CN113151146A - Ginseng stem cell separation culture method using biological reaction device - Google Patents

Abstract

The invention discloses a ginseng stem cell separation culture method by utilizing a biological reaction device, which comprises the following steps: (1) preparing the adventitious roots of the ginseng by adopting a one-step method; (2) taking a root tip part of the ginseng adventitious root prepared in the step (1), and dissecting and separating to obtain a stem cell area; (3) inoculating the stem cells obtained by separation in the step (2) into a stem cell induction culture medium for dark culture to obtain a stem cell mass, and transferring the stem cell mass into a subculture medium for dark culture; (4) inoculating the ginseng stem cells subcultured in the step (3) into a stem cell liquid culture medium shake flask, and culturing under the condition of 100-140rpm in a dark place; (5) and (4) inoculating the ginseng stem cells cultured in the shake flask in the step (4) into a biological reaction device, and culturing in a dark place to obtain the ginseng stem cells. By adopting the liquid culture medium and the culture method, the adventitious roots can be directly generated by the induction of each part of the mature ginseng, the content of ginsenoside in the adventitious roots is improved while the induction steps are simplified and the induction time is shortened, and finally the ginseng stem cells with good indexes are obtained.

Description

Ginseng stem cell separation culture method using biological reaction device

Technical Field

The invention belongs to the technical field of plant tissue culture, and particularly relates to a ginseng stem cell separation culture method 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 ginseng cultivated in the 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 ginseng callus induction, 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 high. 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 method for inducing the proliferation of adventitious roots of ginseng, 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 three-dimensional culture (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 the industrial production, cell line degeneration is easy to occur, and the division capability is weak. In addition, the culture period is long, and the content of effective components in the obtained stem cells needs to be improved.

The ginseng adventitious root has meristem at the root tip part and strong division capability. Therefore, if a method for obtaining ginseng stem cells from the culture of mature ginseng with shorter time and simpler steps can be explored, and the content of effective components in the ginseng stem cells can be increased, the method is beneficial to clinical application and has wide significance.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the defects of the prior art and provide a ginseng stem cell separation culture method by using a biological reaction device. The invention obtains the method for obtaining the ginseng stem cells from the culture of the mature ginseng, which has shorter required time and simpler steps, and particularly selects a specific liquid culture medium to culture the expanded adventitious roots in the process of preparing the adventitious roots by a one-step method, then dissects and separates the stem cell area of the tip part of the obtained adventitious roots, matches the stem cell induction culture medium and the stem cell subculture medium with proper component proportion, and places the stem cell liquid culture process in a biological reaction device for carrying out, thereby greatly improving the growth speed of the separated stem cells, having simpler steps and having high content of various ginsenosides in the obtained stem cells.

In order to solve the technical problems, the invention adopts the technical scheme that:

the invention provides a ginseng stem cell separation culture method by utilizing a biological reaction device, which comprises the following steps:

(1) cleaning and disinfecting mature ginseng, slicing, and inoculating the ginseng to an adventitious root induction culture medium to induce adventitious roots of the ginseng; inoculating the obtained ginseng adventitious root to an adventitious root induction culture medium again for subculture and propagation; then inoculating the ginseng adventitious roots obtained by propagation into a liquid culture medium for culture to obtain the ginseng adventitious roots;

(2) taking a root tip part of the ginseng adventitious root prepared in the step (1), and dissecting and separating to obtain a stem cell area;

(3) inoculating the stem cells obtained by separation in the step (2) into a stem cell induction culture medium for dark culture to obtain a stem cell mass, and transferring the stem cell mass into a subculture medium for dark culture;

(4) inoculating the ginseng stem cells cultured in the step (3) to a stem cell liquid culture medium shake flask, and culturing under the condition of 100-140rpm in a dark place;

(5) inoculating the ginseng stem cells cultured in the shake flask in the step (4) into a biological reaction device, and culturing in a dark place to obtain the ginseng stem cells;

the liquid culture medium used in the step (1) comprises: 15-50g/L of sucrose, 0.6-2.4g/L of WPM culture medium, 1-2g/L N6 culture medium, 0-225mg/L of citric acid and 0-7mg/L of indolebutyric acid.

In the scheme, the method adopts a one-step method to culture the adventitious roots of the ginseng from the mature ginseng, so that the steps can be saved, and the induction time can be shortened; the root tip part of the ginseng adventitious root has more meristematic tissues and strong activity. The existing adventitious roots are generally generated by ginseng callus induction, 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 high. In addition, in the process of inducing the callus to generate adventitious roots, the metabolism degree of the ginseng tissue is higher, so that the content of the generated saponin is reduced, and the ginseng obtained by callus induction culture is difficult to meet the requirements of clinical application and the like.

