JP2010252641A - Method for producing polysaccharide derived from lactic bacterium - Google Patents
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Abstract
Description
本発明は、多糖類の製造方法に関する。 The present invention relates to a method for producing a polysaccharide.
乳酸菌は菌の体外に多糖類を生産することが知られており、菌の種類によって多種多様な多糖類を生産する。このような多糖類は、その物理化学的性質や生理機能から食品や化粧品、医療用途など幅広い用途で用いられることから、生産性を上げるための醗酵生産方法について研究が盛んになされている。 Lactic acid bacteria are known to produce polysaccharides outside the body of bacteria, and produce a wide variety of polysaccharides depending on the type of bacteria. Since such polysaccharides are used in a wide range of applications such as foods, cosmetics, and medical uses because of their physicochemical properties and physiological functions, research on fermentation production methods for increasing productivity has been actively conducted.
なかでも、ヒアルロン酸は、一定の品質の製品が容易に得られることから、醗酵法によって製造されている。その醗酵法において、効率よくヒアルロン酸を製造する方法がいくつか検討されている。例えば、界面活性剤を添加して培養する方法（特許文献１参照）、グルタミンやグルタミン酸を添加して培養する方法（非特許文献１参照）、ウリジンを添加して培養する方法（特許文献２参照）である。
いずれの方法を用いたとしても、微生物を培養するための培地は、コンタミネーションを防ぐためにも加熱殺菌しなければならない。その際に、培地組成に含まれる糖類の濃度が濃い場合は加熱により糖とアミノ酸が反応する褐変反応が生じ、ヒアルロン酸の生産が妨げられる。そこで、糖類の濃度が濃い場合は培地中の糖とその他の成分を別々に加熱殺菌して培養前に混合する必要がある。
しかしながら、糖のみの水溶液の加熱殺菌においても、糖濃度が濃い場合には加熱により糖が変性するため、微生物の生育阻害、ヒアルロン酸合成阻害が引き起こされ、培養中のヒアルロン酸を高濃度に蓄積することができない点が問題である。
Among these, hyaluronic acid is produced by a fermentation method because a product of a certain quality can be easily obtained. In the fermentation method, several methods for efficiently producing hyaluronic acid have been studied. For example, a method in which a surfactant is added and cultured (see Patent Document 1), a method in which glutamine and glutamic acid are added (see Non-Patent Document 1), and a method in which uridine is added and cultured (see Patent Document 2) ).
Regardless of which method is used, the medium for culturing microorganisms must be heat sterilized to prevent contamination. At that time, when the concentration of the saccharide contained in the medium composition is high, a browning reaction in which the saccharide and the amino acid react with each other occurs by heating, thereby preventing the production of hyaluronic acid. Therefore, when the concentration of saccharide is high, it is necessary to separately sterilize the saccharide and other components in the medium and mix them before culturing.
However, even in the heat sterilization of an aqueous solution containing only sugar, if the sugar concentration is high, the sugar is denatured by heating, causing microbial growth inhibition and hyaluronic acid synthesis inhibition and accumulating high concentrations of hyaluronic acid in the culture. The problem is that it cannot be done.
そこで、本発明は、培養液中に多糖類を高濃度に蓄積させ、効率よく多糖類を得ることを目的とする。 Accordingly, an object of the present invention is to efficiently obtain a polysaccharide by accumulating the polysaccharide in a culture solution at a high concentration.
すなわち、本発明は、ｐＨを４以下に調整した後に加熱処理した糖溶液を含む培地中で乳酸菌を培養することにより多糖類を生産する方法に関する。 That is, the present invention relates to a method for producing a polysaccharide by culturing lactic acid bacteria in a medium containing a sugar solution that has been heat-treated after adjusting the pH to 4 or less.
本発明によれば、培養液中に高濃度の多糖類を蓄積することができ、より効率良く多糖類を生産することができる。 According to the present invention, a high concentration of polysaccharides can be accumulated in the culture solution, and the polysaccharides can be produced more efficiently.
