CN104509441B - Dynamic equalization alimentation post-directed training ginseng adventitious root system - Google Patents
Dynamic equalization alimentation post-directed training ginseng adventitious root system Download PDFInfo
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Abstract
The invention discloses dynamic equalization alimentation and cultivate the bioreactor of ginseng adventitious root, it includes: flow container is added in initial medium retort, air pressure pump and nutrition；Initial medium retort is bubble type biological reactor, and reaction cover is additionally provided with nutrition annex solution dropping mouth；Described nutrition annex solution pot bottom is provided with band regulation valve discharging opening, top is provided with cover and band filtering air inlet, and nutrition annex solution dropping mouth is connected with discharging opening by pipeline；Additionally provide bioreactor and produce ginseng adventitious root system, it is to be made up of 1 nutrition interpolation flow container and 3 initial medium retort, this bioreactor, can in time drip nutrition annex solution, increase saponin content, particularly can add the culture medium of the high concentration sucrose of flow container, the sucrose concentration of dynamic equilibrium retort with nutrition, the addition that can strengthen sucrose improves adventitious root yield, the problem solving the suppression adventitious root growth of sucrose concentration height.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of dynamic equalization alimentation and train ginseng adventitious root system for a certain area
System.
Background technology
Radix Ginseng (Panax ginsengC.A.Meyer) it is Araliaceae (Araliacede) Panax (Panax
L.) plant, is one of well-known " northeast three essentials-essence ", and the rare Chinese medicine that China is traditional especially has won fame both at home and abroad.The Ming Dynasty of China
Effect of Radix Ginseng, in its epic monumental work Compendium of Material Medica one book, has been made detailed by the physician of greatness, pharmacy man Li Shizhen (1518-1593 A.D.)
Introduction, established the good reputation of Radix Ginseng " king of BAICAO ".Radix Ginseng is grown on that day and night temperature is little, height above sea level is 500～1100 meters more
In the hillside of Coniferous forest or mixed forest, its main producing region is mainly distributed on 40 °～45 ° (N), between 117.5 °～134 ° (E),
China is mainly distributed on the area, mountain region of three provinces in the northeast of China and northern Hebei, especially with Mountain area, Changbai as main producing region.China is
One of research Radix Ginseng country the earliest, describes Radix Ginseng in detail in the pharmacology monograph Shennong's Herbal of earliest extant and has
" tonifying five ZANG-organs, peace spirit, determine soul, spasmolytic is throbbed with fear, evil removing gas, eyesight-improving intelligence-developing, for a long time take to make light of one's life by commiting suicide and prolong life " effect[7].Modern pharmacology
Research shows, Radix Ginseng has strongly invigorating primordial QI, multiple arteries and veins takes off admittedly, effect of invigorating the spleen to benefit the lung, promoting the production of body fluid and inducing sedation of the mind, and Radix Ginseng has the anti-rhythm of the heart simultaneously
Many pharmacological activities such as not normal, anti-hemolysis, regulation immune system, regulation central nervous system[8].The chemistry contained in Radix Ginseng
Component comparison is complicated, in addition to containing ginsenoside, possibly together with the active component of multiple non-saponin in Radix Ginseng, as containing saccharide
(monosaccharide, oligosaccharide, oligosaccharide, polysaccharide etc.), aminoacid, fatty acid, pyrite, vitamin (VB1, VB2, VB12, nicotinic acid, pantothenic acid etc.)
And the component such as volatile oil.Along with modern pharmacology in recent years, the deepening continuously of pharmacokinetic, the medical value of Radix Ginseng and
Its health-care effect has obtained certification further, and constantly rising violently in price and the status of Radix Ginseng, nowadays Radix Ginseng serves not only as the name of China
Your Chinese herbal medicine, has obtained being widely recognized as of the people of the world especially.
Ginsenoside has good pharmacologically active and health-care effect, shows that ginsenoside is for heart and brain through pharmaceutical research
Blood vessel, central nervous system, immune system, digestive system etc. have good pharmacological activity.And for monomer ginsenoside
The research of pharmacological activity is the most active, the rare ginsenoside that particularly some content are little, has high medical value.
Owing to Radix Ginseng is considered treasure for a long time, wild resource is the most exhausted, and artificial culture occupying woodland causes water and soil stream
Losing, cycle length, management cost height, disease are serious.
