CN106754633A - A kind of regulation and control method of pilot scale culture Herba Saussureae Involueratae cell high yield flavones - Google Patents
A kind of regulation and control method of pilot scale culture Herba Saussureae Involueratae cell high yield flavones Download PDFInfo
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- CN106754633A CN106754633A CN201710079108.8A CN201710079108A CN106754633A CN 106754633 A CN106754633 A CN 106754633A CN 201710079108 A CN201710079108 A CN 201710079108A CN 106754633 A CN106754633 A CN 106754633A
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Abstract
The invention provides a kind of regulation and control method of pilot scale culture Herba Saussureae Involueratae cell high yield flavones, to initial cell strain fast culture first in the way of liquid suspension, once, the increment of cell fresh weight is at 3~5 times for passage in every 7~10 days for the technical scheme；Then use two benches cultivation：Growth phase makes cell fast breeding, obtain maximum cell amount within 7~10 days, the production phase under the conditions of certain 150~400g/l of high density inoculum concentration (fresh weight), adds precursor phenylalanine, derivant coronatine, 35 days harvestings are cultivated, flavones content is improved to more than 13%.This technique uses new derivant coronatine, and consumption is small, and effect is notable, has no toxic side effect, and can rapidly improve the flavones content of saussurea involucrata cell；The present invention is successfully cultivated using the bioreactor of our company in 20L, 100L, 200L, 1000L scale suitable for the Large Scale Biology reactor of more than 20L, and cell state is good, stable yield, and flavones content is more than 11%.
Description
Technical field
The present invention relates to plant cell culture technology field, further to secondary during plant cell pilot scale culture
A kind of induced expression of metabolite, and in particular to regulation and control method of pilot scale culture Herba Saussureae Involueratae cell high yield flavones.
Background technology
Plant cell culture technology is with vitro plant cell or protoplast as object, with cell concentration amplification and target generation
Thank to the culture process for the purpose of product accumulation.For the cell culture for the purpose of obtaining specific metabolite, its is main
Process procedure is not only in that the amplification of cell concentration, meanwhile, the induction synthesis of target metabolic product is even more important to culture benefit.
In the incubation of plant cell, condition of culture and nutritional condition are the most important factors for influenceing yield, and wherein condition of culture is
Refer to the control of the environmental conditions such as temperature, dissolved oxygen, illumination, pH, and nutritional condition refers mainly to the culture medium of plant cell.
Herba Saussureae Involueratae also known as saussurea involucrata, Tianshan Mountains involucre saussurea involucrata, are composite family phoenix hair Dendranthema, are one kind of China alpine region
Rare rare Chinese medicine, containing plurality of active ingredients such as flavones, alkaloid, polysaccharide, with invigorate blood circulation, dispelling cold and removing dampness, strong muscle
The effects such as establishing-Yang, it is used for the treatment of rheumatic arthritis, irregular menstruation, impotence, traumatic injury.But saussurea involucrata growing environment is special
Different, growth cycle is long, the long-term blindness excavation of the mankind, the natural resources of saussurea involucrata is bordering on exhaustion, by the culture next life that suspends
The active ingredient of saussurea involucrata is produced, wild resource can be both protected, growing Chinese medicine demand can be met again.But saussurea involucrata cell is trained
The condition of supporting is harsh, high to nutritional requirement, the in vitro suspension culture techniques of the prior art target although making some progress
The yield of product Xuelianhuangtong is in reduced levels all the time.
In the research of saussurea involucrata cell culture, how to improve main effective medicinal ingredient flavones in culture content and
Quality, Amplification Culture scale, finally realizes industrialized production, is the research emphasis in this field.Domestic and international researcher is in saussurea involucrata
The aspects such as callus tissue culture, cell suspension cultures, fermented and cultured are done a lot of work, but the culture effect of saussurea involucrata cell at present
The yield of fruit and general flavone still has to be hoisted.
