CN113046292B - Method for culturing ginseng stem cells by using biological reaction device - Google Patents

Method for culturing ginseng stem cells by using biological reaction device Download PDF

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CN113046292B
CN113046292B CN202110361867.XA CN202110361867A CN113046292B CN 113046292 B CN113046292 B CN 113046292B CN 202110361867 A CN202110361867 A CN 202110361867A CN 113046292 B CN113046292 B CN 113046292B
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ginseng
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stem cells
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stem cell
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CN113046292A (en
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刘冰
高秀君
闫培生
张明臣
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Shandong Anran Nanometre Ind Development Co ltd
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Abstract

The invention discloses a method for culturing ginseng stem cells by using a biological reaction device, which comprises the following steps: (1) culturing adventitious roots of ginseng by adopting a one-step method; (2) taking the apical part of the adventitious root of the ginseng, and placing the apical part in a penetrant solution for treatment; (3) putting the treated ginseng adventitious root tip part into a stem cell induction culture medium for culture, and then transferring the ginseng adventitious root tip part into a stem cell propagation solid culture medium for culture; (4) and (4) collecting the ginseng stem cells obtained in the step (3), inoculating the ginseng stem cells into a biological reaction device containing a stem cell propagation liquid culture medium, and culturing in a dark place to obtain the ginseng stem cells. The invention obtains the method for obtaining the adventitious roots from the mature ginseng with shorter time and simpler steps so as to obtain the ginseng stem cells, adopts a proper biological reaction device for amplification culture, and has high growth multiple of the stem cells and high content of active ingredients in the stem cells.

Description

Method for culturing ginseng stem cells by using biological reaction device
Technical Field
The invention belongs to the technical field of plant tissue culture, and particularly relates to a method for culturing ginseng stem cells by using a biological reaction device.
Background
Ginseng (Panax ginseng c.a. mey.) is a plant of the genus Panax of the family araliaceae, distributed in china, japan and korea, and its rhizome is a rare Chinese medicinal material, called "king of herbaceous plant". Ginseng is sweet, slightly bitter and slightly warm in taste, has the effects of greatly invigorating primordial qi, recovering pulse, relieving depletion, invigorating spleen, benefiting lung, promoting fluid production, nourishing blood, tranquilizing mind, and improving intelligence, and is mainly used for treating loss of body-shirt, spleen deficiency, anorexia, lung deficiency, cough, body fluid deficiency, thirst, palpitation, insomnia, etc. Ginsenoside is the main active component of ginseng, and has the effects of resisting fatigue, delaying aging, regulating central nervous system, improving immunity, improving cardiovascular and cerebrovascular insufficiency, inhibiting tumor cell production, etc. In recent years, ginseng has been widely used in various cosmetics, health products, and drinks, and has a very wide market prospect.
At present, due to excessive mining, environmental damage and the like, wild ginseng resources are almost exhausted, and field cultivation is a main source of ginseng. However, ginseng grows slowly, the planting years are long, the requirements on environmental conditions are strict, the quality of ginseng is easily influenced by climate, cultivation conditions and plant diseases and insect pests, the cultivation technology is complex, and the development prospect of artificial cultivation of ginseng is greatly limited by the problems of pesticide residue exceeding the standard, ginseng land and the like. The supply of ginseng cultivated in a field is difficult to meet the market demand. The tissue culture technology of ginseng has short period, is not limited by seasons, is easy to carry out large-scale industrial production, and has great development prospect.
The existing adventitious roots are generally generated by the callus induction of the ginseng, the callus needs to be induced firstly and then the adventitious roots need to be induced, the required experimental period is long, the operation steps are complex, and the pollution risk is higher. In addition, the cultured ginseng also has the problem that the content of ginsenoside is low, so that the clinical application requirement is difficult to meet.
The Chinese patent with the application number of 201410698528.0 discloses a ginseng adventitious root induced proliferation method, which comprises the following steps: cutting tissue culture seedlings of ginseng into tissue small blocks, inoculating the tissue small blocks into a solid induction culture medium to induce and form adventitious roots, cutting the adventitious roots into adventitious root small blocks, and inoculating the tissue small blocks into a liquid multiplication culture medium to carry out multiplication culture of the adventitious roots; wherein the solid induction culture medium and the liquid proliferation culture medium are 1/2MS (-N) culture medium which is used as a basic culture medium and contains indolebutyric acid with the concentration of 1-10 mg/L. In the scheme, the tissue culture seedling with the age of 28-32 days is cut into small blocks to directly induce adventitious roots, the tissue culture seedling is tender and has strong differentiation capability, the tissue culture seedling is actually obtained by seed germination or explant culture, at least 28-32 days are still needed, and callus induction is still needed for the explant culture. Therefore, the cycle is not actually shortened.
Ginseng stem cells are undifferentiated cells having an unlimited division ability, and currently, ginseng-related pharmaceutically active ingredients can be produced by isolating and culturing ginseng stem cells. In the prior art, differentiated tissue organs are used as explants when ginseng stem cells are separated, and the differentiated tissue organs are induced into callus cells with differentiation capacity through the culture in a three-dimensional culture stage (dedifferentiation process). However, the cell is essentially derived from differentiated somatic cells, so that the cell has limited division capability and weak stress resistance. In industrial production, cell line degeneration is easy to occur, and the division capability is weak.
Therefore, if a method for obtaining an adventitious root from a mature ginseng, which requires a shorter time and simpler steps, and further rapidly obtaining ginseng stem cells, and a biological reaction device suitable for stem cell culture are searched, it is advantageous to expand the culture of ginseng stem cells, and it is of great significance.
The present invention has been made in view of this point.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects of the prior art and provide a method for culturing ginseng stem cells by using a biological reaction device. The invention obtains the method for obtaining the ginseng stem cells from the mature ginseng with shorter time and simpler steps, adopts a proper biological reaction device for amplification culture, and has high growth multiple of the stem cells and high content of active ingredients in the stem cells.
In order to solve the technical problems, the invention adopts the technical scheme that:
the first object of the present invention is to provide a method for culturing ginseng stem cells using a bioreactor, comprising the steps of:
(1) culturing the adventitious root of the ginseng by adopting a one-step method;
(2) taking the root tip part of the adventitious root of the ginseng, and placing the root tip part in a penetrant solution for treatment;
(3) putting the treated adventitious root tip part of the ginseng into a stem cell induction culture medium for culture, and then transferring the ginseng into a stem cell propagation solid culture medium for culture;
(4) collecting the ginseng stem cells obtained in the step (3), inoculating the ginseng stem cells into a shake flask containing a stem cell liquid culture medium, and culturing at 100-140rpm in the dark;
(5) and (5) inoculating the stem cells obtained in the step (4) into a biological reaction device containing a stem cell liquid culture medium, and culturing in a dark place to obtain the ginseng stem cells.
The invention adopts a one-step method to culture the ginseng adventitious root, can treat each part of mature ginseng and inoculate the treated part into the induction culture medium to directly induce the ginseng adventitious root, does not need the intermediate step of callus induction, can simplify the induction step, shorten the induction time and reduce the pollution risk. The obtained Ginseng radix adventitious root has vigorous activity, and is favorable for producing Ginseng radix stem cell.
The root tip part of the ginseng adventitious root has more meristems and strong activity, after being treated by the penetrant solution, other cells are not resistant to the death of the hypertonic solution, only stem cells can grow, the growth speed of the stem cells is extremely high, and the content of active ingredients in the obtained ginseng stem cells is improved.
The biological reaction device provided by the invention is matched with a stem cell liquid culture medium and control conditions, so that the ginseng stem cells can be rapidly cultured and obtained, and the content of active ingredients in the stem cells is high.
Further, in the step (4), when the culture is carried out in the bioreactor, the ventilation amount is 0.02-0.2 vvm;
preferably, the aeration is 0.05 to 0.15 vvm.
In a further scheme, the biological reaction device comprises a tank body, wherein the bottom of the tank body comprises at least two air inlet devices;
preferably, the height of the bottom wall of the tank body is gradually reduced from the periphery to the center to form an inverted cone with a large upper part and a small lower part; the discharge gate sets up in the minimum position in center, and air inlet unit surrounds the discharge gate interval evenly distributed on by all around to the diapire that the center gradually reduces.