In the above scheme, 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 directly induced from mature ginseng at present. However, the inventors of the present invention have unexpectedly found that the mature ginseng slices can be directly induced to produce adventitious roots on a specific induction medium. Therefore, the intermediate step of callus induction is omitted, the adventitious roots can be directly obtained from the mature ginseng by one-step induction, the induction step is simplified, the induction time is shortened, and the problem of saponin content reduction caused by the metabolic process from generation to differentiation of the callus is also avoided.

The further scheme of the invention is as follows: the liquid culture medium in the step (1) also comprises 0-150mg/L ascorbic acid.

The further scheme of the invention is as follows: the liquid medium of step (1) comprises: 35-45g/L of sucrose, 1g/L N6 of culture medium, 0.6-1.2g/L of WPM culture medium, 0-50mg/L of ascorbic acid, 75-225mg/L of citric acid and 1-3mg/L of indolebutyric acid; preferably, the liquid medium in step (1) comprises: 35g/L of sucrose, 1g/L N6 of culture medium, 1.2g/L of WPM culture medium, 50mg/L of ascorbic acid, 225mg/L of citric acid and 3mg/L of indolebutyric acid.

In the scheme, the components of the liquid culture medium after the propagation step are adjusted in the adventitious root induction process of the ginseng slices, wherein the WPM culture medium and the N6 culture medium with higher content are adopted, the content of a nitrogen source in the liquid culture medium is improved, and the content of ammonium nitrogen is higher, so that the dedifferentiation process of the ginseng cells is inhibited to a certain extent, and the tissue after propagation keeps the original functions of the cells at the root under the action of the liquid culture medium and continues to grow. In order to promote the growth of isolated tissues, the liquid culture medium provided by the invention is also added with a citric acid component properly to promote the TCA cycle of cells, and meanwhile, the introduced ascorbic acid can properly reduce the pH of a culture system, so that the adventitious root is biased to absorb a nitrogen source in nitrate nitrogen in a lower pH environment, and the phenomenon of cell poisoning caused by excessive absorption of ammonium nitrogen is avoided; citric acid may also be used to adjust the pH to facilitate the process. In conclusion, the adventitious root liquid culture medium provided by the invention adjusts the absorption balance of the adventitious root on nitrate nitrogen and ammonium nitrogen through pH, and simultaneously introduces citric acid to promote TCA cycle of adventitious root cells under the action of inhibiting dedifferentiation by the ammonium nitrogen, so that the adventitious root is directly generated without forming callus in the whole preparation process, and the growth multiple of the adventitious root can be greatly improved.

The further scheme of the invention is as follows: in the step (5), when the stem cells are inoculated into a stem cell liquid culture medium of a biological reaction device, the mass of the inoculated stem cells accounts for 1.0-10.0% of the volume of the stem cell propagation liquid culture medium; the ventilation volume for culturing the stem cells in the biological reaction device is 0.02-0.2 vvm; preferably, the aeration is 0.05 to 0.15 vvm.

The further scheme of the invention is as follows: the biological reaction device comprises a tank body, and 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 above scheme, in the biological reaction device provided by 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 inlet air cannot vertically rise, and further, two or more air inlet devices are uniformly distributed around the center at intervals, which is beneficial to uniform distribution of air in the tank body and uniform growth of stem cells in the tank body.

The further scheme of the invention is as follows: in the step (3), the stem cell induction culture medium comprises 2-4mg/L gibberellin, 0.6-1mg/L kinetin, 2-4mg/L indoleacetic acid, 15-75mg/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 1-2.5g/L B5 culture medium; preferably, the stem cell induction medium comprises 3mg/L gibberellin, 0.8mg/L kinetin, 2.5mg/L indoleacetic acid, 50mg/L ascorbic acid, 100mg/L citric acid, 40g/L sucrose, 3g/L plant gel, 1.8 g/L1/2 MS medium, and 1.5g/L B5 medium.

In the scheme, the stem cell induction culture medium provided by the invention contains indoleacetic acid, gibberellin and kinetin, can induce the growth of stem cells, and ascorbic acid and citric acid can generate a synergistic antioxidant effect. The components in the induction culture medium have synergistic effect, and can promote the rapid growth of stem cells in the initial stage.

The further scheme of the invention is as follows: in the step (3), the stem cell subculture medium comprises 2-4mg/L2, 4-dichlorophenoxyacetic acid, 1-3mg/L gibberellin, 0.8-1.2mg/L kinetin, 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 subculture medium comprises 3 mg/L2, 4-dichlorophenoxyacetic acid, 2mg/L gibberellin, 1mg/L kinetin, 35g/L sucrose, 3g/L plant gel, 1.8 g/L1/2 MS medium and 0.8g/L WPM medium.