（１）乳酸菌及び乳酸菌由来の多糖類
本発明における乳酸菌とは、通常の培養により菌体外に多糖類を生産することができる乳酸菌のことである。当該乳酸菌は多糖生産能を有していれば種類は限定されない。例えば、Ｌａｃｔｏｂａｃｉｌｌｕｓ属に属する微生物、Ｌａｃｔｏｃｏｃｃｕｓ属に属する微生物、Ｌｅｕｃｏｎｏｓｔｏｃ属に属する微生物、Ｐｅｄｉｏｃｏｃｃｕｓ属に属する微生物及びＳｔｒｅｐｔｏｃｏｃｃｕｓ属に属する微生物等を挙げることができる。
乳酸菌の生産する乳酸菌由来の多糖類には、単一の糖からなるホモ多糖と、複数の単糖や単糖誘導体からなるヘテロ多糖がある。ホモ多糖には、グルコースからなるデキストラン、βグルカン、ムタン、アルテルナン；フルクトースからなるレバン、イヌリン；ガラクトースからなるガラクタン等が知られている。
デキストランを生産する乳酸菌としてＬａｃｔｏｂａｃｉｌｌｕｓ ｈｉｌｇａｒｄｉｉ、Ｌａｃｔｏｂａｃｉｌｌｕｓ ｃｏｎｆｕｓｕｓ、Ｌａｃｔｏｂａｃｉｌｌｕｓ ｖｉｒｉｄｅｓｃｅｎｓ、Ｌｅｕｃｏｎｏｓｔｏｃ ｍｅｓｅｎｔｅｒｉｏｄｅｓ等、βグルカン生産乳酸菌としてＰｅｄｉｏｃｏｃｃｕｓ ｄａｍｎｏｓｕｓ等、ムタン生産乳酸菌としてＳｔｒｅｐｔｏｃｏｃｃｕｓ ｍｕｔａｎ、Ｓｔｒｅｐｔｏｃｏｃｃｕｓ ｓｏｂｒｉｕｓ等、アルテルナン生産菌としてＬｅｕｃｏｎｏｓｔｏｃ ｍｅｓｅｎｔｅｒｉｏｄｅｓ等、レバン生産乳酸菌としてＳｔｒｅｐｔｏｃｏｃｃｕｓ ｍｕｔａｎ、Ｓｔｒｅｐｔｏｃｏｃｃｕｓ ｓａｌｉｖａｒｉｕｓ等、イヌリン生産乳酸菌としてＳｔｒｅｐｔｏｃｏｃｃｕｓ ｍｕｔａｎ等、ガラクタン生産乳酸菌としてＬａｃｔｏｃｏｃｃｕｓ ｌａｃｔｉｓ等が挙げられる。
ヘテロ多糖は、単糖のグルコース、ガラクトース、ラムノース、フコース、糖誘導体であるＮ−アセチルグルコサミン、Ｎ−アセチルガラクトサミン、グルクロン酸などが少なくとも２種類以上で構成されるユニットが連なったものである。
代表的なヘテロ多糖はN−アセチルグルコサミンとグルクロン酸からなるヒアルロン酸、グルコースとガラクトースからなるケフィランが挙げられる。ヒアルロン酸生産乳酸菌としてＳｔｒｅｐｔｏｃｏｃｃｕｓ ｅｑｕｉ、Ｓｔｒｅｐｔｏｃｏｃｃｕｓ ｚｏｏｅｐｉｄｅｍｉｃｕｓ、Ｓｔｒｅｐｔｏｃｏｃｃｕｓ ｐｙｏｇｅｎｅｓ、Ｓｔｒｅｐｔｏｃｏｃｃｕｓ ｕｂｅｒｉｓ、Ｓｔｒｅｐｔｏｃｏｃｃｕｓ ｔｈｅｒｍｏｐｈｉｌｕｓ等、ケフィラン生産乳酸菌としてＬａｃｔｏｂａｃｉｌｌｕｓ ｋｅｆｉｒａｎｏｆａｃｉｅｎｓ等が挙げられる。
ヘテロ多糖生産菌としてはＬａｃｔｏｂａｃｉｌｌｕｓ ｐａｒａｃａｓｅｉ、Ｌａｃｔｏｂａｃｉｌｌｕｓ ｒｈａｍｎｏｓｕｓ、Ｌａｃｔｏｂａｃｉｌｌｕｓ ｄｅｌｂｒｕｅ ｓｕｂｓｐ．ｂｕｌｇａｒｉｃｕｓ、Ｌａｃｔｏｂａｃｉｌｌｕｓ ｓａｋｅｉ、Ｌａｃｔｏｂａｃｉｌｌｕｓ ａｃｉｄｏｐｈｉｌｕｓ、Ｌａｃｔｏｂａｃｉｌｌｕｓ ｈｅｌｖｅｔｉｃｕｓ、Ｌａｃｔｏｂａｃｉｌｌｕｓ ｋｅｆｉｒａｎｏｆａｃｉｅｎｓ、Ｌａｃｔｏｃｏｃｃｕｓ ｌａｃｔｉｓ ｓｕｂｓｐ．ｃｒｅｍｏｒｉｓ、Ｓｔｒｅｐｔｏｃｏｃｃｕｓ ｔｈｅｒｍｏｐｈｉｌｕｓ、Ｓｔｒｅｐｔｏｃｏｃｃｕｓ ｍａｃｅｄｏｎｉｃｕｓ、Ｓｔｒｅｐｔｏｃｏｃｃｕ ｅｑｕｉ、Ｓｔｒｅｐｔｏｃｏｃｃｕｓ ｚｏｏｅｐｉｄｅｍｉｃｕｓ、Ｓｔｒｅｐｔｏｃｏｃｃｕｓ ｐｙｏｇｅｎｅｓ、Ｓｔｒｅｐｔｏｃｏｃｃｕｓ ｕｂｅｒｉｓ等が挙げられる。
