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1 生物工程学报 Chin J Biotech 2009, June 25; 25(6): journals.im.ac.cn Chinese Journal of Biotechnology ISSN cjb@im.ac.cn 2009 Institute of Microbiology, CAS & CSM, All rights reserved 工业生物技术刘高强 1, 丁重阳 2, 章克昌 2, 王晓玲 1, 韩文军 1, , : 采用液体深层发酵方式, 研究了几种药用昆虫对灵芝多糖生物合成的影响结果表明, 药用昆虫蜣螂在添加量为 5 g/l 时能显著促进灵芝胞内多糖 (IPS) 和胞外多糖 (EPS) 的形成 (P<0.05) 胞内多糖和胞外多糖的产量分别由对照的 (1.93 ± 0.09) g/l 和 (520.3 ± 20.2) mg/l 提高到 (2.41 ± 0.12) g/l 和 (608.9 ± 20.2) mg/l 灵芝胞内多糖和胞外多糖在 DEAE 纤维素柱上都可分离得到 5 种主要组分, 其中 IPS-1 和 EPS-1 分别为 2 类多糖的主要组分进一步用凝胶柱分离显示, IPS-1 由 3 个单个的组分组成, EPS-1 由 2 个单个的组分组成添加蜣螂发酵后, 灵芝胞内多糖和胞外多糖中没有出现新的组分, 且各组分的相对含量也没有显著变化 (P>0.05), 提示添加蜣螂发酵后, 灵芝胞内多糖和胞外多糖主要组分的合成途径并未改变 : 灵芝, 液体发酵, 胞内多糖, 胞外多糖, 药用昆虫 1 Effects of medicinal insect, Catharsius molossus on biosynthesis of polysaccharides from Ganoderma lucidum in submerged culture Gaoqiang Liu 1, Chongyang Ding 2, Kechang Zhang 2, Xiaoling Wang 1, and Wenjun Han 1 1 College of Life Science and Technology, Central South University of Forestry and Technology, Changsha , China 2 State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi , China Abstract: We studied the effects of several medicinal insects on biosynthesis of polysaccharides from Ganoderma lucidum in submerged culture. The results showed that the medicinal insect, Catharsius molossus at 5 g/l significantly promoted the biosynthesis of intracellular polysaccharides (IPS) and extracellular polysaccharides (EPS) of G. lucidum, and compared with control, IPS and EPS yields markedly enhanced from (1.93 ± 0.09) g/l to (2.41 ± 0.12) g/l and (520.3 ± 20.2) mg/l to (608.9 ± 20.2) mg/l, respectively (P < 0.05). Both IPS and EPS consisted of five kinds of components, and IPS-1 and EPS-1 were the major components of IPS and EPS, respectively. Further separation studies showed that IPS-1 was made up of three single compounds, while EPS-1 was made up of two single compounds. There were no new components in both IPS and EPS obtained from G. lucidum in submerged culture by the addition of the insect, C. molossus, suggesting the biosynthetic pathways of the major components of IPS and EPS had not been changed. Keywords: Ganoderma lucidum, submerged fermentation, intracellular polysaccharides, extracellular polysaccharides, medicinal insects Received: March 29, 2009; Accepted: April 29, 2009 Supported by: National Natural Science Foundation of China (No ). Corresponding author: Gaoqiang Liu. Tel: ; gaoliuedu@yahoo.com.cn 国家自然科学基金 (No ) 资助

2 : 915 [Ganodema 1ucidum (W. Curtis.: Fr.) P. Karst.], [1],, [1,2], [1 5],,, [6 12],, [6 12],,, ; [13],, / ( ),, 1 材料和方法 1.1 主要仪器 HYG2, ; UNICO2100 -, ; CR22G, ; BUCHI-R200, BUCHI ÄKTA Explorer( ) 1.2 药用昆虫 (Eupolyphaga sinensis Walker) (Aspongopus chinensis Dallas) (Mylabris phalerata Pall.) (Catharsius molossus) ; (Dendrolimus houi),, 灵芝培养 Ganoderma lucidum, (g/l): 30, 10, 10, 3, KH 2 PO 4 1.5, MgSO , VB ,, [80 : =1: 3(W/V)] : 24 h ; : 500 ml 140 ml : 30 C 170 r/min, 7 d 1.4 生物量的测定 40,, 2,, 60 C, 1.5 灵芝多糖的测定 : [8], 1 mol/l NaOH 60 C 1 h, 3, - [8,10] :, 2/5~3/5, 4 95%, 24 h, 1.6 多糖的组成分析 : DEAE [14] ÄKTA Explorer( ) : HiPrep 16/10 DEAE : mol/l NaCl, ( - ), [14] ; : 2 ml/min; : 5 ml/ ; 2 ml( 9~10 mg ) : DEAE, Superdex 200 HR 10/30, [14] : 0.1 mol/l NaCl; : 0.25 ml/min; : 1 ml/ [14] : -

