WO2010010631A1 - ヒアルロン酸の製造方法 - Google Patents
ヒアルロン酸の製造方法 Download PDFInfo
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- WO2010010631A1 WO2010010631A1 PCT/JP2008/063427 JP2008063427W WO2010010631A1 WO 2010010631 A1 WO2010010631 A1 WO 2010010631A1 JP 2008063427 W JP2008063427 W JP 2008063427W WO 2010010631 A1 WO2010010631 A1 WO 2010010631A1
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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
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
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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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- 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
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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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
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
- C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
- C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
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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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- 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
Definitions
- the present invention relates to a method for producing hyaluronic acid using a microorganism.
- Hyaluronic acid is used as a medicine in cosmetics, moisturizers, ophthalmology, orthopedics, dermatology and the like.
- Hyaluronic acid can be produced from extracts from animal tissues such as chicken crowns, vitreous bodies of cattle eyes, etc., but chondroitin sulfate etc. are mixed as contaminants, hyaluronidase contained in the tissues, etc. Therefore, it is easy to reduce the molecular weight by culturing microorganisms having the ability to produce hyaluronic acid and producing hyaluronic acid from the culture solution (fermentation method) (Patent Document 1).
- the hyaluronic acid produced by the fermentation method is produced with a certain raw material and with a certain method as compared with the extraction method, and therefore, the product quality is kept constant, and thus the industrial utility value is great.
- Patent Documents 2 to 4 describe a fermentation method using a medium in which eight kinds of amino acids essential for growth are increased as an effective component for hyaluronic acid production.
- Patent Document 3 describes a fermentation method using a medium in which arginine and / or glutamic acid is increased.
- Patent Document 4 describes a fermentation method using a medium with an increased amount of arginine.
- the present invention has been made in view of the above circumstances, and an object thereof is to provide a method for easily producing hyaluronic acid in a high yield. Another object of the present invention is to provide a method for producing hyaluronic acid in a short time.
- the inventors have improved hyaluronic acid production by adding arginine and glutamine to a culture solution in combination at a specific time of culture. As a result, the present invention has been completed.
- a method for producing hyaluronic acid comprising a step of culturing a microorganism having hyaluronic acid-producing ability and a step of adding glutamine and arginine to the culture solution in the late logarithmic growth phase of the microorganism.
- hyaluronic acid can be easily produced in a high yield and in a short time by combining glutamine and arginine and adding them to the culture solution at a specific stage of culture.
- hyaluronic acid can be obtained in a high yield and in a short time by adding glutamine and arginine in combination to a culture solution at a specific time of cultivation of a microorganism capable of producing hyaluronic acid. It can be easily manufactured.
- FIG. 1 is a graph of amino acid concentration in a culture solution when a hyaluronic acid-producing microorganism is cultured.
- FIG. 2 is a graph of comparison between the case where arginine is added all at once and the case where it is added afterwards.
- the left graph represents the time course of the bacterial cell concentration, and the right graph represents the time course of the viscosity (an index of the hyaluronic acid concentration).
- the term “late logarithm phase” in the present specification refers to a state in which microorganisms are sufficiently grown, from the latter half of the logarithmic phase to the stationary phase (stationary phase).
- the logarithmic growth phase refers to a time when the logarithm of the number of cells is linear with respect to time when the microorganism proliferates in half at regular intervals.
- the late phase of the logarithmic growth phase can be determined using any index / method known to those skilled in the art. For simplicity, it is not limited to this, but turbidity or specific growth rate may be used as an index, for example.
- Turbidity in the present specification is the most common simple and rapid measurement method for measuring the growth of microorganisms by the turbidity of the culture solution (the amount of cells in the culture solution). Light is applied to the culture medium to determine how much the transmitted light is blocked by scattering and absorption. Since microorganisms that produce hyaluronic acid are fine particles, the amount of microorganisms and the turbidity of the culture solution are in a proportional relationship. Turbidity is measured as follows, for example.
- the intensity of incident light and transmitted light is defined as I 0 , I, the thickness of the transmissive layer is L, the absorbance is ⁇ , and the absorbance obtained by the following formula is defined as the turbidity (OD) of the culture solution.
- I the intensity of incident light and transmitted light
- L the thickness of the transmissive layer
- ⁇ the absorbance obtained by the following formula
- OD the absorbance obtained by the following formula
- the logarithmic growth period is preferably a period showing turbidity of 1.0 or more, more preferably a period showing turbidity of 2.0 or more, and further preferably a period showing turbidity of 3.0 or more.
