JPH01228413A - Production of purified oil - Google Patents

Abstract

PURPOSE:To obtain a purified oil readily and in high yield without being influenced by weather conditions by cultivating a tissue of plant belonging to the genus Anthemis differentiating and introducing an adventitious bud from the culture mixture and collecting a purified oil component produced and accumulated in the adventitious bud. CONSTITUTION:A tissue of a plant belonging to the genus Anthemis is cultivated, an adventitious bud is differentiated and introduced from the culture mixture, the adventitious bud is multiplied by subculture in a medium containing the optimum plant hormone, a strain to highly produce a purified oil is selected from the prepared adventitious bud and a purified oil component is collected from the adventitious bud. In order to select the strain to highly produce a purified oil from strains obtained by multiplying the adventitious bud, a method such as macroscopic selection through shape of the adventitious bud or selection by head space gas chromatography is used.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for producing essential oil components from adventitious buds that have been cultured and differentiated from plant tissues.

[Conventional technology]

Essential oils are substances obtained from plants by squeezing, extraction, etc., and are widely used in the fields of cosmetics, fragrances, edible flavors, and the like. This essential oil is generally produced using cultivated plants as raw materials.

In recent years, plant tissue culture has become relatively easy, and this method is being considered as a method for producing plant components. And genus Belargonium, genus Geranium, genus Ruta, genus Viola,
A method for producing essential oil from adventitious buds of plants of the genus Melissa and Iris has been proposed (Japanese Unexamined Patent Application Publication No. 1983-1999).
No. 213393, JP-A-60-71699, JP-A-6
0-248188). However, there have been no reports on plants belonging to the genus Anthemis.

[Problem to be solved by the invention]

The present inventors conducted research to develop a method for producing essential oil easily and at a high yield in a completely controlled environment that is not affected by weather conditions, etc., compared to conventional plant cultivation.
The present invention has now been completed.

[Failure to solve the problem]

The present invention is a method for producing essential oil, in which tissue of a plant belonging to the genus Anthemis is cultured, adventitious buds are induced to differentiate from the culture, and essential oil components produced and accumulated in the adventitious buds are collected. These adventitious buds include those grown by subculture in a medium containing plant hormones that are optimal for proliferation, and essential oil producing strains selected from the subcultured buds, as well as high-producing strains grown in a liquid medium. Each is used after being cultured in large quantities. Furthermore, the present invention includes visual selection based on morphology from subcultured adventitious buds and headspace gas chromatography method (hereinafter referred to as M
This includes a method for obtaining an essential oil producing strain by selecting an essential oil producing strain by selection (referred to as S-GC method).

The present invention will be described in detail below.

In the present invention, tissue of a plant belonging to the genus Anthemis is cultured in an agar medium, and adventitious buds are induced to differentiate from the culture,
These adventitious buds are subcultured and propagated in the presence of an optimal plant hormone, essential oil-producing strains are selected from the obtained adventitious buds, and essential oil is produced from the adventitious buds. Furthermore, as the adventitious buds used as raw materials, those obtained by culturing the above-mentioned essential oil producing strain in a large scale in a liquid medium can be used.

The plant belonging to the genus Anthemis that can be used in the present invention is Anthemis nobilis.
is).

Anthemis tin
ctoria).

Anthemis alpensis (8nthemis arv)
ensis) + Anthemis cotula (8nthemi)
s cotula). As the plant body, any of leaves, stems, and root fragments can be used. As a medium for inducing differentiation of adventitious buds from plant tissues, those commonly used in plant tissue culture, such as Linsmeyer-Skoog (LS) medium, Murashige-Skoog medium, B5 medium, Niche & Niche medium, and White medium. etc., but LS medium is preferably used. When actually culturing, it is added to these basic media.
! I, vitamins and plant hormones are added and used.

