JP3704664B2 - Novel microorganism and method for producing heteropolysaccharide by microorganism - Google Patents

Description

【００１５】
(d) その他の性質
(1) レバンの生成 ：生成する。
(2) アルギニンの加水分解：分解しない。
(3) Tween 80の分解 ：分解する。
(4) レシチナーゼ ：陽性。
(5) 炭素源，窒素源としてのアスパラギンの利用：利用しない。
(6) ペクチンの溶解 ：溶解する。
(7) エスクリンの加水分解：分解する。
(8) アセトイン試験 ：陰性。
(9) 食塩耐性 ：３％。
(10)0.1％トリフェニルテトラゾリウムクロライド含有培地での増殖：増殖しない。
(11)ヘテロ多糖類の生成 ：多量のキサンタンガムを生成する。
(e) 分離源 静岡県磐田市大藤地区のキャベツの葉組織
【００１６】
本菌株の菌学的性質を「バージーズ マニュアル Bergey's Manual」第８版における菌学的性質と詳細に比較検討した結果、リトマス・ミルク試験での結果が、「脱色し、消化する」であり、硫化水素を生成し、ウレアーゼが陰性であり、食塩耐性が３％であり、かつ、Ｌ−アラビノーズ、Ｄ−グルコース、Ｄ−マンノース、Ｄ−ガラクトースおよびトレハロースのそれぞれの糖から酸を生成することから、本菌株はキサントモナス キャンペストリス（Xanthomonas campestris）に属するものであると判断した。また、澱粉を強く加水分解し、他の炭素源を共存させなくても液化澱粉を強力に資化し、多量のキサンタンガムを生成する点で、キサントモナス キャンペストリスに属する公知の菌株と相違していることから、本菌株はキサントモナス キャンペストリスに属する新菌株と判断し、本菌株をキサントモナス キャンペストリス（Xanthomonas campestris）ＴＳＫ−００１と命名した。
なお、本菌株はＦＥＲＭ Ｐ−１３９９７として、工業技術院生命工学工業技術研究所に寄託されている。
【００１７】
この菌株を、炭素源として液化澱粉のみを含有する培地、さらには、栄養成分として液化澱粉および魚肉質成分のみを含有する培地で好気的に培養することによって、キサントモナス属に属する公知の菌株に比して多量のヘテロ多糖類を生産せしめ、以て、ヘテロ多糖類を容易に、かつ、効率よく製造することが可能となる。
このように本菌株を培養することにより、本菌株によって生産されたヘテロ多糖類は菌体外へ排出され培養液中に蓄積される。
【００１８】
本発明において液化澱粉とは、水性液に溶存している澱粉と定義される。
液化澱粉を生成せしめるためには、常法によって、澱粉を化学的に、または、酵素的に処理して低分子化せしめて水溶性が増大せしめられた、所謂、デキストリン類および可溶性澱粉などが使用される。この原料および処理条件には特に制限はない。
原料の澱粉としては、その代表例として、小麦、とうもろこし、米、ライ麦およびオート麦などから得られた穀物澱粉ならびに馬鈴薯、キャッサバ、タピオカおよび甘藷などの芋類から得られた澱粉などがある。
【００１９】
また、処理条件としては、(1) １９０〜２３０℃程度で加熱する(2)塩酸などの希酸の存在化で１１０〜１４０℃程度で加熱するなどがある。また、酵素としては、通常は、アミラーゼが使用される。市販の可溶性澱粉もそのまま使用することができる。
【００２０】
培地または培養液中の液化澱粉の濃度は、実用上、通常は、培地または培養液１リットル（l）あたり１０〜５０g程度、好ましくは、２０〜４０g程度とされる。この液化澱粉の濃度が約５０gを越えて高くなるに伴って培地乃至は培養液が高粘度となり、攪拌効率の低下から生産性の低下を来す危険性がある。
【００２１】
短時間で培養液の単位容積あたりのヘテロ多糖類の収量を向上させて、効率よくヘテロ多糖類を製造するためには、培養初期においては液化澱粉の濃度を低くすることが好ましく、実用上、通常は、培地または培養液１lあたり２０〜３０g程度とされる。培養終了後の液は高粘度となっており、そのままの状態で菌体を除去することが困難となるので、菌体の分離に先立って、培養液を希釈することが好ましい。この希釈の程度には特に制限はないが、実用上、通常は、培養液中のヘテロ多糖類の濃度が０.１〜１.０（重量／容量）％程度となるように希釈される。
【００２２】
ヘテロ多糖類の生産性をさらに増大させるために、培地または培養液に炭素源以外の成分として魚肉質成分を含有せしめることが好ましい。
魚肉質成分として、魚粕、魚粉、荒粕およびフィッシュソリュブルなどの魚粉類、生鮮魚肉および魚粉などから熱水もしくは水蒸気で抽出され、または、酵素処理によって抽出された抽出物ならびに魚肉汁の濃縮・精製物である魚エキスなどが培地または培養液に含有せしめられる。
