Production of Secondary Metabolites By Large-scale Plant Cell Culture

The traditional extraction and processing of plant active ingredients has the following deficiencies: limited by the distribution area of plants; affected by seasons, climate, disease and pest disasters, etc.; occupying a large area. The industry that adopts large-scale plant cell culture technology to produce useful ingredients is mainly concentrated on some compounds with high price, low output and large demand, such as drugs, spices, food additives, pigments, etc. Through the research and development of plant cell biological reaction technology and large-scale reaction equipment, the yield and quality of these secondary metabolites can be improved and their production costs can be reduced.

Principle

Plant cell bioreactor is an essential equipment used in the large-scale cultivation of plant cells. According to different structures, plant cell bioreactors mainly include mechanically stirred bioreactors, air-lift bioreactors, bubble column reactor, packed bed bioreactors, fluidized bed reactors, and hollow fiber reactors. Different bioreactors have different characteristics, and should be designed and selected according to different cell types and characteristics in practical application.

Procedures

Arnebia euchroma cells were cultured to produce naphthoquinone pigment components by a two-step method in a bioreactor.

1. Shaker culture of plant cells
   The calli cultured for about 15 d were transferred to liquid medium for shaker culture. After 2-3 generations of culture, a suspension culture line of Arnebia euchroma cells with rapid growth, uniformity and good dispersion was obtained. AG-7 culture medium, shaker speed 110 r/min, (25±1)℃, dark cultivation.
2. Bioreactor Culture of Plant Cells
   The first step of culturing is to effectively promote the growth of cells. The air-lift bioreactor was used to transfer the cells suspended for 2-3 generations into the reactor, and the growth medium AG-7 was used to culture for about 12 d. The second step of cultivation is to effectively promote the production of shikonin. First filter out the AG-7 growth medium, add M-9 production medium, and harvest after 16 d of cultivation. Samples were taken regularly during the cultivation process for the determination of biomass and other parameters.
3. Collection of Cell Cultures
   Cultured cells were collected by centrifugation and filtration.
4. Extraction of Secondary Metabolites
   Use organic solvent petroleum ether (60-90℃) to extract and concentrate.

Note:

If there is no bioreactor, shake flask culture can be used. After the first step of cultivation, replace the medium in the shake flask with the production medium and continue the cultivation.

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