JP3785686B2 - Production method of bacterial cellulose with high oxygen transfer capacity coefficient by aeration and agitation culture - Google Patents

Description

ここで、ＣＬは培養液中の溶存酸素濃度（mmol／ｌ）、ｔは時間（hr）、
【数２】

は一定時間における溶存酸素濃度の変化、すなわち酸素移動速度（mmol／ｌ・hr）、ＫＬは液境膜の酸素移動速度係数（cm／hr）、ａは単位体積当たりの気液界面積（cm² ／cm³)、Ｃ^* は気泡の酸素分圧と平衡な溶存酸素濃度（mmol／ｌ）である。
ＫＬは酸素の気相から液相への移動の抵抗の逆数であり、（Ｃ^* −ＣＬ）は酸素が抵抗に逆らって移動するための推進力(driving force）であるとみなすことができる。発酵系のＫＬとａを測定することは大変困難なことであるから、この２項を掛け合わせたＫＬａを酸素移動容量係数と呼んで用いている。ＫＬａのディメンジョンは時間の逆数で、通常hr^-1で表す。酸素移動容量係数は発酵槽の酸素移動能を表す目安となるもので、同じ条件ではＫＬａの大きいもののほうが酸素移動の能力が大きいことを示す。
【０００５】
【発明が解決しようとする課題】
従来の、特に通気攪拌培養における回分及び流加発酵法にあっては、セルロース生産菌の培養によって培養液中にＢＣが蓄積されてくるのに伴い、培養期間の後半には培養液の粘度が増加し、その結果、培養系全体を均一かつ充分に攪拌混合することが著しく困難になる為、酸素の供給（通気）が不充分となって菌によるＢＣの生産速度が低下していたのである。
そこで、本発明者等は、ＢＣを含む培養液において、酸素供給能力の指標であるＫＬａが高い値を示すような培養条件下でＢＣを製造する方法を開発し、本発明を完成するに至った。
即ち、これまで消泡剤の存在は、一般にＫＬａを低下させるものと報告されているが、横型培養装置を使用することで、ＢＣの培養系に於いて、消泡剤の存在により逆にＫＬａの値が増大しＢＣの生産速度を向上し得ることが判明したのである。これは、ＢＣを生産する為のセルロース生産菌の通常の培養液中では、微細な気泡が液中に長く残存するために培養液の流動性が大きく変化し、通気により発生する新しい気泡の液中への分散が阻害されＫＬａが低下するが、消泡剤を従来使用されてきた濃度（気液界面より気相側へ発泡現象により泡が上昇し、最後には通気とともに装置外へ液の一部が出る現象を制止できる濃度）以上に添加することにより、液相中の微細な気泡の界面更新が促進され、その結果、ＫＬａが増加するものと考えられる。
【０００６】
【課題を解決するための手段】
即ち、本発明は、攪拌羽根を槽内水平軸上に備えて成る横型培養装置を使用し、２重量％のバクテリアセルロースを含み、かつ塑性粘度が１５〜２０ポイズであるような模擬液を用いて測定した酸素移動容量係数（ＫＬａ）が約５０〜１００／hrである培養条件下で少なくとも一定期間セルロース生産菌を培養してセルロース性物質を製造する方法に係わるものである。
本発明の具体的態様の一つとして、攪拌羽根を槽内水平軸上に備えて成る横型培養装置を使用し、消泡剤の存在下に培養する方法がある。消泡剤としては従来公知の消泡剤、例えば、ポリプロピレングリコール(PPG) 、シリコン、エステル、アルコール類及び脂肪酸とその誘導体等を使用することができ、その培養液中の濃度は消泡剤の種類にも依るが、好ましくは０．００１重量％以上、より好ましくは０．００５重量％以上である。
又、その他の具体的態様として攪拌羽根を槽内水平軸上に備えて成る横型培養装置を使用し、気泡比率が１０％以下となる条件下で培養する方法がある。尚、気泡比率は以下の方法で算出することができる。
気泡比率の算出方法：
気泡を含む状態の模擬液の見掛け比重をＡ、模擬液の真比重をＢとすると
