JPH07255463A - Highly chlorophyll-containing chlorella variant and method for producing the same - Google Patents

Abstract

PURPOSE:To provide a chlorella variant with a new characteristic obtained, namely, high chlorophyll content, and easy in the culture, and to provide a method for producing the chlorella variant, capable of eliminating the complexity of the maintenance on the industrial culture of the chlorella, and enhancing the industrial producibility of the chlorella. CONSTITUTION:This method for producing a highly chlorophyll-containing chlorella variant comprises irradiating a parent strain comprising a chlorella alga multiplying on a heterotrophical culture using an organic compound as a carbon source with ultraviolet rays, subjecting the irradiated chlorella alga to the heterotrophical nutritive culture, selectively separating a strain having a more green color than that of the parent strain, repeating the ultraviolet rays-irradiation treatment and the selective separation treatment once or more, and subsequently selectively separating the variant higher in the producibility of the chlorophyll than that of the parent strain.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a high chlorophyll-containing chlorella mutant strain containing a high proportion of chlorophyll, which is useful as a natural coloring agent, a cell activator, etc., and a method for producing the mutant strain.

[0002]

Chlorella is a unicellular alga belonging to the class Chlorophyta, Chlorococcus, Oocystis, and Chlorella, and its algal body contains about 60% protein and many essential amino acids such as lysine. Are known. In addition, chlorella contains vitamins, minerals, fibers, and a growth-promoting substance for organisms called chlorella gross factor (CGF), and since its growth rate is also high, it is a vegetable protein food source, health food, and natural coloring. Agent,
It is used as a raw material for drugs such as cell activating agents and anti-cancer agents.

Conventionally, as a method for industrially culturing chlorella, an autotrophic culturing method in which sunlight is used as an energy source and carbon dioxide is used as a carbon source for photosynthesis, and an organic compound is used as a carbon source and an energy source in a tank. There is a heterotrophic culture method that can be cultured in.

The autotrophic culturing method can obtain chlorella having a relatively high chlorophyll content, but it requires a vast amount of land to be exposed to the sun's rays in an outdoor open culture pond, and the yield is high. However, it has the drawback of being affected by the climate and the season. In addition, since it is exposed to the outside and cultured, it is easily contaminated by microorganisms such as bacteria, and the quality is not stable.

On the other hand, the heterotrophic culturing method can be cultivated in a closed tank, and is not contaminated with microorganisms such as various bacteria as compared with the autotrophic culturing method exposed to the outside air, so that the quality is good. , Its yield is also stable, but because the photosynthetic organs in the cultured chlorella cells do not develop sufficiently, only chlorophyll content lower than that of chlorella by the autotrophic culture method can be obtained. There is a problem.

[0006] Techniques for improving the above-mentioned problems in the heterotrophic culture method include dissolved oxygen concentration and pH of the culture solution.
Japanese Patent Publication No. 58-40462 discloses a technique for adjusting the chlorophyll content in chlorella by adjusting the above conditions to a predetermined range and adding an amino acid and glucose.

[0007]

However, in the above-mentioned improved technique, it is necessary to adjust dissolved oxygen and other conditions and add a predetermined amino acid and the like, so that the production control is complicated and the culture solution preparation cost is reduced. In terms of industrial productivity, I was not satisfied.

[0008] Therefore, an object of the present invention is to solve the above-mentioned problem of productivity of the heterotrophic chlorella culture technique,
The purpose of the present invention is to provide an easily cultivated mutant strain of Chlorella that has acquired a new trait of high chlorophyll content, eliminates the complexity of management when industrially culturing Chlorella, and enhances industrial productivity.

[0009]

In order to solve the above-mentioned problems, the present invention has a growth ability in heterotrophic culture using an organic substance as a carbon source and has a total chlorophyll content of 3 by dry weight. % Or more of high chlorophyll-containing chlorella sp. RK-7111 (This strain is stored at 1-8-10 Jiyugaoka, Kanazu-cho, Sakai-gun, Fukui Prefecture, at Rengo Co., Ltd. Kanazu Chemical Bio Plant, and the applicant guarantees the sale.) is there.

Such a high chlorophyll-containing Chlorella spp. Mutant strain has a parent strain of Chlorella algae that grows in a heterotrophic culture using organic matter as a carbon source, and the parent strain is irradiated with ultraviolet rays and then subjected to a heterotrophic culture. By carrying out an operation of selectively isolating a strain that is darker in green than the parent strain, and selectively isolating a mutant strain having higher chlorophyll productivity than the parent strain.

