KR100338393B1 - Bacteriocin-producing microorganism isolated from Jeot-gal and process for preparation bacteriocin using the same - Google Patents

Abstract

The present invention relates to a bacteriocin producing strain and a bacteriocin production method therefrom. The present invention strain Lactococcus lactis SA72 (Accession No. KFCC-11117) isolated and selected from salted fish has excellent antibacterial activity and produces useful bacteriocins as a non-toxic natural preservative. Bacteriocin has an excellent effect that is stable over a wide range of pH and temperature.

Description

Bacteriocin-producing microorganism isolated from Jeot-gal and process for preparation bacteriocin using the same}

The present invention relates to a bacteriocin producing strain and a bacteriocin production method therefrom. More specifically, the present invention relates to a bacteriocin producing strain Lactococcus lactis SA72 (Accession No. KFCC-11117) obtained by separating and identifying from salted fish and a method for producing bacteriocin therefrom.

In order to prevent the deterioration of food and improve shelf life, at home or in food companies, physical methods such as heating, freezing, drying, etc., microbial suppression methods, acidification of foods, pickling with salt and sugar, or chemical synthetic preservatives are used. However, as interest in food safety and naturality has recently increased, bacteriocin has become a concern as a natural food preservative without toxicity.

Bacteriocin is a bacterium-producing protein or protein-based antimicrobial agent, which generally has bactericidal mechanisms for bacteriocin-producing bacteria and systematically similar strains and is directly biosynthesized from their genes. It is a natural antimicrobial protein that is distinguished from ordinary antibiotics in that. These bacteriocins are mostly produced by lactic acid bacteria which are commonly present in various fermented foods and consumed through daily diet.

Lactobacillus uses carbohydrates anaerobicly to produce lactic acid and is widely distributed in nature. In addition, it produces taste traces in various fermentation processes such as dairy products, jang, liquor, and kimchi to improve taste and flavor, and to grow harmful bacteria. It has been widely used since ancient times as a means of preserving foods by preventing the corruption of foods by inhibiting them.

Therefore, the bacteriocin produced by the lactic acid bacterium is recently decomposed by proteolytic enzymes unlike conventional antibiotics, and thus is non-toxic and non-residual to humans, and has high pH and heat stability and is currently applied to various food manufacturing processes and finished products. Attempts are being made.

Salted fish occupies an important position in the diet of Koreans and is a traditional fermented food obtained by the action of various microorganisms and enzymes using fish and shellfish as raw materials. It is rich in unique flavors, free amino acids and taste, and is a food that is closely related to our diet, not only as a protein source, but also as a side ingredient or seasoning of kimchi, but the research on microorganisms related to fermentation of salted fish is still insignificant.

The present inventors completed the present invention by identifying useful bacteriocin-producing strains from the salted fish such as above, and examining the antimicrobial activity of the bacteriocins from which the identified strains are produced.

Accordingly, it is an object of the present invention to provide Lactococcus lactis SA72 (Accession No. KFCC-11117) which produces a large amount of bacteriocin. Another object of the present invention is to ferment the culture Lactococcus lactis SA72 to produce a large amount of bacteriocin.

The above object of the present invention was obtained by separating, selecting and identifying a strain having antimicrobial activity from salted fish and partially purifying bacteriocin from the culture solution in which the strain was fermented. Subsequently, the antibacterial activity of the bacteriocin obtained by the partial purification was investigated, and the sensitivity and pH and heat stability of various enzymes and organic solvents were examined, and then the molecular weight was determined by SDS-PAGE.

1 is a graph showing the amount and activity of the present invention strain Lactococcus lactis SA72 (Accession No. KFCC-11117) cultured in a fermentor over time.

Figure 2 is a graph showing the degree of death of the strain over time after the bacteriocin treatment to Micrococcus flavus ATCC 10240 strain of logarithmic growth stage.

Figure 3 shows the result of determining the molecular weight of the partially purified bacteriocin produced by the strain Lactococcus lactis SA72 (Accession No. KFCC-11117) of the present invention by SDS-PAGE.