The further scheme of the invention is as follows: in the steps (4) and (5), the stem cell liquid culture medium comprises 2-4mg/L2, 4-dichlorophenoxyacetic acid, 1-3mg/L gibberellin, 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 gibberellin, 1mg/L kinetin, 35g/L sucrose, 1.8 g/L1/2 MS culture medium and 0.8g/L WPM culture medium.

The further scheme of the invention is as follows: the pH of the stem cell induction culture medium, the stem cell subculture medium and the stem cell liquid culture medium is 5.6-6.0. The induction medium, the subculture medium and the stem cell liquid medium are adjusted in pH using NaOH or KOH.

The further scheme of the invention is as follows: in the step (2), the root tip part of the adventitious root of the ginseng cultured in the step (1) is taken, the root tip part is observed under a microscope, a stem cell area is determined according to the characteristics of stem cells, and the stem cell area at the root tip is obtained by cutting through a scalpel through micromanipulation.

In the above scheme, the root tip part is observed under a microscope, and the irregular part of the cell shape and arrangement is a growing point, namely a meristem cell aggregation region, wherein the cell contains a plurality of vacuoles. And cutting by a micro-operation scalpel to obtain a root tip stem cell area.

The further scheme of the invention is as follows: in the step (3), the ginseng stem cells after dissection are respectively and sequentially cultured in a stem cell induction culture medium and a stem cell subculture medium at the temperature of 20-25 ℃ in a dark place until a large number of cell clusters grow from the inoculated stem cells.

The further scheme of the invention is as follows: in the step (1), the adventitious root induction culture medium comprises 1-6mg/L of naphthylacetic acid, 0.1-0.6mg/L of kinetin, 0.2-1mg/L of gibberellin, 0.075-1.5g/L of citric acid, 0.03-1g/L of ascorbic acid, 20-60g/L of sucrose, 1-6g/L of plant gel, 1-4g/L B5 of culture medium and 1-2.4g/L of WPM culture medium. Preferably, the adventitious root induction medium includes 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.

In the scheme, the naphthylacetic acid is a plant growth regulator, the gibberellin is a plant hormone, and the formation of adventitious roots can be promoted under the combined action. 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. The induction culture medium under the preferable 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 the scheme, 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, so that the technical purpose of shortening the induction time can be realized by adopting the one-step method for inducing the adventitious roots in the invention under the condition of adopting the conventional culture medium for culture, and meanwhile, the pollution risk is reduced compared with the two-step method in the prior art.

In the present invention, WPM medium, B5 medium, 1/2MS medium, and the like are known in the art.

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.

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.

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.

N6 culture medium

Ingredient (mg/L): potassium nitrate 2800, ammonium sulfate 463, potassium dihydrogen sulfate 400, magnesium sulfate heptahydrate 185, calcium chloride dihydrate 165, disodium ethylenediaminetetraacetate 37.3, ferrous sulfate heptahydrate 27.8, manganese sulfate hydrate 4.4, zinc sulfate heptahydrate 1.5, boric acid 1.6, potassium iodide 0.8, vitamin B11.0, vitamin B60.5, hydrochloric acid 0.5, glycine 2.0, sucrose 2000, pH 5.8(25 ℃).

The further scheme of the invention is as follows: in the step (1), the inoculating the ginseng adventitious roots obtained by propagation to the liquid medium comprises: cutting the ginseng adventitious roots obtained by propagation into small segments, inoculating the small segments into a liquid culture medium, and culturing the small segments on a shaking table at the temperature of 22 +/-1 ℃ and the rotation speed of 110-.

The further scheme of the invention is as follows: in the step (1), the step of inoculating the ginseng adventitious roots obtained by propagation to a liquid culture medium further comprises: inoculating the adventitious roots cultured on the shaking table for 3-4 weeks into a biological reaction device containing a liquid culture medium, and continuously culturing for 3-4 weeks in a dark place, wherein the ventilation volume of the biological reaction device is 0.01-0.4vvm, and the culture temperature is 22 +/-1 ℃; preferably, the aeration is from 0.15 to 0.25 vvm.

In the scheme, the volume of the sterilized liquid culture medium accounts for 20-80% of the volume of the biological reaction device; the mass of the inoculated adventitious roots accounts for 0.5-2.5% of the volume of the liquid culture medium. The biological reaction device is used for culturing the ginseng adventitious roots and comprises: 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.

In the above scheme, the tank body of the biological reaction device 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, and the growth is uniform, and the fast growth of adventitious roots is promoted.

The bioreactor provided by the invention can be used for culturing adventitious roots in the step (1) and can also be used for liquid culture of stem cells in the step (5).

In the above embodiment, the volume of the bioreactor provided by the present invention is 1-50L, preferably 2-20L, preferably 3-10L, preferably 5L.

The bioreactor comprises the following further scheme: the center of the bottom of the tank body is provided with a discharge hole, 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.

