外循环气升式反应器培养新疆紫草细胞

采用两步培养法进行新疆紫草细胞悬浮培养及5L外循环气升式反应器扩大培养,探讨了培养过程中细胞生长、紫草色素合成与培养液的电导率、可溶性糖含量变化之间的关系。第一步培养时细胞生长迅速,但也有一部分色素合成,电导率及可溶性糖含量迅速下降;第二步培养初期电导率也开始下降,但当色素合成达到高峰并有一部分外泌到培养基后,电导率又开始回升。可溶性糖捎耗很快,到后期巳测不出其存在。因此通过监测培养液中电导率及可溶性糖的变化情况,可以为新疆紫草细胞大规模培养与色素合成提供有用的参数指标。     

硬紫草细胞悬浮培养和紫草宁及其衍生物的形成

硬紫草细胞悬浮培养形成紫草宁及其衍生物时．细胞生长曲线呈扁平的s形。细胞停止生长后,紫草宁及其衍生物大量形成,二者的动态变化呈负相关。测定丁此过程中培养液的无机元素和可溶性糖含量、溶氧、pH值以及细胞形态的变化情况,可作为硬紫草细胞大规模培养的参考。     

新疆紫草细胞生产阶段培养中细胞生长及产物合成的研究Ⅱ:发酵培养

用内循环气升式反应器进行新疆紫草细胞生产阶段培养,当培养基浓度增加时,色素产率也随之提高,其数量及增长的幅度远大于悬浮培养。当接种量为180gFW／1,培养基为3倍的M9时能获得最大的色素产率：2．28g／l,是培养基非加倍时的5．4倍,太高的接种量不利于色素的合成。为降低成本而减少培养基中某些成份的用量时,要得到较高的色素产量,需同时调整其它成份的浓度。     

新疆紫草细胞悬浮培养过程研究

新疆紫草细胞生长和紫草素合成之间属非生长偶联型,所以采用二步培养法研究悬浮培养过程。新疆紫草细胞悬浮培养的生长周期约为21 d,紫草素合成周期约为16 d。新疆紫草细胞生长阶段培养液的电导率与生物量呈线性负相关,随着生物量的增加,培养液的电导率降低。因此,可以通过测量电导率来预测培养体系中生物量的变化情况。细胞生长过程中硝酸盐、铵盐和可溶性糖的消耗与生物量的变化具有很好的线性相关性,基于硝酸盐、铵盐和可溶性糖的细胞收率系数分别为8.64、104.3和0.68 g/g。     

真菌诱导物在滇紫草细胞培养中对紫草色素形成的影响

在滇紫草细胞悬浮培养中,真菌诱导物可抑制细胞生长,促进草色素的合成,将培养６ｄ的曲霉菌丝体的粗提物以６００μｇ碳水化合物／５０ｍｌ培养液的浓度加入到处于指数生长初期的滇紫草细胞悬浮培养物中,诱导物促进紫草色素合成的作用最大,紫草色素含量为对照的两倍,经高压锅处理２０ｍｉｎ到２ｈ不影响诱导物的活性,真菌诱导物还影响了紫草色素各衍生物的相对含量。     

真菌诱导物在滇紫草细胞培养中对紫草色素形成的影响

在滇紫草细胞悬浮培养中,真菌诱导物可抑制细胞生长,促进紫草色素的合成。将培养６ｄ的曲霉菌丝体的粗提物以６００μｇ碳水化合物／５０ｍｌ培养液的浓度加入到处于指数生长初期的滇紫草细胞悬浮培养物中,诱导物促进紫草色素合成的作用最大,紫草色素含量为对照的两倍。经高压锅处理２０ｍｉｎ到２ｈ不影响诱导物的活性。真菌诱导物还影响了紫草色素各衍生物的相对含量。     

