JPH06261775A - Production of red pigment by tissue culture of rosemary - Google Patents

Abstract

PURPOSE:To economically mass-produce an anthocyanin-based pigment on an industrial scale in high yield by mass culturing a cultured cell of a rosemary. CONSTITUTION:This method for producing a red pigment by the tissue culture of a rosemary is to induce a callus from a plant tissue of the rosemary which is a plant of the family Labiatae, subsequently select a callus of a cell aggregate capable of producing a rosemary pigment from the resultant callus, transplant the selected callus to another culture medium, then culture the transplanted callus, subculture the callus obtained by the culturing and provide an anthocyanin-based pigment as a secondary metabolite. The method further comprises a culturing step in the first step for subculturing the callus and subsequently mass culturing the callus obtained in the subculture in the first liquid culture medium prepared by modifying the subculture medium and a culturing step in the second stage for mass culturing the callus in the second liquid culture medium obtained by modifying the subculture medium, continuing the culture of the callus mass cultured in the second liquid culture medium and producing the anthocyanin-based pigment.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention
inus officinalis L.) and a method for producing a red pigment, particularly an anthocyanin pigment used for coloring foods, cosmetics, etc., by tissue culture. In the present specification, "callus" refers to an atypical cell obtained by culturing a tissue piece cut out from a plant in a solid medium for tissue culture, and "anthocyanin" refers to a plant flower or fruit skin, etc. Is a glycoside that shows red, blue, purple, etc., which are often found in the part with a beautiful color.

[0002]

2. Description of the Related Art Conventionally, various kinds of synthetic pigments and natural pigments have been used for coloring foods, cosmetics, etc. However, synthetic pigments have problems of mutagenicity, carcinogenicity, etc. It is desired to use pigments derived from natural products. However, the production of pigments by extraction from natural products is easily affected by the natural environment such as season, climate and temperature, and stable supply is difficult. Therefore, production of natural pigments by tissue culture has been attempted. The tissue culture method has an advantage that the target dye can be produced in a short time and the planned production can be efficiently performed on an industrial scale.

As a method for producing anthocyanin pigments by culturing tissue of a plant (callus), callus of Akamega wrinkle (Japanese Patent Application Laid-Open No. 49-94897) and callus of an eye plant belonging to Polygonum (Japanese Patent Application Laid-Open No. 53-840).
26), a callus derived from the leaves of Delis (JP-A-54-54).
11281), callus of a genus of grapes (Japanese Patent Laid-Open No. 55-1).
18392), a euphorpia hanakirin (Japanese Patent Laid-Open No. Sho 5)
7-2696), Roselle's callus (JP-A-64-30)
594) and the like are known. In addition, in tissue culture of perilla plants, after tissue culture under irradiation with blue light having a wavelength of 190 to 500 nm or tissue irradiation under irradiation with red light having a wavelength of 600 to 900 nm, after culturing red pigment-forming callus, respectively. , A method for producing a red pigment from a culture has been proposed (Japanese Patent Laid-Open Nos. 61-195688 and 63-63).
156865). The above-mentioned perilla plant is a red-purple ordinary red radish, whose leaves contain anthocyanin pigments.

The present inventors tissue-cultured an undifferentiated cell group (callus) of an evergreen rosemary, which belongs to the Labiatae family but is different from the above red-purple red radish, and tissue-cultured the anthocyanin pigment, which is its secondary metabolite. He invented a method for obtaining the same and applied for a patent (Japanese Patent Application No. 4-100355). This method induces callus from the plant tissue of rosemary of the Lamiaceae plant, selects the callus of the rosemary pigment-producing cell mass from this callus, transplants the selected callus to a solid medium and cultures this culture. This is a method of subculturing the callus obtained in step 1 to obtain anthocyanin dye as a secondary metabolite.

[0005]

However, since the above method uses only a single medium for subculture, the growth amount of callus and the production amount of anthocyanin pigment, which is a secondary metabolite, are both It was low, and it was insufficient to mass-produce the anthocyanin dye and industrially use it.
The present inventors further studied, and after subculture, if the tissue culture was carried out stepwise in two liquid media having a specific relationship, anthocyanin which is a secondary metabolite obtained from an undifferentiated cell group. The present invention has been accomplished by finding that the amount of the system dye is remarkably increased.

An object of the present invention is to provide a method for mass-culturing rosemary cultured cells having a high ability to produce anthocyanin pigments in a large amount at a high yield economically and on an industrial scale. Especially.