【数３】

【０００７】
該横型培養装置に備える門型羽根又はタービン羽根等それ自体は従来公知の種々の形態のものを使用することができる。
例えばタービン羽根としては、一般的なラシュトンタービンの他に、ディスクのないパドル型やパドル型の直上にディスクを備えたもの、湾曲パドル型、スカバー型及びマリンインペラー型等を使用することができる。
本発明の培養装置に於いて、門型羽根、タービン羽根等の同種及び別種の羽根を、同一槽内に於いて同一軸又は別個の軸に備えることができる。
水平軸は約８０°まで傾けて使用することができる。
ＫＬａは培養において重要で一般的な装置性能の指標であるが、対照となる液の性質や攪拌羽根の形状、回転数により変化する。本発明者等は目的とするバクテリア・セルロースの液性（セルロース生産菌の培養液）に近い模擬液を設定し、そこでＫＬａで評価することにより、生産性の向上に直接関係した因子として評価することができる。この評価系で測定すれば、攪拌羽根の形状と回転数は任意の条件を選ぶことができる。
【０００８】
ＫＬａの測定方法：
２重量％のバクテリアセルロースを含み、かつ塑性粘度が１５〜２０ポイズであるような模擬液を全量２Ｌのガラス製ジャーファーメンター（横型培養装置）の 1／3 容量に張り込み、これに適宜消泡剤を添加した状態で測定する。攪拌羽根を回転させながら窒素を通気することにより溶存酸素濃度を０〜１０％飽和状態とした模擬液に、次に酸素分圧２０〜２１％の空気を通気し、これによって上昇する溶存酸素濃度を溶存酸素電極を用いて測定する。
ＫＬａは前記（数１）式より求められるが、簡便には、５〜３０秒毎に溶存酸素濃度を測定し、時間ｔ１での溶存酸素濃度ＤＯ１と時間ｔ２での溶存酸素濃度ＤＯ２から以下の式でＫＬａを求める。
（（ＤＯ２−ＤＯ１）／（ｔ２−ｔ１））／（Ｃ^* −（ＤＯ１＋ＤＯ２）／２）、単位（／hr） （但し、式中Ｃ^* は気泡の酸素分圧と平衡な溶存酸素濃度）
【０００９】
本明細書中、「ＫＬａが約５０〜１００／hrである培養条件」とは、本明細書中で定義した前述の測定系で得られるところの数値である。
培養に際してどの時期にどの程度の期間をかかる特定の装置条件下に制御するかは菌体の種類、培地の組成及び培養装置の種類等に応じて等業者が適宜選択しうる。菌体の増殖と共に培地の粘度が増加してくるため、通常は少なくとも、培養液中のセルロース濃度が５ｇ／Ｌ以上となる時期、又は生産速度が０．２ｇ／Ｌ／hrとなる時期に一定期間上記特定の範囲のＫＬａを達成して培養系に充分な酸素の供給を行なうようにすることが好ましい。この特定の装置条件を培養期間中、断続的に設けることも可能である。
本発明の方法によって、極めて高いバイオセルロースの生産速度が得られるのである。
【００１０】
【発明の実施の形態】
本発明方法を実施するに際しては、前述の培養形式・培養操作法に加えて、特願平６−１９２２８７号に記載されている「培養装置と浮上分離装置及びエッジフィルター等の分離装置の間で菌体を含む培養液を循環させるセルロース性物質の製造方法であって、該分離装置に於いて、生産物であるセルロース性物質を菌体及び培養液から分離することを特徴とする、前記方法」及び特願平６−１９２２８８号に記載されている「セルロース生産菌を培養してセルロース性物質を製造する方法であって、培養期間中、培養系からの培養液の引き抜き及び該引き抜き量とほぼ等容量の新たな培養液の供給を連続的に行なうことによって、培養中の培養液に於けるセルロース性物質の濃度を例えば１０ｇ／Ｌ以下、又は酸素消費速度が１５μmol ／Ｌ／hr以上に保つことができるように低く維持することを特徴とする前記製造方法」を採ることもできる。
【００１１】
