霍山石斛类原球茎二步法培养细胞生长和多糖合成的动力学研究

磷是调控类原球茎细胞生长和多糖积累的有效因素。为了获得较高的多糖产量,根据霍山石斛类原球茎生长和多糖积累的动力学特性,提出了二步培养方式,采用了补料策略,研究了其培养过程的动力学特性,并建立了相关模型。结果表明,采用二步法培养,生物量从28.7g DW/L提高到44.2g DW/L, 多糖产量从1.86g/L提高到5.22g/L,多糖含量从6.4%提高到11.9%。建立的模型基本反映了类原球茎生长和多糖积累的动力学机制。     

磷对霍山石斛类原球茎悬浮培养细胞生长和多糖合成的影响

在确定了最适接种量和外植体细胞生理时间的基础上,研究了在不同起始磷浓度下,霍山石斛类原球茎生长、碳、氮消耗和多糖积累的动力学特性。以生长30d的类原球茎为材料,在接种量为100g/L时,类原球茎生长的最佳起始磷浓度为2.5mmol/L,培养36d时,类原球茎鲜重达496.5g/L。动力学分析表明,磷是霍山石斛类原球茎生长的限制性因素,胞内磷的积累水平与细胞生长具有相关性,2.5mmol/L的磷酸盐有利于碳、氮等营养物质的吸收;而多糖积累的最佳起始磷浓度为0.312mmol/L,培养36d时,其产量为2.22g/L。     

精胺对霍山石斛类原球茎悬浮培养细胞生长和多糖合成的影响

研究了在添加外源精胺时,霍山石斛类原球茎细胞生长、多糖积累、主要营养物质消耗以及细胞内多胺含量的变化。结果表明,0.6mmol/L的精胺明显促进霍山石斛类原球茎细胞的生长和多糖的合成。细胞的比生长速率从0.046d-1提高到0.054d-1。培养30d时,类原球茎干重达32.4gDW/L,多糖总产量为2.46g/L,分别是对照的1.32和1.31倍。添加外源精胺能够提高内源多胺的含量,同时,蔗糖酶和硝酸还原酶等相关代谢酶的活性增强,促进了碳、氮的吸收和利用。     

水杨酸对霍山石斛类原球茎细胞生长及多糖合成的影响

本研究考察了水杨酸对霍山石斛类原球茎悬浮培养细胞生长、多糖合成、碳代谢的影响,并研究了细胞生长、多糖合成以及蔗糖消耗的动力学。结果表明,水杨酸对霍山石斛类原球茎细胞生长有轻微的抑制作用,但能够显著改善类原球茎对碳源的利用,提高胞内可溶性糖的含量,从而促进多糖的合成,其中以添加100μmol/L浓度的水杨酸效果最好,培养18d时,多糖产量达到3.129g/L,为对照的1.63倍。建立的培养动力学模型能较好地反映水杨酸调控霍山石斛类原球茎悬浮培养过程中细胞生长、多糖合成和蔗糖消耗特性。     

精胺对霍山石斛类原球茎悬浮培养细胞生长和多糖合成的影响

研究了在添加外源精胺时,霍山石斛类原球茎细胞生长、多糖积累、主要营养物质消耗以及细胞内多胺含量的变化。结果表明,0.6mmol/L的精胺明显促进霍山石斛类原球茎细胞的生长和多糖的合成。细胞的比生长速率从0.046d^-1提高到0.054d^-1。培养30d时,类原球茎干重达32.4g DW/L,多糖总产量为2.46g/L ,分别是对照的1.32和1.31倍。添加外源精胺能够提高内源多胺的含量,同时,蔗糖酶和硝酸还原酶等相关代谢酶的活性增强,促进了碳、氮的吸收和利用。     

氮源和真菌诱导子对铁皮石斛原球茎悬浮培养的影响

利用正交实验设计研究不同种类和浓度的氮源对铁皮石斛原球茎生长的影响,利用完全随机实验设计研究不同真菌诱导子对铁皮石斛原球茎生长和多糖积累的影响.结果表明硝态氮促进铁皮石斛原球茎鲜重和干重的增加( P ＜0.05);铵态氮促进铁皮石斛原球茎鲜重增加( P ＜0.05).无论鲜重还是干重,硝态氮和铵态氮的影响均不存在互作,最佳组合为:67-V培养液 100 mg/L (NH4)2SO4 800 mg/L KNO3 30 g/L蔗糖 200 g/L马铃薯提取汁.F检验的结果表明,14种真菌诱导子对铁皮石斛原球茎生物量的增加(鲜重与干重)均无显著性影响( P ＞0.05).但与对照铁皮石斛原球茎的多糖含量相比,真菌诱导子g6、g14、g5、g4处理可使其多糖含量分别提高14%、9%、6%、4%.本研究获得了有利于铁皮石斛原球茎生长的硝态氮和铵态氮的最佳搭配方案以及有利于铁皮石斛原球茎积累多糖的四种真菌诱导子,表明通过液体悬浮培养生产铁皮石斛原球茎及其多糖成分具有较好的开发应用前景.     

