JP2006014700A - New strain of chlorella - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a natural variant of Chlorella, having high proliferation ability and chlorophyll content in a dark culture. <P>SOLUTION: The natural variant of Chlorella is a strain of the genus Chlorella having high proliferation ability and chlorophyll content in a dark culture and is selected from the group consisting of Chlorella M15, M19, M34, M46 and M3. The strain exhibits about ≥20 times proliferation ratio in the vicinity of a room temperature in a culture period of about 3 days and has ≥about 30 mg/g total chlorophyll content on a dry weight. Since the strain is wholly the natural variant, the strain is different from a variant by forced variation and is highly safe. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a strain of genus Chlorella that can be used for health foods and the like. More specifically, the present invention relates to a natural mutant of the genus Chlorella having a high growth ability and high chlorophyll content in dark culture.

  Chlorella is a single cell that is classified as a green algae and is widely distributed in rivers, lakes, and puddles. Although the size varies depending on the species and the developmental stage, it has a spherical shape of about 3 to 10 microns and can be observed with an optical microscope.

  Chlorella contains chlorophyll, protein, vitamins, minerals, dietary fiber, and the like in a well-balanced manner and has been used regularly as a health food. Among them, chlorophyll is considered to be an important component of chlorella and is considered to be effective for maintaining and promoting health. In the field of health food, chlorella having a high chlorophyll content has been desired.

  As for chlorella, physiological activities such as animal growth promoting effect, lactic acid bacteria growth promoting effect, leukocyte depletion preventing effect by irradiation, wound healing effect, detoxification effect, food seasoning effect, etc. have been reported so far. It has not yet been clarified what the body of the physiologically active substance that produces such an effect is. However, it has been reported that chlorophyll contained in chlorella has an antioxidant action, an antimutagenic action, etc., and it is highly possible that chlorophyll is also involved in the above physiological activities, and in fields other than health foods There is a demand for chlorella with high chlorophyll content.

  Natural chlorella grows by autotrophic photosynthesis. However, when chlorella is proliferated as an industry, there are problems that the amount of sunlight irradiation varies greatly depending on the weather and season, and that the cultivated chlorella is susceptible to contamination by microorganisms. For this reason, in the industrial world, a method is desired in which chlorella is placed in a tank, cut off from the outside world, and cultured with heterotrophic nutrients using glucose or the like as a carbon source. However, there is a problem that chlorella grown by heterotrophic in the dark has a lower chlorophyll content and fades green than chlorella grown by autotrophic irradiation by sunlight. For this reason, a chlorella strain having a high chlorophyll content even in dark culture has been demanded.

  As a solution to this problem, a high chlorophyll-containing chlorella sp. Having a growth ability in heterotrophic culture using an organic substance as a carbon source and having a total chlorophyll content of 3% or more by dry weight. RK-7111 is disclosed (Patent Document 1). In addition, when heterotrophic culture is carried out in a medium containing an organic substance as a carbon source, the content of high chlorophyll and high carotenoid with a chlorophyll content of 35 mg / g or more and a total carotenoid content of 5.0 mg / g or more with respect to the dry alga body A chlorella mutant of has been disclosed (Patent Document 2).

JP, 7-255463, A High chlorophyll-containing chlorella mutant JP, 11-75823, A Chlorella mutant containing high chlorophyll and high carotenoid content

  However, the Chlorella genus mutant described in the publication is a strain artificially mutated by chemical treatment and physical treatment. Therefore, there is a possibility that multiple mutations have occurred in some part, and there is a risk including harmfulness. Since chlorella is ingested as a food, it is preferably safer, and a natural mutant is desired rather than a strain that has been forced to cause mutation. In other words, chlorella is relatively stable and difficult to mutate, so if it is a naturally mutated strain that has been screened for naturally occurring mutants over a long period of time, the mutation is point mutation-like, and other parts Is considered to be highly safe with little change from the parent strain. In addition, when continuous tank culture is performed, artificial mutants are likely to undergo mutations such as returning to their original properties, but natural mutants have the advantage that they are stable.

