KR101121159B1 - Novel Cryoprotective Exopolysaccharide - Google Patents

Abstract

The present invention Pseudoaltermonas Pseudoaltermonas arctica) KOPRI 21653 and relates to the strain-derived exopolysaccharide (exopolysaccharide), more detail is seen in Pseudomonas Pseudomonas Alter urticae (Pseudoaltermonas arctica ) KOPRI 21653 and a novel exopolysaccharide having the anti-freezing ability derived from the strain, a method for producing the same and an anti-freezing composition containing the same.
The novel exopolysaccharides and antifreeze compositions containing them are very useful for protecting or preserving biological samples, including cells and tissues, in a refrigerated, frozen and vitrified state with minimal damage to the tissues.

Description

Novel Cryoprotective Exopolysaccharide with Antifreezing Activity {Novel Cryoprotective Exopolysaccharide}

The present invention Pseudoaltermonas arctica ) KOPRI 21653 and a novel exopolysaccharide having anti-freezing activity derived from the strain, a preparation method thereof, and an anti-freezing composition containing the same.

Exopolysaccharide is a polymer composed of sugar molecule units produced by microorganisms.It is glucose, galactose, rhamnose, fucose, and acetyl galactose. Acetylated galactose), acetyl-galactosamine (N-acetyl-galactosamine) is composed of a single sugar molecule to form homopolysaccharides (Homopolysaccharides), or it is classified as heteropolysaccharides (heteropolysacchardes) composed of several kinds of sugar molecules.

In general, exopolysaccharides are secreted to the outside of the cells and surround the cells to protect the cells from environmental growth inhibition requirements due to humidity and temperature and antagonistic mechanisms including toxic substances. It is involved in sequestering, colonizing, or recognizing as homologous cells (Roberts, IS, Annu . Rev. Microbio ., 50:285, 1996; DeVuyst, L. et al. al . , Int . Dairy J. , 11:687, 2001; Krinos, CM et al . , Nature , 414:555, 2001; Looijesteijn, PJ et al . , Int . J. Food Sci . , 64:71, 2001). In addition, it has high adhesion to form a biofilm, and Escherichia coli , Salmonella and Haemophilus Pathogenic Gram-negative bacteria including influenza play a role in conferring pathogenicity (Cerning, J., FEMS Microbiol . Rev. , 87:113, 1990).

The physiological, rheological, and chemical properties of exopolysaccharides differ depending on the length of the polymer chain that determines the molecular weight. The longer the polymer, the more active internal bonds between polymer molecules occur. Determine the quaternary structure and physical properties (Sutherland, IW, Biotechnol . Adv ., 12:393, 1994).

In extreme climatic environments such as the Arctic and Antarctic, the cycle of freezing and thawing occurs continuously, so microorganisms, some diatoms, and cyanobacteria living in this area are large amounts of exopolysaccharides. ) To form a thick gel around the cells to protect the cells, thereby adapting to the harsh environment that is fatal to cell growth (Palmisano, AC and Sullivan, CW, Growth , Metabolism and Dark Survival in Sea Ice Microalgae , p131-146, CRC Press, 1985; Cooksey, KE and Cooksey, B., Aqua . Micro . Ecolo . , 9:87, 1995; Costerton, JW, et al . , Microbiol . , 49:711, 1995; Stoderegger, K. and Herndl, G., Limnol . Oceanol . , 43:877, 1998).

Phomaherbarum , a fungus isolated from Antarctic soil, secretes 7.4 x 10 ⁶ kDa glucose homosaccharide, and this exopolysaccharide is believed to have a function as a cryoprotectant in the cold of Antarctica. (Selbmann, L. et al . , Res . Microbiol ., 153:585, 2002).

Anti-freezing substances are substrates that protect body tissues from tissue damage caused by the formation of ice crystals caused by low temperatures. Insects, fish, amphibians and reptiles living in polar regions produce these substrates in the body to prevent tissue destruction in winter. Insects use sugar as an anti-freezing substrate, and polar frogs use glucose as an anti-freezing substrate.

Some anti-freezing substances have a function of lowering the glass transition temperature of a solution or substance to prevent freezing, and form a hydrogen bond with a biomolecule to replace the binding site with a water molecule. With this principle, proteins or DNA are preserved while maintaining the unique structure of the molecule by hydrogen bonding.

