甘草细胞放大培养的过程工程研究
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摘要
甘草是中成药及配方制剂中用量最大的一味药，居国药之首，素有“十方九草”之说。甘草的化学成分复杂，具有多种生物学与药理学活性，广泛应用于食品、保健品、化妆品等行业。近年来野生甘草面临严重的资源问题，而栽培品种周期长、品质低。利用细胞培养技术直接生产植物有效成分是解决野生植物资源短缺有效的方法之一。在实际培养过程中，尚存在很多需要解决的问题，本文以胀果甘草为材料，对甘草细胞大规模培养生产有效成分的可行性、关键问题如甘草细胞悬浮培养体系的优化控制及反应器放大培养等方面开展了较为系统的研究，取得了以下的研究成果：
（1）系统分析了胀果甘草悬浮细胞合成的次生代谢产物。利用HPLC指纹图谱结合相似度评价系统，构建了胀果甘草悬浮细胞提取物的指纹图谱，标定指纹峰22个，与野生甘草提取物的指纹图谱相似度为58.4%；并建立了悬浮细胞中黄酮类化合物的指纹图谱，标定指纹峰16个，与野生甘草中黄酮类化合物指纹图谱相似度为75.2%，采用LC-ESI-MS技术初步确定两个主要黄酮类指纹峰分别为甘草苷与甘草素，同时分别采用HPLC和LC-MS/MS技术检测了悬浮细胞中查尔酮A和甘草酸的合成。
（2）研究了甘草细胞悬浮体系中酶促褐化反应发生的机理及其有效控制策略。甘草细胞固-液转化后受到液体悬浮环境的渗透胁迫和流体剪切压力，引起细胞膜受伤，导致酚类化合物和多酚氧化酶结合造成酶促褐化的发生。对于甘草细胞来说，比较有潜力的PPO抑制剂是抗坏血酸和L-半胱氨酸，当0.1mM的这两种抑制剂分别被添加到悬浮体系中，褐化现象明显得到控制，细胞活力得到维持。
（3）分析了甘草细胞悬浮体系的流变学特性。获得不同发酵时间的流变参数，建立流变本构方程，为甘草细胞放大培养过程的优化操作以及相关的反应器设计和改造提供参考。
（4）建立了甘草细胞在搅拌式生物反应器中的放大培养工艺。并对甘草细胞在反应器中悬浮培养的基本特性进行了研究，评价了反应器特有条件下，细胞活力、生物量积累、过氧化氢产量和甘草黄酮合成的变化情况。分别以单因素实验和正交试验所得数据为样本，运用BP神经网络耦合遗传算法获得反应器中细胞生长的最佳操作参数。经实验验证，这种基于神经网络耦合遗传算法的优化方法与传统的正交试验方法相比，反应器中细胞的生物量积累提高了6.9%。
（5）建立了根癌由农杆菌介导的胀果甘草悬浮细胞遗传转化体系。并利用该体系，将黄酮类合成途径中的一个关键酶基因-查尔酮异构酶基因，通过构建过表达的pCAMBIA1303-chi重组质粒转化到胀果甘草悬浮细胞中，得到一个稳定的、过表达chi基因，黄酮产量提高的转基因甘草细胞系，甘草黄酮的产量平均提高了0.84倍。
Licorice has been used as a traditional Chinese medicine extensively for over2000years. It is one of the largest consumption kinds of herbs in formulations. The chemicalcomposition of licorice is very complex, so it has a variety of biological andpharmacology activities. It is also widely used in the food, cosmetic industries. In recentyears, the yield of the wild licorice is severely reduced owing to the immoderate andruinous utilization, and cultivated licorice is poor quality and long growth cycle. As analternative approach, cell suspension systems are generally considered as the most suitablemethod for large-scale applications to produce valuable secondary metabolites. But thereare still a lot of problems to solve in the process of culture, the present study mainlyfocused on optimization of licorice cell suspension culture system and large-scale cellculture in bioreactor, etc. The main results were shown as follows:
(1) The secondary metabolites produced from the G.inflata suspension cell wereanalyzed. The fingerprint with22fingerprint peaks of G.inflata suspension cells extractswas constructed by HPLC fingerprint linking with the similarity evaluation system, andthe similarity is58.4%compared with the fingerprint of wild licorice extracts. Using thesame method, the fingerprint was established with16fingerprint peaks of flavonoidsextracted from G.inflata suspension cells, and the similarity is75.2%compared with thefingerprint of wild licorice flavonoids extracts. The two main fingerprints among commonHPLC peaks were identified as liquiritin and liquiritigenin by LC-ESI-MS technology. Inaddition, the content of licochalcone A in suspension cells of G.inflata was determined byHPLC. Glycyrrhizin extracted from G.inflata suspension cells was also analyzed byLC-MS/MS using purchased Glycyrrhizin as standard sample.
