JPH0439998B2 - - Google Patents
Info
- Publication number
- JPH0439998B2 JPH0439998B2 JP5488089A JP5488089A JPH0439998B2 JP H0439998 B2 JPH0439998 B2 JP H0439998B2 JP 5488089 A JP5488089 A JP 5488089A JP 5488089 A JP5488089 A JP 5488089A JP H0439998 B2 JPH0439998 B2 JP H0439998B2
- Authority
- JP
- Japan
- Prior art keywords
- hyaluronic acid
- streptococcus
- medium
- glucose
- sodium hyaluronate
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 claims description 21
- 229920002674 hyaluronan Polymers 0.000 claims description 21
- 229960003160 hyaluronic acid Drugs 0.000 claims description 21
- 241000194048 Streptococcus equi Species 0.000 claims description 8
- 238000004519 manufacturing process Methods 0.000 claims description 8
- 238000012258 culturing Methods 0.000 claims description 5
- 229920002385 Sodium hyaluronate Polymers 0.000 description 11
- 239000002609 medium Substances 0.000 description 11
- 229940010747 sodium hyaluronate Drugs 0.000 description 11
- YWIVKILSMZOHHF-QJZPQSOGSA-N sodium;(2s,3s,4s,5r,6r)-6-[(2s,3r,4r,5s,6r)-3-acetamido-2-[(2s,3s,4r,5r,6r)-6-[(2r,3r,4r,5s,6r)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2- Chemical compound [Na+].CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 YWIVKILSMZOHHF-QJZPQSOGSA-N 0.000 description 11
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 10
- 239000008103 glucose Substances 0.000 description 10
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 8
- 241000194017 Streptococcus Species 0.000 description 7
- 229940041514 candida albicans extract Drugs 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 239000012138 yeast extract Substances 0.000 description 5
- 235000019441 ethanol Nutrition 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
- 238000005273 aeration Methods 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 3
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 3
- 235000011121 sodium hydroxide Nutrition 0.000 description 3
- AKHNMLFCWUSKQB-UHFFFAOYSA-L sodium thiosulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=S AKHNMLFCWUSKQB-UHFFFAOYSA-L 0.000 description 3
- 235000019345 sodium thiosulphate Nutrition 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 108010003272 Hyaluronate lyase Proteins 0.000 description 2
- 102000001974 Hyaluronidases Human genes 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 241000187747 Streptomyces Species 0.000 description 2
- 238000000862 absorption spectrum Methods 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 229960001927 cetylpyridinium chloride Drugs 0.000 description 2
- YMKDRGPMQRFJGP-UHFFFAOYSA-M cetylpyridinium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCC[N+]1=CC=CC=C1 YMKDRGPMQRFJGP-UHFFFAOYSA-M 0.000 description 2
- 238000000354 decomposition reaction Methods 0.000 description 2
- 239000000645 desinfectant Substances 0.000 description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 229960002773 hyaluronidase Drugs 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 1
- VZUNGTLZRAYYDE-UHFFFAOYSA-N N-methyl-N'-nitro-N-nitrosoguanidine Chemical compound O=NN(C)C(=N)N[N+]([O-])=O VZUNGTLZRAYYDE-UHFFFAOYSA-N 0.000 description 1
- 241000194049 Streptococcus equinus Species 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229940127554 medical product Drugs 0.000 description 1