pH值、激素对新疆紫草悬浮培养细胞生长及紫草宁衍生物合成的影响

报道了不同ｐＨ值、激素对新疆紫草悬浮培养细胞生长及紫草宁衍生物合成的影响。结果表明,新疆紫草细胞具有自我调节其培养液ｐＨ值的功能。适合于细胞生长及紫草宁衍生物形成的ｐＨ值为５．６±０．４０。ＢＡＰ、２,４－Ｄ、ＮＡＡ或ＩＢＡ对细胞生长无显著的促进作用,且都会抑制紫草宁衍生物的形成。在生长培养基中添加１．０ｍｇ／ｌ　ＩＡＡ和０．５ｍｇ／ｌＫＴ可促进细胞生长,而在生产培养基中附加０．１ｍｇ／ｌＫＴ和０．７５─１．０ｍｇ／ｌＩＡＡ则有利于紫草宁衍生物含量及产量的提高。     

米根霉诱导因子对紫草细胞培养中紫草宁色素分泌的影响

在紫草细胞培养中,加入米根霉粗提物可显著提高紫草宁色素产量,并可加快胞内色素分泌到培养液中的速率和数量。在细胞培养的第６天加入米根霉诱导因子时其促进紫草宁色素分泌的作用最大,培养液中紫草宁色素含量对照的２．２４倍。此外,同时加入正十六烷和米根霉诱导因子对紫草宁色素的分泌具有协同作用。     

米根霉诱导因子对紫草细胞培养中紫草宁色素分泌的影响

在紫草细胞培养中,加入采根霉粗提物可显著提高紫草宁色素产量,并可加快胞内色素分泌到培养液中的速率和数量。在细胞培养的第6天加入米根霉诱导因子时,其促进紫草宁色素分泌的作用最大,培养液中紫草宁色素含量是对照的2．24倍。此外,同时加入正十六烷和米根霉诱导因子对紫草宁色素的分泌具有协同作用。     

真菌诱导物对滇紫草细胞色素形成的影响（简报）

在滇紫草细胞培养中加入真菌诱导物后紫草色素的合成增强,培养液的电导率和pH值在24h内分别下降和上升,且三者均以指数生长初期加入时作用最为明显。     

Studies on Cell Suspension Culture and Fermentation Culture in Arnebia euchroma

This paper reports some characteristics of cell suspension and fermentation culture in Arnebia euchroma (Royle) Johnst. The yield of suspension culture reached 22.0g dry wt/L per month when inoculum quantity was 2.50 g dry wt/L. Time-course study showed that cell growith lagged in 0–3 days and enhanced greatly in 3–12 days, and almost ceased after 12 days of culture, pH value changed during the culture period and peaked on the 12th day after inoculation. When cells were cultured in liquid production medium, the contents of shikonin derivatives increased quickly and reached to the maximum about the 25th day. The cell yield of 9.47 and 9.34 g dry wt/L per month was obtained in fermentation culture. Timecourse of cell growth in fermentation culture was similar to that in suspension culture. The total content of shikonin derivatives in fermentation culture was 14.26% dry weight from 10 L bioreactor. The yield of shikonin derivatives was 1.93 g/L.     

Physico-chemical factors influencing the shikonin derivatives production in cell suspension cultures of Arnebia euchroma (Royle) Johnston, a medicinally important plant species

Cell suspension cultures of Arnebia euchroma were raised from in vitro leaf-derived friable callus on liquid MS [Murashige and Skoog] medium supplemented with BAP (6-benzylaminopurine) (10.0 μM) and IBA (indole-3-butyric acid) (5.0 μM). A two-stage culture system was employed using growth and production medium for cell biomass and shikonin derivatives, respectively. Factors such as light, temperature, sucrose and pH (hydrogen ion concentration) were studied to observe their effect on the shikonin derivative production. Light conditions completely inhibited shikonin derivative production. Out of different temperature regimes tested, the highest yield (586.17 μg/g FW) was found at 25°C. Maximum production (656.14 μg/g FW) was observed in 6% sucrose. An alkaline pH (7.25-9.50) favoured shikonin derivative production. The results showed that physical and chemical factors greatly influence the production of shikonin derivatives in cell suspension cultures of A. euchroma. Therefore, by employing optimum culture conditions, it is possible to enhance the production of secondary compounds from the cells. The factors optimized for in vitro production of shikonin derivatives during the present study can successfully be employed for their large-scale production in bioreactors.     

