JPS62253386A - Method for increasing yield of red pigment of safflower - Google Patents

Abstract

PURPOSE:To increase the yield of red pigment of safflower, by culturing the cell of safflower in a liquid medium containing a color-development promoting substance. CONSTITUTION:A cell (or cell group) of safflower is cultured in a liquid medium containing a color-development promoting substance (preferably cellulose, etc.) to effect the increase of the yield of red pigment of safflower. A preferable concrete example of the titled method is the cultivation of a safflower cell in a solid medium followed by the spin culture of the resultant callus in a liquid medium in the presence of a color-development promoting substance. The medium is free from NH4<+> and contains naphtaleneacetic acid at a concentration corresponding to <=1/10 of the concentration in the above solid medium.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for increasing the yield of red pigment from safflower.

More specifically, the present invention relates to a method for increasing the yield of red pigment by culturing safflower callus in a liquid medium containing a color-promoting substance.

In general, safflower is a plant of the Chrysanthemum family that is commonly cultivated in the Akita region.The flowers that are harvested contain beautiful red pigments (carthamine) and yellow pigments, as well as other medicinal ingredients in traditional Chinese medicine. The flowers are prized as tea.

In addition, the red pigment extracted from the flower is sold as red dye and as a natural lipstick.

Since the red pigment contained in safflower is extremely useful as a natural pigment, the present inventors previously conducted intensive research on safflower callus culture in order to mass-produce the red pigment of safflower. By lowering the concentration of at least one component in a two-stage culture of solid culture and liquid culture, they succeeded in producing red-colored callus.

However, even with this method, the amount of red pigment produced is small, and mass production has not yet been possible.

The present inventors conducted research to find a method for mass-producing the red pigment of safflower, and found that polymeric substances, enzymes, ion exchange resins, polysaccharides, grains, starches, etc. have a color-promoting effect. It is.

The present invention is a method for increasing the yield of safflower red pigment, which is characterized by culturing safflower cells or cell groups in a liquid medium containing a color development promoting substance.

The color development promoting substances used in the present invention are cellulose, nylon fiber, pulp, chitin, chitosan, rice, rice flour, wheat, wheat flour, potato starch, cotton, glass, yeast extract, cellulase, Dowex 2-X8, Dowex 1 -1 selected from x2, alumina, pectin
This is a method for increasing the yield of safflower red pigment by adding the above-mentioned color development promoting substance to a liquid medium.

Until now, it has not been known that the yield of red pigment in safflower can be increased by the presence of a color-promoting substance.

In the present invention, safflower cells or cell groups are cultured in a solid medium, and the resulting callus is cultured in a liquid medium containing a component with a reduced concentration of at least one of the components of the solid medium. preferable.

Safflower cells or cell groups used in the present invention are collected from growing points, but can also be collected from flower buds.

The term "flower bud" as used herein refers to a young tissue that is undifferentiated or is about to differentiate, and is formed in the leaf sap of a leaf that is lower than the bud at the top after the safflower plant has grown and has a bud at the top. This has the ability to turn into a flower by itself when the top bud is gone.

In the present invention, safflower cells or cell groups are first cultured in a solid medium, and the resulting callus is cultured in a liquid medium containing a color-promoting substance. It is preferable to use a component composed of a component with a reduced concentration of one component.

Furthermore, in the present invention, it is also possible to carry out solid culture, then liquid culture, and then repeat liquid culture in order to further increase the size of the callus. In this case, it is also possible to use a liquid medium in which the concentration of at least one of the solid medium components is further reduced. Alternatively, a color development promoting substance may be contained only in the final liquid medium.

The culture medium used in the present invention is the usual one used for plant tissue culture such as Murashige-Skoog, White, Gamborg, etc. Among the ingredients used here, the inorganic ingredients include nitrogen, phosphorus, potassium, calcium, magnesium,
Sulfur, iron, manganese, zinc, boron, copper, molybdenum, chlorine, sodium, iodine, cobalt, etc., specifically potassium nitrate, sodium nitrate, calcium nitrate, monopotassium phosphate, disodium phosphate, potassium chloride, Examples include calcium chloride, magnesium sulfate, sodium sulfate, ferrous sulfate, ferric sulfate, manganese sulfate, zinc sulfate, boric acid, copper sulfate, sodium molybdate, molybdenum dioxide, potassium iodide, and cobalt chloride.

Examples of carbon sources include hydrocarbons such as sucrose, derivatives thereof, organic acids such as fatty acids, and primary alcohols such as ethanol.

Plant hormones include indole acetic acid (IAA).

Naphthaleneacetic acid (NAA), P-chlorophenoxyisobutyric acid, 2,4-dichlorophenoxyacetic acid (2,4-D
), and cytokinins such as kinetin, zeatin, dihydrozeatin, and benzyladenine.

Vitamins include biotin, thiamin (vitamin B1)
, pyridoxine (vitamin B6), pantothenic acid, ascorbic acid (vitamin C), inositol, nicotinic acid, and the like.

Examples of amino acids include glycine, alanine, glutamine, and cysteine.

In the component composition of the liquid medium in the present invention, it is necessary to reduce the concentration of at least one component among the medium components of the solid medium.

In a liquid medium, at least one component selected from inorganic components, plant hormones, vitamins, and amino acids is preferable as a component that lowers the concentration more than in a solid medium.