これらの魚粉類、抽出物および魚エキスなどの魚肉質成分は、その原料の魚の種類および処理条件などには特に制限はなく、また、市販品をそのまま使用することができる。なお、培地中または培養液中に原料の澱粉と澱粉液化酵素を共存せしめて澱粉液化酵素により原料の澱粉を液化せしめることができる。
【００２３】
これらの魚粉類、抽出物および魚エキスなどの魚肉質成分の使用量は、使用される魚肉質成分の原料の魚の種類および処理方法などによって、多岐にわたる品質、品位のものが存在し、その成分の種類、含有率が大きく変動しており、また、培養条件などによって大きく異なるため、一概に特定し得ないが、実用上、通常は、その全窒素量で特定することができる。この魚肉質成分の使用量は、全窒素量として、培地または培養液１リットル(l)あたり、実用上、通常は、０.１〜１g程度、好ましくは、０.２〜０.５g程度とされる。
なお、魚肉質成分の全窒素含有率は、常法の如くケルダール法により測定される。
【００２４】
培養は常法により好気的に行われる。そのためには、通常は、空気などの酸素含有ガスで培養液は通気攪拌される。空気の通気量は培養液中の溶存酸素濃度が律速とならなければよく、通常は、１分あたり培養液１lにつき０.３〜１.５l（標準状態）程度とされる。
培養液の液性は、ほぼ微酸性乃至微アルカリ性でよく、６〜８.５程度が好適である。培養温度は常温乃至室温でよく、２８〜３５℃程度が好適である。また、培養液中の菌体濃度（乾燥菌体換算で表示 以下同様）は、培養液１lあたり、乾燥菌体換算で１〜３g程度に保つことが好適である。
【００２５】
培養の経過に伴って、培養液の液性はアルカリ性に大きく傾くことがあるが、この場合には、フマール酸および乳酸などの有機酸の添加によって、培養液の液性は所定の値に保持される。なお、培地にこれらの有機酸の塩を予め添加しておけば、培養の経過中で液性はほぼ微酸性乃至微アルカリ性の所定の値に保たれる。また、培養液の発泡を予防・防止するために、培地または培養液にシリコーンのような消泡剤を添加することもできる。
培養は、回分法および連続法のいずれでも行うことができる。
【００２６】
また、ヘテロ多糖類を生産するための培養に先立って、予備培養することができ、しかも好ましい。この培養においては、キサントモナス キャンペストリスに属する公知の菌株の培養に使用される培地をも使用することができる。
【００２７】
このようにして、微生物によって生産されたヘテロ多糖類は菌体外に排出され培養液中に蓄積される。従って、培養の経過に伴って、培養液中のヘテロ多糖類の濃度が上昇すれば、培養液の粘度もこれに伴って増大するので、培養の経過は培養液の粘度を測定することによって知ることができる。
このようにして培養することによって、たとえば、回分培養の場合には、培養液中のヘテロ多糖類の濃度は、培養条件などにもよるが、通常は、約４８時間以内で、最大に達する。
【００２８】
このようにして得られた培養液または所望により希釈された培養液から、常法によって菌体を除去し、ついで、目的物であるヘテロ多糖類が分離・回収される。
すなわち、たとえば、遠心分離および各種の濾過によって培養液または所望により希釈された培養液から菌体が除去される。このようにして得られた清澄液に、貧溶媒であるプロピルアルコールおよびイソプロピルアルコールのような低級アルコールまたは無機塩を添加して、目的物であるヘテロ多糖類を沈殿せしめ、この沈殿を、遠心分離および各種の濾過によって分離・回収する。このようにして得られた粗製のヘテロ多糖類は、さらに所望により各種の精製を経て、製品とされる。なお、培養液が希釈された場合には、貧溶液などの添加によるヘテロ多糖類の沈殿に先立って、除菌後の清澄液を濃縮することが好ましい。
【００２９】
【実施例】
本発明を以下の実施例でさらに具体的に説明するが、これらの実施例は単なる例示であって、本発明を何等制限するものではない。
なお、実施例において培養液中のキサンタンガムは、適宜希釈された培養液から遠心分離（１０,０００×ｇで１時間）によって菌体を分離除去して得られた清澄液を濃縮し、この濃縮液に過剰（２〜３倍量）の貧溶媒であるアルコールを添加して、キサンタンガムを沈殿させる。このキサンタンガムを濾取し、これを乾燥してこの重量をキサンタンガムの量とすることによって定量される。
また、キサンタンガムは、キサンタンガムを酸で加水分解し、この分解物について、グルコース、マンノース、グルクロン酸およびピルビン酸の組成を高速液体クロマトグラフィーにより分析することによって同定される。
【００３０】
実施例１