In the invention, the air inlet devices are arranged on the inclined plane of the bottom wall of the inverted cone-shaped tank body with a large upper part and a small lower part, so that the entering air cannot vertically rise, and further, two or more air inlet devices are uniformly distributed around the center at intervals, which is beneficial to the uniform distribution of air in the tank body and the uniform growth of stem cells in the tank body.
In a further scheme, in the step (3), the volume of the sterilized stem cell liquid culture medium accounts for 20-80% of the volume of the biological reaction device,
preferably, the volume of the bioreactor is 30-70%.
In a further scheme, in the step (4), when the stem cells are inoculated into the stem cell liquid culture medium, the mass of the inoculated stem cells accounts for 1.0-10.0% of the volume of the stem cell liquid culture medium;
preferably, the mass of the inoculated stem cells accounts for 2.0-8.0% of the volume of the stem cell liquid culture medium.
In the further scheme, in the step (4), in a biological reaction device, the culture is carried out for 3-4 weeks in a dark place at the temperature of 20-25 ℃.
When the stem cell liquid culture medium is used for culturing in a bioreactor, the growth effect of the ginseng stem cells is good by adopting the culture medium addition amount and the stem cell inoculation amount.
In the step (2), the root tip part of the adventitious root of the ginseng is taken and placed in a penetrant solution, and low-temperature treatment is carried out for 1-10 hours;
preferably, the low temperature is 1-6 ℃, preferably 4 ℃;
preferably, the length of the apical part of the adventitious root of ginseng is 0.3mm to 0.8 mm.
In a further scheme, in the step (2), the osmotic agent solution is at least one selected from sucrose solution, potassium chloride solution and mannitol solution;
Preferably, the osmotic agent solution is a sucrose solution with the concentration of 1 mol/L.
In a further scheme, in the step (3), the stem cell induction culture medium comprises 0.6-1mg/L kinetin, 2-4mg/L indoleacetic acid, 0.5-1.5mg/L naphthylacetic acid, 25-100mg/L ascorbic acid, 50-150mg/L citric acid, 20-60g/L sucrose, 1-6g/L plant gel, 1-2.4 g/L1/2 MS culture medium and 0.6-1.4g/L WPM culture medium;
preferably, the stem cell induction medium comprises 1mg/L kinetin, 2.5mg/L indoleacetic acid, 1mg/L naphthylacetic acid, 75mg/L ascorbic acid, 75mg/L citric acid, 30g/L sucrose, 3g/L plant gel, 1.8 g/L1/2 MS medium and 0.8g/L WPM medium.
The stem cell induction culture medium contains indoleacetic acid, naphthylacetic acid and kinetin, can induce the growth of stem cells, and the ascorbic acid and the citric acid can generate a synergistic antioxidant effect. The components in the induction medium act synergistically to enable stem cells to grow rapidly in the early stage.
In a further scheme, in the step (3), the stem cell propagation solid culture medium comprises 2-4mg/L2, 4-dichlorophenoxyacetic acid, 1-3mg/L naphthylacetic acid, 0.8-1.2mg/L kinetin, 20-60g/L sucrose, 3g/L plant gel, 1-2.4 g/L1/2 MS culture medium and 0.6-1.4g/L WPM culture medium;
Preferably, the stem cell propagation solid culture medium comprises 3 mg/L2, 4-dichlorophenoxyacetic acid, 2mg/L naphthylacetic acid, 1mg/L kinetin, 35g/L sucrose, 3g/L plant gel, 1.8 g/L1/2 MS culture medium and 0.8g/L WPM culture medium.
In a further scheme, in the step (3), the stem cell liquid culture medium comprises 2-4mg/L2, 4-dichlorophenoxyacetic acid, 1-3mg/L naphthylacetic acid, 0.8-1.2mg/L kinetin, 20-60g/L sucrose, 1-2.4 g/L1/2 MS culture medium and 0.6-1.4g/L WPM culture medium;
preferably, the stem cell liquid culture medium comprises 3 mg/L2, 4-dichlorophenoxyacetic acid, 2mg/L naphthylacetic acid, 1mg/L kinetin, 35g/L sucrose, 1.8 g/L1/2 MS culture medium and 0.8g/L WPM culture medium.
The stem cell expanding propagation solid culture medium and the stem cell liquid culture medium contain 2, 4-dichlorophenoxyacetic acid, naphthylacetic acid and kinetin, so that the growth speed of the stem cells is greatly increased.
In a further embodiment, the stem cell induction medium, the stem cell propagation solid medium and the stem cell propagation liquid medium have a pH of 5.6 to 6.0.
In a further scheme, the pH values of the stem cell induction culture medium, the stem cell propagation solid culture medium and the stem cell propagation liquid culture medium are adjusted by NaOH or KOH, so that the stem cell growth is facilitated.
In the further scheme, in the step (3), the root tip part of the adventitious root of the ginseng is respectively cultured for 25 to 35 days in a dark place at the temperature of between 20 and 25 ℃ on a stem cell induction culture medium and a stem cell propagation solid culture medium.
The second purpose of the invention is to provide a method for culturing ginseng adventitious roots, which adopts a one-step method to culture the ginseng adventitious roots and specifically comprises the following steps:
cleaning and disinfecting mature ginseng, slicing, and inoculating the ginseng into an induction culture medium to induce adventitious roots of the ginseng; inoculating the obtained ginseng adventitious root to an induction culture medium again for relay culture and propagation; then the obtained ginseng adventitious root is cut into segments, and the segments are inoculated into a liquid culture medium for culture to obtain the adventitious root.
The existing adventitious roots are generally generated by the callus induction of the ginseng, the callus needs to be induced firstly and then the adventitious roots need to be induced, the required experimental period is long, the operation steps are complex, and the pollution risk is higher. In addition, the cultured ginseng also has the problem that the content of ginsenoside is low, so that the clinical application requirement is difficult to meet.
In view of the fact that mature ginseng is long in age, high in maturity and not easy to differentiate, there is no report that adventitious roots can be induced directly from mature ginseng at present. Through a large number of experiments, the invention unexpectedly discovers that the mature ginseng slices can be directly induced to generate adventitious roots on a specific induction culture medium, so that the adventitious roots can be directly obtained from the mature ginseng by one-step induction without an intermediate step of callus induction, thereby simplifying the induction step and shortening the induction time.
In a further scheme, the induction culture medium comprises 1-6mg/L naphthylacetic acid, 0.2-1mg/L gibberellin, 0.1-0.6mg/L kinetin, 0.075-1.5g/L citric acid, 0.03-1g/L ascorbic acid, 20-60g/L sucrose, 1-6g/L plant gel, 1-4g/L B5 culture medium and 1-2.4g/L WPM culture medium.
The induction culture medium of the scheme realizes that each part of the mature ginseng is directly induced to generate adventitious roots without an intermediate step of inducing callus, thereby simplifying the induction step and shortening the induction time.
In the scheme, the naphthylacetic acid is a plant growth regulator, and the gibberellin is a plant hormone, so that the formation of adventitious roots can be promoted. Kinetin is a cytokinin that promotes cell division. Citric acid and ascorbic acid can generate a synergistic antioxidation effect, prevent the in vitro tissue of the mature ginseng from browning, and are beneficial to directly inducing adventitious roots from the in vitro tissue of the mature ginseng. The components in the induction culture medium have synergistic effect, and the aim of directly inducing each part of the mature ginseng to generate adventitious roots is finally realized without an intermediate step of inducing callus.
In a further embodiment, the induction medium comprises 4mg/L naphthylacetic acid, 0.6mg/L gibberellin, 0.4mg/L kinetin, 0.1g/L citric acid, 0.05g/L ascorbic acid, 30g/L sucrose, 3g/L plant gel, 1.55g/L B5 medium and 1.21g/L WPM medium.
The induction culture medium under the component proportion has the best induction effect on the mature ginseng to generate the adventitious roots, has a large quantity of the generated adventitious roots and good quality, is beneficial to the next step of propagation expansion, and improves the content of active components in the adventitious roots.
