KR100506625B1 - Mass production method of cultured mountain ginseng by tissue culture - Google Patents

Abstract

The present invention relates to a mass production method of wild ginseng cultured muscle by tissue culture, and in the method of growing the wild ginseng root muscle in a large amount by tissue culture comprising a step of material selection-sterilization-callus induction-culture-mass growth. Selected by the DNA fingerprinting method, the step of inducing callus is omitted, characterized in that induction of ginseng cultured muscle directly from the material in the medium, the step of inducing callus according to the present invention is not secured 2, The use of growth regulators such as 4-D can be excluded, and in particular, the content of the active ingredient, which is a common problem with cultured triceps, can be significantly increased.

Description

Mass production method of cultured mountain ginseng by tissue culture

The present invention relates to a mass production method of wild ginseng cultured muscle by tissue culture, and more particularly, to a mass production method of wild ginseng cultured muscle with increased content of ginsenoside, an effective bioactive substance.

Ginseng is botanically belonging to the genus Araliacea, Panax ginseng (Panax) and uses the root mainly for medicinal purposes. Six to seven species of plants belong to the world are known, but ginseng species that are economically cultivated and distributed as commodities in the ginseng market are mainly domestic, Chinese, and American. Panax ginseng CAMeyer's botanical name, which is geographically distributed and cultivated in the Far East of Asia, has traditionally been the most important medicinal ingredient among Chinese herbal medicines.

Ginseng has been incredibly effective in treating various diseases and promoting energy recovery since ancient times, and continues to research extensively to explore the efficacy and pharmacological effects of ginseng. It can be said to maintain the homeostasis of body control function, and based on this action, it can be anti-fatigue and anti-stress action, anti-diabetic action, blood pressure control action, anti-cancer action and brain function enhancement. It is judged that it is a result. The main active ingredients of ginseng are known saponins, sapongenins, polyacetylenes, pyrazine derivatives, maltol, and the like. In particular, the ginseng line is known to have many beneficial ingredients or contents that are not included in the ginseng line.

Although ginseng derived from wild ginseng and wild ginseng has the same origin as ginseng, ginseng has a short breeding period and has been screened for the purpose of quantitative productivity rather than effect. On the contrary, it is recognized that falling ginseng and wild ginseng derived from wild ginseng, which are close to their origin, maintain a complex genetic structure. In this regard, the difference between wild ginseng or wild ginseng derived from wild ginseng is not only phenomenal by the word of mouth, but also by modern analytical methods.

Therefore, if the ginseng plant that is superior to ginseng in terms of efficacy and high value-added wild ginseng line is provided to consumers through normal cultivation or harvesting ginseng, it has no choice but to provide limited time, cost and opportunities available to consumers. Can be solved through biotechnological methods.

Prior arts related to this include Korean Patent Application Nos. 2001-0003285 and 2001-0031029. These techniques are methods for mass production of wild ginseng roots (seeds) using bioreactors. These techniques allow mass growth of wild ginseng, but there are problems in terms of quality. That is, the above techniques are essential to the process of organically callus, and since growth regulators such as 2,4-D are used in this process, there is a risk of adversely affecting the human body. 2,4-D is a substance used as a component of the defoliant, and is a substance which is not guaranteed to be stable to the human body. In addition, the wild ginseng obtained by the above techniques have a problem that the content of ginsenoid, a bioactive substance, is relatively low.

The present invention is to solve the problems as described above, by mass-producing wild ginseng using a bioreactor, but omitting the use of toxic substances such as 2,4-D by eliminating the step of organically callus, the qualitative aspect The aim is to provide a method for producing excellent wild ginseng in large quantities. Another object of the present invention is to provide a method for producing wild ginseng having a high content of a bioactive substance.

Mass production method of wild ginseng cultured root according to the present invention is made of wild ginseng tissue native to the mountain,

1) selecting individuals holding the native genes of wild ginseng (AFLPs analysis),

2) aseptic material preparation step by surface disinfection of wild ginseng sample,

3) inducing wild ginseng cultured root directly from sterile material,

4) adapting wild ginseng cultured roots to liquid culture for mass production;

5) the initial culture step of wild ginseng cultured root using a bioreactor,

6) Mass production step using bioreactor to increase the content of ginsenoside, an effective bioactive substance.

Hereinafter, the present invention will be described in detail.