The present invention comprises the steps of separating and selecting a strain having antimicrobial activity from commercial salted salt; Investigating the characteristics of the isolated and selected strains using an API CHL kit, identifying the isolated and selected strains as Lactococcus lactis according to Bergey's mannual, and naming and depositing the strains as Lactococcus lactis SA72; Culturing the isolated and identified strain Lactococcus lactis SA72 in a lactobacilli MRS medium and then culturing in a 5 L fermenter; Removing the cells of the cultured strain Lactococcus lactis SA72 of the present invention, filtering and catalase treatment to prepare a cell-free-supernatant for measuring bacteriocin activity; Partially purifying the bacteriocin by ethanol precipitation of the prepared cell-free-supernatant; Determining the antimicrobial activity of the partially purified bacteriocin by the deferred method and the spot-on-lawn method; Protease XIII, Protease XIV, α-chymotrypsin, Trypsin, Proteinase K and various types of organic solvents Investigating the effects of enzymes and organic solvents on the activity of bacteriocin by measuring the activity after the reaction with; Investigating the effects of pH and temperature on the activity of partially purified bacteriocin by reacting the partially purified bacteriocin with various pH buffers and then measuring the activity and then measuring the activity after bathing at various temperatures; Investigating the antimicrobial activity of the partially purified bacteriocin by treating the bacteriocin partially purified in the logarithmic growth stage and then measuring the amount of the target strain over time; SDS-PAGE the partially purified bacteriocin to determine the molecular weight and to determine the antimicrobial activity through activity staining (activity staining).

According to the above configuration, the bacteriocin producing strain Lactococcus lactis SA72 of the present invention isolated and identified in salted fish reached the maximum activity (2,400AU / ml) in only 7 hours when cultured in fermenter, and the bacteriocins obtained by fermentation culture had many food decay. And antimicrobial activity against pathogenic bacteria and molecular weight was about 6.5kDa.

In the present invention, 27 strains including Clostridum perfringens ATCC 3624 among strains used to determine the antimicrobial range of strains Lactococcus lactis SA72 (Accession No. KFCC-11117) and bacteriocin were lyophilized in the National Institutes of Health, and others such as Salmonella typhi . Known strains were directly isolated from humans, seawater, and fish and observed under microscope after gram staining.

Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.

Example 1 Bacteriocin Producing Strains Lactococcus lactis SA72 Isolation, Identification

Step 1: Isolation of Antimicrobial Strains from Salted Fish

Salted fish, which was the source of fungal separation, was purchased at department stores in Masan. Lactobacillus isolated by normal smearing method showed antibacterial activity against 6 target strains ( Lactococcus lactis CA170-12, Lactococcus lactis ATCC4797, Lactococcus lactis KCCM 40104, Leuconostoc mesenteroides KCCM 11324, Escherichia coli KCCM 32396, Escherichia coli JM109). Confirmed. Unlike general smearing method, high activity bacteriocin producing strains were selected using the triple agar layer method, which is a method for separating strains producing antimicrobial substances directly from an agar plate. At this time, the medium used was MRS medium and the culture temperature was 32 ℃. As a result of the experiment, the isolates with primary antimicrobial activity were obtained and the culture solution was obtained.Then, the culture supernatant was obtained by centrifugation, and the pH was adjusted to 6.5 to determine the antimicrobial activity of the indicator by the spot-on-lawn method. The SA72 strain having the highest activity was selected among them.

Second Process: Identifying SA72 Strains

The SA72 strain selected in the first step was reviewed for the availability of SA72 strains for 49 carbon sources by the API CHL kit (bioMereux, France), using the motility test, API20E kit and ATB expression (bioMereux, France). Were identified according to Bergey's Manual of Determinative Bacteriology. At this time, the bacteria were used in three passages for 24 hours in 37 ℃ and 30 ℃, TSB and NA medium, the mold 30 ℃, PDB medium. As a result, as shown in Table 1, the strain of the present invention was gram positive and had a sugar availability of 99.8%, showing Lactococcus lactis subsp. It was identified as lactis . Accordingly, the present inventors named the strain Lactococcus lactis SA72, and deposited it on December 2, 1999 with the deposit number KFCC-11117 at the Korea Microbiological Conservation Center in the Korean spawn association.