Moreover, although the optimal culture | cultivation temperature is set to 36 degreeC as for the chlorella genus mutant described in patent document 2, in order to industrialize, the strain | stump | stock which can culture | cultivate at the temperature nearer room temperature is more economical. It is.
On the other hand, the chlorella genus mutant described in Patent Document 1 is cultured at 30 ° C., which is closer to room temperature, but spends 5 days in culture. For industrialization, a strain that can be cultured in a shorter period is preferred. Therefore, a strain of the genus Chlorella, which can grow within a shorter period at a temperature close to room temperature, has been demanded.

  The present invention has been made in consideration of the above-mentioned problems of the prior art, and an object of the present invention is to provide a natural mutant of the genus Chlorella having high growth ability and high chlorophyll content in dark culture.

  As a result of repeated studies to solve the above problems, the present inventors have succeeded in isolating a natural mutant of the genus Chlorella having a high growth ability and high chlorophyll content under dark culture, and completed the present invention. did.

  As a result of screening chlorella collected from the field over a long period of time, the present inventors succeeded in obtaining 42 strains of chlorella spp. That grow in dark culture. Further research was then continued, and among these 42 strains, 5 mutant strains of the genus Chlorella having high growth ability and high chlorophyll content were isolated under dark culture, and these were isolated as M 15 strains (Accession Number: FERM P-). 20034), M19 strain (Accession number: FERM P-20033), M34 strain (Accession number: FERM P-20066), M46 strain (Accession number: FERM P-20067) and M3 strain (Accession number: FERM) P-20035). These strains reach a plateau in a dark culture at 30 ° C. for 3 days. Moreover, as a result of measuring the chlorophyll content in the said culture | cultivation, the total chlorophyll content showed the high value with 30 mg / g or more by dry weight. Furthermore, since they are all naturally-mutated strains, they are highly safe and highly suitable for use as health foods, etc., unlike those that have been forcibly mutated.

  As is clear from the above explanation, the new strain of genus Chlorella according to the present invention is a highly safe natural mutant strain, has an excellent ability to grow in dark culture, and chlorophyll in the culture. At a high content. Therefore, it can be safely mass-produced in the tank, and a high chlorophyll content can be maintained even without sunlight.

 Dark culture means heterotrophic culture in which sunlight is not used and organic compounds are used as a carbon source and an energy source.

 In the present invention, “high growth ability” means a growth rate of about 10 times or more at a culture period of about 3 days at about 30 ° C., more preferably about 15 times or more, and more preferably about 20 times. It means that the growth rate is more than double. Further, “high chlorophyll content” means that the total chlorophyll content is about 25 mg / g or more by dry weight, more preferably about 30 mg / g or more.

Measurement of proliferative capacity Proliferative capacity was determined by liquid medium (glucose 2%, NaNO ₃ 0.2%, MgSO ₄ · 7H ₂ O 0.02%, CaCl ₂ · 2H ₂ O 0.005%, ammonium iron citrate 0.002%, K ₂ HPO ₄ 0.08%, KH ₂ PO ₄ 0.02%, yeast extract 0.1%, A-5 solution ^* 0.125% dissolved in tap water) was measured at 30 ° C. by shaking culture.
A-5 solution ^* : H ₃ BO ₃ 2.86g, MnCl ₂ · 4H ₂ O 1.81g, ZnSO ₄ · 7H ₂ O 0.22g, CuSO ₄ · 5H ₂ O 0.08g, Na ₂ MoO ₄ 0.021g pure water 1 Dissolved in 1 liter and added 1 drop of concentrated sulfuric acid

Measurement of chlorella content The total chlorophyll content in the present invention was measured by a simple measurement method described in the Algae Research Method. The absorbance of the chlorella alga body methanol and acetone extract was measured at 650 nm and 665 nm, and the total amount of chlorophyll was calculated by the following formula.
Total amount of chlorophyll (μg / ml) = 25.5 x 650 nm absorbance measurement + 4.0 x 665 nm absorbance measurement