In a further scheme, the liquid culture medium is B5 culture medium, WPM culture medium or 1/2MS culture medium as basal culture medium, and contains 4mg/L indolebutyric acid, 0.1g/L citric acid, 0.05g/L ascorbic acid and 30g/L sucrose.
In the invention, when the adventitious roots generated by induction are further subjected to liquid culture, a culture medium for conventional culture, such as 1/2MS culture medium, can be adopted and is consistent with a culture medium for common adventitious roots, which shows that the adventitious roots generated by one-step induction of the invention have no difference with other two-step methods, can be cultured by the conventional culture medium, and simultaneously, the induction time is shortened, and the pollution risk is reduced.
In a further scheme, the slices are slices with the width of 0.5-0.7cm, the length of 0.5-0.7cm and the thickness of 0.2-0.5 mm.
In a further embodiment, the pH of the induction medium and the liquid medium of the present invention is 5.6 to 6.0.
In a further scheme, the pH of the induction culture medium and the liquid culture medium is adjusted by NaOH or KOH, so that the growth of adventitious roots is facilitated.
In a further scheme, the age of the mature ginseng is more than 3 years;
preferably, the age of the mature ginseng is more than 6 years;
as a preferred embodiment, the mature ginseng is centennial ginseng.
The century ginseng is rare in nature, has high edible and medicinal values, and can tonify five internal organs, calm spirit, calm soul, stop palpitation, remove pathogenic qi, improve eyesight, and benefit mind and intelligence; the value of the ginseng planting method is far higher than that of planted ginseng with short age. The invention does not need the intermediate step of inducing callus, can directly obtain the adventitious roots by one-step induction from the hundred-year ginseng cut blocks, not only can simplify the induction step and shorten the induction time, but also can obtain the specific functional components in the female parent hundred-year old ginseng, thereby obtaining the adventitious roots with better nutritive value.
Further, the main root, or the reed head, or the part, or the rootlet, or the fibrous root of the mature ginseng are cleaned, disinfected, sliced and inoculated into an induction culture medium to induce the adventitious root of the ginseng.
In a further scheme, the ginseng is selected from wild ginseng, mountain ginseng, ginseng under forest and garden ginseng;
preferably, the ginseng is wild ginseng.
As a specific preferred embodiment, the step of culturing stem cells using a bioreactor according to the present invention specifically comprises:
One-step method for culturing adventitious roots of ginseng
(1) Induction of adventitious roots
Cleaning main root of mature ginseng, sterilizing, cutting into slices with width of 0.5-0.7cm, length of 0.5-0.7cm and thickness of 0.2-0.5mm, inoculating to induction culture medium containing 4mg/L naphthylacetic acid, 0.6mg/L gibberellin, 0.4mg/L kinetin, 0.1g/L citric acid, 0.05g/L ascorbic acid, 30g/L sucrose, 3g/L plant gel, 1.55g/L B5 culture medium and 1.21g/L WPM culture medium, and dark culturing at 22 + -1 deg.C for 4-5 weeks to induce adventitious root of ginseng.
(2) Subculture of adventitious roots
Inoculating the ginseng adventitious roots obtained in the step (1) into the same induction culture medium as that in the step (1), and carrying out dark culture for 4-5 weeks under the same conditions;
(3) cultivation of adventitious roots
And (3) cutting the ginseng adventitious roots obtained in the step (2) into tissues with the length of about 1-2cm, inoculating the tissues into a liquid culture medium containing 4mg/L of indolebutyric acid, 0.1g/L of citric acid, 0.05g/L of ascorbic acid, 1/2MS culture medium and 30g/L of cane sugar, and culturing for 3-4 weeks on a shaking table at the temperature of 22 +/-1 ℃ to obtain the adventitious roots.
(II) culturing the ginseng stem cells by using a bioreactor
(1) Taking the root tip part of the cultured ginseng adventitious root, and placing the root tip part in 1mol/L sucrose solution for treatment;
(2) Placing the treated root tip part of the adventitious root of the ginseng into a stem cell induction culture medium for culture, and culturing for 25-35 days at 22 ℃ in a dark place; the dry cell induction culture medium comprises 1mg/L kinetin, 2.5mg/L indoleacetic acid, 1mg/L naphthylacetic acid, 75mg/L ascorbic acid, 75mg/L citric acid, 30g/L sucrose, 3g/L plant gel, 1.8 g/L1/2 MS culture medium and 0.8g/L WPM culture medium;
(3) then transferring the stem cells into a solid culture medium for propagation, and culturing for 25-35 days at 22 ℃ in a dark place; the stem cell propagation solid culture medium comprises 3 mg/L2, 4-dichlorophenoxyacetic acid, 2mg/L naphthylacetic acid, 1mg/L kinetin, 35g/L cane sugar, 3g/L plant gel, 1.8 g/L1/2 MS culture medium and 0.8g/L WPM culture medium.
(4) Collecting the ginseng stem cells obtained in the step (3), and inoculating the ginseng stem cells into a biological reaction device containing a stem cell liquid culture medium, wherein the mass of the inoculated stem cells accounts for 1-5.0% of the volume of the stem cell liquid culture medium; the ventilation volume is 0.02-0.2 vvm; culturing at 22 deg.C in dark to obtain Ginseng radix stem cells;
the stem cell liquid culture medium comprises 3 mg/L2, 4-dichlorophenoxyacetic acid, 2mg/L naphthylacetic acid, 1mg/L kinetin, 35g/L cane sugar, 1.8 g/L1/2 MS culture medium and 0.8g/L WPM culture medium.
In the present invention, WPM medium, B5 medium, 1/2MS medium, and the like are known in the art.
Woody Plant Medium (WPM)
Ingredient (mg/L): 400 parts of ammonium nitrate, 556 parts of tetrahydrate calcium nitrate, 990 parts of potassium sulfate, 72 parts of anhydrous calcium chloride, 170 parts of monopotassium phosphate, 0.25 part of sodium molybdate dihydrate, 180 parts of anhydrous magnesium sulfate, 22.4 parts of manganese sulfate monohydrate, 8.6 parts of zinc sulfate heptahydrate, 0.25 part of copper sulfate pentahydrate, 27.8 parts of ferrous sulfate heptahydrate, 37.3 parts of disodium ethylenediamine tetraacetic acid, 100 parts of inositol, 11 parts of vitamin B, 0.5 part of nicotinic acid, 60.5 parts of vitamin B, 2 parts of glycine and 5.2 parts of pH.
B5 Medium
Ingredient (mg/L): potassium nitrate KNO3 2500，MgSO4·7H2O 250，CaCl2·2H2O 150，(NH4)2SO4134，NaH2PO4.H2O 150，KI 0.75，H3BO3 3.0，MnSO4·4H2O 10，ZnSO4·7H2O 2.0，Na2MoO4·2H2O 0.25，CoCl2·6H2O 0.025，CuSO4·5H2O 0.025，Na2-EDTA 37.3，FeSO4·7H2O27.8, inositol 100, nicotinic acid 1.0, pyridoxine hydrochloride 1.0, and ammonium sulfate hydrochloride 10.
1/2MS culture medium
Ingredient (mg/L): potassium nitrate 950, ammonium nitrate 825, calcium chloride dihydrate 220, magnesium sulfate 185, monopotassium phosphate 85, manganese sulfate 11.15, zinc sulfate 4.3, boric acid 3.1, potassium iodide 0.415, sodium molybdate 0.125, copper sulfate 0.0125, cobalt chloride 0.0125, disodium ethylenediaminetetraacetate 37.3, ferrous sulfate 27.8, inositol 100, glycine 2, hydrochloric acid 0.5, pyridoxine hydrochloride 0.5 and ammonium sulfate hydrochloride 0.1.
It is a third object of the present invention to provide a bioreactor for culturing ginseng stem cells, comprising: the tank body is provided with a cover body which can be opened and closed at the top, and an exhaust device is arranged on the cover body or the top of the tank body; at least two air inlet devices are arranged at the bottom of the tank body, and air enters the tank body through the air inlet devices.
The bioreactor is a small bioreactor, has simple structure, convenient operation and easy observation, can be used for conveniently producing the ginseng stem cells, and has high propagation speed and high growth multiple of the stem cells.