本発明の新規な微生物であるキサントモナス キャンペストリス（Xanthomo-nas campestris）ＴＳＫ−００１（ＦＥＲＭ Ｐ−１３９９７）を、ブイヨン寒天斜面培地で培養・賦活された菌体の１白金耳を後記の培地１００mlに接種し、これを３００rpmの回転培養機よって３０℃で２４時間予備培養して、菌体濃度０.１５（重量／容量％）の培養液を得、これを種菌とした。
【００３１】
培地組成
液化澱粉（純正化学製） ３０g
魚エキス（焼津水産化学製 カルチベータ） ５g（全窒素量として０.４g）
フマール酸ナトリウム（純正化学製） ３g
水 １l
pH ７.０
３０l容ジャーファーメンターに前記の培地１０lを仕込み、殺菌後、これに前記の種菌培養液５００mlを接種し、３００rpmで攪拌しつつ、１分間あたり１００容量／容量％で空気を通気し、３０℃で好気的に回分培養を行った。
【００３２】
培養時間の経過に伴って菌は増殖し、培養液のpHは約７〜８の範囲に保持され、また、培養液の粘度は培養の経過に伴って徐々に増大した。培養開始から３２時間経過後には、培養液の粘度は８４００cP（センチポアズ）に達した。
培養期間における培養液中の澱粉濃度および菌体濃度ならびに培養液のpHおよび粘度のそれぞれの経時変化を図１に示す。
培養開始４８時間後には、培養液中のキサンタンガムの濃度は１６.５g／lとなった。
この培養液からプリコートフィルタープレスによって菌体を除去して、清澄粘稠液を得た。
この清澄粘稠液１lを採り、これと２lのプロピルアルコールとを混合して沈殿を生成せしめた。沈殿を遠心分離によって回収し、デシケーター中で乾燥してキサンタンガム１５.３gを得た。
【００３３】
比較例１
本発明の新規な微生物であるキサントモナス キャンペストリス（Xanthomo-nas campestris）ＴＳＫ−００１（ＦＥＲＭ Ｐ−１３９９７）に代えて、公知のキサントモナス キャンペストリス（Xanthomonass campestris）ＮＲＲＬ Ｂ−１４５９を使用した以外は、実施例１と同様にして行ったが、キサンタンガム５gを得たに止まった。
【００３４】
実施例２
魚エキスに代えて市販の飼料用フィッシュソリブルを半量となるまで濃縮した液を培地１lあたり全窒素量として０.４gとなるように添加した以外は実施例１と同様にして行って、キサンタンガム１４.５gを得た。
【００３５】
実施例３
コーンスターチ（純正化学製）３０g、市販の魚粉４g（全窒素量として０.４４g）および水５００mlから成る混合物に、１.５gの澱粉液化酵素タマミール１２０Ｌ（NOVO社製）を添加し、８５℃で３０分間加熱して、このコーンスターチの殆ど全量を可溶化せしめこれを殺菌冷却して可溶化澱粉含有物を得た。
この可溶化澱粉混合物の全量に、３gのフマール酸ナトリウムを添加し、これにさらに水を加えて１lとし、これを培地として使用した以外は、実施例１と同様にして行って、キサンタンガム１６.２gを得た。
【００３６】
実施例４
市販の魚粉（鰯の削り粉）を熱水で６０分間抽出して全窒素含有率が６.２g／lの魚粉熱水抽出物を得た。
魚エキスに代えて、この魚粉熱水抽出物を培地１lあたり６４ml添加した以外は実施例１と同様にして行って、キサンタンガム１３.７gを得た。
【００３７】
実施例５
魚粉（鰯の削り粉）を酵素（天野製薬製 パパイン）で分解し、全窒素含有率２６g／lの魚粉分解抽出物を得た。
魚エキスに代えて、この魚粉分解抽出物を培地１lあたり１５ml添加した以外は実施例１と同様にして行って、キサンタンガム１４.０gを得た。
【００３８】
比較例２
次の培地組成を有する培地を使用した以外は実施例１と同様にして行ったが、キサンタンガム９.４gを得たに止まった。
培地組成
液化澱粉（純正化学製） ３０ g
酵母エキス（日本製薬製） １ g
コーンスチーブリカー（純正化学製） １ ml
硫酸アンモニウム（純正化学製） １ g
硫酸マグネシウム（７水塩）（純正化学製） ０.２g
りん酸二カリウム（純正化学製） １ g
水 １ l
pH ７.０
【００３９】
【発明の効果】
本発明により、低廉な液化澱粉を使用し、工業上の利用価値が高いキサンタンガムのようなヘテロ多糖類が、容易に、しかも効率よく得られる。
【図面の簡単な説明】
【図１】培養期間における培養液中の澱粉濃度および菌体濃度ならびに培養液のpHおよび粘度のそれぞれの経時変化を示すグラフである。[0001]
[Industrial application fields]
The present invention relates to a novel microorganism and a method for producing a heteropolysaccharide using a microorganism such as the novel microorganism.