锗对霍山石斛类原球茎悬浮培养细胞生长和多糖合成及氧化还原态的影响

为解决霍山石斛类原球茎液体培养细胞生长缓慢和代谢水平低下的问题,研究了不同浓度的锗(GeO2)对霍山石斛类原球茎增殖、多糖合成及碳氮利用的影响,分析了类原球茎细胞内还原糖、可溶性蛋白质含量、抗氧化酶活性以及细胞氧化还原态的变化。结果表明,适当浓度的二氧化锗(4.0mg/L)显著促进霍山石斛类原球茎的增殖和多糖的积累,最大细胞干重为32.6g/L,最大多糖产量为3.78g/L;显著提高胞内还原糖和可溶性蛋白质含量,超氧化物歧化酶和过氧化氢酶的活性明显升高,而过氧化物酶的活性则有所降低;氧化还原态分析发现,二氧化锗处理的细胞内还原型谷胱甘肽/氧化型谷胱甘肽的值明显提高,谷胱甘肽还原酶活性升高。添加适量的二氧化锗有利于细胞生长和多糖合成。     

建立金线莲原球茎悬浮体系生产次生代谢产物

研究植物激素浓度和培养周期对金线莲原球茎悬浮培养生长及其代谢产物积累的影响,以增加金线莲悬浮培养的生长量,提高次生代谢产物的生产。结果表明,MS培养基添加S-3307 1.0mg/L,6-BA0.5mg/L和3%的蔗糖适合总生物量的生长(214.45g/L,FW和18.23g/L DW)。而MS培养基添加S-3307 1.0mg/L,6-BA 3.0mg/L和5%的蔗糖,总黄酮,总酚和多糖的干重(5.43mg/g,2.87mg/g和243.23mg/g)达到最大化。研究原球茎悬浮培养过程,发现经过7个星期培养就能获得最大的生物质总量(225.98 g/L的FW和18.53 g/L的DW)、总黄酮干重(5.09mg/g)和总酚干重(2.04mg/g),而多糖生产达到其峰值(229.36mg/g干重)是在培养后5个星期。     

四种添加物对铁皮石斛原球茎生长及多糖含量的影响

为探讨铁皮石斛(Dendrobium officinale)培养基中添加物的作用,在1/2MS培养基中加入椰肉、甘蔗渣、香蕉皮和麦麸等4种添加物,研究不同浓度添加物和培养时间对原球茎生长和多糖含量的影响。结果表明,4种添加物对铁皮石斛原球茎的增殖、分化和多糖含量均有一定影响,其中添加15.0 g L–1甘蔗渣,培养60 d能明显促进铁皮石斛原球茎的增殖与分化(146.1%);而添加20.0 g L–1甘蔗渣,培养40 d能显著提高铁皮石斛原球茎多糖含量(50.4%)。这说明甘蔗渣是培养铁皮石斛原球茎的适宜添加物,既能促进铁皮石斛原球茎的生长发育,还能降低生产成本。     

天然有机物对铁皮石斛原球茎生长及有效成分的影响

为铁皮石斛原料药的生产提供一种新方法。以MS为基本培养基,探讨天然有机附加物与培养时间对铁皮石斛原球茎(PLBs)生长及主要药用成分石斛多糖和总生物碱的影响。研究结果表明:在添加不同浓度土豆汁的培养基中,以250 g/L效果最好,其干物率和多糖含量均在30天时达最大值,分别为8. 944%和17. 921%,增殖率在培养50天时最高,为1 028. 94%,而总生物碱含量在40天时积累最多,达0. 031 61%。增殖率、多糖和总生物碱含量均显著高于添加激素的对照组。添加番茄汁的培养基中各指标变化与土豆汁的类似,但原球茎长势差,颜色泛黄,增殖能力较添加土豆汁的差。综上,MS+250 g/L土豆汁+30 g/L蔗糖+7 g/L琼脂条有利于铁皮石斛PLBs生长和有效成分的积累,可考虑作为石斛多糖和石斛碱的生产来源,对铁皮石斛主要药用成分的生产具有重要的实践意义。     

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.     

Significance of inoculation density control in production of polysaccharide and ganoderic acid by submerged culture of Ganoderma lucidum

Control of inoculation density was significant for cell growth, morphology, and production of polysaccharide and ganoderic acid in submerged culture of the higher fungus Ganoderma lucidum. A maximal cell concentration of 15.7 g dry cell weight (DW)/l was obtained at an inoculation density of 330 mg DW/l. For inoculation density within the range of 70–670 mg DW/l, a large inoculation density led to a small pellet size and high production of extracellular and intracellular polysaccharides, while a relatively big pellet size and high accumulation of ganoderic acid were observed at a low inoculation density. It was also shown that small pellet size resulted in high polysaccharide production, while large pellet size led to high production of ganoderic acid.     