In the invention, the tank body is hollow and is used for containing a liquid culture medium. The bioreactor can be made of any material which is suitable for preparing a fermentation tank and can be used for high-temperature sterilization, such as glass, stainless steel, high-temperature resistant plastic and the like; the stainless steel material is optimized, and the device is durable and long in service life. The cover body on the top of the tank body can be opened or closed and is used for adding liquid culture medium inwards, and the cover body is connected with the tank body in a sealing mode after the liquid culture medium is added. The bottom of the tank body is provided with at least two air inlet devices, and sterile air is introduced into the tank body from different positions, so that cultures in the tank can be fully contacted with the air, the ginseng stem cells can grow uniformly, and the rapid growth of the ginseng stem cells can be promoted.
Furthermore, the biological reaction device also comprises a supporting structure for supporting the tank body to be vertically placed on a plane. Specifically, the supporting structure comprises supporting legs, the supporting legs are connected with the lower portion of the tank body or integrally arranged, and the tank body is placed on a plane or a platform through the supporting legs.
Preferably, the conical bottom of the tank body is positioned in a space surrounded by the supporting feet.
In a further scheme, the volume of the biological reaction device can be set according to the requirement; as an embodiment, the bioreactor of the present invention has a volume of 1 to 50L, preferably 2 to 20L, preferably 3 to 10L, preferably 5L.
In a further scheme, a discharge hole is formed in the center of the bottom of the tank body, and the air inlet devices are uniformly distributed around the discharge hole at intervals;
preferably, the height of the bottom wall of the tank body is gradually reduced from the periphery to the center to form an inverted cone with a large upper part and a small lower part; the discharge hole is arranged at the lowest position in the center, and the air inlet devices are uniformly distributed on the bottom wall which is gradually reduced from the periphery to the center at intervals around the discharge hole.
In the invention, the air inlet devices are arranged on the inclined plane of the bottom wall of the inverted cone-shaped tank body with a large upper part and a small lower part, so that the entering air cannot vertically rise, and further, two or more air inlet devices are uniformly distributed around the center at intervals, which is beneficial to the uniform distribution of air in the tank body and the uniform growth of stem cells in the tank body.
In a further scheme, the air inlet device comprises an air inlet, an air inlet pipe and an air filter, the air inlet is located on the bottom wall of the tank body, the air inlet pipe is connected with the air inlet in a sealing mode, and the air filter is arranged on the air inlet pipe to filter air.
In a further scheme, the exhaust device comprises an exhaust port, an exhaust pipe and a filtering device, the exhaust port is arranged on the cover body or the top of the tank body, the exhaust pipe and the exhaust port are communicated in a sealing mode, and the filtering device is arranged on the exhaust pipe;
preferably, the filter device is an air filter or a liquid filter.
In a preferred embodiment, the vent is provided in the center of the cover.
In the above scheme, when the filtering device arranged on the exhaust pipe is an air filter, external air can be prevented from entering from the top, and the sterile culture environment in the tank body is ensured. In addition, when the filtering device on the exhaust pipe is replaced with a liquid filter, liquid can be added from the top.
Further, an inoculation port is arranged on the cover body or the top of the tank body and used for inoculation.
In a further scheme, a handle is also arranged on the tank body; preferably, the handle comprises at least two.
The handles arranged on the tank body can be symmetrically arranged, so that a user can conveniently take and move the biological reaction device.
In a further scheme, a plurality of transparent observation windows are arranged on the tank body; for observing the growth of the internal stem cells;
Preferably, the observation window is arranged on the side wall of the middle part and/or the lower part of the tank body.
When the jar body adopts opaque stainless steel material, the inside of jar body can be followed different angles to a plurality of transparent observation windows of different positions that set up, in time masters jar internal stem cell's growth condition.
After adopting the technical scheme, compared with the prior art, the invention has the following beneficial effects:
1. the invention relates to a method for culturing ginseng stem cells by utilizing a biological reaction device, which adopts the biological reaction device with an improved structure, the bottom of the biological reaction device is in a smooth transition cone shape, and a plurality of air vents of an air breather are arranged on an inclined plane and evenly ventilate in all directions, so that the air ventilation is even and sufficient, the rapid growth of the ginseng stem cells is facilitated, and the production is expanded.
2. The invention is matched with a liquid culture medium for culturing the ginseng stem cells, adopts proper control conditions, controls the inoculation amount, the ventilation amount and the like, and is not only beneficial to the rapid growth of the ginseng stem cells, but also beneficial to improving the content of ginsenoside in the ginseng stem cells.
3. The invention places the ginseng adventitious roots prepared by the one-step method into the penetrant solution for low-temperature treatment, leads the cells which are not resistant to the hypertonic solution to die, only the stem cells grow, thereby separating and obtaining the stem cells, and the stem cells grow at a very high speed by matching with the stem cell induction culture medium and the stem cell propagation culture medium with proper component proportion, and the content of active ingredients in the obtained ginseng stem cells is improved.
4. In the invention, the method for culturing the adventitious roots of the ginseng adopts a one-step method, each part of the mature ginseng is treated and then inoculated into the induction culture medium to directly induce the adventitious roots of the ginseng, the intermediate step of inducing callus is not needed, the induction step can be simplified, the induction time is shortened, and the pollution risk is reduced.
5. In the method for culturing the ginseng adventitious roots, the adopted induction culture medium comprises 1-6mg/L of naphthylacetic acid, 0.2-1mg/L of gibberellin, 0.1-0.6mg/L of kinetin, 0.075-1.5g/L of citric acid, 0.03-1g/L of ascorbic acid, 20-60g/L of cane sugar, 1-6g/L of plant gel, 1-4g/L B5 of culture medium and 1-2.4g/L of WPM culture medium, all components have synergistic effect, the problem that the adventitious roots are directly induced from mature ginseng in a one-step method is solved, and the produced adventitious roots are large in quantity.
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention, are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention without limiting the invention to the right. It is obvious that the drawings in the following description are only some embodiments, and that for a person skilled in the art, other drawings can be derived from them without inventive effort. In the drawings:
FIG. 1 is a schematic view of the structure of a bioreactor of the present invention;
FIG. 2 is a schematic view of induction of adventitious roots using the induction medium of example 1 of the present invention;
FIG. 3 is a schematic diagram of induction of adventitious roots using the induction medium of comparative example 1;
the device comprises a tank body 1, a cover body 2, an exhaust device 3, an air inlet device 4, a discharge hole 5, an air inlet pipe 6, an air filter 7, an exhaust pipe 8, a filter device 9, an observation window 10, a handle 11 and supporting legs 12.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and the following embodiments are used for illustrating the present invention and are not intended to limit the scope of the present invention.
In the present invention, the preparation method or the detection method is not particularly limited, and any method that can achieve the object in the prior art can be used.
As shown in FIG. 1, the present invention provides a bioreactor for culturing ginseng stem cells, comprising: the tank comprises a tank body 1, wherein the top of the tank body 1 is provided with an openable cover body 2, and an exhaust device 3 is arranged on the cover body 2 or the top of the tank body 1; the bottom of the tank body 1 is provided with at least two air inlet devices 4, and air enters the tank body 1 through the air inlet devices 4.
The bioreactor is a small bioreactor, has simple structure and convenient operation, can conveniently produce the ginseng stem cells by utilizing the bioreactor, and has fast stem cell growth and high growth multiple.
In the invention, the tank body 1 is hollow and is used for containing liquid culture medium. The bioreactor can be made of any material which is suitable for preparing a fermentation tank and can be used for high-temperature sterilization, such as glass, stainless steel, high-temperature resistant plastic and the like; the stainless steel material is optimized, and the device is durable and long in service life. The cover body 2 on the top of the tank body 1 can be opened or closed for adding liquid culture medium inwards, and the cover body 2 is connected with the tank body 1 in a sealing way after the liquid culture medium is added. The bottom of the tank body 1 is provided with at least two air inlet devices 4, and sterile air is introduced into the tank body 1 from different positions, so that cultures in the tank can be fully contacted with the air, the growth is uniform, and the rapid growth of ginseng stem cells is promoted. The bioreactor of the present invention further comprises a support structure for supporting the tank 1 to be vertically placed on a plane. Specifically, the supporting structure comprises supporting legs 12, the supporting legs 12 are connected with the lower part of the tank body 1 or integrally arranged, and the tank body 1 is placed on a plane or a platform through the supporting legs 12. Preferably, the conical bottom of the tank 1 is located in the space enclosed by the support feet 12.
