KR100842420B1 - Method of bioreactor culture of echinacea purpurea adventitious roots - Google Patents

Abstract

A method for culturing Echinacea purpurea adventitious roots is provided to mass-produce the adventitious roots from induced and proliferated adventitious roots by in vitro culturing them under proper condition using a bio-reactor and produce useful ingredients therefrom. A method for mass-producing Echinacea sp. adventitious roots using a bioreactor comprises the steps of: (a) sterilizing and inoculating an Echinacea sp. plant such as Echinacea purpurea to induce and proliferate callus; (b) induce and proliferate the adventitious roots from the callus; and (c) culturing the adventitious roots in the bioreactor to harvest the adventitious roots, wherein the culturing of the step(c) is done at a temperature of 20 deg.C by using a 1/2mMS culture medium[modified MS medium: 1/2 MS(NH^4+:NO^3-=5:25)+sucrose 50g/L+IBA 2.0mg/L] with an adventitious roots inoculation amount of 7.0g/L and an air supply amount of 0.1 vvm for 5 weeks and the 1/2mMS culture medium is further added after 2 weeks of the culture start to increase the growth amount of the adventitious roots.

Description

Method of bioreactor culture of Echinacea purpurea adventitious roots}

1 is a photograph showing the culture of Echinacea purpurea and roots of Echinacea purpurea and roots of Echinacea purpurea in 5L, 20L, 500L, and 1000L bioreactor and harvested roots.

2 is a graph showing the activity of antioxidant enzymes according to temperature in culture of adventitious muscle,

Figure 3 is a graph showing the effect of nitric oxide (NO) on SOD activity in the root muscle culture, b) is a graph showing the effect of nitrogen monoxide (NO) on G-POD activity, c) is APX Is a graph showing the effect of nitrogen monoxide (NO) on the activity,

4 is a graph comparing chromosomal drainage of Echinacea abscess and leaf,

5 is a photograph analyzing the number of chromosomes of Echinacea abscess;

6 is a graph of HPLC analysis of caffeic acid of Echinacea species,

(a) E. palida Cultivation b) E. angustiforlia roots c) E. purpurea roots d) E. purpurea Negative root)

FIG. 7 is a chromatogram comparing and accurately analyzing caffeic acid derivatives, a standard material, for HPLC analysis of Echinacea cultured intramuscular muscles.

The present invention relates to a method for culturing Echinacea purpurea arrhythmia, inducing and propagating the adventitious root, identifying appropriate incubation conditions therefrom, establishing a mass production system using a bioreactor, and culturing the root muscle producing useful components therefrom. It is about.

Echinacea is a medicinal plant native to North America that has been used for centuries by natives to treat wounds, burns, snakes, insects, or colds, infections, and inflammations. . Representative species include E. purpurea , E. angustifolia , and E. pallida , each with different chemical constituents. Echinacea is used in the form of extracts, teas, tonics, tablets and ointments, and is currently best known as an immune booster and is used as a nonspecific immunostimulator for respiratory infections, urinary tract infections, cancer, skin diseases and many other inflammatory processes. It has the effect of increasing cells and is known to kill cancer cells.

Echinacea's immune-promoting function has been reported to be due to the lipophilic and polar fractions of extracts containing alcamides, cichoric acid derivatives, glycoproteins, and polysaccharides. It mainly contributes to the immunostimulatory action of the extract, and it has been reported that the alkamide fraction promotes phagocytosis in vivo and in vitro. Alkamides of echinacea are mainly isobutylamides of highly unsaturated carboxylic acids with olefin bonds or acetylene bonds and have been reported to inhibit cyclooxygenase and 5-lipoxygenase as anti-inflammatory agents. In many studies, echinacoside and the polysaccharide echinacin have antimicrobial and antifungal activity, and many studies have reported that the immune system is regulated. For example, Korean Patent Registration No. 10-0497152 discloses an ethyl acetate fraction of methanol extract from the root of Echinacea angustifolia, which has anticancer activity against blood cancer cells.

Echinacea, however, is difficult to meet demand by harvesting wild species, and more efficient production methods are required. Mass production by tissue culture is currently being applied to many plants because they select genetically superior individuals and can grow in a short period of time. Therefore, in the present invention, Echinacea purpurea is used as a material to induce and propagate the roots of the roots, to determine the appropriate incubation conditions from this, to establish a mass production system using a bioreactor and to produce useful components therefrom.

Therefore, in the present invention, Echinacea purpurea is used as a material to induce and propagate the roots of the roots, to investigate the appropriate incubation conditions, and to establish a mass production system using a bioreactor and to produce useful components therefrom. To this end, the constituents in the medium affecting the growth of the root muscle were investigated and the optimum growth conditions were identified in the bioreactor. In addition, to increase the content of polyphenolic compounds (secondary metabolites) produced by the adventitious roots, a method for increasing productivity by using physical and chemical elicitors was identified.

An object of the present invention is to sterilize and inoculate the seedlings to induce and proliferate callus;

Inducing and propagating adventitious roots from callus; And

In the method of cultivating the root of the root muscle to harvest the root of the root muscle by culturing in the bioreactor,

In the root muscle cultivation method,

(1) The medium was prepared using 1 / 2mMS medium [modified MS medium: 1/2 MS (NH ₄ ⁺ : NO ₃ ⁻ = 5: 25) + sucrose 50 g · L ⁻¹ + IBA 2.0 mg · L ⁻¹ ] ,

(2) The amount of inoculation of the anus muscles assumes 7.0 g / L,

(3) Air supply amount is 0.1vvm,

(4) The culture period is 5 weeks,

(5) The culture temperature is to provide a method for culturing the root muscle, characterized in that the culture at 20 ℃.

Still another object of the present invention is to provide a method for culturing the root muscles, which further comprises adding 1 / 2mMS medium after 2 weeks of the start of the cultivation method to increase the growth of the root muscles.

Another object of the present invention is a method for increasing the content of polyphenol compound, a secondary metabolite, to a photoperiod of 3h / 21h (light / dark) on growth controlled at 30 μmol m ^-2 s ^{- 1} brightness from the beginning of growth. It is to provide a method for culturing the root muscle, characterized in that it comprises culturing after treatment.

Another object of the present invention is to increase the content of polyphenolic compound, which is the secondary metabolite of the root of the root, bioreactor for raising the UV-A fluorescent lamp (20W, Sankyo Denki, Japan) as a physical attractant from the fourth week of culture. It provides a method of culturing the root muscles, characterized in that it comprises a 1 cm treatment for 1 week each day as 50 cm.

