JP2620045B2 - High Chlorophyll-Containing Chlorella Mutants - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION This invention is a natural coloring agent, about the chlorophyll-rich soluble genus Chlorella mutant strains containing useful chlorophyll a high rate as a cell-activating agent.

[0002]

BACKGROUND OF THE INVENTION Chlorella is a unicellular algae belonging to the class Chlorophyceae, Chlorococcus, Occystis, and Chlorella. Are known. Chlorella also contains vitamins, minerals, fiber, and biological growth-promoting substances called chlorella gross factor (CGF), and its growth rate is high. Agent,
It is used as a raw material for pharmaceuticals such as cell activators and anticancer agents.

Conventionally, chlorella is industrially cultivated as an autotrophic cultivation method in which sunlight is used as an energy source and photosynthesis is performed using carbon dioxide as a carbon source, and an organic compound in a tank is used as a carbon source and an energy source. Heterotrophic culture methods that can be cultured in

In the autotrophic culture method, chlorella having a relatively high chlorophyll content can be obtained. However, a large amount of land is required to be exposed to sunlight in an outdoor open culture pond, and the yield is limited by the weather. The disadvantage is that it depends on the climate and the season. In addition, since the culture is open to the outside and cultivated, there is a disadvantage that the microorganism is easily contaminated by microorganisms such as bacteria and the quality is not stable.

On the other hand, the heterotrophic cultivation method allows cultivation in a closed tank and is not contaminated with microorganisms such as germs as compared with the autotrophic cultivation method exposed to the outside air. Although the yield is stable, the photosynthetic organs in the cultured chlorella cells are not sufficiently developed, so that only those having a lower chlorophyll content than chlorella obtained by the autotrophic culture method can be obtained. There is a problem.

Techniques for solving the above problems in the heterotrophic culture method include a dissolved oxygen concentration of a culture solution, pH
Japanese Patent Publication No. 58-40462 discloses a technique in which conditions such as the above are adjusted to a predetermined range and an amino acid and glucose are added to increase the chlorophyll content in chlorella.

[0007]

However, according to the above-mentioned improved technology, it is necessary to prepare dissolved oxygen and other conditions and to add a predetermined amino acid and the like, so that production control is complicated and the cost of preparing a culture solution is reduced. It was not satisfactory in terms of industrial productivity.

[0008] Accordingly, an object of the present invention is to solve the productivity problem of the heterotrophic chlorella culture technique described above,
An object of the present invention is to provide an easily cultivated mutant strain of chlorella that has acquired a new trait of high chlorophyll content, eliminate the complexity of management when culturing chlorella industrially, and increase industrial productivity.

[0009]

In order to solve the above-mentioned problems, the present invention has a growth ability in a heterotrophic culture using an organic substance as a carbon source, and the total chlorophyll content in the culture has a dry weight. High chlorophyll content of 3% or more by weight
Chlorella sp. RK-7111
(This strain is located at 1-8-1 Jiyugaoka, Kanazu-cho, Sakai-gun, Fukui Prefecture)
No. 0, which is stored in the Kanazu Chemicals Bio-factory of Rengo Co., Ltd., and its distribution under Article 27-3 of the Ordinance for Enforcement of the Patent Act is guaranteed by the applicant. ).

[0010] Such a high chlorophyll-containing mutant of Chlorella spp. Is a parent strain of Chlorella algae which grows in heterotrophic culture using an organic substance as a carbon source. And performing an operation to selectively isolate strains that are darker green than the parent strain, and selectively isolate mutants having higher chlorophyll productivity than the parent strain.

[0011] The conditions of the ultraviolet treatment in the production method are preferably such that one ultraviolet irradiation time is 10 to 100 seconds.