[0002]
[Prior art, problems to be solved by the invention]
Xanthan gum is known as a representative example of a heteropolysaccharide. Since this xanthan gum has useful effects such as viscosity enhancement and quality stability, it is widely used as a food additive, emulsifier for printing ink, and thickener for printing paste. It is a useful substance that is also used in large quantities in the recovery of by-product oil.
[0003]
A method for producing such useful xanthan gum using a microorganism has been widely known for a long time, and bacteria belonging to the genus Xanthomonas are known as this microorganism.
The production, utilization technology and chemical properties of xanthan gum are described in many literatures. Pee. A. (Sanford. PA) et al. Describes in particular in detail in the “Extracellular Microbial Polysaccharide of Microorganisms”.
[0004]
A practical method for producing a heteropolysaccharide such as xanthan gum using a microorganism is disclosed, for example, in US Pat. No. 3,485,719. In this method, corn sugar, glucose and other carbohydrates are used as a carbon source, and Xanthomonas campestris NRRL B-1459 is used for continuous culture over a period of about 2 weeks or more. Monosaccharides such as glucose used in this method can be obtained by hydrolyzing starch or the like chemically or enzymatically, but this requires considerable cost and time.
Accordingly, it is practically significant that the raw material for such a monosaccharide can be used as it is as a carbon source for producing a polysaccharide without hydrolysis.
[0005]
Furthermore, for example, in the method described in JP-A-6-30600, starch is used as a carbon source. However, in this method, saccharified starch enzymes such as α-amylase, β-amylase, glucoamylase and isoamylase and pullulanase are contained in an aqueous nutrient medium together with starch, and the starch is previously hydrolyzed by the enzyme to produce glucose. The monosaccharide is used as a carbon source for bacteria such as Xanthomonas.
[0006]
In this publication, starch includes not only starch itself but also starch hydrolysis products. In the method disclosed in this publication, since the carbon source utilized by the microorganism is glucose in the end, this method is not essentially different from the method disclosed in the aforementioned US patent specification. In the method described in this publication , the concentration of glucose in the aqueous nutrient medium is set to 1 to 15%. Thus, since the concentration of glucose differs depending on the type of microorganism used, the concentration of starch and enzyme in the aqueous nutrient medium must be changed according to the type of microorganism used. This is complicated and becomes an obstacle to practical use. In addition, when this method is industrially performed by continuous culture, the glucose concentration in the aqueous nutrient medium must be controlled to be a predetermined concentration during the operation period in order to enable stable operation. This must be easy.
[0007]
Furthermore, in any of the methods described in the above-mentioned US Pat. No. 3,485,719 and JP-A-6-30600, suitable for each microorganism depending on the microorganism used. It is necessary to add other nutrient sources such as organic components and inorganic components other than sugar, and the medium preparation is complicated.
Recently, as part of the effective use of milk whey (milk) containing lactose produced as a by-product in large quantities with the expansion of dairy farming, it is proposed to be used as a carbon source in the production of heteropolysaccharides by microorganisms. Has been.
However, even in such a case, other organic nutrient components are required, and for example, addition of a corn soluble extract (Constiti-prica-CSL), casein, glutamic acid, and the like is required.
[0008]
On the other hand, the bacteriological properties of bacteria belonging to the genus Xanthomonas are generally said to “assimilate starch”, but it is known that this assimilation property is not strong enough to be practically satisfactory.
That is, for example, Xanthomonas campestris NRRL B-1459 used in the above-mentioned U.S. Pat. No. 3,485,719 is replaced with glucose as a carbon source used here, and soluble starch of a commercial reagent which is a liquefied starch is used. When used, the present inventors have confirmed that the growth and proliferation of this strain itself is poor and, for example, the production amount of polysaccharides such as xanthan gum is small and not suitable for practical use.