In a further scheme, the volume of the biological reaction device can be set according to the requirement; as an embodiment, the bioreactor of the present invention has a volume of 1-50L, preferably 2-20L, preferably 3-10L, preferably 5L.
A discharge port 5 is formed in the center of the bottom of the tank body 1, and the air inlet devices 4 are uniformly distributed around the discharge port 5 at intervals;
preferably, the height of the bottom wall of the tank body 1 is gradually reduced from the periphery to the center to form an inverted cone with a large upper part and a small lower part; the discharge ports 5 are arranged at the lowest position in the center, and the air inlet devices 4 are uniformly distributed on the bottom wall which is gradually reduced from the periphery to the center at intervals around the discharge ports 5.
In the invention, the air inlet devices 4 are arranged on the inclined plane of the bottom wall of the inverted cone-shaped tank body 1 with a large upper part and a small lower part, so that the entering air cannot vertically rise, and further, two or more air inlet devices 4 are uniformly distributed around the center at intervals, which is beneficial to the uniform distribution of air in the tank body 1 and the uniform growth of stem cells in the tank body 1.
Air inlet unit 4 includes air inlet, intake pipe 6 and air cleaner 7, the air inlet is located the diapire of jar body 1, intake pipe 6 and air inlet sealing connection, air cleaner 7 sets up filters the institute through the air on intake pipe 6.
The exhaust device 3 comprises an exhaust port, an exhaust pipe 8 and a filtering device 9, the exhaust port is arranged on the cover body 2 or the top of the tank body 1, the exhaust pipe 8 is communicated with the exhaust port in a sealing way, and the filtering device 9 is arranged on the exhaust pipe 8;
preferably, the filtering device 9 is an air filter 7 or a liquid filter.
In a preferred embodiment, the vent is provided in the center of the lid body 2.
In the above scheme, when the filtering device 9 arranged on the exhaust pipe 8 is the air filter 7, the external air can be prevented from entering from the top, and the sterile culture environment in the tank body 1 is ensured. In addition, when the filter device 9 on the exhaust pipe 8 is replaced with a liquid filter, it is possible to achieve liquid addition from the top.
A plurality of transparent observation windows 10 are arranged on the tank body 1; is used for observing the growth condition of the ginseng stem cells in the tank body 1.
The observation window 10 is arranged on the side wall of the middle part and/or the lower part of the tank body 1.
When the tank body 1 is made of opaque stainless steel, the inside of the tank body 1 can be observed from different angles through the plurality of transparent observation windows 10 arranged at different positions, and the growth condition of the ginseng stem cells in the tank body 1 can be mastered in time.
An inoculation port is arranged on the cover body 2 or the top of the tank body 1 and is used for inoculation.
The tank body 1 is also provided with a handle 11; preferably, the handle 11 comprises at least two.
The handles 11 arranged on the tank body 1 can be symmetrically arranged, so that a user can conveniently take and move the biological reaction device.
The bioreactor used in the examples of the present invention (e.g., examples 7 to 9) is the bioreactor according to the above-mentioned embodiment.
EXAMPLE 1 one-step cultivation of ginseng adventitious roots
(1) Induction of adventitious roots
Removing rhizoma Phragmitis and parts of wild ginseng of 20 ages, cleaning main root, sterilizing, cutting into slices with width of 0.6cm, length of 0.7cm and thickness of 0.3mm, inoculating into induction culture medium, and dark culturing at 22 + -1 deg.C for 4-5 weeks to induce adventitious root of wild ginseng; wherein the induction culture medium comprises 4mg/L naphthylacetic acid, 0.6mg/L gibberellin, 0.4mg/L kinetin, 0.1g/L citric acid, 0.05g/L ascorbic acid, 30g/L sucrose, 3g/L plant gel, 1.55g/L B5 culture medium and 1.21g/L WPM culture medium, and the pH value is 5.8.
(2) Subculture of adventitious roots
Inoculating the mountain ginseng adventitious roots obtained in the step (1) into the same induction culture medium as that in the step (1), and carrying out dark culture for 4-5 weeks under the same conditions;
(3) cultivation of adventitious roots
Shearing the mountain ginseng adventitious roots obtained in the step (2) into tissues with the length of about 1cm, inoculating the tissues into a liquid culture medium, and culturing for 3-4 weeks on a shaking table at the temperature of 22 +/-1 ℃ to obtain adventitious roots; wherein the liquid culture medium contains 4mg/L indolebutyric acid, 0.1g/L citric acid, 0.05g/L ascorbic acid, 1/2MS culture medium and 30g/L sucrose, and has pH value of 5.8.
In this example, the adventitious roots produced on the induction medium in the step (1) are shown in FIG. 2, in which A is a photograph of 1 week of culture, B is a photograph of 3 weeks of culture, and C is a photograph of 5 weeks of culture. As can be seen, after 5 weeks, adventitious roots were induced directly on the mature wild ginseng slices.
EXAMPLE 2 one-step cultivation of adventitious roots of Panax ginseng
(1) Induction of adventitious roots
Cleaning reed heads of 6-age garden ginseng, sterilizing, cutting into slices with the width of 0.5cm, the length of 0.6cm and the thickness of 0.3mm, inoculating the slices into an induction culture medium, and performing dark culture at the temperature of 22 +/-1 ℃ for 4-5 weeks to induce adventitious roots; wherein the induction culture medium comprises 6mg/L naphthylacetic acid, 0.2mg/L gibberellin, 0.4mg/L kinetin, 1.2g/L citric acid, 0.1g/L ascorbic acid, 20g/L sucrose, 5g/L plant gel, 4g/L B5 culture medium and 1.8g/L WPM culture medium, and the pH value is 5.6.
(2) Subculture of adventitious roots
Inoculating the adventitious roots obtained in the step (1) into the same induction culture medium as that in the step (1), and carrying out dark culture for 4-5 weeks under the same conditions;
(3) cultivation of adventitious roots
Shearing the adventitious roots obtained in the step (2) into tissues with the length of about 1cm, inoculating the tissues into a liquid culture medium, and culturing for 3-4 weeks on a shaking table at the temperature of 22 +/-1 ℃ to obtain the adventitious roots; wherein the liquid culture medium contains 4mg/L indolebutyric acid, 0.1g/L citric acid, 0.05g/L ascorbic acid, 1/2MS culture medium and 30g/L sucrose, and has pH value of 5.6.
Similar to the results of example 1, the adventitious roots can be induced by one step in step (1) of this example, number of adventitious roots in one step in example 3
(1) Induction of adventitious roots
Cleaning parts of age 10 of ginseng under forest, sterilizing, cutting into slices with width of 0.7cm, length of 0.7cm and thickness of 0.5mm, inoculating into induction culture medium, and dark culturing at 22 + -1 deg.C for 4-5 weeks to induce adventitious roots; wherein the induction culture medium comprises 5mg/L naphthylacetic acid, 1mg/L gibberellin, 0.1mg/L kinetin, 0.075g/L citric acid, 0.03g/L ascorbic acid, 40g/L sucrose, 4g/L plant gel, 2g/L B5 culture medium and 1g/L WPM culture medium, and the pH value is 6.0.
(2) Subculture of adventitious roots
Inoculating the adventitious roots obtained in the step (1) into the same induction culture medium as that in the step (1), and carrying out dark culture for 4-5 weeks under the same conditions;
(3) cultivation of adventitious roots
Shearing the adventitious roots obtained in the step (2) into tissues with the length of about 2cm, inoculating the tissues into a liquid culture medium, and culturing for 3-4 weeks on a shaking table at the temperature of 22 +/-1 ℃ to obtain the adventitious roots; wherein the liquid culture medium contains 4mg/L indolebutyric acid, 0.1g/L citric acid, 0.05g/L ascorbic acid, WPM culture medium and 30g/L sucrose, and has pH of 6.0.