Herba Saussureae Involueratae is difficult to artificial growth, and resource is persecuted, and supply falls short of demand；Culture plant cell aspect cell line is unstable
Fixed, secondary metabolite yields poorly, and large-scale production is difficult.At present reported in terms of Herba Saussureae Involueratae megatechnics culture it is less,
The reactor of 10L or so, and the application without two benches culture technique in terms of saussurea involucrata cell production flavones are related only to, regulates and controls skill
Art application is few etc., causes the modulation process for lacking a kind of stable, high-yielding and quick large-scale culture Herba Saussureae Involueratae cell now.
The content of the invention
It is contemplated that for the technological deficiency of prior art, there is provided a kind of pilot scale culture Herba Saussureae Involueratae cell high yield is yellow
The regulation and control method of ketone, is asked with solving the relatively low technology of yield of flavone obtained by the Herba Saussureae Involueratae cell culture processes of prior art
Topic.
Another technical problem to be solved by the present invention is that the Herba Saussureae Involueratae cell culture processes of prior art are difficult to scale
Amplify.
To realize above technical purpose, the present invention uses following technical scheme：
A kind of regulation and control method of pilot scale culture Herba Saussureae Involueratae cell high yield flavones, comprises the following steps：
1) Herba Saussureae Involueratae cell is taken to be inoculated in liquid growth media, with 20~25 DEG C of temperature, daily illumination 14~
18h, 6~10h of dark, the CMC model of 1000~3000lux of intensity of illumination are passed on once, until in nutrient solution for every 7~10 days
Cell fresh weight reaches 3~5 times of initial cell inoculum concentration；
2) take step 1) culture obtained by Herba Saussureae Involueratae cell, liquid growth is inoculated in the inoculum concentration of fresh weight 30-80g/L
In culture medium, with 20~25 DEG C of temperature, the rotating speed shaking table culture of 100~130rpm 5~8 days；Herba Saussureae Involueratae is taken from product thin
Born of the same parents, are inoculated in liquid production culture medium with the inoculum concentration of 150~400g/L of fresh weight, with 24h full exposures, intensity of illumination 2000~
The rotating speed shaking table culture of 3000lux, 100~130rpm 1~4 day；
3) take step 2) culture obtained by Herba Saussureae Involueratae cell, with the inoculum concentration of 50~100g/L of fresh weight be inoculated in liquid give birth to
In culture medium long, with 0.01~1m3The Ventilation Rate culture of/h 5~8 days；Herba Saussureae Involueratae cell is taken from product, with fresh weight 150
The inoculum concentration of~300g/L is inoculated in liquid production culture medium, with 24h full exposures, intensity of illumination 2000~3000lux, 0.01
~1m3The Ventilation Rate culture of/h 1~4 day；
Wherein described growth medium is to contain 0.4~0.6mg/L plant hormones 6-BA, 0.8~1.2mg/L plant hormones
The MS culture mediums of NAA, 20~40g/l sucrose；
The production medium be containing 0.4~0.6mg/L plant hormones 6-BA, 0.8~1.2mg/L plant hormones NAA,
0.1~100 μm of ol/l phenylalanines, 0.5~10 μm of ol/l coronatines, MS culture mediums of 15~25g/l sucrose.
Preferably, step 1) in for be inoculated with Herba Saussureae Involueratae cell be prepared by the following：Take natural snow
Lotus test tube seedling carries out explant induction, obtains callus, utilizes the gamma-rays of Ser 137, uv-B mutagenesis to solid cell, then
0-8 DEG C for the treatment of, recycles the MS solid mediums containing sucrose 50-100g/L, with 20~25 DEG C of temperature, daily 14~18h of illumination,
6~10h of dark, the condition of 1000~3000lux of intensity of illumination is cultivated repeatedly, and screening obtains natural snow lotus cell line.
Preferably, step 1) described in cultivate repeatedly during, subculture is once every two weeks.
Preferably, step 1) in for the Herba Saussureae Involueratae cell that is inoculated with, its yield of flavone is not less than cell gross dry weight
7%.