Antifreeze substances are used in automobile antifreeze, cell preservation, and food preservation, but some antifreeze substances are toxic despite their excellent antifreeze ability, so rather than single use, other antifreeze substrates are used in combination. When added to food, sucrose is mainly used, which is not toxic.

Conventional cryoprotectants used industrially include glycols including ethylene glycol, propylene glycol, and glycerol, and in the case of ethylene glycol, As an antifreeze for automobiles, propylene glycol is also used as an emulsifier to suppress ice formation when making ice cream, and glycerol or dimethyl sulfoxide (DMSO) is used for cell preservation. This material minimizes the moisture content of the cells, allows vitrification to proceed, and improves the protective function of the intracellular polymer (Dumont, F. et. al . , Cryobiol , 46:33, 2003; Ablett, S. et al . , J. Chem . Soc . Faraday Trans., 88:789, 1992; Adam, MM et al . , Cryobiol . , 32:92, 1994; Anchordoguy, TJ et al . , Cryobiol . , 24:324, 1987).

In recent years, in particular, as interest in cryopreservation of cells has increased due to the improvement of medical technology such as stem cell research, transplantation and cell therapy technology, cryopreservation aims at 100% preservation technology in a freeze-thaw environment for various cells. Technology development is actively in progress (Albrecht, RM et al . , Cryobiol . , 10:233, 1973; Dumont, F. et al . , Cryobiol. , 46:33, 2003; Hubalek, Z., Cryobiol . , 46:205, 2003), therefore, the development of novel anti-freezing substances that are environmentally friendly and have low toxicity to cells is constantly required.

Accordingly, the present inventors isolated exopolysaccharide from Pseudoalteromonas arctica isolated from the soil of Antarctica, and measured cell viability in an environment where E. coli is frozen and thawed using the same. As a result, cell viability was measured. The present invention was completed by confirming that the storage power was excellent.

An object of the present invention is Pseudoaltermonas altica (Pseudoaltermonas) having exopolysaccharide production ability arctica ) KOPRI 21653 and to provide a method for producing an exopolysaccharide using the strain.

Another object of the present invention is to provide an anti-freezing composition containing the strain-derived exopolysaccharide and the exopolysaccharide.

In order to achieve the above object, the present invention is Pseudoaltermonas altica (Pseudoaltermonas) having exopolysaccharide producing ability. arctica ) KOPRI 21653 KCTC 11233BP is provided.

The present invention also provides an exopolysaccharide having anti-freezing ability, having a glucose:galatose of about 1.5:1 (molar ratio).

The present invention is also, (a) Pseudoalteromonas altica (Pseudoalteromonas arctica ) culturing KOPRI 21653; (b) adding alcohol to the culture medium of the microorganism to obtain a precipitate; (c) removing proteins from the precipitate and then reprecipitating by adding alcohol; And (d) dialysis of the reprecipitate and then collecting the material in the dialysis membrane.

The present invention also provides a composition for anti-freezing containing the exopolysaccharide.

The exopolysaccharide according to the present invention and the anti-freezing composition containing the same exhibit excellent cell preservation effects at low temperatures, so that biological samples including cells and tissues are refrigerated, frozen, and vitrified while minimizing tissue damage. Very useful for protection or preservation.

1 is a Pseudoalteromonas altica (Pseudoalteromonas arctica) with pseudo Alter contains KOPRI 21653 Monastery in (Pseudoalteromonas sp .)
Figure 2 shows a scanning electron micrograph of E. coli (a: E. coli not treated with P-21653 ; b: E treated with 0.1 (w/v)% P-21653) . coli)
3 shows the results of measuring the survival rate of E. coli after each treatment with 0.2% (w/v) crude exopolysaccharide derived from microorganisms isolated from the soil.
4 shows the results of GC/MS (Gas chromatography/Mass Selective Detector) analysis for confirming the sugar composition of the new exopolysaccharide.
5 is a confocal fluorescence micrograph showing the survival degree of E. coli measured using the LIVE/DEAD BacLight™ Bacterial Viablity kit.
6 shows the results of measuring the survival rate of E. coli after repeating a number of freeze-thawing in a state treated with various concentrations of P-21653.
Figure 7 shows the results of comparing the anti-freezing ability of the conventional anti-freezing substrate and P-21653.