The bioreactor comprises the following further scheme: air inlet unit sets up on the jar body diapire inclined plane of back taper shape big-end-up, so, the gas that gets into can not rise perpendicularly, and is further, and two and more than two air inlet unit distribute around the center interval uniformly, are favorable to the air at jar internal evenly distributed, are favorable to the internal adventitious root of jar to grow evenly.

The bioreactor comprises the following further scheme: 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.

The bioreactor comprises the following further scheme: air inlet unit includes air inlet, intake pipe and air cleaner, the air inlet is located the diapire of the jar body, intake pipe and air inlet sealing connection, air cleaner sets up and filters institute through the air in the intake pipe.

The bioreactor comprises the following 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.

The bioreactor comprises the following further scheme: an inoculation port is arranged on the cover body or the top of the tank body and used for inoculation.

The bioreactor comprises the following further scheme: a plurality of transparent observation windows are arranged on the tank body; preferably, the observation window is arranged on the side wall of the middle part and/or the lower part of the tank body.

In the above scheme, when the jar body adopts opaque stainless steel material, the internal portion of jar can be observed from 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.

In a further scheme, a handle is also arranged on the tank body; preferably, the handle comprises at least two.

In the above scheme, the handles arranged on the tank body can be symmetrically arranged, so that a user can conveniently take and move the biological reaction device.

The further scheme of the invention is as follows: in the step (1), 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; the ginseng age of the mature ginseng is more than 3 years, preferably, the ginseng age of the mature ginseng is more than 6 years.

As a preferred embodiment, the mature ginseng is a centennial ginseng.

The century ginseng is rare in nature, has high edible and medicinal values, can tonify five internal organs, calm spirit, calm soul, stop palpitation, remove pathogenic qi, improve eyesight, and benefit heart and intelligence; the value of the ginseng cultivation method is far higher than that of planted ginseng with short ginseng 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 branch root, 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 the scheme, the saponin content in different parts of the ginseng is different, wherein the root part has the highest content, so the part is preferably sliced to improve the saponin content in the induced adventitious root.

In a further scheme, the ginseng is selected from wild ginseng, transplanted ginseng, ginseng under forest and garden ginseng; preferably, the ginseng is wild ginseng.

After adopting the technical scheme, compared with the prior art, the invention has the following beneficial effects:

1. the ginseng stem cell separation culture method utilizing the biological reaction device provided by the invention adopts the biological reaction device with an improved structure, so that the ventilation is uniform and sufficient, the rapid growth of stem cells is facilitated, and the production is expanded;

2. the invention provides a ginseng stem cell separation culture method by utilizing a biological reaction device, which is characterized in that a stem cell area is obtained by carrying out microscopic observation and dissection on a ginseng adventitious root prepared by adopting a one-step method, the separated stem cell is cultured by matching with a stem cell induction culture medium and a stem cell subculture medium with proper component proportions, and meanwhile, the biological reaction device is combined to be used, so that the stem cell growth speed is extremely high, and the content of active ingredients in the obtained ginseng stem cell is improved;

3. the method for separating and culturing the adventitious roots of the ginseng by using the biological reaction device realizes that the adventitious roots of the ginseng are directly induced by adopting a one-step method and inoculating each part of the mature ginseng after being processed into the induction culture medium without the intermediate step of inducing callus, so that the induction step can be simplified, the induction time can be shortened, and the pollution risk can be reduced;

4. according to the ginseng stem cell separation culture method using the biological reaction device, in the process of culturing the adventitious root by the one-step method, the liquid culture medium adjusts the absorption balance of the adventitious root on nitrate nitrogen and ammonium nitrogen through pH, and simultaneously, under the action of inhibiting dedifferentiation by the ammonium nitrogen, citric acid is introduced to promote TCA circulation of the adventitious root cell, so that generation of dedifferentiation callus is avoided, and the growth multiple of the adventitious root can be greatly improved while the saponin content is ensured.

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 induction of adventitious roots using the induction medium of example 1 of the present invention;

FIG. 2 is a schematic diagram of induction of adventitious roots using the induction medium of comparative example 1;

FIG. 3 is a schematic view showing the structure of a bioreactor of the present invention.

The main components in the figure are: 1-jar body, 2-lid, 3-exhaust apparatus, 4-air inlet unit, 5-discharge gate, 6-intake pipe, 7-air cleaner, 8-blast pipe, 9-filter equipment, 10-observation window, 11-handle, 12-supporting legs.

It should be noted that the drawings and the description are not intended to limit the scope of the inventive concept in any way, but to illustrate it by a person skilled in the art with reference to specific embodiments.

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.

As shown in FIG. 3, the present invention provides a bioreactor for culturing ginseng adventitious roots, 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 be used for conveniently producing the ginseng stem cells, 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, 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, and the growth is uniform, and the fast growth of adventitious roots is promoted.

