JPH03269060A - Production of red natural coloring matter - Google Patents

Abstract

PURPOSE:To obtain with industrially advantageous way the title coloring matter useful as a nontoxic colorant for food and medical items etc. by incubating red coloring matter-productive dispersed cells in a liquid medium. CONSTITUTION:Tissue fragments obtained from a plant tissue such as of apple are put to culture under illumination in a liquid medium such as Murashige Skoog medium to induce callus. The callus is then separated from the tissue and put to subculture repeatedly into stable callus with high red coloring matter concentration. The resultant callus is similarly put to subculture pref. at a dark place to effect cell proliferation into fragile, homogeneous dispersed cells. This cell aggregate is then crushed into cell particles and put to suspension culture (pref, in a B5 medium of gamboge) followed by further subculture to collect initiation cells. The resulting cells are then incubated pref. in a HC medium under an irradiation of blue rays 220-500nm in wavelength for 5-10 days at 10-30 deg.C, squeezed etc., and then extracted with a solvent in a darkness, thus obtaining the objective coloring matter.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for producing a red natural pigment by using callus derived from the tissues of plants such as Malus. Since the red natural pigment obtained by the method of the present invention is harmless to the human body, it is used as a coloring agent for foods, medicines, etc.

(Prior art) In recent years, the use of synthetic colorants in food, medicine, etc. has decreased in both type and amount due to safety concerns.
Natural colorants are on the rise. In particular, the development of red natural pigments is desired.

On the other hand, natural colorants are largely dependent on imports, and supply and prices are unstable. As a means to compensate for this drawback, attempts have been made in recent years to produce natural pigments through tissue culture.

According to this method, planned production can be carried out in a shorter period of time than by cultivating plants, and without being affected by the weather.

Among red pigments, anthocyanin pigments (cyanidin-3
-galactoside) are well studied.

Especially apples (Malus pumila Mill v.
ar dome-stica C! , to 5chn
) contains anthocyanin-based pigments in the pericarp and is attracting attention as a raw material for red pigments, but the pigments are only localized in a few layers of epidermal cells, making it difficult to use this as a coloring agent.

Regarding apple tissue culture, R.K. Ibrahim et al.
Lloydia), 34, 175-182 (19
71)), Ota et al. [Horticultural Journal, 52, 117-122
(1983) J et al., it is known that callus derived from cotyledons or pulp produces anthocyanin pigments.

(Problems to be Solved by the Invention) However, all of these studies used solid media and did not use liquid media suitable for mass culture. In general, it is necessary to obtain homogeneous dispersed cells when culturing in a liquid medium, and when it comes to pigment production, it is necessary to clarify the optimal culture conditions to improve productivity. conditions are unknown.

That is, a method for effectively producing red pigment in apples,
In particular, there is no known method for producing it in large quantities in a short period of time using a liquid medium.

(Means for Solving the Problems) The present invention solves the above-mentioned problems by inducing culture of a plant such as an apple that produces a red pigment, and preferably culturing the resulting red pigment-producing callus in a liquid medium. This is a method for producing a red natural pigment, which comprises culturing with shaking in the dark to obtain uniformly dispersed cells, then culturing them preferably under irradiation with blue light, and collecting the red pigment from the culture.

That is, the present invention provides: (1) a method for producing a red natural pigment, which comprises culturing dispersed cells that produce a red pigment in a liquid medium to produce a red pigment, and collecting the red pigment; (2) a method for producing a red pigment; The production method described in (1) above, characterized in that dispersed cells produced by subculturing and subculturing callus producing 100% by shaking in a liquid medium are used; (3) culturing the dispersed cells under blue light irradiation; (4) A method for producing dispersed cells, which comprises subculturing a callus that produces a red pigment with shaking in a liquid medium; (5) The production method described in (4) above, which is characterized by culturing callus with shaking in the dark, (6) the food and drink containing the red natural pigment obtained by the method (1) above, and (7) the above ( The present invention relates to a pharmaceutical product containing a red natural pigment obtained by the method of 1).

