JPH0463599A - Production of betacyanin-type natural red pigment - Google Patents

Abstract

PURPOSE:To obtain a betacyanin-type natural red pigment suitable for the coloring of foods by inducing a callus from Joseph's coat amaranth, subculturing the callus in light place in a medium containing a plant-growth regulator and recovering the produced red pigment from the cell. CONSTITUTION:A betacyanin-type natural pigment is produced by inducing a callus by the tissue-culture of Joseph's coat amaranth, subculturing the callus in a medium containing a plant-growth regulator in light place while selecting the red pigment-producing cell and recovering the red pigment from the obtained red pigment-producing cell. The growth regulator is preferably auxins or cytokinins.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application J This invention relates to a method for producing a betacyanine-based natural red pigment.

[Prior Art] Among the red natural pigments currently in use, betacyanin-based red pigments have a vivid reddish-purple color, and among the natural red pigments, they exhibit the most beautiful strawberry color, so they are used in the largest amount. ing. On the other hand, with regard to food coloring, there is a shift from synthetic colorants to natural colorants due to stricter legal regulations on synthetic colorants and safety of synthetic products, and demand for natural colorants is on the rise. be.

Betacynian red pigments are found in the phylum Explanatory Plants - Subphylum Apophyta -
It has been found that plants in <10 families of the Centrospermaelales, excluding Caryophyllaceae, contain this pigment.

Conventionally, betacyanin has been mainly produced by extracting it from plants containing the component. Currently, when producing betacinian pigments industrially, red sugar beet (red beet), which is a variety of the family Chenopodiaceae, genus Euphorbia, sugar beet,
Rubra is used as raw material. This red beet, also known as Sangojuna, is cultivated in the United States, the Soviet Union, Eastern European countries, Israel, Sudan, Morocco, South America, etc., both for food and as feed for livestock. Cultivation is carried out twice a year, and the harvest is imported into Japan as a raw material for pigments, either in crushed and dried form or in the form of concentrated squeezed juice.

However, while the shift to natural colorants is progressing and demand is increasing, the supply of natural plant resources that serve as raw materials is unstable in terms of quantity, quality, and price, mainly due to unseasonable weather around the world. The situation continues.

[Problems to be Solved by the Invention] Therefore, an object of the present invention is to provide a method for producing betacyanin red pigments stably and in a high yield, regardless of natural environmental conditions. .

Means for Solving Problems j As a result of intensive research into a method for mass-producing betacyanin-based red pigments, the present inventors have developed
By subculturing calli induced by tissue culture from Ranthus tricolor plants under specific culture conditions, it is possible to select cells that are highly productive of betacyanin-based natural red pigments, and from the culture thus obtained. They discovered that betacyanin-based red pigments can be recovered and completed this invention.

That is, the present invention comprises a step of inducing callus by tissue culturing bulrushes, and a step of subculturing the callus in a medium containing a plant growth regulator in the light while selecting red pigment-producing cells. , provides a method for producing a betacyanin-based red pigment, which includes a step of recovering the red pigment from the red pigment-producing cells obtained by the subculture.

[Effects of the Invention] The present invention provides a highly productive method for producing a betacyanin-based natural red pigment. When using the method of the present invention, compared to the method of cultivating and extracting red pigment-containing plants,
Not affected by natural environmental conditions such as weather (stable,
Moreover, it is possible to produce a large amount of betacyanin-based red pigment useful as a food coloring agent that is safe and harmless to the human body in a short period of time.

[Detailed Description of the Invention] In the method of the present invention, first, callus is induced from the plant tissue of the staghorn beetle. Although the tissue piece for inducing callus may be derived from any tissue of the bulrush plant, roots, stems, and leaves are preferable, and recently-expanded petioles are particularly preferable.