In order to obtain wild ginseng with a high content of effective bioactive substances, the selection process of individuals holding a gene unique to wild ginseng is required. The most effective individual selection method is a DNA fingerprint analysis method, which is described in detail in Korean Patent Application No. 2002-36091 (wild ginseng identification method using DNA fingerprint analysis method) filed by the present applicant.

Selected individuals require a sterilization step through surface disinfection. Culture of plant tissues is slower than that of microorganisms or fungi, so sterile conditions should be premised upon induction of tissue samples. Induction of wild ginseng culture roots also requires disinfection of wild ginseng plants. At the time of strong disinfection to reduce the contamination rate, the tissues of the dentate bodies are destroyed, and the roots of the culture roots are not induced. do.

Therefore, it is desirable to disinfect a weak disinfection with different disinfectants. As a specific example, the first disinfection is first washed with distilled water to remove the soil around the roots, and then immersed in 70% ethanol for 30-120 seconds, washed with sterile water, and the second disinfection is a 2% hypochlorous acid solution. Immerse in 1-10 minutes.

More preferably, after the second disinfection, the disinfectant on the surface is absorbed into the absorbent. This sterilization method results in more than two times faster growth in solid culture.

The biggest feature of the present invention is to directly induce wild ginseng cultured root from sterile material, and the medium for direct induction of wild ginseng cultured root from sterile material uses WPM (Lloyd and McCown) or MS (Murashige and Skoog) medium. At this time, the plant growth regulator is treated with 0.1-10 ppm, preferably 2-5 ppm of IAA (Indole 3-acetic acid) in oxins, and the induction effect of IAA is more effective than other growth regulators. Most stable in terms of differentiation rate.

The growth of wild ginseng root culture in suspension culture was performed using MS, WPM (Lloyd and McCown), and SH (Schenk and Hildebrandt) medium as liquid suspension culture medium. It is effective, and four weeks of culture in a subculture that exchanges the medium at intervals of two weeks is more than three times the increase in bio weight.

At the flask level, the induction of culture roots can be derived from liquid suspension culture tissues. The secondary induction of wild ginseng culture roots is performed by cutting only newly generated culture roots from sterile materials from which the ginseng culture roots have been grown in the tissues, and injecting them into the flask, Inoculation of the whole tissue into the flask, WPM (Lloyd and McCown), mB5 (modified Gamberg), MS (Murashige and Skoog), 1/2 MS, B5 (Gamberg) with 0.1 to 10 ppm of IAA, a growth regulator ), SH (Schenk and Hildebrandt), LP (Quoirin and Lepoivre), Whit, GD (Gresshoff and Doy), DKW (Driver and Kuniyuki) or DCR (Gupfa and Durzan) medium. In this case, it is most effective to add 2 ppm to 5 ppm of IAA or IBA or 2 ppm to 4 ppm of NAA in the culture medium of WPM.

When cultured in a general air cultured bioreactor, the culture medium is the most effective WPM medium in the medium, it is necessary to add about 0.5 ppm to 5 ppm of IAA or IBA to induce wild ginseng root culture from the tissue. Cell inoculation at the time of inoculation is adjusted to 0.1% to 0.5% of the total amount of the medium, and then maintained in the medium of the same composition for 4 to 6 weeks of culture in order to facilitate the differentiation of wild ginseng cultured roots.

The culture of wild ginseng roots using bioreactors is scaled up in stages. When the wild ginseng roots are incubated in a 5 L air-cultured bioreactor, the initial inoculation is about 0.5%.

This ratio applies equally to the 10L and 20L bioreactors.

The medium can be supplied 7-10 days before and when the culture roots are not agglomerated in the medium solution, and the method of completely replacing the medium in the bioreactor during the initial cultivation is more effective in increasing yield and quality than the method of continuously adding the medium. Wild ginseng cultured seeds were secured. Inoculation of wild ginseng cultured roots within 1-3cm length during inoculation showed the largest increase in total fresh weight at the final harvest.

When culturing on board, the incubation period is about 45 days, so a method of effectively adsorbing cell secretions in the culture solution is necessary, especially a mechanism for adsorbing growth regulators. Activated charcoal (activated carbon) It is most effective.

The cultivation temperature can achieve rapid growth at a temperature of 25 ° C or below, which is a general plant tissue or cell culture temperature, especially at 20-25 ° C.