Collection from the field and establishment of the strain Green algae collected from the field were cultured in a medium for separation (2 g of potassium nitrate, 0.1 g of yeast extract, 30 g of calcium carbonate, 1 L of tap water) under low light for 2 weeks. This was planted on an agar medium for separation (sodium acetate 5 g, yeast extract 1 g, polypeptone 2 g, tap water 1 L, agar 15 g, pH 7.4), a single colony was collected, and a new agar medium was streaked. A single colony was picked again.
These colonies were grown on agar medium for growth (glucose 2%, NaNO ₃ 0.2%, MgSO ₄ · 7H ₂ O 0.02%, CaCl ₂ · 2H ₂ O 0.005%, ammonium iron citrate 0.002%, K ₂ HPO ₄ 0.08%, KH ₂ PO ₄ 0.02%, yeast extract 0.1%, A-5 solution 0.125%, agar 1.5% dissolved in tap water) were streaked and dark culture was performed. Large colonies and green ones were collected from the colonies that had grown. This was repeated several times under dark culture, and a single colony was transferred to a slant medium having the same composition as the agar medium for growth. After confirming that these strains were free from bacterial contamination in the bacterial growth medium, the storage slope medium (glucose 1.5%, NaNO ₃ 0.2%, MgSO ₄ · 7H ₂ O 0.02%, CaCl ₂ · 2H ₂ O 0.005%, Aqueous solution of ammonium ammonium citrate 0.002%, K ₂ HPO ₄ 0.08%, KH ₂ PO ₄ 0.02%, yeast extract 0.1%, A-5 solution 0.125%, agar 1.5% dissolved in tap water) at 30 ° C After 5 days of dark culture, it was stored at 14 ° C.

Medium The following growth medium was used.
Slope medium: glucose 2%, NaNO ₃ 0.2%, MgSO ₄ · 7H ₂ O 0.02%, CaCl ₂ · 2H ₂ O 0.005%, ammonium iron citrate 0.002%, K ₂ HPO ₄ 0.08%, KH ₂ PO ₄ 0.02% , Yeast extract 0.1%, A-5 solution 0.125% Agar 1.5%
Liquid medium: slant medium without agar

  Forty-two isolates were obtained by the above method. These 42 strains were cultured with shaking at 30 ° C. in the dark for 5 days, and on the first, fourth and fifth days, the color density and the cell amount of the cell pellet were evaluated relative to each other. Proliferation and color intensity were considerably different in each strain. By relative evaluation, we selected 5 strains that are both excellent. These five shares will be transferred to the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology as of April 28, 2004 or May 28, 2004, with accession numbers FERM P-20033, FERM P-20034, and FERM P, respectively. -20035, FERM P-20066 and FERM P-20067.

  The mycological properties of M15, M19, M34, M46 and M3 strains are shown below.

Morphological and staining single cells, spherical, 2.8-3.9 x 3.2-4.2μ in size. Has one cup-shaped chloroplast and one pyrenoid in the cell. It is dyed red with ruthenium red.

Proliferation (1) Growth temperature is 20-40 ° C, optimal temperature is 30 ° C. Growth pH 5-9, optimum pH is 7.2.
(2) Proliferate by forming 2-6 daughter cells by cell division by asexual reproduction.
(3) In light culture, photosynthesis is performed and it grows by autotrophic, but it grows also by heterotrophic using glucose as a carbon source in dark culture.
(4) It is much better to grow in heterotrophic culture than to grow by photosynthesis alone.
(5) When normal chlorella grows in dark culture, the amount of chlorophyll produced decreases, and the green color fades. However, these strains are also grown in light culture and dark green. The feature is that there is no inferiority and the production amount of chlorophyll does not fall.

All five strains are classified as Chlorella based on their morphological characteristics, stainability, growth pattern, and DNA analysis.
Classified as vulgaris.

  As a result of DNA analysis, the above 5 strains were different from any known strains, and thus were judged to be new strains and named as M15 strain, M19 strain, M34 strain, M46 strain and M3 strain. .

  In the following Examples 2 to 7, the growth ability, chlorophyll content, antioxidant activity, antimutagenicity, macrophage bactericidal activity for each of the M15, M19, M34, M46 and M3 strains The ability-promoting action and gene sequence were examined.

Growth potential Each strain of Chlorella was transferred from slant to liquid medium, and then cultured overnight in the dark before shaking. The solution was adjusted to 5.0 × 10 ⁷ / ml with a liquid medium, shake-cultured in the dark at 30 ° C. and 170 rpm, a part was collected every 24 h, the number of cells was counted, and the cell concentration was calculated. The experiment was performed twice.

