JPH0130474B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a cultured cell belonging to the genus Euphorbia Hanakirin, and more particularly to a cultured cell belonging to the genus Euphorbia Hanakirin, which has an undifferentiated cell morphology that has a high ability to produce red pigment, is stable, is highly adaptable to the environment, and has a vigorous growth. Pigments are often used as food coloring agents to add aesthetic appeal to foods, but synthetic colorants pose various problems due to their toxicity, such as mutagenicity and carcinogenicity. . Therefore,
From the viewpoint of safety, it is desired to use pigments derived from natural products as coloring agents for foods and the like. However, natural cultivation is subject to constraints from the natural environment such as season, climate, temperature, latitude, etc.
It is not possible to maintain a stable supply by collecting from natural plants. In addition, large-scale cultivation using arable land naturally competes with food production, so there is a limit to its supply, and there is also a limit to its productivity, making it extremely expensive. However, in recent years, research on plant cell culture has been progressing as a method for producing plant components. Plant cell culture grows at a much faster rate than natural plants, which grow on a yearly or monthly basis, so it is possible to produce the desired ingredients in a short period of time, and unlike natural cultivation, it is less susceptible to the effects of weather etc. It has the advantage of being able to be produced in a planned manner on an industrial scale without the need for much labor for collection. From this point of view, the present inventors have carried out intensive research in order to industrially advantageously produce natural pigments by culturing plant cells, and as a result, as mentioned above, the leaves of Euphorbia sp. We have succeeded in creating cultured cells belonging to the Euphorbia genus Hanakirin, which have a high ability to produce red natural pigments obtained by culturing in a medium containing euphorbia, are stable, highly adaptable to the environment, and have an undifferentiated cell morphology that grows vigorously. This led to the present invention. The conventional variety Euphorbia Hanakirin has no ability to produce useful natural red pigments, and therefore tissue culture of this variety could not be used to produce red natural pigments. However, the present inventors have prepared cell tissues of Hanakirin of the genus Euphorbia, preferably using 2,4-dichlorophenoxyacetic acid (2,4-D).
By culturing in a medium containing 10 ^-6 to 10 ^-7 M and sucrose at a concentration of 4 to 8%, undifferentiated cells with red natural pigment production ability were induced, and then red natural pigment productivity was used as an indicator. , undifferentiated cells with excellent red natural pigment production ability and stable production ability, preferably 2,4-D10 ^-6 to 10 ^-7 M and sucrose 4 to 8
As a result of selecting and fixing its characteristics on a medium containing a concentration of We created cultured cells belonging to the Euphorbia genus Hanakirin with fixed characteristics. The cultured cells belonging to the Euphorbia plant Hanakirin according to the present invention were created, for example, as follows. First, the leaves of the commercially available garden variety Hanakirin are thoroughly washed with deionized water, and then immersed in 70% ethyl alcohol for 5 to 10 minutes and then in a 10% bleaching powder solution for 5 to 10 minutes to remove any bacteria that may have adhered to the surface. After sterilization, remove residual disinfectant by washing with sterile distilled water. Next, the sterilized leaves are cut into small pieces with a sterile scalpel to an appropriate size, and the pieces are placed on a synthetic medium containing 2,4-dichlorophenoxyacetic acid (2,4-D). An example of a synthetic medium is 2,4-D5× inorganic synthetic culture solution with the composition shown in Table 1 below.
10 ^-7 M and organic matter and agar with the composition shown in Table 2.
A medium solidified by adding 0.8% w/v can be preferably used.

【table】

【table】

[Table] After placing on the bed, store in a bright place under constant temperature conditions of 20 to 30℃, preferably around 25℃, preferably 100 Lux or more.
More preferably, the culture is carried out under light irradiation of 3,000 to 100,000 lux. After one week of such culturing, cultured cells that produce the red pigment are formed from the leaves of Hanakirin. The conventional cultivar A, which was induced with a medium containing the commonly used naphthalene acetic acid (NAA) as an auxin agent instead of 2,4-D, has no ability to produce the useful natural pigment chrysanthemine. The medium used for inducing or culturing the cultured cells is based on various known inorganic synthetic agar media, to which trace organic substances such as vitamins, carbon sources, plant hormones, and various natural extracts are added. use Representative examples of the inorganic synthetic agar medium are shown in Table 1.