The advantage that ginseng-cell is cultivated, root is cultivated is that it has the huge biosynthesis energy that primordial plant root is had
Power, in root cultivation, the height differentiation of cell makes the synthesis capability of secondary metabolite significantly improve, and table goes out obvious generation simultaneously
Thank to stability.
The inducing culture technology of ginseng adventitious root reaches its maturity, commonly used, washes fresh of perennial Radix Ginseng
Only, sterilize, cut after in the MS solid medium containing 2,4-D, KT and sucrose, carry out callus induction.To induce again
Callus move to the MS of IBA and sucrose induce, breeds adventitious root.
Cultivating compared with ginseng-cell, the advantage that root is cultivated is that it has the huge biosynthesis that primordial plant root is had
Ability, in root cultivation, the height differentiation of cell makes the synthesis capability of secondary metabolite significantly improve, and table goes out significantly simultaneously
Metabolic stability and the stability of vigor.
Ginseng adventitious root bioreactor culture, commonly used sucrose provides carbon source, along with the increase of sucrose concentration, Radix Ginseng
The yield of adventitious root is consequently increased, and in culture medium the concentration of sucrose 60g more than L, have suppression to make to the growth of adventitious root
With.
Breeding ginseng adventitious root for bioreactor culture at present, most employing will prepare culture medium and disposably add
In reactor, inoculate adventitious root, cultivate exhausting to nutrition.Results adventitious root.And the mistake of bioreactor culture adventitious root
Cheng Zhong, is a dynamic growth course, grows each stage and is not quite similar the demand of nutrition, the battalion of culture medium theoretically
Forming part is a process gradually decreased.Disposably add culture medium, it is impossible to the concrete needs adapting to each growth stage.
Having been reported that and use same culture medium, adding in reactor at twice or three times, number of times is the most, and growth coefficient is more
Little.
Summary of the invention
It is an object of the invention to as solving bioreactor culture ginseng adventitious root, the unbalanced problem of each Stage Nutrition,
Improve yield and the content of secondary metabolite of ginseng adventitious root, and provide dynamic equalization alimentation post-directed training Radix Ginseng indefinite
The bioreactor of root.
Dynamic equalization alimentation trains the bioreactor of ginseng adventitious root for a certain area, and it includes: initial medium retort
4, flow container 6 is added in air pressure pump 5 and nutrition；Initial medium retort 4 is bubble type biological reactor, is provided with gas bottom it
Dynamic device, reaction cover 403 is additionally provided with nutrition annex solution dropping mouth 401；Air pressure pump 5 is connected with pneumatic means；Described
Be provided with band regulation valve discharging opening 64 bottom nutrition interpolation flow container 6, top is provided with cover 62 and band filtering air inlet 61, air inlet
61 are located on cover 62, and nutrition annex solution dropping mouth 401 is connected with discharging opening 64 by pipeline 7；
The shower nozzle of pneumatic means is many micropores shower nozzle 405；
A diameter of the 2.0～0.2mm of described nutrition annex solution dropping mouth 401；
Described nutrition is added the air inlet 61 of flow container 6 and is connected by air inlet regulating device with air pressure pump 5.
Another object of the present invention is to provide dynamic equalization alimentation post-directed training ginseng adventitious root system, and it includes: 1
Flow container 6 and 3 initial medium retort 4 are added in nutrition, and the discharging opening 64 that flow container 6 is added in nutrition is anti-with 3 initial mediums
The foster annex solution dropping mouth 401 answering tank 4 connects；
Described initial medium retort 4 pneumatic means, nutrition add the air inlet 61 of flow container 6 by pipeline and air
Compression pump is connected；
Described initial medium retort 4 and nutrition are added flow container 6 and are 18L, and initial medium addition is 8-
10L, the addition of nutrition annex solution is 12-15L；
Described initial medium cane sugar content is 20-35g/L, and nutrition annex solution cane sugar content is 90-120g/L.
