甘草细胞在搅拌式生物反应器中的放大培养
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摘要
在建立了稳定的甘草细胞搅拌式生物反应器放大培养体系的基础上,本文研究了甘草细胞在搅拌式反应器中悬浮培养的生长特性,包括细胞生长、细胞膜的透性、培养体系的p H变化及甘草黄酮合成情况等,并与摇瓶培养作比较。结果发现,同等条件下,反应器中培养细胞生物量的积累低于摇瓶培养,整个培养周期较摇瓶培养缩短。培养过程中同一时间段反应器中的p H值略低于摇瓶中的p H,细胞中H2O2的浓度是摇瓶中的1.8倍,甘草黄酮的产量是摇瓶培养的1.5倍,表明反应器中机械搅拌与流体剪切的培养环境对细胞生长起到一定程度的抑制作用,但刺激了细胞次生代谢产物甘草黄酮较高水平的合成。
Based on the establishment of a stable Glycyrrhiza uralensis(licorice) cells amplifi cation culture system in stirring bioreactor, the growth characteristics of cell suspension culture in stirring bioreactor were studied, including the cell growth, cell membrane permeability, the p H change of the culture system and licorice fl avonoids synthesis, and compared with the shake fl ask culture. The results showed that cell biomass accumulation in the bioreactor was less than that in shake fl ask under the same conditions, and the whole cultivation period was shortened. At the same time in the culture period, p H was slightly lower than that in the shake fl ask, the H2O2 concentration in cells was 1.8 times of that in shake fl ask, and the production of licorice fl avonoids was 1.5 times of that in the shake fl ask. These indicated that the cultivation environment with mechanical agitation and fl uid shear in the bioreactor inhibited the cells growth at a certain level, but stimulated secondary metabolites licorice fl avonoids synthesis cell in higher levels.
Based on the establishment of a stable Glycyrrhiza uralensis(licorice) cells amplifi cation culture system in stirring bioreactor, the growth characteristics of cell suspension culture in stirring bioreactor were studied, including the cell growth, cell membrane permeability, the p H change of the culture system and licorice fl avonoids synthesis, and compared with the shake fl ask culture. The results showed that cell biomass accumulation in the bioreactor was less than that in shake fl ask under the same conditions, and the whole cultivation period was shortened. At the same time in the culture period, p H was slightly lower than that in the shake fl ask, the H2O2 concentration in cells was 1.8 times of that in shake fl ask, and the production of licorice fl avonoids was 1.5 times of that in the shake fl ask. These indicated that the cultivation environment with mechanical agitation and fl uid shear in the bioreactor inhibited the cells growth at a certain level, but stimulated secondary metabolites licorice fl avonoids synthesis cell in higher levels.
引文
董静洲,易自力,蒋建雄(2006).我国药用植物资源研究概况.医学研究杂志,35(1):67~69
杨英(2007).甘草细胞培养合成甘草黄酮及其调控研究.武汉:华中科技大学
Alvarez ME,Pennell RI,Meijer PJ,Ishikawa A,Dixon RA,Lamb C(1998).Reactive oxygen intermediates mediate a systemic signalnetwork in the establishment of plant immunity.Cell,92(6):773~784
Chong TM,Abdullah MA,Fadzillah NM,Lai OM,Lajis NH(2005)Jasmonic acid elicitation of anthraquinones with some associated enzymic and non-enzymic antioxidant responses in Morindaelliptica.Enzyme Microb Technol,36(4):469~477
Chen SY,Huang SY(2000).Shear stress effects on cell growt h andL-DOPA production by suspension culture of Stizolobiumhassjoo cells in an agitated bioreactor.Bioprocess Eng,22(1):5~12
Gong YW,Li SY,Han RB,Yuan YJ(2006).Age-related responsesof suspension cultured Taxus cuspidata to hydrodynamic shearstress.Biochem Eng J,32(2):113~118
Han RB,Yuan YJ(2004).Oxidative burst in suspension cultu re ofTaxus cuspidata induced by a laminar shear stress in short-term.Biotechnol Progr,20(2):507~513