本明細書では、これらの多糖類のうち、代表してヒアルロン酸について述べる。
（２）ヒアルロン酸生産能を有する微生物
本発明では、ヒアルロン酸生産能を有する微生物としては、Ｓｔｒｅｐｔｏｃｏｃｃｕｓ属に属する微生物が好ましい。ヒアルロン酸生産能を有するＳｔｒｅｐｔｏｃｏｃｃｕｓ属に属する微生物は、一般に牛鼻腔粘膜、牛眼球に存在していることが知られている。本発明ではそこから単離された微生物を利用することもできる。また、Ｓｔｒｅｐｔｏｃｏｃｃｕｓ属に属しない微生物でも、通常の遺伝子工学的手法を用いてヒアルロン酸生産能を得た微生物も使用することができる。
(1) Lactic acid bacteria and polysaccharides derived from lactic acid bacteria The lactic acid bacteria in the present invention are lactic acid bacteria capable of producing polysaccharides outside the cells by normal culture. The type of lactic acid bacteria is not limited as long as it has a polysaccharide-producing ability. Examples include microorganisms belonging to the genus Lactobacillus, microorganisms belonging to the genus Lactococcus, microorganisms belonging to the genus Leuconostoc, microorganisms belonging to the genus Pediococcus, microorganisms belonging to the genus Streptococcus, and the like.
Polysaccharides derived from lactic acid bacteria produced by lactic acid bacteria include homopolysaccharides composed of a single sugar and heteropolysaccharides composed of a plurality of monosaccharides and monosaccharide derivatives. Known homopolysaccharides include dextran composed of glucose, β-glucan, mutan, alternan; levan composed of fructose, inulin; galactan composed of galactose, and the like.
