KR101671229B1 - Strain for Producing Hyaluronic Acid and Preparing Method of Hyaluronic Acid Using the Same - Google Patents

Abstract

The present invention relates to a strain producing hyaluronic acid and a method for producing hyaluronic acid using the same, and more particularly, to a novel strain of Streptococcus species capable of producing hyaluronic acid at a high concentration and a method for producing hyaluronic acid using the same.
Streptococcus epidemicus KL0101J (KCCM 11360P) according to the present invention not only has excellent hyaluronic acid producing ability, but also is a high-priced, allergen-inducing animal raw material, such as tryptone and soy peptone, in place of Yeast Extract ) Can be used in a wide range of food, cosmetic and pharmaceutical fields because it can produce hyaluronic acid at a high concentration.

Description

TECHNICAL FIELD The present invention relates to a strain for producing hyaluronic acid and a method for producing hyaluronic acid using the same,

The present invention relates to a strain producing hyaluronic acid and a method for producing hyaluronic acid using the same, and more particularly, to a novel strain of Streptococcus species capable of producing hyaluronic acid at a high concentration and a method for producing hyaluronic acid using the same.

Hyaluronic acid is a polymer (0.5 ~ 1.3MDa) polysaccharide and has a strong water-binding ability. It is widely used as a moisturizer in cosmetics. Recently, it has been widely used as a raw material for eyedrops, joint injections and molding fillers in ophthalmology, orthopedics and dermatology . Generally, hyaluronic acid is produced by extraction from a chicken broth (J. General Microbiology, 85, 372 to 375, 1976), or by culturing and purifying a microorganism having hyaluronic acid at a high concentration on a cell wall under certain conditions .

In general, Streptococcus sp. Strains are known to have hyaluronic acid producing ability. These strains are known to be capable of optimal culture in an animal-derived tryptone-containing medium and soybean-derived soy peptone-containing medium, and most strains having hyaluronic acid- And cultured in a medium containing tryptone and soy peptone.

However, the animal medium, tryptone, has a concern about mad cow disease, soybean-derived soy peptone is limited in its use due to allergy induction and GMO, and even when cultured in a medium containing tryptone and soy peptone, hyaluronic acid productivity Is low to 2 to 4 g / L, which makes it difficult to industrialize (Korean Patent No. 0250573 and US Patent No. 6090596).

Recently, a strain having increased productivity of hyaluronic acid has been reported (Korean Patent No. 0047007). However, since this patent also uses an expensive animal medium, it is difficult to expand the market by industrializing in terms of economy In fact.

Therefore, it is possible to obtain a strain capable of producing hyaluronic acid at a high concentration under non-animal medium conditions which do not use soy peptone, which is expensive and has limited use, and soy peptone which is a cause of allergy, Development of conditions is required.

In order to solve the above problems, the present inventors have conducted extensive studies in order to solve the above-mentioned problems. In order to solve the above-mentioned problems, the present inventors have found that Streptococcus japonidis ( Streptococcus japonicus), which is produced by mutagenizing Streptococcus equi subsp. Zooepidemicus (KCCM 40304 and 40305) It was found that the hyaluronic acid producing ability of Streptococcus equi subsp. Zooepidemicus KL0101J was excellent and that when the strain having hyaluronic acid producing ability was cultured in a medium containing glucose and a non-animal yeast extract, Lonic acid, and the present invention was completed.

It is an object of the present invention to provide a strain capable of producing hyaluronic acid at a high concentration and a method for producing hyaluronic acid using the same.

It is another object of the present invention to provide a non-animal culture medium composition for culture of strains having hyaluronic acid-producing ability.

In order to attain the above object, the present invention provides Streptococcus equi subsp. Zooepidemicus KL0101J, which is capable of producing hyaluronic acid, KL0101J (KCCM 11360P).

The present invention also provides a method for producing hyaluronic acid, which comprises separating hyaluronic acid from the culture solution obtained by culturing said Streptococcus japonidemicus KL0101J (KCCM 11360P) ( Streptococcus equi subsp. Zooepidemicus KL0101J).

In the present invention, the Streptococcus equi subsp. Zooepidemicus KL0101J is prepared by a method comprising: (a) 200-300 g / L of glucose; and (b) 15 g / L of tryptone , 5 g / L of soytone and 5 g / L of sodium chloride).

In the present invention, the Streptococcus equi subsp. Zooepidemicus KL0101J is prepared by a method comprising: (a) 200-300 g / L of glucose and (b) 25-30 g / L of yeast extract; In the culture medium.