Another object of the present invention is a method for increasing the content of polyphenolic compound, which is a secondary metabolite of arrhythmia, which comprises culturing after treatment with nitrogen monoxide with 100 μmol as a chemical attractant in the fourth week of culture. It is to provide a culture method.

Still another object of the present invention is to provide a method for culturing abscess muscles, wherein salicylic acid is treated with 200 μmol in the fourth week of cultivation as a method for increasing the content of polyphenolic compound, which is a secondary metabolite of the root muscle.

Still another object of the present invention is to provide a method for culturing the root of root, characterized in that the parent material is selected from ginseng, wild ginseng and camphor ginseng.

Still another object of the present invention is to provide a method for culturing the root muscle, characterized in that the mother plant is Echinacea purpurea.

It is still another object of the present invention to provide a method for culturing neglected roots, which is genetically stable due to the same mass as the parental strain in chromosomal ploidy and chromosome number.

The present invention relates to an in vitro culture method of Echinacea purpurea malaria, to determine the appropriate incubation conditions from the induction and proliferation of malaria root, to establish a mass production system using a bioreactor and to produce useful components therefrom. For this purpose, the constituents in the medium affecting the growth of adventitious roots were investigated, and the optimal conditions of the bioreactor for the growth and useful components of the adventitious roots were investigated. In addition, to increase the productivity of polyphenolic compounds and caffeic acid, the secondary metabolite of gluteus larvae, physical and chemical elicitors were investigated.

The present invention sterilizes and inoculates the parent plants to induce and proliferate callus;

Inducing and propagating adventitious roots from callus; And

In the method of cultivating the root of the root muscle to harvest the root of the root muscle by culturing in the bioreactor,

In the root muscle cultivation method,

(1) The medium was prepared using 1 / 2mMS medium [modified MS medium: 1/2 MS (NH ₄ ⁺ : NO ₃ ⁻ = 5: 25) + sucrose 50 g · L ⁻¹ + IBA 2.0 mg · L ⁻¹ ]

(2) The amount of inoculation of the anus muscles assumes 7.0 g / L,

(3) Air supply amount is 0.1vvm,

(4) The culture period is 5 weeks,

(5) The culture temperature includes a method for culturing the root muscle, characterized in that the culture at 20 ℃.

The present invention includes a method for culturing the root muscle, characterized in that it further comprises adding 1 / 2mMS medium after 2 weeks from the start of cultivation as a method for increasing the growth of the root muscle.

The present invention is a method for increasing the content of polyphenolic compound, the secondary metabolite of inferior root muscle, and treated with photoperiod at 3 h / ²¹ h (light / dark) on growth controlled at 30 μmol m ^-2 s ^{- 1} brightness from the beginning of daily growth. It includes a method of culturing the root muscle, characterized in that it comprises culturing after.

The present invention is a method for increasing the content of polyphenolic compound, which is a secondary metabolite of inferior root muscle, is a UV-A fluorescent lamp (20W, Sankyo Denki, Japan) as a physical attractant from the fourth week of culture. It includes a method of culturing the root muscles, characterized in that it comprises culturing by 1h for one week.

The present invention includes a method for increasing the content of the polyphenolic compound, which is a secondary metabolite of arrhythmia, comprising a method of culturing the root of negative root, characterized in that it comprises culturing after treating with nitrogen monoxide with 100 μmol as a chemical attractant in the fourth week of culture. do.

The present invention includes a method for culturing the root muscles, characterized in that the treatment of salicylic acid with 200 μmol in the fourth week of the culture as a method for increasing the content of the polyphenolic compound, which is a secondary metabolite of the root muscle.

The present invention includes a method of culturing the root of negative root, characterized in that the parent material is selected from ginseng, wild ginseng and camphor ginseng.

The present invention includes a method of culturing the root of roots, characterized in that the phenotype is Echinacea purpurea.

The present invention includes a method for culturing the root muscles, characterized in that the mass rooted inferior root muscles are genetically stable in terms of chromosomal ploidy and chromosome number.

Hereinafter, specific features of the present invention will be described in detail with reference to the following examples. However, these Examples are only examples of the present invention, and the scope of the present invention is not limited thereto.

Example 1 Induction of Callus and Abdominal Muscle

Echinacea purpurea used in the present invention used seedlings germinating seeds purchased from native Canada. For sterilization of the surface before inoculation, cut the stem and leaf parts of the plant to the appropriate size, soak for 30 seconds in 70% ethanol, shake for 5 minutes in 4% sodium hypochlorite, and then wash with sterilized water. This was made into small pieces (1 × 0.5 × 0.3cm) and used as a culture material.

In order to induce the callus samples from the stems and leaves of a plant seed germination naphthalene acetic acid (NAA) 2㎎ ^-1 · L, benzyl adenine (BA) 0.1㎎ · L ^{- 1} Growth Regulators and 30g · L ^-1 of sucrose (sucrose) and a light gel (gelrite) 2.0g · L ^{- 1} a MS (Murashinge and Skoog, 1962) containing 8 weeks in a growth chamber under dark condition of 22 ± 1 ℃ were inoculated in basal medium and cultured. 30 g · L ⁻¹ of sucrose and 2.0 g · L ⁻¹ were added to MS basal medium to induce the root muscle from the obtained callus, and the growth regulator was NA ± 0.5 mg · L ⁻¹ and 22 ± 1 ° C. The culture was carried out for 8 weeks in the dark condition of the culture room. Each medium was adjusted to pH 5.8 using 1N sodium hydroxide (NaOH), sterilized using autoclave at 121 ° C. for 20 minutes, and then dispensed in 30 ml portions in plastic cell culture dishes.

IBA 1㎎ · L a jeongdanbu of inducing adventitious root to about 1㎝ degree cut in 1 / 2MS ^medium-1 with the addition of sucrose, 50g · L ^-1 were inoculated into the liquid medium. The medium was dispensed in 14L into a balloon-type bubble bioreactor (BTBB) with a total volume of 20L and cultured for 4 weeks in a culture room with a dark condition of 22 ± 1 ℃.