[0009]
[Means and Actions for Solving the Problems]
The present inventors only use liquefied starch as a carbon source, and without using a complex medium containing enzymes such as saccharified starch degrading enzymes and other nutrient components such as inorganic components, As a result of diligent and intensive studies on methods that can efficiently produce such heteropolysaccharides, saccharified starch-degrading enzymes, etc. can be obtained simply by using a medium with a simple composition containing only liquefied starch as a carbon source. We succeeded in isolating new microorganisms that can efficiently produce heteropolysaccharides such as xanthan gum without coexisting other nutrients such as enzymes and inorganic components. When producing saccharides, heteropolysaccharides can be easily and efficiently produced simply by using liquefied starch as a carbon source. By occupying Sonse, it discovered that can made to increase the production of heteropolysaccharides markedly, reaching the present invention based on this discovery.
[0010]
That is, the present invention is Xanthomonas campestris TSK-001 (FERM P-13997) which can grow and proliferate in a medium containing only liquefied starch as a carbon source and produce a large amount of xanthan gum, and Xanthomonas campestris. A method for producing a heteropolysaccharide comprising culturing TSK-001 (FERM P-13997) in a medium containing only liquefied starch as a carbon source and recovering the heteropolysaccharide from the obtained culture. further, belonging to the genus Xanthomonas, the only liquefied starch as a carbon source, a bacteria capable of producing heteropolysaccharide, and cultured in a medium containing a liquefied starch alone and fish meat Ingredients as a carbon source, resulting culture A method for producing a heteropolysaccharide comprising recovering a heteropolysaccharide from
[0011]
The mycological properties of the novel microorganism of the present invention are as follows. That is,
(a) Form
(1) Cell shape and size: Neisseria gonorrhoeae, 0.6-0.8 μm × 2-3 μm.
(2) Presence / absence of motility: Yes, with one flagella.
(3) Gram staining: Negative.
(4) Acid resistance: None.
[0012]
(b) Physiological properties
(1) Meat agar plate culture: Grows well. The shape of the colony is circular and the surface and periphery are both smooth. The color is yellow translucent and glossy.
(2) Meat broth agar slope culture: Grows. The state is filamentous, glossy and yellow translucent.
(3) Meat broth liquid culture: Grows in a cloudy state.
(4) Meat broth gelatin puncture culture: Grows in the surface layer and liquefaction of gelatin is observed.
(5) Litmus milk: Decolorized and digested after 7 days of culture.
[0013]
(c) Physiological properties
(1) Reduction of nitrate: No reduction.
(2) Denitrification reaction: Negative.
(3) MR test: Negative.
(4) VP test: Negative.
(5) Generation of indole: not generated.
(6) Generation of hydrogen sulfide: Generated.
(7) Starch hydrolysis: Strongly decomposes.
(8) Use of citric acid (Combination of Koser's medium and Christensen's medium): Use.
(9) Use of inorganic nitrogen source: Do not use nitrate. Use ammonium salt.
(10) Urease: Negative.
(11) Oxidase: positive.
(12) Catalase: Positive.
(13) Range of growth: pH 6 to 8.5, temperature 28 to 35 ° C.
(14) Attitude toward oxygen: aerobic.
(15) Presence or absence of acid and gas generation from saccharides: as shown in Table 1.
[0014]
[Table 1]

[0015]
(d) Other properties
(1) Generating levan: Generate.
(2) Arginine hydrolysis: Does not decompose.
(3) Decomposition of Tween 80: Decompose.
(4) Lecithinase: positive.
(5) Use of asparagine as a carbon and nitrogen source: Not used.
(6) Dissolution of pectin: Dissolve.
(7) Esculin hydrolysis: Decomposes.
(8) Acetoin test: negative.
(9) Salt tolerance: 3%.
(10) Growth on a medium containing 0.1% triphenyltetrazolium chloride: No growth.
(11) Production of heteropolysaccharide: A large amount of xanthan gum is produced.
(e) Separation source Cabbage leaf tissue in Oto district, Kamata City, Shizuoka Prefecture [0016]
As a result of a detailed comparison of the mycological properties of this strain with those in the 8th edition of the Bergey's Manual, the result of the litmus milk test was “decolorizing and digesting” Since it produces hydrogen, urease is negative, salt tolerance is 3%, and it produces acids from the respective sugars of L-arabinose, D-glucose, D-mannose, D-galactose and trehalose, This strain was judged to belong to Xanthomonas campestris . It is also different from the known strains belonging to Xanthomonas campestris in that starch is strongly hydrolyzed and liquefied starch is strongly assimilated without coexisting with other carbon sources to produce a large amount of xanthan gum. Therefore, this strain was judged to be a new strain belonging to Xanthomonas campestris , and this strain was named Xanthomonas campestris TSK-001.