The center of the bottom of the tank body 1 is provided with a discharge hole 5, and the air inlet devices are uniformly distributed around the discharge hole 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.

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.

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 manner, and the filtering device 9 is arranged on the exhaust pipe 8;

preferably, the filter device 9 is an air filter 7 or a liquid filter.

In a preferred embodiment, the exhaust port 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.

An inoculation port is arranged on the cover body 2 or the top of the tank body 1 and is used for inoculation.

A plurality of transparent observation windows 10 are arranged on 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.

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.

Example 1

In this embodiment, the preparation of the ginseng adventitious roots by the one-step method and the specific liquid medium culture specifically comprises the following steps:

(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. 1, wherein A is a photograph of 1 week of cultivation, B is a photograph of 3 weeks of cultivation, and C is a photograph of 5 weeks of cultivation. As can be seen, after 5 weeks, adventitious roots were induced directly on the slices of the mature wild ginseng.

Example 2

In this embodiment, the preparation of the ginseng adventitious roots by the one-step method and the specific liquid medium culture specifically comprises the following steps:

(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.

Example 3

In this embodiment, the preparation of the ginseng adventitious roots by the one-step method and the specific liquid medium culture specifically comprises the following steps:

(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, 0.75g/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 adventitious roots can be induced by one step in step (1) of this example.

Example 4

In this embodiment, the preparation of the ginseng adventitious roots by the one-step method and the specific liquid medium culture specifically comprises the following steps:

(1) induction of adventitious roots

Cleaning main root 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 induction culture medium, and dark culturing at 22 + -1 deg.C for 4-5 weeks to induce mountain ginseng adventitious root; 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, in step (1) of this example, adventitious roots can be induced directly by one step.

Example 5

In this embodiment, the preparation of the ginseng adventitious roots by the one-step method and the specific liquid medium culture specifically comprises the following steps:

(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, in step (1) of this example, adventitious roots can be induced directly by one step.

Example 6

In this example, the adventitious roots of ginseng were prepared by further combining the bioreactor with the basis of example 1, and the adventitious roots prepared in step (3) of example 1 were further processed as follows:

adding a liquid culture medium into a tank body (the volume is 5L) of a biological reaction device, wherein the volume of the liquid culture medium in the biological reaction device accounts for 70 percent of the volume of the biological reaction device; sterilizing at 121 deg.C for 20 min;

the adventitious roots obtained in example 1 were cut into a tissue having a length of about 1cm, and inoculated into a liquid medium in a bioreactor, the mass of the inoculated adventitious roots occupying 1.0% of the volume of the liquid medium; ventilating the biological reaction device, wherein the ventilation rate is 0.15vvm, and culturing in the dark at the temperature of 22 +/-1 ℃ for 3-4 weeks to obtain the adventitious roots.

The liquid medium used in this example was the same as that used in example 1.

Examples 7 to 14

Examples 7 to 14 are based on example 1, wherein the liquid medium in step (3) was replaced with a medium containing 15 to 50g/L sucrose, 0.6 to 2.4g/L WPM medium, 1 to 2g/L N6 medium, 0 to 150mg/L ascorbic acid (Vc), 0 to 225mg/L citric acid, 0 to 7mg/L indolebutyric acid (IBA) and having a pH of 5.8. The specific content ratio of the medium in each example is shown in Table 1.

Example 15

In this example, the isolation and culture of ginseng stem cells using the adventitious roots of ginseng obtained in example 11 and the above-mentioned bioreactor specifically comprises the following steps:

(1) the ginseng adventitious roots prepared in example 11 were used;

(2) taking 0.5mm of the apical part of the adventitious root of the ginseng in the step (1), and dissecting and cutting a stem cell area of the apical part by micromanipulation;

(3) inoculating the stem cells obtained by separation in the step (2) into a stem cell induction culture medium, and carrying out dark culture at the temperature of 22 ℃ until a large number of cell clusters grow out from the inoculated stem cells; the stem cell induction medium comprises: 3mg/L gibberellin, 0.8mg/L kinetin, 2.5mg/L indoleacetic acid, 50mg/L ascorbic acid, 100mg/L citric acid, 40g/L sucrose, 3g/L plant gel, 1.8 g/L1/2 MS culture medium, 1.5g/L B5 culture medium, and the pH is 5.8; then collecting the stem cells, transferring the stem cells into a stem cell subculture medium, and continuously culturing the stem cells in a dark place at 22 ℃ until a large number of cell clusters grow from the inoculated stem cells; the stem cell subculture medium comprises 3 mg/L2, 4-dichlorophenoxyacetic acid, 2mg/L gibberellin, 1mg/L kinetin, 35g/L cane sugar, 3g/L plant gel, 1.8 g/L1/2 MS medium and 0.8g/L WPM medium, and the pH is 5.8;

