WO2008153255A1

WO2008153255A1 - Spirulina platensis m20cjk3 [kctc11127bp] characterized by enhanced floatation of its algal clump

Info

Publication number: WO2008153255A1
Application number: PCT/KR2007/006146
Authority: WO
Inventors: Hee-Mock Oh; Choong-Jae Kim; Chan Yoo; Chi-Yong Ahn; Hee-Sik Kim
Original assignee: Korea Research Institute Of Bioscience And Biotechnology
Priority date: 2007-06-14
Filing date: 2007-11-30
Publication date: 2008-12-18
Also published as: KR100844189B1

Abstract

The present invention relates to Spirulina platensis M20CJK3 (KCTC 11127BP) having enhanced floatability of algal clumps. In particular, the present invention relates to a mutant strain Spirulina platensis M20CJK3 (KCTC 11127BP) which is prepared by treating cyanobacteria Spirulina platensis AG20590 (KCTC 11039BP) with a mutagen, EMS (ethylmethane sulfonate), wherein the cyanobacteria has been used as health supporting foods, physiologically active materials, feeds for livestocks, pets, etc. The mutant strain Spirulina platensis M20CJK3 of the present invention has enhanced floatability of algal clumps, leading to easy culture and economic harvest, and morphologically has a trichrome structure on which to wind coils close together.

Description

[DESCRIPTION] [Invention Title]

SPIRULINA PLATENSIS M20CJK3 [KCTC11127BP] CHARACTERIZED BY ENHANCED FLOATATION OF ITS ALGAL CLUMP

[Technical Field]

The present invention relates to Spirulina platensis M20CJK3 (KCTC 11127BP) having enhanced floatability of algal clumps. In particular, the present invention relates to a mutant strain Spirulina platensis M20CJK3 (KCTC 11127BP) which is prepared by treating cyanobacteria Spirulina platensis AG20590 (KCTC 11039BP) with a mutagen, EMS (ethylmethane sulfonate), wherein the cyanobacteria is used as health supporting foods, physiologically active materials, feeds for livestocks, pets, etc. The mutant strain Spirulina platensis M20CJK3 of the present invention has enhanced floatability of algal clumps, thus providing easy and cost-effective culture, and also has a trichrome structure with tightly wound coils.

[Background Art]

Spirulina is filamentous monocellular cyanobacteria and is characterised by its active proliferation under an alkaline environment where tropical and subtropical bicarbonates are abundant. Since Spirulina is rich in protein (60~70%), vitamins (vitamin Bl 2, provitamin A), essential amino acids, minerals, essential fatty acid- containing and unsaturated fatty acids GLA (gamma-linolenic acid), it can be used as a blood circulating agent and an immunopotentiator. Thus, it has been widely used as feed supplements for pets and marine farming, and health supporting foods [Belay, A., 2004, The potential application of Spirulina (Arthrospira) as a nutritional and therapeutic supplement in health management].

Spirulina forms a long, coil-shaped trichromes where the distance between coils is in the range of from 10 to 70 μm. Since Spirulina has intracellular air vesicles, it floats on the surface of the sea during the day and sinks to the depths at night. A domestic cyanobacteria strain, Spirulina AG20590 (KCTC 11039BP) is characterized by a loosely coiled morphology (Fig. 1) and very low floatability. In the case of mass production, such a low floatability of Spirulina makes it difficult to effectively and economically harvest. If the floatability is insignificant, the expense of harvesting the mass-produced cells is greatly increased. Further, the Spirulina strain with tightly wound coils has an advantage over that with loosely wound coils when culturing it in large quantity. That is, in case of tight coiling, the length of trichome becomes very short, thus reducing the self-shading effect caused by algal clumps in high density culture, and thus enables culturing of the Spirulina strain at a relatively higher concentration. [Disclosure] [Technical Problem]

The inventors of the present invention have endeavored to overcome the problems of the above-mentioned Spirulina stain, and finally succeeded in developing Spirulina platensis M20CJK3 (KCTC 11127BP), which is suitable for preparing a large scale culture, by improving the algal clump's floatability and morphological improvement by treating a local strain Spirulina platensis AG20590 (KCTC 11039BP) with a mutagen EMS (ethylmethane sulfonate).

Therefore, the object of the present invention is to provide Spirulina platensis M20CJK3 (KCTC 11127BP) suitable for large scale culture by improving the algal clump's floatability and morphological features.

