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030 0 2 9搅拌式生物反应器悬浮培养水母雪莲细胞的研究〔中〕/黄艳… //生物工程学报 .- 2 0 0 1,17(5 ) .- 5 6 1～ 5 6 5应用 2L通气搅拌式生物反应器一步批式培养水母雪莲细胞。采用倾斜式搅拌桨代替透平桨 ,研究了搅拌转速、通气量和接种量对细胞生长和黄酮合成的影响 ,发现在 75r/min、70 0～ 10 0 0L/min和 4 .0～ 5 .0gDCW /L接种量下细胞生长和黄酮合成比较好。经过 12d培养细胞干重达 13.8gDCW /L ,黄酮产量 4 16mg/L ,黄酮含量占细胞干重的 3.0 %。水母雪莲细胞生长及黄酮合成的进程表明 ,黄酮积累与细胞生长呈正相关。对细…     

水母雪莲细胞悬浮培养合成黄酮及抗氧化活性

对水母雪莲细胞悬浮培养过程中细胞生长、黄酮积累和底物消耗的动力学过程进行了研究.经15 d液体培养可获得最大生物量干重和黄酮产量分别为17.2 g·L-1和607.8 mg·L-1,通过调控基本培养基种类和有机添加物可提高雪莲细胞的生长和黄酮积累.获得的水母雪莲细胞培养物具有明显的抗氧化活性,其抗氧化活性与雪莲细胞中的黄酮含量相关.     

水母雪莲悬浮培养细胞生长和黄酮类活生成分合成

在ＭＳ培养基上进行水母雪莲细胞悬浮培养,研究了摇床转速、接种量、培养液初始ｐＨ、碳源等的影响。结果表明,摇床转速为９０￣１２０ｒ／ｍｉｎ,接种量为５０￣８０ｇＦＷ／Ｌ,培养液初始ｐＨ５．５￣６．０,对水母雪莲悬浮培养细胞生长和黄酮合成最有利。碳源以蔗糖最适合,蔗糖浓度则以４０ｇ／Ｌ较好,此时细胞生长量为１８￣１９ｇＤＷ／Ｌ,总黄酮合成量可达１４２３．２５ｍｇ／Ｌ。用ＨＰＬＣ检测显示４＇,５,７－三     

水母雪莲悬浮培养细胞生长和黄酮类活性成分合成

在ＭＳ培养基上进行水母雪莲（ＳａｕｓｕｒｅａｍｅｄｕｓａＭａｘｉｍ．）细胞悬浮培养,研究了摇床转速、接种量、培养液初始ｐＨ、碳源等的影响。结果表明,摇床转速为９０～１２０ｒ／ｍｉｎ,接种量为５０～８０ｇＦＷ／Ｌ,培养液初始ｐＨ５．５～６．０,对水母雪莲悬浮培养细胞生长和黄酮合成最有利。碳源以蔗糖最适合,蔗糖浓度则以４０ｇ／Ｌ较好,此时细胞生长量为１８～１９ｇＤＷ／Ｌ,总黄酮合成量可达１４２３．２５ｍｇ／Ｌ。用ＨＰＬＣ检测显示４′,５,７三羟基３′,６二甲氧基黄酮（ｊａｃｅｏｓｉｄｉｎ）和４′,５,７三羟基６甲氧基黄酮（ｈｉｓｐｉｄｕｌｉｎ）的含量分别达到总黄酮含量的２２．１１％和０．１５％。     

甘草细胞在搅拌式生物反应器中的放大培养

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。     

碳源和氮源对水母雪莲悬浮培养细胞生长和黄酮合成的影响

在MS培养基上进行水母雪莲(Saussurea medusa Maxim)细胞悬浮培养时研究了碳源和氮源的影响。结果表明碳源以蔗糖最适合,蔗糖浓度则以40g/L较好,细胞生长量干重为18.12g/L,总黄酮合成量达到1423.25mg/L。在培养过程中,水母雪莲细胞能够快速将蔗糖水解为葡萄糖和果糖,并首先利用葡萄糖。氮源总浓度(包括NH⁺₄和NO^-₃)为60～120mmol/L,NH⁺₄/NO^-₃比例为20/40有利于雪莲细胞生长和黄酮合成。用HPLC检测显示4′,5,7-三羟基3′,6-二甲氧基黄酮(Jaceosidin)和4′,5,7-三羟基-6-甲氧基黄酮(Hispidulin)2种黄酮的含量分别达到细胞干重的1.46%和0.010%     