Lactobacillus Hilgardii as lactic acid bacteria which produce dextran, Lactobacillus confusus, Lactobacillus viridescens, Leuconostoc mesenteriodes like, beta glucan Pediococcus damnosus such as producing lactic acid bacterium, Streptococcus Mutan as mutan producing lactic acid bacterium, Streptococcus Sobrius like, Leuconostoc Mesenteriodes such as alternan-producing bacteria, levan producing lactic acid bacterium Streptococcus mutan, Streptococcus salivarius, etc., Streptococcus mutan, etc. as inulin-producing lactic acid bacteria, Lactococcus lactis etc. are mentioned as lactic acid bacteria produced.
The heteropolysaccharide is a series of units composed of at least two kinds of monosaccharides such as glucose, galactose, rhamnose, fucose, and sugar derivatives N-acetylglucosamine, N-acetylgalactosamine, glucuronic acid and the like.
Representative heteropolysaccharides include hyaluronic acid composed of N-acetylglucosamine and glucuronic acid, and kefiran composed of glucose and galactose. Examples of hyaluronic acid-producing lactic acid bacteria include Streptococcus equi, Streptococcus zooepidemicus, Streptococcus pyogenes, Streptococcus uberis, Streptococcus thermophilus, etc.
Examples of heteropolysaccharide-producing bacteria include Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus delbrue subsp. bulgaricus, Lactobacillus sakei, Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus kefiranofaciens, Lactococcus lactis subsp. Cremoris, Streptococcus thermophilus, Streptococcus macedonicus, Streptococcus equi, Streptococcus zooepidemicus, Streptococcus pyogenes, Streptococcus, etc.
In this specification, hyaluronic acid is described as a representative of these polysaccharides.
(2) Microorganism having hyaluronic acid producing ability In the present invention, a microorganism belonging to the genus Streptococcus is preferred as the microorganism having hyaluronic acid producing ability. It is known that microorganisms belonging to the genus Streptococcus having the ability to produce hyaluronic acid are generally present in the bovine nasal mucosa and the bovine eyeball. In the present invention, microorganisms isolated therefrom can also be used. Further, even microorganisms that do not belong to the genus Streptococcus can be used microorganisms that have obtained the ability to produce hyaluronic acid using ordinary genetic engineering techniques.
Ｓｔｒｅｐｔｏｃｏｃｃｕｓ属に属する微生物としては、例えば、Ｓｔｒｅｐｔｏｃｏｃｃｕｓ ｚｏｏｅｐｉｄｅｍｉｃｕｓ、Ｓｔｒｅｐｔｏｃｏｃｃｕｓ ｅｑｕｉ、Ｓｔｒｅｐｔｏｃｏｃｃｕｓ ｐｙｏｇｅｎｓ等が挙げられる。その中でも、Ｓｔｒｅｐｔｏｃｏｃｃｕｓ ｚｏｏｅｐｉｄｅｍｉｃｕｓがより好ましい。 Examples of microorganisms belonging to the genus Streptococcus include Streptococcus zooidemicus, Streptococcus equi, Streptococcus pyogens, and the like. Of these, Streptococcus zooepidemicus is more preferable.
さらに、Ｓｔｒｅｐｔｏｃｏｃｃｕｓ属の属する微生物等のヒアルロン酸生産能を有する微生物を、紫外線、ＮＴＧ（Ｎ‐メチル−Ｎ´−ニトロ−Ｎ−ニトロソグアニジン）、メチルメタンスルホン酸等で処理することにより、ヒアルロニダーゼ非生産菌や非溶血性菌に改良することがより好ましい。人体または動物に悪影響を及ぼす可能性が低くなるからである。 Further, by treating a microorganism having hyaluronic acid producing ability such as a microorganism belonging to the genus Streptococcus with ultraviolet light, NTG (N-methyl-N′-nitro-N-nitrosoguanidine), methylmethanesulfonic acid, etc., hyaluronidase It is more preferable to improve to production bacteria or non-hemolytic bacteria. This is because the possibility of adverse effects on the human body or animals is reduced.