The method of the present invention involves culturing callus derived from plants such as apples that produce red pigment under light to select a red pigment-producing strain, and culturing this culture in liquid suspension in the dark to obtain uniformly dispersed cells. , this cell is cultured under blue light to produce red pigment, and can be broadly divided into callus induction, selection, dispersed cell proliferation,
It consists of four steps in the production of pigments.

Hereinafter, the present invention will be explained in order of steps.

Callus induction step Callus is induced and cultured from plant tissues such as apples by a known method. Plant tissues include, for example, branches of plants,
Tissue pieces such as leaves, fruits, roots, etc. are preferably used, preferably from growing parts such as shoot tips, cotyledons, hypocotyls, etc. This tissue piece is placed on a medium, for example, a liquid medium or a solid medium, and a callus is induced under illumination.

The above-mentioned plants are preferably sterilized with disinfectant alcohol, sodium hypochlorite solution, P solution of bleaching powder suspension, etc., washed with sterile water, cut into small pieces, and placed on a culture medium.

In addition to normal solid media, the media include liquid media used for plant tissue culture, such as Murashige-Skoog's (hereinafter referred to as MS) medium, Gamborg's BS (hereinafter referred to as B).
5) medium, Linsmeyer-Skoog's medium, White's medium, Heller's medium, Schenku-Hildegrant's medium, Nichni-Nilachie's medium, and modified media thereof. Alternatively, a solid medium obtained by adding a solidifying agent (agar, agarose, gelrite, etc.) to these liquid media may be used.

Such a medium may contain a carbon source, a nitrogen source, inorganic salts, organic substances, etc. as appropriate.

Examples of carbon sources used include sugars such as sucrose, glucose, galactose, fructose, and maltose, and soluble starch. As the nitrogen source, for example, nitrates, ammonium salts, etc. are used. Examples of inorganic salts used include those containing elements such as phosphorus, potassium, calcium, magnesium, manganese, copper, zinc, molybdenum, boron, iron, cobalt, and nickel. Examples of organic substances include inositol, nicotine, pyridoxine hydrochloride, thiamine hydrochloride, calcium pantothenate, folic acid, p-aminobenzoic acid, biotin, choline chloride, riboflavin, ascorbic acid, vitamins such as vitamin A1, vitamin DBs, and vitamin BI2, such as pyruvin. organic acids such as sodium chloride, citric acid, malic acid, fumaric acid, e.g. coconut milk,
Natural substances such as casein hydrolyzate and yeast extract are used.

The medium may further contain, as appropriate, auxins, cytokinins, plant growth regulators such as Todoroki W, agar, and the like. Such auxins include, for example, 2,4-dichlorophenoxyacetic acid, 2,4-dichlorophenylacetic acid, indole-3-acetic acid, indole-3-butyric acid, 1-naphthaleneacetic acid (hereinafter abbreviated as NAA), 2- naphthoxyacetic acid, parachlorophenoxyacetic acid,
2.4.5-Trichlorophenoxyacetic acid, 1-naphthaleneacetamide, etc. are used. Further, as cytokinins, for example, 6-benzyladenine (hereinafter referred to as B
(abbreviated as AP), 2-isopentyladenine, 2-isopentenyladenine, kinetin, zeatin, dihydrozeatin, zeatin riboside, diphenylurea, etc. are used.

Among them, examples of auxins include NAA,
For example, BAP is frequently used as a cytokinin.

Usually, auxins are added to the medium at a rate of about 0.01-20 ppm, and cytokinins are added at a rate of about 0.01-15 ppm. When cultured in the light at about 15 to 35°C, callus is induced from the tissue piece after about 10 to 40 days.

Of course, inorganic or organic acids, alkalis, buffers, etc. may be added for the purpose of adjusting the pH of the medium, or an appropriate amount of fats and oils, surfactants, etc. may be added for the purpose of defoaming.

The culturing method may be static culture, shaking culture, or aeration/agitation culture. For large-scale processing, it is desirable to use so-called shaking culture. Although the culture conditions naturally vary depending on the composition of the medium, the culture method, etc., it is generally preferable to select a temperature of 10° C. to 45° C. and an initial pH of around neutrality. In particular, it is desirable that the temperature during the middle stage of the culture is 20° C. to 37° C., and that the initial p and u values are 5.0 to 85. The culture time may be about 1 day to 3 months, but 1 week to 4 weeks is particularly preferable.

