JP5093796B2 - Enzyme composition, low molecular weight hyaluronic acid and method for producing the same - Google Patents
Enzyme composition, low molecular weight hyaluronic acid and method for producing the same Download PDFInfo
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Description
本発明は、酵素組成物、低分子化ヒアルロン酸及びその製造法に関する。 The present invention relates to an enzyme composition, a low molecular weight hyaluronic acid and a method for producing the same.
ヒアルロン酸は酸性ムコ多糖（グリコサミノグリカン）の一種であり、現在、医薬品、飲食品、化粧品、美容の分野で広く用いられている。ヒアルロン酸の吸収性を高めるためには、低分子化ヒアルロン酸を使用することが有用である。低分子化の方法としては、煮沸による加水分解（例えば、特許文献１参照）、酵素処理と高温高圧処理を組合わせる方法（例えば、特許文献２参照）等が公知である。なお、ヒアルロン酸を分解する酵素であるヒアルロニダーゼとしては、牛精巣由来（例えば、非特許文献１参照）の他、Streptomyces属、Propionibacterium属、Staphylococcus属、Peptostreptococcus属、Streptococcus属の微生物由来（例えば、非特許文献２参照）のものが知られている。 Hyaluronic acid is a kind of acidic mucopolysaccharide (glycosaminoglycan) and is currently widely used in the fields of pharmaceuticals, foods and drinks, cosmetics, and beauty. In order to increase the hyaluronic acid absorbability, it is useful to use low molecular weight hyaluronic acid. Known methods for reducing the molecular weight include hydrolysis by boiling (for example, see Patent Document 1), a method for combining enzyme treatment and high-temperature and high-pressure treatment (for example, see Patent Document 2), and the like. The hyaluronidase, which is an enzyme that degrades hyaluronic acid, is derived from bovine testes (see, for example, Non-patent Document 1), as well as from Streptomyces genus, Propionibacterium genus, Staphylococcus genus, Peptostreptococcus genus, Streptococcus genus (eg, non-patent document 1). Patent Document 2) is known.
本発明の課題は、低分子化ヒアルロン酸及びその製造法を提供することにある。また本発明の課題は、低分子化ヒアルロン酸の製造に好適に使用できる酵素組成物を提供することにある。 The subject of this invention is providing the low molecular-weight hyaluronic acid and its manufacturing method. Moreover, the subject of this invention is providing the enzyme composition which can be used conveniently for manufacture of low molecular-weight hyaluronic acid.
本発明者らは、Penicillium属の微生物を由来とするヒアルロニダーゼが、低分子化ヒアルロン酸の製造のために好適であることを見出し本発明を完成した。
すなわち、本発明は以下に関する。
（１）ヒアルロン酸に、Penicillium属の微生物を由来とするヒアルロニダーゼ、又はヒアルロニダーゼを含有する酵素組成物を作用させることを特徴とする、低分子化ヒアルロン酸の製造法。
（２）ヒアルロン酸に、Penicillium属の微生物を由来とするヒアルロニダーゼ、又はヒアルロニダーゼを含有する酵素組成物を作用させ、次いで、ゲルろ過法による分子量分布曲線において分子量5,000〜15,000の範囲内にピークを有する低分子化ヒアルロン酸を採取することを特徴とする、低分子化ヒアルロン酸の製造法。
（３）Penicillium属の微生物を培地で培養し、培養物からヒアルロニダーゼを含有する酵素組成物を採取することにより得られる、ヒアルロニダーゼ含有酵素組成物。
（４）Penicillium属の微生物を培地で培養し、培養物からヒアルロニダーゼを含有する酵素組成物を採取することを特徴とする、ヒアルロニダーゼ含有酵素組成物の製造法。
The present inventors have found that hyaluronidase derived from a microorganism belonging to the genus Penicillium is suitable for the production of low molecular weight hyaluronic acid, and completed the present invention.
That is, the present invention relates to the following.
(1) A method for producing a low molecular weight hyaluronic acid, comprising causing hyaluronic acid to act on hyaluronidase derived from a microorganism belonging to the genus Penicillium, or an enzyme composition containing hyaluronidase.
(2) Hyaluronic acid is allowed to act on hyaluronidase derived from a microorganism belonging to the genus Penicillium, or an enzyme composition containing hyaluronidase, and then has a peak within a molecular weight range of 5,000 to 15,000 in a molecular weight distribution curve by gel filtration. A method for producing low molecular weight hyaluronic acid, comprising collecting low molecular weight hyaluronic acid.