[0007]

Means for Solving the Problems The present invention induces callus from a plant tissue of rosemary of a Lamiaceae plant, and selects a callus of a rosemary pigment-producing cell mass from this callus,
After transplanting the selected callus into another medium and culturing, the callus obtained by this culture is subcultured to obtain anthocyanin pigment as a secondary metabolite is there. The characteristic constitution is that, after the subculture, the first-stage culturing step of mass-culturing the callus obtained by the subculture in the first liquid medium in which the subculture medium is modified, and the subculture medium are modified. The second step of culturing the callus, which has been cultivated in a large amount in the second liquid medium, to produce an anthocyanin dye as a secondary metabolite.

The present invention will be described in detail below. (a) Plant Material Examples of the plant material used in the present invention include leaves, stems, flowers, roots, seeds and other tissues of the Lamiaceae plant. Such rosemary plants are sterilized by a commonly used bactericide such as a sodium hypochlorite solution to make them sterile.

(B) Induction of Callus from Plant Tissue Next, the sterilized plant material is cut into small pieces of appropriate size with a sterile scalpel, and the cut pieces are placed on a medium. After bed placement, 2
Callus is induced from plant tissue under constant temperature conditions of 0 to 30 ° C, preferably 24 to 26 ° C. As a medium for inducing the callus of the plant body, an ordinary inorganic synthetic agar medium for tissue culture is used. For example, Murashige
Murashige-Skoog Medium, Gamboug-B5
(Gamborg-B5) medium, White medium, Heller medium and modified mediums thereof are used. Such a medium may contain a carbon source, a nitrogen source, inorganic salts, organic substances, plant hormones and the like.

As the carbon source, sugars such as sucrose, glucose, maltose and fructose, starch and the like are used. As the nitrogen source, nitrates, ammonium salts,
Glycine, urea, etc. are used. As inorganic salts,
Those containing elements such as phosphorus, potassium, calcium, magnesium, manganese, copper, zinc, molybdenum, boron, iron and cobalt are used. Organic substances include inositol, nicotinic acid, pyridoxine hydrochloride, thiamine hydrochloride, calcium pantothenate, p-aminobenzoic acid,
Folic acid, biotin, riboflavin, ascorbic acid, citric acid, malic acid, coconut milk, yeast extract and the like are used.

As plant hormones, 2,4-dichlorophenoxyacetic acid (hereinafter abbreviated as 2,4-D), α-
Auxins such as naphthalene acetic acid (hereinafter abbreviated as NAA), indole acetic acid (hereinafter abbreviated as IAA), indole butyric acid (hereinafter abbreviated as IBA), and N
Cytokinins such as ⁶ -furfurylaminopurine (hereinafter abbreviated as kinetin), N ⁶ -benzyladenine (hereinafter abbreviated as BA) and the like can be used. The use concentration of auxin is 10 ⁻⁷ to 10 ⁻⁴ M, preferably 10 ^{−5 to} 5 × 10 ⁻⁵ M, and the use concentration of cytokinin is 10 ⁻⁷ to 10 ⁻⁵ M, preferably 10 ⁻⁶ to ⁵ M. × 1
It is 0 ^-6 M. If the concentration of auxin or cytokinin used exceeds the above range, callus formation (induction of callus) may not occur, which is not preferable. After a culture period of about 2 to 4 weeks under the above culture conditions, callus is induced from each cut piece.

(C) Selection of Callus of Rosemary Pigment-Producing Cell Mass and Culture of this Callus From this induced callus, a callus, which is a red-purple dark rosemary pigment-producing cell mass, is selected. This is transplanted onto another fresh medium having an appropriate composition, and the culture is continued at a constant temperature of 20 to 30 ° C, preferably 24 to 26 ° C. As this other medium, besides the solid medium such as the agar medium described above, a liquid medium containing no agar may be used. A subculture of this callus in a solid medium or a liquid medium is used as a callus for the next large-scale culture.

(D) First Stage Culture The first liquid medium used in the characteristic first stage culture of the present invention is such that the composition ratio of the medium components is modified so as to be suitable for promoting the growth and proliferation of callus for mass culture. The medium is basically the same as the medium for inducing callus from the plant tissue of (b) above, except that agar is not included for liquefaction. Specifically, the first liquid medium is modified so as to contain at least one component of the nitrogen source or the organic component in an amount larger than that of the same component in the subculture medium of the callus at the subculture of (c) above. To increase the nitrogen source, it is desirable to increase the concentration of either or both of the nitrate ion concentration and the ammonium ion concentration, and to increase the organic component, it is desirable to increase the concentration of inositol. The initial callus concentration of the first liquid medium can be changed over a wide range, but it is desirable to add about 1 g to about 200 g of fresh callus to 1 liter of the liquid medium.

