真菌诱导子对悬浮培养西洋参细胞的生理效应

报道了不同真菌诱导子对悬浮培养的西洋参（Ｐａｎａｘｑｕｉｎｑｕｅｆｏｌｉｕｍ）细胞生长、皂甙和多糖合成,以及细胞内和培养液中过氧化物酶活性的生理效应。悬浮培养的西洋参细胞经刺盘孢菌（Ｃｏｌｌｅｔｏｔｒｉｃｈｕｍｎｉｃｏｌｔｉａｎａｅ）丝体诱导子处理后,总皂甙产率可由对照的２９６ｍｇ／Ｌ增加到６７９ｍｇ／Ｌ（约占细胞干重的（１６．３％）,比对照提高约１．３倍,而且总皂甙的８５％排放在培养液中;经黑曲霉（Ａｓｐｅｒｇｉｌｌｕｓｎｉｇｒａｎ）诱导子处理后,细胞多糖含量可达到１１．７９％（细胞干重）,比对照增加１倍多。初步纯化的刺盘孢菌丝体诱导子和尖孢镰刀菌（Ｆｕｓｕｒｉｕｍｏｘｙｓｐｏｒｕｍ）滤液诱导子在诱导处理前期能明显促进西洋参细胞生长,同时细胞内及培养液中过氧化物酶活性显著增加;随时间延长,细胞生长和酶活性逐步受到抑制。     

三七.人参和西洋参细胞悬浮培养的比较研究

用薄层层析对三七、人参和西洋参愈伤组织进行的初步鉴定表明,三种愈伤组织都含有皂甙和主要皂甙成分Rb_1、Rg_1,三七愈伤组织还含有一种抗癌皂甙Rh_1。对愈伤组织的生长,三七低于人参高于西洋参;对愈伤组织中总皂甙含量,三七均高于人参和西洋参。三种植物细胞悬浮培养结果类似于他们的愈伤组织培养,但生长又进一步提高。三七细胞悬浮培养中皂甙产生的时间进程几乎与生长平行,合适的收获期为培养30天。寡糖素不仅增强三七培养细胞的皂甙形成而且促进细胞生长,较合适的浓度为1.25 ppm。通过以上研究,使三七悬浮培养细胞的生长(干重增加178毫克)为最初培养愈伤组织的4倍以上,总皂甙产率高达20.6毫克,为最初培养愈伤组织的8.5倍。     

西洋参细胞大量培养的研究

当西洋参细胞培养在Ms培养液中,KN0₃含量提高一倍而去掉NH₄NO₃。时细胞生长速率和皂甙产量分别比在正常培养液中提高65．1％和166．2％。黑节草寡糖素和人参寡糖素均有利于西洋参细胞的生长和皂甙含量的提高,尤其能增加Rg组皂甙的含量。西洋参细胞悬浮培养以生产皂甙收获的最佳时期为培养25天以上,其合成皂甙的高峰在细胞生长的对数期稍后出现。细胞悬浮培养和发酵培养过程中均未见pH值回升的现象。pH值稳定的发酵培养和pH值任其变化的培养相比,其皂甙含量,生长速率和生物量均要高。最后对细胞的培养方式进行了比较。     

西洋参细胞悬浮培养中皂甙生物合成的代谢调节

七种真菌菌丝体诱导因子中,六种促进培养细胞的皂甙生物合成。尤其葡枝根霉作用更为明显,提高皂甙含量二倍。人参寡糖素是一种既能诱导皂甙生物合成,又能促进细胞生长的新型诱导因子,浓度为50mg／L寡糖素既能维持细胞较好生长又能提高皂甙含量,因而得到较高的皂甙产率。用四种条件培养液进行培养,结果均促进悬浮培养细胞的皂甙生物合成,其中丹参条件培养液使悬浮培养细胞的皂甙含量提高二倍多。加入皂甙生物合成主途径的三种前体,都不同程度地促进皂甙的生物合成,前体中以角鲨烯作用最为明显。     

真菌激发子对人参悬浮培养细胞的生长和人参皂甙生物合成的影响

真菌诱导子处理人参悬浮培养细胞后,人参皂甙的合成有明显增加,诱导处理改变人参皂甙的积累时程,促进人参细胞培养物中次生产物的外泌,同时增强细胞对蔗糖的摄取、吸收并引起细胞H~ 流的变化。     

三七细胞大量（发酵）培养的研究

三七(Panax notoginseng)细胞发酵培养的皂甙含量为干重的11.21%,皂甙产率为1513.3mg·L~(-1),细胞培养物产率为每月13.58g干重·L~(-1),均比悬浮培养高。接种量的增加能明显地提高生长速率、皂甙产率以及细胞培养物产率。通气速率0.8vvm较适合于三七细胞的发酵培养。在发酵培养过程中pH值由5.8逐渐降至3.92,没有出现回升。     

西洋参细胞大量培养的工艺学研究

较适合于植物细胞悬浮培养的培养液体积为三角瓶总容量的1／5至2／5。氧溶浓度的输出随着温度的增加而增加。西洋参细胞培养液的粘性明显大于无细胞培养液。细胞发酵培养较合适的搅拌速度为60rpm。渗透压的增大会明显地提高细胞培养物的皂甙含量,但对细胞的生长不利。本研究为今后设计出适合于西洋参细胞大量培养的发酵罐提供了重要的依据。     