Preferably, step 2) yield of flavone is not less than the 13% of cell gross dry weight in products therefrom.
Preferably, step 3) in the culture that is carried out using growth medium be in Publication No. CN103224882A or
Carried out in bioreactor disclosed by the Chinese patent literature of CN204385208U, the frequency that shakes of reactor is in incubation
1~20 time/min.
Preferably, step 3) in the culture that is carried out using production medium be in Publication No. CN103224882A or
Carried out in bioreactor disclosed by the Chinese patent literature of CN204385208U, the frequency that shakes of reactor is in incubation
5-20 times/min.
Preferably, step 3) yield of flavone is not less than the 11% of cell gross dry weight in products therefrom.
Preferably, step 2) described in growth medium be contained in 100mL triangular flasks；Step 2) described in produce training
Foster base is contained in 100mL triangular flasks.
Preferably, step 2) described in Herba Saussureae Involueratae cell is taken from product, be that the screen filtration of 50~150 mesh is removed
What nutrient solution was obtained.
Preferably, step 3) in the culture of Herba Saussureae Involueratae cell be in the bioreactor of 20~2000L volumes
Carry out.
The invention provides a kind of regulation and control method of pilot scale culture Herba Saussureae Involueratae cell high yield flavones, the technical scheme is led to
The improvement for crossing training method and inducer composition significantly improves the yield of flavone of cell, while being applicable to putting for scale
Greatly.From the point of view of specifically, the present invention, to initial cell strain fast culture, is passed on once for every 7~10 days first in the way of liquid suspension,
The increment of cell fresh weight is at 3~5 times；Then use two benches cultivation：Growth phase makes cell fast breeding, 7~10 days
Maximum cell amount is obtained, the production phase under the conditions of certain 150~400g/l of high density inoculum concentration (fresh weight), adds precursor benzene
Alanine, derivant coronatine, cultivate 3-5 days harvestings, and flavones content is improved to more than 13%.This technique is lured using new
Agent coronatine is led, consumption is small, effect is notable, has no toxic side effect, and can rapidly improve the flavones content of saussurea involucrata cell；The present invention is suitable
For the Large Scale Biology reactor of more than 20L, the bioreactor of our company has been utilized to be advised in 20L, 100L, 200L, 1000L
Mould is successfully cultivated, and cell state is good, stable yield, and flavones content is more than 11%.
Specific embodiment
Specific embodiment of the invention will be below described in detail.In order to avoid excessive unnecessary details,
Be will not be described in detail to belonging to known structure or function in following examples.In addition to being defined, institute in following examples
Technology and scientific terminology have the identical meanings being commonly understood by with those skilled in the art of the invention.
Embodiment 1 (growth of Herba Saussureae Involueratae cell line and production dynamics are investigated)
The MS culture mediums after screening and optimize through hormone are configured, i.e. growth medium LG contains 0.5mg/l6-BA, 1mg/
LNAA, 30g/l sucrose, 100ml triangle shaking flasks, liquid amount is 20ml, takes Herba Saussureae Involueratae of one plant of culture to a cycle end thin
Born of the same parents are (being named as SE-5), in accessing above-mentioned triangle shaking flask with 40g/l concentration (fresh weight), respectively culture 0 day, 2 days, 4 days, 6
My god, 8 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days detection cell fresh weight and yield of flavone, as a result as shown in table 1.