When cultivating wild ginseng roots in a large-scale bioreactor of 5 tons or more, a major factor in large-scale cultivation is to inject a large amount of air into the entire reactor so that the end of the culture root can be continuously stimulated. In addition, the content of ginsenoside, an effective physiologically active substance, increases or decreases depending on the effects of air pressure and water pressure.

In order to further increase the content of ginsenoid which is an effective bioactive substance, the irradiated ginsenoside or ginsenoside component may be extracted from ginseng, and the extract may be increased in the culture solution in a mass culture step. More preferably, the ground portion of the ginseng is collected to extract the active ingredient. The ground portion of ginseng is not currently used for food, but only for livestock feed.

An embodiment of the present invention is as follows.

Example 1

* Selection of material

Amplified Fragment Length Polymorphism (AFLP)-Silver Staining (Silver Coloring Reaction), a DNA fingerprinting method for the selection of materials, was carried out by the wild ginseng identification method using the DNA fingerprinting method filed by the applicant [(02-36091). Ho) -Patent Application] is used.

In the above method, wild ginseng, which is 80-year-old from Gangwon-do, which maintains the intricate genetic structure of wild ginseng, was selected and used as a source material for mass production of wild ginseng culture root by inducing the individual into tissue.

Example 2

Surface sterilization of samples and direct induction of wild ginseng cultured roots

The selected wild ginseng was separated from the roots for cultivation, and then washed several times with tap water containing several drops of a washing agent (Tween-20) to remove soil and contaminants attached to the roots first.

After washing, it was immersed in 70% ethanol for 1 minute and washed with sterile water, and then immersed in 2% hypochlorous acid solution for 5 minutes to perform secondary sterilization and absorbed the disinfectant on the surface as an absorbent.

The sterilized sample was divided into 4 portions, and then inoculated into a medium containing plant growth material to observe the differentiation state. At this time, the culture medium used was MS medium, and plant growth regulators were treated with 0.5 ppm gradient from 0.5 ppm (mg / L) to 10 ppm of IBA in oxins.

When the subculture was cultured three times at two week intervals, it was observed that growth differentiation progressed rapidly.

Example 3

* Suspension cultured mountain ginseng root growth

When the ginseng cultured muscle was induced from the tissue, the cultured muscle-induced tissue was suspended in the liquid culture medium like the culture root and subcultured at about 1 month intervals. At this time, the passages were stimulated at the end of tissue growth using scalpels and tweezers for continuous passage, and cutting was minimized by releasing the tangled tissue using tweezers.

It was effective to adjust the initial tissue inoculation amount to 10% of the total amount of the medium in liquid suspension culture, and it was observed that the biomass weight increased more than three times after 4 weeks of culture. In liquid suspension culture, MS, WPM (Lloyd and McCown), and SH (Schenk and Hildebrandt) medium showed little difference between the medium in the growth of wild ginseng root, but WPM was the best.

In order to adapt the liquid culture of wild ginseng roots, the culture roots were grown in 5L, 10L, and 20L air-cultured bioreactors in order, and then the roots of the wild ginseng cultured in the same bioreactor were used for comparative experiments. .

Example 4

Derivation of secondary aides from suspension culture culture roots

Suspension-cultivated wild ginseng roots were not found to increase numerically when cultured at the flask level or in an airborne bioreactor, but the second growth of cultured roots was clearly observed. The culture medium was the most effective WPM medium.

In order to induce the secondary culture root from the culture root, 3ppm of IAA was added, and the concentration of wild ginseng cultured muscle was adjusted to about 7% of the total media volume at the time of inoculation, and then maintained in the same medium for 5 weeks to induce secondary differentiation. .

Example 5

* Ginseng culture root culture using small scale bioreactor

The ginseng cultured roots derived directly from the culture tissue were generally 1 cm to 10 cm in length, and the number was about 20 to 40 from the tissue. This sample was used to examine the effect of culture root size on the growth in order to find the optimal conditions for continuous production in small scale bioreactors. When harvesting wild ginseng roots from 0.5 cm to 2.0 cm in length, A significant increase in weight could be observed. When incubating in a 5L bioreactor, the initial inoculum was about 5%, and the medium supply was replaced by 7-10 days, which is the time when the culture roots began to entangle. It was found to be more effective than the one batch culture method.