The present invention also provides a culture medium for culture of a strain, which comprises (a) 200-300 g / L of glucose and (b) 25-30 g / L of yeast extract.

Streptococcus epidemicus KL0101J (KCCM 11360P) according to the present invention not only has excellent hyaluronic acid producing ability, but also is a high-priced, allergen-inducing animal raw material, such as tryptone and soy peptone, in place of Yeast Extract ) Can be used in a wide range of food, cosmetic and pharmaceutical fields because it can produce hyaluronic acid at a high concentration.

In the present invention, it has been confirmed that a mutant strain capable of producing hyaluronic acid can be produced by causing a mutation in Streptococcus jupiondemicus strain known to have hyaluronic acid producing ability.

In the present invention, random mutagenesis was performed on a known Streptococcus japonicus strain. As a result, it was confirmed that a strain having increased hyaluronic acid production ability was present in the mutant strain pool.

That is, in one embodiment of the present invention, EGF (ethane methyl sulfonate) was treated with streptococcus juepidemicus (KCCM 40304 and 40305) at various concentrations and then the hyaluronic acid production ability of the mutant strains was examined. As a result, (KCCM 11360P) ( Streptococcus equi subsp. Zooepidemicus KL0101J) was deposited in the Korean Microorganism Preservation Center.

Accordingly, the present invention relates, in one aspect, to Streptococcus equi subsp. Zooepidemicus KL0101J, which is capable of producing hyaluronic acid, KL0101J (KCCM 11360P).

The present invention also relates to a method for producing hyaluronic acid, which comprises separating hyaluronic acid from the culture medium obtained by culturing Streptococcus equipidemicus KL0101J (KCCM 11360P) ( Streptococcus equi subsp. Zooepidemicus KL0101J).

Streptococcus equi subsp . Zooepidemicus KL0101J) can be cultured under aerobic conditions at 35-37 ° C, and prevent the culture of the strain from being inhibited by lactic acid produced during cultivation It is preferable to maintain the pH at 6.5 to 7.5. The pH can be adjusted without any particular limitation using a pH adjustable agent without directly affecting the culture of the strain, and NaOH and the like can be exemplified.

(A) glucose 200-300 g / L, and (b) TSB medium [tryptone 15 g / L, sucrose (100 g / L)], which is known as a medium for culture, and Streptococcus juepidemicus KL0101J 5 g / L of soytone, 5 g / L of sodium chloride and 5 g / L of sodium chloride, and then hyaluronic acid can be isolated from the culture.

From the culture solution, hyaluronic acid is removed from impurities firstly by simple filtration using diatomaceous earth as known in the art, and then filtered with activated charcoal or the like to further remove other impurities and the like, and then finally crystallized using ethanol .

On the other hand, in the present invention, tryptone and soyipeptones, which are essential medium for culturing strains having a hyaluronic acid producing ability, are expensive and have problems such as influx of animal-derived disease factors, allergen and GMO In an effort to develop alternative media compositions.

As a result, it was confirmed that when a strain having hyaluronic acid-producing ability was cultured in a medium containing glucose as a carbon source and a yeast extract as a nitrogen source, the growth of the strain and hyaluronic acid production ability were further increased.

Accordingly, in another aspect, the present invention provides a culture medium for culture of a strain for hyaluronic acid production, which comprises (a) 200-300 g / L of glucose and (b) 25-30 g / L of yeast extract .

Since the culture composition according to the present invention contains a non-animal yeast extract as a nitrogen source, there is no limitation in use, such as peptone and soy peptone, which are widely used in the past, and the cost is low, The hyaluronic acid production ability can be further increased.

When the content of glucose in the culture medium exceeds 200-300 g per liter and the content of yeast extract is outside the range of 25-30 g per liter, the growth of the strain is inhibited and the hyaluronic acid production ability is decreased There is a problem.

In another aspect of the present invention, there is provided a method for preparing a yeast extract, comprising the steps of: (a) preparing 200-300 g / L glucose and (b) yeast extract; And 30 g / L, and separating hyaluronic acid from the obtained culture medium. The present invention also relates to a method for producing hyaluronic acid.

The method of producing hyaluronic acid may be carried out by using Streptococcus jupipemicus KL0101J (manufactured by Takara Shuzo Co., Ltd.) in a medium containing a non-animal ingredient and a low-cost yeast extract as a nitrogen source, instead of an animal- KCCM 11360P), it is possible to produce hyaluronic acid at a high concentration of 9 ~ 10g / L, and thus it can be widely used for existing foods, cosmetics and pharmaceuticals.