(2) At the beginning of the establishment of G.inflata cell suspension culture system,when the cells was transferred from solid culture medium to liquid medium, osmotic stressand hydrodynamics shear from the liquid culture environment cause cell membrane injury,then phenolic compounds and polyphenol oxidase would react resulting in enzymaticbrowning. For G.inflata cells, more potential PPO inhibitors are ascorbic acid andL-homocysteine, when0.1mM of these two kinds of inhibitors were added to the suspension system, the browning was controlled obviously.
(3) The rheological characteristics of G.inflata cells suspension culture system wasstudied, the rheological parameters of different fermentation time were obtained andrheological constitutive equation was established, these results provide the reference foroptimized operation and bioreactor design of licorice cells amplifying culture.
(4) G. inflata cells was cultured in the stirred tank bioreactor successfully, and thebasic characteristics of G. inflata cells cultured in bioreactor were studied (control withculture in Erlenmeyer flasks), including the changes of the cell vitality, biomassaccumulation, hydrogen peroxide production and flavonoids synthetic under the bioreactorcondition. Using single factor and orthogonal test data as samples respectively, the bestoperating conditions was obtained by neural network coupling genetic algorithm. Theresults indicated that the optimization method based on neural network coupling geneticalgorithm improved the accumulation of cells biomass in bioreactor compared with thetraditional orthogonal test method.
(5) In this study, the transformation protocol for G. inflata suspension cells wassuccessfully developed with A. tumefaciens strain LBA4404harboring the binary vectorpCAMBIA1303by optimizing several important parameters of the transformation system.The chi gene was introduced into G. inflata suspension cells by the transformationprotocol to overexpress the chi gene, which encodes a key regulatory enzyme in theflavonoid synthesis pathway, resulting in a stable, maintainable, flavonoid-yield-increasedtransgenic G. inflata cell line. The study provides an effective approach to efficientlyincrease the end products of secondary metabolic pathways by appropriate geneticengineering strategy.
（1）系统分析了胀果甘草悬浮细胞合成的次生代谢产物。利用HPLC指纹图谱结合相似度评价系统，构建了胀果甘草悬浮细胞提取物的指纹图谱，标定指纹峰22个，与野生甘草提取物的指纹图谱相似度为58.4%；并建立了悬浮细胞中黄酮类化合物的指纹图谱，标定指纹峰16个，与野生甘草中黄酮类化合物指纹图谱相似度为75.2%，采用LC-ESI-MS技术初步确定两个主要黄酮类指纹峰分别为甘草苷与甘草素，同时分别采用HPLC和LC-MS/MS技术检测了悬浮细胞中查尔酮A和甘草酸的合成。
（2）研究了甘草细胞悬浮体系中酶促褐化反应发生的机理及其有效控制策略。甘草细胞固-液转化后受到液体悬浮环境的渗透胁迫和流体剪切压力，引起细胞膜受伤，导致酚类化合物和多酚氧化酶结合造成酶促褐化的发生。对于甘草细胞来说，比较有潜力的PPO抑制剂是抗坏血酸和L-半胱氨酸，当0.1mM的这两种抑制剂分别被添加到悬浮体系中，褐化现象明显得到控制，细胞活力得到维持。
（3）分析了甘草细胞悬浮体系的流变学特性。获得不同发酵时间的流变参数，建立流变本构方程，为甘草细胞放大培养过程的优化操作以及相关的反应器设计和改造提供参考。
（4）建立了甘草细胞在搅拌式生物反应器中的放大培养工艺。并对甘草细胞在反应器中悬浮培养的基本特性进行了研究，评价了反应器特有条件下，细胞活力、生物量积累、过氧化氢产量和甘草黄酮合成的变化情况。分别以单因素实验和正交试验所得数据为样本，运用BP神经网络耦合遗传算法获得反应器中细胞生长的最佳操作参数。经实验验证，这种基于神经网络耦合遗传算法的优化方法与传统的正交试验方法相比，反应器中细胞的生物量积累提高了6.9%。
（5）建立了根癌由农杆菌介导的胀果甘草悬浮细胞遗传转化体系。并利用该体系，将黄酮类合成途径中的一个关键酶基因-查尔酮异构酶基因，通过构建过表达的pCAMBIA1303-chi重组质粒转化到胀果甘草悬浮细胞中，得到一个稳定的、过表达chi基因，黄酮产量提高的转基因甘草细胞系，甘草黄酮的产量平均提高了0.84倍。
Licorice has been used as a traditional Chinese medicine extensively for over2000years. It is one of the largest consumption kinds of herbs in formulations. The chemicalcomposition of licorice is very complex, so it has a variety of biological andpharmacology activities. It is also widely used in the food, cosmetic industries. In recentyears, the yield of the wild licorice is severely reduced owing to the immoderate andruinous utilization, and cultivated licorice is poor quality and long growth cycle. As analternative approach, cell suspension systems are generally considered as the most suitablemethod for large-scale applications to produce valuable secondary metabolites. But thereare still a lot of problems to solve in the process of culture, the present study mainlyfocused on optimization of licorice cell suspension culture system and large-scale cellculture in bioreactor, etc. The main results were shown as follows:
(1) The secondary metabolites produced from the G.inflata suspension cell wereanalyzed. The fingerprint with22fingerprint peaks of G.inflata suspension cells extractswas constructed by HPLC fingerprint linking with the similarity evaluation system, andthe similarity is58.4%compared with the fingerprint of wild licorice extracts. Using thesame method, the fingerprint was established with16fingerprint peaks of flavonoidsextracted from G.inflata suspension cells, and the similarity is75.2%compared with thefingerprint of wild licorice flavonoids extracts. The two main fingerprints among commonHPLC peaks were identified as liquiritin and liquiritigenin by LC-ESI-MS technology. Inaddition, the content of licochalcone A in suspension cells of G.inflata was determined byHPLC. Glycyrrhizin extracted from G.inflata suspension cells was also analyzed byLC-MS/MS using purchased Glycyrrhizin as standard sample.
(2) At the beginning of the establishment of G.inflata cell suspension culture system,when the cells was transferred from solid culture medium to liquid medium, osmotic stressand hydrodynamics shear from the liquid culture environment cause cell membrane injury,then phenolic compounds and polyphenol oxidase would react resulting in enzymaticbrowning. For G.inflata cells, more potential PPO inhibitors are ascorbic acid andL-homocysteine, when0.1mM of these two kinds of inhibitors were added to the suspension system, the browning was controlled obviously.
(3) The rheological characteristics of G.inflata cells suspension culture system wasstudied, the rheological parameters of different fermentation time were obtained andrheological constitutive equation was established, these results provide the reference foroptimized operation and bioreactor design of licorice cells amplifying culture.
(4) G. inflata cells was cultured in the stirred tank bioreactor successfully, and thebasic characteristics of G. inflata cells cultured in bioreactor were studied (control withculture in Erlenmeyer flasks), including the changes of the cell vitality, biomassaccumulation, hydrogen peroxide production and flavonoids synthetic under the bioreactorcondition. Using single factor and orthogonal test data as samples respectively, the bestoperating conditions was obtained by neural network coupling genetic algorithm. Theresults indicated that the optimization method based on neural network coupling geneticalgorithm improved the accumulation of cells biomass in bioreactor compared with thetraditional orthogonal test method.
(5) In this study, the transformation protocol for G. inflata suspension cells wassuccessfully developed with A. tumefaciens strain LBA4404harboring the binary vectorpCAMBIA1303by optimizing several important parameters of the transformation system.The chi gene was introduced into G. inflata suspension cells by the transformationprotocol to overexpress the chi gene, which encodes a key regulatory enzyme in theflavonoid synthesis pathway, resulting in a stable, maintainable, flavonoid-yield-increasedtransgenic G. inflata cell line. The study provides an effective approach to efficientlyincrease the end products of secondary metabolic pathways by appropriate geneticengineering strategy.
引文
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