916 ISSN1000-3061 CN11-1998/Q Chin J Biotech June 25, 2009 Vol.25 No.6 1.7 统计分析 3 Excel 2000 SAS 8.1 t 2 结果和讨论 2.1 不同昆虫粉对细胞生长的影响 1, 4 g/l, (P<0.01); ;, (CM) (ES) 5 g/l,, (520.3 ± 20.2) mg/l (608.

3 916 ISSN CN /Q Chin J Biotech June 25, 2009 Vol.25 No 统计分析 3 Excel 2000 SAS 8.1 t 2 结果和讨论 2.1 不同昆虫粉对细胞生长的影响 1, 4 g/l, (P<0.01); ;, (CM) (ES) 5 g/l,, (520.3 ± 20.2) mg/l (608.9 ± 20.2) mg/l (581.3 ± 19.1) mg/l (P < 0.05) 2 图 2 各虫不同添加量对灵芝胞内多糖的的影响 Fig. 2 Effects of insect concentration on production of IPS production. IPS: intracellular polysaccharides; : E. sinensis; : D. houi; : C. molossus; : A. chinensi. 图 1 几种昆虫 (4 g/l) 对灵芝细胞生长的影响 Fig. 1 Effect of various insects (4 g/l) on cell growth of Ganoderma lucidum. CM: Catharsius molossus; MP: Mylabris phalerata; AC: Aspongopus chinensis; DH: Dendrolimus houi; ES: Eupolyphaga sinensis. 2.2 对灵芝多糖产物形成的影响 [13],, (Intracellular polysaccharides, IPS) 2, (C. molossus) 5 g/l, [ (1.93 ± 0.09) g/l (2.41 ± 0.12 )g/l](p < 0.05),, (Extracellular polysaccharides, EPS) 图 3 各虫不同添加量对灵芝胞外多糖的的影响 Fig. 3 Effects of insect concentration on production of EPS production. EPS: extracellular polysaccharides; : E. sinensis; : D. houi; : C. molossus; : A. chinensi. 2.3 蜣螂对灵芝胞内和胞外多糖组成的影响 DEAE- 4 (5 g/l) ÄKTA Explorer DEAE- 4, 5, IPS-1 IPS-2 IPS-3 IPS-4

4 : 917 图 4 灵芝胞内多糖在 DEAE 柱上的洗脱曲线 Fig. 4 HiPrepTM 16/10 DEAE elution profile of IPS. : sample of containing C. molossus in medium; : control. IPS-5, 0.06 mol/l 0.12 mol/l 0.25 mol/l 0.5 mol/l NaCl IPS-1, IPS %, IPS % IPS-1, Superdex 200 HR 10/30 ( 0.1 mol/l NaCl ), 2 IPS-1 3, 3, ( 5) 5, mol/l NaCl, EPS-1 EPS-2 EPS-3 EPS-4 EPS-5 EPS-1 EPS %, EPS % EPS-1, Superdex 200 HR 10/30 ( 0.1 mol/l NaCl ), 2 EPS-1 2,, ( 7) 2 3,,, 4~7,,, 3 讨论图 5 IPS-1 在 Superdex 200 HR 10/30 凝胶柱上的洗脱曲线 Fig. 5 Superdex 200 HR 10/30 elution profile of IPS-1. : sample of containing C. molossus in medium; : control. 6 ÄKTA Explorer DEAE-, ph, - [6 8, 12]