Example 2 Investigation of Conditions on Growth of Abdominal Muscle in Bioreactor Culture

In all experiments, 4L of medium was added to the air-floating CTBB having a total volume of 5L, and the culture was carried out under 22 ± 1 ° C. and dark conditions for 5 weeks, and each treatment was repeated twice. The medium used was a medium consisting of mMS medium [modified MS medium: 1/2 MS (NH ₄ ⁺ : NO ₃ ⁻ = 5: 25) + sucrose 50 g · L ⁻¹ + IBA 2.0 mg · L ⁻¹ ] The effects of medium concentration, involuntary root inoculum, air supply and incubation period on the growth of larvae, phenol, flavonoids and caffeic acid derivatives were investigated.

In each step, the method for measuring the root growth, phenol, flavonoid and caffeic acid content is as follows.

The fresh weight of the root muscle was collected by filtering the harvested root roots with stainless steel, and then the weight was measured after removing enough moisture with a clean tissue, and the weight was measured after drying for 2 days at 60 ° C (growth ratio = [(final growth (In the building of the intestinal root inoculated)] (in the building of the involuntary root inoculated)] / (in the building of the involuntary root inoculated early)). The content of phenol flavonoid caffeic acid derivatives and the like was analyzed with the extract extracted from the dried plant in 80% methanol. The phenol content was measured by absorbing at 760 nm based on gallic acid with Folin-Ciocalteu reagent and flavonoids at 510 nm based on catechin. Measured. HPLC analysis of caffeic acid derivative content was measured at 330 nm with water and acetonitrile as mobile phase in an XTerra® RP 18 column.

<2-1. Effect of Medium Concentration on Bioreactor Growth on Abdominal Muscle>

4L each of the 5L bioreactor was inoculated with medium of 1/4, 1/2, 3/4 and 1 MS concentrations and then inoculated with 7.0 g of root muscle. The air injection in the bioreactor was performed through an air compressor, and a device for condensing compressed air, a filter for removing impurities such as dust, and an air dryer were sequentially connected for a constant temperature of the supplied air. The air injection amount was adjusted to 0.1 vvm after passing through the air flow controller and the micro filter. Cultivation was carried out in two repeated treatments under 22 ± 1 ° C. and dark conditions for 5 weeks.

[Table 1] Growth and Contents of Polyphenolic Compounds of Abdominal Muscle

Medium concentration Live weight (g / L) Under building (g / L) Growth rate Total phenolic content (㎎ · g ^-1 · DW) The total flavonoid content (㎎ · g ^-1 · DW) 1 / 4MS 50.9 d ^z 7.10 c 8.2 57.28 b 35.14 b 1 / 2MS 73.6 a 10.03 a 12.4 61.14 a 38.30 a 3 / 4MS 67.2 b 9.23 b 11.0 37.78 c 22.61 c 1MS 59.6 c 6.7 d 7.7 21.66 d 11.50 d

The measured values were verified by Duncan's multiple test using SAS (SAS Institute, Cary, NC) program.

[Table 2] Caffeic acid derivative content

Medium concentration Caffeic acid derivatives derivatives (mg g- ¹ DW) Captaric acid Chlorogenic acid Chicorylic acid all 1 / 4MS 2.19 ± 0.01 ^z 5.02 ± 0.01 28.06 ± 0.10 35.27 ± 0.08 1 / 2MS 4.35 ± 0.01 4.87 ± 0.09 29.06 ± 0.08 38.28 ± 0.16 3 / 4MS 3.03 ± 0.02 4.70 ± 0.01 19.93 ± 0.01 27.66 ± 0.01 1MS 1.46 ± 0.01 2.86 ± 0.01 8.16 ± 0.02 12.48 ± 0.04

  Investigation of the concentration of minerals in the medium suitable for Echinacea purpurea irregular root growth showed that 73.6g / L of live weight, 10.03g / L of dry matter, total phenolic content 61.14mg.g DW, total flavonoid content 38.30mg / g The highest content of DW and total caffeine derivative was 38.28mg / g DW. Therefore, it could be confirmed that the mineral concentration in the medium best suited for growth of Echinacea purpureas was 1 / 2MS (Table 1, Table 2).

<2-2. Influence of Involuntary Inoculation

Culture conditions were carried out in the same manner as in the above example, and inoculated with 2.5, 5.0, 7.0, 10.0, 15.0 g / L of inferior root in a bioreactor containing 4L of medium, and then irradiated.

[Table 3] Growth and Contents of Polyphenolic Compounds of Abdominal Muscle

Inoculation amount (g / L) Live weight (g / L) Under building (g / L) Growth rate Total phenolic content (㎎ · g ^-1 · DW) The total flavonoid content (㎎ · g ^-1 · DW) 2.5 55.6 e ^z 10.58 e 15.4 34.2 e 25.8 d 5.0 59.2 d 10.17 d 10.5 55.2 b 33.8 b 7.0 67.2 c 10.80 c 10.8 58.5 a 38.6 a 10.0 74.8 b 10.17 b 8.2 45.4 c 27.6 c 15.0 79.0 a 10.48 a 5.4 40.6 d 23.4 e

The measured values were verified by Duncan's multiple test using SAS (SAS Institute, Cary, NC) program.

[Table 4] Caffeic acid derivative content

Inoculation amount (g / L) Caffeic Acid Derivatives (mgg- ¹ DW) Captaric acid Chlorogenic acid Chicorylic acid all 2.5 2.35 ± 0.02 ^Z 4.24 ± 0.27 16.56 ± 0.06 23.15 ± 0.35 5.0 2.48 ± 0.02 4.56 ± 0.02 27.27 ± 0.09 34.31 ± 0.14 7.0 4.14 ± 0.06 5.14 ± 0.08 28.15 ± 0.47 37.42 ± 0.34 10.0 2.70 ± 0.01 4.58 ± 0.04 27.25 ± 0.06 34.53 ± 0.10 15.0 2.39 ± 0.02 2.79 ± 0.08 25.21 ± 0.08 30.39 ± 0.17

  As a result of incubation with different inoculum in the 5L bioreactor, the in vivo and dry weight tended to increase as the inoculum was increased. However, the inoculation amount of more than 7.0g / L tended to decrease the content of all secondary metabolites, such as the total caffeic acid derivatives. From the above results, the optimal inoculation amount of Echinacea abscess was 7.0g / L, and the body weight was 67.2g / L, the dry weight was 10.80g / L, and the total caffeine derivative content was 37.42mg / g DW (Table 3, Table 4). ).

