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Regulation and control method for culturing saussurea involucrata cells in scale manner for high-producing flavone

A cell and snow lotus technology is applied in the field of regulation of high-yield flavonoids in large-scale cultivation of Tianshan snow lotus cells, which can solve problems such as low flavonoid yield, achieve the effects of no toxic and side effects, improve flavonoid yield, and stabilize yield

Inactive Publication Date: 2017-05-31
TIANJIN ACELBIO BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The present invention aims at the technical defects of the prior art, and provides a method for regulating the large-scale cultivation of Saussurea chinensis cells for high-yield flavonoids, so as to solve the technical problem of low yield of flavonoids obtained by the cell culture method of Saussurea chinensis in the prior art

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1 (the investigation of growth and production kinetics of Saussurea tianshanensis cell line)

[0028] Configure the MS medium after hormone screening and optimization, that is, the growth medium LG, containing 0.5mg / l6-BA, 1mg / lNAA, 30g / l sucrose, 100ml Erlenmeyer shaker flask, the liquid volume is 20ml, take one plant for culture The Saussurea tianshanensis cell line (named SE-5) at the end of a cycle was inserted into the above-mentioned Erlenmeyer shaker flasks with a concentration of 40g / l (fresh weight), and cultured for 0 days, 2 days, 4 days, 6 days, and 8 days respectively. The fresh weight and flavonoid production of the cells were detected on the 1st, 10th, 12th, 14th, 16th, 18th, and 20th day, and the results are shown in Table 1.

[0029] Table 1 The growth kinetics of the Saussurea tianshanensis cell line SE-5 in the growth medium LG

[0030] Training time \ inspection project Cell fresh weight (g) Flavonoid production (μg) 0 d...

Embodiment 2

[0031] Embodiment 2 (realization of two-stage regulation and control technology of Saussurea sauraceae cell in small test shake flask scale)

[0032] Prepare growth medium LG (containing 0.5mg / l 6-BA, 1mg / lNAA, 30g / l sucrose) and production medium LP (containing 0.5mg / l6-BA, 1.0mg / lNAA, 20g / l, 100umol / l phenylalanine, 5umol / l coronatine, 20g / l sucrose), inoculate 60g / l (fresh weight) SE-5 cells in a 100ml Erlenmeyer shaker flask with 20mlLG medium, and the culture temperature is 23°C , the shaker speed is 120rpm, cultivated for 10 days; use 100 mesh screen to filter out the culture medium, inoculate 150 / l (fresh weight) cells in a 100ml Erlenmeyer shake flask, equipped with 20ml LP production medium, 24h full light, light The intensity was 2000lux, the rotational speed was 130rpm, and the cells were harvested after 3 days of cultivation.

[0033] Results: After the growth and culture stage, the fresh weight of the Snow Lotus cells in Tianshan Mountains doubled to 5 times, an...

Embodiment 3

[0034] Example 3 (realization of two-stage regulation process of Snow Lotus cell in Tianshan in pilot scale 1000L reactor)

[0035] Disposable bioreactor is used, equipped with 1000L LG growth medium (containing 0.5mg / l6-BA, 1.0mg / lNAA, 30g / l sucrose), the vibration frequency is 5 times / min, and the air is 0.3m 3 / h, the inoculum size is 50g / l, and the culture period is 7 days; inoculate 200g / l (fresh weight) cells in 1000lLP production medium (comprising 0.5mg / l6-BA, 1.0mg / lNAA, 100umol / l benzene Alanine, 5umol / l coronatine, 20g / l sucrose) in a one-time biological reaction, 24h full light, light intensity 2000-3000lux, vibration frequency 7 times / min, air ventilation 0.6m 3 / h, cultivated for 5 days and harvested the cells.

[0036] Results: After the growth and cultivation stage, the fresh weight of Saussurea sauraceae cells doubled to 4 times, and the growth amount reached 200g / l (fresh weight). At the same time, the production and cultivation of a batch of 1000L reactors w...

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Abstract

The invention provides a regulation and control method for culturing saussurea involucrata cells in a scale manner for high-producing flavone. According to the technical scheme, first, an initial cell strain is quickly cultured in a liquid suspension way, passage is carried out once for every 7 to 10 days, and the increments of the fresh weights of cells are 3 to 5 times; subsequently, a two-stage culture method is adopted, the cells are enabled to quickly proliferate at a growth stage, the maximum cell concentration is obtained within 7 to 10 days, a precursor phenylalanine and an inducer coronatine are added into the cells in the condition of a certain high-density inoculum concentration of 150g / l to 400g / l (a fresh weight) at a production stage, the cells are harvested after being cultured for 3 to 5 days, and the flavone content is increased to 13 percent or above. The novel inducer coronatine is adopted for the process; the use level is few; the effect is obvious; no toxic or side effects exist; the flavone content of the saussurea involucrata cells can be quickly improved; the regulation and control method is applicable to a large-scale bioreactor of 20L or larger; the cells are already successfully cultured on scales of 20L, 100L, 200L and 1,000L by utilizing a bioreactor from the company of the inventor, furthermore, the cellular state is good, the yield is stable and the flavone content is 11 percent or above.

Description

technical field [0001] The invention relates to the technical field of plant cell culture, and further relates to the induced expression of secondary metabolites in the process of large-scale plant cell culture, and in particular to a method for regulating the high-yield flavonoids in the large-scale culture of Saussurea chinensis cells. Background technique [0002] Plant cell culture technology is a culture process aimed at the expansion of cell mass and the accumulation of target metabolites based on isolated plant cells or protoplasts. For cell culture for the purpose of obtaining specific metabolites, the main process link is not only the expansion of cell mass, but also the induction of synthesis of target metabolites is more important for culture benefits. In the process of cultivating plant cells, culture conditions and nutritional conditions are the most important factors affecting yield. The culture conditions refer to the control of environmental conditions such a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/04C12P17/06
Inventor 刘晓月张玉洁丁靖志宋静静张桂才王海燕刘永彬张卫
Owner TIANJIN ACELBIO BIOTECH
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