The condition of the ultraviolet treatment in the above-mentioned production method is preferably such that one ultraviolet irradiation time is 10 to 100 seconds.

[0012]

Examples As a heterotrophic culture method in which the carbon source in the present invention depends on the extracorporeal organic matter, sugars, organic acids, etc. are added as carbon sources to an inorganic salt medium, and nitrates, ammonia, ammonium salts as nitrogen sources, Add urea etc.,
Add proteins as needed, in the dark at 20-30 ° C
Well-known culture methods such as culturing in a tank while shaking or aerating with agitation (for example, Japanese Patent Publication No. 45-1714
No. 6, etc.) can be adopted.

The wavelength of the ultraviolet irradiation treatment for the strain capable of growing by such a heterotrophic culture method is not particularly limited as long as it is in the ultraviolet region. For example, the wavelength is 254n.
It is preferable to use ultraviolet rays of m and UV intensity of 400 μW / cm ² (15 cm directly below the ultraviolet lamp) for 10 to 100 seconds. This is because the mutation effect is scarcely recognized when the ultraviolet irradiation treatment per time is less than 10 seconds, and the chlorella algal cells are entirely killed by the ultraviolet irradiation treatment exceeding 100 seconds, which is not preferable.

The series of operations including the above-mentioned ultraviolet irradiation treatment and the subsequent selective separation treatment can be carried out once or more.

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 paddy fields in various parts of Japan and placed in a culture flask containing medium A described below. After inoculating and culturing autonomically for 7 days at 30 ° C. under sunlight, this culture solution was appropriately diluted with sterile water (10 to 100 times),
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 the light for 7 days. Then, this culture can be spread on an agar plate of medium B having the following composition, and can be heterotrophically grown by statically culturing for 7 days in the dark.
0 shares 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 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 0.021 g, [Medium B composition] pH 6.5 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 Each of the 20 strains of chlorella obtained by the above isolation treatment had a diameter of about It is a spherical shape with a diameter of 5 μm.
6) pyrenoids and 1 shallow cup-shaped chloroplast, and the availability of carbon and nitrogen compounds was investigated.
idea, C. luteoviridis, C. miniata, C. protothecoi
des, C. saccharophila, C. sorokiniana, C. variega
Since this strain is different from any of the algal cells of ta, C. vulgaris, C. xanthella, and C. zopfingiensis, this strain was chlorella s
p. It was named RK-7000.

The mycological properties of RK-7000 are shown below. 1. Microscopic findings (1) Size about 5 μm, (2) Shape sphere,
(3) No motility 2. Physiological properties (1) Growth temperature 25-40 ° C, (2) Optimal growth temperature 35 ° C, (3) Growth pH 5.5-7.5, (4) Optimal growth p
H 6.5-7.0 (5) KNO ₃ utilization + (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 source

[0019]

[Table 1]

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

[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: a solution prepared by dissolving the following components 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 upper lid of the dish was removed to expose the medium surface. UV lamp (made by Funakoshi: UVGL-58)
Type, wavelength of 254 nm), and the resulting agar medium in a petri dish coated with the suspension is subjected to UV treatment for 40 seconds, and then the petri dish is covered with an aluminum foil to prevent light reactivation and shielded from light. In this state, static culture was carried out at 30 ° C. for 10 days in a constant temperature incubator.

(Selective isolation and culture of chlorella mutant)
The 112 colonies obtained by this method were visually identified, and 6 white colonies (5%) and 8 yellow colonies (7) were identified.
%), Pale yellow colonies 28 strains (25%), green colonies 52 strains (46%) having the same color as the parent strain, and green colonies 18 strains (16%) darker than the parent strain.

After subculturing the green colony 18 strains darker than the parent strain three times, heterotrophic culture in liquid medium C was performed under the following conditions. That is, an Erlenmeyer flask (100 ml containing 30 ml of the liquid medium C was
After autoclaving at 121 ° C. for 20 minutes, inoculate the alga cells of 18 strains of each colony at 30 ° C. and 190
The culture was carried out by shaking at rpm for 5 days.

(Recovery of Chlorella Mutant) The culture solution after the above culture was centrifuged at 4000 rpm for 15 minutes to separate and collect the precipitated Chlorella algal cells. This recovery Chlorella 3
Wash once with 0 ml distilled water, centrifuge it,
Suspend in ml of distilled water and dry it in a lyophilizer.
About 0.5 g of dried chlorella was obtained for each of the 8 strains.