<2-3. Effect of Air Supply in Bioreactor>

In order to determine the effect of the amount of air supplied into the bioreactor on the growth of adventitious roots, the air injection was treated at 0.05, 0.1, 0.2, 0.3, 0.05-0.1, 0.05-0.2 and 0.05-0.3vvm. The bioreactor and the culture conditions were the same as in the above example, and the inoculation amount of the root muscle was 7.0 g · L ⁻¹ .

Table 5 Growth and Contents of Polyphenolic Compounds of Abdominal Muscle

Air supply amount (vvm) Live weight (gL- ¹ ) Under building (gL- ¹ ) Growth rate Total phenolic content (㎎ · g ^-1 · DW) The total flavonoid content (㎎ · g ^-1 · DW) 0.05 70.5 ab ^z 8.95 ab 10.6 60.9 a 37.8 a 0.1 70.1 ab 9.06 ab 10.8 60.7 a 38.8 a 0.2 63.1 de 8.20 c 9.7 61.1 a 38.3 a 0.3 61.4 e 8.10 c 9.5 58.7 b 35.6 b 0.05-0.1 66.1 CD 8.76 b 10.4 60.2 a 38.2 a 0.05-0.2 67.5 bc 8.84 b 10.5 61.1 a 38.9 a 0.05-0.3 71.9 a 9.19 a 10.9 61.1 a 38.7 a

The measured values were verified by Duncan's multiple test using SAS (SAS Institute, Cary, NC) program.

Table 6 Contents of Caffeic Acid Derivatives

Air supply amount (vvm) Caffeic acid derivatives (mg g- ¹ DW) Captaric acid Chlorogenic acid Chicorylic acid all 0.05 4.76 ± 0.07 ^z 5.67 ± 0.01 25.04 ± 0.01 35.47 ± 0.08 0.1 4.72 ± 0.15 5.99 ± 0.02 26.64 ± 0.07 37.39 ± 0.24 0.2 3.82 ± 0.01 5.59 ± 0.02 24.17 ± 0.09 33.59 ± 0.07 0.3 3.63 ± 0.04 5.29 ± 0.01 22.50 ± 0.62 31.42 ± 0.57 0.05-0.1 4.89 ± 0.06 5.87 ± 0.06 27.60 ± 0.05 38.36 ± 0.07 0.05-0.2 4.82 ± 0.13 6.02 ± 0.06 28.37 ± 0.25 39.21 ± 0.32 0.05-0.3 4.92 ± 0.07 6.01 ± 0.01 28.16 ± 0.37 39.09 ± 0.44

Investigation of the proper air supply for growth of Echinacea purpurea malaria muscle showed that the air supply of 0.1vvm or more decreased the growth of secondary muscle and secondary metabolite. As the growth of the adventitious roots progressed, the secondary metabolites increased slightly in vivo, dry weight, and dry matter, but gradually increased to 0.3vvm. Therefore, the suitable air supply for growth of Echinacea purpurea was found to be 0.1vvm, and 70.1g / L in live weight, 9.06g / L in dry matter and 37.39mg / g DW in total caffeine derivatives (Table 5, Table 6).

<2-4. Variation of Abdominal Muscle Growth by Bioreactor Culture Period

Inoculum was inoculated at 7.0 g · L ⁻¹ and air was injected at 0.1 vvm. The composition of the supplied air was the same as that of the atmosphere (N ₂ 78%, 0 ₂ 20.8%, CO ₂ 0.03%, Ne, He) and incubated for a total of 8 weeks. The content of phenolic compound and caffeic acid derivative, pH of the medium, electrical conductivity (EC), sugar, cation and anion content were examined at intervals of one week of culture. Culture conditions were carried out in the same manner as in Example 2-1.

The growth and secondary metabolite contents were examined at weekly intervals to determine the growth and phenol content of the root muscle with the bioreactor incubation period. The growth rate was gradually increased from 3 weeks to 5 weeks, and after 5 weeks, growth slowed down and growth was almost stopped after 7 weeks. Secondary metabolite content such as total phenol content, total flavonoid content, and total caffeic acid derivatives increased rapidly from 1 to 3 weeks, gradually increased from 3 to 5 weeks, and then increased slowly after 5 weeks. A trend was shown. From this, it can be seen that 5 weeks is appropriate for the culture cycle of Echinacea abscess in the 5L bioreactor. Surose content in the medium was rapidly dropped from 2 weeks after incubation to glucose and fructose, which was found to be used in plants. The cationic anion content in the medium was rapidly depleted from 2 weeks, resulting in an imbalance of nutrients in the medium.

Example 3. Effects of Different Media Concentrations, Culture Temperature, and Photoperiod on Growth of Abdominal Muscle in Bioreactor Culture

All experiments were basically treated with 4L of medium in a 5L air-lifted CTBB and cultured under 22 ± 1 ° C., dark conditions for 5 weeks, and each treatment was repeated twice. The medium used was a medium consisting of 1 / 2mMS medium [modified MS medium: 1/2 MS (NH ₄ ⁺ : NO ₃ ⁻ = 5: 25) + sucrose 50 g · L ⁻¹ + IBA 2.0 mg · L ⁻¹ ] The effects of medium addition, culture temperature, and photoperiod on the growth of adventitious root, phenol, flavonoid and caffeic acid derivatives were investigated.

<3-1. Effect of Different Media on Growth of Abdominal Muscles>

After incubation under the above culture conditions, the effects of growth on the growth of adventitious roots were examined when the cells were further cultured with concentration mediums of 1/4, 1/2, 3/4 and 1 MS at 2 and 3 weeks.

Table 7 Growth and Contents of Polyphenolic Compounds of Abdominal Muscle

Medium concentration Live weight (g) (G) in building Growth rate Total phenolic content (㎎ · g ^-1 · DW) The total flavonoid content (㎎ · g ^-1 · DW) Control 65.3 f ^y 9.80 g 11.7 60.29 a 36.96 a after 2 weeks 1 / 4MS 78.8 c 12.65 c 15.4 59.22 a 34.38 b 1 / 2MS 83.1 a 14.76 a 18.2 59.21 a 34.19 b 3 / 4MS 82.4 a 14.78 a 18.2 56.77 b 31.94 CD 1MS 83.6 a 14.94 a 18.4 54.78 b 30.56 e 3 weeks later 1 / 4MS 63.8 g 10.62 f 12.8 60.43 a 34.10 b 1 / 2MS 70.0 d 11.97 e 14.5 60.49 a 35.06 b 3 / 4MS 67.8 e 12.32 d 15.0 59.26 a 32.28 c 1MS 80.5 b 13.71 b 16.8 56.50 b 31.10 de

The measured values were verified by Duncan's multiple test using SAS (SAS Institute, Cary, NC) program.