银杏液体悬浮细胞培养产生黄酮的研究

探讨了光照与黑暗、培养时间、细胞聚集体大小、前体物对银杏液体悬浮细胞系产生黄酮的影响。试验结果表明:光照条件下培养14 d黄酮含量最高,3～4 mm粒径的细胞聚集体是黄酮合成的适宜细胞聚集体,前体物L-苯丙氨酸和乙酸钠对黄酮合成无明显的促进作用,且对细胞生长有抑制作用。     

前体对水母雪莲悬浮培养细胞黄酮合成的影响

在水母雪莲（Saussurea medusa Maxim)细胞悬浮体系中加入苯丙氨酸、肉桂酸和乙酸钠3种前体。结果显示,3种前体均能促进细胞内黄酮的生物合成,但它们对细胞的生长也有一定的抑制作用。实验表明,3种前体的添加时间均以第6天为宜。苯丙氨酸的最佳添加浓度为0.05mmol/L,肉桂酸、乙酸钠的最佳添加浓度都是0.1mmol/L。3种前体中,难溶于水的肉桂酸对黄酮合成的促进作用最强,它可使培养物的黄酮产量高达1801mg/L,是对照1．98倍。苯丙氨酸、乙酸钠两种前体协同添加,比它们单独加入更能促进细胞的黄酮合成。     

用多孔微载体大规模培养rCHO细胞

多孔微载体是近年来发展起来的一种用于大规模高密度培养动物细胞的支持物,具有许多优点,如：容易固定细胞,适合于贴壁细胞和悬浮细胞的固定化连续灌流培养;细胞生长在载体内部,增加了细胞固定化的稳定性,可降低血清用量,适合长期培养;能保护细胞免受机械损伤,增加搅拌强度和通气量,强化反应器的传质;比表面积大,为细胞提供了充分的生长空间;细胞固定化过程简单无害,细胞能从长满细胞的微载体中自动转移到未长细胞的新载体中生长,接种方便,操作简单。特别适合于搅拌式、气升式、周定床和流化床等生物反应器的大规模培养〔1,2。尿激酶原(pro-UK)是一种重要的溶栓药物．与一般的生物医药制品相比,pro-UK给药量较大(约20mg／人～80mg／人),小规模生产不能满足市场需求。本文报道利用20L搅拌式反应器培养分泌pro-UK的重组CHO细胞的工艺条件,取得了初步结果。     

培养基及其组成对水母雪莲悬浮培养细胞生长及黄酮形成的影响

8种不同的基本培养基对水母雪莲(Saussurea medusa)细胞生长和黄酮形成的影响不同。实验结果表明,MS培养基较有利于细胞生长和黄酮形成。从MS培养基修饰得到的MG和MP培养基比前者细胞生长量与黄酮产量分别提高32%和70%。碳源、氮源、植物激素对细胞生长及黄酮形成的影响较为明显。用HPLC对细胞培养物中两种有效黄酮(Jaceosidin和Hispidulin)作了定性定量分析。     

Adventitious root culture of <Emphasis Type="Italic">Polygonum multiflorum</Emphasis> for phenolic compounds and its pilot-scale production in 500 L-tank

Polygonum multiflorum Thunb. is an important medicinal plant that synthesizes an array of phenolic compounds. Its roots are used in a variety of pharmacological and cosmetic formulations, notably as hair dye. In the present study, the inoculum density (3–15 g/L) and culture period (1–7 weeks) were optimized in a 3 L bioreactor. High root biomass (14.18 g/L dry weight (DW)) was recorded with an inoculum of 7 g/L (p?≤?0.05), which is consistent with the results for 5 and 10 g/L. However, significantly higher yield of bioactive compounds (53.87 mg/g DW total phenolics and 27.96 mg/g DW total flavonoids) with high free radical scavenging activity was obtained in root samples from 5 g/L inoculum density. A 4 week culture period was sufficient for optimum root growth and metabolite production. The optimized conditions were used for large-scale (5 and 20 L) and pilot-scale (500 L) studies. Considering that the continuous aeration of root cultures may lead to oxidative stress, antioxidant enzyme activity and lipid peroxidation also were studied. The results revealed high catalase (CAT) and guaiacol peroxidase (G-POD) activities, and low malondialdehyde (MDA) production, with increasing culture scale (20 and 500 L), which may indicate low-level oxidative damage to the cultures. An optimal yield of 4.01 kg dry root biomass with 287.12 mg/L of total phenolic productivity was achieved in a 500 L pilot-scale bioreactor. This work can pave the way for commercial production of biomass and secondary metabolites at the industrial level, and meet the rising demand for natural ingredients, especially in the pharmaceutical and cosmetic industries.     

