Study On Process Engineering Of Glycyrrhiza Inflata Cell In Large-Scale Culture

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.