- 239000012533 medium component Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 229910000160 potassium phosphate Inorganic materials 0.000 description 1
- 235000011009 potassium phosphates Nutrition 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 229940115921 streptococcus equinus Drugs 0.000 description 1
- 210000003954 umbilical cord Anatomy 0.000 description 1
- 210000004127 vitreous body Anatomy 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
〔産業上の利用分野〕
本発明は、醗酵法によるヒアルロン酸の製造法
に関する。さらに詳しくは、栄養要求性が部分的
に解除されたストレプトコツカス・エキを培養
し、ヒアルロン酸を生成蓄積せしめることを特徴
とするヒアルロン酸の製造法に関する。
〔従来の技術〕
従来ヒアルロン酸はニワトリのトサカ、牛の眼
の硝子体又は臍帯等より抽出によつて得られてい
た。しかしながら抽出法によるヒアルロン酸製造
は、分離精製が非常に繁雑等の課題を有してい
た。
その課題を改良するために、ヒアルロン酸を生
産する能力を有する微生物を培養し、その培養液
から直接ヒアルロン酸を採取する方法が提案され
てきたが収量のバラツキがあり、生産性が不安定
であつた。
本発明者らも先にヒアルロン酸生産を工業的に
実施すべく種々研究を行つた結果、栄養要求性が
部分的に解除されたストレプトコツカス・エキの
変異株FM・100が親株よりも高収量で、しかも
収量のバラツキが少なく安定的にヒアルロン酸を
生成することを見い出した（特開昭63−123392号
公報）。
〔発明が解決しようとする課題〕
しかしストレプトコツカス・エキの変異株
FM・100を用いてヒアルロン酸生産を行うに際
し、長期間にわたつて何回も培養を行なう場合、
あるいは、培養液の一部を残し、そこに新たな培
地を添加し培養を継続する半連続培養を行なう場
合、ヒアルロン酸の収量が徐々に低下し、バラツ
キも大きく、安定なヒアルロン酸の製造を行うこ
とが困難であつた。
〔課題を解決するための手段〕
本発明者らは、かかる課題を解決すべく、種々
研究を行なつた結果、ストレプトコツカスエキの
変異株FM・100から、更に栄養要求性が解除さ
れた株として誘導された変異株FM・300（微工研
条寄第2319号）がヒアルロン酸の生産安定性の面
で大きく改善されていることを見い出し、本発明
を完成するに至つた。
すなわち本発明は、ストレプトコツカス・エキ
（Streptococcus equi）の変異株FM・300（微工
研条寄第2319号）を培養し、ヒアルロン酸を生成
蓄積せしめることを特徴とする該ヒアルロン酸の
製造方法である。
以下、本発明について具体的に説明する。
本発明の栄養要求性が部分的に解除されたスト
レプトコツカス・エキFM・300（微工研条寄第
2319号）は、ヒアルロン酸生成能を有するストレ
プトコツカス・エキFM・100の突然変異株の中
から取得することが出来る。
例えば、ストレプトコツカス・エキFM・100
を用い、ポリペプトン1.5％、酵母エキス0.5％、
グリコース２％の培地にて、33℃で培養し、対数
増殖期の菌を、低温で遠心分離により集菌し、生
理食塩水を用いて、無菌的に３回洗浄する。Ｎ−
メチル−N′−ニトロ−Ｎ−ニトロソグアニジン
50μg／mlを含むPH5.0、0.05Mリン酸緩衝液中、
30℃で１時間振盪したのち、氷冷する。ついで、
生理食塩水を用いて、低温で菌体を３回洗浄した
後、ポリペプトン1.5％、酵母エキス0.5％、グル
コース２％の培地で、33℃、３時間培養し、また
生理食塩水を用いて、低温で菌体を３回洗浄す
る。表１に示す人工合成培地で、33℃、７日間液
体培養し、増殖してきた培養液をさらに、新しい
同じ人工合成培地にうえつぎ、この操作を３回く
りかえす。
次に寒天を含む同じ組成の培地上に塗布し、コ
ロニーを分離し、ストレプトコツカス・エキ
FM300を得る。
本菌株は、工業技術院微生物工業技術研究所
に、微工研条寄第2319号として受託されている。
ストレプトコツカスFM・300はストレプトコツ
カス・エキFM・100から更にスレオニン及びフ
エニルアラニンの栄養要求性が解除され、ストレ
プトコツカス・エキFM・100が生育できない表
１に示す培地成分だけからなる人工合成培地によ
く生育することができる。
[Industrial Field of Application] The present invention relates to a method for producing hyaluronic acid by fermentation. More specifically, the present invention relates to a method for producing hyaluronic acid, which is characterized by culturing Streptococcus equi whose auxotrophy has been partially released, and producing and accumulating hyaluronic acid. [Prior Art] Hyaluronic acid has conventionally been obtained by extraction from chicken crests, cow eye vitreous bodies, umbilical cords, and the like. However, the production of hyaluronic acid by the extraction method has problems such as extremely complicated separation and purification. In order to solve this problem, a method has been proposed in which microorganisms capable of producing hyaluronic acid are cultivated and hyaluronic acid is directly collected from the culture solution, but the yield varies and productivity is unstable. It was hot. The present inventors also previously conducted various studies to implement hyaluronic acid production industrially, and as a result, a mutant strain of Streptococcus equi, FM-100, in which auxotrophy was partially abolished, was found to have higher levels of growth than the parent strain. It has been found that hyaluronic acid can be produced stably in a high yield with little variation in yield (Japanese Patent Laid-Open Publication No. 123392/1983). [Problem to be solved by the invention] However, mutant strains of Streptococcus equi