In one aspect, the present invention, Pseudoalteromonas altica having exopolysaccharide production ability ( Pseudoalteromonas arctica ) KOPRI 21653 KCTC 11233BP and glucose: galactose is about 1.5:1 (molar ratio) to an exopolysaccharide having an anti-freezing ability (cryoprotective) (exopolysaccharide).

As used herein, the term'antifreeze activity' refers to the ability to protect against damage caused by ice crystal formation in tissues or molecules at low temperatures, and this effect can be obtained by exposure to a substrate having antifreeze activity.

As used herein, the term'vitrification' is a solidified state in which crystals are not formed in a liquid state but supercooled.

Pseudoaltermonas altica of the present invention (Pseudoaltermonas arctica ) KOPRI 21653 is a microorganism isolated from soil in Antarctica. Microorganisms that secrete exopolysaccharides were selected for a number of microorganisms isolated from soil samples in the Antarctic region. Among them, strains with excellent exopolysaccharide production and antifreezing ability against E. coli were isolated. Sequencing was performed. As a result, the isolated strain was Pseudoaltermonas altica (Pseudoaltermonas arctica ) It was found to be 100% consistent with the 16S rRNA of ^{A37-1-2 T.}

The exopolysaccharide of the present invention is Pseudoaltermonas altica isolated from Antarctic soil. arctica ) It is produced in KOPRI 21653 and secreted to the outside of the cells, and is a heteropolysaccharide composed of a mixture of glucose and galactose in a ratio of about 1.5:1. The exopolysaccharide has excellent anti-freezing ability compared to conventional commercial anti-freezing agents in anti-freezing ability.

As a result of mixing the solution in which E. coli was suspended and the diluent of 0.001 (w/v)% to 1 (w/v)% of the exopolysaccharide of the present invention, the cells were repeatedly freeze-thaw 3 to 7 times, When the content of the exopolysaccharide of the present invention is increased, the survival rate of E. coli that has undergone freeze-thaw increased from 18.41±0.42% to 100.57±6.7%, and also freeze-thaw up to 7 times. Under the repeated conditions, the antifreeze activity against the same amount of exopolysaccharide was decreased by 30% to 40% compared to the case of 5 freeze-thaw, but specifically the content of exopolysaccharide. In the case of 0.5% to 1%, there was no significant effect.

In addition, as a result of comparing the antifreeze activity of the exopolysaccharide of the present invention with the antifreeze agent commercialized under the same freeze-thaw conditions as described above for E. coli, the exopolysaccharide of 0.1 (w/v)% or more was excellent. It was analyzed as having antifreeze activity.

The present invention from another viewpoint, (a) Pseudoalteromonas altica (Pseudoalteromonas arctica) culturing KOPRI 21653; (b) adding alcohol to the culture medium of the microorganism to obtain a precipitate; (c) removing proteins from the precipitate and then reprecipitating by adding alcohol; And (d) dialysis of the reprecipitate, and then collecting the material in the dialysis membrane.

In the manufacturing method of the present invention, it is preferable to additionally add 10% CPC (cetylpyridinium chloride) during the reprecipitation of step (c).

The process of culturing the strain to prepare the exopolysaccharide of the present invention may be performed according to a suitable medium and culture conditions known in the art. Such a culture process can be easily adjusted and used by those skilled in the art according to the culture conditions selected. Examples of the culture method include, but are not limited to, batch, continuous, and fed-batch culture.

After culturing the microorganism, a method of separating and purifying the desired exopolysaccharide may be isolated and purified by a conventional method known in the art. Methods such as precipitation, centrifugation, filtration, exchange chromatography, and lyophilization may be used for such separation and purification methods. For example, after centrifugation to remove biomass, alcohol and/or metal ions are added to the microbial culture to precipitate polysaccharides, a process of removing proteins from the obtained precipitate can be added. However, it is not limited to these examples.

In another aspect, the present invention relates to an anti-freezing composition containing the exopolysaccharide.

It is preferable that the anti-freezing composition of the present invention contains 0.0001% to 50% by weight of the exopolysaccharide.

The anti-freezing composition of the present invention protects the activity of the sample while the refrigeration, freezing, or vitrification of a biological sample including cells, embryos, human or animal-derived tissues, microorganisms, plants, etc. is maintained ( It can be used for cryoprotection and preservation.