Examples of plant hormones include indole-3-acetic acid (
IAA), indole-3-butyric acid (TBA), α-naphthaleneacetic acid (NAA), auxins such as 2,4-dichlorophenoxyacetic acid (2°4-D), kinetin (
Kn), 6-benzyladenine (6-BA), and other cytokinins, and these auxins and cytokinins are used in combination. A particularly preferred combination is auxin:cytokinin is 10-6M: 10-
'M. By culturing in this medium under light conditions at 20 to 30° C., preferably 25° C., for 2 to 3 weeks, callus formation occurs, and adventitious shoots and adventitious roots are induced. Light conditions are 200
A light irradiation of 0-3000 lux is used.

These adventitious buds are divided into small plants of 2 to 3 leaves each containing new buds and subcultured to proliferate, and the above-mentioned basic medium is used as the medium at that time. Adventitious buds with different forms (leaf shape, color, etc.) can be obtained depending on the composition of the plant hormone used. That is, IBA as a plant hormone
10-'M, BA10-'M; IBA10-
'M, BA 10-6M, NAA t
o-'M, BA 10-'M, etc., the morphology of the adventitious buds was classified into three types.

There are three types: Type 1, which has a morphology similar to that of the mother plant; Type 1, which has few leaf branches; and Type 2, where the leaves and stems are indistinguishable. According to the morphological classification, as shown in Table 1, good essential oil components were produced only in type (2). Therefore, type l
Plant hormones suitable for the proliferation of adventitious buds include, for example, IBA
: 10-'M and BA:10-'' to 10-7M are combined.

In order to select high essential oil producing strains from the strains obtained by propagating adventitious buds, the morphology and essential oil components of the adventitious buds are analyzed by MS-
GC method (Nobuhiko Onda: Bunseki, average.

12 (1987)). Conventionally, analysis of essential oil components requires complicated operations such as distillation and solvent extraction, as well as large amounts of samples, but with this MS-GC method, analysis can be performed easily and in a short time using a small amount of sample. .

This high essential oil producing strain can be cultured in large quantities even in a liquid medium. This liquid medium consists of the following composition.

(Liquid medium composition) (1) Basic medium 1 to 1/2 times the concentration of LS medium (2
) Sucrose 3% (3) Hormone IBA 10-'MBA
10-'M (4) Nitrogen source Ammonia nitrogen/Nitrate nitrogen = 1
75-1/11 Culture at 25°C for 7-10 days in the light (2000-30
00 lux).

The adventitious buds obtained in this way contain the same essential oil components as plants of the genus Anthemis, and extraction methods such as extraction using a solvent such as petroleum ether or steam distillation are commonly used. Essential oil components can be easily obtained by known methods.

(Effects of the Invention) The method of the present invention is less affected by weather conditions than conventional cultivation methods, and can produce essential oil in large amounts at high yields in a short period of time in a completely controlled environment. In addition, the essential oil high school graduate adventitious bud strain used as a raw material can be easily selected by visual selection of morphology and HS-GC method.

Example 1 (1) Induction of differentiation of adventitious buds Washed leaves of Anthemis nobilis were pre-sterilized with 70% ethanol for 1 minute, and then sterilized with 1% antiformin for 5 to 10 minutes. Next, the leaves from which the drug had been completely removed with sterile distilled water were aseptically chopped into pieces of about 5+mn square to form explants. This shredded leaf is used as a plant hormone, auxin (
10-5M to 10-6 of IBA or NAA) and 10-'M to 10 of cytokinin (B8 or kinetin)
– 3% Si=J added in combination with 6M, respectively.
The cells were placed on an LS medium adjusted to pH 5.6 containing 1% agar and cultured at 25°C under light irradiation of 2,000 to 3,000 lux to form callus, and after 2 to 3 weeks, adventitious shoots and , or adventitious roots were induced to differentiate.

(2) Subculture of adventitious buds and selection of high essential oil producing strains From these adventitious buds, small plants with new buds containing 2 to 3 leaves were selected and subcultured for 3 to 4 weeks in the same medium and culture conditions. Morphology of adventitious buds obtained in each medium (leaf shape, color, etc.) and H3-
The GC results are shown in Table 1. Regarding this result, the component N was determined from the peak height of the chromatogram obtained by H3-GC, and was expressed as a percentage of the total amount of components appearing in the chromatogram. From this, it was found that type I was a high-producing strain of essential oil. As can be seen from this result, high essential oil producing strains can be easily visually selected from the subcultured adventitious buds.