In addition to the synthetic media shown in , White's medium, Hildebrand's medium, Linsmeyer-Skoog's medium, etc. can be mentioned. In addition, these media with improved compositions can also be used. Examples of trace organic substances such as vitamins include vitamins such as thiamine hydrochloride, pyridoxine hydrochloride, and nicotinic acid; amino acids such as glycine and asparagine; and hexahydric alcohols such as inosite and sorbitol. It may show good growth even without being added to the medium. As the carbon source, it is particularly preferable to use sucrose because it has high pigment productivity. The concentration used is 2-10% w/
v, preferably 4 to 8% w/v. If this concentration is too high, growth will be slow, and if it is too low, pigment production will be suppressed, which is preferable. As mentioned above, as plant hormones, 2,4
Although the use of -D is essential, this does not preclude the concomitant use of other plant hormones other than auxin-acting substances, such as cytokinins such as chimetin and gibberellins such as gibberellin _A3 . 2,
The concentration of 4-D used is between 10 ^-4 and ^{10 -7} M, preferably
The concentration is 10 ^-6 to ^{10 -7} M, and if this concentration is too high, the cells will not grow, which is undesirable, and if it is too low, the cells will turn green or not grow, which is undesirable. Examples of the various natural extracts include casein hydrolyzate (0-2% w/v), coconut milk (0-25% w/v), yeast extract (0-2%
w/v), malt extract (0 to 2% w/v), etc. can be used alone or in any combination. In the present invention, as described above, cell culture is carried out by placing a culture vessel containing cells under light irradiation of 100 lux or more, preferably 3,000 to 100,000 lux. As a light source, sunlight, a fluorescent lamp, an incandescent lamp, a mercury lamp, etc. can be used. If no light irradiation is performed, no dye will be produced. Among the cultured cells having the ability to produce red natural pigment induced in this way, undifferentiated cells with excellent red natural pigment production ability and stable production ability are selected using red natural pigment productivity as an index. To obtain a new variety B with fixed characteristics, the following steps (a) to (d) can be carried out, for example. Step (a): In this step, the cultured Hanakirin cell mass formed above is loosened, dispersed in the above-mentioned medium, and grown preferably by a suspension culture method. Suspension culture is carried out at a temperature of 20 to 30°C, preferably around 25°C, in the light, preferably under light irradiation of 3000 lux or more for about 3 to 14 days. If the culture is continued for a long period of time, exceeding 14 days, it will be in the latter half of the stationary phase, and conversely, if it is less than 3 days, it will still be in the lag phase, so normal growth in the following steps cannot be expected. Step (b): In this step, the suspension culture obtained in step (a) above is passed through two filters having two different mesh sizes, preferably a double filter. Cultured cell clusters of various sizes are selectively collected to uniformize the size of the cultured cell clusters within a predetermined range. The mesh size of the filter can be selected arbitrarily, but in order to prevent the contamination of broken cell debris and giant cell clusters, the mesh size of the smaller filter should be 50 to 100μ, preferably 100μ, and the mesh size of the larger filter should be 50 to 100μ, preferably 100μ. It is desirable that the mesh size is 150-200μ, preferably 150μ. The material of the filter is not particularly limited as long as it does not affect cells, but stainless steel and nylon are preferable, for example. Step (c): In this step, the cultured cells obtained in step (b) above with a size within a predetermined range are dispersed in a dissolved agar medium, poured into a flat container such as a Petri dish, and dispersed. The cultured cells are grown to form colonies. In order to grow cultured cells, the cell concentration when dispersing cultured cells on agar medium must be 10 ⁴
A value of ~10 ⁶ cells/ml is desirable; if the concentration is higher than this, the colonies that form will be too close together;
On the other hand, if it is too low, it will be difficult to grow, which is not preferable. The agar medium used in this step is preferably the same medium as the suspension culture medium described above, or an appropriately modified medium to which, for example, 0.6 to 0.8% agar is added. Cultivation is carried out at a temperature of 20 to 30°C, preferably around 25°C, in the light, preferably under light irradiation of 3000 lux or more for about 14 to 28 days. If the culture period exceeds 28 days, the stationary phase will be exceeded, and conversely,
If it is less than 14 days, it is still in the lag phase or early logarithmic growth phase, and the colony size is too small, which is not preferable. Step (d): In this step, a cell mass with a high content of red pigment chrysanthemin is selected from the proliferated cell mass. Desired cell clusters may be selected by visually determining the red pigment concentration and selecting one or more cells that are considered to have the highest pigment content; Taking advantage of this fact, it is also possible to divide each cell mass in half, extract the pigment contained in one half, measure the pigment content, and select the other half with the highest pigment content. .
It goes without saying that one or more cell clusters with high pigment content may be selected in addition to the cell cluster with the highest content. For extraction and quantification of the above pigments, use 0.01 to 5% hydrochloric acid (or formic acid, acetic acid, etc.)