- the “specific growth rate” in the present specification is a value defined by the following formula.
- the cell generation time is the time required for the microorganisms producing hyaluronic acid to double.
- the growth rate decreases due to depletion of nutrients, accumulation of metabolites, and the like, and the specific growth rate also decreases.
- a time when the specific growth rate is 0.5 h ⁇ 1 or less is used.
- time showing a 0.4 h -1 or less specific growth rate more preferably time showing a 0.3h -1 following specific growth rate, 0.2 h -1 following ratio
- a period showing a growth rate is more preferable, and a period showing a specific growth rate of 0.1 h ⁇ 1 or less is even more preferable.
- the specific growth rate is greater than 0h- 1 .
- Streptococcus bacterium includes, but is not limited to, any bacterium belonging to the genus Streptococcus capable of producing hyaluronic acid, and its mutant strains. Exhibition (Streptococcus equi), Streptococcus zooepidemicus (Streptococcus zooepidemicus), Streptococcus Ekishimirisu (Streptococcus equisimilis), Streptococcus dysgalactiae (Streptococcus dysgalactiae), and the like Streptococcus pyogenes (Streptococcus pyogenes) and their mutants
- the definition of chemical substances such as “glutamine”, “arginine”, “hyaluronic acid” and the like is not limited to any salt that can be used within the range not impairing the object of the invention.
- metal salts such as sodium salt and potassium salt, acid adducts such as hydrochloride, phosphate and citrate) and hydrates, and mixtures thereof are also included.
- each numerical range in this specification includes an upper limit value and a lower limit value indicated by “ ⁇ ”.
- An embodiment of the present invention is a method for producing hyaluronic acid, comprising a step of culturing a microorganism having hyaluronic acid-producing ability and a step of adding glutamine and arginine to the culture solution in the late logarithmic growth phase of the microorganism.
- this production method by adding arginine together with glutamine at the later stage of culture, the growth inhibitory action of microorganisms at a high concentration of arginine can be suppressed, and hyaluronic acid can be produced in a high yield and in a short time.
- the medium includes, for example, a carbon source composed of sugar components such as glucose, fructose, galactose, sucrose, monopotassium phosphate, dipotassium phosphate, magnesium nitrate, sodium sulfite, sodium thiosulfate, ammonium phosphate.
- Inorganic salts such as polypeptone, casamino acid, yeast extract, corn steep liquor, soybean hydrolyzate, and various vitamins are suitably used.
- the culture can be performed using a known method such as an aerobic culture method such as aeration stirring culture.
- the culture temperature is preferably 30 to 35 ° C., but is not limited thereto. Since the pH of the culture solution decreases with the growth of the microorganism, a pH adjuster such as sodium hydroxide, potassium hydroxide, or ammonia may be added to control the pH to 7.0 to 9.0.
- Glutamine and arginine may be added simultaneously or separately, and other components such as buffers and salts may be added together as long as the object of the present invention is not impaired. In consideration of the process and the like, it is preferable that the number of components that need to be purified is small.
- the nutritional component or active component other than glutamine and arginine is 0.001% or less, 0.0005% or less, or is not included. preferable.
- a further embodiment of the present invention is the above production method, wherein the late logarithmic growth phase is a time when the turbidity at 660 nm of the culture solution shows 0.5 or more.
- glutamine and arginine are added when the turbidity is 2.0 or more.
- glutamine and arginine are added when the turbidity is 3.0 or more. It is more preferable to add glutamine and arginine at a time when the turbidity is 3.0 or more, and it is possible to reliably improve the yield and shorten the time.
- the turbidity of the culture solution can be used as it is, but the turbidity of a control medium such as a medium before culturing is so large that it cannot be ignored (not limited to this, for example, 0.01 or 0.05 or more), and the turbidity obtained by subtracting the turbidity of the control medium from the turbidity of the culture solution may be used as an index.
- a control medium a medium before culture or a culture solution not inoculated with microorganisms may be used.
- Yet another embodiment of the present invention is the above production method, wherein the late logarithmic growth phase is a time when the specific growth rate is 0.5 h ⁇ 1 or less.
- the yield of hyaluronic acid is more reliably improved.