(E) Second Stage Culture The second liquid medium used in the characteristic second stage culture of the present invention is also suitable for the grown large-scale callus to produce the anthocyanin pigment of the secondary metabolite. As described above, the medium is basically the same as the medium for inducing callus from the plant tissue of (a) above, except that the composition ratio of the medium component is modified and agar is not included for liquefaction. Specifically, the second
Liquid medium contains at least one of nitrogen source or phosphate ion
It is modified to contain less than the same component in the passage medium, or at least one component of iron ion, organic component or carbon source more than the same component in the passage medium.

In order to reduce the nitrogen source, it is desirable that either one or both of the nitrate ion concentration and the ammonium ion concentration be low, and the ammonium ion concentration is 1/10 or less of the total nitrogen amount in the second liquid medium. Is particularly desirable. Further, in order to reduce phosphate ions, it is desirable that the concentration is 1/2 or less of the concentration in the passage medium.
In order to increase the iron ion, it is desirable to make the concentration twice or more the concentration in the subculture medium, and to increase the organic component, it is desirable to increase the concentration of either or both of thiamine and inositol. In order to further increase the carbon source, it is desirable to increase the concentration of sugars such as sucrose, glucose and maltose.

The callus tissue-cultured in the first liquid medium of the first stage culture of (d) above is continued to be cultured in the second liquid medium of the second stage culture. The initial concentration of callus of the second liquid medium can be changed over a wide range like the first liquid medium, but about 1 g of fresh callus per 1 liter of liquid medium is added.
It is desirable to add about 200 g. In the second-stage culture, the growth rate of callus is lower than that in the first-stage culture, but the production amount of the anthocyanin pigment, which is a secondary metabolite, is significantly increased. At this time, it is desirable to irradiate with light. The light irradiation is preferably 100 lux or more,
More preferably, it is performed by irradiating daylight color light of 3,000 to 100,000 lux. As the light source, sunlight, fluorescent lamp, incandescent lamp, mercury lamp and the like can be used.

(F) Extraction of red pigment After completion of the second stage culture, 0.1 to 0.1% of the extraction solvent is used as an extraction solvent.
The anthocyanin red dye is obtained by extracting the dye with 1.0% by weight hydrochloric acid-methanol and then removing the solvent by filtration or the like.

[0018]

INDUSTRIAL APPLICABILITY As described above, according to the present invention, callus derived from the plant tissue of rosemary of the Lamiaceae plant is subjected to stepwise tissue culture in two liquid media having a specific relationship. To be able to use a tank incubator as compared with a conventional solid medium, and to produce a large amount of anthocyanin pigment, which is a natural red pigment suitable as a colorant for foods and cosmetics, in a pure form and on an industrial scale. You can As a result, it can be economically produced with extremely high yield as compared with the case of producing from a natural plant.

[0019]

EXAMPLES Examples of the present invention will be described below. The present invention is not limited to the examples below. <Callus induction from rosemary> First, the leaves of rosemary were washed with running water, and then washed with 70% ethyl alcohol.
After immersing for 2 seconds, it was sterilized by immersing in an aqueous solution of sodium hypochlorite (effective chlorine concentration: 1.5%) for 15 minutes, and then washed 3 times with sterilized water. The sterile leaves sterilized in this way were cut into small pieces having an area of about 1 cm ² using a sterile knife. 2,4-D is 2ppm, kinetin is 0.2ppm, sucrose is 3%, and agar is 0.8%.
The plate was placed on a Murashige-Skoog medium whose pH was adjusted to 5.8. After placing the bed, 25 ℃
The cells were cultured under white light irradiation with a fluorescent lamp of 3,000 lux. Three weeks after the placement, callus tissue was induced from each cut piece, and a reddish purple part (rosemary pigment-producing cell mass) was confirmed in the tissue.

<Rosemary pigment-producing cell mass (callus)
Selection and subculture> Of this rosemary pigment-producing cell mass, a particularly dark portion was selected, and p of the composition shown in Table 1 was selected.
It was transplanted to a solid medium of H5.8 and cultured at 25 ° C. for 3 weeks under irradiation of white light with a fluorescent lamp of 3,000 lux.
The callus obtained by this culture was subcultured in the subculture medium shown in Table 1 consisting of the same components as the solid medium to obtain a fresh callus of rosemary. Note that this passage medium may not contain agar in some cases.