(3) A hyaluronidase-containing enzyme composition obtained by culturing a microorganism belonging to the genus Penicillium in a medium and collecting an enzyme composition containing hyaluronidase from the culture.
(4) A method for producing a hyaluronidase-containing enzyme composition, wherein a microorganism belonging to the genus Penicillium is cultured in a medium, and an enzyme composition containing hyaluronidase is collected from the culture.
本発明によれば、低分子化ヒアルロン酸を効率よく製造することができる。 According to the present invention, low molecular weight hyaluronic acid can be efficiently produced.
〔低分子化ヒアルロン酸及びその製造法〕
本発明の低分子化ヒアルロン酸は、ヒアルロン酸にPenicillium属の微生物を由来とするヒアルロニダーゼ又はヒアルロニダーゼ含有酵素組成物を作用させることにより製造される。
ヒアルロン酸は、その由来や平均分子量は特に限定されず、各種の動物（鶏冠等）又は微生物（Streptococcus zooepidemicus、Streptococcus equismilis等）から得られるものが使用できる。また、ヒアルロン酸としては市販品（紀文フードケミファ社製「ヒアルロン酸ＦＣＨ」、キューピー社製「ヒアルロンサンＨＡ−Ｆ」等）も使用できる。
[Low molecular hyaluronic acid and method for producing the same]
The low molecular weight hyaluronic acid of the present invention is produced by allowing hyaluronic acid or a hyaluronidase-containing enzyme composition derived from a microorganism belonging to the genus Penicillium to act on hyaluronic acid.
The origin and average molecular weight of hyaluronic acid are not particularly limited, and those obtained from various animals (such as chicken crowns) or microorganisms (Streptococcus zooepidemicus, Streptococcus equismilis, etc.) can be used. As hyaluronic acid, commercially available products (“Hyaluronic acid FCH” manufactured by Kibun Food Chemifa, “Hyaluronic sun HA-F” manufactured by Kewpie, etc.) can be used.
本発明のヒアルロニダーゼは、Penicillium属の微生物を由来とする。Penicillium属の微生物とは、例えば、Penicillium purpurogenumやPenicillium funiculosumであり、より具体的にはPenicillium purpurogenum (IAM 13753)、Penicillium purpurogenum(IAM 13754)、Penicillium purogenum(IAM 7095)、Penicillium funiculosum(IAM13752)である。Penicillium属の微生物は、例えば、東京大学分子細胞生物学研究所 細胞機能情報研究センター ＩＡＭカルチャーコレクション[IAM Culture Collection（略号 IAM）]から入手することができる。ヒアルロニダーゼは、Penicillium属の微生物を培地で培養し、培養物から酵素組成物としてヒアルロニダーゼを採取することにより得られる。 The hyaluronidase of the present invention is derived from a microorganism belonging to the genus Penicillium. The microorganisms of the genus Penicillium are, for example, Penicillium purpurogenum and Penicillium funiculosum, more specifically Penicillium purpurogenum (IAM 13753), Penicillium purpurogenum (IAM 13754), Penicillium purogenum (IAM 7095), Penicillium funiculosum (IAM13752). . Microorganisms belonging to the genus Penicillium can be obtained, for example, from the IAM Culture Collection (abbreviated as IAM), Cell Function Information Research Center, Institute for Molecular Cell Biology, The University of Tokyo. Hyaluronidase can be obtained by culturing a microorganism belonging to the genus Penicillium in a medium and collecting hyaluronidase as an enzyme composition from the culture.