Identification of the bacteriocin-producing strain Lactococcus lactis SA72 Test Items SA72 Test Items SA72 Morphology coccus Gram staining + ^a Eaculine + Glycerol - Salicine + Erythritol - Cellobiose + D-Arabinose - Maltose + L-Arabinose + Lactose + Ribose + Melibiose - D-Xylose + Saccharose + L-Xylose - Trehalose + Adonitol - Inuline - β -methylxyloside (β- Methylxyloside) - Melezitose - Galactose + D-raffinose - D-Glucose + Amidon + D-Fructose + Glycogen - D-Mannose + Xylitol - L-Sorbose - β- Gentibiose + Rhamnose - D-Turanose - Dulcitol - D-Lyxose - Inositol - D-Tagatose - Mannitol + D-Fucose - Sorbitol - L-Fucose - α- methyl -D- manno side (α -methyl-D-Mannoside) - D-Arabitol - α - methyl -D- glucoside (α -methyl-D-Glucoside) - L-Arabitol - N-Acetylglucosamine + Gluconate + Amygdaline + 2-cetogluconate - Arbutine + 5-cetogluconate - Idenfitication Lactococcus lactis (99.8%) (Note) a: Data obtained by API50 CHL kit. +: Positive,-: negative

Example 2: Inventive Strains Lactococcus lactis Bacteriocin Production by Culture of SA72

First step: strain of the present invention Lactococcus lactis SA72 (Accession No. KFCC-11117) Culture

In this example, lactobacilli MRS medium (Difco Laboratories, USA) was used for culturing the strain Lactococcus lactis SA72 of the present invention. Culture conditions and methods were purely isolated from the Lactococcus lactis SA72 seedlings and preserved in MRS agar plate, and these strains were inoculated with one platinum in MRS medium in a 10 mL test bube and cultured at 30 ~ 35 ℃ It was. The main culture in the fermenter was cultured using a 5L fermenter (5L-jar fermenter; Korea Fermentor Co., Ltd.), the culture conditions are 30 ~ 35 ℃, pH 6.0 ± 0.5, stirring speed 180 ~ 220rpm, The inoculation ratio was 0.8 ~ 1.2% (v / v), the working volume was 3L and incubated for 9 hours under anaerobic conditions. At this time, the growth of lactic acid bacteria and the activity of bacteriocin were measured while incubating at 32 ° C for a certain time. As a result, as shown in Fig. 1, bacteriocin producing strains were detected for the first time at 4 hours after cultivation and showed a maximum at 7-9 hours.

Second Process: Preparation of Cell-Removing Supernatant

In this step, the strain Lactococcus lactis SA72 of the present invention obtained in the first step was centrifuged at 8,000 × g for 20 minutes at 4 ° C. to remove the cells. The obtained culture supernatant was filtered through a 0.22 μm-pore-size cellulose acetate filter to obtain a filtrate, the pH was adjusted with 3N NaOH solution and 3N HCl solution, and a catalase was added at a final concentration of 1 mg / mL. After incubation at 37 ° C. for 30 minutes to remove hydrogen peroxide, a cell-free supernatant was prepared to measure bacteriocin activity.

Process 3: partial purification of bacteriocin

Cold ethanol was slowly added at 4 ° C. and stirred constantly to 40% saturated concentration in the cell-free supernatant prepared in the second step. The precipitated protein was precipitated by centrifugation at 12,000 × g for 20 minutes at 4 ° C. and resuspended in 0.1 M phosphate buffer (pH 7.0). Ethanol was evaporated at 30 ° C. for 2 hours and partially purified bacteriocin was stored at −70 ° C.

Experimental Example 1: Antimicrobial Activity of Bacteriocin

In this experiment, the deferred method and the spot-on-lawn method were used to measure the antimicrobial activity against bacteriocin-producing strains. Bacteriocin was a bacteriocin produced in L. lactis SA72 strain partially purified in Example 2, gram-negative bacteria and gram-positive bacteria were used after culturing at optimal temperature for 12 hours using TSB medium and fungi PDB medium. . The antimicrobial activity of the deferred method is expressed in diameter (mm) measured using an antibiotic zone reader (Fischer Scientific Co., USA), and the activity of the spot-on-lawn method (AU / mL) is bacteriocin After diluting the stock solution containing 2 times with phosphate buffer solution, each diluting solution was spotted and the maximum dilution factor showing obvious inhibition was inversely calculated and compared with the representative bacteriocin nisin. As a result, as shown in Table 2, partially purified bacteriocin produced by the strain L. lactis SA72 of the present invention by the Deferred method showed activity against all target strains. It was similar to KCCM40104, a nisin-producing bacterium. As a result of measuring the antibacterial activity of the partially purified bacteriocin produced by the L. lactis SA72 strain of the present invention by spot-on-lawn assay, it showed a broad antimicrobial range against Gram-positive bacteria as shown in Table 3. It showed little activity.