(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 carrying out dark culture on the ginseng stem cells under the conditions of 22 +/-2 ℃ and 120rpm shaking tables;

(5) adding a 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 percent of the volume of the culture medium; sterilizing at 121 deg.C for 20 min; collecting the ginseng stem cells obtained in the step (4), 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;

the stem cell liquid culture medium in the steps (4) and (5) comprises: 3 mg/L2, 4-dichlorophenoxyacetic acid, 2mg/L gibberellin, 1mg/L kinetin, 35g/L sucrose, 1.8 g/L1/2 MS culture medium and 0.8g/L WPM culture medium, and the pH is 5.8.

Example 16

In this example, the isolation and culture of ginseng stem cells using the adventitious roots of ginseng obtained in example 12 and the above-mentioned bioreactor specifically comprises the steps of:

(1) the ginseng adventitious roots prepared in example 12 were used;

(2) taking the apical part of the adventitious root of the ginseng in the step (1) to be 0.8mm, and dissecting and cutting the stem cell area of the apical part by micromanipulation;

(3) inoculating the stem cells obtained by separation in the step (2) into a stem cell induction culture medium, and carrying out dark culture at 25 ℃ until a large number of cell clusters grow out from the inoculated stem cells; the stem cell induction medium comprises: 4mg/L gibberellin, 0.6mg/L kinetin, 2mg/L indoleacetic acid, 75mg/L ascorbic acid, 50mg/L citric acid, 20g/L sucrose, 6g/L plant gel, 2.4 g/L1/2 MS culture medium, 1g/L B5 culture medium, and the pH is 5.8; then collecting the stem cells, transferring the stem cells into a stem cell subculture medium, and continuously culturing in a dark place at 25 ℃ until a large number of cell clusters grow from the inoculated stem cells; the stem cell subculture medium comprises 2 mg/L2, 4-dichlorophenoxyacetic acid, 3mg/L gibberellin, 1.2mg/L kinetin, 60g/L cane sugar, 1g/L plant gel, 1 g/L1/2 MS medium and 1.4g/L WPM medium, and the pH is 5.8;

(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 carrying out dark culture on the ginseng stem cells under the conditions of 22 +/-2 ℃ and 120rpm shaking tables;

(5) 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; collecting the ginseng stem cells obtained in the step (4), 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 0.05vvm, and the ginseng stem cells are cultured in a dark place at 22 ℃ to obtain the ginseng stem cells;

the stem cell liquid culture medium in the steps (4) and (5) comprises: 2 mg/L2, 4-dichlorophenoxyacetic acid, 3mg/L gibberellin, 1.2mg/L kinetin, 60g/L sucrose, 1 g/L1/2 MS culture medium and 1.4g/L WPM culture medium, and the pH is 5.8.

Example 17

In this example, the isolation and culture of ginseng stem cells using the adventitious roots of ginseng obtained in example 13 and the above-mentioned bioreactor specifically comprises the steps of:

(1) the ginseng adventitious roots prepared in example 13 were used;

(2) taking the apical part of the adventitious root of the ginseng in the step (1) to be 0.3mm, and dissecting and cutting the stem cell area of the apical part by micromanipulation;

(3) inoculating the stem cells obtained by separation in the step (2) into a stem cell induction culture medium, and carrying out dark culture at the temperature of 20 ℃ until a large number of cell clusters grow out from the inoculated stem cells; the stem cell induction medium comprises: 2mg/L gibberellin, 1mg/L kinetin, 4mg/L indoleacetic acid, 15mg/L ascorbic acid, 150mg/L citric acid, 60g/L sucrose, 1g/L plant gel, 1 g/L1/2 MS culture medium, 2.5g/L B5 culture medium, and the pH is 5.8; then collecting the stem cells, transferring the stem cells into a stem cell subculture medium, and culturing at 22 ℃ in a dark place until a large number of cell clusters grow from the inoculated stem cells; the stem cell subculture medium comprises 4mg/L2, 4-dichlorophenoxyacetic acid, 1mg/L gibberellin, 0.8mg/L kinetin, 20g/L sucrose, 6g/L plant gel, 2.4 g/L1/2 MS medium and 0.6g/L WPM medium, and the pH is 5.8;

(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 carrying out dark culture on the ginseng stem cells under the conditions of 22 +/-2 ℃ and 120rpm shaking tables;

(5) adding a 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 percent of the volume of the biological reaction device; sterilizing at 121 deg.C for 20 min; collecting the ginseng stem cells obtained in the step (4), 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 0.2vvm, and the ginseng stem cells are cultured in a dark place at 22 ℃ to obtain the ginseng stem cells;

the stem cell liquid culture medium in the steps (4) and (5) comprises: 4mg/L of 2, 4-dichlorophenoxyacetic acid, 1mg/L of gibberellin, 0.8mg/L of kinetin, 20g/L of cane sugar, 6g/L of plant gel, 2.4g/L of 1/2MS culture medium and 0.6g/L of WPM culture medium, and the pH value is 5.8.