前記ヒアルロニダーゼ活性及び溶血性を欠損させた菌株としては、Ｓｔｒｅｐｔｏｃｏｃｃｕｓ ｚｏｏｅｐｉｄｅｍｉｃｕｓＮＨ−１３１（ＦＥＲＭ Ｐ−７５８０）、Ｓｔｒｅｐｔｏｃｏｃｃｕｓ ｚｏｏｅｐｉｄｅｍｉｃｕｓＨＡ−１１６（ＡＴＣＣ３９９２０）、Ｓｔｒｅｐｔｏｃｏｃｃｕｓ ｚｏｏｅｐｉｄｅｍｉｃｕｓＭＫ５（ＦＥＲＭ Ｐ−２１４８７）、Ｓｔｒｅｐｔｏｃｏｃｃｕｓ ｚｏｏｅｐｉｄｅｍｉｃｕｓＹＴＴ２０３０（ＦＥＲＭ ＢＰ−１３０５）が好ましく、その中でもＳｔｒｅｐｔｏｃｏｃｃｕｓ ｚｏｏｅｐｉｄｅｍｉｃｕｓＭＫ５（ＦＥＲＭ Ｐ−２１４８７）、Ｓｔｒｅｐｔｏｃｏｃｃｕｓ ｚｏｏｅｐｉｄｅｍｉｃｕｓＹＴＴ２０３０（ＦＥＲＭ ＢＰ−１３０５）が特に好ましい。 The strains deficient in the hyaluronidase activity and hemolysis include Streptococcus zooepidemicus NH-131 (FERM P-7580) Streptococcus zoepidemicus HA-116 (ATCC39920), Streptococcus zoMp14 Among them, Streptococcus zooepidemicus MK5 (FERM P-21487) and Streptococcus zooepidemicus YTT2030 (FERM BP-1305) are particularly preferable.
これらのうち、ＦＥＲＭ株については、独立行政法人産業技術総合研究所特許生物寄託センターより入手可能である。また、ＡＴＣＣ株については、Ａｍｅｒｉｃａｎ Ｔｙｐｅ Ｃｕｌｔｕｒｅ Ｃｏｌｌｅｃｔｉｏｎから入手可能である。 Among these, the FERM strain can be obtained from the National Institute of Advanced Industrial Science and Technology Patent Organism Depositary. The ATCC strain is also available from the American Type Culture Collection.
（３）培地
培地は、上記微生物がヒアルロン酸を生産できる培地であれば限定されず、微生物の種類に応じた通常の培地を用いることができる。例えば、炭素源としてグルコース、フルクトース等の単糖類、乳糖、スクロース、マルトース等の二糖類、オリゴ糖類等；窒素源としてポリペプトン、酵母エキス等の有機窒素源；アルギニン、グルタミン酸、グルタミン等の遊離アミノ酸；ビタミン；無機塩類等；タンニン等のフェノール性水酸基を有するヒアルロニダーゼ阻害剤を含む（水に溶解した）培地を使用することができる。
(3) The medium is not limited as long as the microorganism can produce hyaluronic acid, and a normal medium according to the type of microorganism can be used. For example, monosaccharides such as glucose and fructose as a carbon source, disaccharides such as lactose, sucrose and maltose, oligosaccharides and the like; organic nitrogen sources such as polypeptone and yeast extract as a nitrogen source; free amino acids such as arginine, glutamic acid and glutamine; Vitamins; inorganic salts and the like; media containing a hyaluronidase inhibitor having a phenolic hydroxyl group such as tannin (dissolved in water) can be used.
当該培地は、加熱処理（加熱殺菌）を行った後に、微生物の培養に用いることができる。微生物がじゅうぶんに殺菌されれば、加熱条件は限定されない。例えば、１００〜１３０℃で５〜３０分間、より好ましくは１２１℃で１５〜３０分間という条件を挙げることができる。
＜糖溶液＞
本発明では、培地の炭素源として用いる糖類を、他の培地成分とは別に糖溶液として調製する。糖類としては、グルコース、ガラクトース、フルクトース等の単糖類、スクロース、マルトース、ラクトース等の二糖類、オリゴ糖類などを使用することができる。これらの中でも、特にグルコース、フルクトースの使用が好ましい。
The medium can be used for culturing microorganisms after heat treatment (heat sterilization). The heating conditions are not limited as long as the microorganisms are fully sterilized. For example, the conditions of 5 to 30 minutes at 100 to 130 ° C., more preferably 15 to 30 minutes at 121 ° C. can be mentioned.