A callus can be induced from a piece of tissue of a staghorn plant according to a conventional method. For example, the tissue of a bulrush plant is cut out and sterilized. Sterilization can be carried out according to conventional methods. The sterilized tissue piece is, for example, 1OIII11
Cut a corner-sized section, implant it on an agar nutrient medium, and culture. At this time, the basic medium used for culture is a medium commonly used for plant tissue culture, such as Murashige
-3koog medium (Murashige, T, an
d Skoog, F., Physiol.

Plant,, 15,473 f196211,
B5 medium (Gamborget al., Exp.
Ce1l Res, 50.151 (196811 and Nitsch's medium (Nitsch, J, P.,
Amer, J, Bot,, 38°566 f19
51+1 as well as modified media thereof can be used. Among them, Murashige-5koog medium (hereinafter referred to as
MS medium) is preferred. Furthermore, known plant growth regulators can be added as needed to further promote callus induction. As a plant growth regulator, 2,4-dichlorophenoxyacetic acid (2,4-D
), indole acetic acid (IAA), indole butyric acid (IB
A), auxins such as naphthalene acetic acid (NNA), and cytokinins such as kinetin and benzyladenine are used. The content of auxins and cytokinins in the medium is usually 0. O1-10ppm and 0
．． It is 1 to 10 ppm. Tissue pieces are usually kept at 15-35℃
The tissue pieces are cultured in a dark place, and a callus is formed from the tissue piece in about 10 to 30 days.

By transplanting callus derived from tissue fragments of a staghorn plant and culturing it in an appropriate nutrient medium, the amount of callus to be subjected to the next subculture can be increased. As the nutrient medium, known media used for plant tissue culture can be used, such as the above-mentioned MS medium, B5 medium, N1tsch medium, and the like. In particular, auxin or auxin and cytokinin were added to the MS medium at 0.00% each.
A medium containing 1 to 10 ppm and 0.1 to 10 ppm is preferably used. In this case, the culture temperature is preferably 15 to 35°C, more preferably around 25°C, and the culture is carried out in the light. The illuminance during culturing is 500 to 10,000 lux.
Preferably it is 3000-6000 lux.

Next, the callus induced as described above is subcultured while selecting red pigment-producing cells. This subculture can be carried out in a medium containing a plant growth regulator under the same conditions as in the above-mentioned nutrient medium. 2.4-D as a plant growth regulator at 0.1 to 5 ppa+
, more preferably about o,t ppm, and benzyladenine at 0.001 to 5 ppm.

More preferably, a medium containing about 1 ppm is particularly preferred. In order to induce red pigment-producing cells particularly effectively, subculturing should be continued every 3 to 4 weeks in a medium containing approximately 1 ppm of 2.4-D, and once the growth has stabilized, Preferably, the cells are transplanted into a medium containing 0.1 ppm of 2.4-D and about 1 ppm of benzyladenine, and cultured at a temperature within the above range and subjected to light bombardment.

Furthermore, as the wavelength of the light source, blue light and pure color light are preferably used because they promote pigment formation.

In addition, instead of auxins and cytokinins,
Alternatively, similar effects can be obtained by using abscisic acid together with these. The content of abscisic acid in the medium is
Preferably 0.001-10.0 ppm, more preferably 0.01-1. It is oppm.

Furthermore, the following components can be further added to the basic medium for the purpose of promoting callus proliferation and increasing productivity of red pigment.

Tryptophan and/or phenylalanine are each 104 to 10-'M, particularly preferably each about 10-'M.
-"M is preferably added.

It is preferred to add sucrose as a carbon source in a concentration of 1-10% by weight, particularly preferably about 4% by weight.

It is preferred to add potassium nitrate as a nitrogen source from 5mM to 50mM, particularly preferably about 40mM.

By continuing the above subculture every 3 to 4 weeks and repeatedly selecting only red pigment-producing cells at each subculture according to the small agglomeration selection method, it is possible to obtain only highly productive betacyanin-based red pigment-producing cells. can.

Although an agar medium can be used as a medium for culturing red pigment-producing cells, using a shaking culture method in a liquid medium will further promote the growth of red pigment-producing cells, making it suitable for large-scale cultivation. is also suitable and therefore preferred.