[Description of Drawings]

Arrangements and embodiments may be described in detail with reference to the following drawings wherein:

Fig. 1 is a microscopic picture showing the morphological feature of Spirulina platensis AG20590 (KCTC 11039BP) (200^χ magnification);

Fig. 2 is a result of comparison between the nucleotide sequence of cpcBA-IGS derived from a mutant strain Spirulina platensis M20CJK3 (KCTC 11127BP) and that derived from a control strain Spirulina platensis AG20590 (KCTC 11039BP) (mismatch parts of the sequence are underlined);

Fig. 3 is a graph comparing the floatability of Sγirulina platensis M20CJK3 (KCTC 11127BP), a mutant strain, with that of Sγirulina platensis AG20590 (KCTC 11039BP); Fig. 4 is a picture showing the floatability of Sγirulina platensis M20CJK3 (KCTC

11127BP), a mutant strain, with that of Spirulina platensis AG20590 (KCTC 11039BP) after 1 hr of stationary culture (A: algal clumps immediately after stationary culture; B: algal clumps after 1 hr of stationary culture);

Fig. 5 is a graph showing the change in the number of coils per trichome through the culture of a control strain Spirulina platensis AG20590 (KCTC 11039BP) and a mutant strain Spirulina platensis M20CJK3 (KCTC 11127BP);

Fig. 6 is a graph comparing the distance between adjacent coils (coil pitch) of a mutant strain Spirulina platensis M20CJK3 (KCTC 11127BP) with that of a control strain Spirulina platensis AG20590 (KCTC 11039BP); and Fig. 7 is a picture comparing the morphological feature of Spirulina platensis

M20CJK3 (KCTC 11127BP), a mutant strain, with that of a control strain Spirulina platensis AG20590 (KCTC 11039BP).

[Best Mode] The present invention relates to a mutant stain Sγirulina γlatensis M20CJK3 (KCTC 11127BP) suitable for large scale culture through the enhancement of algal clump's floatability and morphological improvement.

Hereinafter, the present invention is described in more detail. The present invention relates to a mutant strain Spirulina γlatensis M20CJK3

(KCTC 11127BP) which is prepared by treating cyanobacteria Spirulina γlatensis AG20590 (KCTC 11039BP) with a mutagen, EMS (ethylmethane sulfonate), wherein the cyanobacteria is used as health supporting foods, physiologically active materials, feeds for livestocks, pets, etc. The mutant strain Sγirulina γlatensis M20CJK3 of the present invention has improved floatability of algal clumps, thus providing easy and cost-effective culture and collection, and has a trichrome structure with tightly wound coils.

A local strain Sγirulina AG20590 (KCTC 11039BP) was treated with a mutagen

EMS at a concentration of 0.24% (v/v), spread onto an agar plate and cultured for a perdetermined period of time. After the culture, colonies that grew on the medium were picked, cultured in a liquid medium, and then the floatability of algal clumps and morphological features were examined.

Thus selected mutant strain Sγirulina γlatensis M20CJK3 exhibited at least 3-fold higher floatability of algal clumps than Sγirulina γlatensis AG20590 (KCTC 11039BP), had a small number of coils per trichome, and showed a tendency to grow shorter the distance between adjacent coils as the culture was in progress.

Spirulina γlatensis M20CJK3 according to the present invention has the following characteristics: floatability (%) of algal clumps is 60~99%, the number of coils per trichome is 5~7.5, and a coil pitch is 1~55 μm long. Therefore, Spirulina γlatensis

M20CJK3 has superior characteristics over to Spirulina platensis AG20590 (KCTC

11039BP).

The mutant strain obtained by EMS treatment according to the present invention was named Spirulina platensis M20CJK3 and deposited to the Korean Collection for Type Cultures (KCTC) in Korea Research Institute of Bioscience and Biotechnology (KRIBB) on May 4, 2007 under the accession number KCTC 11127BP.

The mutant strain Spirulina platensis M20CJK3 (KCTC 11127BP) having such characteristics as described above has several advantages that its high floatability of algal clumps provides an easy and cost-effective method in case of conducting a large scale culture, and the short coil pitch of trichome can reduce the self -shading effect of algal clumps, thereby enabling a high density culture, and increasing productivity therefrom. Further, the improved floatability, more tightly coiled morphological feature and shorter trichome of Spirulina platensis M20CJK3 (KCTC

11127BP) as compared to those of Spirulina platensis AG20590 (KCTC 11039BP) are considered very advantageous to be used as a model for molecular biology study. The present invention will now be described in detail with reference to the following examples, however, they should not be construed as limiting the scope of the present invention.

Example 1: EMS treatment for mutation of Spirulina platensis AG20590 (KCTC 11039BP) and viability test

A Spirulina strain used in the following experiment was Spirulina platensis AG20590 (KCTC 11039BP) which was obtained from the Korean Collection for Type Cultures.