<Sugar solution>
In the present invention, the saccharide used as the carbon source of the medium is prepared as a sugar solution separately from the other medium components. As the saccharide, monosaccharides such as glucose, galactose and fructose, disaccharides such as sucrose, maltose and lactose, oligosaccharides and the like can be used. Among these, the use of glucose and fructose is particularly preferable.
糖溶液の濃度は特に限定されるものではないが、１０〜５０質量％の糖溶液を作製して加熱殺菌を行った後、糖以外の培地成分を溶解した培地に添加することにより、最終的に糖の濃度が１〜１０質量％になるように調製すればよい（終濃度）。より好ましくは３０質量%濃度の糖溶液を加熱殺菌し、終濃度４〜６質量％となるよう調製して培養培地とする。 The concentration of the sugar solution is not particularly limited. After preparing a 10-50 mass% sugar solution and sterilizing by heating, the final solution is obtained by adding medium components other than sugar to the dissolved medium. The sugar concentration may be adjusted to 1 to 10% by mass (final concentration). More preferably, a sugar solution having a concentration of 30% by mass is sterilized by heating, and adjusted to a final concentration of 4-6% by mass to obtain a culture medium.
本発明における糖溶液は、５N水酸化ナトリウム、５N硫酸を用いて、ｐＨを４．３以下、好ましくは０．１〜４．３、より好ましくは１〜４に調整した後に加熱殺菌する。糖溶液を当該ｐHに調整してから加熱殺菌することにより、糖溶液の加熱による変性を抑制することができ、微生物によるヒアルロン酸の生産が促進される。 The sugar solution in the present invention is sterilized by heating after adjusting pH to 4.3 or less, preferably 0.1 to 4.3, more preferably 1 to 4 using 5N sodium hydroxide and 5N sulfuric acid. By adjusting the sugar solution to the pH and then sterilizing by heating, denaturation due to heating of the sugar solution can be suppressed, and production of hyaluronic acid by microorganisms is promoted.
糖溶液の加熱条件は、微生物がじゅうぶんに殺菌されれば限定されない。例えば、１００〜１３０℃で１０〜３０分間、より好ましくは１２１℃で１５〜３０分間という条件を挙げることができる。加熱殺菌された糖溶液は、糖以外の成分の殺菌された培地中に添加すればよい。
（４）培養
培養条件は、上記微生物がヒアルロン酸を生産できる条件であれば限定されず、微生物の種類に応じた通常の培養条件を用いることができる。通常、ｐＨを４〜８、好ましくは７〜７．５、温度を３０℃〜３７℃、好ましくは、３３℃〜３７℃に制御して行うことが好ましい。
The heating conditions for the sugar solution are not limited as long as the microorganisms are thoroughly sterilized. For example, the conditions of 10 to 30 minutes at 100 to 130 ° C., more preferably 15 to 30 minutes at 121 ° C. can be mentioned. The heat-sterilized sugar solution may be added to a medium in which components other than sugar are sterilized.
(4) Culture conditions are not limited as long as the microorganism can produce hyaluronic acid, and normal culture conditions according to the type of microorganism can be used. Usually, it is preferable to carry out by controlling the pH to 4 to 8, preferably 7 to 7.5, and the temperature to 30 to 37 ° C, preferably 33 to 37 ° C.
本発明における培養方法としては、特に限定されるものではないが、回分培養とする方法と、連続培養とする方法、流加培養とする方法の何れも可能である。 The culture method in the present invention is not particularly limited, and any of a batch culture method, a continuous culture method, and a fed-batch method is possible.