本発明において使用されるセルロース生産菌は、例えば、ＢＰＲ２００１株に代表されるアセトバクター・キシリナム・サブスピーシーズ・シュクロファーメンタンス（Acetobacter xylinum subsp. sucrofermentans）、アセトバクター・キシリナム（Acetobacter xylinum ）ＡＴＣＣ２３７６８、アセトバクター・キシリナムＡＴＣＣ２３７６９、アセトバクター・パスツリアヌス（A. pasteurianus ）ＡＴＣＣ１０２４５、アセトバクター・キシリナムＡＴＣＣ１４８５１、アセトバクター・キシリナムＡＴＣＣ１１１４２及びアセトバクター・キシリナムＡＴＣＣ１０８２１等の酢酸菌、その他に、アグロバクテリウム属、リゾビウム属、サルシナ属、シュードモナス属、アクロモバクター属、アルカリゲネス属、アエロバクター属、アゾトバクター属及びズーグレア属並びにそれらをＮＴＧ（ニトロソグアニジン）等を用いる公知の方法によって変異処理することにより創製される各種変異株である。
尚、ＢＰＲ２００１株は、平成５年２月２４日に通商産業省工業技術院生命工学工業技術研究所特許微生物寄託センターに寄託され（受託番号ＦＥＲＭ Ｐ−１３４６６）、その後１９９４年２月７日付で特許手続上の寄託の国際的承認に関するブダペスト条約に基づく寄託（受託番号ＦＥＲＭ ＢＰ−４５４５）に移管されている。
【００１２】
ＮＴＧ等の変異剤を用いての化学的変異処理方法には、例えば、Bio Factors, Vol. l, p.297−302 (1988)及び J. Gen. Microbiol, Vol. 135, p.2917−2929 (1989) 等に記載されているものがある。従って、当業者であればこれら公知の方法に基づき本発明で用いる変異株を得ることができる。また、本発明で用いる変異株は他の変異方法、例えば放射線照射等によっても得ることができる。
本発明の製造方法に用いる培地の組成物中、炭素源としてはシュクロース、グルコース、フラクトース、マンニトール、ソルビトール、ガラクトース、マルトース、エリスリット、グリセリン、エチレングリコール、エタノール等を単独或いは併用して使用することができる。更にはこれらのものを含有する澱粉水解物、シトラスモラセス、ビートモラセス、ビート搾汁、サトウキビ搾汁、柑橘類を始めとする果汁等をシュクロースに加えて使用することもできる。 また、窒素源としては硫酸アンモニウム、塩化アンモニウム、リン酸アンモニウム等のアンモニウム塩、硝酸塩、尿素等有機或いは無機の窒素源を使用することができ、或いはＢａｃｔ−Ｐｅｐｔｏｎｅ、Ｂａｃｔ−Ｓｏｙｔｏｎｅ、Ｙｅａｓｔ−Ｅｘｔｒａｃｔ、豆濃などの含窒素天然栄養源を使用してもよい。有機微量栄養素としてアミノ酸、ビタミン、脂肪酸、核酸、２，７，９−トリカルボキシ−１Ｈピロロ〔２，３，５〕−キノリン−４，５−ジオン、亜硫酸パルプ廃液、リグニンスルホン酸等を添加してもよい。
【００１３】
生育にアミノ酸等を要求する栄養要求性変異株を使用する場合には、要求される栄養素を補添することが必要である。無機塩類としてはリン酸塩、マグネシウム塩、カルシウム塩、鉄塩、マンガン塩、コバルト塩、モリブデン酸塩、赤血塩、キレート金属類等が使用される。
更に、前述のセルロース生成促進因子を適宜培地中に添加することもできる。
例えば、酢酸菌を生産菌として用いる場合には、培養のｐＨは３ないし７に、好ましくは５付近に制御する。培養温度は１０〜４０℃、好ましくは２５〜３５℃の範囲で行う。培養装置に供給する酸素濃度は１〜１００％、望ましくは２１〜８０％であれば良い。これら培地中の各成分の組成割合及び培地に対する菌体の接種等は培養方法に応じて当業者が適宜選択し得るものである。
【００１４】
本発明の方法によって製造されるＢＣは菌体はそのまま回収してもよく、さらに本物質中に含まれる菌体を含むセルロース性物質以外の不純物を取り除く処理を施すことが出来る。
不純物を取り除くためには、水洗、加圧脱水、希酸洗浄、アルカリ洗浄、次亜塩素酸ソーダ及び過酸化水素などの漂白剤による処理、リゾチームなどの菌体溶解酵素による処理、ラウリル硫酸ソーダ、デオキシコール酸などの界面活性剤による処理、常温から２００℃の範囲の加熱洗浄などを単独及び併用して行い、セルロース性物質から不純物をほぼ完全に除去することができる。