Similar to the results of example 1, the generation of adventitious roots can be induced by one step in step (1) of this example.
EXAMPLE 4 one-step cultivation of Panax ginseng adventitious roots
(1) Induction of adventitious roots
Cleaning main roots of 15-age mountain ginseng, sterilizing, cutting into slices with width of 0.5cm, length of 0.6cm and thickness of 0.4mm, inoculating into an induction culture medium, and performing dark culture at 22 +/-1 ℃ for 4-5 weeks to induce mountain ginseng adventitious roots; wherein the induction culture medium comprises 1mg/L naphthylacetic acid, 0.5mg/L gibberellin, 0.6mg/L kinetin, 1.5g/L citric acid, 1g/L ascorbic acid, 50g/L sucrose, 6g/L plant gel, 1g/L B5 culture medium and 2.4g/L WPM culture medium, and the pH value is 5.7.
(2) Subculture of adventitious roots
Inoculating the mountain ginseng adventitious roots obtained in the step (1) into the same induction culture medium as that in the step (1), and carrying out dark culture for 4-5 weeks under the same conditions;
(3) cultivation of adventitious roots
Shearing the mountain ginseng adventitious roots obtained in the step (2) into tissues with the length of about 1cm, inoculating the tissues into a liquid culture medium, and culturing for 3-4 weeks on a shaking table at the temperature of 22 +/-1 ℃ to obtain adventitious roots; wherein the liquid culture medium contains 4mg/L indolebutyric acid, 0.1g/L citric acid, 0.05g/L ascorbic acid, B5 culture medium and 30g/L sucrose, and has pH value of 5.7.
Similar to the results of example 1, the step (1) of this example can directly induce the generation of adventitious roots in one step.
EXAMPLE 5 one-step cultivation of ginseng adventitious roots
(1) Induction of adventitious roots
Removing rhizoma Phragmitis and parts of wild ginseng, cleaning main root, sterilizing, cutting into slices with width of 0.6cm, length of 0.7cm and thickness of 0.3mm, inoculating into induction culture medium, and dark culturing at 22 + -1 deg.C for 4-5 weeks to induce adventitious root of wild ginseng; wherein the induction culture medium comprises 4mg/L naphthylacetic acid, 0.6mg/L gibberellin, 0.4mg/L kinetin, 0.1g/L citric acid, 0.05g/L ascorbic acid, 30g/L sucrose, 3g/L plant gel, 1.55g/L B5 culture medium and 1.21g/L WPM culture medium, and the pH value is 5.8.
(2) Subculture of adventitious roots
Inoculating the mountain ginseng adventitious roots obtained in the step (1) into the same induction culture medium as that in the step (1), and carrying out dark culture for 4-5 weeks under the same conditions;
(3) cultivation of adventitious roots
Shearing the mountain ginseng adventitious roots obtained in the step (2) into tissues with the length of about 1cm, inoculating the tissues into a liquid culture medium, and culturing for 3-4 weeks on a shaking table at the temperature of 22 +/-1 ℃ to obtain adventitious roots; wherein the liquid culture medium contains 4mg/L indolebutyric acid, 0.1g/L citric acid, 0.05g/L ascorbic acid, 1/2MS culture medium and 30g/L sucrose, and has pH value of 5.8.
Similar to the results of example 1, the step (1) of this example can directly induce the generation of adventitious roots in one step.
Example 6 culturing of Ginseng Stem cells Using a bioreactor
(1) The ginseng adventitious roots prepared in example 1 were used;
(2) taking the apical part of 0.5mm ginseng adventitious root, putting the apical part into 1mol/L sucrose solution, and taking out after 6 hours in a refrigerator at 4 ℃;
(3) placing the treated root tips into a stem cell induction medium comprising: 1mg/L kinetin, 2.5mg/L indoleacetic acid, 1mg/L naphthylacetic acid, 75mg/L ascorbic acid, 75mg/L citric acid, 30g/L sucrose, 3g/L plant gel, 1.8 g/L1/2 MS culture medium, 0.8g/L WPM culture medium, and the pH is 5.8; culturing at 22 + -2 deg.C in dark for 25-35 days;
(4) then collecting the stem cells, transferring the stem cells into a stem cell propagation solid culture medium, and continuously culturing for 25-35 days at 22 +/-2 ℃ in a dark place to enable the stem cells to enter a rapid growth period; the stem cell propagation solid culture medium comprises 3 mg/L2, 4-dichlorophenoxyacetic acid, 2mg/L naphthylacetic acid, 1mg/L kinetin, 35g/L cane sugar, 3g/L plant gel, 1.8 g/L1/2 MS culture medium and 0.8g/L WPM culture medium, and the pH value is 5.8;
(5) collecting the ginseng stem cells obtained in the step (4), inoculating the ginseng stem cells into a shake flask containing a stem cell liquid culture medium, and performing dark culture at the speed of 120rpm and the temperature of 22 +/-2 ℃;
The liquid culture medium of the stem cells comprises 3mg/L of 2, 4-dichlorophenoxyacetic acid, 2mg/L of naphthylacetic acid, 1mg/L of kinetin, 35g/L of cane sugar, 1.8g/L of 1/2MS culture medium and 0.8g/L of WPM culture medium, and the pH value is 5.8;
(6) adding stem cell liquid culture medium into a tank body (the volume is 5L) of a biological reaction device, wherein the volume of the culture medium in the biological reaction device accounts for 70% of the volume of the biological reaction device; sterilizing at 121 deg.C for 20 min; the stem cell liquid culture medium comprises 3 mg/L2, 4-dichlorophenoxyacetic acid, 2mg/L naphthylacetic acid, 1mg/L kinetin, 35g/L cane sugar, 1.8 g/L1/2 MS culture medium and 0.8g/L WPM culture medium, and the pH is 5.8;
collecting the ginseng stem cells obtained in the step (5), and inoculating the ginseng stem cells into a biological reaction device containing a stem cell liquid culture medium, wherein the mass of the inoculated stem cells accounts for 8.0% of the volume of the stem cell liquid culture medium; the ventilation volume is 0.15 vvm; culturing at 22 deg.C in dark to obtain Ginseng radix stem cells.
Example 7 culturing of Ginseng Stem cells Using a bioreactor
(1) The ginseng adventitious roots prepared in example 2 were used;
(2) taking the apical part of 0.8mm ginseng adventitious root, putting the apical part into 1mol/L mannitol solution, and taking out after 6 hours in a refrigerator at 6 ℃;
(3) placing the treated root tips into a stem cell induction medium comprising: 1mg/L kinetin, 2mg/L indoleacetic acid, 1.5mg/L naphthylacetic acid, 25mg/L ascorbic acid, 50mg/L citric acid, 20g/L sucrose, 6g/L plant gel, 2.4 g/L1/2 MS culture medium, 1.4g/L WPM culture medium, and the pH is 5.8; culturing at 22 + -2 deg.C in dark for 25-35 days;
(4) Then collecting the stem cells, transferring the stem cells into a stem cell propagation solid culture medium, and continuously culturing for 25-35 days at 22 +/-2 ℃ in a dark place to enable the stem cells to enter a rapid growth period; the stem cell propagation solid culture medium comprises 2 mg/L2, 4-dichlorophenoxyacetic acid, 3mg/L naphthylacetic acid, 1.2mg/L kinetin, 20g/L cane sugar, 1g/L plant gel, 1 g/L1/2 MS culture medium and 1.4g/L WPM culture medium, and the pH value is 5.8;
(5) collecting the ginseng stem cells obtained in the step (4), inoculating the ginseng stem cells into a shake flask containing a stem cell liquid culture medium, and carrying out dark culture at 130rpm and 22 +/-2 ℃; the stem cell liquid culture medium comprises: 2 mg/L2, 4-dichlorophenoxyacetic acid, 3mg/L naphthylacetic acid, 1.2mg/L kinetin, 20g/L sucrose, 1 g/L1/2 MS culture medium and 1.4g/L WPM culture medium, wherein the pH is 5.8;
(6) adding stem cell liquid culture medium into a tank body (the volume is 5L) of a biological reaction device, wherein the volume of the culture medium in the biological reaction device accounts for 30% of the volume of the biological reaction device; sterilizing at 121 deg.C for 20 min; the stem cell liquid culture medium comprises: 2 mg/L2, 4-dichlorophenoxyacetic acid, 3mg/L naphthylacetic acid, 1.2mg/L kinetin, 20g/L sucrose, 1 g/L1/2 MS culture medium and 1.4g/L WPM culture medium, wherein the pH is 5.8;
collecting the ginseng stem cells obtained in the step (5), and inoculating the ginseng stem cells into a biological reaction device containing a stem cell liquid culture medium, wherein the mass of the inoculated stem cells accounts for 2% of the volume of the stem cell liquid culture medium; the ventilation volume is 0.05 vvm; culturing at 22 deg.C in dark to obtain Ginseng radix stem cells.