Schizophyllan production by newly isolated fungus <Emphasis Type="Italic">Schizophyllum commune</Emphasis> IBRC-M 30213: optimization of culture medium using response surface methodology

Schizophyllan (SPG) is a commercially attractive biopolymer produced by Schizophyllum commune. An investigation on the potential for SPG production by Iranian native S. commune was conducted based on culture medium, fermentation conditions and bioreactor type, . Nine native fungal strains were isolated from the northern forest of Iran at different times. Based on growth rate and SPG production, one strain was selected for further study. Optimal medium composition and inoculum size for maximizing SPG production and minimizing biomass were determined using central composite design by setting sucrose, yeast extract, inoculum size, carboxymethyl cellulose and oleic acid in the ranges of 50–200 g/L, 1–4 g/L, 2–10%, 2–12 g/L and 0.032–0.222%, respectively. The results showed that optimal results were obtained at 93.47 g/L sucrose, 1.87 g/L yeast extract, 7.68% inoculum size, 9.07 g/L carboxymethyl cellulose and 0.13% oleic acid, with maximum SPG production of 9.97 g/L and minimum biomass of 35.18 g/L. Under these optimal conditions, the production of SPG was studied in stirred tank and bubble column bioreactors. The results revealed greater production in the stirred tank because of better mixing of the culture medium. The SPG produced was characterized using rheometery, Fourier transform infrared spectroscopy, nuclear magnetic resonance), scanning electron microscopy and gel permeation chromatography. The results of these characterizations demonstrated the similarity of the SPG produced by S. commune IBRC-M 30213 to commercial SPG. Thus, the SPG produced shows good potential as a polysaccharide for use in various industries.     

Comparative evaluation of modified bioreactors for enhancement of growth and secondary metabolite biosynthesis usingPanax ginseng hairy roots

Hairy root cultures have demonstrated great promise in terms of their biosynthetic capability toward the production of secondary metabolites, but continue to constitute a major challenge with regard to large-scale cultures. In order to assess the possibility of conducting mass production of biomass, and the extraction of useful metabolites fromPanax ginseng. P. ginseng hairy roots, transformed byRhizobium rhizogenes KCTC 2744, were used in bioreactors of different types and sizes. The most effective mass production of hairy roots was achieved in several differently sized air bubble bioreactors compared to all other bioreactor types. Hairy root growth was enhanced by aeration, and the production increased with increasing aeration rate in a 1 L bioreactor culture. It was determined that the hairy root growth rate could be substantially enhanced by increases in the aeration rate upto 0.5 wm, but at aeration rates above 0.5 wm, only slight promotions in growth rates were observed. In 20 L air bubble bioreactors, with a variety of inoculum sizes, the hairy roots exhibited the most robust growth rates with an inoculum size of 0.1% (w/v), within the range 0.1 to 0.7% (w/v). The specific growth rates of the hairy roots decreased with increases in the inoculum size.     

The feasibility of growing cells of Saccharomyces cerevisiae for citronellol production in a continuous-closed-gas-loop bioreactor (CCGLB)

The present work aims to address the gas-phase biotransformation of geraniol into citronellol using growing cells of Saccharomyces cerevisiae (baker's yeast) in a continuous-closed-gas-loop bioreactor (CCGLB). This study revealed that the gaseous geraniol had a severe effect on the production of biomass during the growing cell biotransformation resulting in the decrease in the specific growth rate from 0.07 to 0.05 h?1. The rate of reaction of the growing cell biotransformation was strongly affected by agitation and substrate flow rates. The highest citronellol concentration of 1.18 g/L and initial rate of reaction of 7.06 × 10?? g/min g(cell) were obtained at 500 rpm and 8 L/min, respectively.     

不同因素对岩黄连悬浮细胞生长的影响

目的:建立一个快速生长的岩黄连悬浮细胞培养体系。方法:研究了接种量、基本培养基、初始pH值、不同碳源对岩黄连悬浮细胞生长的影响。结果:合适的接种量是7.5~10%(FW),接种量过少会抑制细胞生长;B5和MS基本培养基均适合岩黄连细胞的生长;最佳初始培养基pH值为6.0,此时获得的细胞生物量最高;岩黄连悬浮细胞培养的生长周期为24d,最大生物量出现在第18d,达到14.1g/l(DW);蔗糖比葡萄糖更有利于岩黄连细胞的生长,添加60g/l蔗糖所获得的生物量最高,达到18.5g/l(DW)。     

营养和环境因子对长春花激素自养型细胞生长和阿玛碱生成的影响

在摇瓶中用液体培养基培养长春花（Catharanthusroseus（L．）G．Don）激素自养型细胞系C20hi,比较了不同初始糖浓度、接种量、初始pH值、光照时间、光质和摇床转速对该培养细胞生长、阿玛碱积累和释放的影响。结果表明,此细胞系具有较强的环境耐受性;一定范围内初始糖浓度增加,有利于细胞生长和生物碱生成;最适的接种量是60gFW／L;一定范围内改变培养基pH值,对生物碱生成没有显著影响;照光后生物碱生成量下降,红光较蓝光更有利于生物碱生成;最适的摇床转速是120r／min。     

