JPH07155190A - Production of yellow pigment - Google Patents

Abstract

PURPOSE:To obtain a large amount of a yellow pigment, having high safety and useful for foods, cosmetics, etc., in a short period without being influenced by weather conditions by carrying out the tissue culture of a plant which belongs to the genus Lactuca and collecting the yellow pigment from the resultant culture. CONSTITUTION:This method for producing the objective yellow pigment is to sterilize a seed of a plant which belongs to the genus Lactuca such as Lactuca angustana or Lactuca sativa, subsequently sow the sterilized seed onto a culture medium for germination, aseptically germinate the seed at 25 deg.C in the dark, collect an aseptic young plant, cut off a hypocotylous part, bed the cut off hypocotylous part on a culture medium for inducing a callus, culture the cut off hypocotylous part in the presence of a phytohormone at 25 deg.C in the dark, induce the yellowish callus, then subculture the callus, subsequently extract the resultant culture with ethyl acetate, concentrate the resultant extract solution to dryness, treat the residue by silica gel column chromatography and purify the residue.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a natural pigment, more specifically, a method for producing a natural pigment by culturing plant tissue.

[0002]

2. Description of the Related Art The pigments used for coloring products such as foods and cosmetics that come into direct contact with the human body are limited in the types that can be used from the viewpoint of their safety, and recently, the use of synthetic coloring agents is limited. There is a tendency. Therefore, there is an urgent need to develop natural pigments that are considered safe for the human body. Since such natural pigments are obtained by extracting from animals and plants,
The amount depends on the animal or plant tissue used as the raw material. Therefore, it has the drawback that it is susceptible to climatic conditions and social influences and production is not constant. In general, pigments are present only in trace amounts in animals and plants, and thus a large amount of raw materials and large-scale equipment are required to obtain them.

On the other hand, in recent years, attempts have been made to efficiently obtain natural pigments by using a plant tissue culture technique. For example, for the red dye, Japanese Patent Laid-Open No. 50-2
4494, JP-A 61-166396 and the like, blue dyes in JP-A 61-2608787 and JP-A 63-42689, and yellow dyes in JP-A-62-257384. Is mentioned.

[0004]

However, the pigment-producing plants conventionally used are not the plants generally ingested by the human body, and although the pigment produced by the plant is a natural pigment, it is safe from the viewpoint of safety. It's still hard to say. Therefore, an object of the present invention is to provide a method for efficiently obtaining a yellow natural pigment by tissue culture of a plant widely used for food.

[0005]

[Means for Solving the Problems] In order to achieve the above object, the present inventors have conducted diligent research and as a result, adopted plants belonging to the genus Lactuca which is an edible plant of the Asteraceae family Upon culturing, it was found that a large amount of yellow pigment was produced even though almost no yellow was observed in the raw material plant itself, and the present invention was completed.

That is, the present invention relates to a method for producing a yellow pigment, which comprises culturing a plant belonging to the genus Lactuca in tissue culture and collecting the yellow pigment from the obtained culture.

Examples of plants belonging to the genus Lactuca used in the production method of the present invention include Lactuca angustana and Lactuca sativa.
Those selected from are listed. Specifically, Chisha (L
actuca sativa L. ), Kakichisha (L
actuca angustana hort. ), Tamachicha (Lactuca sativa var.c)
apitata L. ), Tachicha (Lactuca)
sativa var. longifolia La
m. ), Chilean Mentischa (Lactuca sativ)
a var. crispa L. ) Are mentioned, and of these, Chile mmentia is particularly preferable.

In the present invention, callus derived from such a plant tissue belonging to the genus Lactuca is cultured in a medium,
It is preferred to collect the yellow pigment from the culture. Therefore, first, callus is induced from a plant tissue belonging to the genus Lactuca.