ヒアルロン酸にヒアルロニダーゼを作用させる条件は、使用するヒアルロン酸及びヒアルロニダーゼの種類に応じて適宜設定すればよい。例えば、微生物由来の平均分子量1,800,000〜2,200,000のヒアルロン酸に、Penicillium purpurogenum由来のヒアルロニダーゼ含有酵素組成物を作用させる場合、０.１％ヒアルロン酸溶液に対して、酵素組成物を添加し、４〜５０℃、好ましくは３５〜４５℃で、１分〜３０日間、好ましくは１〜４８時間程度反応させればよい。
上記の反応によって低分子化ヒアルロン酸が生成したか否かは、ゲルろ過法又は極限粘度法等により確認できる。ゲルろ過法による分子量分布曲線が、低分子化ヒアルロン酸に起因するピークを有する場合は、低分子化ヒアルロン酸が生成したといえる。また、極限粘度法を用いる場合は、酵素組成物による処理前後のヒアルロン酸の粘度から平均分子量を算出する。酵素組成物による処理後のヒアルロン酸の方が平均分子量が小さい場合には、低分子化ヒアルロン酸が生成したといえる。
ヒアルロン酸とヒアルロニダーゼの比率、反応温度、反応時間等の反応条件を変えることにより、ヒアルロン酸の低分子化の程度を調整することができる。反応条件の変更により、例えば、ゲルろ過法による分子量分布曲線において分子量約400〜300,000、好ましくは分子量約400〜30,000、より好ましくは5,000〜15,000、最も好ましくは6,000〜12,000の範囲内にピークを有する低分子化ヒアルロン酸を得ることができる。
The conditions for causing hyaluronidase to act on hyaluronic acid may be appropriately set according to the types of hyaluronic acid and hyaluronidase used. For example, when a hyaluronidase-containing enzyme composition derived from Penicillium purpurogenum is allowed to act on hyaluronic acid having an average molecular weight of 1,800,000-2,200,000 derived from microorganisms, the enzyme composition is added to a 0.1% hyaluronic acid solution, and 4-50 The reaction may be carried out at a temperature of 35 ° C., preferably 35 to 45 ° C., for 1 minute to 30 days, preferably about 1 to 48 hours.
Whether or not a low molecular weight hyaluronic acid is generated by the above reaction can be confirmed by a gel filtration method or an intrinsic viscosity method. When the molecular weight distribution curve by the gel filtration method has a peak due to the low molecular weight hyaluronic acid, it can be said that the low molecular weight hyaluronic acid was generated. When the intrinsic viscosity method is used, the average molecular weight is calculated from the viscosity of hyaluronic acid before and after the treatment with the enzyme composition. When the average molecular weight of hyaluronic acid after treatment with the enzyme composition is smaller, it can be said that low molecular weight hyaluronic acid was produced.
By changing the reaction conditions such as the ratio of hyaluronic acid and hyaluronidase, reaction temperature, reaction time, etc., the degree of molecular weight reduction of hyaluronic acid can be adjusted. By changing the reaction conditions, for example, in the molecular weight distribution curve by gel filtration, the molecular weight is about 400 to 300,000, preferably the molecular weight is about 400 to 30,000, more preferably 5,000 to 15,000, and most preferably 6,000 to 12,000. Low molecular weight hyaluronic acid can be obtained.
〔ヒアルロニダーゼ含有酵素組成物及びその製造法〕
本発明のヒアルロニダーゼ含有酵素組成物は、Penicillium属の微生物を培地で培養し、培養物からヒアルロニダーゼを含有する酵素組成物を採取することにより製造される。
Penicillium属の微生物とは、例えば、Penicillium purpurogenumやPenicillium funiculosumであり、より具体的にはPenicillium purpurogenum (IAM 13753)、Penicillium purpurogenum(IAM 13754)、Penicillium purpurogenum(IAM 7095)、Penicillium funiculosum(IAM13752)である。
ヒアルロニダーゼ含有酵素組成物が得られる限り、微生物の培養方法及び酵素組成物の採取方法は特に限定されないが、例えば、以下の方法が採用できる。Penicillium purpurogenumを、適当な培地に植菌し、２５〜３５℃、３〜７日間、静地培養する。培地としては、例えば合成培地、デンプン含有培地、米飯等の穀物蒸煮物が使用できる。得られた培養物を０．２Ｍ ＮａＣｌ水溶液を用いて抽出すれば、抽出液として、ヒアルロニダーゼ含有酵素組成物が採取できる。更に通常の酵素精製方法を用いて、該ヒアルロニダーゼ含有酵素組成物中のヒアルロニダーゼ含有量を高めることができる。この様にして得られたヒアルロニダーゼ含有酵素組成物は、本発明の低分子化ヒアルロン酸を製造する際に使用できる。
[Hyaluronidase-containing enzyme composition and its production method]
The hyaluronidase-containing enzyme composition of the present invention is produced by culturing a microorganism of the genus Penicillium in a medium and collecting the enzyme composition containing hyaluronidase from the culture.
The microorganism of the genus Penicillium is, for example, Penicillium purpurogenum or Penicillium funiculosum, more specifically Penicillium purpurogenum (IAM 13753), Penicillium purpurogenum (IAM 13754), Penicillium purpurogenum (IAM 7095), Penicillium funiculosum (IAM13752). .