内生真菌和诱导子对长春花悬浮细胞及生物碱合成的影响

目的:探讨内生真茵和诱导子对长春花悬浮细胞生长及生物碱合成的影响.方法:接种内生真茵与悬浮细胞的共同培养,添加诱导子到细胞培养液中对长春花悬浮细胞进行诱导处理,检测实验处理后长春花悬浮细胞各项生化指标.结果:培养液pH值上升,悬浮细胞MDA舍量增加,细胞抗氧化酶(POD、CAT)和生物碱合成的关键性酶(PAL、TDC)活性升高.生物碱产量得到提高,诱导组悬浮细胞生物碱产量达到770.36μg/gFW,共培养组生物碱产量为693.76 μg/gFW,分别比对照组提高了48%和32%.结论:真菌及其诱导子能改变长春花悬浮培养细胞的态势,导致细胞代谢结构的改变,使生物碱的产量提高.     

不同诱导子对枇杷悬浮细胞生长及熊果酸、齐墩果酸含量的影响

在已建立的稳定枇杷细胞悬浮培养系的试验中,研究加入诱导子水杨酸（SA）、茉莉酸甲酯（MJ）、水解酪蛋白（CH）和酵母提取物（YE）对枇杷悬浮细胞生长及次生代谢产物熊果酸（UA）、齐墩果酸（OA）含量的影响。结果表明,从细胞生长、UA及OA含量三方面综合考虑,以MJ为100.0 mg.L^-1时培养最佳,此时细胞生长、UA及OA含量分别为对照的1.081、2.540及2.590倍。     

碳源对K.fragilis LFS-8611β-D-半乳糖苷酶合成的影响

探讨了碳源对脆壁克鲁维酵母(Kluyveromyces fragilis)LFS-8611生长、β-D-半乳糖苷酶合成的影响及碳源对该酶合成的诱导作用。脆壁克鲁维酵母(K,fragilis)LFS-8611生长与β-D-半乳糖苷酶合成同步。该菌株生长和产酶的最适碳源为半乳糖,乳糖次之。菌体生物量和酶活力随着培养基中乳糖浓度的增加而增加,乳糖浓度为12mg／mL,菌体生物量和酶活力达到峰值,分别为5．84g／L、19,12U／mL。半乳糖和乳糖对β-D-半乳糖苷酶合成具有诱导作用。诱导物浓度对β-D-半乳糖苷酶的诱导合成有较大影响。半乳糖诱导以山梨醇为碳源预培养的K．fragilis LFS-8611细胞合成β-D-半乳糖苷酶的最适浓度为10mg／mL。     

Effects of oligosaccharins on callus growth and saponin content of Panax notoginseng

This work provides some evidences for the saponinproduction of Panax notoginseng callus by using biologi-cally active,wall-related oligosaccharins.In anappropriate concentration,three kinds of oligosaccharinsstimulated saponin formation or callus growth.Theconcentration of DO,GO and CO for saponin productionof Panax notoginseng callus culture were 15ppm,15ppmaud 20ppm respectively by comparing saponin yield.Itwas very obvious for DO to increase saponin contentwhen the concentration was 10ppm,and for GO tostimulate callus growth when the concentration was20ppm.It would be a good way to produce saponin byusing oligosaccharins in large scale culture in thefuture.     

Studies on Fermentation Culture of Cells of Panax notoginseng

The saponin content of Panax notoginseng cell cultures-was 11.14% of dry weight, the saponin yield was 1513.3mg· L-1, and yield of cell cultures was 13.58g dry wt · L-1 per month in fermentation culture, which were all better than those obtained from in suspension culture. Increasing inoculum quantity could obviously increase growth rate, saponin content and yield of cell cultures. An aeration rate of 0.8vvm was optimal for fermentation culture of the cells. The pH value of the culture broth went down from 5.80 to 3.92 gradually and never returned in fermentation culture of P. notoginseng cells.     

Enhanced taxol production and release in Taxus chinensis cell suspension cultures with selected organic solvents and sucrose feeding

Suspension culture of Taxus chinensis cells was carried out in aqueous-organic two-phase systems for the production and in situ solvent extraction of taxol (paclitaxel). Three organic solvents, hexadecane, decanol, and dibutylphthalate, were tested at 5-20% (v/v) in the culture liquid. All of these solvents stimulated taxol release and the yield per cell, though decanol and higher concentrations of the other two solvents depressed biomass growth significantly. Ten percent dibutylphthalate was the optimal solvent for improving taxol production and release with minimal cell growth inhibition. The time of solvent addition to the culture also affected taxol production, with the addition during the late-log growth phase being most favorable. By feeding sucrose to the culture near the stationary growth phase, the cell growth and taxol production period was extended from 27 to 42 days. The combining of the two-phase culture and sucrose feeding increased the taxol yield by about 6-fold compared with the single-phase batch culture, to 36.0 +/- 3.5 mg/L, with up to 63% taxol released. This study shows that in situ solvent extraction combined with nutrient feeding is an effective process strategy for production and recovery of secondary metabolites in plant cell suspension culture.     