As described in Fig. 1, the strain has a loosely coiled shape of trichome and exhibits insignificant floatability in a stationary culture. Spirulina platensis AG20590 (KCTC 11039BP) was cultured in a SOT liquid medium having the following composition described in Table 1 until the cells entered the stationary phase of growth, and then, its algal clumps were collected. [Table 1]

Composition of a SOT medium used for the culture of Spirulina platensis

Thus collected algal clumps were centrifuged three times at 10,000 rpm for 5 min and washed with distilled water and suspended in distilled water. After the suspension was treated by ultrasonication in an ice-bath for 10 sec to cut the long trichomes into shorter ones, EMS was added thereto at a concentration of 0.24% (v/v). The resulting suspension was distributed to a microtube (1.5 mL) by 1 mL, and 3 microtubes were assigned as a repetitive group. Each microtube was incubated in a water bath kept at 35 ^°C for 20 min to induce mutation by EMS. A control was according to the same method as the repetitive group (experimental group) except for the EMS treatment. After the above incubation, the microtube was centrifuged at 10,000 rpm for 5 min to remove supernatant. To thus separated pellet was added 1 mL of 5% sodium thiosulfate to terminate the mutation reaction and the mixture was centrifuged at 10,000 rpm for 5 min and the resulting supernatant was removed. To remove EMS and sodium thiosulfate, 1 mL of the SOT medium was added to the pellet and centrifuged at 10,000 rpm for 5 min. The entire procedure was repeated 3 times and the resulting pellet was resuspended in 1 mL of SOT medium and cultured at 30 ^°C for 3 days under 24 hr-light condition. 100 μL of the culture solution was collected at each growth phase and observed its viability under a microscope. To separate viable cells, the culture was spread on a 1.5% agar plate containing the SOT medium. Colonies that grew on the medium were picked and cultured in 10 mL of a liquid SOT medium.

After culturing for 3 days following the treatment of a mutagen, the number of cells in the control was 740 cells/ mL, while that in the experimental group was 40 cells/mL, thus showing 5% of viability.

Example 2: Characteristics of mutant strain M20CJK3 (KCTC 11127BP)

The mutant strain M20CJK3, which was prepared by treating Spirulina platensis AG20590 (KCTC 11039BP) obtained from the Korean Collection for Type Cultures with EMS, belongs to Spirulina platensis with a genetic mutation occurred in a specific gene domain. Morphological features of Spirulina platensis M20CJK3 are shown in Table 2. Further, Spirulina platensis M20CJK3 was identified according to a molecular biological identification technique, and the result thereof is shown in Table 3. As illustrated in Tables 2 and 3, the mutant strain Spirulina platensis M20CJK3 exhibited significantly different morphological features from Spirulina platensis AG20590. As a result of analyzing a phycocyanin-related gene, a characteristic dye of cyanobacteria, the mutant strain showed 97.5% sequence homology with the wild type AG20590, showing that there is indeed a little difference between two strains (Table 3, Fig. 2). [Table 2] Morphological features of Spirulina platensis M20CJK3 (KCTC 11127BP)

[Table 3] Molecular biological identification of Spirulina platensis M20CJK3 (KCTC 11127BP)

Example 3: Floatability and morphological analysis of Spirulina platensis M20CJK3 (KCTC 11127BP) Spirulina platensis AG20590 (KCTC 11039BP) was treated with EMS at a concentration of 0.24% for 20 min, to thereby separate 40 strains. Thus separated strains had common characteristics that they can float even in stationary culture conditions due to their high floatability and morphologically form tightly coiled trichomes. Therefore, a representative strain of them was selected as an experimental group and subjected to analyses of floatability and morphological features as compared with a control strain Spirulina platensis AG20590 (KCTC11039BP). The strain used as an experimental group was a mutant strain Spirulina platensis M20CJK3 (KCTC 11127BP).

Each of AG20590 (KCTC 11039BP) and M20CJK3 (KCTC 11127BP) culture solutions cultured in a test tube was inoculated in a conical flask filled with 150 mL of SOT medium and cultured for 10 days. Thus obtained culture solution was inoculated in a 2 L glass container filled with 1.2 L of the SOT medium as an inoculum. The culture was carried out at 30 ± 2°C under light condition with luminance of 200 μmol photons/ m²/s. During the culture, the culture solution was stirred by using a magnetic bar, and air was injected thereinto at air pressure of 2.5 L/min for smooth circulation. Floatability of algal clumps, coil morphology, a coil pitch and a number of coils per trichome were measured at intervals of 2 days during the culture. The floatability of algal clumps was measured as follows: collecting 6 mL of algal clumps from the culture solution, transferring it to a 15 mL plastic container, stationary culturing for 1 hr at light condition of 200 μmol photons/ m²/ s, and comparing an initial absorbance at 680 ran with an absorbance 1 hr after the stationary culture at 680 nm. The absorbance 1 hr after the stationary culture was measured after removing 4 mL of the culture solution from the upper portion of the container, and mixing the remaining algal clumps. The floatability of algal clumps was calculated by the following Equation 1. [Equation 1] Floatability(%) = ( 1 - later OD/ initial OD) x 100 (1)