Growth kinetics performances of the Herba Saussureae Involueratae cell line SE-5 of table 1 in growth medium LG
| Incubation time investigate project | Cell fresh weight (g) | Yield of flavone (μ g) |
| 0 day | 40 | 2.1 |
| 2 days | 45.7 | 2.4 |
| 4 days | 72.5 | 3.6 |
| 6 days | 110.8 | 5.9 |
| 8 days | 159.5 | 7.8 |
| 10 days | 165.2 | 8.2 |
| 12 days | 140.1 | 6.8 |
| 14 days | 100.6 | 5.2 |
| 16 days | 68.3 | 3.5 |
| 18 days | 65.8 | 2.4 |
| 20 days | 53.2 | 1.8 |
Embodiment 2 (realization of the Herba Saussureae Involueratae cell two benches modulation process in lab scale shake flask scale)
It is respectively configured growth medium LG (containing 0.5mg/l 6-BA, 1mg/lNAA, 30g/l sucrose) and production medium
LP (contains 0.5mg/l6-BA, 1.0mg/lNAA, 20g/l, 100umol/l phenylalanine, 5umol/l coronatines, 20g/l sucrose),
In 100ml triangle shaking flasks, equipped with 20mlLG culture mediums, cultivation temperature is 23 DEG C to the SE-5 cells of inoculation 60g/l (fresh weight), is shaken
Bed rotating speed is 120rpm, is cultivated 10 days；Remove nutrient solution using the screen filtration of 100 mesh, inoculation 150/l (fresh weight) cells in
In 100ml triangle shaking flasks, equipped with 20mlLP production mediums, 24h full exposures, intensity of illumination is 2000lux, and rotating speed is
130rpm, cultivates 3 days harvestings.
As a result：By the Herba Saussureae Involueratae cell in grown cultures stage, the double amount of fresh weight is 5 times, and increment reaches 300g/l
(fresh weight), while starting the productive culture of two batches, culture terminates after 3 days, and the flavones content of the saussurea involucrata cell of results is 13%.
Embodiment 3 (realization of the Herba Saussureae Involueratae cell two benches modulation process in pilot scale 1000L reactor scales)
Using disposable bioreactor, (0.5mg/l6-BA, 1.0mg/ are included equipped with 1000L LG growth mediums
LNAA, 30g/l sucrose), for 5 times/min, blowing air is 0.3m to the frequency that shakes3/ h, inoculum concentration is 50g/l, and cultivation cycle is 7 days；Connect
Kind of 200g/l (fresh weight) cells equipped with 1000lLP production mediums in (including 0.5mg/l6-BA, 1.0mg/lNAA, 100umol/
L phenylalanines, 5umol/l coronatines, 20g/l sucrose) disposable biological reaction in, 24h full exposures, intensity of illumination is
2000-3000lux, shake 7 times/min of frequency, and blowing air is 0.6m3/ h, cultivates 5 days harvestings.
As a result：By the Herba Saussureae Involueratae cell in grown cultures stage, the double amount of fresh weight is 4 times, and increment reaches 200g/l
(fresh weight), while the productive culture of startup-batch 1000L reactors, terminates, the flavones of the saussurea involucrata cell of results after cultivating 5 days
Content reaches 11%.
Embodiment 4
A kind of regulation and control method of pilot scale culture Herba Saussureae Involueratae cell high yield flavones, comprises the following steps：
1st, Herba Saussureae Involueratae cell line selection：Explant induction is carried out using natural snow lotus test tube seedling, callus is obtained, it is right
Solid cell carries out Ser-137, uv-B mutagenesis, and 4 DEG C for the treatment of of low temperature, the MS culture mediums containing sugar 50-100g/l high are repeated sieve
Choosing, obtains one plant of Herba Saussureae Involueratae cell line, is named as SE-5, and condition of culture is 23 DEG C, and the photoperiod is 16h/8h, and intensity of illumination is
1000-3000lux, subculture is once every two weeks.
2nd, liquid suspension Herba Saussureae Involueratae cell line SE-5：Select growth conditions good, color is vivid, the solid of quality consolidation
Cell is put into the MS growth mediums LG containing 0.5mg/l6-BA, 1.0mg/lNAA, 20-40g/l sucrose, and condition of culture is
23-25 DEG C, the photoperiod is 16h/8h, and intensity of illumination is 1000-3000lux, is passed on once per 7-10 days, the double amount of cell fresh weight
At 3-5 times, flavones content is more than 7%.