When producing hyaluronic acid using FM-100, if culturing is performed many times over a long period of time,
Alternatively, when performing semi-continuous culture in which a portion of the culture medium is left and a new medium is added to continue culturing, the yield of hyaluronic acid gradually decreases and is highly variable, making it difficult to produce stable hyaluronic acid. It was difficult to do. [Means for Solving the Problem] In order to solve the problem, the present inventors conducted various studies, and as a result, the auxotrophy was further removed from the mutant strain FM-100 of Streptococcus nigra. The inventors discovered that the derived mutant strain FM-300 (Feikoken Jokyo No. 2319) was greatly improved in terms of production stability of hyaluronic acid, leading to the completion of the present invention. Specifically, the present invention provides a method for producing hyaluronic acid, which is characterized by culturing Streptococcus equi mutant strain FM-300 (Feikoken Jokyo No. 2319) and producing and accumulating hyaluronic acid. It's a method. The present invention will be explained in detail below. Streptococcus Eki FM 300 with partially released auxotrophy according to the present invention
No. 2319) can be obtained from a mutant strain of Streptococcus equi FM 100 that has the ability to produce hyaluronic acid. For example, Streptococcus Eki FM 100
using polypeptone 1.5%, yeast extract 0.5%,
The cells are cultured at 33°C in a medium containing 2% glycose, and the bacteria in the logarithmic growth phase are collected by centrifugation at a low temperature and washed three times aseptically with physiological saline. N-
Methyl-N'-nitro-N-nitrosoguanidine
In PH5.0, 0.05M phosphate buffer containing 50μg/ml,
After shaking at 30°C for 1 hour, cool on ice. Then,
After washing the bacterial cells three times at low temperature with physiological saline, they were cultured at 33°C for 3 hours in a medium containing 1.5% polypeptone, 0.5% yeast extract, and 2% glucose. Wash the bacterial cells three times at low temperature. The cells were cultured in liquid at 33°C for 7 days in the artificial synthetic medium shown in Table 1, and the grown culture was then transferred to the same new artificial synthetic medium, and this operation was repeated three times. Next, it was plated on a medium of the same composition containing agar, the colonies were isolated, and Streptococcus equinus
Get FM300. This strain has been entrusted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, as FAIKEN Article No. 2319.
Streptococcus FM-300 has the nutritional requirements for threonine and phenylalanine removed from Streptococcus Eki FM-100, and consists only of the medium components shown in Table 1, in which Streptococcus Eki FM-100 cannot grow. It can grow well in artificial synthetic media.