The invention provides dynamic equalization alimentation and cultivate the bioreactor of ginseng adventitious root, it includes: initial incubation
Flow container is added in base retort, air pressure pump and nutrition；Initial medium retort is bubble type biological reactor, reacts cover
It is additionally provided with nutrition annex solution dropping mouth；Described nutrition annex solution pot bottom is provided with band regulation valve discharging opening, top is provided with cover
With band filtering air inlet, nutrition annex solution dropping mouth is connected with discharging opening by pipeline；Additionally provide dynamic equalization alimentation
Cultivating ginseng adventitious root system, it is to be made up of 1 nutrition interpolation flow container and 3 initial medium retort, and described is initial
Culture medium retort and nutrition are added flow container and are 18L, and initial medium addition is 8-10L, the addition of nutrition annex solution
For 12-15L, this bioreactor, can in time drip nutrition annex solution, increase saponin content, particularly can use high concentration sugarcane
The culture medium of sugar, the sucrose concentration of dynamic equilibrium retort, the addition that can strengthen sucrose improves adventitious root yield, solves sugarcane
The problem of sugar concentration height suppression adventitious root growth.Initial medium retort and nutrition are added flow container and are 18L, initial medium
Addition is 8-10L, and the addition of nutrition annex solution is the culture systems of 12-15L, provides optimal life for ginseng adventitious root
Long environment.
Accompanying drawing explanation
Fig. 1 is that the initial medium retort of the bioreactor of dynamic equalization alimentation post-directed training ginseng adventitious root is shown
It is intended to；
Fig. 2 is that flow container signal is added in the nutrition of the bioreactor of dynamic equalization alimentation post-directed training ginseng adventitious root
Figure；
Fig. 3,4 be dynamic equalization alimentation post-directed training ginseng adventitious root system schematic.
Detailed description of the invention
The acquisition of embodiment 1 ginseng adventitious root
Choosing the 6 years raw Park Ginseng in Changbaishan area (An County, fair, Tonghua, Jilin Province), stripping and slicing, by MS culture medium callus induction group
Knit, by induction callus with 34 SH culture medium+IBA5.0mg/L+ sucrose 30g/L, inducing adventitious root, successive transfer culture.
Embodiment 2 initial medium and the preparation of nutrition annex solution
1, the preparation of culture medium
1) initial medium
3/4SH culture medium+butyric acid potassium (K-IBA) 3mg/L+ sucrose 30g/L
2) nutrition annex solution
Nutrition annex solution (1):
In every liter of 3/4SH culture medium, add naphthalene acetic acid (C12H10O2) 0.5-0.9mg × 3/L, (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate
(C13H20O3) 50-80mg × 3/L, salicylic acid 50-80 mg × 3/L, sucrose (C12H22O11) 30g × 3/L.
Nutrition annex solution (2):
In every liter of 3/4SH culture medium, add naphthalene acetic acid (C12H10O2) 0.5-0.9g × 3/L, (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate
(C13H20O3) 50-80g × 3/L, salicylic acid 50-80, sucrose (C12H22O11) 40g × 3/L.
Embodiment 3 bioreactor dynamic equalization alimentation post-directed training ginseng adventitious root
1) adding in 18L bubble type biological reactor by initial medium 12L, nutrition annex solution 6L adds in liquid replenisher,
Bioreactor and liquid replenisher sterilizing under the conditions of temperature 120 DEG C, pressure 0.08Mpa1；
2) adventitious root cuts 1cm block, is inoculated in biological reactor；
3) ventilation, 23 ± 2 DEG C of cultivations；
4) cultivate by the 10th day, drip nutrition annex solution (1) 6L, within 3-5 days, drip off；
5) continue to cultivate 42 days, gather in the crops adventitious root.
Embodiment 4 bioreactor dynamic equalization alimentation post-directed training ginseng adventitious root
1) adding in 18L bubble type biological reactor by initial medium 10L, nutrition annex solution 5L adds in filling tank,
Bioreactor and filling tank sterilizing under the conditions of temperature 120 DEG C, pressure 0.08Mpa1；
2) adventitious root cuts 1cm block, is inoculated in biological reactor；
3) ventilation, 23 ± 2 DEG C of cultivations；
4) cultivate by the 5th day, drip nutrition annex solution (2) 2L to biological reactor, drip 1 day, cultivate by the 10th day, drip
Ensure Liquid annex solution (2) 3L, drips off for 4 days；Ventilation is strengthened during dropping.
5) continue to cultivate 42 days, gather in the crops adventitious root.