In addition to the exopolysaccharide of the present invention, the anti-freezing composition includes conventional EBS (Earle's Buffered salts), CZB, T6, Earle's MTF, KSOM, SOF medium, in order to maintain the optimal refrigeration state of biological samples in addition to the exopolysaccharide of the present invention. A commercially available balanced salt media (BSM) such as tyrode's albumin lactate phosphate (TALP), non-electrolyte, citric acid, magnesium kelate, or an anion having a high molecular weight may be included.

In addition to the exopolysaccharide of the present invention, the anti-freezing composition is used to prevent acidosis, generation of free radicals in cells, and contracture that may occur during refrigeration of biological samples in addition to the exopolysaccharide of the present invention. It may contain a buffer solution (Buffer), mannitol (manitol), glutathione (glutathione), glutamate, and the like, but is not limited thereto.

In addition to the exopolysaccharide of the present invention, the composition for anti-freezing of the present invention is DMSO, glycerol, propylene glycol, ethylene glycol, propanediol, Antifreezing agents such as dimethylformamide and acetamide may be additionally included.

The anti-freezing composition of the present invention may additionally include polyvinyl alcohol, polyvinyl pyrrolidine, fish or plant-derived anti-freezing proteins, carboxymethylcellulose, serum albumin, hydroxyethyl starch ( hydroxylethyl starch), Ficoll, dextran, gelatin, milk protein, lipid vesicles, and permeating agents such as lecithin, but are limited thereto. no.

A method of preserving a biological sample by refrigerating, freezing, or vitrifying using the anti-freezing composition of the present invention can be performed using a conventional technique in the art.

Example

Hereinafter, the present invention will be described in more detail by examples. These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited to these examples.

Isolation and identification of microorganisms

Coastal soils and sediments collected from King George Island's Barton Peninsula were diluted in saline (0.85 NaCl), followed by ZoBell medium (Zobell, CE, Marine Microbiology XV , p240, Chronica Botanica, 1946). And cultured at 25° C. for 3 days. After incubation, only the colonies producing the mucous polymer were selected, suspended in 20% (v/v) glycerol, and stored at -80°C. In order to isolate the strain producing exopolysaccharide, the frozen colonies were inoculated in 20 mL of Zobell medium, cultured with shaking at 120 rpm at 25°C for 3 days, and centrifuged at 10,000 g for 20 minutes to remove the cells. Only the removed supernatant was collected, and ethanol was added in an amount of twice the volume to the supernatant and left at 4° C. for 24 hours to precipitate the polymer complex.

After extracting DNA from cells selected with a precipitation polymer using AccuPrep genomic DNA extraction kit (Bioneer, Korea), 27F and 1492R primer sets (Lane, DJ, Nucleic Acid Techniques in Bacterial Systematics , p. 115, Wiley, 1991) was used to amplify 16S rRNA from bacterial DNA, and the nucleotide sequence of the obtained PCR product was analyzed.

27F: 5'-AGAGTTTGATC(C/A)TGGCTCAG-3'

1492R: 5'-GGTTACCTTGTTACGACTT-3'

The analyzed nucleotide sequence was first sequenced using CLUSTALW (Thompson et al, 1994), a multi-sequence nucleotide analysis tool, and analyzed based on the secondary structure information of 16S rRNA using PHYDIT (Chun, 1995). The structure of the base sequence was estimated. Phylogenetic analysis was performed using PHYLIP (Felsenstein, 1993), and a phylogenetic tree using Kimura's two-parameter model and neighboring coupling method was prepared (Saitou and Nei, 1987). The phylogenetic tree shape derived from the above process was evaluated by bootstrap analysis of neighboring bonds.

It was confirmed that the exopolysaccharide-producing strain produced mucous polysaccharides in 7 strains from about 25 strains that were found to secrete mucous substances based on the thickness of the precipitated polymer complex. Ride-producing strain KOPRI 21653 was isolated (Table 1).