microbe Culture medium Incubation temperature Bacteriocin Producing Strains Nisin (KCCM40104) L. lactis SA72 Gram-positive microorganisms Bacillus cereus NB 30 ℃ 25 20 Bacillus cereus ATCC 11778 NB 30 ℃ 20 22 Bacillus pumilis NB 30 ℃ 41 39 Bacillus subtilis ATCC 6633 TSB 37 ℃ 30 30 Clostridium perfringens ATCC 3624 TSB 37 ℃ 10 14 Enterococcus faecalis ATCC 19433 TSB 37 ℃ 15 15 Lactobacillus delbrueckii ATCC 4797 MRS 37 ℃ 32 28 Leuconostoc mesenteroides KCCM 11324 MRS 25 ℃ 30 25 Listeria innocua TSB 37 ℃ 23 28 Listeria monocytogenes ATCC 15313 TSB 37 ℃ 21.5 30 Listeria monocytogenes TSB 37 ℃ 25 27 Micrococcus flavus ATCC 10240 NB 37 ℃ 34 37 Propionibacterium acnes P3 NLB 32 ℃ 11 7 Propionibacterium acidipropionici P9 NLB 32 ℃ 30 30 Propionibacterium acidipropionici P200910 NLB 32 ℃ 30 30 Propionibacterium thoenii P127 NLB 32 ℃ 19 21 Rodococcus equi TSB 37 ℃ 17 30 Staphylococcus aureus ATCC 6538 TSB 37 ℃ 23 27 Staphylococcus aureus ATCC 25923 TSB 37 ℃ 27 22 Streptococcus bovis ATCC 9809 TSB 37 ℃ 27 26 Gram negative microorganism Escherichia coli ATCC 8739 TSB 37 ℃ 30 30 Escherichia coli ATCC 25922 TSB 37 ℃ 24 25 Escherichia coli O157: H7 TSB 37 ℃ 20 26 Escherichia coli KCCM 32396 LB 37 ℃ 23 26 Escherichia coli JM109 LB 37 ℃ 9 10 Pseudomonas aeruginosa ATCC 15422 TSB 37 ℃ 20 21 Pseudomonas syringae ATCC 12885 TSB 37 ℃ 16 18 Salmonella enteritidis TSB 37 ℃ 21 33 Salmonella london E TSB 37 ℃ 18 27 Salmonella paratyphi TSB 37 ℃ 30 30 Salmonella typhi TSB 37 ℃ 27 30 Salmonella typhimurium TSB 37 ℃ 24 27

microbe Culture medium Incubation temperature Bacteriocin Producing Strains Nisin (KCCM40104) L. lactis SA72 Shigella flexneri TSB 37 ℃ 21 27 Shigella sonnei TSB 37 ℃ 25 20 Vibrio cholerae O139 TSB 37 ℃ 17 25 Vibrio parahaemolyticus ATCC 17802 TSB 37 ℃ 21 26 Vibrio parahaemolyticus TSB 37 ℃ 17 26 Vibrio vulnificus TSB 37 ℃ 30 30 Yersinia enterocolitica ATCC 27729 TSB 37 ℃ 18 28 mold Aspergillus niger KCCM 11239 PDB 25 ℃ ND ND Aspergillus oryzae KCCM 11371 PDB 25 ℃ ND ND [Note] ND: Not detected

