KR20170060627A - Method of Producing High Molecular Weight Hyaluronic Acid by Bio Fermentation - Google Patents
Method of Producing High Molecular Weight Hyaluronic Acid by Bio Fermentation Download PDFInfo
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- KR20170060627A KR20170060627A KR1020150164392A KR20150164392A KR20170060627A KR 20170060627 A KR20170060627 A KR 20170060627A KR 1020150164392 A KR1020150164392 A KR 1020150164392A KR 20150164392 A KR20150164392 A KR 20150164392A KR 20170060627 A KR20170060627 A KR 20170060627A
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- hyaluronic acid
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- zooepidemicus
- molecular weight
- streptococcus
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
- C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/34—Sugars
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/60—Buffer, e.g. pH regulation, osmotic pressure
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/70—Undefined extracts
- C12N2500/74—Undefined extracts from fungi, e.g. yeasts
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2523/00—Culture process characterised by temperature
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- C12R1/46—
Abstract
The present invention relates to a method of producing hyaluronic acid by microbial fermentation, and more particularly, to a method of producing hyaluronic acid from hyaluronic acid (Hyaluronic Acid) using Streptococcus equi subsp. Zooepidemicus capable of producing hyaluronic acid (HA). ≪ / RTI >
The production method of the polymer hyaluronic acid is carried out by culturing Streptococcus equi subsp. Zooepidemicus in a culture medium containing 200 to 300 g / L of glucose and 25 to 30 g / L of yeast extract at 30 to 33 ° C Lt; / RTI >
The method of producing a polymeric hyaluronic acid according to the present invention can produce HA of 3.0 MDa or more at a high yield by optimizing culture conditions of a strain having hyaluronic acid production ability and can produce hyaluronic acid (HA) or hyaluronic acid (HA) Are more widely available in the cosmetics and pharmaceuticals field.
Description
The present invention relates to a method of producing hyaluronic acid by microbial fermentation, and more particularly, to a method of producing hyaluronic acid from hyaluronic acid (Hyaluronic Acid) using Streptococcus equi subsp. Zooepidemicus capable of producing hyaluronic acid (HA). ≪ / RTI >
HA is a major component of the skin in vivo, and is involved in skin moisturization and skin aging. HA, a main component of articular cartilage, is a polymeric polysaccharide widely used for cosmetics, foods and pharmaceuticals. In the early 1970s, HA was extracted from animal materials such as chicken eggs and chrysanthemums, and was extracted in the form of ultrahigh molecular weight of 300 to 9 million units. Due to its difficulty in extraction and purification due to its high molecular weight, Of natural cosmetics and medicines.
However, since the 1990s, there has been a growing concern about the incidence of animal - derived diseases such as mad cow disease and avian influenza, and the method of producing HA by microbial fermentation has been developed and the market expanded. However, in the case of HA by microbial fermentation, the molecular weight is up to 200 to 5 million as compared with the animal-derived material, and there are restrictions on the use of joint injections, molding fillers, and ophthalmic cataract operations as high molecular weight HA.
Therefore, it is necessary to develop a high molecular weight HA production method by microbial fermentation.
Korean Patent No. 0047007 relates to a strain having increased productivity of some hyaluronic acid, but it has been difficult to expand the market by industrializing it in view of economical efficiency because it uses an expensive animal medium.
The present inventor has found that Streptococcus equi subsp. Zooepidemicus KL0101J is prepared by a method comprising the steps of (a) 200-300 g / L glucose and (b) TSB medium (15 g / L tryptone, (Patent Application No. 10-2014-A) discloses a method for producing hyaluronic acid by inoculating a medium containing 5 g / L of soytone and 5 g / L of sodium chloride, 0107835), and further studies were conducted to increase the production yield of hyaluronic acid.
As a result, it was confirmed that high molecular weight HA can be produced with high yield by controlling the incubation temperature of Streptococcus japonidemicus KL0101J (KCCM 11360P) ( Streptococcus equi subsp. Zooepidemicus KL0101J).
It is an object of the present invention to provide a method for producing hyaluronic acid in a maximum yield using a strain having hyaluronic acid-producing ability.
In order to accomplish the above object, the present invention provides a method for producing Streptococcus equi subsp. Zooepidemicus in a medium containing 200 to 300 g / L of glucose and 25 to 30 g / L of yeast extract, And culturing the cells at a temperature of < RTI ID = 0.0 > 33 C < / RTI >
In the present invention, the Streptococcus juepidemicus is Streptococcus equipidemicus KL0101J (KCCM 11360P) ( Streptococcus equi subsp. Zooepidemicus KL0101J).
In the present invention, the culture medium contains 300 g / L of glucose and 25 g / L of yeast extract, the pH is 7.0 or more, and the culture temperature is 33 ° C.
The method of producing a polymeric hyaluronic acid according to the present invention can produce HA of 3.0 MDa or more at a high yield by optimizing culture conditions of a strain having hyaluronic acid production ability and can produce hyaluronic acid (HA) Are more widely available in the cosmetics and pharmaceuticals field.