The table of the culture time and the number of cells at the time of shaking culture is shown below (Table 1-1 shows the results of the first experiment and Table 1-2 shows the results of the second experiment).

When the average value of the growth rate after 72 hours is calculated from the above two measurement results, the M15 strain is 34 times, the M19 strain is 29 times, the M34 strain is 26 times, the M46 strain is 20 times, and the M3 strain is 22 times. Met. Thus, it was found that each of the above strains is a strain having excellent potentiating ability that can be sufficiently cultured in dark culture at about 30 ° C. for about 3 days. In particular, the M15 and M19 strains showed excellent growth rates.
This result is based on shaking culture in an Erlenmeyer flask, and it can be expected that the growth rate will be considerably higher when tank culture is performed with a concentrated medium.

  Growth curves were prepared for the above five strains (see FIG. 1). Comparing the 24th hour, in the first time (growth curve A), M 19, M 46, M 34, M 3, M 15, and in the second time (growth curve B), M 19, M 46, M 34 , M 15 and M 3 were proliferating in this order.

Measurement of the amount of chlorophyll 1) Extraction of chlorophyll Weighed chlorella dry powder was put in a mortar, 5 times the amount (weight) of quartz sand was added, and stirred uniformly. To this, 50 mM phosphate buffer, pH 7.5 was added, moistened and ground.
4 times the amount of methanol in the buffer was gradually added while grinding (80% methanol extraction), and the supernatant was placed in a volumetric flask. While further pulverizing, 80% methanol (50 mM phosphate buffer, pH 7.5) was added for extraction, and the supernatant was added to the volumetric flask. After performing this extraction operation again, it extracted similarly with the same amount of acetone, and added to the methanol extract.
2) Chlorophyll measurement
According to the method of Arnon et al. (Algae Research Method, Kazutoshi Nishizawa, Mitsuo Chihara, Kyoritsu Shuppan, 1979), it was calculated using the following formula.
Total amount of chlorophyll (μg / ml) = 25.5 x 650 nm absorbance measurement + 4.0 x 665 nm absorbance measurement

From the total amount of chlorophyll in the extract (μg / ml) calculated by the above formula, the total amount of chlorophyll per mg of chlorella algal body dry weight (mg / g) was determined by the following formula.
Total amount of chlorophyll per gram dry weight of chlorella algae (mg / g)
= Total chlorophyll in extract (μg / ml) ÷ 1000 × Extract (ml) ÷ Algae dry weight (g)
The results are summarized in Table 2.

  As shown in Table 2, all the above five strains had a total chlorophyll content of 30 mg / g or more by dry weight in dark culture. Thus, it was found that the above five strains produced a large amount of chlorophyll even in dark culture. In particular, the chlorophyll content of M15, M19 and M34 strains was high.

Measurement of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging ability of chlorella by antioxidant activity spectrophotometer

  Using freeze-dried chlorella algae, measurement was performed by a test tube method and a microplate method.

Test tube method
1) Chlorella 50 mg was extracted with 1 ml of 80% ethanol, and the extract was further diluted 5-fold with 80% ethanol to prepare a sample.
2) 50%, 100, 150, 200, 250, and 300 μl of 80% ethanol were added to 0.9 ml of an equal mixture of 400 μM DPPH, 200 mM MES buffer (pH 6.0) and 20% ethanol, respectively.
3) 250, 200, 150, 100, 50, 0 μl of each analytical sample was added every 30 seconds and vortexed.
4) Absorbance was sequentially measured at 520 nm in the order of addition 20 minutes after addition of the analysis sample.
All measurements were done in duplicate.

The results are shown in Table 3 and FIG. In proportion to the downward slope of the line graph in FIG. 2, the DPPH radical scavenging ability is high, that is, the antioxidant activity is high.

Microplate method
1) Chlorella 50 mg was extracted with 1 ml of 80% ethanol, and the extract was further diluted 5-fold with 80% ethanol to prepare a sample.
2) The analysis sample was diluted in two steps with 80% ethanol, and 50 μl of each diluted solution and 80% ethanol alone were added to a 96-well plate.
3) 150 μl of an equal volume of 400 μM DPPH, 200 mM MES buffer (pH 6.0), 20% ethanol was added to all wells except the blank.
4) The reaction solution was set in a plate reader, and the absorbance was measured at 540-630 nm 20 minutes after addition of the analysis sample.
All measurements were done in duplicate.