Example 18

In this example, the isolated culture of ginseng stem cells was carried out using the ginseng adventitious roots obtained in example 1 and the bioreactor provided above, and other embodiments of this example are the same as in example 15.

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. 2 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 picture display of comparative example 1.

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.

Test example 1

In this test example, the fold increase and ginsenoside content of the ginseng adventitious roots obtained in examples 7 to 14 were measured by the following methods:

a method for detecting ginsenoside in adventitious roots of ginseng comprises the following steps:

(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

(3) Analytical procedure

Taking about 6g (accurate to 0.01g) of the uniformly mixed sample, grinding the sample in a 150mL mortar, transferring the sample into a 50mL centrifuge tube, adding 10mL of water, uniformly mixing, breaking the wall for 3 minutes by 400W on an ultrasonic cell crusher, and freezing the sample in a refrigerator at the temperature of 18 ℃ below zero for 3 hours. Freeze-drying in a freeze-drying machine for 48 hours until no water drops exist outside the cup body.

The sample was ground in a mortar and 50mg was weighed accurately into a 10ml centrifuge tube, 70% methanol solution was added and vortexed. Sonicate on a sonicator for 10 minutes, repeat twice, filter for use.

(4) Reference conditions of the apparatus

A) A 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) the column temperature is 30 ℃;

E) the detection wavelength is 203 nm;

F) the injection volume is 10 muL.

(5) Presentation of analytical results

The content of each component of the ginsenoside in the sample is calculated according to the formula (1):

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 obtained by adding the components.

The content of ginsenoside in the sample is the sum of the components to be detected.

Secondly, the calculation mode of the increase multiple is as follows:

the growth factor is the weight of adventitious roots after the end of growth/the weight of inoculated adventitious root seeds.

The results are shown in table 1:

TABLE 1

As is clear from Table 1, the liquid culture media prepared in examples 11 to 13 showed relatively high growth factors in adventitious roots and high total saponins. The group with the advantage of the fold increase can be partially ignored due to the need for subsequent stem cell culture of adventitious roots, whereas examples 11-13 with higher total saponins were used. Among them, the adventitious roots obtained in example 11 were the best in terms of both the fold increase and the saponin content.

Test example 2

In this test example, saponin detection was performed on the wild ginseng adventitious root stem cells obtained in examples 15 to 17 and the wild ginseng slices used in example 1, respectively.

The detection method is as follows:

(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 samples obtained in examples 15-17 were washed three times with water, ground into a paste in a mortar, broken by ultrasound, lyophilized, and then the samples were porphyrized in a mortar, 50mg were 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 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, Rg1, Ra3, Rb1, Rf, Rb2, Rb3, F3, Rg2, Rd and F1 in a 10ml volumetric flask, and fixing the volume by using high-grade pure methanol.

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 of the apparatus

A) A 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):

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.

The results are shown in table 2:

TABLE 2

From the above results, it can be seen that the contents of various ginsenosides in the wild ginseng adventitious root stem cells cultured by the present invention are all increased compared with the mature wild ginseng slices, and the total content of ginsenosides is further increased under the addition effect of the bioreactor. 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.

In the liquid phase detection, some ginsenosides peaks were relatively close and could not be separated, and therefore some ginsenosides were mixed and calculated in the table.

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 (10)

1. A method for separating and culturing ginseng stem cells by using a biological reaction device is characterized by comprising the following steps:

(1) cleaning and disinfecting mature ginseng, slicing, and inoculating the ginseng to an adventitious root induction culture medium to induce adventitious roots of the ginseng; inoculating the obtained ginseng adventitious root to an adventitious root induction culture medium again for subculture and propagation; then inoculating the ginseng adventitious roots obtained by propagation into a liquid culture medium for culture to obtain the ginseng adventitious roots;

(2) taking a root tip part of the ginseng adventitious root prepared in the step (1), and dissecting and separating to obtain a stem cell area;

(3) inoculating the stem cells obtained by separation in the step (2) into a stem cell induction culture medium for dark culture to obtain a stem cell mass, and transferring the stem cell mass into a subculture medium for dark culture;

(4) inoculating the ginseng stem cells subcultured in the step (3) into a stem cell liquid culture medium shake flask, and culturing under the condition of 100-140rpm in a dark place;

(5) inoculating the ginseng stem cells cultured in the shake flask in the step (4) into a biological reaction device, and culturing in a dark place to obtain the ginseng stem cells;

the liquid culture medium used in the step (1) comprises: 15-50g/L of sucrose, 0.6-2.4g/L of WPM culture medium, 1-2g/L N6 culture medium, 0-225mg/L of citric acid and 0-7mg/L of indolebutyric acid.