- glutamine and arginine are added at a time when the specific growth rate is 0.4 h ⁇ 1 or less.
- glutamine and arginine are added at a time when the above-mentioned specific growth rate is exhibited in a state where microorganisms are sufficiently grown, hyaluronic acid can be obtained with a higher yield. It is more preferable to add glutamine and arginine at a time when the turbidity is 0.3 h ⁇ 1 or less, and it is possible to reliably improve the yield and achieve a short time.
- the definition of the addition time based on the above specific growth rate is a specific example of the time range by a certain index, and the step of measuring the specific growth rate under the above conditions is necessarily required. is not.
- a further embodiment of the present invention is the above production method, wherein the microorganism having the ability to produce hyaluronic acid is a Streptococcus bacterium.
- the microorganism having the ability to produce hyaluronic acid is a Streptococcus bacterium.
- a Streptococcus bacterium that has hyaluronic acid production ability and is generally used, hyaluronic acid can be industrially produced easily and reliably.
- the Streptococcus bacteria used here include Streptococcus bacteria isolated from nature and mutants thereof.
- the Streptococcus bacterium in the above embodiment includes Streptococcus equi, Streptococcus equi mutant strain FM-100 (Microtechnical Research Institute No. 9027) or Streptococcus ex mutant strain FM-300 (Microtechnological Institute Research Institute No. 2319) can be preferably used.
- Streptococcus equi Streptococcus equi mutant strain FM-100 (Microtechnical Research Institute No. 9027) or Streptococcus ex mutant strain FM-300 (Microtechnological Institute Research Institute No. 2319)
- hyaluronic acid can be produced with a higher yield and stable productivity.
- the amount of glutamine added in the above embodiment is not particularly limited as long as the object of the present invention is not impaired. However, when the amount is excessively added, the effect is reduced, so 0.01 to 0.3% is preferable. More preferably, they are 0.05% or more, 0.1% or more, and / or 0.2% or less. By using glutamine in this concentration range, the effects of yield improvement and production stabilization can be achieved more reliably. Further, by using glutamine below the above upper limit value, the burden on the subsequent separation / purification process can be reduced.
- the amount of arginine added in the above embodiment is not particularly limited as long as the object of the present invention is not impaired. More preferably, they are 0.05% or more, 0.1% or more, and / or 0.1% or less. By using arginine within this concentration range, the effects of yield improvement and production stabilization can be more reliably achieved. Moreover, by using arginine below the above upper limit value, it is possible to reduce the burden of the subsequent separation / purification process, and it is possible to further prevent the growth inhibitory action by arginine.
- the manufacturing method may include other steps, or further other steps / methods may be performed following the manufacturing method.
- processes / methods include removal / purification of impurities such as endotoxin, protein, nucleic acid, metal, etc. by sterilization process by activated carbon or filtration, neutralization process, crystallization process, chromatography, centrifugation, etc. The process etc. are mentioned.
- hyaluronic acid is “HA”
- glutamine is “Gln”
- arginine is “Arg”
- adding a certain component to the medium from the beginning of the culture is “batch addition”
- post-addition The addition of a component at a later stage (particularly at the later stage of the logarithmic growth phase) to the culture medium was referred to as “post-addition”.
- post-addition hyaluronic acid production was evaluated using the viscosity correlated with the hyaluronic acid concentration as an index.
- Example 1 One liter of medium consisting of 1.5% polypeptone and 0.5% yeast extract is heat-sterilized and then added to 6% glucose, 0.1% dipotassium hydrogen phosphate, and 0.06% glutamine as initial culture conditions. Inoculated with Streptococcus ex FM-100 (90th craftsmanship of fine work laboratory), aerated at 500 rpm with aeration of air at 1 vvm, temperature 33 ° C, pH 8.0 (25% sodium hydroxide auto The culture was started by dripping control). The culture solution was collected over time, and when the turbidity at 660 nm reached 3.6 and the specific growth rate reached 0.34 h ⁇ 1 , glutamine 0.1% and arginine 0.05% equivalent were added. .
- the culture was continued until the increase in the viscosity of the culture broth due to the accumulation of hyaluronic acid stopped. After stopping the increase in viscosity of the culture solution, the culture solution was adjusted to pH 3 with nitric acid, the cells were removed by centrifugation, and the supernatant was collected.