Enhanced production of taxol in suspension cultures of Taxus chinensis by controlling inoculum size

Inoculum size (1.5-6.0g dry weight/l) significantly affected cell growth and accumulation of intracellular and extracellular taxol in Taxus chinensis. A shorter cultivation time and a higher biomass productivity were achieved using inoculum size of 6.0g DW/l. Both the intracellular content and total production of taxol were increased almost 30% with an increase of inoculum size from 1.5 to 3.0g DW/l, while an even higher inoculum size decreased taxol formation. The extracellular taxol concentration was relatively higher (0.091mg/l) at low inoculum sizes of 1.5 and 2.0g DW/l; and in various cases it was less than 25% of the total amount of taxol produced.     

Production of somatic embryos in a helical ribbon impeller bioreactor

Embryogenic cultures of a transformed Eschscholtzia californica cell line were carried out in a 11-L helical ribbon impeller bioreactor operated under various conditions to evaluate the performance of this equipment for somatic embryo (SE) production. All bioreactor cultures produced SE suspensions with maximum concentrations at least comparable to those obtained from flask control cultures ( approximately 8-13 SE . mL(-;1)). However, an increase of the mixingspeed, from 60 to 100 rpm, and low sparging rate ( approximately 0.05 VVM, k(L) a approximately 6.1 h(-;1)) for dissolved oxygen concentration (DO) control yielded poorer quality embryogenic cultures. The negative effects on SE production were attributed mainly to the low but excessive shear experienced by the embryogenic cells and/or embryoforming aggregates. High DO ( approximately 60% of air saturation) conditions favored undifferentrated biomass production and high nutrient uptake rates at the expense of the slower SE differentiation process in both flask and bioreactor cultures. Too low DO (-5-10%) inhibited biomass and SE production. The best production of SE ( approximately 44 SE . mL(-1) or approximately 757 SE . g dw(-1) . d(-1)) was achieved by operating the bioreactor at 60 rpm while controlling DO at approximately 20%by surface oxygenation only (0.05 VVM, k(L) a approximately 1.4 h(-;1)). This production was found to be a biomass production/growth-associated process and was mainly limited by the availability of extracellular phosphate, magnesium, nitrogen salts, and carbohydrates. (c) 1994 John Wiley & Sons, Inc.     

Inoculum production of the ectomycorrhizal fungus Pisolithus microcarpus in an airlift bioreactor

Many important tree species in reforestation programs are dependent on ectomycorrhizal symbiosis in order to survive and grow, mainly in poor soils. The exploitation of this symbiosis to increase plant productivity demands the establishment of inoculum production methods. This study aims to propose an inoculum production method of the ectomycorrhizal fungus Pisolithus microcarpus (isolate UFSC-Pt116) using liquid fermentation in an airlift bioreactor with external circulation. The fungus grew as dark dense pellets during a batch fermentation at 25.5 degrees C and air inlet of 0.26-0.43 vvm. The maximum biomass (dry weight) achieved in the airlift bioreactor was approximately 5 g.l(-1) after 10-11 days. The specific growth rate (micro(x)) in the exponential phase was 0.576 day(-1), the yield factor (Y(X/S)) 0.418, and the productivity (P(X)) 0.480 g.l(-1).day(-1). This specific growth rate was higher than that observed by other authors during fermentation processes with other Pisolithus isolates. The method seems to be very suitable for biomass production of this fungus. However, new studies on the fungus growth morphology in this system, as well as on the efficiency of the process for the cultivation of other ectomycorrhizal fungi, are necessary. It is also necessary to test the infectivity and efficiency of the inoculum towards the hosts.     

胀果甘草悬浮培养细胞合成甘草总黄酮

比较了胀果甘草(Glycyrrhiza inflata)悬浮细胞在逐级放大摇瓶中的生长、黄酮产量以及营养消耗过程,以便了解其放大规律。结果表明,在250和500mL摇瓶中,细胞的最大生物量、黄酮产量以及最大比生长速率没有显著性差异,但是在1L的摇瓶中,这三种参数都显著地降低,分别比250mL摇瓶中降低了27%,30%和27%。在逐级放大的摇瓶中,氮、磷、铵浓度都随着培养时间延长而逐渐降低,尽管在1L的摇瓶中磷消耗得最慢,但三种摇瓶中磷在细胞生长对数期基本都被消耗尽了。此外,硝态氮在第18天时基本被消耗完,而铵态氮在细胞收获时仍能维持在100mg/L。因此在反应器中培养时,主要的培养条件还需进一步优化。