[0021]

[Table 1]

<First Stage Culture> Next, the first stage culture was performed using the fresh callus. That is, a sterilized Gamboug-B5 first liquid medium having the composition shown in Table 2 in which a 100 ml Erlenmeyer flask was modified from the above passage medium (however, NAA 2 mg / l, kinetin 0.2 mg /
1 and sucrose 30 g / l. ) 0.4 g of the above-mentioned rosemary callus was added to 25 ml, and cultivated at 25 ° C. for 14 days on a rotary shaker at 100 rpm.

<Second-stage culture> Further, second-stage culture was carried out. That is, a second liquid medium having the composition shown in Table 2 which is suitable for the production of anthocyanin dyes obtained by modifying the above passage medium (however, NAA 2 mg / l, kinetin 0.2 mg).
/ L, sucrose 50 g / l. ) In 25 ml, 0.4 g of the callus obtained in the first stage culture was put, and cultivated by rotation in the same manner as in the first stage culture under the irradiation of white light with a fluorescent lamp of 3,000 lux. In Table 2, the concentrations of the first and second liquid culture media are the volume concentration (mg / l) and the molar concentration (mM).
/ l). In addition, KCl 3 was newly added in the second liquid medium to supplement the potassium component due to the decrease in KNO ₃ .

<Comparative Example> For comparison, callus was grown in the same manner as in Example except that the first liquid medium was continuously cultured as it was without performing the second-stage culture to produce anthocyanin pigment. (Comparative example 1). For another comparison, callus was grown only in the subculture medium without performing the first-stage culture and the second-stage culture to produce anthocyanin pigment (Comparative Example 2).

<Measurement of Callus Growth Amount and Calculation of Anthocyanin Dye Production Amount> Cultured cells (callus) of rosemary after culturing in Examples and Comparative Examples were collected by suction filtration, and their weight (fresh weight ) Is measured,
The amount of callus growth per liter of liquid medium was determined. Further, the finally obtained callus of Examples and Comparative Examples was immersed in 1% hydrochloric acid-acidic methanol at 5 ° C. for 24 hours to extract anthocyanin dye, and the absorbance of the extract at 530 nm was measured. The total anthocyanin production per liter was calculated. The results are shown in Table 3.

[0026]

[Table 2]

[0027]

[Table 3]

As is apparent from Table 3, the production amount of the anthocyanin dye in the example was increased by about 5 times as compared with the comparative example.

Claims (6)

[Claims] 1. A callus is induced from a plant tissue of a rosemary of a Lamiaceae plant, a callus of a rosemary pigment-producing cell mass is selected from this callus, and the selected callus is transplanted to another medium and cultured, A method for producing a red pigment by tissue culture of rosemary, wherein the callus obtained by this culture is subcultured to obtain an anthocyanin pigment as a secondary metabolite, wherein the subculture medium is used after the subculture. The first-step culturing step of mass-culturing the callus obtained by the subculture in the modified first liquid medium, and the callus mass-cultured in the modified second liquid medium of the subculture medium are continued to the above 2 And a second stage culture step for producing an anthocyanin-based pigment, which is a secondary metabolite, wherein the first liquid medium contains at least one component selected from a nitrogen source and an organic component from the same component in the subculture medium. A large amount, the second liquid medium contains at least one component of a nitrogen source or a phosphate ion in a smaller amount than the same component in the passage medium, or at least one component of an iron ion, an organic component or a carbon source. A method for producing a red pigment by tissue culture of rosemary, characterized in that it contains more than the same kind of component in the subculture medium. 2. The increase of the nitrogen source in the first liquid medium or the decrease of the nitrogen source in the second liquid medium increases either or both of the nitrate ion concentration and the ammonium ion concentration to a high concentration or a low concentration. Method for producing red pigment. 3. The method for producing a red pigment according to claim 1, wherein the increase of the organic component in the first liquid medium increases the concentration of inositol. 4. The method for producing a red pigment according to claim 1, wherein the increase of the organic component in the second liquid medium increases the concentration of either or both inositol and thiamine. 5. The method for producing a red dye according to claim 1, wherein the reduction of phosphate ion in the second liquid medium is made to be 1/2 or less of the phosphate ion concentration in the passage medium. 6. The method for producing a red pigment according to claim 1, wherein the iron ion concentration in the second liquid medium is increased to twice or more the iron ion concentration in the subculture medium.