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

Moisture (%): normal pressure heat drying method (105
℃, 5 hours), Ash content (%): Direct ashing method, Protein: Semi-micro Kjeldahl method, Iron content (mg%): Colorimetric method specified by Japan Health Food Association, Chlorophyll: Alkaline pyridine method specified by Japan Health Food Association, [Example 2] Chlorella sp. Mutant treatment by ultraviolet irradiation (ultraviolet irradiation time was 60 seconds), selective isolation and culture of chlorella mutant under the same conditions as those used in Example 1 described above except that RK-7100 was used as a parent strain. Each treatment operation for recovery of the Chlorella mutant strain was repeated.

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

Example 3 Next, chlorella sp. RK-
Using 7110 as a parent strain, mutation treatment by ultraviolet irradiation (ultraviolet irradiation time was 20 seconds) under the same conditions as those used in Example 1 described above except for special mention, selective isolation and culture of a chlorella mutant, and chlorella mutation Each processing operation for recovering the strain was repeated.

Among the 16 strains thus obtained, strains having a chlorophyll content of 3% or more were chlorella sp. RK-71
It was named 11. Moisture, ash, protein content of this strain,
The contents of iron and chlorophyll were measured by the above method, and the results are shown in Table 2.

[0030]

[Table 2]

The mutant strains obtained by the above-mentioned UV treatment all have the mycological properties except for the chlorophyll content, which is the parent strain Chlorella sp. It was the same as RK-7000.

The algal cells obtained by the mutation treatment were passaged several tens of times, but no decrease in chlorophyll content was observed.

Comparative Example 1 The parent strain Chlorella sp. Using RK-7000 as Comparative Example 1, 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 foods was used as Comparative Example 2, and its water content, ash content, protein content,
The contents of iron and chlorophyll were measured by the above-mentioned method, and the results are also shown in Table 2.

As is clear from the results in Table 2, Example 1 can be cultivated in the general culturing state of 5 days at 30 ° C. using the medium C containing no special additives such as amino acids, and The metabolite, chlorophyll, was contained in a high proportion of 3% or more by dry weight.

Further, by the method adopted in Examples 1 to 3, it was possible to selectively isolate mutant strains having higher chlorophyll productivity than the parent strain.

On the other hand, in Comparative Example 1 or Comparative Example 2, since the ultraviolet treatment was not performed, the chlorophyll content was 1.7.
It was as low as 7% or 1.89%, and it was impossible to expect a chlorophyll content higher than this by the usual culture method.

[0038]

[Effect] As described above, the present invention is a novel mutant strain of the genus Chlorella which has a growth ability in normal heterotrophic culture and a high total chlorophyll content of 3% or more by dry weight. Can be provided. This has the advantage that it can be efficiently grown on an industrial scale by a normal heterotrophic culture method in a tank and chlorophyll can be efficiently produced with a stable quality.

The high chlorophyll content chlorella is
It can be said to be an efficient method for producing a mutant strain, because it can be produced by a relatively simple operation of ultraviolet irradiation treatment.

Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI Technical indication C12R 1:89) (72) Inventor Yuki Takeyasu 1-8-10 Jiyugaoka, Kanazu-cho, Sakai-gun, Fukui Prefecture Rengo stock Company Kanazu Chemicals Bio Plant (72) Inventor Michiaki Tanimoto 1-8-10 Jiyugaoka, Kanazu-cho, Fukui Prefecture Rengo Co., Ltd. Kanazu Chemicals Bio-plant

Claims (3)

[Claims] 1. It has the ability to grow in a heterotrophic culture using organic matter as a carbon source, and the total chlorophyll content is 3 by dry weight.
% Or more of high chlorophyll-containing chlorella sp. RK
-7111. 2. A parent strain is a chlorella alga that grows in a heterotrophic culture using organic matter as a carbon source, and the parent strain is irradiated with ultraviolet rays, and then heterotrophic culture is performed to select a darker green strain than the parent strain. A method for producing a high chlorophyll-containing chlorella mutant strain, which comprises performing a separation operation and selectively separating a mutant strain having a higher chlorophyll productivity than the parent strain. 3. The method for producing a high chlorophyll-containing chlorella mutant strain according to claim 2, wherein a single ultraviolet irradiation time is 10 to 100 seconds. How to make.