3rd, lab scale modulation process (liquid-liquid two benches cultivation)：Growth phase is the SE-5 for being inoculated with 30-80g/l (fresh weight)
In 100ml triangle shaking flasks, equipped with 20mlLG growth mediums, cultivation temperature is 23 DEG C to cell, and shaking speed is 100-
130rpm, cultivates 7-10 days；Production phase is to remove nutrient solution using the screen filtration of 50-150 mesh, and 150-400/l is (fresh for inoculation
Cell, equipped with 20mlLP production mediums, includes 0.5mg/l6-BA, 1.0mg/ in 100ml triangle shaking flasks again) in culture medium
LNAA, 0-100umol/l phenylalanine, 0.5-10umol/l coronatines, 20g/l sucrose, 24h full exposures, intensity of illumination is
2000-3000lux, rotating speed is 100-130rpm, cultivates 3-5 days harvestings, and now the flavones content of saussurea involucrata cell is more than
13%.
4th, Large Scale Biology reactor modulation process：Using the disposable bioreactor reactor of our company's independent research
(see patent ZL 201210021722.6 and 201420705851.1), equipped with 20-1000L LG growth mediums, the frequency that shakes is for 1-
20 times/min, blowing air 0-1m3/ h, inoculum concentration is 50-100g/l, and cultivation cycle is 7-10 days, flavones content more than 6%；Connect
150-300/l (fresh weight) cells are planted in the disposable biological reaction equipped with 20-1000LP production mediums, is included in culture medium
0.5mg/l6-BA, 1.0mg/lNAA, 100umol/l phenylalanine, 0.5-10umol/l coronatines, 20g/l sucrose, the full light of 24h
According to intensity of illumination is 2000-3000lux, and the frequency that shakes is for 5-20 times/min, blowing air 0-1m3/ h, cultivates 3-5 days harvestings, this
The flavones content of Shi Xuelian cells is more than 11%.
Embodiment 5
A kind of regulation and control method of pilot scale culture Herba Saussureae Involueratae cell high yield flavones, comprises the following steps：
1) Herba Saussureae Involueratae cell is taken to be inoculated in liquid growth media, with 20 DEG C of temperature, daily illumination 14h, dark 6h,
The CMC model of intensity of illumination 1000lux, passes on once for every 7 days, until cell fresh weight reaches initial cell inoculum concentration 3 in nutrient solution
Times；
2) take step 1) culture obtained by Herba Saussureae Involueratae cell, with the inoculum concentration of fresh weight 30g/L be inoculated in 15mL liquid give birth to
In culture medium long, with 20 DEG C of temperature, the rotating speed shaking table culture of 100rpm 5 days；Herba Saussureae Involueratae cell is taken from product, with fresh weight
The inoculum concentration of 150g/L is inoculated in 15mL liquid production culture mediums, with 24h full exposures, intensity of illumination 2000lux, 100rpm's
Rotating speed shaking table culture 1 day；
3) take step 2) culture obtained by Herba Saussureae Involueratae cell, 20L liquid growths are inoculated in the inoculum concentration of fresh weight 50g/L
In culture medium, with 0.01m3The Ventilation Rate culture of/h 5 days；Herba Saussureae Involueratae cell is taken from product, with connecing for fresh weight 150g/L
The amount of kind is inoculated in 20L liquid production culture mediums, with 24h full exposures, intensity of illumination 2000lux, 0.01m3The Ventilation Rate of/h
Culture 1 day；
Wherein described growth medium is to contain 0.4mg/L plant hormone 6-BA, 0.8mg/L plant hormones NAA, 20g/l
The MS culture mediums of sucrose；
The production medium is to contain 0.4mg/L plant hormone 6-BA, 0.8mg/L plant hormones NAA, 0.1 μm of ol/l
Phenylalanine, 0.5 μm of ol/l coronatines, MS culture mediums of 15g/l sucrose.