次に実施例により、本発明を詳しく説明する
が、本発明はこれに限定されるものではない。
実施例 １
グルコース２％、リン酸第１カリウム0.2％、
硫酸マグネシウム７水塩0.05％、チオ硫酸ソーダ
0.1％、ポリペプトン1.0％酵母エキス0.5％からな
るPH8.5の培地 1lに同一培地からなるストレプ
トコツカス・エキFM・300の前培養液10mlを接
触し、通気量1.5vvm（volume volume minute）、
攪拌200回転／分、温度33℃でカ性ソーダでPHを
8.5にコントロールしながら培養し、15時間後に、
グルコース２％を分添し、グルコースが全部消費
された時点で培養液900mlを抜き出した。さらに
グルコース２％、リン酸第１カリウム0.2％、硫
酸マグネシウム７水塩0.05％、チオ硫酸ソーダ
0.1％、ポリペプトン1.0％酵母エキス0.5％からな
るpH8.5の培地900mlを添加し、通気量1.5vvm、
攪拌200回転／分、温度33℃でカ性ソーダでPHを
8.5にコントロールしながら培養し、５時間後に、
グルコース２％を分添し、グルコースが全部消費
された時点で培養液900mlを抜き出した。
上記の半連続操作をあと４回くりかえし、計６
バツチ行つた結果を表２に示す。
培養液は塩酸でPH４に調整後、蒸留水で２倍希
釈し、遠心分離により除菌した。得られた除菌液
をエチルアルコールを加え、ヒアルロン酸ソーダ
を析出せしめる。これをろ別した後、水に溶解
し、セチルピリジニウムクロライドを加え、生じ
た沈殿をろ取し、２％食塩水に再溶解後、再びエ
チルアルコールによる析出をくり返す。得られた
ヒアルロン酸ソーダを室温で減圧乾燥して、白色
のヒアルロン酸ソーダを得た。
得られたヒアルロン酸ソーダは、赤外線吸収ス
ペクトル、Ｃ−13核磁気共鳴スペクトル、ストレ
プトミセスのヒアルロニダーゼによる分解実験で
ヒアルロン酸ソーダであることが確認された。
Next, the present invention will be explained in detail with reference to Examples, but the present invention is not limited thereto. Example 1 Glucose 2%, potassium monophosphate 0.2%,
Magnesium sulfate heptahydrate 0.05%, sodium thiosulfate
0.1%, polypeptone 1.0%, yeast extract 0.5%, pH 8.5 medium 1l was contacted with 10ml of a preculture of Streptococcus equi FM 300 made of the same medium, and the aeration rate was 1.5vvm (volume volume minute).
Stir at 200 rpm and adjust the pH with caustic soda at a temperature of 33°C.
Cultured under control at 8.5, and after 15 hours,
2% glucose was added in portions, and when all the glucose was consumed, 900 ml of the culture solution was taken out. In addition, glucose 2%, potassium monophosphate 0.2%, magnesium sulfate heptahydrate 0.05%, sodium thiosulfate
Add 900 ml of pH 8.5 medium consisting of 0.1% polypeptone, 1.0% yeast extract, 1.5 vvm aeration volume,
Stir at 200 rpm and adjust the pH with caustic soda at a temperature of 33°C.
Cultured under control at 8.5, 5 hours later,
2% glucose was added in portions, and when all the glucose was consumed, 900 ml of the culture solution was taken out. Repeat the above semi-continuous operation 4 more times, for a total of 6
Table 2 shows the batch results. The culture solution was adjusted to pH 4 with hydrochloric acid, diluted 2 times with distilled water, and sterilized by centrifugation. Ethyl alcohol is added to the obtained disinfectant solution to precipitate sodium hyaluronate. After filtering this, it is dissolved in water, cetylpyridinium chloride is added, and the resulting precipitate is collected by filtration, redissolved in 2% saline, and the precipitation with ethyl alcohol is repeated again. The obtained sodium hyaluronate was dried under reduced pressure at room temperature to obtain white sodium hyaluronate. The obtained sodium hyaluronate was confirmed to be sodium hyaluronate by an infrared absorption spectrum, a C-13 nuclear magnetic resonance spectrum, and a decomposition experiment using Streptomyces hyaluronidase.