Table 8 Contents of Caffeic Acid Derivatives

Medium concentration Caffeic Acid Derivatives (mg / g DW) Captaric acid Chlorogenic acid Chicorylic acid all Control 4.67 ± 0.08 ^z 5.73 ± 0.11 25.68 ± 0.06 36.07 ± 0.13 2 weeks later 1 / 4MS 5.88 ± 0.01 4.60 ± 0.23 27.24 ± 0.04 37.72 ± 0.18 1 / 2MS 5.76 ± 0.01 4.26 ± 0.06 26.12 ± 0.05 36.15 ± 0.02 3 / 4MS 5.59 ± 0.08 4.03 ± 0.05 25.99 ± 0.06 35.61 ± 0.07 1MS 4.87 ± 0.08 3.83 ± 0.01 24.30 ± 0.11 33.04 ± 0.18 3 weeks later 1 / 4MS 6.98 ± 0.02 4.93 ± 0.01 26.11 ± 0.02 38.07 ± 0.02 1 / 2MS 6.14 ± 0.08 5.10 ± 0.10 25.18 ± 0.01 36.52 ± 0.18 3 / 4MS 5.43 ± 0.01 4.73 ± 0.26 24.78 ± 0.08 34.94 ± 0.10 1MS 5.16 ± 0.02 4.61 ± 0.04 19.08 ± 0.47 28.84 ± 0.45

In order to increase the growth and the content of secondary metabolites of the roots of the roots, medium of different concentrations were added at the second and third weeks of culture. In the medium and dry weight, all medium added treatments were higher than the control, and the content of secondary metabolites tended to decrease compared to the control. At 2 weeks, the media addition treatment showed better growth than the 3 week media addition treatment. The treatment group with both good fresh weight, dry weight, and total secondary metabolites content was added to 2 weeks 1 / 2MS medium, and the weight could be increased to 83.1g / L (1.27 times) and dry weight to 14.76g / L (1.5 times). The total phenol content was 59.21 mg / g DW, the total flavonoid 34.19 mg / g DW, and the total caffeic acid derivative content were 36.15 mg / g DW (Table 7, Table 8).

<3-2. Effect of Culture Temperature on Growth of Abdominal Muscle

2 L of 1/2 mM MS medium [modified MS medium: 1/2 MS (NH ₄ ⁺ : NO ₃ ⁻ = 5: 25) + sucrose 50 g · L ⁻¹ + IBA 2.0 mg · L ⁻¹ ] was added to the ³ L bioreactor. The incubation temperature of the varicose root was incubated at 10, 15, 20, 25 and 30 ° C.

Table 9 Growth and Contents of Polyphenolic Compounds of Abdominal Muscle

Temperature (℃) Live weight (g / L) Under building (g / L) Growth rate Total Phenolic Content (mgg- ¹ ) Total Flavonoid Content (mgg- ¹ ) 10 19.6 e ^y 2.75 e 2.6 31.5 e 23.9 a 15 39.3 d 6.50 d 7.4 45.9 d 27.7 b 20 65.5 a 10.37 a 12.5 61.5 a 38.0 a 25 60.3 b 9.52 b 11.4 58.6 b 28.9 b 30 56.1 c 8.32 c 9.8 50.6 c 29.5 b

The measured values were verified by Duncan's multiple test using SAS (SAS Institute, Cary, NC) program.

Table 10 Contents of Caffeic Acid Derivatives

Temperature (℃) Caffeic Acid Derivatives (mgg- ¹ DW) Captaric acid Chlorogenic acid Chicorylic acid all 10 1.90 ± 0.01 4.59 ± 0.01 13.42 ± 0.02 19.91 ± 0.03 15 4.38 ± 0.03 4.37 ± 0.06 22.01 ± 0.57 30.76 ± 0.48 20 4.70 ± 0.13 5.16 ± 0.05 28.42 ± 0.14 38.28 ± 0.04 25 4.04 ± 0.01 5.01 ± 0.01 25.16 ± 0.09 34.22 ± 0.07 30 4.14 ± 0.01 4.46 ± 0.04 25.26 ± 0.19 33.86 ± 0.21

The growth of Echinacea abscess was temperature-sensitive, which was measured at 20 ° C in vivo (65.5 g / L), dry matter (10.37 g / L), total phenol content (61.5 mg / g DW), and total flavonoid content (38.0). mg / L), the total content of caffeic acid derivatives (38.28 mg / g DW) was the highest, and the lowest growth was at 10 ° C. In addition, the activity of SOD, APX, CAT, G-POD and DPPH was investigated after incubation for 5 weeks at 10, 25, 20, 25 and 30 ℃ to determine the activity of antioxidant enzymes according to temperature. The activity of sulfated enzyme SOD did not change significantly with temperature, and APX activity decreased with increasing temperature. G-POD activity was low at 20 ℃ and 25 ℃, and CAT activity was the highest at 10 ℃. High. The activity of radical scavenging ability DPPH was highest at the optimum growth temperature (Table 9, Table 10, Figure 2).

SOD, APX, CAT, and G-POD coenzyme extracts were pulverized with 1.0 g of inferior rhizome cultured for 5 weeks using liquid nitrogen with phosphate buffer, followed by centrifugation. The activity of SOD was measured by comparing the absorbance with the control at 560 nm after irradiation of the coenzyme solution and the reaction solution in a transparent test tube with a fluorescent lamp. G-POD activity is H ₂ O ₂ The oxidation of guaiacol in the presence was measured as a decrease in absorbance at 470 nm. Activity of CAT (catalase) was measured at 240nm to put a certain amount of the enzyme extract in the reaction containing the H ₂ O ₂ solution in a concentration reduction of H ₂ O _2. Activity of APX (Ascorbate peroxidase) in H ₂ O ₂ The reaction was initiated by addition and the oxidation of ascorbic acid was measured by a decrease in absorbance at 290 nm. Radical scavenging ability was measured using the DPPH method. DPPH was added to the dried methanol extract of dry root, and left for 10 minutes, and then the absorbance was measured at 520 nm.