Bioprocess engineering of <Emphasis Type="Italic">Echium italicum</Emphasis> L.: induction of shikonin and alkannin derivatives by two-liquid-phase suspension cultures

An in vitro cell suspension culture of Echium italicum was established and assayed for the production of shikonin and alkannin derivatives. Callus tissues were induced from cotyledon explants of the plant incubated onto the solidified B5 medium. A two-liquid-phase system suspension culture was then established to elicit pigments of shikonin and alkannin derivatives using liquid paraffin. The presence of liquid paraffin efficiently induced production of pigments in cultured cells. The production and/or accumulation of these compounds in the E. italicum cells was examined using fluorescence microscopy as the naphthoquinone molecules display autofluorescent properties. Phytochemical analysis of the n-hexane extract of the medium was also carried out using preparative HPLC. The chemical structure of shikonin and alkannin derivatives were characterized by UV, 1H-NMR, and 13C-NMR techniques. Based on our findings, this bioprocess engineering approach resulted in induction of shikonin and alkannin derivatives, whereupon it may be recruited for production of these important secondary metabolites.     

藻类活性物质对紫草细胞生长和色素形成的影响

本文研究了螺旋藻、栅列藻和织线藻的水提物对新疆紫草和硬紫草细胞生长和色素形成的作用。结果表明不同种类的藻的提取物对不同紫草细胞作用呈现差异。在生长阶段,对新疆紫草,上述3种藻的低浓度提取物促进生长但作用不大,高浓度的螺旋藻和栅列藻提取物强烈抑制生长;对硬紫草,各种藻提取液的所有浓度处理均有促生长作用。在色素形成阶段,连续用高浓度织线藻提取物处理可以加速新疆紫草色素形成,同时提高色素含量。低浓度的织线藻提取液处理能提高硬紫草色素含量。栅列藻的水提物对两种紫草的色素形成均起抑制作用。螺旋藻水提物的适当浓度可加速两种紫草的色素形成。B_5培养基中加高浓度织线藻水提物,可抑制新疆紫草生长阶段的色素形成。     

Regulation of shikonin production by glutamine in Lithospermum erythrorhizon cell cultures

Amino acid analysis has shown that Lithosperum erythrorhizon cell suspension cultures which are unable to produce shikonin derivatives in LS medium containing ammonium accumulate a large quantity of glutamine, as compared with shikonin-producing cells cultured in the production medium M9 containing nitrate as the sole nitrogen source. The addition of glutamine to M9 medium proved to be strongly inhibitory to shikonin production. Furthermore, culture experiments using an inhibitor of glutaminase suggested that shikonin synthesis is not inhibited by ammonium released from glutamine but by glutamine itself. These findings indicate that the repression of shikonin synthesis occurs in close association with an accumulation of glutamine in cultured cells grown in a medium containing ammonium.     

The Effective Production of Shikonin by Cultures with an Increased Cell Population

We have studied the efficient production of shikonin derivatives by suspension cultures of Lithospermum erythrorhizon with an increased cell population. The yield of shikonin derivatives was highest (800 mg/liter) when 2.8 g dry wt/liter of the cells was inoculated into the M-2 medium which we had developed for the production, but the excess inoculum lowered the yield.

We investigated suitable conditions for production with the increased cell population. The optimum amount of inoculum rose to 4.9 g dry wt/liter when the concentrations of all the components contained in the M-8 medium, which we developed for increasing the productivity by modification of the M-2 medium, were increased in proportion to the amount of inoculum, and consequently we could increase the yield of the shikonin derivatives from 1400 mg/liter to 1900 mg/liter. Moreover, the increased rate of oxygen supply in addition to the enrichment of the medium made it possible to produce 2300 mg/liter of the shikonin derivatives from a culture for which 5.6 g dry wt/liter of the cells was inoculated.