Since the betacyanin-producing cells obtained as described above accumulate red pigment inside the cells, they are collected after completion of the culture, and are crushed and dried if necessary. The betacyanin red pigment can be extracted from the dried callus by solvent extraction using a conventional method. As the extraction solvent, cold water or hot water is used, but the extraction solvent is not limited thereto. For example, an acetic acid aqueous solution or the like can also be used.

[Examples] Hereinafter, the present invention will be specifically explained with reference to Examples, but the Examples of the present invention are not limited to these.

Fruit "L Example" 2 Seeds of sagebrush were placed in a Miracloth bag according to the usual method and immersed in a 70% ethanol solution for several tens of seconds.
% sodium subzinc oxide solution for 5 to 15 minutes and washed three times with sterile water. The sterilized seeds were placed on an agar medium (agar content 0.8%) containing 3% sucrose (W/Vl) in a 2-fold diluted MS medium and cultured at 25°C in the dark. 7 days after germination. The fully expanded cotyledons of the eyes were removed and placed on an agar medium supplemented with MS medium containing 2.4-D O,lppm and benzyladenine]ppa+, and incubated for 25 minutes.
A callus was formed from the tissue piece by culturing it in the light at ℃ for 2 weeks. However, the illuminance under bright conditions is approximately 3.
It was set to 500 lux.

Next, the calli were selected and subcultured every 3 weeks under light (approximately 3,500 lux) in the same medium or the same MS medium supplemented with only 2.4-Do, lppm to proliferate. . A part of the callus cultured in this way was collected in a test tube, and -
After freezing at 30°C, the mixture was thawed at room temperature, a small amount of 3.77mM acetic acid was added, and the mixture was frozen again at -30°C. After further melting at room temperature, 3.77 mM acetic acid was added in a volume 9 times the fresh weight of the callus to extract the red pigment.

The obtained extract was filtered through a filter paper, and the absorption spectrum of the dye was measured using a dual wavelength spectrophotometer (manufactured by Hitachi, Model 200-10) for quantitative determination. The measurement result of the absorption spectrum was consistent with that of the betacyanin-based red pigment extracted from the germination of the bulrush seeds shown in Figure 1, so it was confirmed that the red pigment contained in the obtained callus was a betacyanin-based pigment. In addition, the content of betacyanin is Piat
(Piattelli, M.
et al.

Phytochemistry, 8nia 31-736
(19691). That is, 3
．． The absorbance at 537 nm in 33 mM acetic acid was measured, and the molar extinction coefficient of betacyanin was 5.66 xlO'
(The betacyanin content contained per ton of callus Ig (fresh weight) was calculated using M-'cm-'l. As a result, 1.4 x 10-' fM/Lf, wl of betacyanin red pigment was contained. I found out that

Red pigment-producing callus was obtained in the same manner as in Example 1, except that 2.4-D, α-naphthaleneacetic acid, or indole-3-acetic acid was added as auxin in amounts shown in Table 1 below. The betacyanin content of the obtained callus was determined in Example 1.
It was measured in the same manner. The results are shown in Table 1. In addition,
Callus cultured on MS medium containing no plant growth regulator served as a control.

From Table 1, it can be seen that both 2.4-D, NAA and IAA are effective in producing betacynine, but 2.4-D
0. It was found that the medium supplemented with lppm was the most effective for both callus growth and betacyanin production.

Red pigment-producing castules were obtained in the same manner as in Example 1, except that tryptophan and/or phenylalanine were further added to the subculture medium used in Example 1 in amounts shown in Table 2 below. The betacyanin content of the obtained callus was measured in the same manner as in Example 1. The results are shown in Table 2. Callus cultured in MS medium containing no plant growth regulator was used as a control.

Table 2 shows that both tryptophan and phenylalanine are effective in producing betacyanin. especially,
It was found that a medium supplemented with lo-3M tryptophan can efficiently promote callus growth and betacyanin production.