5 918 ISSN CN /Q Chin J Biotech June 25, 2009 Vol.25 No.6 图 6 灵芝胞外多糖在 DEAE 柱上的洗脱曲线 Fig. 6 HiPrepTM 16/10 DEAE elution profile of EPS. : sample of containing C. molossus in medium; : control. Tang & Zhong α-, [15] Yang, 1.5%, 2.0%,, [14],,, [11],,,,, REFERENCES 图 7 EPS-1 在 Superdex 200 HR 10/30 凝胶柱上的洗脱曲线 Fig. 7 Superdex 200 HR 10/30 elution profile of EPS-1. : sample of containing C. molossus in medium; : control.,, [1] Lin ZB. Modern Research of Ganoderma. 2nd Ed. Beijing: Beijing Medical University Press, :, [2] Shiao MS. Natural products of the medicinal fungus Ganoderma lucidum: Occurrence, biological activities, and pharmacological functions. Chem Record, 2003, 3: [3] Baek S, Kim Y, Yong H, et al. Antitumor activities of the proteoglycans from the mycelium of Ganoderma lucidum IY009. Yakhak Hoechi, 2001, 45 (6): [4] Lee S, Lee P, Chen C. Antitumor effects of polysaccharides of Ganoderma lucidum (Curt.: Fr.) P. Karst. (Ling Zhi, Reishi mushroom) (Aphyllophoromycetideae). Int J Med Mushrooms, 2003, 5(1): [5] Kimura Y, Taniguchi M, Baba K. Antitumor and antimetastatic effects on liver of triterpenoid fractions of Ganoderma lucidum: Mechanism of action and isolation of an active substance. Anticancer Res, 2002, 22:

6 : [6] Fang QH, Zhong JJ. Two-stage culture process for improved production of ganoderic acid by liquid fermentation of higher fungus Ganoderman lucidum. Biotechnol Prog, 2002, 18: [7] Chang MY, Tsai GJ, Houng JY. Optimization of the medium composition for the submerged culture of Ganoderma lucidum by Taguchi array design and steepest ascent method. Enzyme Microb Tech, 2006, 38: [8] Tang YJ, Zhong JJ. Fed-batch fermentation of Gaoderma lucidum for hyperproduction of polysaccharide and ganoderic acid. Enzyme Microb Technol, 2002, 31: [9] Liu GQ, Zhang KC. Effects of water and ethanol extracts of insects on polysaccharides production by submerged fermentation of Ganoderma lucidum. Mycosystema, 2006, 25(2): [10] Liu GQ, Zhang KC. Enhancement of polysaccharides production in Ganoderma lucidum by the addition of ethyl acetate extracts from Eupolyphaga sinensis and Catharsius molossus. Appl Microbiol Biotechnol, 2007, 74(3): [11] Liu GQ, Ding CY, Zhang KC. Effects of powdered dung beetle (Catharsius molossus) on cell growth and triterpenoid production of Ganoderma lucidum. Mycosystema, 2008, 27(5): ,,.., 2008, 27(5): [12] Tang YJ, Zhang W, Zhong JJ. Performance analyses of a ph-shift and DOT-shift integrated fed-batch fermentation process for the production of ganoderic acid and Ganoderma polysaccharides by medicinal mushroom Ganoderma lucidum. Bioresource Technol, 2009, 100(5): [13] Jiang SJ. Integration of Chinese Medicinal Insects. Beijing: China Forestry Publishing House, :, [14] Yang HL, Wu TX, Zhang KC. Enhancement of mycelial growth and polysaccharide production in Ganoderma lucidum by the addition of ethanol. Biotechnol Lett, 2004, 26: [15] Tang YJ, Zhong JJ. Exopolysaccharide biosynthesis and related enzyme activities of the medicinal fungus, Ganoderma lucidum, grown on lactose in a bioreactor. Biotechnol Lett, 2002, 24: 更正 H 6 M P H M H P

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc 微生物学通报 Microbiology China tongbao@im.ac.cn Apr. 20, 2016, 43(4): 793 797 http://journals.im.ac.cn/wswxtbcn DOI: 10.13344/j.microbiol.china.150504 高校教改纵横生物工程专业发酵课程群建设探索 * 赵宏宇