Example 8 culturing of Ginseng Stem cells Using a bioreactor
(1) The ginseng adventitious roots prepared in example 3 were used;
(2) taking the root tip part of 0.3mm ginseng adventitious root, putting the root tip part into a 1mol/L sucrose solution, and taking out the ginseng after 6 hours in a refrigerator at 1 ℃;
(3) placing the treated root tips into a stem cell induction medium comprising: 0.6mg/L kinetin, 4mg/L indoleacetic acid, 1.5mg/L naphthylacetic acid, 100mg/L ascorbic acid, 150mg/L citric acid, 60g/L sucrose, 1g/L plant gel, 1 g/L1/2 MS culture medium, 0.6g/L WPM culture medium, and the pH is 5.8; culturing at 22 + -2 deg.C in dark for 25-35 days;
(4) then collecting the stem cells, transferring the stem cells into a stem cell propagation solid culture medium, and continuously culturing for 25-35 days at 22 +/-2 ℃ in a dark place to enable the stem cells to enter a rapid growth period; the stem cell propagation solid culture medium comprises 4 mg/L2, 4-dichlorophenoxyacetic acid, 1mg/L naphthylacetic acid, 0.8mg/L kinetin, 20g/L cane sugar, 6g/L plant gel, 2.4 g/L1/2 MS culture medium and 0.6g/L WPM culture medium, and the pH value is 5.8;
(5) collecting the ginseng stem cells obtained in the step (4), inoculating the ginseng stem cells into a shake flask containing a stem cell liquid culture medium, and carrying out dark culture at 130rpm and 22 +/-2 ℃; the stem cell liquid culture medium comprises: 4mg/L of 2, 4-dichlorophenoxyacetic acid, 1mg/L of naphthylacetic acid, 0.8mg/L of kinetin, 20g/L of cane sugar, 2.4g/L of 1/2MS culture medium and 0.6g/L of WPM culture medium, wherein the pH is 5.8;
(6) Adding stem cell liquid culture medium into a tank body (the volume is 5L) of a biological reaction device, wherein the volume of the culture medium in the biological reaction device accounts for 50% of the volume of the biological reaction device; sterilizing at 121 deg.C for 20 min; the liquid culture medium of the stem cells comprises 4mg/L of 2, 4-dichlorophenoxyacetic acid, 1mg/L of naphthylacetic acid, 0.8mg/L of kinetin, 20g/L of cane sugar, 2.4g/L of 1/2MS culture medium and 0.6g/L of WPM culture medium, and the pH value is 5.8;
collecting the ginseng stem cells obtained in the step (3), and inoculating the ginseng stem cells into a biological reaction device containing a stem cell liquid culture medium, wherein the mass of the inoculated stem cells accounts for 10% of the volume of the stem cell liquid culture medium; the ventilation volume is 0.2 vvm; culturing at 22 deg.C in dark to obtain Ginseng radix stem cells.
Test example 1
In this test example, the wild ginseng stem cells prepared in example 6 and the wild ginseng slices used in example 1 were subjected to saponin detection, respectively.
1. Detection method
(1) Principle of
The sample is separated by a C18 chromatographic column after pretreatment such as extraction, and is detected by a high performance liquid chromatography-ultraviolet detector, and the content of each component of the ginsenoside is quantitatively measured by an external standard method.
(2) Reagent
Methanol (CH)4O): chromatographically pure acetonitrile (C)6H11N): pure chromatography
Standard reagents: ginsenoside Re, Rg1, Ra3, Rb1, Rf, Rb2, Rb3, F3, Rg2, Rd, F1
(3) Analytical procedure
Preparing a ginseng stem cell sample:
the sample obtained in example 6 was washed three times with water, ground into paste in a mortar, subjected to ultrasonic wall breaking and freeze-drying, then the sample was porphyrized in the mortar, 50mg was accurately weighed into a 10ml centrifuge tube, 70% methanol solution was added, and vortexed. Sonicate on a sonicator for 10 minutes, repeat twice, filter for use. The latter method is the same as the adventitious root detection method.
Preparing a wild ginseng sample:
washing the sliced wild ginseng sample with water for three times, grinding the sliced wild ginseng sample into paste in a mortar, ultrasonically breaking the wall, freeze-drying, grinding the sample on the mortar, accurately weighing 50mg in a 10ml centrifuge tube, adding 70% methanol solution, and vortexing. Sonicate on a sonicator for 10 minutes, repeat twice, filter for use.
Preparing a standard substance:
preparation of stock solution (0.8 mg/ml): respectively weighing 11 kinds of standard substances including 8.00mg of ginsenoside Re, Rb1, Rg1, Rd, Rf, F3, Rk2, Rb2, Rb3, Rg2 and F1 in a 10ml volumetric flask, and adding high-grade pure methanol to the volume.
Preparation of working solution (32 ug/ml): accurately sucking 1ml of stock solution (0.8mg/ml) into a 25ml volumetric flask, fixing the volume with high-grade pure methanol, and filtering through an organic filter membrane of 0.22um for later use.
(4) Reference conditions for the apparatus
A) And (3) chromatographic column: c18 column with column length of 150mm, column inner diameter of 4.6mm, column packing particle size of 5 μm, or equivalent;
B) mobile phase: a: acetonitrile, b: filtering water with 0.45 μm microporous membrane;
C) flow rate: 0.7 mL/min; gradient elution procedure: 0-13 min, 23% -46% acetonitrile, and the volume flow rate is 0.7 mL/min; 13-33 min, 46-68% acetonitrile, and the volume flow rate is 0.7 mL/min; 33-46.5 min, 68-85% acetonitrile, and the volume flow rate is 0.7 mL/min;
D) column temperature: 30 ℃;
E) detection wavelength: 203 nm;
F) sample introduction volume: 10 μ L.
(5) Presentation of analytical results
The content of each component of the ginsenoside in the sample is calculated according to the formula (1):
Figure BDA0003005919200000191
in the formula:
x is the content of each component of ginsenoside in the sample, and the unit is milligram per kilogram (mg/kg) or milligram per liter (mg/L);
a1-area of peak of ginsenoside component in sample
A2 peak area of ginsenoside component in standard
Rho-concentration of each component of ginsenoside in standard (ug/ml)
V — final volumetric volume of sample solution in milliliters (mL);
m-sample mass in grams (g);
the content of ginsenoside in the sample is the sum of the components to be detected.
2. And (3) detection results: as shown in table 1.
TABLE 1
Figure BDA0003005919200000192
From the above results, it can be seen that the content of various ginsenosides in the wild ginseng stem cells cultured by the bioreactor in example 6 of the present invention is increased, and the total content of ginsenosides is also increased, compared to the mature wild ginseng slices. Therefore, the culture method of the ginseng stem cells is simple and convenient in steps, suitable for large-scale production and high in active ingredient content.
Note that some ginsenosides peaks were relatively close to each other and could not be separated in the liquid phase detection, and therefore some ginsenosides were calculated in table 1.
Comparative example 1
This comparative example differs from example 1 in the induction medium used and the other steps are carried out with reference to example 1. The induction culture of this comparative example included: 30g/L of sucrose, 0.5mg/L of kinetin, 3mg/L of indolebutyric acid, 1.5mg/L of 2, 4-dichlorophenoxyacetic acid, 1/2MS culture medium, 3g/L of plant gel and pH value of 5.8.