Luna-Palencia GR,Cerda-Garcia-Rojas CM,Rodriguez-Monroy M,Ramos-Valdivia AC(2005).Influence of auxins and sucrose inmonoterpenoid oxindole alkaloid production by Uncaria tomentosa cell suspension cultures.Biotechnol Progr,21(1):198~204
Mur LAJ,Kenton P,Draper J(2005).In planta measurements ofoxidative bursts elicited by avirulent and virulent bacterialpathogens suggests that H2O2 is insufficient to elicit cell death intobacco.Plant Cell Environ,28(4):548~561
Pan ZW,Wang HQ,Zhong JJ(2000).Scale-up study on suspen sioncultures of Taxus chinensis cells for production of taxane diterpene.Enzyme Microb Technol,27(9):714~723
Trejo-Tapia G,Cerda-Garcia-Rojas CM,Rodriguez-Monroy MRamos-Valdivia AC(2005).Monoterpenoid oxindole alkaloidproduction by Uncaria tomentosa(Willd)DC cell suspensioncultures in a stirred tank bioreactor.Biotechnol Progr,21(3):786~792
Xu QM,Cheng JS,Ge ZQ,Yuan YJ(2004).Effects of organic solvents on membrane of Taxus cuspidata cells in two-liquid-phasecultures.Plant Cell Tiss Org Cult,79(1):63~69
Zhang QY,Ye M(2009).Chemical analysis of the Chinese herbal medicine Gan-Cao(licorice).J Chromatogr,1216(11):1954~1969
Zhao J,Fujita K,Sakai K(2005).Oxidative stress in plant cell culture:A role in production of beta-thujaplicin by Cupresssus lusitanicasuspension culture.Biotechnol Bioeng,90(5):621~631
杨英(2007).甘草细胞培养合成甘草黄酮及其调控研究.武汉:华中科技大学
Alvarez ME,Pennell RI,Meijer PJ,Ishikawa A,Dixon RA,Lamb C(1998).Reactive oxygen intermediates mediate a systemic signalnetwork in the establishment of plant immunity.Cell,92(6):773~784
Chong TM,Abdullah MA,Fadzillah NM,Lai OM,Lajis NH(2005)Jasmonic acid elicitation of anthraquinones with some associated enzymic and non-enzymic antioxidant responses in Morindaelliptica.Enzyme Microb Technol,36(4):469~477
Chen SY,Huang SY(2000).Shear stress effects on cell growt h andL-DOPA production by suspension culture of Stizolobiumhassjoo cells in an agitated bioreactor.Bioprocess Eng,22(1):5~12
Gong YW,Li SY,Han RB,Yuan YJ(2006).Age-related responsesof suspension cultured Taxus cuspidata to hydrodynamic shearstress.Biochem Eng J,32(2):113~118
Han RB,Yuan YJ(2004).Oxidative burst in suspension cultu re ofTaxus cuspidata induced by a laminar shear stress in short-term.Biotechnol Progr,20(2):507~513
Luna-Palencia GR,Cerda-Garcia-Rojas CM,Rodriguez-Monroy M,Ramos-Valdivia AC(2005).Influence of auxins and sucrose inmonoterpenoid oxindole alkaloid production by Uncaria tomentosa cell suspension cultures.Biotechnol Progr,21(1):198~204
Mur LAJ,Kenton P,Draper J(2005).In planta measurements ofoxidative bursts elicited by avirulent and virulent bacterialpathogens suggests that H2O2 is insufficient to elicit cell death intobacco.Plant Cell Environ,28(4):548~561
Pan ZW,Wang HQ,Zhong JJ(2000).Scale-up study on suspen sioncultures of Taxus chinensis cells for production of taxane diterpene.Enzyme Microb Technol,27(9):714~723
Trejo-Tapia G,Cerda-Garcia-Rojas CM,Rodriguez-Monroy MRamos-Valdivia AC(2005).Monoterpenoid oxindole alkaloidproduction by Uncaria tomentosa(Willd)DC cell suspensioncultures in a stirred tank bioreactor.Biotechnol Progr,21(3):786~792
Xu QM,Cheng JS,Ge ZQ,Yuan YJ(2004).Effects of organic solvents on membrane of Taxus cuspidata cells in two-liquid-phasecultures.Plant Cell Tiss Org Cult,79(1):63~69
Zhang QY,Ye M(2009).Chemical analysis of the Chinese herbal medicine Gan-Cao(licorice).J Chromatogr,1216(11):1954~1969
Zhao J,Fujita K,Sakai K(2005).Oxidative stress in plant cell culture:A role in production of beta-thujaplicin by Cupresssus lusitanicasuspension culture.Biotechnol Bioeng,90(5):621~631