このようにして得られた本発明でいうセルロース性物質とは、セルロース及び、セルロースを主鎖としたヘテロ多糖を含むもの及びβ−１，３、β−１，２等のグルカンを含むものである。ヘテロ多糖の場合のセルロース以外の構成成分はマンノース、フラクトース、ガラクトース、キシロース、アラビノース、ラムノース、グルクロン酸等の六炭糖、五炭糖及び有機酸等である。
尚、これ等の多糖が単一物質である場合もあるし２種以上の多糖が水素結合等により混在してもよい。
【００１５】
【実施例】
以下の実施例により、本発明をさらに詳細に説明する。
実施例１
図１に示した横型培養装置を使用して、前述のＫＬａ測定系を用いて、所要動力を約４０〜１７０kg・cm/sec・L の範囲で変化させて、それに伴いＫＬａの値が消泡剤の有無でどのように変化するかを測定した。その結果を図２に示す。所要動力が比較的高い場合に、特に消泡剤の添加による効果が顕著になることが判る。
又、同様な系で、消泡剤の濃度がＫＬａの値に与える影響を測定した。その結果を図３に示す。回転数の比較的高い場合に、特に消泡剤の添加による効果が顕著になることが判る。
更に、同様な系（回転数：２００rpm ）で、消泡剤の濃度が気泡比率に与える影響を測定した。その結果を図４に示す。
【図面の簡単な説明】
【図１】 ＫＬａ測定系に用いた横型培養装置を示す。
【図２】 所要動力の変化に伴うＫＬａの値の変化を示す。
【図３】 消泡剤の濃度がＫＬａの値に与える影響を示す。
【図４】 消泡剤の濃度が気泡比率及びＫＬａの値に与える影響を示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a cellulosic property using a cell belonging to a microorganism (hereinafter referred to as “cellulose-producing bacterium”) having an ability to produce a cellulosic substance under a culture condition capable of achieving a high oxygen transfer capacity coefficient (KLa). The present invention relates to a method for producing a substance (hereinafter referred to as “bacterial cellulose” or “BC”).
[0002]
[Prior art]
BC (bacterial cellulose) is edible and is used in the food field and has excellent water-based dispersibility, so it retains the viscosity of food, cosmetics, paints, etc., strengthens the raw material dough, retains moisture, and improves food stability. Industrially useful as a low calorie additive or an emulsion stabilization aid.
BC is characterized in that the fibril fragment width is about two digits smaller than cellulose produced from wood pulp or the like.