The wild ginseng roots grown in a 5 L bioreactor were sub-cultured again in the 20 L bioreactor 4 weeks after the culture, and the harvested roots were 0.5 cm to 2.0 cm using tweezers and blades. Inoculations were divided by size.

Example 6

* Mass culture of large ginseng cultured roots (more than 5 tons)

After transferring the culture roots to the large-scale bioreactor using a sterile transfer device, the aseptically transferred wild ginseng cultured roots were propagated after about 45 days of culture, and new ginseng cultured root tissues did not develop. , As a result of the growth of the after-life, such as will increase the weight. The most important in mass culture of wild ginseng roots is to ensure that the ginseng cultured tissues receive a minimal amount of water pressure from large liquid cultures. It is important to directly monitor the growth end of the ginseng cultured roots by temperature and water pressure by monitoring and installing a window in the bioreactor. Temporary growth disorder due to water temperature or water pressure is the most important factor in mass cultivation because it leads to growth stop if left for a long time.

Example 7

* Mass culture of wild ginseng cultured roots (more than 5 tons) for increasing active ingredient content I

Cultured wild ginseng produced according to the general wild ginseng culture method as in Example 6 showed relatively low results in ginsenoside content and flavor compared to raw ginseng, and in order to improve this, the ground part of ginseng (including camphor ginseng) (leaf, Stem, root, and the like), and extract and concentrate the ginsenoside component 70% ethanol from it, and then adjust the leaf component extract ginsenoside component solution to be 99% water content. The culture was carried out by adjusting the dilution level to 1-5ppm level from the time of.

The resulting ginseng cultured roots were found to significantly increase the content of ginsenoside, an active bioactive component, and are summarized in Table 1 below.

Leaf extract ingredient-free treatment (general culture) 1 ppm of leaf extract component 2 ppm of leaf extract components 5ppm leaf extract component Ginsenosides content (as irradiated ponin) (in dry weight) 3.6 (g / 100g) 4.0 (g / 100g) 4.5 (g / 100g) 5.5 (g / 100g)

Example 8

* Mass culture of wild ginseng cultured roots (more than 5 tons) to increase the active ingredient content II

In order to increase the content of the active bioactive components of cultured wild ginseng, total ginseng extract of ginseng was extracted and concentrated, and then the extracted ginsenoside component solution was adjusted so that the moisture content was 99%. The culture was carried out by adjusting the dilution level to 0.1-0.5ppm level from the time of.

In the above experimental results, the treated ginseng cultured root was confirmed to increase the content of ginsenoside, which is an active bioactive component, to a certain level, and the results are summarized in Table 2 below.

No treatment (general culture) Irradiated Ponine 0.1ppm Treatment Irradiated Ponine 0.25 ppm Treatment Irradiated Ponine 0.5ppm Treatment Ginsenoside content (as total amount) (in dry weight) 3.6 (g / 100g) 4.1 (g / 100g) 5.3 (g / 100g) 6.5 (g / 100g)

(Experimental treatment of irradiated with 0.5ppm or more of irradiated phononin reached an effective level, but meaningless due to the increase in treatment cost)

As can be seen through the above embodiment, the step of inducing callus by the present invention can be omitted, so that the use of growth regulators, such as 2,4-D, which is not secured, may be excluded. It is possible to increase the content of the active ingredient, which is a common problem with it.

Claims (6)

In the method of mass growth of wild ginseng root muscle by tissue culture consisting of material selection-sterilization-callus induction-culture-mass growth, the material is selected by DNA fingerprinting method, and the step of inducing callus is omitted Mass propagation method of wild ginseng cultured root, characterized in that inducing ginseng cultured root directly from the material. The method of claim 1, wherein the medium for inducing ginseng cultured muscle directly from the material is WPM or MS medium. The method of claim 1, wherein the medium for inducing ginseng cultured muscle directly from the material contains 0.1-10 ppm of IAA or IBA as a growth regulator. The method of claim 1, wherein the sterilization method is a method of mass propagation of wild ginseng cultured roots, characterized in that the disinfection in a double with different disinfectants. The method for mass propagation of wild ginseng cultured root according to claim 4, characterized in that after disinfection, an antiseptic solution present on the surface of the material is removed with an absorbent.  The method of mass growth of wild ginseng cultured roots according to claim 1, wherein the solution containing ginseng's irradiated saponin or ginsenoside extract is increased in the culture medium.