【表】
比較例 １
ストレプトコツカス・エキFM・100を実施例
１と同様にして６回半連続培養したが、得られた
ヒアルロン酸ソーダはそれぞれ、7.1g，5.0g，
4.5g，4.0g，2.5g，2.0gであり収量は実施例１に
比較して、低く、またばらついていた。
実施例 ２
グルコース２％、リン酸第１カリウム0.2％、
硫酸マグネシウム７水塩0.05％、チオ硫酸ソーダ
0.1％、ポリペプトン1.0％酵母エキス0.5％からな
るpH8.5の培地 1lに同一培地からなるストレプ
トコツカス・エキFM・300の前培養液10mlを接
触し、通気量1.5vvm、攪拌200回転／分、温度33
℃でカ性ソーダでPHを8.5にコントロールしなが
ら培養し、15時間後に、グルコース２％を分添
し、グルコースが全部消費された時点で培養を停
止した。
培養液を塩酸でPH４に調整後、蒸留水で２倍希
釈し、遠心分離により除菌した。得られた除菌液
をエチルアルコールを加え、ヒアルロン酸ソーダ
を析出せしめる。これをろ別した後、水に溶解
し、セチルピリジニウムクロライドを加え、生じ
た沈殿をろ取し、２％食塩水に再溶解後、再びエ
チルアルコールによる析出をくり返す。得られた
ヒアルロン酸ソーダを室温で減圧乾燥して、培養
液1lあたり7.7gの白色ヒアルロン酸ソーダを得
た。
得られたヒアルロン酸ソーダは、赤外線吸収ス
ペクトル、Ｃ−13核磁気共鳴スペクトル、ストレ
プトミセスのヒアルロニダーゼによる分解実験で
ヒアルロン酸ソーダであることが確認された。
上記と同様の培養を14回くりかえし、全部で15
バツチ行なつた結果を表３に示す。[Table] Comparative Example 1 Streptococcus Eki FM 100 was continuously cultured six and a half times in the same manner as in Example 1, but the obtained sodium hyaluronate was 7.1 g, 5.0 g,
The yields were 4.5g, 4.0g, 2.5g, and 2.0g, which were lower than in Example 1 and varied. Example 2 Glucose 2%, potassium phosphate 0.2%,
Magnesium sulfate heptahydrate 0.05%, sodium thiosulfate
0.1%, polypeptone 1.0%, yeast extract 0.5%, pH 8.5 medium (1 liter) was contacted with 10 ml of Streptococcus equi FM 300 preculture solution made of the same medium, aeration rate 1.5 vvm, stirring 200 rpm/min. , temperature 33
The cells were cultured at ℃ while controlling the pH to 8.5 with caustic soda, and after 15 hours, 2% glucose was added in portions, and the culture was stopped when all the glucose was consumed. The culture solution was adjusted to pH 4 with hydrochloric acid, diluted 2 times with distilled water, and sterilized by centrifugation. Ethyl alcohol is added to the obtained disinfectant solution to precipitate sodium hyaluronate. After filtering this, it is dissolved in water, cetylpyridinium chloride is added, and the resulting precipitate is collected by filtration, redissolved in 2% saline, and the precipitation with ethyl alcohol is repeated again. The obtained sodium hyaluronate was dried under reduced pressure at room temperature to obtain 7.7 g of white sodium hyaluronate per liter of culture solution. The obtained sodium hyaluronate was confirmed to be sodium hyaluronate by an infrared absorption spectrum, a C-13 nuclear magnetic resonance spectrum, and a decomposition experiment using Streptomyces hyaluronidase. The same culture as above was repeated 14 times, for a total of 15
Table 3 shows the batch results.
本発明によれば、ヒアルロン酸生産のために数
多く繰り返し培養を行つたり、半連続培養を行つ
ても、ヒアルロン酸を高収率で、しかも収量にほ
とんどばらつきなく、安定に生産することができ
る。また菌株管理も容易である。
本発明によつて製造されたヒアルロン酸は、化
粧品、医療品に配合して使用できる。
According to the present invention, hyaluronic acid can be stably produced at a high yield with almost no variation in yield even if many repeated cultures or semi-continuous cultures are performed for hyaluronic acid production. . In addition, strain management is easy. The hyaluronic acid produced according to the present invention can be used in cosmetics and medical products.
