state and submerged cultures. Biochem Eng J, 37. (2007) 139-143. [43] Yang, F.C. and C.B. Liau, The influence of environmental conditions on polysaccharide formation by Ganoderma lucidum in submerged culture. Process Biochem., 33. (1998) 547-553. [44] Wu, J.Z., P.C.K. Cheung, K.H. Wong, and N.L. Huang, Studies on submerged fermentation of Pleurotus tuber-regium (Fr.). Food Chem, 81. (2003) 389-393. [45] Lee, K.C., J.T. Bae, H.B. Pyo, T.B. Choe, S.W. Kim, H.J. Hwang, and J.W. Yun, Submerged culture conditions for the production of mecelial biomass and exopolysaccharides by the edible Basidiomycete Grifola frondosa. Enzyme Microb. Technol., 35. (2004) 369-376. [46] Liu, G.Q. and X.L. Wang, optimization of critical medium components using response surface methodology fro biomass and extracellular polysaccharide production by Agaricus blazei. Appl Microbiol Biotechnol, 74. (2007) 78-83. [47] Tang, Y.J. and J.J. Zhong, Fed-batch fermentation of Ganoderma lucidum for hyperproduction of polysaccharide and ganoderic acid. Enzyme Microb Technol, 31. (2002) 20-28. [48] Shih, I., K. Pan, and C. Hsieh, Inference of nutritional components and oxygen supply on the mycelial growth and bioactive metabolites production in submerged culture of Antrodia cinnamomea. Process Biochem, 41. (2006) 1129-1135. [49] Chang, M.Y., G.J. Tsai, and H.J. Y., Optimization of the medium composition for the submerged culture of Ganoderma lucidum by Taguchi array design and steepest ascent method. Enzyme Microb Technol, 38. (2006) 407-414. [50] Burn, P.J., P. Yeo, T. Keshevarz, S. Roller, and C.S. Evans, Physiological studies of exopolysaccharide production from basidiomycetes Pleurotus sp. Florida; effect of C and N sources on polysaccharide production for potential as a hypocholesterolemic, antimutor and a fat mimetic. Enzyme Microb Technol, 34. (1994) 566-572. [51] Shu, C.Y. and M.Y. Lung, Effect of pH on the production and molecular weight distribution of exopolysaccharide by Antrodia camphorata in batch cultures. Process Biochem., 39. (2004) 931-937. [52] Lee, K.M., S.Y. Lee, and H.Y. Lee, Bistage control of pH for improving exopolysaccharide production from mycelia of Ganoderma lucidum in an air-lift fermentor. J Biosci Bioeng, 88. (1999)6 646-50. [53] Fang, Q.H. and K.C. Zhang, Effect of initial pH on production of ganoderic acid and polysaccharide by submerged fermentation of Ganoderma lucidum. Process Biochem, 37. (2002) 769-774. [54] Souw, P. and A.L. Demain, Role od citrate in xanthan production production by Xamthomonas campestris. J. Ferment. Technol., 58. (1980) 411-416. [55] Haq, I., S. Ali, M.A. Qadeer, and J. Iqbal, Stimulatory effect of alcohols on citric acid productivity by a 2-deoxy D-glucose resistant culture of Aspergillus niger GCB-47. Bioresource Technol., 86. (2003) 227-233. [56] Silva, C.C., R.F. Dekker, R.S.S. Silva, M.L. Corradi da Silva, and A.M. Barbosa, Effect of soybean oil and Tween 80 on the production of botryosphaeran by Botryophaeria rhodina MAMB-05. Process Biochem., 42. (2007) 1254-1258. [57] Hsieh, C., H.L. Wang, C.C. Chen, T.H. Hsu, and M.H. Tseng, Effect of plant oil and surfacant on the production of mycelial biomass and polysaccharides in submerged culture of Grifola frondosa. Biochem. Eng. J. (2007). [58] Lim, J.M. and J.W. Yun, Enhanced production of exopolysaccharides by supplementation of toluene in submerged culture of an edible mushroom Collybia maculata TG1. Process Biochem, 41. (2006) 