Strain name Derived sample Collection location KOPRI 21650 Sediment King George Island, 62° 13'S, 58° 47'W KOPRI 21653 Sediment King George Island, 62° 14'S, 58° 44'W KOPRI 21654 Seaside soil King George Island, 62° 13'S, 58° 44'W KOPRI 21663 Sediment King George Island, 62° 13'S, 58° 46'W EP 144 Seaside soil King George Island, 62° 13'S, 58° 47'W EP 205 Seaside soil King George Island, 62° 13'S, 58° 47'W EZ 291 Seaside soil King George Island, 62° 14'S, 58° 43'W

KOPRI 21653 was observed as a bacillus 3 to 5 µm in length and 0.3 to 0.5 µm in width, and 1400 bp of 16S rRNA sequence in KOPRI 21653 was analyzed using Advanced BLAST (www.ncbi.nlm.nih.gov) search tool. As a result, Pseudoalteromonas arctica ) A 37-1-2 ^T was found to have 100% homology with 16S rRNA.

Other Pseudoalteromonas strains (Pseudoalteromonas elyakovii KMM162 ^T , 99.64%; Pseudoalteromonas distincta KMM638 ^T , 99.64%; ^{Pseudoalteromonas nigrifaciens NCIMB-8614 T, 99.57} %; Pseudoalteromonas paragorgicola KMM3548 ^T , 99.50%) showed somewhat less homology, and as shown in the schematic diagram of FIG. 1, the strain used in this experiment showed a close relationship with the pseudoalteromonas strains.

As a result of the above results, the strain producing exopolysaccharide having anti-freezing ability was developed as Pseudoalteromonas altica (Pseudoalteromonas). arctica ) named KOPRI 21653, and deposited with the Korea Research Institute of Bioscience and Biotechnology on November 13, 2007 (KCTC11233BP).

Exopolysaccharide Pure separation

The microbial culture was separated at 4°C for 30 minutes at 12,000g to remove the cells, and then twice the volume of ethanol was added to the culture supernatant obtained here, and then precipitated at 4°C for 24 hours to extract exopolysaccharide. I did. The precipitated exopolysaccharide was collected by centrifugation at 4° C., 10,000 g for 20 minutes _{, dissolved in dH 2} O, and then lyophilized. To remove the protein from the crude exopolysaccharide obtained by lyophilization, proteolytic enzyme treatment was performed at 37°C for 30 minutes, and dialyzed at _{dH 2 O using Viva-Flow (Sartorius, Germany),} 10% cetylpyridinium chloride (CPC, Cetylpyridinium Chloride) was mixed and reprecipitated. The precipitated CPC-zoexopolysaccharide complex was collected by centrifugation at 4° C., 10,000 g for 20 minutes, and dissolved again in 10% NaCl. The precipitated polysaccharides were finally obtained after re-extracted material by addition of an amount of ethanol corresponding to three times the volume, dissolved in dH ₂ O 2 times dialysis in dH ₂ O and was freeze-dried again.

Exopolysaccharide Antifreeze ability

Exopolysaccharide obtained by adding the obtained exopolysaccharide to E. coli and confirming its effect on bacterial growth by freeze-thawing using the bacterial growth identification kit LIVE/DEAD BacLight™ Bacterial Viablity kit (Molecular Probes, USA) The antifreeze activity was confirmed.

E. coli was inoculated in 20 ml of medium, cultured until the late log phase, and centrifuged at 10,000 g for 5 minutes to collect the cells. The collected cells were washed three times with 20 ml of saline (0.85% NaCl), and the number of bacteria ^{was diluted to 2 x 10 8} cells/ml (OD ₆₇₀ = 0.06), and then 100 µl of E. coli. The diluent and the same amount of exopolysaccharide were added and mixed. Repeat freeze-thaw of the mixture 3-4 times, and take 100 µl and dispense it into a 96-well microtiter plate (Nunc, USA) and include it in the LIVE/DEAD BacLight™ Bacterial Viablity kit. 100 µl of the stained solution was mixed and reacted for 15 minutes in the dark at room temperature. Fluorescence was measured at 485 nm/530 nm (Emission 1, G) and 485 nm/630 nm (Emission 2, R) using a fluorescence reader (Perkin elmer, USA) at which the reaction was terminated. It was calculated using the equation.

Cell viability (%) = (A/B) x 100

A = fluorescence ratio of Emission 1/Emission 2 after freeze-thaw treatment

B = fluorescence ratio of initial Emission 1/Emission 2

As a result, it was investigated that P-21653 has the highest antifreezing ability among the finally obtained exopolysaccharides (FIGS. 2 and 3).

Characterization of P-21653

In order to analyze the sugar composition of exopolysaccharides, a GC-MS (Gas Chromatography/Mass selective detector, Perkin Elmer) method was used.