<3-3. Influence of photoperiod

2 L 1 / 2mMS medium [modified MS medium: 1/2 MS (NH ₄ ⁺ : NO ₃ ⁻ = 5: 25) + sucrose 50 g · L ⁻¹ + IBA 2.0 mg · L ⁻¹ ] was added to a ³ L bioreactor and 20 ℃ culture temperature and 30μmol m ^-2 s ^- and incubated after treatment with the growth on the control light intensity of ¹ Gwangju group 0h / 24h (light / dark), 3h / 21h, 6h / 18h and 12h / 12h.

[Table 11] Growth and Content of Polyphenolic Compounds of Abdominal Muscle

Light / dark Live weight (g / L) Under building (g / L) Growth rate Total phenolic content (㎎ · g ^-1 · DW) The total flavonoid content (㎎ · g ^-1 · DW) 0h / 24h 67.0 a ^y 9.82 a 10.6 53.00 b 28.81 b 3h / 21h 72.6 a 9.78 a 8.8 56.64 a 36.41 a 6h / 18h 68.5 a 9.65 a 9.6 56.37 a 36.30 a 12h / 12h 51.6 b 6.60 b 6.5 45.18 c 31.04 b

The measured values were verified by Duncan's multiple test using SAS (SAS Institute, Cary, NC) program.

Table 12 Contents of Caffeic Acid Derivatives

Light / dark Caffeic acid derivatives (mg g- ¹ DW) Captaric acid Chlorogenic acid Chicorylic acid all 0h / 24h 4.45 ± 0.34 ^z 5.02 ± 0.60 27.85 ± 0.73 38.53 ± 1.18 3h / 21h 6.49 ± 0.26 5.16 ± 0.02 34.18 ± 0.68 47.27 ± 0.94 6h / 18h 6.28 ± 0.32 4.48 ± 0.16 33.12 ± 0.52 45.23 ± 1.06 12h / 12h 3.09 ± 0.04 3.18 ± 0.18 29.91 ± 0.13 36.97 ± 0.28

After 5 weeks of incubation, the 3h / 21h and 6h / 18h treatments had higher fresh weight and dry weight than the control 0h / 24h, but there was no significant difference. The total phenol content was 56.64 mg / g DW (1.07 fold), the total flavonoid 36.41 mg / g DW (1.26 fold) and the total caffeic acid derivative content 47.27 mg / g DW (1.23 fold). Long time photoperiod treatment 12h / 12h tended to reduce both growth and secondary metabolite content compared to the control (Table 11, Table 12).

Example 4 Growth of Nerve Root by Contents of Elicitors and Investigation of Contents of Phenolic Compounds and Caffeic Acid Derivatives

Two-stage cultures were performed in the bioreactor to maximize the growth of the abscess and the accumulation of secondary metabolites. That is, after incubation for 4 weeks in the bioreactor to achieve a constant growth from the fifth week treated with a different treatment of the attractant was incubated for one more week.

<4-1. Effect of UV-A as a Physical Attractant>

The adventitious roots of Echinacea a 3L bioreactor 2L 1 / 2mMS [modified MS medium : 1/2 MS (NH 4 +: NO 3 - = 5: 25) + sucrose ^{50g · L -1 + IBA 2.0㎎ ·} L -1 ] The medium was added and cultured for 5 weeks at 20 ° C. under dark conditions. In the fourth week of culture, UV-A fluorescent lamp (20W, Sankyo Denki, Japan) was used to irradiate with 0h, 0.5h, 1h, 4h daily, and the height of the lamp was 50 cm in the bioreactor and repeated twice.

Table 13 Growth and Contents of Polyphenolic Compounds of Abdominal Muscle

UV-A Live weight (g / L) Under building (g / L) Growth rate Total Phenolic Content (mgg- ¹ ) Total Flavonoid Content (mgg- ¹ ) 0h 63.2 b ^y 9.48 c 11.3 51.54 b 32.15 b 0.5h 67.1 a 9.86 bc 11.8 61.36 a 37.35 a 1h 67.7 a 10.59 a 12.8 61.72 a 36.52 a 4h 67.0 a 10.17 b 12.2 60.72 a 35.73 a 8h 62.5 b 9.53 c 11.4 60.97 a 32.85 b

The measured values were verified by Duncan's multiple test using SAS (SAS Institute, Cary, NC) program.

Table 14 Contents of Caffeic Acid Derivatives

UV-A Caffeic Acid Derivatives (mgg- ¹ DW) Captaric acid Chlorogenic acid Chicorylic acid all 0h 1.82 ± 0.18 ^z 4.00 ± 0.03 28.41 ± 1.50 34.23 ± 1.65 0.5h 2.24 ± 0.26 4.83 ± 0.30 34.61 ± 0.62 41.68 ± 0.51 1h 2.48 ± 0.07 4.15 ± 0.19 35.74 ± 1.36 42.38 ± 1.48 4h 2.95 ± 0.29 3.66 ± 0.19 35.26 ± 1.47 41.87 ± 2.20 8h 2.61 ± 0.13 3.92 ± 0.14 35.08 ± 0.01 41.62 ± 0.28

The fresh weight, dry weight, total phenol content, total flavonoid content and total caffeic acid derivative content of UV-A treatment were increased compared to the control. Among them, the 1h treatment group was the best, with a live weight of 67.7g / L, dry matter of 10.59g / L, total phenol content of 61.72mg / g DW (1.20 times), total flavonoid content of 36.52mg / g DW (1.14 times), Total caffeic acid derivative content is 42.38 mg / g DW (1.24 fold) (Table 13, Table 14).

<4-2. Effect of Nitrogen Monoxide as a Chemical Attractant>

The adventitious roots of Echinacea to 5L bioreactor 4L 1 / 2mMS [modified MS medium : 1/2 MS (NH 4 +: NO 3 - = 5: 25) + sucrose ^{50g · L -1 + IBA 2.0㎎ ·} L -1 ] The medium was added and cultured for 5 weeks at 20 ° C. under dark conditions. Nitrogen monoxide (NO) was treated at concentrations of 0, 50, 100, 250 and 500 μmol, respectively, at the fourth week of culture, and was repeated twice for each treatment.