microbe Culture medium Incubation temperature Bacteriocin Nisin L.lactis SA72 Gram-positive microorganisms Bacillus cereus NB 30 ℃ + + Bacillus cereus ATCC 11778 NB 30 ℃ + + Bacillus pumilis NB 30 ℃ + + Bacillus subtilis ATCC 6633 TSB 37 ℃ + + Clostridium perfringens ATCC 3624 TSB 37 ℃ + + Enterococcus faecalis ATCC 19433 TSB 37 ℃ + + Lactobacillus delbrueckii ATCC 4797 MRS 37 ℃ + + Leuconostoc mesenteroides KCCM 11324 MRS 25 ℃ + + Listeria innocua TSB 37 ℃ +/- ^a) + Listeria monocytogenes ATCC 15313 TSB 37 ℃ +/- + Listeria monocytogenes TSB 37 ℃ + + Micrococcus flavus ATCC 10240 NB 37 ℃ + + Propionibacterium acnes P3 NLB 32 ℃ + + Propionibacterium acidipropionici P9 NLB 32 ℃ + + Propionibacterium acidipropionici P200910 NLB 32 ℃ + + Propionibacterium thoenii P127 NLB 32 ℃ + + Rodococcus equi TSB 37 ℃ + + Staphylococcus aureus ATCC 6538 TSB 37 ℃ + + Staphylococcus aureus ATCC 25923 TSB 37 ℃ +/- +/- Streptococcus bovis ATCC 9809 TSB 37 ℃ + + Gram negative microorganism Escherichia coli ATCC 8739 TSB 37 ℃ - - Escherichia coli ATCC 25922 TSB 37 ℃ - - Escherichia coli O157: H7 TSB 37 ℃ - - Escherichia coli KCCM 32396 LB 37 ℃ + + Escherichia coli JM109 LB 37 ℃ - - Pseudomonas aeruginosa ATCC 15422 TSB 37 ℃ - - Pseudomonas syringae ATCC 12885 TSB 37 ℃ - - Salmonella enteritidis TSB 37 ℃ - - Salmonella london E TSB 37 ℃ - - Salmonella paratyphi TSB 37 ℃ - - Salmonella typhi TSB 37 ℃ - - Salmonella typhimurium TSB 37 ℃ - -

Organisms Culturemedium Incubationtemp. Bacteriocin Nisin L. lactis SA72 Shigella flexneri TSB 37 ℃ - - Shigella sonnei TSB 37 ℃ - - Vibrio cholerae O139 TSB 37 ℃ - - Vibrio parahaemolyticus ATCC 17802 TSB 37 ℃ - - Vibrio parahaemolyticus TSB 37 ℃ - - Vibrio vulnificus TSB 37 ℃ - - Yersinia enterocolitica ATCC 27729 TSB 37 ℃ - - mold Aspergillus niger KCCM 11239 PDB 25 ℃ - - Aspergillus oryzae KCCM 11371 PDB 25 ℃ - - [Note] ND: Not detected

Experimental Example 2: Sensitivity of Bacteriocin to Organic Solvents and Enzymes

In this experimental example, the bacteriocin produced by the L. lactis SA72 strain of the present invention, which was partially purified, was first reacted with various enzymes at a final concentration of 1 mL / mg for 24 hours to investigate the effects of enzymes and organic solvents on bacteriocin activity. ⅩⅢ enzyme protease (protease ⅩⅢ), ⅩⅣ protease (protease ⅩⅣ), α- chymotrypsin (α -chymotrypsin), trypsin (trypsin), the better protein K (proteinase K) was dissolved in a pH buffer solution, pH buffer The enzyme-treated bacteriocin dissolved in was used as a control. The effect of the organic solvent on the bacteriocin was mixed with various organic solvents in a 1: 1 ratio and used for 1 hour incubation at 30 ° C. As a result, as shown in Table 4, the bacteriocins were very stable against organic solvents and their activity was completely destroyed by protease XIV. The enzyme did not lower the antimicrobial activity. Thus it was demonstrated that this bacteriocin is a protein.

Sensitivity to Bacteriocins from Organic Solvents and Enzymes Produced by Partially Purified L. lactis SA72 Strains Treatment Remaining Activity (AU / mL) Treatment Remaining Activity (AU / mL) Control 51,200 Control 12,800 ethanol 51,200 Protease XIII 12,800 Methanol 51,200 Protease XIV 0 Acetone 51,200 Trypsin 12,800 toluene 51,200 α -chymotrypsin 12,800 chloroform 51,200 Proteinase K 12,800 Isopropyl Alcohol 51,200

Experimental Example 3: Stability against pH and heat of bacteriocin

Stability against pH was determined by bacteriocin produced by partially purified L. lactis SA72 strain (50 mM citrate buffer, pH 2-6; 50 mM phosphate buffer, pH 7.0; 50 mM tris-HCl buffer, pH 8-9). ) Was mixed at a 1: 1 ratio, and then measured at 4 ° C. for 4 hours. To investigate thermal stability, partially purified L. lactis SA72 strain-producing bacteriocin was reacted by agitation at various temperatures for 30 minutes, and at 121 ° C. for 15 minutes. As a result, as shown in Table 5, the range of pH 2-9 and the heat treatment were stable at 40 ° C. to 80 ° C., and gradually decreased from 90 ° C. to 121 ° C., and activity was also observed at 121 ° C.