The results are shown in Table 4 and FIG. As in FIG. 2, the DPPH radical scavenging ability is high, that is, the antioxidant activity is high in proportion to the lower right of the line graph of FIG. 3.

  As shown in Tables 3 and 4 and FIGS. 2 and 3, all the strains were found to have antioxidant activity. Of these, M 15 and M 19 were found to be particularly strong.

Anti-mutagenicity test with chlorella extract An experiment was conducted using an extract of chlorella, Salmonella typhimurium TA98, and Trp-P2 (mutagenic substance).

[experimental method]
The chlorella extract was prepared by the following two methods.
1) 15 g of quartz sand was added to 3 g of chlorella algae, and 30 ml of DW was gradually added while grinding with a mortar. The mixture was placed on ice for 15 minutes and centrifuged at 6000 rpm at 10'4 ° C. The supernatant was collected and lyophilized. The lyophilized product was dissolved in DW at a concentration of 10 mg / ml and used for the AMES test.
2) To 50 mg of chlorella algae, 500 μl of 80% ethanol was added and placed on ice for 60 minutes, and then centrifuged at 6000 rpm for 2 ′ to collect the supernatant. Ethanol was blown off by air-drying and dissolved in DMSO at half the volume of the supernatant, and then DW in an amount equal to DMSO was added to prepare a sample.

The following mixtures were made:
(1) Sample extracted from quartz sand
A: Trp-2 (0.01 μg / 1 ml DMSO) 100 μl, Chlorella extract 100 μl (10 mg / ml)
B: DMSO 100μl, Chlorella extract 100μl (10mg / ml)
C: Trp-2 (0.01 μg / 1 ml DMSO) 100 μl, DW 100 μl
D: DMSO 100μl, DW 100μl
(2) Sample extracted with 80% ethanol
A: Trp-2 (0.02μg / 1ml DMSO) 50μl, Chlorella extract 100μl (10mg / ml)
B: DMSO 50μl, Chlorella extract 100μl (10mg / ml)
C: Trp-2 50 μl (0.02 μg / 1 ml DMSO), 50% DMSO (in DW) 100 μl
D: DMSO 50 μl, 50% DMSO (in DW) 100 μl

 Extraction sample (1) with quartz sand (1) contains 600 μl of 0.1M Na-PB and 100 μl of S-9 Mix. Extraction sample with 80% ethanol (2) contains 650 μl of 0.1M Na-PB and 100 μl of S-9 Mix. After the addition, 100 μl of Salmonella typhimurium TA98 culture solution that had been subjected to shaking culture for 18 hours was added, followed by shaking at 37 ° C. for 20 minutes.

  To each of the above mixtures, add 2 ml of Top agar (0.75 g of agar powder, 0.54 g of NaCl, 100 ml of deionized water, 10 ml of biotin-histidine solution), and a minimum glucose plate (30 g of agar powder, 1,860 ml of deionized water, 50 times concentrated Vogel- After seeding in Bonnner salt solution (40 ml, 40% glucose solution 100 ml), the cells were cultured at 37 ° C. for 2 days. The experiment was performed twice for each of the extraction sample (1) using quartz sand and the extraction sample (2) using ethanol.

The number of colonies was determined, and antimutagenicity (%) was determined by the following calculation method.

  The results are shown in Table 5 (quartz sand extract. Table 5-1 shows the first result, Table 5-2 shows the second result) and Table 6 (ethanol extract. Table 6-1 shows the first result, Table 5-2. 6-2 is shown in the second result). Moreover, the average value of 2 times about each extract is shown on a graph (refer FIG. 4. FIG. 4A is a quartz sand extract, FIG. 4B is an ethanol extract).

  As shown in Tables 5 and 6, and FIG. 4, antimutagenicity was observed in all strains. In particular, the extract with 80% ethanol showed high antimutagenicity. This suggests that the antimutagenic action is due to Chlorella pigments. In the comparison between strains, M 34 and M 3 were superior in the chlorella quartz sand extract, and M 15, M 34, and M 3 were superior in the ethanol extract.