FIG. 3 shows the results of induction of adventitious roots on the medium in step (1) of this comparative example, wherein A is a photograph taken after 1 week of culture, B is a photograph taken after 3 weeks of culture, and C is a photograph taken after 5 weeks of culture. As can be seen, after 5 weeks, the culture medium of comparative example 1 failed to induce the generation of adventitious roots directly from the mature wild ginseng slices.
Comparative example 2
This comparative example differs from example 1 in the induction medium used and the other steps are carried out with reference to example 1. The induction culture of this comparative example included: 30g/L of sucrose, 0.5mg/L of kinetin, 3mg/L of indolebutyric acid, 1/2MS culture medium, 3g/L of plant gel and pH value of 5.8.
As a result, the adventitious roots cannot be induced directly from the mature wild ginseng slice, similarly to the comparative example 1 picture demonstration.
Comparative example 3
This comparative example differs from example 1 in the induction medium used and the other steps are carried out with reference to example 1. The induction culture of this comparative example included: 30g/L of sucrose, 0.5mg/L of kinetin, 3mg/L of indoleacetic acid, 3g/L of WPM and 3g/L of plant gel, and the pH value is 5.8.
As a result: the whole body turns yellow in the first week, the color deepens in the third week, the middle part begins to turn brown, and the whole body turns brown and is dried up in the fifth week.
Comparative example 4
This comparative example differs from example 7 in the bioreactor used.
In the comparative example, the biological reaction device is only provided with an air inlet device (common fermentation tank) at the right center of the conical bottom of the tank body, and the bottom wall of the tank body is in a downward-sunken arc shape; the culture method and the liquid medium were the same as in example 7.
Thus, examining the cases of culturing ginseng adventitious roots using example 7 (using the bioreactor of the present invention, volume: 5L) and the ordinary fermentation tank of the present comparative example (volume: 5L), the ginseng adventitious roots growth results were as follows:
TABLE 2
Figure BDA0003005919200000201
Therefore, compared with a common fermentation tank, the biological reaction device has the advantages that the growth multiple of stem cells is higher, the stem cells grow better, the efficiency is improved, and higher content of ginsenoside is obtained.
Although the present invention has been described with reference to a preferred embodiment, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (17)

1. A method for culturing ginseng stem cells by using a biological reaction device is characterized by comprising the following steps:
(1) The method for culturing the ginseng adventitious root by adopting a one-step method comprises the following steps: cleaning and disinfecting mature ginseng, slicing, and inoculating the ginseng into an induction culture medium to induce adventitious roots of the ginseng; inoculating the obtained ginseng adventitious root to an induction culture medium again for relay culture and propagation; then cutting the obtained ginseng adventitious roots into segments, and inoculating the segments into a liquid culture medium for culture to obtain the adventitious roots;
the induction culture medium comprises 1-6mg/L naphthylacetic acid, 0.2-1mg/L gibberellin, 0.1-0.6mg/L kinetin, 0.075-1.5g/L citric acid, 0.03-1g/L ascorbic acid, 20-60g/L sucrose, 1-6g/L plant gel, 1-4g/L B5 culture medium and 1-2.4g/LWPM culture medium;
(2) taking the apical part of the adventitious root of the ginseng, and placing the apical part in a penetrant solution for treatment;
(3) putting the treated ginseng adventitious root tip part into a stem cell induction culture medium for culture, and then transferring the ginseng adventitious root tip part into a stem cell propagation solid culture medium for culture; the dry cell induction culture medium comprises 0.6-1mg/L kinetin, 2-4mg/L indoleacetic acid, 0.5-1.5mg/L naphthylacetic acid, 25-100mg/L ascorbic acid, 50-150mg/L citric acid, 20-60g/L cane sugar, 1-6g/L plant gel, 1-2.4 g/L1/2 MS culture medium and 0.6-1.4g/L WPM culture medium;
the stem cell propagation solid culture medium comprises 2-4mg/L2, 4-dichlorophenoxyacetic acid, 1-3mg/L naphthylacetic acid, 0.8-1.2mg/L kinetin, 20-60g/L sucrose, 3g/L plant gel, 1-2.4 g/L1/2 MS culture medium and 0.6-1.4g/L WPM culture medium;
(4) Collecting the ginseng stem cells obtained in the step (3), inoculating the ginseng stem cells into a shake flask containing a stem cell liquid culture medium, and culturing at 100-140rpm in the dark; the stem cell liquid culture medium comprises 2-4mg/L2, 4-dichlorophenoxyacetic acid, 1-3mg/L naphthylacetic acid, 0.8-1.2mg/L kinetin, 20-60g/L sucrose, 1-2.4 g/L1/2 MS culture medium and 0.6-1.4g/L WPM culture medium;
(5) inoculating the stem cells obtained in the step (4) into a biological reaction device containing a stem cell liquid culture medium, and carrying out dark culture to obtain the ginseng stem cells.
2. The method according to claim 1, wherein in the step (4), the aeration amount is 0.02 to 0.2vvm when the culture is carried out in the bioreactor.
3. The method of claim 2, wherein the ventilation is from 0.05 to 0.15 vvm.
4. The method of claim 1, wherein the bioreactor comprises a tank, the bottom of which comprises at least two gas inlet means.
5. The method according to claim 4, wherein the height of the bottom wall of the tank body is gradually reduced from the periphery to the center to form an inverted cone with a large upper part and a small lower part; the discharge gate sets up in the minimum position in center, and air inlet unit surrounds the discharge gate interval evenly distributed on by all around to the diapire that the center gradually reduces.
6. The method according to any one of claims 1 to 5, wherein in the step (5), when the stem cells are inoculated into the stem cell liquid medium of the bioreactor, the mass of the inoculated stem cells accounts for 1.0 to 10.0 percent of the volume of the stem cell propagation liquid medium.
7. The method of claim 6, wherein the mass of the seeded stem cells is 2.0% to 8.0% of the volume of the stem cell expansion liquid medium.
8. The method according to any one of claims 1 to 5, wherein in the step (2), the apical part of the adventitious root of a ginseng is taken, placed in a penetrant solution, and cryogenically treated for 1 to 10 hours.
9. The method of claim 8, wherein the low temperature is 1-6 ℃.
10. The method of claim 9, wherein the low temperature is 4 ℃.
11. The method according to claim 8, wherein the length of the apical part of the adventitious root of ginseng is 0.3mm to 0.8 mm.
12. The method for culturing ginseng stem cells according to any one of claims 1 to 5, wherein in the step (2), the osmotic agent solution is at least one selected from the group consisting of a sucrose solution, a potassium chloride solution and a mannitol solution.
13. The method for culturing the ginseng stem cells according to claim 12, wherein the osmotic agent solution is a sucrose solution of 1 mol/L.
14. The method for culturing the ginseng stem cells, according to any one of claims 1 to 5, wherein in the step (3), the stem cell induction medium comprises 1mg/L of kinetin, 2.5mg/L of indoleacetic acid, 1mg/L of naphthylacetic acid, 75mg/L of ascorbic acid, 75mg/L of citric acid, 30g/L of sucrose, 3g/L of plant gel, 1.8g/L of 1/2MS medium and 0.8g/L of WPM medium.
15. The method for culturing the ginseng stem cells, according to any one of claims 1 to 5, wherein in the step (3), the solid culture medium for expanding the stem cells comprises 3mg/L of 2, 4-dichlorophenoxyacetic acid, 2mg/L of naphthylacetic acid, 1mg/L of kinetin, 35g/L of sucrose, 3g/L of plant gel, 1.8g/L of 1/2MS culture medium and 0.8g/L of WPM culture medium.
16. The method for culturing ginseng stem cells according to any one of claims 1 to 5, wherein the liquid culture medium for stem cells comprises 3 mg/L2, 4-dichlorophenoxyacetic acid, 2mg/L naphthylacetic acid, 1mg/L kinetin, 35g/L sucrose, 1.8g/L1/2MS medium and 0.8g/L WPM medium.
17. The method for culturing the ginseng stem cells according to any one of claims 1 to 5, wherein in the step (3), the root tip parts of the adventitious roots of ginseng are cultured on the stem cell induction medium and the stem cell propagation solid medium respectively at 20 to 25 ℃ for 25 to 35 days in the absence of light.
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