The bioreactor of embodiment 5 dynamic equalization alimentation post-directed training ginseng adventitious root
Refer to Fig. 1,2, dynamic equalization alimentation post-directed training ginseng adventitious root bioreactor, it includes: just
Flow container 6 is added in beginning culture medium retort 4, air pressure pump 5 and nutrition；Initial medium retort 4 is bubble type biological respinse
Tank, is provided with pneumatic means bottom it, pneumatic means includes many micropores shower nozzle 405, retort pneumatic means interface 406, air mistake
Filter 407, mass air flow sensor 408, air intake control valve 409, top sets reaction cover 403 and band filter exhaust mouth 402, band mistake
Filter air vent 402 is located on reaction cover 403, and reaction cover 403 is additionally provided with nutrition annex solution dropping mouth 401；Air pressure
Pump 5 is connected with pneumatic means；
Be provided with band regulation valve discharging opening 64 bottom described nutrition interpolation flow container 6, top is provided with cover 62 and band filter
Air inlet 61, air inlet 61 is located on cover 62, and nutrition annex solution dropping mouth 401 is connected with discharging opening 64 by pipeline 7；
A diameter of the 2.0～0.2mm of described nutrition annex solution dropping mouth 401；
Described nutrition is added the air inlet 61 of flow container 6 and is connected by air inlet regulating device with air pressure pump 5.
During use, closing each terminal valve, added by 10L initial medium in initial medium retort 4, nutrition is added
Flow container 6 adds 5L nourishing additive agent, and nutrition is added the discharging opening 64 of flow container 6 and is connected with retort nutrition annex solution dropping mouth 401,
Put into high-pressure sterilizing pot inner high voltage sterilizing, be inoculated in sterilizing room in bubble type biological reactor 4, move to culturing room, start
Air pressure pump, opens band filter exhaust mouth 402 and air shut-off valve, and the air after filtration sprays through many micropores shower nozzle 405,
Adventitious root is suspended in culture medium, and air is discharged through air vent 42,23 ± 2 DEG C of cultivations, in cultivating 5-10 days, opens nutrition and adds
The band filtering air inlet 61 of flow container 6 and band regulation valve discharging opening 64, regulation charge flow rate and discharging opening regulation valve, control dropping
Speed, drips nutrition annex solution, drips off within 3-5 day.
Embodiment 6 dynamic equalization alimentation post-directed training ginseng adventitious root system
Referring to Fig. 1,2,3,4, dynamic equalization alimentation trains ginseng adventitious root for a certain area, and it includes: 1 nutrition annex solution
Tank 6 and 3 initial medium retort 4, nutrition is added the discharging opening 64 of flow container 6 and is added with the foster of 3 initial medium retort 4
Liquid feeding dropping mouth 401 connects；
Initial medium retort 4 air intake installation, nutrition add the air inlet 61 of flow container 6 by pipeline and air pressure pump
It is connected；
Described bubble type biological reactor 4 and nutrition are added flow container 6 and are 18L, and initial medium addition is 8-
10L, the addition of nutrition annex solution is 12-15L.
Embodiment 7 bioreactor dynamic equalization orientation alimentation cultivates ginseng adventitious root yield and the mensuration of saponin content
1, with embodiment 4,5 as experimental group, with 3/4SH culture medium+IBA5mg/L+ sucrose 50g/L for culture medium routine side
Formula is cultivated as compareing, found that:
Matched group starts by the 10th day from cultivation, and adventitious root increasess slowly, but 10-20 days rapid developments, 20-30 days also in
Growth trend, but slower；Within 30-40 days, do not find to increase.
Cultivate ginseng adventitious root method by embodiment 4 bioreactor dynamic equalization alimentation and cultivate adventitious root, from cultivation
Starting by the 10th day, within the 10th-20 day, increase than matched group and dramatically speed up, the is slightly above matched group for 20-25 days, and 25-40 days with several
Do not increase.
Cultivate ginseng adventitious root method by embodiment 5 bioreactor dynamic equalization alimentation and cultivate adventitious root, each stage
Relatively comparison and embodiment 4 growth significantly improve, until 35 days have growth trend, within 35 days-42 days, increase hardly.
Embodiment 4 relatively matched group dry weight slightly improves, and nearly 5%, embodiment 5 improves 20%.
2, will cultivate 6 weeks ginseng adventitious roots by embodiment 4 method, and send Ginseng In Jilin Province inspection center to detect, result is shown in
Table 2
Claims (7)
1. the bioreactor of dynamic equalization alimentation post-directed training ginseng adventitious root, it is characterised in that it includes: initial incubation
Flow container (6) is added in base retort (4), air pressure pump (5) and nutrition；Initial medium retort (4) is that bubble type is biological anti-
Answering tank, be provided with pneumatic means bottom it, reaction cover (403) is additionally provided with nutrition annex solution dropping mouth (401)；Air pressure pump
(5) it is connected with pneumatic means；Described nutrition interpolation flow container (6) bottom is provided with band regulation valve discharging opening (64), top is provided with tank
Lid (62) and band filtering air inlet (61), air inlet (61) is located on cover (62), and nutrition annex solution dropping mouth (401) passes through
Pipeline (7) is connected with discharging opening (64)；
Initial medium is 3/4SH culture medium+butyric acid potassium 3mg/L+ sucrose 30g/L；
Nutrition annex solution is addition naphthalene acetic acid 0.5-0.9mg × 3/L, (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate 50-in every liter of 3/4SH culture medium
80mg × 3/L, salicylic acid 50-80 mg × 3/L, sucrose 30g × 3/L.