Cell selection process The reddish callus formed is separated from the tissue piece.
Transplant into solid or liquid medium similar to that used for callus induction. Cultivation can be performed under the same conditions as in the callus induction step.

After culturing, the dark red callus portion is cultured again to obtain a red pigment culture with high pigment concentration. If this operation is repeated every 5 to 60 days, preferably every 14 days, and subculture selection is performed, calli that stably produce red pigment can usually be obtained after 1 to 5 months.

This is selected and used in the next step.

Dispersed Cell Proliferation Process The obtained callus stably producing red pigment is transferred to the above liquid medium, preferably in the dark, and grown every 5-14 days for 2 to 14 days.
Cell proliferation is performed by repeating subculturing 10 times. When this cell proliferation is carried out for about 3 weeks, a flimsy and homogeneous dispersed cell mass is obtained. This cell mass is crushed into fine cell particles in a liquid and then cultured in suspension for further cell proliferation. As a medium,
A liquid medium similar to that used for callus induction, preferably B5 medium, is used. Culture can be performed under the same conditions as those used for callus induction.

This culture allows cells to proliferate rapidly, and this culture can be carried out in large quantities.

The resulting cell mass is subcultured every 5 to 14 days.

At this time, it is preferable to use the cell mass after passing it through a sieve.

After about 2 to 3 weeks, the proliferated cells are collected as initial cells and used in the next step of producing red pigment.

If this step is performed in the light, the pectin-like substance will gradually increase and the proportion of red cells will decrease, so it is preferable to perform it in the dark.

The initial cells obtained in the previous step of the red pigment production process are cultured in the same solid medium or liquid medium as used for callus induction. A preferred medium is a liquid medium, such as MO medium.

The culture period is 3 to 14 days, preferably 5 to 10 days. The culture temperature is about 10-30°C.

Cultivation is performed under illumination, preferably under blue light irradiation with a wavelength of 220 to 500 nm. This culture yields red to dark red callus. These calli contain large amounts of red pigment. After collecting the callus, squeezing or crushing as necessary, extracting with a suitable solvent and removing the solvent, a red pigment is obtained. The pigment may be extracted from the collected callus without drying it, or it may be extracted after being dried by a conventional drying method such as vacuum drying. As the extraction solvent, alcohols such as methanol, ethanol, etc. are preferred, and these preferably contain hydrochloric acid. Particularly preferred is 0.05-2
, Ow/w% methanol containing hydrochloric acid. The preferred temperature for extraction is about 0-20°C and the time is about 16 hours to one week.

This extraction is preferably carried out in the dark.

The red natural pigment produced in this way may optionally be
For example, dissolution, concentration (preferably vacuum concentration).

Further purification may be performed by means known per se such as chromatography and crystallization.

Method for producing food and beverages containing red natural pigments The food and beverages containing red natural pigments produced by the method of the present invention include the red pigment powder obtained by the above method as an ingredient for the production of ordinary foods and beverages. For example, it can be easily produced by blending it with fruit juice, glucose, fructose, liquid sugar, starch syrup, etc.

Any food or drink can be used as the food or drink containing the red natural pigment produced by the method of the present invention, such as juice, ice cream, jelly, jam,
Examples include drops.

The amount of red natural pigment used in the food and drink containing red natural pigment produced by the method of the present invention is - Although it is not possible to say generally, the amount of red natural pigment used is usually about 0.001% to 20% based on the finished food and beverage.
It is about %.

Method for producing a pharmaceutical formulation containing a red natural pigment The pharmaceutical formulation containing a red natural pigment produced by the method of the present invention is produced in exactly the same manner as the conventional method for producing a pharmaceutical formulation, except that the red pigment is used as a coloring agent. Manufactured.

Pharmaceutical preparations used in the present invention include tablets, granules, granules, sugar-coated tablets, film tablets, hard capsules, and the like.

The amount of red natural dye used in a pharmaceutical formulation containing a red natural dye prepared by the method of the invention is - although not generally stated, usually about 0.001 to 2.0
It is about %.

(Example) The invention will be further explained below in the form of examples.

In each example, q indicates weight unless otherwise specified.