Each cell mass is immersed in a certain amount of methanol (or ethanol, water, etc.) containing the following at a temperature of -5 to 10%, and after 1 to 2 hours, a certain amount of the extract is quantified colorimetrically, or A method may be used in which a fixed amount of the extract is quantified using high performance liquid chromatography or a thin layer chromatodensitometer using an internal standard method or an external standard method. The cell mass selected in this manner can be further subjected to selection steps (a), (b), (c), and (d) as many times as desired to obtain a stable cell line with a high pigment content, if necessary. When producing the red pigment chrysanthemine by culturing the selected cell mass, the produced red pigment can be separated and collected by a known method, such as a solvent extraction method. An example will be explained below. First, cells are cultured in a solvent containing an acid such as hydrochloric acid, acetic acid, or formic acid at a concentration of about 0.01 to 5% by weight, preferably water or an alcoholic solvent such as methanol or ethanol, and the culture is preferably lyophilized according to a conventional method. The cells are immersed at a temperature of -5 to 10°C and the produced red pigment is extracted. Next, after removing the solid content from the obtained extract through operations such as filtration, it is concentrated under reduced pressure at a temperature of 40°C or lower (too high a temperature is not preferable because the red pigment easily decomposes),
Transfer this concentrated solution to a separating funnel, add ether (hexane, heptane, petroleum ether, chloroform, methylene dioxide, ethyl acetate, etc. can also be used in place of ether), shake it, and remove oil-soluble impurities with ether. Dissolve and separate and remove. After repeating this ether washing operation several times, the desired red pigment can be obtained by drying the pigment extract under reduced pressure. It can be purified by e.g. It is not possible to directly prove whether or not the genetic structure of the plant has been changed by the above-mentioned generation means, that is, whether or not the genetic structure is different between the conventional variety A and the cultured cells of the present invention. However, even if the cultured cells of the present invention are grown in a medium containing NAA, the ability to produce the natural red pigment chrysanthemine does not disappear, and even if the conventional variety A is grown in a medium containing 2,4-D, the ability to produce the pigment does not disappear. Considering these two points, it can be concluded that these two varieties are not simply mutant strains but have different genetic compositions. Since the cultured cells of the present invention are plants that proliferate in the form of undifferentiated cells, the cultivation method is different from that of plants that proliferate in the form of differentiated cells. That is, for example, add 0.1 ppm of 2,4-D and an organic substance with the composition shown in Table 2 to an inorganic synthetic culture solution with the composition shown in Table 1, autoclave it at pH 6.0, and place it in a triangular chamber with a cotton plug. Pour into a flask or Sakaguchi flask or a tank equipped with a stirrer, and cultivate this cultivar B in the culture solution. Cultivation conditions are, for example, temperature 20~
Under a constant temperature of 35℃, preferably around 25℃, in a bright place,
Preferably under light irradiation of 3000 to 100000 lux,
For example, this can be carried out by shaking a 300 ml Erlenmeyer flask containing 100 ml of culture solution at 120 rpm on a reciprocating shaker having a shaking width of 4 cm. Next, the characteristics of the cultured cells of the present invention will be explained below while comparing them with the characteristics of conventional variety A. Regarding the growth rate, as shown in Figure 1, when the growth ratio is used as an indicator, the cultured cells of the present invention enter the stationary phase after 14 days of cultivation, and show a growth ratio of about 10 times, whereas the conventional variety A It enters the stationary phase in 28 days and exhibits a growth ratio of about 8 times. In this way, the cultured cells of the present invention are of the conventional variety A.
The lag period is shorter and the growth rate is faster than that of
Moreover, the growth rate during the stationary phase was also large, so it was observed that the growth rate was extremely high. Next, regarding environmental adaptability, the results of examining PH, temperature, and shaking number are shown in Figures 2 and 3, respectively.