As long as the hyaluronidase-containing enzyme composition is obtained, the method for culturing microorganisms and the method for collecting the enzyme composition are not particularly limited. For example, the following methods can be employed. Penicillium purpurogenum is inoculated into an appropriate medium and cultured at 25 to 35 ° C. for 3 to 7 days. As the medium, for example, a synthetic medium, a starch-containing medium, a cooked cereal such as cooked rice can be used. If the obtained culture is extracted using a 0.2 M NaCl aqueous solution, a hyaluronidase-containing enzyme composition can be collected as the extract. Furthermore, the hyaluronidase content in the hyaluronidase-containing enzyme composition can be increased by using a normal enzyme purification method. The thus obtained hyaluronidase-containing enzyme composition can be used for producing the low molecular weight hyaluronic acid of the present invention.
以下により、Penicillium属の微生物を由来とするヒアルロニダーゼ酵素組成物が、低分子化ヒアルロン酸の製造のために使用できることを確認した。
１．試験菌株（４種）
（１）Penicillium purpurogenum(IAM 13753)
（２）Penicillium purpurogenum(IAM 13754)
（３）Penicillium purpurogenum(IAM 7095)
（４）Penicillium funiculosum(IAM 13752)
２．ヒアルロン酸の調製
Streptococcus zooepidemicus由来のヒアルロン酸（紀文フードケミファ社製「ヒアルロン酸ＦＣＨ ＦＣＨ２００」、平均分子量：1,800,000〜2,200,000）を０．１％の濃度で蒸留水に溶解させ、ヒアルロン酸水溶液とした。
３．ヒアルロニダーゼ含有酵素組成物の調製
試験菌株を米飯培地に植菌し、３０℃、３日間静置培養した。培養物に０．２Ｍ ＮａＣｌ水溶液を添加し、１時間振とうした後、遠心により培養上清を調製し、これをヒアルロニダーゼ含有酵素組成物とした。コントロールとして、米飯培地を３０℃、３日間静置培養し、０．２Ｍ ＮａＣｌ水溶液を添加し、１時間振とうした後、遠心により遠心上清を得た。
４．ヒアルロン酸の低分子化処理
ヒアルロン酸水溶液とヒアルロニダーゼ含有酵素組成物とを任意の割合で混合した後、混合物を３７℃で２４時間静置し、ヒアルロン酸の低分子化処理を行った。
５．ゲルろ過法による低分子化ヒアルロン酸の分析
低分子化処理後の試料中に含まれるヒアルロン酸の分子量について、ゲルろ過カラムを用いて分析した。ゲルろ過の条件は以下の通り。
・カラム： G6000PWXL, G4000PWXL, G2500PWXL （東ソー社製 7.8 x 300mm）
・溶離液： 0.2M NaCl 1ml/min.
・検 出： 吸光度（210nm）
・分子量標準物質： Shodex STANDARD P-82（昭和電工社製、物質名:プルラン、分子量範囲：5,000〜800,000）
６．低分子化ヒアルロン酸の分析結果
４種の試験菌株全てについて、ゲルろ過法による分子量分布曲線を分析したところ、低分子化処理後の試料に含まれるヒアルロン酸の分子量分布曲線のピークは、処理前のピークよりも、低分子量側に移動したことが確認された。また、酵素組成物の添加量を増やした場合、又は処理時間を長くした場合は、分子量分布曲線のピークは更に低分子量側に移動することが確認された。上記４．の処理による低分子化ヒアルロン酸の生成が確認された。
図１に、Penicillium purpurogenum由来のヒアルロニダーゼ含有酵素組成物を用いて得られた低分子化ヒアルロン酸の、ゲルろ過法による分子量分布曲線を示す。低分子化処理前のヒアルロン酸の分子量のピークは1,000,000以上であった（図１上段）。一方、３７℃で２４時間で低分子化処理した後のヒアルロン酸の分子量のピーク（溶出時間：２６．３３３分）は、約12,000であった（図１中段）。図１中段に示す低分子化ヒアルロン酸の、ゲルろ過法での溶出時間は２４．５〜３０分である。分子量標準物質「Shodex STANDARD P-82」（昭和電工社製）を用いてゲルろ過を行った結果によれば、この溶出時間は分子量約900〜47,000に相当する。
一方、上記３．でコントロールとして調製した遠心上清については、処理の前後でヒアルロン酸の分子量分布曲線のパターンは変化しなかった。
以上のことから、４種の試験菌株全てがヒアルロニダーゼ生産能を有し、その培養物からヒアルロニダーゼ含有酵素組成物が得られること、及び該酵素組成物が低分子化ヒアルロン酸の製造に使用できることが示された。
７．低分子化ヒアルロン酸の吸光度分析
Penicillium purpurogenum由来のヒアルロニダーゼ含有酵素組成物による低分子化反応の解析のため、以下の方法により、低分子化ヒアルロン酸の吸光度分析を行った。
Streptomyces属由来のヒアルロニダーゼ（リアーゼ型）、牛精巣（Bovine Testes）由来のヒアルロニダーゼ（加水分解型）及びPenicillium purpurogenum由来のヒアルロニダーゼ含有酵素組成物を用いて、上記と同様にヒアルロン酸の低分子化処理を行った。次いで、ゲルろ過カラムにより分離を行い、分子量約10,000に相当する溶出時間で、多波長検出器（日本分光社製 MD2010plus）により吸光度スペクトルを得た。結果を図２に示す。
吸光度パターンの比較により、Penicillium purpurogenum由来のヒアルロニダーゼ含有酵素組成物による低分子化反応は、リアーゼ反応ではなく、加水分解反応であることが示唆された。