1620-1626. [59] Shou, C.H., Y.C. Chen, and Y.C. Hsu, Effects of citric acid on cell growth and schizophyllan formation in submerged culture of Schizophyllum commune. J. Chin. Chem. Engrs., 33. (2002) 315-325. [60] Shu, C.Y., M.Y. Lung, and X.C. J., Effect of sopplementation of succinic acid on the production and molecular weight discribution of exopolysaccharides by Antrodia camphorata in batch culture. J. Chem. Technol. Biotechnol., 80. (2005) 216-222. [61] Yang, H.L., T.X. Wu, and K.C. Zhang, Enhancement of mycelial growth and polysaccharide production in Ganoderma lucidum (the Chinese medicinal fungus, ''Lingzhi'') by the addition of ethanol. Biotechnol. Lett., 26. (2004)10 841-4. [62] Yang, F., Y. Ke, and S. Kuo, Effect of fatty acids on the mycelial growth and polysaccharide formation by Ganoderma lucidum in shake flask cultures. Enzyme Microb Technol, 27. (2000)3-5 295-301. [63] Park, J.P., S.W. Kim, H.J. Hwang, Y.J. Cho, and J.W. Yun, Stimulatory effect of plant oils and fatty acids on the exo-biopolymers production in Cordyceps militaris. Enzyme Microb Technol, 31. (2002) 250-255. [64] Hsieh, C., C.J. Liu, M.H. Tseng, C.T. Lo, and Y.C. Yang, Effect of olive oil on the production of mycelial biomass and polysaccharides of Grifola frondosa under high oxygen concentration aeration. Enzyme Microb. Technol., 39. (2006) 434-439. [65] Katic, M., J. Frantar, I. Grgic, H. Podgornik, and A. Perdih, Polyethylene stimulates lignin peroxidase production in Phanerochaete chrysosporium. Folia Mocrobiol., 43. (1998) 631-634. [66] Grgic, I. and A. Perdih, Stimulation of ligninolytic enzyme production in Phanerochaete chrysosporium by polyoxyalkanes. J. Appl. Microbiol., 94. (2003) 360-368. [67] Kos, N. and A. Perdih, Polyoxirane distribution in a Phanerochate chrysosporium culture. Filia Microbiol., 44. (1999) 527-529. [68] Wu, J., Q. Ruan, and H.Y.P. Lam, Effect of surface active medium additive on insert cell surface hydrophibicity relating to cell protection against bubble demage. Enzyme Microb Technol, 21. (1997) 341-348. [69] Dlamini, A.M. and P.S. Peiris, Production of exopolysaccharide by Pseudomonas sp. ATCC31461 using whey as fermentation substrate. Appl Microbiol Biotechnol, 47. (1997) 52-57. [70] Lee, H.Y., M.K. Song, Y. Yu, and H.J. Hwang, Production of Ganoderma lucidum mycelium using cheese whey as an alternative substrate: response surface analysis and biokinetics. Biochem Eng J, 2003. (2003) 93-99. [71] Mukhopadhyay, R., B.P. Chatterjee, and A.K. Guha, Effect od nitrogenous sources on the growth of Pleurotus sajor-caju, an edible mushroom, grown on whey. Biochem Arch, 15. (1999) 393-398. [72] Dlamini, A.M. and P.S. Peiris, Biopolymers production by a Klebsiella oxytoca isolate using whey as fermentation substrate. Biotechnol Lett, 19. (1997) 127-130.
摘要: 
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。
本論文主要包含四個部分。第一部分,針對豬苓在液態醱酵培養過程中,提出以類似動植物培養之調整培養基概念,收集培養豬苓過後之黑褐色培養液(GBC),並將其添加入培養基中,觀察GBC對豬苓菌絲體生長之影響。研究結果顯示,於固態培養中添加GBC可促進豬苓菌絲體生長。在液態培養中添加GBC亦可促進豬苓菌絲體生長並縮短菌絲體生長之滯留期時間。最大菌體生成量可提升至12.78g/L,約為比較組的1.18倍。
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。

第四部分以廢棄物再利用的觀點,將液態醱酵之培養基以乳漿蛋白(whey)取代,評估其對豬苓液態醱酵培養菌絲體生長與多醣體生產之可行性。實驗結果顯示,以乳漿蛋白為醱酵培養豬苓所需之氮源基質,可成功地培養豬苓液態菌絲體生長與多醣體生產。

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.
URI: http://hdl.handle.net/11455/3657
其他識別: U0005-2901200814512200
Appears in Collections:化學工程學系所

Show full item record
 

Google ScholarTM

Check


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.