[0012]

DETAILED DESCRIPTION OF THE INVENTION As a heterotrophic cultivation method according to the present invention which depends on an extracorporeal organic substance as a carbon source, a saccharide, an organic acid or the like is added as a carbon source to an inorganic salt medium, and a nitrate, ammonia, ammonium salt or the like is added as a nitrogen source. Add urea etc.
If necessary, add proteins and keep at 20-30 ° C in the dark.
A well-known culturing method such as culturing in a tank while shaking or aeration with stirring (for example, Japanese Patent Publication No. 45-1714)
No. 6 and the like) can be employed.

The ultraviolet irradiation treatment of the strain capable of growing in such a heterotrophic culture method is not particularly limited as long as it is in the ultraviolet region.
It is preferable to use ultraviolet light of m for 10 to 100 seconds at a UV intensity of 400 μW / cm ² (15 cm immediately below the ultraviolet lamp). This is because if the ultraviolet irradiation treatment per one time is less than 10 seconds, the mutation effect is hardly recognized, and if the ultraviolet irradiation treatment exceeds 100 seconds, all the Chlorella alga bodies are killed, which is not preferable.

A series of operations including the above-described ultraviolet irradiation treatment and the subsequent selective separation treatment can be performed one or more times.

The parent strain of Chlorella used in the invention of the method for producing a mutant strain is not particularly limited as long as it belongs to the genus Chlorella.

[Example 1] (Isolation of parent strain) In July 1993, water containing green algae was collected from rivers, lakes and marshes and paddy fields in various parts of Japan, and placed in a culture flask containing the following medium A. After inoculation, the cells were autotrophically cultured at 30 ° C. for 7 days under sunlight, and the culture was diluted appropriately (10 to 100 times) with sterile water.
Penicillin 10 μg / ml and erythromycin 10
The medium A having the following composition to which μg / ml was added was spread on an agar plate, and cultured in a light place for 7 days. Next, this culture is spread on an agar plate of a medium B having the following composition, and is cultivated by heterotrophic stationary culture in a dark place for 7 days to allow heterotrophic growth.
0 strains were obtained.

[Medium A composition] pH 6.5 KNO ₃ 0.25 g / l KH ₂ PO ₄ 0.175 g / l K ₂ HPO ₄ 0.075 g / l MgSO ₄ .7H ₂ O 0.075 g / l _{l FeSO 4 · 7H 2 O 5.0} mg / l Arnon's A 5 solution _{* 1.0 ml / l CaCl 2 0.01} g / l agar (20.0) g / l deionized water 1.000 l <* Arnon's a ₅ solution: the following components that dissolved in 1 liter of _{water> H 3 BO 3 2.86 g,} MnCl 2 · 4H 2 O 1.81 g, ZnSO 4 · 7H 2 O 0.22 g, CuSO 4 · _{5H 2 O 0.88 g, Na 2} MoO 4 0.021g, [ medium B _{composition] pH6.5 KH 2 PO 4 1.0 g} / l MgSO 4 · 7H 2 O 1.0 g / l FeSO 4 · 7H _{2 O 5.0 mg / l Arnon's} A 5 solution * 1.0 ml / l Isutoe 5.0 g / l glucose 20.0 g / l agar (20.0) g / l deionized water 1.000 l All of the 20 strains of chlorella obtained by the above-mentioned isolation treatment have a diameter of about 20%. It is a 5 μm sphere, and several of these (3 to
6) and one shallow cup-shaped chloroplast. As a result of examining the availability of carbon and nitrogen compounds, C. ellipso, a chlorella strain distributed by the ATCC, was obtained.
idea, C. luteoviridis, C. miniata, C. protothecoi
des, C. saccharophila, C. sorokiniana, C. variega
ta, C. vulgaris, C. xanthella, and C. zopfingiensis.
p. RK-7000.