Example 1 [Callus induction process] 70 apical or axillary buds of apple (variety Nistarking)
% ethanol for 5 minutes, then immersed in 1% sodium hypochlorite solution for 10 minutes, and then washed five times with sterile water. Next, the shoot apex was removed under a stereomicroscope and placed on MS agar medium (
NAA 2. Omg/l! , BaF2.5mg/l
, agar (9 g/l, pH 5.8).

Under lighting (white fluorescent light, 2000 Lux, 16 hour day length)
After culturing at 25°C for 3 weeks, a reddish callus formed on the cross section of the shoot apex was separated and transplanted onto the above MS agar medium.

[Cell selection process] Thereafter, the deep red callus portion was subplanted every 7 days, and after 2 months, a callus that stably produced red pigment was obtained.

[Dispersed cell expansion step] The callus obtained above was placed in a growth medium (NAA2.Omg/
1XBAP2.5mg/l! , B5 liquid medium supplemented with 5% sucrose) and cultured in the dark with shaking (60 rpm).

Cells were repeatedly subcultured every 7 days, and uniformly dispersed cells were obtained after one month.

[Red pigment manufacturing process]

The dispersed cells were plated onto a nylon mesh and treated with chromogenic medium (NAA 2.0 mg/CBiF 3.5 mg/l).

The cells were transplanted into a 9 cm plastic petri dish containing 20 ml of MB medium supplemented with 3% sucrose. Initial cell amount is 20mg
/ml. Cultivation was performed using a blue fluorescent lamp (product name: FL-
408B (manufactured by NEC Corporation) under lighting at 25°C for 8 days with shaking (60 rpm).

In addition, green (product name: FL40SG) is used as a control.

Shaking culture at 25°C for 8 days under the illumination of red (product name: FL40SPK) and fluorescent lamps for plant growth (product name: FL408BR-A, manufactured by NEC Corporation)
6 Orpm) I.

To measure the content of anthocyanin in the obtained callus,
A portion of the callus (400 mg) was collected and extracted with 0.1 dihydrochloric acid methanol (4 ml) in the dark at 4°C for 48 hours. After removing impurities, the absorbance of the liquid (wavelength 530
nm) was measured. The product of the absorbance and the wet callus weight was defined as the pigment production amount (AXB).

Table 1 shows the amount of pigment produced under each fluorescent lamp.

Table 1 As is clear from Table 1, the amount of andocyanin produced was highest under blue light irradiation, and the amount produced was more than twice that in other cases.

In addition, the callus was filtered from the culture solution containing the callus obtained by shaking culture under blue fluorescent lamp illumination using a to μm nylon mesh and freeze-dried. After degreasing 24 g of the obtained dry callus with acetone, it was dissolved in 300 ml of methanol containing 1% hydrochloric acid, and this was applied to an ion exchange column (Dowex 50W-X2, manufactured by Dow Chemical Company).
The dye was adsorbed onto the resin. After washing this sequentially with water and methanol to remove impurities such as sugar and lipids, 5%
The dye was eluted with 350 ml of methanol containing hydrochloric acid. 20ml of the obtained dye fraction! Concentrate under reduced pressure until
), and after washing with water and methanol, the dye was eluted with 170 ml of methanol containing 0.4% hydrochloric acid. The eluate was 2
The absorbance was monitored at 64 nm. The obtained main fraction was concentrated to dryness using a rotary evaporator, and the residue was dissolved in 5 ml of methanol. The operation of adding 3 ml of ethyl ether thereto was repeated 10 times to precipitate the pigment, yielding 60 mg of purified pigment powder.

2 Dissolve 1 mg of the purified pigment powder obtained above in 200 μl of methanol containing 1% hydrochloric acid, add 2 ml of 10% formic acid to prepare a test solution, and perform high performance liquid chromatography (HPLC).
The dye was analyzed using YoMogawa LC100). The above test solution was injected into a YMCPack A3120D8 column (6 mm x 150 mm: Yamamura Kagaku Kenkyusho fR), and a 10% formic acid aqueous solution and a methanol mixture containing 10% formic acid were flowed at a rate of 1.4 ml/min. After 10% formic acid content of the entire mixed liquid: 30%. The eluent is
Absorbance was monitored at 535 nm. The results are shown in Figure 1.