[Example]

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for illustrating the present invention only, and the scope of the present invention is not limited to these examples.

Example 1: Selection of high-concentration HA-expressing mutant strains

In order to induce the mutation of Streptococcus epidemicus (KCCM 40304 and 40305), which has been distributed from the Korean Microorganism Conservation Center, the mutation rate according to the concentration of EMS was measured using the chemical substance EMS (ethane methyl sulfonate) .

The mutation rate was predicted based on the survival rate of the microorganism under each condition. In particular, in this example, the mutation rate of the strain capable of selecting a single colony forming a mucous substance in the medium was confirmed.

In order to select strains having high concentration of hyaluronic acid producing ability in a strain pool induced by mutation by EMS (ethane methyl sulfonate), the chromogenic method (Asian Blue Stain) (Biotechnol. Bioeng. 101 (4), 788-796 , 2008).

Step 1: Production of mutant strain pool

Random mutagenesis was performed using EMS (Sigma-Aldrich, St. Louis, MO, USA). First, Streptococcus epidemicus (KCCM 40304 and 40305) cultured at 37 占 폚 in 10 mL of Todd Hewitt liquid medium (THB) was centrifuged and precipitated. After washing with PBS buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na ₂ HPO _4, 1.8 mM KH ₂ PO ₄ ), the cells were centrifuged again under the same conditions and this procedure was repeated twice. After addition of PBS buffer, EMS was added and cultured at 37 ° C for 30 min. After washing three times with PBS buffer, 100 μl aliquots were added to the THB agar plate. The mortality rate by EMS was calculated as shown in equation (1).

[Equation 1]

When the mortality rate was more than 50%, the mutation occurred more frequently.

Step 2: Selection of mutant strains capable of producing high-concentration HA

Quantitative determination of hyaluronic acid was performed using alcian blue (Sigma-Aldrich), a colorant used to test acidic mucopolysaccharides. Since the culture medium was not blue due to the color of the medium itself, it was diluted 10 ^{- 1 with} distilled water to make 400 μL, and then divided into 550 μL of 3% acetic acid (Daejung). 50 μL alcian blue staining reagent was added and heated by microwave for 30 seconds after voltexing. Centrifuged at 13,000 rpm for 1 minute, cooled at room temperature for 2.5 hours, and centrifuged again at 13,000 rpm for 1 minute. The optical density (OD) was measured at 540 nm using an infinite 200 pro (TECAN, Mannedorf, Switzerland) after 96 well microplates. Standard curves were used with different concentrations of purified hyaluronic acid. The strains with OD values significantly lower than the original strains (KCCM 40304 and 40305) were selected first.

Step 3: Additional verification

Streptococcus epidemicus (KCCM 40304 and 40305) and primary selected strains were inoculated into Todd Hewitt liquid medium (THB) and cultured for 18 hours. The cultured strains were plated on a blood agar medium and cultured for 18 hours to confirm that hemolysis was not observed.

Example 2: Final discovery of Streptococcus jupy demicus KL0101J

The strain selected in Example 1 was finally cultured in a 5 L jar fermenter to confirm the final production ability of hyaluronic acid. At this time, the total volume of the medium was 2.5 liters. Seed culture was performed in TSB medium (50 mL) and incubated for 6 hours at 37 ° C at 200 rpm. The same TSB medium (tryptone 15 g / L, soytone 5 g / L, and sodium chloride 5 g / L) Two 125 mL aliquots were inoculated 5 mL each and cultured under the same conditions.

Finally, strains selected in fermentor (Fermentor) supplemented with 250 g / L of glucose sterilized separately in TSB medium were inoculated and cultured for 24 hours at a culture temperature of 35 ° C and pH 7.

After completion of cultivation, hyaluronic acid contained in the culture solution was quantified by the carbazole method (European Pharmacoepia 6.0 p2706). At this time, one strain in which hyaluronic acid production ability was repeatedly confirmed to be 7 to 8 g / L was finally selected, and Streptococcus equi (strain KL0101J subsp . zooepidemicus KL0101J) and deposited on Jan. 30, 2013 with the Korean Society for Microbiological Care (Accession No .: KCCM 11360P).

EXPERIMENTAL EXAMPLE 1: Verification of production ability of hyaluronic acid

Streptococcus epidemicus (KCCM 40304 and 40305), the original strain, was cultured under the same conditions as in Example 1, and the hyaluronic acid production ability of the strain was confirmed.