This strain is deposited as FERM P-13997 at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology.
[0017]
By aerobically culturing this strain in a medium containing only liquefied starch as a carbon source, and further in a medium containing only liquefied starch and fish meat components as nutrient components, a known strain belonging to the genus Xanthomonas is obtained. Compared with this, it is possible to produce a larger amount of heteropolysaccharide, and thus it is possible to easily and efficiently produce the heteropolysaccharide.
By culturing the present strain in this way, the heteropolysaccharide produced by the present strain is discharged out of the cells and accumulated in the culture solution.
[0018]
In the present invention, liquefied starch is defined as starch dissolved in an aqueous liquid.
In order to produce liquefied starch, so-called dextrins and soluble starch, etc. that have been increased in water solubility by treating starch chemically or enzymatically to increase the water solubility are used to produce liquefied starch. Is done. There are no particular restrictions on the raw materials and processing conditions.
Representative examples of the raw material starch include cereal starch obtained from wheat, corn, rice, rye and oats, and starch obtained from potatoes such as potato, cassava, tapioca and sweet potato.
[0019]
The processing conditions include (1) heating at about 190 to 230 ° C. (2) heating at about 110 to 140 ° C. in the presence of dilute acid such as hydrochloric acid. As the enzyme, amylase is usually used. Commercially available soluble starch can also be used as it is.
[0020]
In practice, the concentration of the liquefied starch in the culture medium or culture medium is generally about 10 to 50 g, preferably about 20 to 40 g per liter (l) of the culture medium or culture medium. As the concentration of the liquefied starch exceeds about 50 g, the medium or the culture solution becomes highly viscous, and there is a risk that the productivity is lowered due to a decrease in stirring efficiency.
[0021]
In order to improve the yield of the heteropolysaccharide per unit volume of the culture solution in a short time and to produce the heteropolysaccharide efficiently, it is preferable to reduce the concentration of the liquefied starch at the initial stage of the culture. Usually, the amount is about 20 to 30 g per liter of the culture medium or culture solution. Since the liquid after completion of the culture has a high viscosity and it is difficult to remove the bacterial cells as they are, it is preferable to dilute the culture liquid prior to the separation of the bacterial cells. The degree of dilution is not particularly limited, but for practical use, it is usually diluted so that the concentration of the heteropolysaccharide in the culture solution is about 0.1 to 1.0 (weight / volume)%.
[0022]
In order to further increase the productivity of the heteropolysaccharide, it is preferable to contain a fish meat component as a component other than the carbon source in the medium or the culture solution.
Concentrates of fish meat juice, extracted from fish meal such as fish bream, fish meal, rough fish and fish solubil, fresh fish meat and fish meal with hot water or steam, or extracted by enzymatic treatment A fish extract or the like, which is a purified product, is contained in the culture medium or culture solution.
The fish meat components such as fish meals, extracts and fish extracts are not particularly limited in terms of the type and processing conditions of the raw fish, and commercially available products can be used as they are. Note that the starch as a raw material and the starch liquefying enzyme can coexist in the culture medium or the culture solution, and the starch as the raw material can be liquefied by the starch liquefying enzyme.
[0023]
The amount of fish meat components such as fish meals, extracts, and fish extracts used varies widely depending on the type of fish used as the raw material for fish meat components and the processing method. However, it can not be specified unconditionally because it varies greatly depending on the culture conditions and the like, but in practice, it can usually be specified by its total nitrogen amount. The amount of the fish meat component used is practically usually about 0.1 to 1 g, preferably about 0.2 to 0.5 g per liter (l) of the medium or culture solution as the total nitrogen amount. Is done.
Note that the total nitrogen content of the fish meat component is measured by the Kjeldahl method as in a conventional method.
[0024]
Cultivation is performed aerobically by conventional methods. For this purpose, the culture medium is usually aerated and stirred with an oxygen-containing gas such as air. The amount of aerated air is not limited if the dissolved oxygen concentration in the culture solution is not rate-limiting, and is usually about 0.3 to 1.5 l (standard state) per liter of culture solution per minute.