As a control, 400 mg of freeze-dried apple peel (variety Nyster King) was purified for pigmentation in the same manner as above, and analyzed by HPLC under the same conditions as above. The results are shown in Figure 2.

In addition, as standard products, cyanidin-15-cy'/lucoside (cyanin; manufactured by Carl Roth), cyanidin-3-galactoside (ivuanin; manufactured by Extra Synthes),
Cyanidin-3-glucoside (chromanin; manufactured by Extra Synthes), cyanidin-3-rutinoside (keracyanin; manufactured by Extra Synthes), and cyanidin (
The mixture was subjected to HPLC using a HPLC (manufactured by Carl Ross). The results are shown in Figure 3.

The retention time of the largest peak of the apple red pigment produced by the method of the present invention and the red pigment purified from apple peel coincided with the peak of cyanidin-3-galactoside. That is, the main component of the apple red pigment produced by the method of the present invention is anthocyanin pigment (
Cyanidin-3-galactoside).

Example 2 Culture was carried out under the same conditions as in Example 1 except for using an agar medium (0.9% agar was added to the pigment production medium in the red pigment production process of Example 1). Table 2 shows the amount of pigment produced after 8 days.

(Margin below) As is clear from Table 2, the amount of pigment produced was about 1/10 of that in Example 1 (suspension culture in liquid), but the amount of pigment produced under blue light irradiation was different. It was better than the case.

Example 3 15 g of agar was soaked in 1 kg of water for 5 hours, and then heated until dissolved. Pour the agar solution into a pot and add 38 ounces of sugar.
0g of glucose, 50g of glucose, and 425g of starch syrup were added, and the mixture was placed in a vacuum concentrator and concentrated at 60°C for 15 minutes. Next, an appropriate amount of the apple red pigment obtained in Example 1 and an appropriate amount of flavor were added, mixed uniformly, and cooled until no steam came out. This liquid was poured into a mold, placed in a coagulation chamber at 15°C, and allowed to cool and solidify. After removing it from the mold and cutting it into thin pieces using a cutting machine, it was wrapped in a wafer and made into a natural apple-colored jelly.

Example 4 500 g of apple fruit was washed with water, cut into 1 cm thick pieces, immersed in saline solution to prevent discoloration, and then boiled in IDDg of water. Boiling is 1/2 to 1/3 of the total amount of sugar added.
The mixture was heated with 400 g of sugar in a jacketed boiling pot while stirring, and after the juice had leached out and the sugar had dissolved, the remaining sugar was added and boiled under normal pressure for 20 minutes. After boiling, the mixture was cooled to 80°C using a cooler, and in order to compensate for the color tone, an appropriate amount of the apple red pigment obtained in Example 1 was added to obtain a red apple jam.

Example 5 65 kg of sugar was dissolved in 15 kg of water, mixed with 50 kg of starch syrup, boiled until the water content was 1 to 2%, and placed on a cooling plate to dissolve 100 g of the apple red pigment obtained in Example 1 and 50 g of acidulant.
After adding and mixing 0 g and fragrance, the mixture was molded using a stamping machine to form apple red drops.

Example 6 Red colored granules and granules 1 kg of powder consisting of acetaminophenone (40% by weight), lactose (30% by weight) and starch (30% by weight)
was charged into a vertical granulator (FM-VG-10; manufactured by Fuji Sangyo ■), and a suspension (01 g of apple pigment powder obtained in Example 1 was dispersed in 150 m13 of water, and a further drop of Roxymethyl cellulose (3 weight t%
(prepared by adding the equivalent)) was added and colored while granulating, to obtain a red-colored granulated product.

This material was dried with a vacuum dryer (manufactured by Kusunoki Seisakusho ■), sized with a power mill (P-3; manufactured by Showa Kagaku Kikai Co., Ltd.), and passed through a sieve to obtain granules with 90% passing through 16 meshes. .

Example 7 Red-colored granules and tablets Powder containing ascorbic acid, sodium ascorbate, and sorbitol as main components (ascorbic acid 15 MIk%)
, sodium ascorbate (15% by weight, sorbitol (60% by weight)) in a fluidized bed granulation dryer (Brat WSQ-16; manufactured by Okawara Seisakusho ■). (prepared by dispersing 20 g of pigment powder in 600 ml of water) was sprayed to color the powder while granulating it.