Among these, inorganic components such as ammonium ions, nitrate ions, phosphate ions, potassium ions, calcium ions, iron ions, manganese ions, cobalt ions, iodine ions, sodium ions, and chloride ions are particularly important as components that lower the concentration. , cytokinins,
At least one selected from vitamins and amino acids
More than one type of component is suitable.

Among these, in the case of ammonium ion, good results can be obtained if the liquid medium does not contain it at all.

Naphthalene acetic acid is required for both solid and liquid media, but when solid media contains about 10-5M, liquid media contains 10-6~to-'M.
It is necessary to reduce the concentration to a certain degree.

In addition, as a solid medium, a liquid medium containing various components can be used.
．． It is sufficient to add about 8% agar.

Color development promoting substances used in the present invention include polymeric substances, enzymes, inorganic substances, ion exchange resins, and polysaccharides.

There are grains, starches, etc., specifically cellulose, nylon fiber, pulp, chitin, and chitosan.

Examples include rice, rice flour, wheat, wheat starch, potato starch, glass, yeast extract, cellulase, Dowex 2-x8, Dowex 1-x2, alumina, and pectin.

If callus is cultured in a liquid medium containing these color development promoting substances, it is possible to elute the red pigment into the culture solution or cause the red pigment to develop within the callus. The color development promoting substance contains 0.1 to 10%, preferably 1% in the liquid medium.
The presence of about 5% is sufficient.

Preferred examples of the culture method in the present invention include the following methods. That is, safflower cells or cell groups are placed on a solid medium and cultured at 10 to 35° C. for about 7 to 30 days to form a callus.

When the callus thus obtained is subcultured, the production rate gradually increases and stable callus is obtained. This callus is grown in a liquid medium containing a component with a reduced concentration of at least one of the components of the solid medium, in particular a component that eliminates ammonium ions and a concentration of naphthalene acetic acid of 171 O or less, and also contains a color development promoting substance. and culture by swirling.

In the culture of the present invention, light is not necessarily required, and the culture temperature is preferably 10 to 35°C, particularly around 25°C.

When cultured in liquid, the culture solution turns red and the callus turns red, indicating that a red pigment is produced.

The red pigment can be extracted from callus or culture fluid using the same method as conventionally used for extracting safflower pigment.

Furthermore, when chitosan is used as a color development promoting substance, the red pigment is eluted into the medium, but the red pigment may bind to chitosan and not be easily separated.

In this case, the chitosan to which the red pigment is bound can be ground as it is in a mill and used as a pigment powder.

Next, an example of the present invention will be shown.
The compositions of medium A and medium O as modified Skoog medium are shown in Table 1 below.

Table 1 In the case of solid medium: Agar 9.5 g IQ Example 1 On the 60th day after sowing safflower, when the flower buds had slightly swelled, a large number of cell groups were separated aseptically.

Separately, a solid medium prepared by adding agar to the A medium in Table 1 was prepared, and safflower flower bud cells were dispersed therein.
After culturing at 25°C for 20 days, a large number of calli were obtained.

Next, 30+nQ of the Otsu medium shown in Table 1 was placed in a 100 mΩ Erlenmeyer flask, and each amount of the color development promoting substance shown in Table 2 was added thereto, followed by sterilization at 120°C for 10 minutes.

After cooling, 1.5 g of wet callus was added and cultured at 25° C. in the dark for 4 days.

If the dye was adsorbed to the additive after culturing, the entire culture was filtered; if the dye was not adsorbed, a small amount of cellulose powder was added to adsorb the dye, then filtered, and the dye on the filter paper was eluted with pyridine. . The formation of the red pigment (calcamine) can be confirmed by thin layer chromatography (cellulose powder coating,
n-butanol:pyridine:water=6:4:3(
Comparison of Rf with standard calsamine (developed using v/v) as a solvent, and ultraviolet absorption spectrum.
The content was estimated from the absorbance at 520 nm. The fresh weight of callus growth was determined by subtracting the weight of the additive from the weight of the mixture of cells and additive after filtration of the culture.

The results are shown in Table 2.

Table 2 Fist Each Log/Q addition m-0, 1gIQ addition Example 2 In the same manner as in Example 1, flower buds were separated, callus was produced, and 2g of each color development promoting substance shown in Table 3 was added in the same liquid culture solution. /1
00IIQ was added, and the cells were cultured for 3 days at 25° C. under room light, and the callus was observed to turn red and the culture solution turned red.

The results are shown in Table 3.

Table 3 - No blushing at all ±: Slightly reddened +: Reddening is observed ++: Significantly reddened +++: Significantly reddening

Claims (3)

[Claims] (1) A method for increasing the yield of red pigment from safflower, which comprises culturing safflower cells or cell groups in a liquid medium containing a color-promoting substance. (2) The method for increasing the yield of red pigment of safflower according to claim 1, wherein the color development promoting substance is a substance insoluble in water. (3) The color development promoting substance is cellulose, nylon fiber, pulp, chitin, chitosan, rice, rice flour, wheat, wheat flour,
Potato starch, cotton, glass, yeast extract, cellulase, Dowex 2-X8, Dowex 1
-X2, alumina, and pectin, the method for increasing the yield of safflower red pigment according to claim 1.