The bioreactor of dynamic equalization alimentation the most according to claim 1 post-directed training ginseng adventitious root, its feature
Being, the shower nozzle of described pneumatic means is many micropores shower nozzle (405).
The bioreactor of dynamic equalization alimentation the most according to claim 1 and 2 post-directed training ginseng adventitious root, it is special
Levy and be, a diameter of the 2.0～0.2mm of described nutrition annex solution dropping mouth (401).
The bioreactor of dynamic equalization alimentation the most according to claim 3 post-directed training ginseng adventitious root, its feature
Being, described nutrition is added the air inlet (61) of flow container (6) and is connected by air inlet regulating device with air pressure pump (5).
5. bioreactor produces ginseng adventitious root system, it is characterised in that it includes: initial medium retort (4), air
Flow container (6) is added in compression pump (5) and nutrition；Initial medium retort (4) is bubble type biological reactor, is provided with gas bottom it
Dynamic device, reaction cover (403) is additionally provided with nutrition annex solution dropping mouth (401)；Air pressure pump (5) is connected with pneumatic means；
Described nutrition interpolation flow container (6) bottom is provided with band regulation valve discharging opening (64), top is provided with cover (62) and band filter enters
QI KOU (61), air inlet (61) is located on cover (62), and nutrition annex solution dropping mouth (401) is by pipeline (7) and discharging opening
(64) it is connected；It is 1 that flow container (6) is added in described nutrition, and described initial medium retort (4) is 3, nutrition annex solution
The discharging opening (64) of tank (6) drips mouth (401) with the nutrition annex solution of 3 initial medium retort (4) and is connected；Initial incubation
The pneumatic means of base retort (4) is connected with air pressure pump by pipeline (7)；
Initial medium is 3/4SH culture medium+butyric acid potassium 3mg/L+ sucrose 30g/L；
Nutrition annex solution is addition naphthalene acetic acid 0.5-0.9mg × 3/L, (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate 50-in every liter of 3/4SH culture medium
80mg × 3/L, salicylic acid 50-80 mg × 3/L, sucrose 30g × 3/L.
Bioreactor the most according to claim 5 produces ginseng adventitious root system, it is characterised in that described nutrition adds
The air inlet (61) adding flow container (6) is connected with air pressure pump by pipeline.
7. produce ginseng adventitious root system according to the bioreactor described in claim 5 or 6, it is characterised in that described drum
Bubble biological reactor (4) and nutrition are added flow container (6) and are 18L, and initial medium addition is 8-10L, nutrition annex solution
Addition be 12-15L.
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| CN104996303A (en) * | 2015-08-20 | 2015-10-28 | 固元本草汉方生物科技股份有限公司 | Efficient reactor for cultivating ginseng adventitious roots |
| CN112931216B (en) * | 2021-04-02 | 2022-08-05 | 山东安然纳米实业发展有限公司 | Biological reaction device and culture method for ginseng adventitious roots |
| CN112806267B (en) * | 2021-04-02 | 2022-08-02 | 山东安然纳米实业发展有限公司 | Method for culturing ginseng adventitious roots by using biological reaction device |
| CN115053810B (en) * | 2022-06-29 | 2023-04-11 | 珲春华瑞参业生物工程股份有限公司 | Culture method for simultaneously increasing contents of multiple ginsenosides in ginseng adventitious roots |
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| CN102559491A (en) * | 2010-10-14 | 2012-07-11 | 杨凯 | A kind of bubble bioreactor for tissue culture of lily bulbs |
| CN102408991B (en) * | 2011-07-28 | 2014-11-05 | 东北林业大学 | Bioreactor system for large scale culture of medicinal plants |
| CN103141394A (en) * | 2013-03-28 | 2013-06-12 | 延边大学 | Method for culturing adventitious roots of oplopanax elatus Nakai by utilizing bioreactor |
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