The adventitious buds obtained by this method were cultured to produce essential oil, and the culture conditions, growth rate, essential oil components, etc. are shown in Table 2.

(Margin below next summer) In addition, in Tables 1 and 2, the degree of proliferation of adventitious buds,
A sensory test for aroma and an analysis of essential oil components were conducted using the following method.

(1) Growth rate of adventitious buds (fold) Fresh type of section (or culture) at the start of culture (g) (2)
Aroma sensory test Adventitious buds were crushed between hands and the aroma was judged by olfactory sense by 10 experts. The control was the original plant. The average value was used as a sensory evaluation.

Evaluation criteria: 1. No smell of refined ingredients of the original plant 2. Slight odor 3.
Available 4・
Strong 5, Very strong 3) Analysis of essential oil components by headspace gas chromatography (Its-GC)
), the amount of the component was determined from the height of the peak in the chromatogram, and expressed as a percentage of the total amount of the component appearing in the chromatogram. Qualitative analysis was performed by comparing retention times and mass spectra with standard substances using gas chromatography and mass spectrometry.

(Measurement conditions) 1) Gas chromatography model: HP5890A gas chromatography column:
DB-WAX fused silica capillary column (diameter 0.25mm x 60m) Detector: FID Injection temperature: 250°C Oven temperature = 70°C-180°C (4°C/min
carrier gas flow 1jt(lle): 0.7
8m/m111 split ratio: 1/20 2) Headspace sampler model: HP19395A Heat retention temperature near 5°C Heat retention time: 90 minutes 3) Mass spectrometer model: HP59970C Ionization voltage: 70eV Ion source temperature: 260”C Mass filter: Hyperbolic Quadrupole Example 2 Two leaves, including new shoots, of the essential oil high-producing strain obtained in Example 1.
〜3 stocks attached, IBAIO-'M, BAIO-'
M.

was added, 3% sucrose was added, and the ratio of ammonia nitrogen and nitrate nitrogen was adjusted to 1:5 to 1:11. pH 5, 6) and 2000 to 3 at 25°C.
100r, p, m under 000 lux light irradiation.

A shaking culture was performed.

Two days after culture, new adventitious buds appeared from the stem base of the adventitious buds, and their number increased thereafter.

The culture results after 10 days are shown in Table 2 as Example 2. From Table 2, the adventitious buds obtained by liquid culture (Example 2) have a better proliferation rate than Example 1.

It is also seen that an essential oil with a composition equivalent to that of plants obtained through cultivation was obtained.

Example 3 The same high-producing strain as in Example 2 was introduced into the same medium as in Example 2. However, in this example, instead of shaking culture, aerated culture was performed using fine bubbles (about 2 glass filters).

The results after 10 days of culture are shown in Table 2 as Example 3. It can be seen that, compared to Example 2, good growth was obtained and essential oil with the same composition as the plant obtained by cultivation was obtained.

Claims (1)

[Claims] 1. An essential oil, which is characterized by culturing the tissue of a plant belonging to the genus Anthemis, inducing differentiation of adventitious buds from the culture, and collecting essential oil components produced and accumulated in the adventitious buds. Production method. 2. The method for producing essential oil according to claim 1, wherein the adventitious buds are subcultured and propagated in a medium containing a plant hormone optimal for proliferation. 3. The method for producing essential oil according to claim 1, wherein the adventitious bud is a high essential oil producing strain selected from subcultures in a medium containing plant hormones that are optimal for proliferation. 4. The method for producing essential oil according to claim 1, wherein the adventitious buds are obtained by mass culturing in a liquid medium. 5. A method for obtaining a high essential oil producing strain, which comprises selecting a high essential oil producing strain from subcultured adventitious buds by visual selection according to their morphology and selection by headspace gas chromatography.