Claims (1)
（Streptococcusequi）の変異株FM・300（微工研
条寄第2319号）を培養し、ヒアルロン酸を生成蓄
積せしめることを特徴とする該ヒアルロン酸の製
造方法。1. A method for producing hyaluronic acid, which comprises culturing a mutant strain of Streptococcusequi FM-300 (Feikoken Joyori No. 2319) to produce and accumulate hyaluronic acid.
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP5488089A JPH02234689A (en) | 1989-03-09 | 1989-03-09 | Production of hyaluronic acid |
| US07/347,337 US4946780A (en) | 1988-10-12 | 1989-05-04 | Method for producing sodium hyaluronate by fermentation method |
| DE68914236T DE68914236T2 (en) | 1988-10-12 | 1989-05-11 | Process for the production of sodium hyaluronate by fermentation. |
| EP89108522A EP0363561B1 (en) | 1988-10-12 | 1989-05-11 | Method for producing sodium hyaluronate by fermentation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP5488089A JPH02234689A (en) | 1989-03-09 | 1989-03-09 | Production of hyaluronic acid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH02234689A JPH02234689A (en) | 1990-09-17 |
| JPH0439998B2 true JPH0439998B2 (en) | 1992-07-01 |
Family
ID=12982901
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP5488089A Granted JPH02234689A (en) | 1988-10-12 | 1989-03-09 | Production of hyaluronic acid |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH02234689A (en) |
Families Citing this family (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100393322C (en) | 2002-08-16 | 2008-06-11 | 电气化学工业株式会社 | Separate medical material |
| EP1681306B1 (en) | 2003-10-29 | 2013-02-20 | Teijin Limited | Hyaluronic acid compound, hydrogel thereof and material for treating joint |
| WO2008041514A1 (en) | 2006-09-22 | 2008-04-10 | Kochi University | Radiation sensitizer or anti-cancer chemotherapy sensitizer |
| JP4920552B2 (en) * | 2007-11-07 | 2012-04-18 | 株式会社ヤクルト本社 | Method for producing hyaluronic acid |
| JP2011195611A (en) * | 2010-03-17 | 2011-10-06 | Denki Kagaku Kogyo Kk | Purification method for hyaluronic acid and/or salt thereof |
| JP2011195604A (en) * | 2010-03-17 | 2011-10-06 | Denki Kagaku Kogyo Kk | Method for removing foreign matter in solution containing hyaluronic acid and/or salt thereof |
| CN102812051B (en) * | 2010-03-17 | 2015-08-26 | 电气化学工业株式会社 | The dissolving method of hyaluronic acid and/or its salt |
| KR101834588B1 (en) | 2010-08-23 | 2018-03-05 | 덴카 주식회사 | Crosslinked hyaluronic acid composition and self-crosslinking hyaluronic acid particles |
| TR201905159T4 (en) | 2013-02-15 | 2019-05-21 | Kortuc Inc | Radiation / chemotherapy sensitizer to be used in intra-tumoral local injection and for the controlled release of hydrogen peroxide with hydrogel as carrier. |
| KR20160029818A (en) | 2013-07-08 | 2016-03-15 | 덴카 주식회사 | Core-shell crosslinked hyaluronic acid gel particles, production method for same, and medical material |
| JP6391956B2 (en) * | 2014-03-20 | 2018-09-19 | コスモＡｌａ株式会社 | Process for producing 5-aminolevulinic acid or a salt thereof |
| JP6391957B2 (en) * | 2014-03-20 | 2018-09-19 | コスモＡｌａ株式会社 | Process for producing 5-aminolevulinic acid or a salt thereof |
| AU2021292668A1 (en) | 2020-06-15 | 2023-02-09 | KORTUC Japan LLC | Sensitiser for cancer treatment |
| JP7684719B2 (en) | 2023-02-17 | 2025-05-28 | 合同会社Kortuc Japan | Pharmaceutical composition for stimulating immunity against tumors and method thereof |
-
1989
- 1989-03-09 JP JP5488089A patent/JPH02234689A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPH02234689A (en) | 1990-09-17 |
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