Strain KCCM 40304 KCCM 40305 KCCM 11360P Hyaluronic acid production ability 2.5 to 3.0 g / L 2.7 to 3.2 g / L 7.0 to 8.0 g / L

From Table 1, it can be seen that, while the original strain has a hyaluronic acid production capacity of about 2.5 to 3.2 g / L, the mutant strain KCCM 11360P has a hyaluronic acid production capacity of 7 to 8 g / L and a hyaluronic acid production capacity of 250% It was able to know that it was high.

Example 3: Investigation of optimal culture composition of strains having hyaluronic acid-producing ability

The optimum medium composition was searched for Streptococcus equipidemicus KL0101J ( Streptococcus equi subsp. Zooepidemicus KL0101J) which was confirmed to have excellent hyaluronic acid producing ability.

Under the same culture conditions as in Example 2, Seed culture was inoculated with 50 ml of TSB and incubated for 6 hours at 37 ° C at 200 rpm. Then, the medium was changed to non-animal medium (Yeast Extract 25-40 g / L and Sodium Chloride 5 g / ) 125 mL were inoculated in 5 mL each and cultured under the same conditions.

Finally, strains selected in a fermenter (Fermentor) supplemented with 250-350 g / L of separately sterilized glucose were inoculated into non-animal medium (25-40 g / L of Yeast Extract and 5 g / L of Sodium Chloride) , pH 7 for 24 hours.

After the completion of cultivation, the amount of hyaluronic acid contained in the culture solution was quantified by the carbazole method (European Pharmacoepia 6.0 p2706), and the results are shown in Table 2.

No. Medium composition Generation Glucose Yeast Extract One 250 30 7.5 to 8.5 g / L 2 300 30 7 ~ 8g / L 3 350 30 6 to 7 g / L 4 300 25 9 to 10 g / L 5 300 30 6.0 to 7.0 g / L 6 300 35 6.5 to 7.0 g / L 7 300 40 7 ~ 8g / L

From Table 2, it was confirmed that when the glucose was 300 g / L and the yeast extract was 25 g / L, the hyaluronic acid production ability of the strain was the highest as 9 to 10 g / L.

On the other hand, when the glucose content exceeds 300 g / L or the yeast extract content exceeds 30 g / L, the productivity of hyaluronic acid is rapidly reduced to about 6.0 to 8.0 g / L.

This is because when the glucose content is higher than 350 g / L or the content of yeast extract is higher than 30 g / L, the energy source necessary for culturing strains such as carbon source and nitrogen source at the initial strain culture is present at a concentration higher than a certain concentration, Rather than promoting it.

Streptococcus epidemicus (KCCM 40304 and 40305), which was the original strain, was inoculated into the culture composition having the composition of No. 4 in Table 2, which was found to have the highest mixing ratio of glucose and yeast extract, and cultured under the same conditions Next, hyaluronic acid contained in the culture solution was quantified by the carbazole method (European Pharmacoepia 6.0 p2706), and the results are shown in Table 3.

Strain KCCM 40304 KCCM 40305 KCCM 11360P Hyaluronic acid production ability 2 to 3 g / L 2.5 to 3.5 g / L 9 to 10 g / L

From Table 3, it can be seen that, in the case of KCCM 40304 and 40305, the productivity of hyaluronic acid in culturing in non-animal medium was similar to that of cultured in animal medium of 2 to 3 g / L and 2.5 to 3.5 g / L, respectively, Streptococcus epidemicus KL0101J (KCCM 11360P), a new strain, was further increased in hyaluronic acid production ability to 9 to 10 g / L.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will readily appreciate that many modifications are possible in the exemplary embodiments without materially departing from the novel teachings and advantages of this invention. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Korea Microorganism Conservation Center KCCM11360P 20130130

Claims (5)

Streptococcus equiepidemicus KL0101J (KCCM 11360P) (Streptococcus equi subsp. Zooepidemicus KL0101J) having hyaluronic acid-producing ability derived from Streptococcus epidemicus (KCCM 40304) treated with EMS (ethane methyl sulfonate).
The method of claim 1, wherein the Streptococcus equi subsp. Zooepidemicus KL0101J is selected from the group consisting of (a) 200-300 g / L glucose and (b) 25-30 g / L yeast extract. A method for producing hyaluronic acid, which comprises separating hyaluronic acid from the culture liquid obtained by culturing in a medium.
delete delete (KCCM 11360P) ( Streptococcus equi subsp. Zooepidemicus KL0101J) having hyaluronic acid- producing ability,
Wherein the culture medium comprises (a) 200-300 g / L of glucose and (b) 25-30 g / L of yeast extract.