Acceleration test of macrophage bactericidal ability Experiments were performed using chlorella algae, Salmonella enteritidis, and C57BL / 6CrSIc mice (female 7-9 weeks old).

(1) Mice were injected intraperitoneally with 2 ml of 4.05% (in DW) TGC medium (Nissui).
(2) Three days later, peritoneal exudate cells (PEC) were collected and adjusted to 2 × 10 ⁶ / ml with 10% FBS RPMI.
(3) PEC was seeded on a 24-well plate at 400 μl / well, and macrophages were allowed to attach at 37 ° C. in a 5% CO ₂ incubator for 1.5 hours.
(4) After removing floating cells by washing, the cells were conditioned for 1 day in a 37 ° C., 5% CO ₂ incubator.
(5) Add each chlorella alga body suspended in 10% FBS RPMI at 0.5 μg / ml or culture medium only as a control at 500 μl / well to wells with macrophages attached overnight. Cultured.
(6) After washing once, 1 × 10 ⁸ / ml of Salmonella enteritidis suspension was added at 250 μl / well, and macrophages were phagocytosed at 37 ° C. under 5% CO ₂ for 30 minutes. After phagocytosis, extracellular bacteria were washed away.
(7) After 0 and 60 minutes, macrophages were lysed with DW to collect intracellular bacteria.
(8) The collected bacteria were plated on a normal agar plate medium and the number of viable bacteria was counted.

  The experiment was conducted for the M15 strain, M3 strain, and M46 strain the first time, for the M19 strain and M34 strain for the second time, and for the M46 strain, M3 strain, and M19 strain the third time. Each sample was tested in duplicate. The sterilization rate for 60 minutes inside the cells was calculated from the number of viable bacteria counted, and the control ratio of each chlorella-treated cell when the control sterilization rate was 1 was determined. The results are shown in Table 7 (Table 7-1 shows the first time, Table 7-2 shows the second time, and Table 7-3 shows the third time). The results are summarized in FIG. 5 (average values were taken for the strains subjected to the two experiments).

  As shown in Table 7 and FIG. 5, the macrophages treated with the chlorella extract showed an increase in the bactericidal rate compared to the untreated control. The sterilization rate was high in M 34 and M 19, and M 46 was not much different from the control.

Analysis of Chlorella 18S rRNA Gene Sequence Genomic DNA was prepared from Chlorella M-15, M-19, M-34 and M-46 strains by extraction with benzyl chloride using ISOPLANT (Nippon Gene), respectively. Next, based on the sequence of the 18S ribosomal RNA gene of Chlorella described in Nakayama et al. (Phycological Reseach A 1996. 44: 47-55), oligonucleotide primer 1 (5'-tacctggttgatcctgccag-3 ') and primer 2 (5'-ccttccgcaggttcacctac-3 ') was synthesized. Using these primers, PCR was performed using TaKaRa LA Taq DNA polymerase (Takara Bio Inc.) with the genomic DNA (about 0.5 μg) prepared from each alga body as described above as a template. The PCR reaction was first incubated at 95 ° C for 6 minutes, then at 58 ° C for 1 minute, then at 68 ° C for 4 minutes, then at 95 ° C for 1 minute, then at 58 ° C for 1 minute, then at 68 ° C for 4 minutes. Incubation for 30 cycles was performed. As a result of agarose gel electrophoresis of the PCR product and staining with ethidium bromide, a DNA fragment of about 1700 bp was detected in any sample. After each gel part containing these fragments was excised, DNA was recovered from the gel part using QIAquick Gel Extraction Kit (Qiagen). The recovered DNA (about 1.0 μg) was inserted into pGEM-T Easy Vector (Promega) by the TA cloning method. After transducing E. coli strain HB101, the E. coli strain was grown in TB medium, and plasmid DNA in E. coli was recovered using the Qiagen Maxi EndoFree plasmid purification Kit. Using the recovered plasmid DNA as a template, primers (5'-tacctggttgatcctgccag-3 ', 5'-agggcaagtctggtgccag-3 ', 5'-ggattgacagattgagagagct-3', 5'-gcgcaaattacccaatcctg-3 ', 5'-gagtaatgattaagagggac-3', 5'-atagtgaggattgacagatt-3 ', 5'-ccttccgcaggttcacctac-3') A sequence sample was prepared by Sequencing Ready Reaction Kit (Perkin Elmer Japan), and this was applied to DNA sequencer PRIZM310 to analyze the base sequence. The base sequence of the obtained 18S ribosomal RNA gene was compared with the base sequence of the previously reported 18S ribosomal RNA gene of Chlorella green algae.