The liquidity of the culture solution may be approximately slightly acidic to slightly alkaline, and is preferably about 6 to 8.5. The culture temperature may be room temperature to room temperature, and preferably about 28 to 35 ° C. In addition, the cell concentration in the culture solution (displayed in terms of dry cells) is preferably maintained at about 1 to 3 g in terms of dry cells per liter of culture solution.
[0025]
As the culture progresses, the liquidity of the culture solution may become significantly alkaline, but in this case, the liquidity of the culture solution is maintained at a predetermined value by adding organic acids such as fumaric acid and lactic acid. Is done. If salts of these organic acids are added to the medium in advance, the liquidity is maintained at a predetermined value of approximately slightly acidic to slightly alkaline during the course of culture. In addition, an antifoaming agent such as silicone can be added to the medium or the culture solution in order to prevent / prevent foaming of the culture solution.
Culturing can be performed by either a batch method or a continuous method.
[0026]
Moreover, prior to the culture for producing the heteropolysaccharide, it can be precultured, and is preferred. In this culture, a medium used for culturing a known strain belonging to Xanthomonas campestris can also be used.
[0027]
In this way, the heteropolysaccharide produced by the microorganism is discharged out of the cell and accumulated in the culture solution. Therefore, if the concentration of heteropolysaccharide in the culture solution increases with the progress of culture, the viscosity of the culture solution also increases with this, so the progress of culture is known by measuring the viscosity of the culture solution. be able to.
By culturing in this way, for example, in the case of batch culture, the concentration of the heteropolysaccharide in the culture solution usually reaches its maximum within about 48 hours, although it depends on the culture conditions.
[0028]
The bacterial cells are removed from the thus obtained culture solution or the culture solution diluted as desired by a conventional method, and then the target heteropolysaccharide is separated and recovered.
That is, for example, the cells are removed from the culture solution or the culture solution diluted as desired by centrifugation and various types of filtration. A lower alcohol or inorganic salt such as propyl alcohol and isopropyl alcohol, which are poor solvents, is added to the clarified liquid thus obtained to precipitate the target heteropolysaccharide, which is centrifuged. And separated and collected by various types of filtration. The crude heteropolysaccharide thus obtained is further subjected to various purifications as desired to obtain a product. In addition, when the culture solution is diluted, it is preferable to concentrate the clarified solution after sterilization prior to precipitation of the heteropolysaccharide by addition of a poor solution or the like.
[0029]
【Example】
The present invention will be described more specifically with reference to the following examples. However, these examples are merely illustrative, and do not limit the present invention in any way.
In the examples, xanthan gum in the culture broth was concentrated by concentrating the clarified liquid obtained by separating and removing cells from the appropriately diluted broth by centrifugation (10,000 × g for 1 hour). An excess (2 to 3 times amount) of an alcohol that is a poor solvent is added to the liquid to precipitate xanthan gum. The xanthan gum is filtered and quantified by drying it to make this weight the amount of xanthan gum.
Xanthan gum is identified by hydrolyzing xanthan gum with an acid, and analyzing the composition of glucose, mannose, glucuronic acid and pyruvic acid by high performance liquid chromatography.
[0030]
Example 1
Xanthomo - nas campestris TSK-001 (FERM P-13997), which is a novel microorganism of the present invention, was cultured and activated in a bouillon agar slant medium, and 1 platinum ear of the cells was cultured in 100 ml of the medium described later. And precultured at 30 ° C. for 24 hours with a rotary incubator at 300 rpm to obtain a culture solution having a cell concentration of 0.15 (weight / volume%), which was used as an inoculum.
[0031]
Medium composition liquefied starch (made by Pure Chemical) 30g
5 g of fish extract (Yakitsu Suisan Chemical cultivator) (0.4 g of total nitrogen)
Sodium fumarate (Pure Chemical) 3g
1 l of water
pH 7.0
Into a 30 liter jar fermenter, 10 liters of the medium was sterilized, inoculated with 500 ml of the inoculum culture solution, stirred at 300 rpm, aerated with 100 volume / volume% per minute, 30 ° C. The batch culture was performed aerobically.
[0032]
The bacteria grew with the passage of the culture time, the pH of the culture broth was maintained in the range of about 7-8, and the viscosity of the culture broth gradually increased with the progress of the culture. After 32 hours from the start of the culture, the viscosity of the culture reached 8400 cP (centipoise).
FIG. 1 shows changes over time in the starch concentration and cell concentration in the culture medium and the pH and viscosity of the culture liquid during the culture period.
After 48 hours from the start of the culture, the concentration of xanthan gum in the culture solution was 16.5 g / l .
From this culture broth by removing the bacterial cells by the pre-coat filter press, to give a clear viscous liquid.