また、前記培養は、通常、前培養を行った後に本培養を行う。前培養の条件としては、グルコース、フルクトース等の炭素源、ポリペプトン、酵母エキス、麦芽エキス等の窒素源、ビタミン、無機塩類を含む培地中で、ｐＨを４〜８、温度を３０〜３７℃に制御して好気的に培養することが好ましい。 In addition, the culture is usually performed after pre-culture. Pre-culture conditions include a carbon source such as glucose and fructose, a nitrogen source such as polypeptone, yeast extract and malt extract, a medium containing vitamins and inorganic salts, pH 4 to 8, temperature 30 to 37 ° C. It is preferable to culture aerobically with control.
以下、実施例、比較例により本発明をさらに詳細に説明する。 Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples.
実施例１〜４
ポリペプトン（和光純薬工業株式会社製）１．５質量％、酵母エキス（オリエンタル酵母工業製）０．５質量％、リン酸第二カリウム０．２質量％、硫酸マグネシウム７水塩０．１質量％、塩化カルシウム０．００５質量％、グルタミン酸ナトリウム０．１質量％、アデカノールＬ６１（消泡剤 旭電化工業製）の組成からなる培地（ｐＨ７．０）を３Ｌのジャーファメンターに１．６Ｌ添加した後、当該培地を殺菌した。
Examples 1-4
Polypeptone (manufactured by Wako Pure Chemical Industries, Ltd.) 1.5 mass%, yeast extract (produced by Oriental Yeast Industry) 0.5 mass%, dipotassium phosphate 0.2 mass%, magnesium sulfate heptahydrate 0.1 mass% %, Calcium chloride 0.005% by mass, sodium glutamate 0.1% by mass, Adecanol L61 (antifoaming agent, manufactured by Asahi Denka Kogyo Co., Ltd.) 1.6 L added to 3 L jar fermenter After that, the medium was sterilized.
一方、グルコースは１２０ｇを水０．４Lに溶解させた後、５N硫酸を用いてｐＨをそれぞれ１．０（実施例１）、２．０（実施例２）、３．０（実施例３）、４．０（実施例４）に調整した。得られた糖溶液を１２１℃で２０分加熱殺菌し、上記殺菌済みの培地に添加した。
糖溶液添加済みの上記培地２Lに、前培養したStreptococcus zooepidemicusＭＫ５（ＦＥＲＭ Ｐ−２１４８７）を１％接種し、１２％水酸化ナトリウム水溶液にて培養液のｐＨを７．４に制御しながら３７℃で２３時間通気攪拌培養を行った（本培養）。
On the other hand, after dissolving 120 g of glucose in 0.4 L of water, pH is adjusted to 1.0 (Example 1), 2.0 (Example 2), and 3.0 (Example 3) using 5N sulfuric acid, respectively. 4.0 (Example 4). The obtained sugar solution was sterilized by heating at 121 ° C. for 20 minutes and added to the sterilized medium.
1 L of precultured Streptococcus zooepidemicus MK5 (FERM P-21487) is inoculated into 2 L of the above-mentioned medium to which a sugar solution has been added, and the pH of the culture solution is controlled at 7.4 with a 12% aqueous sodium hydroxide solution at 37 ° C. The culture was aerated and stirred for 23 hours (main culture).
なお、前培養は、グルコース０．２質量％、ポリペプトン２．０質量％、酵母エキス０．５質量％、硫酸マグネシウム７水塩０．０５質量％の組成からなる培地（ｐＨ７．０）を綿栓付き５００ｍｌ容三角フラスコに１００ｍｌ添加し、加熱殺菌後、同一組成の寒天プレート上に形成したコロニーを一白菌耳植菌し、３０℃で１８時間培養した。 In the pre-culture, a medium (pH 7.0) comprising 0.2% by mass of glucose, 2.0% by mass of polypeptone, 0.5% by mass of yeast extract, and 0.05% by mass of magnesium sulfate heptahydrate is cotton 100 ml was added to a 500 ml Erlenmeyer flask with a stopper, and after heat sterilization, a colony formed on an agar plate of the same composition was inoculated with a white fungus and cultured at 30 ° C. for 18 hours.