From the following, it was confirmed that a hyaluronidase enzyme composition derived from a microorganism belonging to the genus Penicillium can be used for the production of low molecular weight hyaluronic acid.
1. Test strains (4 types)
(1) Penicillium purpurogenum (IAM 13753)
(2) Penicillium purpurogenum (IAM 13754)
(3) Penicillium purpurogenum (IAM 7095)
(4) Penicillium funiculosum (IAM 13752)
2. Preparation of hyaluronic acid
Hyaluronic acid derived from Streptococcus zooepidemicus ("Hyaluronic acid FCH FCH200" manufactured by Kibun Food Chemifa Co., Ltd., average molecular weight: 1,800,000-2,200,000) was dissolved in distilled water at a concentration of 0.1% to obtain a hyaluronic acid aqueous solution.
3. Preparation of Hyaluronidase-Containing Enzyme Composition The test strain was inoculated into a cooked rice medium and statically cultured at 30 ° C. for 3 days. A 0.2 M NaCl aqueous solution was added to the culture and shaken for 1 hour, and then a culture supernatant was prepared by centrifugation, which was used as a hyaluronidase-containing enzyme composition. As a control, the cooked rice medium was statically cultured at 30 ° C. for 3 days, a 0.2 M NaCl aqueous solution was added, and the mixture was shaken for 1 hour, and then a centrifugal supernatant was obtained by centrifugation.
4). Hyaluronic acid molecular weight reduction treatment A hyaluronic acid aqueous solution and a hyaluronidase-containing enzyme composition were mixed at an arbitrary ratio, and then the mixture was allowed to stand at 37 ° C. for 24 hours to perform a hyaluronic acid molecular weight reduction treatment.
5. Analysis of low molecular weight hyaluronic acid by gel filtration The molecular weight of hyaluronic acid contained in the sample after the low molecular weight treatment was analyzed using a gel filtration column. The conditions for gel filtration are as follows.
・ Column: G6000PWXL, G4000PWXL, G2500PWXL (Tosoh 7.8 x 300mm)
・ Eluent: 0.2M NaCl 1ml / min.
・ Detection: Absorbance (210nm)
・ Molecular weight standard substance: Shodex STANDARD P-82 (manufactured by Showa Denko KK, substance name: pullulan, molecular weight range: 5,000 to 800,000)
6). Results of analysis of low molecular weight hyaluronic acid When all four test strains were analyzed for molecular weight distribution curves by gel filtration, the peak of the molecular weight distribution curve of hyaluronic acid contained in the sample after low molecular weight treatment was It was confirmed that it moved to the lower molecular weight side than the peak. Moreover, when the addition amount of the enzyme composition was increased or when the treatment time was lengthened, it was confirmed that the peak of the molecular weight distribution curve further moved to the low molecular weight side. 4. above. Formation of low molecular weight hyaluronic acid by the treatment of was confirmed.