It is shown in FIG. Regarding adaptability to pH changes, as shown in Figure 2, the cultured cells of the present invention grow stably in the pH range of 4.5 to 7, whereas the conventional variety A grows stably in a narrow range of pH 5.5 to 6.5. does not grow. In this way, the cultured cells of the present invention have wide adaptability to PH changes. Regarding adaptability to temperature changes, as shown in Figure 3, the cultured cells of the present invention grow stably in a temperature range of 20 to 35°C, whereas the conventional variety A grows stably only in a narrow temperature range of 25 to 30°C. It doesn't grow. In this way, the cultured cells of the present invention have wide adaptability to temperature changes. As shown in Figure 4, the adaptability to changes in the shaking rate of the shaking culture machine is as follows:
While it grows stably in a wide range of shaking speeds from 100 to 180 rpm, conventional variety A grows stably only in a narrow range of shaking speeds from 100 to 120 rpm. In this way, the cultured cells of the present invention have wide adaptability to changes in the shaking rate. Regarding the high productivity and stable productivity of the red pigment chrysanthemine of the cultured cells of the present invention, as shown by the curves □-□ in Figures 1 to 4, the productivity is 10%/dry weight or more, and the red natural pigment production ability is 10%/dry weight or more. Of course, compared to the conventional variety A, which has no red pigment, the cultured cells of the present invention have a red pigment productivity of 1.4%/dry weight, which is about 8 times more than the red pigment productivity of the cultured cells before selection. It is clear that the cultured cells of the present invention have an extremely high ability to produce red natural pigment. Furthermore, as is clear from the results shown in Figures 2 to 4, the cultured cells of the present invention have PH4
6.5, it stably produces red natural pigment in the range of temperature 15-30℃ and shaking number 60-180rpm, and has an extremely wide stable productivity. Regarding the morphological characteristics of the cultured cells of the new variety B of the present invention, the size and shape are similar to those of the conventional variety A, and the size is about 30 to 50μ, the shape is almost spherical, and the color is red throughout. In this respect, conventional variety A
It's completely different. As described above, the characteristics of the cultured cells of the present invention and the conventional variety A are completely different, and from this point as well, it can be concluded that the two varieties A and B have different genetic compositions. It should be noted that an attempt was made to deposit the cultured cells of the present invention at the Microtech Institute, but the request was not accepted. However, variety B of the present invention can be repeatedly and reliably created from the conventional variety Hanakirin by the above-described creation means. Examples of the present invention will be described below, but it goes without saying that the scope of the present invention is not limited to the following examples. Example Leaves of a commercially available garden cultivar Hanakirin (tree height: approx. 30 cm, leaf length: approx. 5 cm) were thoroughly washed with water and cut into approximately 4 cm ² pieces. These sections were then immersed in 70% ethyl alcohol for 5 minutes, and then in a 10% bleaching powder solution.
After being sterilized by immersion for 10 minutes, it was washed by immersion in sterile distilled water several times in a sterile box to thoroughly remove any remaining sterilizer. These leaf sections were cut into small pieces with a width of about 1 cm ² using a sterile scalpel, and the obtained small pieces of Hanakirin leaves were placed aseptically on a synthetic agar medium having the following composition. As a medium, the inorganic salt medium shown in Table 1 above, 6% w/v of sucrose, 0.2% w/v of malt extract,
2,4-D5×10 ^-7 M, thiamine hydrochloride 0.1ppm,
Pyridoxine hydrochloride 0.5ppm, nicotinic acid
0.5ppm, glycine 2ppm and inositol
Add 100ppm and adjust to PH6.0, agar 0.8%w/
A medium was used which had been sterilized in a conventional manner by adding V. Small pieces of Hanakirin leaves placed on such a medium were cultured at a culture temperature of 25°C under light irradiation of 3000 lux. After one week, a red mass of cultured cells appeared around the cut end of the leaf. One month later, 1 g (fresh weight) of the cultured cell mass, which had grown significantly, was dispersed in a 100 ml Erlenmeyer flask containing 50 ml of a liquid medium having the following composition. As a medium, see Table 1 above.
4% w/v of sucrose, 0.2% w/v of malt extract, 2,4-D5×10 ^-7 M, 0.1 ppm of thiamine hydrochloride, and pyridoxine hydrochloride in the inorganic salt medium shown in .
The pH was adjusted to 6.0 by adding 0.5 ppm, nicotinic acid, 0.5 ppm, glycine 2 ppm, and inositol 100 ppm, and the pH was sterilized in the usual manner. The flask containing the cell culture medium was then shaken several times.
The cells were fixed on a reciprocating shaking incubator at 120 rpm and a shaking width of 4 cm, and cultured for 7 days at a temperature of 25° C. under light irradiation of 3000 lux. After 7 days, the culture solution became a mixed suspension of destroyed cells, minute cell clumps, and giant cell clumps. The resulting suspension is mixed with a mesh size of
Passed through two stainless steel filters of 100μ and 150μ, and accumulated between the two filters.