P-21653 (10 ㎍) was put in a 10 ml Pyrex tube, and 0.5 ml of Methanolysis solution mixed with 100 ml of anhydrous methanol and 4.56 ml of acetyl chloride (Sigma, USA) was mixed at 80℃ for 20 hours. Reacted for a while. When the reaction was completed, the sample was dried by adding nitric acid, dissolved in 200 µl of anhydrous acetonitrile, and then 25 µl of anhydrous heptaflurobutyric acid (HFBAA, anhydrous heptaflurobutyric acid) was added at 150°C. After heating for 30 minutes and cooling to room temperature, nitric acid gas was added weakly to dry it, and finally dissolved in 100 µl of anhydrous acetonitrile and used for GC-MS analysis.

The hydrolysis product of P-21653 was analyzed by GC-MS to confirm the sugar composition of the exopolysaccharide produced by Pseudoalteromonas altica (Zanetta, JP et al ., Glycobiol. 9:255, 1999). According to the results of GC-MS analysis, four peaks appeared on the GC chromatogram, and the two peaks at 11.6 and 11.7 minutes showed the same retention time and cleavage pattern as those of glucose, so they were identified as glucose. In addition, the two peaks appearing at 11.2 minutes and 12.2 minutes were identified as galactose because they showed the same retention time and cleavage pattern as galactose (FIG. 4). As a result of calculating the area ratio by integrating the areas of the peaks of glucose (11.6 minutes and 11.7 minutes) and galactose (11.2 minutes and 12.2 minutes), it was confirmed that glucose: galactose = 1.5:1.

In addition, in order to confirm the antifreeze ability of P-21653 in the same process as above, analysis of freeze-thaw three times in repeated conditions within the range of 0.01 to 1% (w/v) content, 0.01 (w/v )%, 0.03(w/v)%, 0.05(w/v)%, 0.1(w/v)%, 0.5(w/v)% and 1(w/v)% of P-21653 , The survival rates of E. coli were 18.41±0.42%, 24.99±1.10%, 58.29±4.46%, 82.62±5.60%, 96.61±4.94%, and 100.57±6.7%, respectively (Figs. 5 and 6).

These values tended to decrease as the number of freeze-thaw cycles increased, and when freeze-thaw was performed 5 to 7 times, 58.29±4.46% to 34.87±% for 0.05% (w/v) of P-21653 was observed. A survival rate of 0.44% was recorded, and the survival rate was from 71.51±9.38% to 48.12±0.35% for 0.1(w/v)% of P-21653. On the other hand, even if freeze-thaw was repeated the same number of times, it was observed that the survival rate of E. coli for P-21653 of 0.5(w/v)% to 1(w/v)% was not affected by the number of times.

In addition, the antifreeze effect of P-21653 is 90% (v/v) VM3 antifreeze, 20%, a combination of 90% (v/v) VEG and 1% (v/v) X-1000 on the market. As a result of comparison with the efficacy of glycerol, the survival rate of E. coli when freeze-thaw 3 times, compared to 92 to 95% of commercially available antifreeze drugs, was 1% (w/v) P-21653 was 98.48±4.37. %, 0.5% were 91.69±5.99%, and 0.1% were 83.12±8.93%, indicating that they had very good antifreeze activity (FIG. 7).

As described above, a specific part of the content of the present invention has been described in detail, and for those of ordinary skill in the art, it is obvious that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Therefore, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents.

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Korea Research Institute of Bioscience and Biotechnology KCTC11233 20071113

Attach electronic file of sequence list

Claims (3)

delete Exopolysaccharide derived from Pseudoalteromonas arctica KOPRI 21653 KCTC 11233BP and having a glucose: galactose having a cryoprotective at a molar ratio of 1.5: 1, comprising the following steps: Method for preparing (exopolysaccharide):
(a) culturing Pseudoalteromonas arctica KOPRI 21653 KCTC 11233BP;
(b) adding alcohol to the culture medium of the microorganism to obtain a precipitate;
(c) removing the protein from the precipitate and then reprecipitating by adding alcohol; And
(d) dialysis of the reprecipitate and then collecting the material in the dialysis membrane. The method of claim 2, wherein 10% CPC (cetylpyridinium chloride) is added during reprecipitation of step (c).

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