On the basis of above technical scheme, following condition is met：
Step 1) in for be inoculated with Herba Saussureae Involueratae cell be prepared by the following：Natural snow lotus test tube seedling is taken to enter
Row explant is induced, and obtains callus, and the gamma-rays of Ser 137, uv-B mutagenesis are utilized to solid cell, then 0 DEG C for the treatment of, then
Using the MS solid mediums containing sucrose 50g/L, with 20 DEG C of temperature, daily illumination 14h, dark 6h, intensity of illumination 1000lux's
Condition is cultivated repeatedly, and screening obtains natural snow lotus cell line.
Step 1) described in cultivate repeatedly during, subculture is once every two weeks.
Step 1) yield of flavone is the 7% of cell gross dry weight in products therefrom.
Step 2) described in growth medium be contained in 100mL triangular flasks；Step 2) described in production medium contain
In 100mL triangular flasks.
Step 2) described in Herba Saussureae Involueratae cell is taken from product, be that the screen filtration of 50 mesh is removed nutrient solution and obtained.
Step 1) yield of flavone is the 13% of cell gross dry weight in products therefrom.
Step 3) in the culture that is carried out using growth medium be Chinese patent in Publication No. CN103224882A
Carried out in bioreactor disclosed by document, the frequency that shakes of reactor is for 1 time/min in incubation.
Step 3) in the culture that is carried out using production medium be Chinese patent in Publication No. CN103224882A
Carried out in bioreactor disclosed by document, the frequency that shakes of reactor is for 5 times/min in incubation.
Step 3) yield of flavone is the 11% of cell gross dry weight in products therefrom.
Embodiment 6
A kind of regulation and control method of pilot scale culture Herba Saussureae Involueratae cell high yield flavones, comprises the following steps：
1) take Herba Saussureae Involueratae cell to be inoculated in liquid growth media, with 25 DEG C of temperature, daily illumination 18h, dark
10h, the CMC model of intensity of illumination 3000lux is passed on once, until cell fresh weight connects up to initial cell in nutrient solution for every 10 days
Plant 5 times of amount；
2) take step 1) culture obtained by Herba Saussureae Involueratae cell, with the inoculum concentration of fresh weight 80g/L be inoculated in 25mL liquid give birth to
In culture medium long, with 25 DEG C of temperature, the rotating speed shaking table culture of 130rpm 8 days；Herba Saussureae Involueratae cell is taken from product, with fresh weight
The inoculum concentration of 400g/L is inoculated in 25mL liquid production culture mediums, with 24h full exposures, intensity of illumination 3000lux, 130rpm's
Rotating speed shaking table culture 4 days；
3) take step 2) culture obtained by Herba Saussureae Involueratae cell, 1000L liquid is inoculated in the inoculum concentration of fresh weight 100g/L
In growth medium, with 1m3The Ventilation Rate culture of/h 8 days；Herba Saussureae Involueratae cell is taken from product, with connecing for fresh weight 300g/L
The amount of kind is inoculated in 1000L liquid production culture mediums, with 24h full exposures, intensity of illumination 3000lux, 1m3The Ventilation Rate training of/h
Support 4 days；
Wherein described growth medium is to contain 0.6mg/L plant hormone 6-BA, 1.2mg/L plant hormones NAA, 40g/l
The MS culture mediums of sucrose；
The production medium is to contain 0.6mg/L plant hormone 6-BA, 1.2mg/L plant hormones NAA, 100 μm of ol/l
Phenylalanine, 10 μm of ol/l coronatines, MS culture mediums of 25g/l sucrose.
On the basis of above technical scheme, following condition is met：
Step 1) in for be inoculated with Herba Saussureae Involueratae cell be prepared by the following：Natural snow lotus test tube seedling is taken to enter
Row explant is induced, and obtains callus, and the gamma-rays of Ser 137, uv-B mutagenesis are utilized to solid cell, then 8 DEG C for the treatment of, then
Using the MS solid mediums containing sucrose 100g/L, with 25 DEG C of temperature, daily illumination 18h, dark 10h, intensity of illumination 3000lux
Condition cultivate repeatedly, screening obtain natural snow lotus cell line.