[Table 15] Growth and Contents of Polyphenolic Compounds of Abdominal Muscle

Nitrogen Monoxide Concentration (μmol) Live weight (g / L) Under building (g / L) Growth rate Total Phenolic Content (mgg- ¹ ) Total Flavonoid Content (mgg- ¹ ) 0 70.1 a ^y 11.17 b 13.5 57.88 b 37.27 b 50 70.4 a 11.33 b 13.7 57.41 b 37.39 b 100 69.8 a 11.31 b 13.7 61.07 a 39.93 a 250 69.4 a 11.99 a 14.6 53.62 c 35.64 c 500 70.1 a 11.77 ab 14.3 52.24 c 35.31 c

The measured values were verified by Duncan's multiple test using SAS (SAS Institute, Cary, NC) program.

[Table 16] Content of Caffeic Acid Derivatives

Nitric oxide concentration (μmol) Caffeic acid derivatives (mg g- ¹ DW) Captaric acid Chlorogenic acid Chicorylic acid all 0 3.22 ± 0.03 ^z 4.73 ± 0.12 27.59 ± 0.16 35.53 ± 0.07 50 3.47 ± 0.02 4.53 ± 0.02 28.82 ± 0.54 36.82 ± 0.58 100 3.71 ± 0.03 4.95 ± 0.04 34.89 ± 0.32 43.55 ± 0.31 250 3.68 ± 0.04 4.60 ± 0.07 33.64 ± 0.44 41.93 ± 0.54 500 3.67 ± 0.05 4.49 ± 0.01 32.88 ± 0.29 41.04 ± 0.35

  The fresh weight and dry weight in all treatments of nitrogen monoxide showed higher or the same growth than the control. The content of secondary metabolite was the highest in 100μmol treatment group, and its biomass was 69.8g / L, dry weight was 11.31g / L, total phenol content 61.07mg / g DW (1.06 times), and total flavonoid content was 39.93mg / g DW ( 1.07 fold), and the total caffeine content was 43.55 mg / g DW (1.23 fold). Treatment with high nitric oxide concentrations inhibited the content of secondary metabolites. SOD antioxidant enzymes showed the same level of activity in all treatments, G-POD antioxidant enzymes were the lowest in 50μmol and APX antioxidant enzymes in the 100μmol treatment. It appeared high (Table 15, Table 16, FIG. 3).

<4-3. Effect of Salicylic Acid>

The adventitious roots of Echinacea to 5L bioreactor 4L 1 / 2mMS [modified MS medium : 1/2 MS (NH 4 +: NO 3 - = 5: 25) + sucrose ^{50g · L -1 + IBA 2.0㎎ ·} L -1 ] The medium was added and cultured for 5 weeks at 20 ° C. under dark conditions. Salicylic acid was incubated by treatment at concentrations of 200, 400, 600, and 800 μmol, respectively, in the fourth week of culture.

[Table 17] Growth and Contents of Polyphenolic Compounds of Abdominal Muscle

Salicylic Acid Concentration (μmol) Live weight (g / L) Under building (g / L) Growth rate Total Phenolic Content (mgg- ¹ ) Total Flavonoid Content (mgg- ¹ ) 0 70.8 a ^y 9.69 a 11.6 53.34 b 31.48 a 200 71.5 a 9.32 a 11.1 60.69 a 32.84 a 400 70.7 a 8.94 a 10.6 60.45 a 33.12 a 600 67.7 a 8.90 a 10.6 59.97 a 31.00 ab 800 61.5 b 7.49 b 8.73 46.43 b 28.42 b

The measured values were verified by Duncan's multiple test using SAS (SAS Institute, Cary, NC) program.

Table 18 Contents of Caffeic Acid Derivatives

Salicylic Acid Concentration (μmol) Caffeic Acid Derivatives (mgg- ¹ DW) Captaric acid Chlorogenic acid Chicorylic acid all 0 2.33 ± 0.30 ^z 4.40 ± 0.03 29.73 ± 0.59 36.47 ± 0.77 200 2.64 ± 0.41 4.34 ± 0.04 33.38 ± 0.15 40.36 ± 0.22 400 2.39 ± 0.33 3.84 ± 0.26 34.65 ± 0.34 40.88 ± 0.41 600 3.82 ± 0.36 2.92 ± 0.26 31.02 ± 1.23 37.76 ± 1.13 800 6.23 ± 0.12 1.67 ± 0.02 26.30 ± 1.28 34.20 ± 1.38

High concentrations of salicylic acid inhibited the growth of Echinacea abscesses and decreased the contents of secondary metabolites. However, low concentrations of salicylic acid showed the same growth as control and secondary metabolites tended to increase than control. Among them, the 200μmol treatment group was the best, with a total weight of 71.5g / L, dry weight of 9.32g / L, total phenol content of 60.69 mg / g DW (1.29 times), total flavonoid content of 32.84 mg / g DW (1.05 times), total The caffeine content was 40.36 mg / g DW (1.11 fold) (Table 17, Table 18).

Example 5 Mass Production of Echinacea Purpurea Malaria for Secondary Metabolites

For the mass production of Echinacea abscess, the characteristics and genetic stability of secondary metabolite content of the abscess were examined first. Secondary metabolite content was compared with the roots of three cultivars grown for 3 years under natural environmental conditions. Genetic stability was verified by chromosome number and ploidy.

<5-1. HPLC Analysis of Caffeic Acid>

The three Echinacea varieties analyzed were three-year-old cultivated in Canada, and Echinacea purpurea malaria was cultured using optimal medium in a 20L bioreactor. The content of caffeic acid derivatives was analyzed by HPLC and the analytical conditions were measured at 330 nm with water and acetonitrile as mobile phase using XTerra® RP 18 column.

Table 19 HPLC Analysis

sample Caffeic Acid Derivative Content (mg / g DW) Captaric acid Chlorogenic acid Caffeic Acid Ekina Coside Cinnarin Chicorylic acid all E. angustifolia - - - 6.70 ± 0.26 - - 6.70 ± 0.26 E. pallida 0.34 ± 0.01 - - 5.96 ± 0.24 - 0.66 ± 0.02 6.97 ± 0.26 E. purpurea 2.39 ± 0.22 - - - - 6.18 ± 0.86 8.57 ± 1.08 E. purpurea Nerve root 4.39 ± 0.52 5.25 ± 0.13 - - - 24.48 ± 0.85 34.12 ± 1.51

The root of E. angustifolia showed only equinacoside, and its content was 6.70 mg / g DW, and the root of E. pallida showed 0.34 mg / g DW of captanic acid, 5.96 mg / g DW, and echinaconic acid 0.66 mg / g. DW, total 6.97 mg / g DW and E.purpuread is 2.39 mg / g DW, capric acid, 6.18 mg / g DW, total 8.57 mg / g DW. E. pupura labia roots were 4.39 mg / g DW in caprateic acid, 5.25 mg / g DW in chlorogenic acid, 24.48 mg / g DW in total cholic acid, and 34.12 mg / g Dw in total, 1.84 times capric acid and 3.96 times more than E.purpurea. Chlorogenic acid material was produced which was high and did not appear in cultivation (Table 19, Figure 6).