培養終了後における培養液中のヒアルロン酸含量は、カルバゾール硫酸法にて測定した。分析結果は表１に示す。ｐＨを４．３以下にして滅菌した糖溶液を使用した場合には、大量のヒアルロン酸が蓄積された。
＜カルバゾール硫酸法＞
培養液を０．２Ｍ ＮａＣｌ水溶液で２０倍に希釈し、その２ｍｌにエタノールを１０ｍｌ加えて攪拌することによりヒアルロン酸を析出させる。遠心分離し、上清を取り除いた後の沈殿に再び０．２Ｍ ＮａＣｌ水溶液を１０ｍｌ加えてヒアルロン酸含有液を作成する。
Ａ液（Ｎａ２Ｂ４Ｏ７・１０Ｈ２Ｏ ０．９５ｇをＨ２ＳＯ４ １００ｍｌで溶解したもの）５ｍｌを試験管に取り、上記のヒアルロン酸含有液１ｍｌを加えて１００℃で１０分湯浴する。室温まで水冷した後に、Ｂ液（カルバゾール０．１２５ｇをエタノール１００ｍｌに溶解したもの）０．２ｍｌを加えて１００℃で１５分湯浴する。室温まで冷却した後に、５３０ｎｍの波長における吸光度を測定してヒアルロン酸含有液中のグルクロン酸量を測定することでヒアルロン酸量を算出する。
The hyaluronic acid content in the culture broth after completion of the culture was measured by the carbazole sulfate method. The analysis results are shown in Table 1. When a sugar solution sterilized with a pH of 4.3 or lower was used, a large amount of hyaluronic acid accumulated.
<Carbazole sulfate method>
The culture solution is diluted 20-fold with 0.2 M NaCl aqueous solution, and 10 ml of ethanol is added to 2 ml thereof and stirred to precipitate hyaluronic acid. Centrifugation and 10 ml of 0.2 M NaCl aqueous solution are again added to the precipitate after removing the supernatant to prepare a hyaluronic acid-containing solution.
5 ml of solution A (0.92 g of Na 2 B 4 O 7 · 10H 2 O dissolved in 100 ml of H 2 SO 4 ) was placed in a test tube, 1 ml of the above-mentioned hyaluronic acid-containing solution was added and hot water was added at 100 ° C. for 10 minutes. Take a bath. After cooling to room temperature with water, 0.2 ml of Liquid B (0.125 g of carbazole dissolved in 100 ml of ethanol) is added and bathed at 100 ° C. for 15 minutes. After cooling to room temperature, the amount of hyaluronic acid is calculated by measuring the absorbance at a wavelength of 530 nm and measuring the amount of glucuronic acid in the hyaluronic acid-containing solution.
比較例１〜３
グルコース１２０ｇを水０．４Lに溶解した後に、ｐＨを４．５（比較例１）、５．０（比較例２）、７．０（比較例３）に調整し（糖溶液）、１２１℃２０分加熱殺菌して培養開始前にその他の培地に添加した以外は、実施例１と同様の操作を行った。ヒアルロン酸含量の分析結果は、併せて表１に示す。
Comparative Examples 1-3
After dissolving 120 g of glucose in 0.4 L of water, the pH was adjusted to 4.5 (Comparative Example 1), 5.0 (Comparative Example 2), 7.0 (Comparative Example 3) (sugar solution), 121 ° C. The same operation as in Example 1 was performed, except that the mixture was sterilized by heating for 20 minutes and added to other medium before the start of culture. The analysis results of the hyaluronic acid content are also shown in Table 1.
ｐＨを４．５以上にして滅菌した糖溶液を使用した場合は、ヒアルロン酸の蓄積が非常に少なかった。 When a sugar solution sterilized with a pH of 4.5 or higher was used, the accumulation of hyaluronic acid was very small.
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