- the supernatant was sequentially diluted, and the concentration of hyaluronic acid contained in the medium was measured by size exclusion chromatography equipped with a differential refraction type detector.
- a high yield of 6.2 g / L was obtained for the hyaluronic acid in the medium, and the time for the hyaluronic acid concentration to reach 4.0 g / L as the culture time was 17 hours, which was a short time.
- Example 2 Culture was started in the same manner as in Example 1, and 0.1% glutamine and 0.1% arginine were added. A high yield of 6.0 g / L was obtained for the hyaluronic acid in the medium, and the time for the hyaluronic acid concentration to reach 4.0 g / L as the culture time was 17 hours, which was a short time.
- Example 3 Culture was started in the same manner as in Example 1, and 0.04% less glutamine than in Example 1 and 0.05% equivalent to arginine were added. The addition was carried out at a bacterial cell concentration of OD5.3 and a specific growth rate of 0.16. Since the amount of added glutamine is small, hyaluronic acid in the medium is less than in Examples 1 and 2, but a high yield of 5.2 g / L was obtained, and the time for the hyaluronic acid concentration to reach 4.0 g / L as the culture time was It was as short as 18 hours.
- Example 4 Culture was started in the same manner as in Example 3, and glutamine and arginine were added as in Example 3. The addition was performed at a bacterial cell concentration of OD 7.0 and a specific growth rate of 0.05. A high yield of 5.0 g / L of hyaluronic acid in the medium was obtained, and the time for the hyaluronic acid concentration to reach 4.0 g / L as a culture time was a short time of 18 hours.
- Example 1 Culture was started in the same manner as in Example 1, and glutamine and arginine were not added.
- the hyaluronic acid in the medium was a low yield of 4.1 g / L, and the time for the hyaluronic acid concentration to reach 4.0 g / L as a culture time was as long as 23 hours.
- Example 2 Culture was started in the same manner as in Example 1, and 0.05% of arginine alone was added. As the culture time, the time when the hyaluronic acid concentration reached 4.0 g / L was 18 hours, but hyaluronic acid in the medium was a low yield of 4.3 g / L.
- Example 3 Culture was started in the same manner as in Example 1, and only 0.1% glutamine was added. Hyaluronic acid in the medium was a low yield of 5.3 g / L. As the culture time, the time when the hyaluronic acid concentration reached 4.0 g / L was as long as 21 hours.
- Example 4 Culture was started in the same manner as in Example 3, and glutamine and arginine were added as in Example 3. The addition was carried out at a bacterial cell concentration of OD0.0001 and a specific growth rate of 1.0. Hyaluronic acid in the medium was a low yield of 4.0 g / L.