Take this clarification viscous liquid 1l, yielding a precipitate by mixing a and 2l of propyl alcohol thereto. The precipitate was collected by centrifugation and dried in a desiccator to obtain 15.3 g of xanthan gum.
[0033]
Comparative Example 1
Instead of Xanthomo - nas campestris TSK-001 (FERM P-13997) which is a novel microorganism of the present invention, the known Xanthomonas campestris NRRL B-1459 was used. This was carried out in the same manner as in Example 1, but only 5 g of xanthan gum was obtained.
[0034]
Example 2
Xanthan gum was carried out in the same manner as in Example 1 except that a solution obtained by concentrating commercially available fish solubil to half the amount in place of the fish extract was added to a total nitrogen amount of 0.4 g per liter of the medium. 14.5 g was obtained.
[0035]
Example 3
To a mixture of 30 g of corn starch (manufactured by Junsei Kagaku), 4 g of commercially available fish meal (0.44 g as the total amount of nitrogen) and 500 ml of water, 1.5 g of starch liquefaction enzyme Tamameal 120 L (NOVO) is added at 85 ° C. It was heated for 30 minutes to solubilize almost the whole amount of the corn starch, which was sterilized and cooled to obtain a solubilized starch-containing material.
Except that 3 g of sodium fumarate was added to the total amount of the solubilized starch mixture, water was further added to make 1 l, and this was used as a medium, the same as in Example 1, xanthan gum 16. 2 g was obtained.
[0036]
Example 4
Commercial fish meal (sardine shavings) was extracted with hot water for 60 minutes to obtain a fish meal hot water extract having a total nitrogen content of 6.2 g / l.
Instead of the fish extract, 13.7 g of xanthan gum was obtained in the same manner as in Example 1 except that 64 ml of this fish meal hot water extract was added per liter of medium.
[0037]
Example 5
Fish meal (meal shaving powder) was decomposed with an enzyme (Papain, Amano Pharmaceutical Co., Ltd.) to obtain a fish meal decomposition extract having a total nitrogen content of 26 g / l.
Instead of the fish extract, 14.0 g of xanthan gum was obtained in the same manner as in Example 1 except that 15 ml of this fish meal decomposition extract was added per liter of the medium.
[0038]
Comparative Example 2
The procedure was the same as Example 1 except that a medium having the following medium composition was used, but only 9.4 g of xanthan gum was obtained.
Medium composition liquefied starch (manufactured by Pure Chemical) 30 g
Yeast extract (Nippon Pharmaceutical) 1 g
Corn steep liquor (Pure Chemical) 1 ml
Ammonium sulfate (Pure Chemical) 1 g
Magnesium sulfate (7 water salt) (made by Junsei) 0.2g
Dipotassium phosphate (Pure Chemical) 1 g
1 l of water
pH 7.0
[0039]
【The invention's effect】
According to the present invention, a heteropolysaccharide such as xanthan gum, which uses inexpensive liquefied starch and has high industrial utility value, can be obtained easily and efficiently.
[Brief description of the drawings]
BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a graph showing changes over time in starch concentration and cell concentration in culture broth and pH and viscosity of culture broth during the culture period.

Claims (8)

Xanthomonas campestris TSK-001 (FERM P-13997) which can grow and proliferate in a medium containing only liquefied starch as a carbon source to produce a large amount of xanthan gum. Xantomonas campestris TSK-001 (FERM P-13997) is cultured in a medium containing only liquefied starch as a carbon source, and the heteropolysaccharide is recovered from the obtained culture. Production method. Belonging to the genus Xanthomonas, the only liquefied starch as a carbon source, a bacteria capable of producing heteropolysaccharide, and cultured in a medium containing a liquefied starch alone and fish meat Ingredients as a carbon source, a heteropolysaccharide from the resulting culture A method for producing a heteropolysaccharide comprising recovering a saccharide. The method for producing a heteropolysaccharide according to claim 3, wherein the bacterium belonging to the genus Xanthomonas is a bacterium belonging to Xanthomonas campestris. The method for producing a heteropolysaccharide according to claim 4, wherein the bacterium belonging to the genus Xanthomonas is Xanthomonas campestris TSK-001 (FERM P-13997) . The method for producing a heteropolysaccharide according to any one of claims 2 to 5, wherein the culture is an aerobic culture. The method for producing a heteropolysaccharide according to any one of claims 3 to 6, wherein fish meal, fish extract or fish extract is contained as a fish meat component. The method for producing a heteropolysaccharide according to any one of claims 2 to 7, wherein the heteropolysaccharide is xanthan gum.

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