Step 2) described in Herba Saussureae Involueratae cell is taken from product, be that the screen filtration of 150 mesh is removed nutrient solution and obtained.
Step 3) in the culture that is carried out using growth medium be Chinese patent in Publication No. CN204385208U
Carried out in bioreactor disclosed by document, the frequency that shakes of reactor is for 20 times/min in incubation.
Step 3) in the culture that is carried out using production medium be Chinese patent in Publication No. CN204385208U
Carried out in bioreactor disclosed by document, the frequency that shakes of reactor is for 20 times/min in incubation.
Embodiment 7
A kind of regulation and control method of pilot scale culture Herba Saussureae Involueratae cell high yield flavones, comprises the following steps：
1) take Herba Saussureae Involueratae cell to be inoculated in growth medium, with 23 DEG C of temperature, daily illumination 16h, dark 8h, illumination
The CMC model of intensity 2000lux, passes on once for every 8 days, until cell fresh weight reaches 4 times of initial cell inoculum concentration in nutrient solution；
2) take step 1) culture obtained by Herba Saussureae Involueratae cell, with the inoculum concentration of fresh weight 55g/L be inoculated in 20mL growth training
In supporting base, with 23 DEG C of temperature, the rotating speed shaking table culture of 115rpm 6 days；Herba Saussureae Involueratae cell is taken from product, with fresh weight 275g/L
Inoculum concentration be inoculated in 20mL production mediums, with 24h full exposures, the rotating speed shaking table of intensity of illumination 2500lux, 115rpm is trained
Support 3 days；
3) take step 2) culture obtained by Herba Saussureae Involueratae cell, 20L grown cultures are inoculated in the inoculum concentration of fresh weight 75g/L
In base, with 0.5m3The Ventilation Rate culture of/h 7 days；Herba Saussureae Involueratae cell is taken from product, is connect with the inoculum concentration of fresh weight 225g/L
Plant in 20L production mediums, with 24h full exposures, intensity of illumination 2500lux, 0.5m3The Ventilation Rate culture of/h 2 days；
Wherein described growth medium is to contain 0.5mg/L plant hormone 6-BA, 1mg/L plant hormone NAA, 30g/l sugarcanes
The MS culture mediums of sugar；
The production medium is to contain 0.5mg/L plant hormone 6-BA, 1mg/L plant hormones NAA, 500 μm of ol/l benzene
Alanine, 5 μm of ol/l coronatines, MS culture mediums of 20g/l sucrose.
Embodiments of the invention have been described in detail above, but the content is only presently preferred embodiments of the present invention,
It is not intended to limit the invention.All any modification, equivalent and improvement made in application range of the invention etc., all should
It is included within protection scope of the present invention.
Claims (9)
1. a kind of regulation and control method of pilot scale culture Herba Saussureae Involueratae cell high yield flavones, it is characterised in that comprise the following steps：
1) Herba Saussureae Involueratae cell is taken to be inoculated in liquid growth media, it is daily 14~18h of illumination, black with 20~25 DEG C of temperature
Dark 6~10h, the CMC model of 1000~3000lux of intensity of illumination is passed on once, until cell is fresh in nutrient solution for every 7~10 days
3~5 times of the initial cell inoculum concentration that weighs；
2) take step 1) culture obtained by Herba Saussureae Involueratae cell, liquid growth culture is inoculated in the inoculum concentration of fresh weight 30-80g/L
In base, with 20~25 DEG C of temperature, the rotating speed shaking table culture of 100~130rpm 5~8 days；Herba Saussureae Involueratae cell is taken from product, with
The inoculum concentration of 150~400g/L of fresh weight is inoculated in liquid production culture medium, with 24h full exposures, intensity of illumination 2000~
The rotating speed shaking table culture of 3000lux, 100~130rpm 1~4 day；
3) take step 2) culture obtained by Herba Saussureae Involueratae cell, with the inoculum concentration of 50~100g/L of fresh weight be inoculated in liquid growth train
In foster base, with 0.01~1m3The Ventilation Rate culture of/h 5~8 days；Herba Saussureae Involueratae cell is taken from product, with fresh weight 150~
The inoculum concentration of 300g/L is inoculated in liquid production culture medium, with 24h full exposures, intensity of illumination 2000~3000lux, 0.01~
1m3The Ventilation Rate culture of/h 1~4 day；
Wherein described growth medium be containing 0.4~0.6mg/L plant hormones 6-BA, 0.8~1.2mg/L plant hormones NAA,
The MS culture mediums of 20~40g/l sucrose；
The production medium is to contain 0.4~0.6mg/L plant hormones 6-BA, 0.8~1.2mg/L plant hormones NAA, 0.1
~100 μm of ol/l phenylalanines, 0.5~10 μm of ol/l coronatines, MS culture mediums of 15~25g/l sucrose.