1 Arashiri A Same as Example 1 except that abscisic acid was added in the amount shown in Table 3 below instead of 2.4-D and 6-benzyladenine to the subculture medium used in Example 1. A red pigment-producing callus was obtained. The betacyanin content of the obtained callus was measured in the same manner as in Example 1. The results are shown in Table 3. Note that callus cultured in MS medium containing no plant growth regulator was used as a control.

Table 3 shows that abscisic acid is effective in producing betacyanin. In particular, abscisic acid of 0.1 to 1
．． When Oppm was added, it was most effective for both callus growth and betacyanin production.

Red pigment-producing callus was obtained in the same manner as in Example 1, except that sucrose was used as a carbon source in the subculture medium used in Example 1 in the amount shown in Table 4 below. The betacyanin content of the obtained callus was measured in the same manner as in Example 1. The results are shown in Table 4.

Table 4 shows that sucrose is effective in producing betacyanin. In particular, when 3% or 4% sucrose was added, it was most effective for both callus growth and betacyanin production.

Red pigment-producing callus was obtained in the same manner as in Example 1, except that potassium silicate was used as a nitrogen source in the subculture medium used in Example 1 in the amount shown in Table 5 below. However, no nitrogen source other than potassium nitrate was added. The betacyanin content of the obtained callus was measured in the same manner as in Example 1. The results are shown in Table 5.

From Table 5, within the added concentration range, the concentration that was most effective in producing betacyanin was 40 mM.

! Red pigment-producing callus was obtained in the same manner as in Example 1, except that red light, green light, blue light, and white light with different wavelengths were used as light sources as the light bombardment conditions during 1U crude culture. . The production of betacyanin was calculated from the following formula, and the results are shown in Table 6.

Betacyanin synthesis: From Table 6, it was found that using blue light as a light source was most effective for callus growth and betacyanin production.

1 Arashi ■1 The red pigment-producing callus obtained in Example 1 was transplanted into a liquid medium from which agar was removed from the same medium, and cultured under rotational shaking under light irradiation (80 r, p, m, l for 3 weeks). A red pigment-producing callus was obtained by culturing under the same conditions as in Example 1 except for the above.The red pigment was extracted and quantified in the same manner as in Example 1.

As a result, the amount of betacyanin red pigment produced was 1.25
x 10-' fM/g, f, wl.

[Brief explanation of the drawing]

The drawings are absorption spectra of red pigments extracted from callus and red pigments obtained from sprouts by the method of the present invention.

Claims (1)

[Scope of Claims] (1) A step of inducing callus by tissue culturing barley plants, and subculturing the callus in a light place in a medium containing a plant growth regulator while selecting red pigment-producing cells. A method for producing a betacyanin-based red pigment, comprising a step of recovering the red pigment from the red pigment-producing cells obtained by the subculture. (2) The method according to claim 1, wherein the medium used for the subculture contains auxins and cytokinins as the plant growth regulators. (3) The above-mentioned subculturing is performed as a plant growth regulator of about 0.
After subculturing every 3 to 4 weeks in a medium containing 1 ppm 2,4-dichlorophenoxyacetic acid, the medium contains about 0.1 ppm 2,4-dichlorophenoxyacetic acid and about 1 ppm benzyladenine at the stage of stable growth. 2. The method according to claim 1, which is carried out by subculturing in a medium. (4) The method for producing a red betacyanine pigment according to any one of claims 1 to 3, wherein the medium used for the subculture contains tryptophan and/or phenylalanine. (5) The method according to any one of claims 1 to 4, wherein the subculture is carried out in a medium containing abscisic acid as a plant growth regulator. (6) The method according to any one of claims 1 to 5, wherein the subculture is carried out in a medium containing about 4% by weight of sucrose as a carbon source. (7) The method according to any one of claims 1 to 6, wherein the illumination intensity during the subculture is 1,500 to 10,000 lux. (8) The method according to any one of claims 1 to 7, wherein the subculture is performed under blue light.