2. The method for separating and culturing ginseng stem cells using a bioreactor according to claim 1, wherein the liquid culture medium of step (1) further comprises 0-150mg/L ascorbic acid.

3. The method for separating and culturing ginseng stem cells using a bioreactor according to claim 2, wherein the liquid culture medium of the step (1) comprises: 35-45g/L of sucrose, 1g/L N6 of culture medium, 0.6-1.2g/LWPM of culture medium, 0-50mg/L of ascorbic acid, 75-225mg/L of citric acid and 1-3mg/L of indolebutyric acid;

preferably, the liquid medium in step (1) comprises: 35g/L of sucrose, 1g/L N6 of culture medium, 1.2g/LWPM of culture medium, 50mg/L of ascorbic acid, 225mg/L of citric acid and 3mg/L of indolebutyric acid.

4. The isolated culture method of ginseng stem cells using a bioreactor according to claim 1, 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% of the volume of the stem cell propagation liquid medium; the ventilation volume for culturing the stem cells in the biological reaction device is 0.02-0.2 vvm;

preferably, the aeration is 0.05 to 0.15 vvm.

5. The method for separating and culturing ginseng stem cells using a bioreactor according to claim 1, wherein the bioreactor comprises a tank body, and 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.

6. The isolated culture method of ginseng stem cells using a bioreactor according to claim 1, wherein in the step (3), the stem cell induction medium comprises 2-4mg/L gibberellin, 0.6-1mg/L kinetin, 2-4mg/L indoleacetic acid, 15-75mg/L ascorbic acid, 50-150mg/L citric acid, 20-60g/L sucrose, 1-6g/L plant gel, 1-2.4 g/L1/2 MS medium, 1-2.5g/L B5 medium;

preferably, the stem cell induction culture medium comprises 3mg/L gibberellin, 0.8mg/L kinetin, 2.5mg/L indoleacetic acid, 50mg/L ascorbic acid, 100mg/L citric acid, 40g/L sucrose, 3g/L plant gel, 1.8 g/L1/2 MS culture medium and 1.5g/L B5 culture medium;

the stem cell subculture medium comprises 2-4mg/L2, 4-dichlorophenoxyacetic acid, 1-3mg/L gibberellin, 0.8-1.2mg/L kinetin, 20-60g/L sucrose, 1-6g/L plant gel, 1-2.4 g/L1/2 MS medium and 0.6-1.4g/L WPM medium;

preferably, the stem cell subculture medium comprises 3 mg/L2, 4-dichlorophenoxyacetic acid, 2mg/L gibberellin, 1mg/L kinetin, 35g/L sucrose, 3g/L plant gel, 1.8 g/L1/2 MS medium and 0.8g/L WPM medium.

7. The isolated culture method of ginseng stem cells using a bioreactor according to claim 1, wherein the liquid culture medium of the stem cells comprises 2-4mg/L2, 4-dichlorophenoxyacetic acid, 1-3mg/L gibberellin, 0.8-1.2mg/L kinetin, 20-60g/L sucrose, 1-2.4 g/L1/2 MS medium and 0.6-1.4g/L WPM medium in steps (4) and (5);

preferably, the stem cell liquid culture medium comprises 3 mg/L2, 4-dichlorophenoxyacetic acid, 2mg/L gibberellin, 1mg/L kinetin, 35g/L sucrose, 1.8 g/L1/2 MS culture medium and 0.8g/L WPM culture medium.

8. The method for separating and culturing ginseng stem cells using a bioreactor according to claim 1, wherein in the step (2), the apical part of the adventitious root of ginseng cultured in the step (1) is taken, the apical part is observed under a microscope, the stem cell region is determined according to the characteristics of the stem cells, and the apical stem cell region is obtained by cutting with a scalpel through micromanipulation.

9. The isolated culture method of ginseng stem cells using a bioreactor according to claim 1, wherein the ginseng stem cells dissected and separated are cultured in the stem cell induction medium and the stem cell subculture medium at 20-25 ℃ in the dark, respectively, until the inoculated stem cells grow a large number of cell clusters.

10. The isolated culture method of ginseng stem cells using a bioreactor according to claim 1, wherein the adventitious root-inducing culture medium in step (1) comprises 1-6mg/L naphthylacetic acid, 0.1-0.6mg/L kinetin, 0.2-1mg/L gibberellin, 0.75-1.5g/L citric acid, 0.03-1g/L ascorbic acid, 20-60g/L sucrose, 1-6g/L plant gel, 1-4g/L B5 medium and 1-2.4g/L WPM medium.

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