Plant tissues used for callus induction include leaves, stems,
Any of a part of a plant such as a root and a cotyledon, a hypocotyl, a root of an aseptically germinated seedling may be used. Further, the medium used is not particularly limited, and may be a medium usually used for plant tissue culture. For example Mur
Ashige-Skoog's medium, Linsmaier
-Skoog's medium, Gamborg's medium, Whit
e medium, Tuleeke medium, Nitsch &
Nitsch medium and modified mediums thereof can be used. A plant hormone is added, if necessary, to further promote callus induction. Examples of plant hormones include 2,4-dichlorophenoxyacetic acid (2,4
-D), α-naphthylacetic acid (NAA), indoleacetic acid (IAA), indolebutyric acid (IBA) and other auxins; furfurylaminopurine (kinetin), benzyladenine (BA), isopentenylaminopurine (2i)
P) and the like cytokinins; gibberellins (GA ₃ ) and the like. Among them, good results are obtained when the auxins are used alone or the auxins and cytokinins are combined. Plant hormone levels auxins necessary callus induction ^{1 × 10 -4 M~1 × 10 -7} M, cytokinin is preferably ^{5 × 10 -5 M~5 × 10 -7} M.

In addition to the above basic medium and plant hormones, sugar is added to the callus induction medium as a carbon source. Examples of sugars include glucose, fructose, mannose,
Xylose, arabinose, rhamnose, fucose, sucrose, etc. are mentioned, and 1 type (s) or 2 or more types can be used among these. The concentration is preferably 1 to 6% by weight. The induced callus can be continuously cultured in the above callus induction medium while maintaining the same morphology.

In order to produce a yellow pigment, callus is cultured in a solid medium such as agar medium or a liquid medium.
It is preferable to culture in a liquid medium. The basal medium is the same as the callus induction medium, that is, Murashige-
Skoog's medium, Linsmaier-Skoog's medium, Gamburg's medium, White's medium, Tu
Leeke's medium, Nitsch &Nitsch's medium and modified mediums thereof can be used. A yellow pigment can be efficiently obtained by adding a plant hormone to the above medium. Examples of plant hormones include 2,4-
Examples include auxins such as D, NAA, IAA and IBA; cytokinins such as kinetin, BA, 2iP; and gibberellins such as GA ₃ . Among them, good results are obtained when the auxins are used alone or when the auxins are used in combination with the cytokinins. The concentration varies slightly depending on the plant hormone used, but basically auxins are 1 × 10 ⁻⁴ M to 1 × 10 ⁻⁷ M, cytokinins are 5 × 10 ⁻⁵ M to ⁵ × 10 ⁻⁷ M, and gibberellins are 5 x 10
^-5 M to 1 x 10 ^-7 M is preferable. In the medium for pigment production, sugar is added as a carbon source in addition to the above basic medium and plant hormones. Examples of the sugar include glucose, fructose, mannose, xylose, arabinose, rhamnose, fucose, sucrose and the like. The pigment production is not strongly influenced by the type of carbon source to be added, and usually one or more of these can be used. The concentration is preferably 1 to 6% by weight. The culturing method is not particularly limited, but is usually 20 to 30 ° C. and 10 in the dark.
It is preferable to perform shaking culture for 30 days.

The cells thus cultivated produce a yellow pigment. The produced yellow pigment is also produced in the medium from the inside of the cell. Therefore, in order to collect the yellow pigment from the obtained culture, for example, by collecting the callus from the culture by centrifugation or filtration, a method of extracting with an organic solvent, etc., after transferring the callus to a new medium, the pigment from the medium A method of extracting, a method of extracting the whole culture with an organic solvent, and the like are possible. As the organic solvent for extracting the yellow pigment, a usual organic solvent such as ether, hexane, ethyl acetate or the like can be used. When culturing is performed in a liquid medium, the cells can be filtered off, and then the dye can be directly extracted from the culture medium using an organic solvent. Therefore, the cells separated by filtration from the liquid medium can be continuously subjected to culture, and continuous pigment production is possible.