Cultured cell clusters with a size of 100-150μ were collected. The isolated cultured cell mass was collected at a cell concentration of 10 ⁶ cells/
ml of the above liquid medium. This cell concentration was mainly confirmed using a Thoma hemocytometer. Next, 1 ml of this cell suspension was suspended in 20 ml of dissolved agar medium having the following composition, and then poured into a 9 cm Petri dish and solidified. As an agar medium,
10 ml of medium with the same composition as used for the suspension culture described above
Combine the above suspension culture solution with 10ml of the solution passed through a 0.2μ membrane filter, add 126mg of agar (0.6% w/v), and after dissolving the agar, heat to 48℃. A maintained medium was used. Next, this Petri dish was left standing under light irradiation of 3000 lux at 25°C, and cultured for 21 days. Of the 26 cell clusters that grew and proliferated in the Petri dish, 2 cell clusters that were determined to be deeper in red by visual inspection were selected.
They were selected and named the X strain and the Y strain, respectively. In this way, we were able to select two types of cultured cells with high red pigment production ability, the X strain and the Y strain, from the original cultured cell mass P. In order to investigate how much the pigment production ability was improved compared to P, the following experiment was conducted. First, strain X (55 mg) and strain Y (52 mg) were each dispersed in test tubes containing 5 ml of the same culture solution used in step (a) above, and suspension culture was performed for 14 days under the same culture conditions as in the above step. Summer. Next, take 2.5 ml of the culture solution and
The cultured cells on the paper were collected by passing through No. 2 paper, and the production amount was weighed. The cells were cooled in 2% hydrochloric methanol for 1 hour, filtered, 2% hydrochloric methanol was added to the solution to make 10 ml, the absorbance at 530 nm was measured, and the dye weight and pigment content were determined from the calibration curve. Pigment content of each strain (%/g fresh weight, same below)
is 0.0862% for X stock and 0.0882% for Y stock, and step (a)
Compared to the pigment content of the cultured cell mass before entering the culture cell mass, which was 0.0708%, an improvement of 21.8% and 24.6%, respectively, was observed. When the cultured cell clusters contained in the remaining 2.5 ml of culture solution were dispersed in a Petri dish in the same manner as in the above step, 25 cell clusters of the X strain and 18 cell clusters of the Y strain grew and proliferated. Among these, cell clusters that were visually determined to have a deeper red color were selected from each of the X and Y strains. These were 2-X strain and 2-X strain, respectively.
-Y strain, and the content of each pigment was measured according to the pigment content measurement method described above.
They were 0.145 and 0.152%. The obtained 2-X strain and 2-Y strain were further subjected to the same selection process for 4 times.
After repeating the process several times, the final strain 6-X (pigment content
Two strains, 0.651% (the same applies hereinafter) and 6-Y strain (0.673%), were obtained. The ratios of improvement in these pigment contents to those of the original cell mass were 9.2 times and 9.5 times, respectively. Remaining culture solution for 6-Y strain with high pigment content
2.5 ml was added to a 100 ml Erlenmeyer flask containing 50 ml of a culture solution with the same composition as above, and cultured for 14 days under the same culture conditions as above. Next, this culture solution was added to a 1 liter Erlenmeyer flask containing 500 ml of a culture solution with the same composition, and after culturing for 14 days under the same culture conditions, the cultured cell mass (158 g) was filtered.
I got it. This was freeze-dried in the usual manner and the dried product was 7.3
I got g. This dried product was ground in a mortar and then immersed in 2% hydrochloric acid methanol in a cold place for 24 hours. The extract obtained after filtration was concentrated to about 50 ml by removing methanol at a temperature below 40° C., transferred to a separating funnel, 100 ml of ether was added, and after thorough shaking, it was separated from the ether layer. After repeating this ether washing operation several times, it was further concentrated under reduced pressure at 40°C or lower to dryness. This dried product was dissolved in a small amount of 0.01% hydrochloric acid methanol, injected into preparative HPLC, and eluted with methanol/formic acid water (formic acid content 0.1%), and a red effluent was fractionated. This effluent was dried under reduced pressure at 40℃ or below to obtain 1030 mg of black-red powder (yield 14.1% vs. dry weight,
0.652% fresh weight) was obtained. Furthermore, when we compared the UV absorption spectra of the obtained dye and cyanidin-3-arabinoside, we found that
As shown in Table 3, both results were almost in agreement.

[Table] Arabinosides
Cyanidin〓7〓 533275 27 131 +
Arabinoside

Claims (1)

[Claims] 1. Undifferentiated cell morphology with high ability to produce red natural pigment, stability, environmental adaptability, and vigorous growth obtained by culturing the leaves of Euphorbia genus Hanakirin in a medium containing 2,4-dichlorophenoxyacetic acid. A cultured cell belonging to the Euphorbia genus Hanakirin having the following.