The bacteriological properties of RK-7000 are shown below. 1. Microscopic findings (1) Size about 5 μm, (2) Shape sphere,
(3) No mobility 2. Physiological properties (1) Growth temperature 25 to 40 ° C, (2) Optimal growth temperature 35 ° C, (3) Growth pH 5.5 to 7.5, (4) Optimal growth p
H 6.5-7.0 (5) KNO ₃ assimilation + (6) (NH ₄ )
₂ SO ₄ assimilation + (7) Decomposition of urea + (8) Glucose concentration 5% growth ++ (9) Glucose concentration 10% growth + (10) Salt concentration 1% growth −3. Utilization of carbon sources

[0019]

[Table 1]

(Mutation treatment by UV irradiation) Next, Chlorella sp. The culture solution containing RK-7000 was diluted with sterile water to prepare a chlorella suspension having about 10 ⁵ cells / ml. Then, the suspension was spread on an agar plate in a petri dish of a medium C having the following composition with a conical bar.

[0021] [Medium C Composition] pH 6.5 Glucose _{25.0 g / l MgSO 4 · 7H} 2 O 1.25 g / l KH 2 PO 4 1.25 g / l yeast extract 6.25 g / l KNO _{3 4.17 g / l Arnon's A} 5 solution * 1) 2.5 ml / l Fe salt solution * 2) 2.5 ml / l agar (20.0) g / l deionized water 1.000 l <* 1) Arnon's a ₅ solution: the following components that dissolved in 1 liter of _{water> H 3 BO 3 2.86 g,} MnCl 2 · 4H 2 O 1.81 g, ZnSO 4 · 7H 2 O 0.22 g, CuSO _{4 · 5H 2 O 0.88 g,} Na 2 MoO 4 0.021g, [* 2) Fe salt solutions: one of the following components dissolved in 1 liter of water] citric acid _{18.9 g, FeSO 4 · 7H 2} O 5.0 g, and the top cover of the Petri dish was removed to expose the medium surface In this state, this is replaced with an ultraviolet lamp (Funakoshi: UVGL-58).
Mold, wavelength 254 nm), and place the obtained suspension-coated agar medium in a petri dish with ultraviolet light for 40 seconds. Then, cover the petri dish with aluminum foil to prevent light reactivation, and shield it from light. In this state, the cells were statically cultured in a thermostat at 10 ° C. for 10 days.

(Selective Isolation and Culture of Chlorella Mutant)
The 112 colonies obtained by this method were identified with the naked eye, and 6 white colonies (5%) and 8 yellow colonies (7
%), 28 light yellow colonies (25%), 52 green colonies (46%) of the same color as the parent strain, and 18 green colonies (16%) darker than the parent strain.

Each of the 18 green colonies darker than the parent strain was subcultured three times, and then subjected to heterotrophic culture in liquid medium C under the following conditions. That is, an Erlenmeyer flask (100 ml) containing 30 ml of the liquid medium C
Autoclaved at 121 ° C. for 20 minutes, and then inoculated with 18 algal cells of each colony.
Shaking culture was performed at rpm for 5 days.

(Recovery of Chlorella Mutant) The culture solution after the culture was centrifuged at 4000 rpm for 15 minutes to separate and recover the precipitated Chlorella algae. This recovered chlorella
It was washed once with 0 ml of distilled water, centrifuged,
suspended in distilled water, and dried with a freeze dryer.
About 0.5 g of dried chlorella was obtained for each of the eight strains.

The chlorophyll content of the 18 strains in the dried chlorella was measured by the alkaline pyridine method specified by Japan Health Foods, and the strain with the highest chlorophyll content (1.89%) was selected from Chlorella sp. RK-7100
It was named. The water, ash, protein, iron, and chlorophyll contents of this strain were measured by the following methods, and the results are shown in Table 2.

Moisture (%): normal pressure heating drying method (105
C, 5 hours), Ash (%): Direct ashing method, Protein: Semi-micro Kjeldahl method, Iron (mg%): Colorimetric method specified by the Japan Health Food Association, Chlorophyll: Alkaline pyridine method specified by the Japan Health Food Association, Example 2 Chlorella sp. Using RK-7100 as a parent strain, mutagenesis treatment by ultraviolet irradiation (ultraviolet irradiation time was set to 60 seconds), selective isolation and culture of chlorella mutant strain under exactly the same conditions as those used in Example 1 except as otherwise specified. Each processing operation of recovering the Chlorella mutant was performed again.