[0013]

[Effects of the Invention] Thus, even if cells containing a yellow pigment are grown and subcultured for a long period of time, the cell morphology and growth rate,
The pigment content does not change. Therefore, a large amount of yellow pigment can be obtained in a short time regardless of climatic conditions. Further, the pigment obtained from cells cultured in the dark can be homogeneous without being contaminated with green pigment such as chloroplast. This yellow pigment is used as a coloring agent for foods, cosmetics, etc.
For example, it can be used for juices, beverages such as wine, confections such as sheepskins, frozen desserts such as jellies, makeup cosmetics such as lipsticks and eye shadows.

[0014]

The present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

Example 1 (a) Induction of callus: Lamentuca (Lactuca)
sativa var. crispa L. ) Was sterilized with a 70% ethanol solution for 1 minute, further sterilized with a 1% sodium hypochlorite solution for 10 minutes, and then washed with sterile water. The sterilized seeds were sown on a germination medium. Li which does not contain plant hormones as a germination medium
A solid medium was prepared by adding 0.8% agar to the nsmaier-Skoog medium, and 3% sucrose was added as a carbon source. Add this medium to 0.1N KOH
The pH was adjusted to 5.7 by means of autoclave sterilization and then used. Aseptic germination was carried out at 25 ° C. in the dark, and aseptic seedlings were collected 3 days after germination, and the hypocotyl portion was cut off and placed on a callus induction medium. As a callus induction medium, a Linsmaier-Skoog medium containing 0.8% agar was used.
As a plant hormone, 10 ⁻⁶ MN as auxin
10 ⁻⁶ M BA was added as AA and cytokinin. 3% sucrose was added as a carbon source. This medium was adjusted to pH 5.7 with 0.1 N KOH and then sterilized by the above-mentioned method. The culture was performed at 25 ° C. in the dark. After culturing for about 30 days, a slightly yellowish callus was induced from the explants.

(B) Subculture of callus: In (a)
Induce the callus using the same medium as the induction medium.
Cultured under one condition and transferred to a new medium about every 30 days.
It was (C) Production culture: 10 or more subcultures in (b)
Shaken callus using a liquid medium for pigment production.
Carrot culture was performed. As a pigment production medium, Linsm
The plant hormone is auxilized in the air-Skoog medium.
10 as Shin ^-FiveMNAA, 1 as cytokinin
0^-6MBA was added. 3% shook as carbon source
Loin was added. PH of this medium with 0.1 N KOH
After adjusting to 5.7, it was sterilized by the above-mentioned method and used. Cultivation
For feeding, add 250 ml of medium to a 500 ml Erlenmeyer flask,
Freshly weighed 5g of callus, 25 ℃, dark place, 100
It was carried out at rpm for 30 days. Within a few days after starting the culture,
Sus became dark yellow and continued to grow. At this time, the medium also turns yellow
The yellow color became darker as the culturing continued.

(D) Extraction of pigment: After culturing in (c) for 30 days, the culture was filtered off and the fresh weight was about 50 g.
I got a callus. The callus was extracted with 200 ml of ethyl acetate. The same extraction operation was repeated twice, and the obtained extracts were combined and concentrated to dryness with a rotary evaporator. The amount of yellow pigment obtained was about 0.1 g.

(E) Purification of dye: 0.1 g of the yellow dye extract obtained in (d) was eluted with hexane and ethyl acetate by silica gel column chromatography,
Fractional purification was performed and 0.002 g of bright yellow-orange crystals were obtained.

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Claims (2)

[Claims] 1. A method for producing a yellow pigment, which comprises subjecting a plant belonging to the genus Lactuca to tissue culture and collecting the yellow pigment from the obtained culture. 2. A plant belonging to the genus Lactuca is Lactuca angustan.
a) and Lactuca sativa (Lactuca s)
The method for producing a yellow dye according to claim 1, which is selected from ativa).