2. a kind of regulation and control method of pilot scale culture Herba Saussureae Involueratae cell high yield flavones according to claim 1, its feature
Be step 1) in for be inoculated with Herba Saussureae Involueratae cell be prepared by the following：Taking natural snow lotus test tube seedling is carried out outward
Implant is induced, and obtains callus, and the gamma-rays of Ser 137, uv-B mutagenesis are utilized to solid cell, then 0-8 DEG C for the treatment of, then profit
With the MS solid mediums containing sucrose 50-100g/L, with 20~25 DEG C of temperature, daily 14~18h of illumination, dark 6~10h, light
Condition according to 1000~3000lux of intensity is cultivated repeatedly, and screening obtains natural snow lotus cell line.
3. a kind of regulation and control method of pilot scale culture Herba Saussureae Involueratae cell high yield flavones according to claim 2, its feature
Be step 1) described in cultivate repeatedly during, subculture is once every two weeks.
4. a kind of regulation and control method of pilot scale culture Herba Saussureae Involueratae cell high yield flavones according to claim 1, its feature
Be step 1) in for the Herba Saussureae Involueratae cell that is inoculated with, its yield of flavone is not less than the 7% of cell gross dry weight.
5. a kind of regulation and control method of pilot scale culture Herba Saussureae Involueratae cell high yield flavones according to claim 1, its feature
It is step 2) yield of flavone is not less than the 13% of cell gross dry weight in products therefrom.
6. a kind of regulation and control method of pilot scale culture Herba Saussureae Involueratae cell high yield flavones according to claim 1, its feature
Be step 3) in the culture that is carried out using growth medium be in Publication No. CN103224882A or CN204385208U
Chinese patent literature disclosed by bioreactor in carry out, the frequency that shakes of reactor is for 1~20 time/min in incubation.
7. a kind of regulation and control method of pilot scale culture Herba Saussureae Involueratae cell high yield flavones according to claim 1, its feature
Be step 3) in the culture that is carried out using production medium be in Publication No. CN103224882A or CN204385208U
Chinese patent literature disclosed by bioreactor in carry out, the frequency that shakes of reactor is for 5-20 times/min in incubation.
8. a kind of regulation and control method of pilot scale culture Herba Saussureae Involueratae cell high yield flavones according to claim 1, its feature
It is step 3) yield of flavone is not less than the 11% of cell gross dry weight in products therefrom.
9. a kind of regulation and control method of pilot scale culture Herba Saussureae Involueratae cell high yield flavones according to claim 1, its feature
Be step 3) in the culture of Herba Saussureae Involueratae cell carried out in the bioreactor of 20~2000L volumes.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110923190A (en) * | 2019-11-28 | 2020-03-27 | 大连普瑞康生物技术有限公司 | Method for improving flavone phenylpropanoid compounds of saussurea involucrate cell culture |
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| CN110923190A (en) * | 2019-11-28 | 2020-03-27 | 大连普瑞康生物技术有限公司 | Method for improving flavone phenylpropanoid compounds of saussurea involucrate cell culture |
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