 As a result of analyzing the base sequence of the 18S ribosomal RNA gene of each strain, M15 is the base sequence shown in SEQ ID NO: 1, M19 is the base sequence shown in SEQ ID NO: 2, M34 is the base sequence shown in SEQ ID NO: 3, and M46 is It had the base sequence shown in SEQ ID NO: 4.

[Conclusion ]
From Examples 2 and 3, it was found that the new strain of chlorella according to the present invention exhibits very high growth ability and chlorophyll productivity even under dark culture. Among them, the M15 and M19 strains were found to have particularly excellent growth rates and chlorophyll contents.

  Moreover, from Example 4, it turned out that all strains have high anti-mutagenicity from Example 5, and all strains have high antimutagenicity. Because of its high antioxidant activity and antimutagenicity, these chlorella strains can be expected to have diabetes-improving effects, anti-tumor effects, etc., and can also be expected to increase the immune action by regulating the whole organism. . Furthermore, from Example 6, it was found that these chlorella strains promote the activity of macrophages that are immunocompetent cells to some extent. However, this degree was not so strong and calm. Therefore, even if the Chlorella strain according to the present invention is used as a food or the like, it can be expected that it does not act directly on the immune cells of normal individuals and can be used safely.

It is a line graph which shows the growth curve of Chlorella genus M15 strain | stump | stock, M19 strain | stump | stock, M34 strain | stump | stock, M46 strain | stump | stock, and M3 strain | stump | stock. A shows the growth curve of the first experiment, and B shows the growth curve of the second experiment. It is a line graph which shows the antioxidant activity of Chlorella genus M15 strain, M19 strain, M34 strain, M46 strain and M3 strain. It is a line graph which shows the antioxidant activity of Chlorella genus M15 strain, M19 strain, M34 strain, M46 strain and M3 strain. It is a bar graph which shows the antimutagenicity of Chlorella genus M15 strain, M19 strain, M34 strain, M46 strain and M3 strain extract. A shows the antimutagenicity of the extract from quartz sand and B the extract from ethanol. It is a bar graph which shows the macrophage bactericidal ability by the Chlorella genus M15 strain | stump | stock, M19 strain | stump | stock, M34 strain | stump | stock, M46 strain | stump | stock, and M3 strain | stump | stock extract. The partial sequence of ribosomal RNA of Chlorella sp. M15 strain is shown. The partial sequence of ribosomal RNA of Chlorella sp. M19 strain is shown. The partial sequence of ribosomal RNA of Chlorella sp. M34 strain is shown. The partial sequence of ribosomal RNA of Chlorella sp. M46 strain is shown.

SEQ ID NO: 1 Partial sequence of ribosomal RNA of Chlorella sp. M15 strain SEQ ID NO: 2 Partial sequence of ribosomal RNA of Chlorella sp. M19 strain SEQ ID NO: 3 Partial sequence of ribosomal RNA of Chlorella sp. Strain M34 SEQ ID NO: 4 of ribosomal RNA of Chlorella sp. M46 strain Partial array

Claims (6)

A chlorella strain having a high growth ability and high chlorophyll content under dark culture, which is selected from the group consisting of M15, M19, M34, M46 and M3. The natural mutant of the genus Chlorella according to claim 1, wherein the strain is Chlorella genus M15 (accession number: FERM P-20034). The natural mutant of the genus Chlorella according to claim 1, wherein the strain is Chlorella genus M19 (accession number: FERM P-20033). The natural mutant of the genus Chlorella according to claim 1, wherein the strain is Chlorella sp. M34 (accession number: FERM P-20066). The natural mutant of the genus Chlorella according to claim 1, wherein the strain is Chlorella sp. M46 (accession number: FERM P-20067). The natural mutant of the genus Chlorella according to claim 1, wherein the strain is Chlorella sp. M3 strain (Accession number: FERM P-20035).