Improvement of Panax notoginseng cell culture for production of ginseng saponin and polysaccharide by high density cultivation in pneumatically agitated bioreactors

A Panax notoginseng cell culture was successfully scaled up from shake flask to 1.0-L bubble column reactor and concentric-tube airlift reactor. High-density bioreactor batch cultivation was carried out using a modified MS medium. The maximum cell density in batch cultures reached 20.1, 21.0 and 24.1 g/L in the shake flask, bubble column and airlift reactors, respectively, and their corresponding biomass productivity was 950, 1140 and 1350 mg/(L x d) for each. The productivity of ginseng saponin was 70, 96 and 99 mg/(L x d) in the flask, bubble column and airlift reactors, respectively; and the polysaccharide productivity reached 104, 119 and 151 mg/(L x d) for each. Furthermore, a fed-batch cultivation strategy was developed on the basis of specific oxygen uptake rate (SOUR), i.e., sucrose feeding before a sharp decrease of SOUR, and the highest cell density of 29.7 g/L was successfully achieved in the airlift bioreactor on day 17 with a very high biomass productivity of 1520 mg/(L x d). The concentrations of ginseng saponin and polysaccharide reached about 2.1 and 3.0 g/L, respectively, and their productivity was 106 (saponin) and 158 mg/(L x d) (polysaccharide). This work successfully demonstrated the high-density bioreactor cultivation of P. notoginseng cells in pneumatically agitated bioreactors and the reproduction of the shake flask culture results in bioreactors. The cell density, biomass productivity, production titer and productivity of both ginseng saponin and polysaccharide obtained here were the highest that have been reported on a reactor scale for all the ginseng species.     

Effects of elicitation on the production of saponin in cell culture of Panax ginseng

This study was initiated to investigate the impacts of elicitor concentration and elicitor-adding time on the saponin synthesis and the cell growth of Panax ginseng cell suspensions. Both of the elicitors tested, yeast extract and methyl jasmonate, significantly improved saponin production. The highest additive level of the seven ginsenosides tested was 2.07% (dry weight basis), which was 28-fold higher than that in the control. The optimum time to add either elicitor was found to be on the day of inoculation. The addition of either elicitor did not show as significant an influence on cell growth as on saponin production. It was advisable to remove 2,4-dichlorophenoxyacetic acid (2,4-D) from the medium when methyl jasmonate was used as the elicitor as methyl jasmonate interacts antagonistically with 2,4-D. These results suggest that the addition of an elicitor to ginseng cell suspension cultures could stimulate saponin production.     

Heuristic optimization of antibody production by Chinese hamster ovary cells

Large-scale production of monoclonal antibodies necessitates the development of a commercially viable process using the appropriate bioreactors, culture medium, and optimal feeding strategies. In the development of feeding strategies for higher antibody titers it is critical to assess the effects of limiting substrates on cell culture longevity and antibody production. In this study, glucose and L-glutamine were identified as limiting substrates and their effects on culture longevity and antibody production were evaluated in small-scale experiments. The results suggested that an optimal feeding strategy should account for the osmolality profile of the culture. The heuristic approach taken to optimize the antibody production showed that the fed-batch cultivation is superior to batch culture and maintaining low osmolality during growth phase increases cumulative viable cell density and thus leads to higher final antibody titer.     

Studies on Cell Suspension Culture of Panax notoginseng

The optimum period of harvesting in cell suspension culture of Panax notoginseng was 30 days. The time course of sap.nih formation proceeded almost in parallel with the cell growth. An appropriate concentration of oligosaccharms from Panax ginseng, precursor fames.l, mannffol and lysozymum which were added into tbe culture broth 10 days before harvesting, all induced saponin biosynthesis significantly. Oligosaecharins at a concentration of 15ppm(it increased 1 fold of saponin yield, and increased 22.7%(of cell growth rate compared with those of the control) and farnesol at 200ppm(it increased 70.5% of sap.nih yield and stimulated cell growth compared with those of the control) were more effective.     

三七愈伤组织的培养

在MS培养基中加入不同浓度的KT和2,4-D,综合考虑三七愈伤组织生长缓慢兼顾皂甙含量,较合适的KT浓度为0.7ppm,较合适的2,4-D浓度在2—3ppm之间。在培养基中补充各种添加剂,结果以椰子乳和水解乳蛋白较好。综合生长和皂甙含量以20％的椰子乳和0.7％的水解乳蛋白较合适。从21个三七愈伤组织无性繁殖系中筛选山了5个较优的无性系,特别是其中04号无性系更优,无论生长速率还是总皂甙含量都更高。通过以上研究,使三七愈伤组织的生长速率达220毫克/升/天,是原初培养愈伤组织(54.0mg干重/升/天)的4倍。愈伤组织中总皂甙含量高达13％,是原初培养愈伤组织(5.37％)的2.4倍,为原植物的3倍。从而证明了三七培养组织次级代谢的全能性是可调节的,为三七细胞工程的工业生产应用打下了初步基础。