PH and Thermal Stability of Partially Purified L. lactis SA72 Strains Producing Bacteriocin Workaround ^a (heat) Remaining activity (AU / mL) Workaround ^a (heat) Remaining activity (AU / mL) Control 25,600 Control 51,200 40 ℃ 25,600 2 51,200 50 ℃ 12,800 3 51,200 60 ℃ 12,800 4 51,200 70 ℃ 12,800 5 51,200 80 ℃ 12,800 6 51,200 90 ℃ 9,051 7 51,200 100 ℃ 4,525 8 51,200 120 ℃ 400 9 25,600 Note: In order to examine the heat resistance in bacteriocin activity, partially purified bacteriocin was incubated at various temperatures (40, 50, 60, 70, 80, 90, 100 ° C) for 30 minutes or at 121 ° C for 15 minutes. .b: In order to examine the pH effect on bacteriocin activity, partially purified bacteriocin was reacted at various pHs (2, 3, 4, 5, 6, 7, 8, 9) for 4 hours.

Experimental Example 4: Mechanism of Action of Bacteriocin

The cells of the logarithmic growth stage of the micrococcus flavus ATCC 10240 strain were suspended in 0.1 M phosphate buffer solution (pH 7.0), and then sampled every hour after treatment with partially purified L. lactis SA72 strain producing bacteriocin at 1,600 AU / mL. , The number of bacteria of the target strain was measured. As a result, as shown in FIG. 2, the bactericidal action of killing susceptible cells in one and a half hours was shown.

Experimental Example 5: Bacteriocin molecular weight

SDS-PAGE of 16% polyacrylamide gel was used to determine the molecular weight of partially purified L. lactis SA72 strain producing bacteriocin. The sample was mixed at a 1: 1 ratio of the purified bacteriocin solution and sample buffer, and then boiled at 100 ° C. for 5 minutes. Electrophoresis was performed for 2 hours at 100V in a vertical slab gel apparatus (protein cell II; Bio-red). For activity staining, electrophoresed gels were fixed in a solution containing 20% isopropanol and 10% acetic acid, and then left in sterile distilled water for at least 4 hours, followed by pre-cultured Lactobacillus on the gel. After delbrueckii ATCC 4797 was poured into 20 mL of soft agar and incubated at 30 ° C. for 12 hours, the presence of clear zone was confirmed. As a result, the molecular weight of the partially purified L. lactis SA72 strain producing bacteriocin was found to be a substance having a molecular weight of about 6.5 kDa, as shown in FIG. 3, and the result of activity staining to determine whether the activity is shown It showed antimicrobial activity. Therefore, it was confirmed that the partially purified material was bacteriocin.

As described above, the present invention strain Lactococcus lactis SA72 (Accession No. KFCC-11117), which is isolated and selected from salted fish as described through the above examples, has excellent antibacterial activity and produces useful bacteriocin as a non-toxic natural preservative, and this bacteriocin has a wide range. It is a very useful invention for the food industry and biopharmaceutical industry because it has an excellent effect of stable at pH and temperature.

Claims (2)

Bacteriocin-producing strain Lactococcus lactis SA72, characterized in that it is isolated from salted fish (Accession No. KFCC-11117) After culturing Lactococcus lactis SA72 (Accession No. KFCC-11117) in MRS medium, the temperature was 30-35 ℃, pH was 6.0 ± 0.5, stirring speed was 180-220rpm, inoculation ratio was 0.8-1.2% (v / v). Culturing under anaerobic conditions; A cell-free supernatant manufacturing step of removing the cells from the culture solution and then filtering the culture supernatant with a cellulose acetate filter, treating the catalase, and removing hydrogen peroxide; Bacteriocin production method using the strain Lactococcus lactis SA72 (Accession No. KFCC-11117), characterized in that the step of removing the protein produced by the addition of ethanol to the cell-removing supernatant, and evaporating the ethanol partially purified.