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Abstract
Description
本明細書における「対数増殖期後期」とは、微生物が十分に増殖した状態であって、対数増殖期の後半~静止期(定常期)にかけてをいう。なお、対数増殖期とは、微生物の培養において、微生物が一定時間ごとに二分して増殖するとき、時間に対して細胞数の対数が直線となる時期のことをいう。対数増殖期の後期であることは、当業者に既知の、任意の指標・方法を用いて判断することができる。簡便には、これに限定されるものではないが、例えば濁度又は比増殖速度を指標として判断してもよい。
以下、本発明の実施の形態について説明する。
従来の標準的な条件でのヒアルロン酸生産微生物の培養における、各種アミノ酸の濃度変化を調べた。結果のグラフを図1に示す。各種アミノ酸は、培養時間経過に伴い、種類によって減少、一定、増加の何れかの挙動を示したが、特に、グルタミンおよびアルギニン量の大幅な減少が観察され、標準的な培養条件では、これらが枯渇することが観察された。
予備実験1の結果に基づき、グルタミン及びアルギニンを添加することを念頭において、さらなる検討を行った。アルギニンは、その菌体内での機構は不明であるが、単体ではヒアルロン酸収量向上には寄与せず、生育阻害をもたらすことが報告されている。そこで、それらの点について、アルギニンの添加方法を改良することでさらに改善しようと考え、検討を行った。
その結果、図2のグラフに示すように、アルギニンを培養時に一括添加せず、後添加することで、ヒアルロン酸の濃度(グラフ中、ヒアルロン酸濃度は粘度により評価される)が飛躍的に高まることが見出された。このアルギニンについての実験結果は、以下の表にまとめることができる。
以上の実験の結果から得られた知見を基に、標準条件に対し、グルタミン及びアルギニンを後添加してヒアルロン酸生産微生物を培養する実験を行った。この実験結果を以下の表2に示す。
以下に示す実施例1~4及び比較例1~4の実験を、実施例1と同様の条件で行い、ヒアルロン酸の濃度及び、一定のヒアルロン酸の濃度(粘度で評価)に達するまでの時間を計測した。結果は、表3に示した。
ポリペプトン1.5%、酵母エキス0.5%からなる培地1リットルを加熱殺菌後、グルコース6%、リン酸水素二カリウム0.1%、グルタミン0.06%となるように加え培養初期条件とし、ストレプトコッカス・エキFM-100(微工研条寄第9027号)を接種し、空気を1vvmで通気しながら、撹拌500回転/分、温度33℃、pH8.0(25%水酸化ナトリウムの自動滴下によるコントロール)で培養を開始した。経時的に培養液を採取し、培養液の660nmにおける濁度が3.6、比増殖速度が0.34h-1に達した時点でグルタミン0.1%、アルギニン0.05%相当を添加した。ヒアルロン酸の蓄積による培養液の粘度上昇が停止するまで培養を継続した。培養液の粘度上昇の停止後、培養液を硝酸でpH3に調整し、遠心により菌体を除き、上清を回収した。
実施例1と同様に培養を開始し、グルタミン0.1%、アルギニン0.1%相当を添加した。培地中のヒアルロン酸は6.0g/Lの高収量が得られ、培養時間としてヒアルロン酸濃度が4.0g/Lに達した時間は17時間と短時間であった。
実施例1と同様に培養を開始し、グルタミンは実施例1より少ない0.04%、アルギニンは0.05%相当を添加した。添加時期は菌体濃度がOD5.3、比増殖速度0.16で行った。添加グルタミン量が少ないため、培地中のヒアルロン酸は実施例1、2より少ないが5.2g/Lの高収量が得られ、培養時間としてヒアルロン酸濃度が4.0g/Lに達した時間は18時間と短時間であった。
実施例3と同様に培養を開始し、実施例3と同様にグルタミンとアルギニンを添加した。添加時期は菌体濃度がOD7.0、比増殖速度0.05で行った。培地中のヒアルロン酸は5.0g/Lの高収量が得られ、培養時間としてヒアルロン酸濃度が4.0g/Lに達した時間は18時間の短時間であった。
実施例1と同様に培養を開始し、グルタミンとアルギニンは添加しなかった。培地中のヒアルロン酸は4.1g/Lと低収量であり、培養時間としてヒアルロン酸濃度が4.0g/Lに達した時間は23時間と長時間であった。
実施例1と同様に培養を開始し、アルギニンのみを0.05%添加した。培養時間としてヒアルロン酸濃度が4.0g/Lに達した時間は18時間であったが、培地中のヒアルロン酸は4.3g/Lと低収量であった。
実施例1と同様に培養を開始し、グルタミンのみを0.1%添加した。培地中のヒアルロン酸は5.3g/Lと低収量だった。培養時間としてヒアルロン酸濃度が4.0g/Lに達した時間は21時間と長時間であった。
実施例3と同様に培養を開始し、実施例3と同様にグルタミンとアルギニンを添加した。添加時期は菌体濃度がOD0.0001、比増殖速度1.0で行った。培地中のヒアルロン酸は4.0g/Lと低収量であった。
上記実施例1~4及び比較例1~4と同様の方法(グルコース濃度は6%)で、さらなる比較実験を行った。結果を以下の表4に示す。この比較実験からも、ヒアルロン酸の生産量(表中HA濃度)がグルタミンとアルギニンとを対数増殖期後期に培養液に添加した場合において、ヒアルロン酸の生産量が増加することが確認された。
以上の実験から、グルタミン及びアルギニンを対数増殖期後期に培養液に添加する方法では、従来技術に比べ、ヒアルロン酸を高収率かつ短時間で製造可能であることが確認された。
Claims (8)
- ヒアルロン酸生産能を有する微生物を培養する工程と、該微生物の対数増殖期後期にグルタミン及びアルギニンを培養液に添加する工程とを含むヒアルロン酸の製造法。
- 前記対数増殖期後期が、培養液の660nmにおける濁度が0.5以上を示す時期である請求項1に記載の製造方法。
- 前記対数増殖期後期が、培養液の660nmにおける濁度が2.0以上を示す時期である請求項1に記載の製造方法。
- 前記対数増殖期後期が、比増殖速度が0.5h-1以下を示す時期である請求項1に記載の製造方法。
- 前記ヒアルロン酸生産能を有する微生物がストレプトコッカス属細菌である請求項1ないし4の何れか一項に記載のヒアルロン酸の製造法。