It was confirmed that maximizing the yield of hyaluronic acid when culturing Streptococcus juepidemicus capable of producing hyaluronic acid by optimizing culture medium and culture medium.
In the present invention, Streptococcus juepidemicus was inoculated into a culture medium containing glucose and yeast extract, cultured at 30 to 35 ° C and pH 7.0, and the viscosity and yield of hyaluronic acid produced were measured. As a result, it was confirmed that high-viscosity hyaluronic acid can be produced at a high yield when cultured at 33 to 35 ° C.
Accordingly, the present invention provides, in one aspect, a method for producing Streptococcus equi subsp. Zooepidemicus in a medium comprising 200-300 g / L of glucose and 25-30 g / L of yeast extract, Lt; RTI ID = 0.0 > C, < / RTI > to a method of producing the hyaluronic acid.
The Streptococcus equi subsp. Zooepidemicus is not particularly limited as long as it has a hyaluronic acid producing ability . However , Streptococcus juepidemicus is not limited to Streptococcus equi subsp. Zooepidemicus , but may be one having Streptococcus equipisemicus KL0101J (KCCM 11360P) ( Streptococcus equi subsp. zooepidemicus KL0101J) is preferable because it can produce hyaluronic acid with high yield.
In the production method of the polymer hyaluronic acid, the culture medium contains 300 g / L of glucose and 25 g / L of yeast extract, the pH is 7.0 or more, preferably 7.0 to 8.0, and the culture temperature is 33 ° C . The pH can be adjusted by using a pH adjustable agent without directly affecting the culture of the strain, and NaOH can be exemplified.
Hyaluronic acid is removed from the culture solution of Streptococcus juepidemicus having hyaluronic acid-producing ability by removing the insoluble impurities primarily by simple filtration using diatomaceous earth, filtering it with activated charcoal, etc., and adding other impurities And then finally crystallizing using ethanol.
[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: Investigation of culture conditions of polymer hyaluronic acid -1
Streptococcus equi subsp. Zooepidemicus KL0101J (accession number: KCCM 11360P) was inoculated in 50 mL of TSB and then cultured at 35 DEG C and 200 rpm for 6 hours. Next, 5 mL of seed culture medium was inoculated into each of 125 mL of the vegetative medium (Yeast Extract 25-40 g / L and Sodium Chloride 5 g / L) as the main medium and cultured under the same conditions.
The strains were inoculated into a fermenter (Fermentor) supplemented with 300 g / L of glucose, separately sterilized, in vegetable medium (Yeast Extract 25 g / L and Sodium Chloride 5 g / L) And cultured for 24 hours.
After completion of the culture, the viscosity of the culture solution was measured using a viscometer (Brookfield Viscometer DV2T), and the amount of hyaluronic acid in the culture solution was quantified by the carbazole method (European Pharmacoepia 6.0 p2706).
From Table 1, it was confirmed that the hyaluronic acid production ability was 9 to 10 g / L when the culture temperature was 33 ° C, and the viscosity of the culture solution was the highest at 7,000 cps or more.
On the other hand, it was confirmed that the HA productivity and the viscosity of the culture liquid were lowered below 33 ° C. At above 34 ℃, the HA productivity was about 9 ~ 10g / L, which was the same as 33 ℃ but the viscosity of the culture solution was lowered.
That is, the productivity of HA at 33 ~ 35 ℃ was the same as that of HA at 9 ~ 10g / L. However, the molecular weight of HA in the culture medium was the highest value due to the highest molecular weight of HA cultured at 33 ℃.
Example 2: Investigation of culture conditions of polymer hyaluronic acid-2
Streptococcus juphidemicus KL0101J ( Streptococcus equi subsp . The strain was cultured under the same conditions except that Streptococcus epidemicus (KCCM 40305) was used instead of zooepidemicus KL0101J (Accession No .: KCCM 11360P), and the viscosity and hyaluronic acid production ability of the culture solution according to temperature were checked. Respectively.
From Table 2, the productivity of HA during incubation at 33-35 ° C is the same as 3.5-4.0 g / L, but the molecular weight of HA in the culture medium is the highest viscosity value because HA is the highest molecular weight at 33 ℃ I could.
That is, from the results shown in Tables 1 and 2, it was confirmed that when Streptococcus juphondemicus was cultured, the HA productivity was excellent at 33 to 35 ° C, and HA having a higher viscosity and molecular weight was produced at 33 ° C.
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.
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| KR102249438B1 (en) | 2020-09-25 | 2021-05-07 | 주식회사 칸젠 | Strain for producing hyaluronic acid and preparing method of hyaluronic acid using the same |
| KR102315133B1 (en) | 2021-03-26 | 2021-10-19 | 주식회사 현대바이오랜드 | Hair cosmetic composition comprising Streptococcus strain culture medium that has beneficial effects on hair |
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