Scale-up of centrifugal impeller bioreactor for hyperproduction of ginseng saponin and polysaccharide by high-density cultivation of panax notoginseng cells

Scale-up of a novel centrifugal impeller bioreactor (CIB) was demonstrated for production of valuable plant-specific secondary metabolites by high-density cell cultures. Initial kLa was identified to be a key factor affecting cell growth and production of ginseng saponin and polysaccharide by high-density cultivation of Panax notoginseng cells in a 3-L CIB. A high level of ginseng saponin and polysaccharide production was obtained at an initial kLa value of 30.2 h(-1). A maximum dry cell weight (DW) and production titer of ginseng saponin and polysaccharide reached 22.0 +/- 0.3, 1.5 +/- 0.1, and 2.7 +/- 0.2 g/L on day 15 with their corresponding productivity of 1140 +/- 42, 81 +/- 8, and 150 +/- 17 mg/(L.d), respectively. Based on initial kLa level, the CIB high-cell-density cultivation process was successfully scaled up from 3 L to 30 L. A maximum DW and production titer of ginseng saponin and polysaccharide in a 30-L CIB reached 25.5 +/- 0.5, 1.7 +/- 0.1, and 2.9 +/- 0.1 g/L (on day 15) at an initial kLa value of 28.7 h(-1), respectively, and their corresponding productivity was 1340 +/- 56, 91 +/- 9, and 164 +/- 15 mg/(L.d). Furthermore, by adopting a fed-batch cultivation strategy, a maximum DW and concentrations of total saponin and polysaccharide in the 30-L CIB were enhanced to 30.3 +/- 1.0, 2.1 +/- 0.1, and 3.5 +/- 0.2 g/L with their corresponding productivity of 1467 +/- 87, 102 +/- 13, and 179 +/- 18 mg/(L.d), respectively. The work suggests that the CIB may have great potential in large-scale high-density plant cell cultures for efficient production of useful secondary metabolites.     

Nitrogen Effect on Water-Soluble Polysaccharide Accumulation in <Emphasis Type="Italic">Streblonema</Emphasis> sp. (Ectocarpales,Phaeophyceae)

The water-soluble polysaccharides of brown algae attract the increasing attention of researchers as an important class of polymeric materials of biotechnological interest. The sole source for production of these polysaccharides has been large brown seaweeds such as members of Laminariales and Fucales. A new source of water-soluble polysaccharides is suggested here: it is a filamentous brown alga Streblonema sp., which can be cultivated under controlled conditions in photobioreactors that allow obtaining algal biomass with reproducible content and quality of polysaccharides. The accumulation of water-soluble polysaccharides can be stimulated by macronutrient limitation. In response to nitrogen deficiency, Streblonema sp. accumulated water-soluble polysaccharides (WSPs) rich in laminaran. WSP accumulation started after 3–4 days following nitrate depletion and reached a plateau at around day 7. Polysaccharide accumulation was related to cellular nitrogen content. The critical internal N level that triggered the onset of polysaccharide accumulation was 2.3% dry weight (DW); at a cellular N concentration less than 1.4% DW, the polysaccharide synthesis stopped. Upon nitrate re-supply, mobilization of WSP occurred after 3 days. These results suggest that a two-stage cultivation process could be used to obtain large algal biomass with high water-soluble polysaccharide production: a first cultivation stage using nitrate-supplemented medium to accumulate algal biomass followed by a second cultivation stage in a nitrate-free medium for 3 to 7 days to enhance polysaccharide content in the alga.     

A promising approach on biomass accumulation and withanolides production in cell suspension culture of Withania somnifera (L.) Dunal

Withanolide is one of the most extensively exploited steroidal lactones, which are biosynthesized in Withania somnifera. Its production from cell suspension culture was analyzed to defeat limitations coupled with its regular supply from the plant organs. In order to optimize the different factors for sustainable production of withanolides and biomass accumulations, different concentrations of auxins or cytokinins and their combinations, carbon sources, agitation speed, organic additives and seaweed extracts was studied in cell suspension culture. Maximum biomass accumulation (16.72 g fresh weight [FW] and 4.18 g dry weight [DW]) and withanolides production (withanolide A 7.21 mg/g DW, withanolide B 4.23 mg/g DW, withaferin A 3.88 mg/g DW and withanone 6.72 mg/g DW) were achieved in the treatment of Gracilaria edulis extract at 40 % level. Organic additive l-glutamine at 200 mg/l in combination with picloram (1 mg/l) and KN (0.5 mg/l) promoted growth characteristics (11.87 g FW and 2.96 g DW) and withanolides synthesis (withanolide A 5.04 mg/g DW, withanolide B 2.59 mg/g DW, withaferin A 2.36 mg/g DW and withanone 4.32 mg/g DW). Sucrose at 5 % level revolved out to be a superior carbon source yielded highest withanolides production (withanolide A 2.88 mg/g DW, withanolide B 1.48 mg/g DW, withaferin A 1.35 mg/g DW and withanone 2.47 mg/g DW), whereas biomass (7.28 g FW and 1.82 g DW) was gratefully increased at 2 % level of sucrose in cell suspension culture. This optimized protocol can be utilized for large scale cultivation of W. somnifera cells in industrial bioreactors for mass synthesis of major withanolides.