Then, of the 10 strains obtained, the strain with the highest chlorophyll content (2.38%) was selected from Chlorella s.
p. RK-7110. Moisture, ash,
The protein content, iron content, and chlorophyll content were measured in the same manner as described above, and the results are shown in Table 2.

Embodiment 3 Next, Chlorella sp. RK-
Mutation treatment by ultraviolet irradiation (ultraviolet irradiation time was 20 seconds) under the same conditions as those used in Example 1 except for special mention, using 7110 as a parent strain, selective separation and culture of Chlorella mutants, Chlorella mutation Each processing operation of the strain recovery was performed again.

Then, of the 16 strains obtained, strains having a chlorophyll content of 3% or more were selected from Chlorella sp. RK-71
It was named 11. Moisture, ash, protein content,
The contents of iron and chlorophyll were measured by the methods described above, and the results are shown in Table 2.

[0030]

[Table 2]

All of the mutants obtained by the above-mentioned ultraviolet ray treatment have the mycological properties except for the chlorophyll content, which is the parent strain Chlorella sp. It was not different from RK-7000.

The alga bodies obtained by the mutation treatment were subcultured several tens of times, but no decrease in the chlorophyll content was observed.

[Comparative Example 1] Chlorella sp. RK-7000 was used as Comparative Example 1, and the water content, ash content, protein content, iron content, and chlorophyll content of this strain were measured by the methods described above, and the results are also shown in Table 2.

Comparative Example 2 A commercially available chlorella powder for health food was used as Comparative Example 2 to determine its water, ash, protein content,
The contents of iron and chlorophyll were measured by the above-described methods, and the results are shown in Table 2.

As is clear from the results in Table 2, Example 1 can be cultured in a general culture state of 30 days at 30 ° C. using the medium C containing no special additives such as amino acids. It contained its metabolite, chlorophyll, in a high proportion of 3% or more by dry weight.

Further, the mutants having higher chlorophyll productivity than the parent strain could be selectively isolated by the method employed in Examples 1 to 3.

On the other hand, in Comparative Example 1 or Comparative Example 2, the chlorophyll content was 1.7 because it had not been subjected to ultraviolet treatment.
It was as low as 7% or 1.89%, and it was not possible to expect a further higher chlorophyll content by the usual culture method.

[0038]

[Effect] The present invention, As described above, has the ability to grow in a normal heterotrophic culture, the addition special amino acids such as
An object of the present invention is to provide a novel mutant of the genus Chlorella which contains a high percentage of total chlorophyll content of 3% or more by dry weight even under culture conditions in which no substance is added . The Chlorella mutant has an advantage that it can be efficiently grown on an industrial scale by a usual heterotrophic culture method in a tank, and can efficiently produce chlorophyll with stable quality.

[0039]

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Yuki Takeyasu 1-8-10 Jiyugaoka, Kanazu-cho, Sakai-gun, Fukui Prefecture Inside the Rengo Co., Ltd. Kanazu Chemicals Bio Plant (72) Inventor Michiaki Tanimoto Kanazu, Sakai-gun, Fukui Prefecture 1-8-10 Jiyugaoka, Machi, Kanazu Chemicals Bio Plant, Rengo Co., Ltd. (56) References JP-A-57-83279 (JP, A) JP-A-57-47476 (JP, A) JP-A 52- 28989 (JP, A) JP 48-43872 (JP, B2)

Claims (1)

(57) [Claims] 1. A chlorophyll-containing mutant having a high chlorophyll content of 3% or more by dry weight in a total chlorophyll content in a heterotrophic culture using an organic substance as a carbon source. Chlorella sp. RK-7111.