Coil morphology was observed with a microscope, the distance between adjacent coils was determined by observing 50 or more of trichomes at 200^χ magnification and calculating an average thereof. The number of coils was determined as an average of 50 trichomes. As shown in Fig. 3, the floatability of the mutant strain Spirulina platensis M20CJK3 (KCTC 11127BP) was started from - 9.3%, drastically increased to 82% on the second day of the culture, and after that, gradually decreased to 71% on the eighth day. In the case of the control strain Spirulina platensis AG20590 (KCTC 11039BP), its initial floatability being -65%, they mostly sank to the lower part. As the culture progressed, they exhibited the maximum floatability of -34% on the sixth day, but have a tendency to sink rather than float on the whole. Fig. 4 is a picture comparing the floatability of two strains on the sixth day of culture. At an initial stage (A of Fig. 4), both strains were distributed throughout the flask container, but in an hour (B of Fig. 4), while 75% of the mutant strain Spirulina platensis M20CJK3 (KCTC 11127BP) floated, the control strain Spirulina platensis AG20590 (KCTC 11039BP) showed low floatability while still distributed throughout.

The number of coils per trichome reflects the growth state of a given cell culture. If growth conditions are favorable, the number of coils per trichome is generally increased, while if cells enter the stationary phase, it is decreased and the length of trichome is shortened. As illustrated in Fig. 5, in the case of Spirulina platensis AG20590 (KCTC 11039BP), the number of coils per trichome was gradually increased during the course of culture, reached the maximum number of 4.9 coils/ trichome on the fourth day, and declined thereafter. On the other hand, in the case of Spirulina platensis M20CJK3 (KCTC 11127BP), it exhibited a smaller number of coils than Spirulina platensis AG20590 (KCTC 11039BP), reached the maximum number of 3.4 coils/ trichome on the fourth day, and then, declined. On the sixth day of culture, the decreased number of coils in both strains was because the number of short trichomes was increased due to the cutting of long trichomes. In the case of Spirulina platensis AG20590 (KCTC 11039BP), its coil pitch was 87.0 μm at the initial stage of culture, increased to 127.6 μm on the fourth day of culture, and then, showed a tendency to decline. On the other hand, in the case of Spirulina platensis M20CJK3 (KCTC 11127BP), 51.4 μm of the initial coil pitch was decreased to 5.5 μm on the sixth day of culture, which was contrary to the pattern of Spirulina platensis AG20590 (KCTC 11039BP) (Fig. 6).

Fig. 7 is a picture of comparing the morphology of both strains on the sixth day of culture. While Spirulina platensis AG20590 (KCTC 11039BP) had loose coils and lengthened trichomes, Spirulina platensis M20CJK3 (KCTC 11127BP) had tight coils and relatively short trichomes. There was a distinct difference between the mutant strain Spirulina platensis M20CJK3 (KCTC 11127BP) and the control strain Spirulina platensis AG20590 (KCTC 11039BP) in terms of floatability and coil morphology.

[Industrial Applicability]

As described above, the mutant strain Spirulina platensis M20CJK3 (KCTC 11127BP) of the present invention shows at least 3-fold higher floatability than Spirulina platensis AG20590 (KCTC 11039BP), has a small number of coils per trichome, and the distance between adjacent coils becomes shorter as the culture progresses. The mutant strain Spirulina platensis M20CJK3 (KCTC 11127BP), with the aforementioned characteristics, has several advantages that its high floatability of algal clumps provides an easy and economical collection in case of preparing a large scale culture, and the short coil pitch of trichome reduces the self-shading effect of algal clumps, thus enabling a high density culture and increasing productivity therefrom. Further, the high floatability, tightly coiled morphological feature and shorter trichome of Spirulina platensis M20CJK3 (KCTC 11127BP) than those of Spirulina platensis AG20590 (KCTC 11039BP) make it useful as a model for a molecular biology study.

Claims

[CLAIMS] [Claim 1]

Spirulina platensis M20CJK3 (KCTC 11127BP) having enhanced floatability of algal clumps.

[Claim 2]

Spirulina platensis M20CJK3 (KCTC 11127BP) according to Claim 1, which has floating activity of algal clumps in the range of from 60 to 99%.

[Claim 3]

Spirulina platensis M20CJK3 (KCTC 11127BP) according to Claim 1, which has 5 to 7.5 coils/ trichome.

[Claim 4] Spirulina platensis M20CJK3 (KCTC 11127BP) according to Claim 1, which has the distance between adjacent coils (coil pitch) in the range of from 1 to 55 μm.