<5-2. Investigation of Chromosomal Drainage in Echinacea>

To determine the drainage variability of long-term passaged varus roots in a bioreactor, we compared leaf and drainage levels of the same variety grown in nature. The root root of the plant was about 1.0cm in size, and the leaves of the plant were 0.5cm × 0.5 (horizontal × vertical) in size, cut finely with a knife, and extracted with nuclei.

As a result of the measurement, the peak of the abscesses appeared at the same position as the relative peak of the parent strain. Therefore, even if the tissues of echinacea were cultured for a long time, no nuclear change was observed and it was found to be genetically stable because it had the same drainage as the parent strain. 4).

<5-3. Analgesia chromosome count analysis>

For chromosome observation, the root end of the plant was taken and pretreated in saturated aqueous solution of 1-bromonaphthalene for 22-24 hours (4 ℃), followed by 1: 3 fixation (glacial acetic acid: ethanol, v / v). After fixing, the solution was softened and washed with 1N HCL (60 ° C.) for 6 minutes, and stained with a Feulgen solution to make a slide and observed under a microscope. As a result of observation, the number of chromosomes of the cultured root muscle was 2X = 22. This genetic in long-term culture of Bata glia (Battaglia) is the same as in 1947 «CHROMOSOME ATLAS OF FLOWERING one chromosome of Echinacea purpurea (E. purpurea) are called in PLANTS» 22 levels, so once again eke Micronesia adventitious root Stability was confirmed (Fig. 5).

<5-4. Involuntary root culture in large-scale bioreactors>

Echinacea mass production of adventitious roots Echinacea is 500L, 1000L of the balloon (balloon) type and drum (drum) type, each creature 300L, 600L 1 / 2mMS [modified MS medium in the ^{_{reactor: 1/2 MS (NH 4 +:}} NO 3 - = 5:25) + sucrose 50g · L ⁻¹ + IBA 2.0mg · L ⁻¹ ] medium was added, and 7g / L inoculum was incubated at 22 ± ° C. for 50 days under dark conditions to examine growth and caffeine content of the root muscle. .

[Table 20] Content of Abdominal Muscle Growth and Caffeic Acid Derivatives

Live weight (kg) Building weight (kg) Caffeic acid derivative content (mg / g dry weight) Captaric acid Chlorogenic acid Chicorylic acid all 20L 1.07 0.14 4.87 5.30 26.51 36.68 500L 26.80 3.62 2.66 4.85 19.33 26.84 1000L 41.02 5.10 4.08 4.85 23.1 32.03

Echinacea abscess was harvested in 20L bioreactor with 1.07Kg of live weight, 0.14Kg in building, total caffeine derivative derivative 36.68mg / g DW, 26.80kg live weight, 3.62Kg in building, 500L in balloon type bioincubator. Caffeic acid derivatives were harvested at 26.84 mg / g DW. In the 1000 L bioreactor, the root muscle culture was grown at 41.02 Kg in vivo, 5.10 kg in dry matter, and the content of caffeic acid derivative was 32.03 mg / g DW. The above results indicate that the roots of Echinacea can be grown without inhibition in large-scale bioreactors, and can produce relatively high contents of this secondary metabolites in a short period of time (Table 20).

As apparent from the above examples, the method of culturing Echinacea purpurea malaria of the present invention can establish a mass production system using a bioreactor by identifying and optimizing the in-flight culture conditions from the induction and proliferation of malaria roots. have. In addition, if the cultivation method of the present invention is used as an optimal in-flight cultivation method that selects genetically superior individuals and enables proliferation within a short period of time, it will be highly useful as a plant tissue culture strategy for producing useful components.

Claims (9)

Sterilizing and inoculating plants of the genus Echinacea to induce and propagate callus; Inducing and propagating adventitious roots from callus; And In the method of cultivating the root of the root muscle to harvest the root of the root muscle by culturing in the bioreactor, In the root muscle cultivation method, (1) the medium 1 / medium ^2mMS-: using the [modified MS medium:: 1/2 MS (NH ₄ NO ₃ ⁺ 5 = 25), + sucrose ^{50g · L -1 + IBA 2.0㎎ ·} L -1] , (2) Inoculation amount of irregular root assumes 7.0 g / L, (3) The air supply amount is 0.1vvm, (4) The incubation period is 5 weeks, (5) the culture temperature is incubated at 20 ℃, (6) a method for cultivating the root muscle of the root muscle, characterized in that it further comprises adding 1 / 2mMS medium 2 weeks after the start of the culture as a method for increasing the growth of the root muscle. delete The photoperiod of 3h / 21h (light / dark) according to claim 1, wherein the photoperiod on the growth is adjusted to 30 μmol m ^-2 s ^{- 1} luminosity every day from the beginning of growth by a method for increasing the content of the polyphenolic compound which is the secondary metabolite of the root canal. Method for culturing the root of neglect, characterized in that it comprises culturing after treatment with. The bioreactor according to claim 1, wherein the UV-A fluorescent lamp (20W, Sankyo Denki, Japan) is used as a physical attractant from the fourth week of culture to increase the content of the polyphenolic compound which is the secondary metabolite of the root canal. Cultivation method of intestinal root, characterized in that it comprises a 1cm treatment for 1 week every day as 50cm. The method of claim 1, wherein the method for increasing the content of the polyphenolic compound, which is a secondary metabolite of the root canal, comprises culturing after treatment with nitrogen monoxide at 100 μmol with a chemical attractant in the fourth week of the culture. Way. The method of claim 1, wherein the salicylic acid is treated with 200 μmol at the fourth week of culture as a method for increasing the content of the polyphenolic compound which is the secondary metabolite of the root canal. delete The method of claim 1, wherein the plant of the genus Echinacea is Echinacea purpurea. 2. The method of culturing the root muscle of claim 1, wherein the involuntary root muscle is genetically stable because of its chromosomal drainage and number of chromosomes equal to the parental strain.

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