FIG. 1 shows a molecular weight distribution curve by gel filtration of a low molecular weight hyaluronic acid obtained using a hyaluronidase-containing enzyme composition derived from Penicillium purpurogenum. The molecular weight peak of hyaluronic acid before the molecular weight reduction treatment was 1,000,000 or more (the upper part of FIG. 1). On the other hand, the molecular weight peak (elution time: 26.333 minutes) of hyaluronic acid after low molecular weight treatment at 37 ° C. for 24 hours was about 12,000 (middle in FIG. 1). The elution time in the gel filtration method of the low molecular weight hyaluronic acid shown in the middle of FIG. 1 is 24.5 to 30 minutes. According to the results of gel filtration using the molecular weight standard substance “Shodex STANDARD P-82” (manufactured by Showa Denko KK), this elution time corresponds to a molecular weight of about 900 to 47,000.
On the other hand, the above 3. In the centrifugal supernatant prepared as a control, the pattern of the molecular weight distribution curve of hyaluronic acid did not change before and after the treatment.
From the above, all four test strains have the ability to produce hyaluronidase, the hyaluronidase-containing enzyme composition can be obtained from the culture, and the enzyme composition can be used for the production of low molecular weight hyaluronic acid. Indicated.
7). Absorbance analysis of low molecular weight hyaluronic acid
In order to analyze the molecular weight reduction reaction using the hyaluronidase-containing enzyme composition derived from Penicillium purpurogenum, the absorbance analysis of the low molecular weight hyaluronic acid was performed by the following method.
Using hyaluronidase (lyase type) derived from Streptomyces genus, hyaluronidase (hydrolyzed type) derived from bovine testes and hyaluronidase-containing enzyme composition derived from Penicillium purpurogenum, hyaluronic acid can be reduced in molecular weight as described above. went. Next, separation was performed with a gel filtration column, and an absorbance spectrum was obtained with a multi-wavelength detector (MD2010plus manufactured by JASCO Corporation) with an elution time corresponding to a molecular weight of about 10,000. The results are shown in FIG.
Comparison of the absorbance patterns suggested that the molecular weight reduction reaction by the hyaluronidase-containing enzyme composition derived from Penicillium purpurogenum was not a lyase reaction but a hydrolysis reaction.
実施例１とは異なる反応条件を用いて、ゲルろ過法による分子量分布曲線において分子量約6,000にピークを有する低分子化ヒアルロン酸を得た。
まず、実施例１の２．及び３．記載の方法により調製した０．１％ヒアルロン酸水溶液及びヒアルロニダーゼ含有酵素組成物を混合した後、３７℃で４８時間静置し、ヒアルロン酸の低分子化処理を行った。
低分子化処理後の混合物について、実施例１の５．記載のゲルろ過法により分析を行った。ゲルろ過法による分子量分布曲線において分子量約6,000にピーク（溶出時間：２７．３０７分）を有する低分子化ヒアルロン酸が生成していることが確認された（図１下段）。図１下段にしめす低分子化ヒアルロン酸のゲルろ過法での溶出時間は２５．５〜３０分である。分子量標準物質「Shodex STANDARD P-82」（昭和電工社製）を用いてゲルろ過を行った結果によれば、この溶出時間は分子量約900〜23,000に相当する。
Using reaction conditions different from those in Example 1, a low molecular weight hyaluronic acid having a peak at a molecular weight of about 6,000 in a molecular weight distribution curve by gel filtration was obtained.
First, in Example 1. And 3. The 0.1% hyaluronic acid aqueous solution prepared by the method described above and the hyaluronidase-containing enzyme composition were mixed, and then allowed to stand at 37 ° C. for 48 hours to perform a hyaluronic acid molecular weight reduction treatment.
Regarding the mixture after the molecular weight reduction treatment, 5. in Example 1. Analysis was performed by the described gel filtration method. It was confirmed that low molecular weight hyaluronic acid having a peak at a molecular weight of about 6,000 (elution time: 27.307 minutes) in the molecular weight distribution curve by gel filtration was generated (lower part of FIG. 1). The elution time of the low molecular weight hyaluronic acid shown in the lower part of FIG. 1 in the gel filtration method is 25.5 to 30 minutes. According to the result of gel filtration using a molecular weight standard substance “Shodex STANDARD P-82” (manufactured by Showa Denko KK), this elution time corresponds to a molecular weight of about 900 to 23,000.
本発明により、医薬品、飲食品、化粧品の分野で有用な低分子化ヒアルロン酸を簡単に得ることができる。 According to the present invention, low molecular weight hyaluronic acid useful in the fields of pharmaceuticals, foods and drinks, and cosmetics can be easily obtained.
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