- 前記ストレプトコッカス属細菌が、ストレプトコッカス・エキ、ストレプトコッカス・エキ変異株FM-100(微工研条寄第9027号)又はストレプトコッカス・エキ変異株FM-300(微工研条寄第2319号)である請求項5に記載のヒアルロン酸の製造方法。
- 前記グルタミンの濃度が、0.01~0.3%である請求項1ないし6の何れか一項に記載のヒアルロン酸の製造法。
- 前記アルギニンの濃度が、0.01~0.2%である請求項1ないし7の何れか一項に記載のヒアルロン酸の製造法。
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PCT/JP2008/063427 WO2010010631A1 (ja) | 2008-07-25 | 2008-07-25 | ヒアルロン酸の製造方法 |
EP08791671A EP2311971B1 (en) | 2008-07-25 | 2008-07-25 | Method for producing hyaluronic acid |
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JP2014197985A (ja) * | 2013-03-29 | 2014-10-23 | 栃木県 | オルニチンを富化した納豆の製造方法 |
JP2014528239A (ja) * | 2011-09-30 | 2014-10-27 | イルドン ファーム カンパニー リミテッド | ストレプトコッカスディスガラクティエID9103菌株及びこれを利用したヒアルロン酸生産方法(StreptococcusdysgalactiaeID9103andmethodforproductionofhyaluronicacidusingthesame) |
US9074000B2 (en) | 2011-04-01 | 2015-07-07 | Memorial Sloan Kettering Cancer Center | T cell receptor-like antibodies specific for a WT1 peptide presented by HLA-A2 |
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ITMI20121184A1 (it) * | 2012-07-05 | 2014-01-06 | Altergon Italia Srl | Processo per la produzione industriale di ialuronato di sodio (hana) altamente purificato a peso molecolare controllato |
KR101598329B1 (ko) * | 2013-03-20 | 2016-02-29 | 일동제약주식회사 | 히알루론산의 제조방법 및 상기 제조방법으로 제조된 히알루론산을 포함하는 유착방지용 조성물 |
CN108611387B (zh) * | 2018-05-07 | 2020-09-08 | 山东焦点生物科技股份有限公司 | 一种利用植物蛋白胨生产医药级透明质酸钠的方法 |
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KR20230034047A (ko) | 2021-09-02 | 2023-03-09 | (주)아모레퍼시픽 | 해조류 추출물을 이용한 히알루론산의 제조방법 |
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
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US9074000B2 (en) | 2011-04-01 | 2015-07-07 | Memorial Sloan Kettering Cancer Center | T cell receptor-like antibodies specific for a WT1 peptide presented by HLA-A2 |
US9540448B2 (en) | 2011-04-01 | 2017-01-10 | Memorial Sloan Kettering Cancer Center | T cell receptor-like antibodies specific for a WTI peptide presented by HLA-A2 |
JP2014528239A (ja) * | 2011-09-30 | 2014-10-27 | イルドン ファーム カンパニー リミテッド | ストレプトコッカスディスガラクティエID9103菌株及びこれを利用したヒアルロン酸生産方法(StreptococcusdysgalactiaeID9103andmethodforproductionofhyaluronicacidusingthesame) |
JP2014197985A (ja) * | 2013-03-29 | 2014-10-23 | 栃木県 | オルニチンを富化した納豆の製造方法 |
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JPWO2010010631A1 (ja) | 2012-01-05 |
US8927234B2 (en) | 2015-01-06 |
US20110207178A1 (en) | 2011-08-25 |
AU2008359822A1 (en) | 2010-01-28 |
KR101443429B1 (ko) | 2014-09-24 |
CA2732092A1 (en) | 2010-01-28 |
CA2732092C (en) | 2015-02-24 |
AU2008359822B2 (en) | 2013-12-05 |
EP2311971A4 (en) | 2012-06-27 |
KR20110036845A (ko) | 2011-04-11 |
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JP5356388B2 (ja) | 2013-12-04 |
CN102124120A (zh) | 2011-07-13 |
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