Subsequently, 100 g of starch was dispersed in 1.900 ml of water, and while spraying a size solution heated to 80°C,
It was further granulated.

The red colored granules were dried, sized, and then compressed to produce tablets.

Example 8 1 kg of red-colored granular crystalline granilla sugar was placed in a coating pan (12 inches; manufactured by Kikusui Seisakusho ■), and 0.1 g of the apple pigment powder obtained in Example 1 was suspended in 500 ml of water. Spray coating was carried out using 1 kg of a mixture of equal amounts of ascorbic acid and powdered sugar (spraying agent) while spraying the liquid.

Coating was carried out until the diameter of the particles was about 5 mm, and then vacuum-dried (Hosui Seisakusho ■*) L to obtain granules.

Example 9 Red-colored tablet (dragage-coated tablet) A tablet (diameter 9.5 mm-, weight 250 mg, s, 000 tablets)
Sugar coating was carried out in a coating pan (12 inches; manufactured by Kikusui Seisakusho ■).

At this time, the sugar coating liquid used was one in which 0.1 g of the apple pigment powder obtained in Example 1 was added to sugar coating liquid 11 consisting of granilla sugar, talc, pullulan, and water.

Finishing was performed with a syrup solution to obtain sugar-coated tablets with a finished weight of 450 mg.

Example 10 Red colored film tablet/Lactose and starch tablet (diameter 9.
5 mm-, weight 250rr+g, 40,000 tablets) were coated with the film solution containing the apple color powder obtained in Example 1 using Accela Coater 24 (manufactured by Manestee).

The film liquid used was hydroxypropyl methyl cellulose [TO-5 (B); manufactured by F-Etsu Kagaku Kogyo ■],
A film liquid 31 consisting of titanium oxide, polyethylene glycol 6,000, and water was used in which 015 g of the apple pigment powder obtained in Example 1 was added.

In this way, a finished film tablet weighing 11,260 mg was obtained.

Example 11 Red colored hard capsule Add 60 parts of water to 30 parts of gelatin and dissolve by heating.

On the other hand, 1 part of titanium oxide and 1 part of the apple pigment powder obtained in Example 1 are dispersed in 10 parts of water using a homomixer (manufactured by Tokushu Kika ■), and added to an aqueous gelatin solution for coloring. .

A capsule bottle is poured into this solution and molded, and after drying, it is made into a hard capsule.

The resulting capsules were colored bright apple red.

(Effects of the Invention) According to the present invention, red pigment can be produced on an industrial scale in a short time without being affected by natural conditions such as climate and soil by tissue culturing apple callus using a specific method. I can do it. This pigment is an anthocyanin pigment and is harmless to the human body, so it is used as a coloring agent for foods, medicines, etc.

[Brief explanation of drawings]

FIG. 1 shows an HPLC chromatogram of the red dye produced by the method of the invention. Figure 2 shows HPLC of red pigment purified from apple peel.
The chromatogram is shown below. Figure 3 shows the HPLC chromatogram of the standard product, in which A is cyanidin-35-diglucoside, B is cyanidin-3-galactoside, and C is cyanidin-3-
glucoside, D represents cyanidin rutinoside, and E represents cyanidin.

Claims (7)

[Claims] (1) A method for producing a red natural pigment, which comprises culturing dispersed cells that produce a red pigment in a liquid medium to produce a red pigment, and collecting the red pigment. (2) The production method according to claim 1, characterized in that dispersed cells produced by culturing and subculturing red pigment-producing calli with shaking in a liquid medium are used. (3) The manufacturing method according to claim 1, characterized in that the dispersed cells are cultured under blue light irradiation. (4) A method for producing dispersed cells, which comprises subculturing callus that produces a red pigment with shaking in a liquid medium. (5) The production method according to claim 4, characterized in that the callus is cultured with shaking in the dark. (6) A food or drink containing a red natural pigment obtained by the method of claim 1. (7) A pharmaceutical product containing the red natural pigment obtained by the method of claim 1.