Accordingly, BC disaggregation has various industrial uses as a reinforcing agent for polymers, particularly aqueous polymers, based on such structural and physical characteristics of microfibrils. A material obtained by solidifying such a cellulosic disaggregate into a paper or solid form exhibits a high tensile elastic modulus, and therefore excellent mechanical properties based on the structural characteristics of microfibrils are expected. is there.
[0003]
Regarding the BC production method, JP-A-62-265990, JP-A-63-202394, JP-B-6-43443, etc. describe the production method of BC.
Suitable nutrient media for culturing cellulosic bacteria include Schramm / Hestrin media (Schramm et al., J. General Biology, ll , pp) consisting of carbon source, peptone, yeast extract, sodium phosphate and citric acid. .123-129, l954) are known. In addition, in such a nutrient medium, inositol, phytic acid, and pyrroloquinoline quinone (PQQ) (Japanese Patent Publication No. 5-1718; Mitsuo Takai, Papa Gijutsu Kyodo Journal) are cellulose production promoting factors by specific nutrients in the medium. 42, No. 3, pages 237 to 244) and the like, and further, carboxylic acid or a salt thereof (Japanese Patent Application No. 5-191467), invertase (Japanese Patent Application No. 5-331491) and It has been found that the addition of methionine (Japanese Patent Application No. 5-335664) improves the productivity of cellulosic substances.
Conventionally, as a culture format for culturing microorganisms, stationary, shaking or aeration-agitation culture has been used. Moreover, as a culture operation method, a so-called batch fermentation method, fed-batch fermentation method, repeated batch fermentation method, continuous fermentation method, and the like have been used.
As the stirring means, for example, an impeller (stirring blade), an air lift fermenter, a pump-driven circulation of fermentation broth, a combination of these means, and the like are used.
Known types of impellers include portal blades, turbine blades, helical ribbon blades, screw blades, and the like.
[0004]
By the way, in general industrial fermentation processes, the oxygen demand of culture is satisfied by aeration and agitation. However, in many fermentation processes, productivity is limited by the oxygen supply capacity of the fermenter, and therefore, it is considered important to examine factors that influence oxygen supply during the cultivation of microorganisms.
When oxygen in the air moves to the cells in the culture system, oxygen transfer from the bubbles to the liquid phase is represented by the following equation.
[Expression 1]

Here, CL is dissolved oxygen concentration (mmol / l) in the culture solution, t is time (hr),
[Expression 2]

Is the change in dissolved oxygen concentration over a period of time, that is, oxygen transfer rate (mmol / l · hr), KL is the oxygen transfer rate coefficient (cm / hr) of the liquid boundary film, and a is the gas-liquid interface area per unit volume (cm ² / cm ³ ), C ^* is the dissolved oxygen concentration (mmol / l) in equilibrium with the oxygen partial pressure of the bubbles.
KL is the reciprocal of the resistance of oxygen from the gas phase to the liquid phase, and (C ^* -CL) can be regarded as a driving force for oxygen to move against the resistance. Since it is very difficult to measure KL and a in a fermentation system, KLa obtained by multiplying these two terms is called an oxygen transfer capacity coefficient. The dimension of KLa is the reciprocal of time and is usually expressed as hr- ¹ . The oxygen transfer capacity coefficient is a measure representing the oxygen transfer ability of the fermenter. Under the same conditions, a larger KLa indicates a greater oxygen transfer ability.
[0005]
[Problems to be solved by the invention]
In the conventional batch and fed-batch fermentation methods, particularly in aeration and agitation culture, as BC is accumulated in the culture solution by culturing cellulose-producing bacteria, the viscosity of the culture solution is increased in the latter half of the culture period. As a result, it becomes extremely difficult to uniformly and sufficiently stir and mix the entire culture system, so that oxygen supply (aeration) is insufficient and the production rate of BC by bacteria is reduced. .
Accordingly, the present inventors have developed a method for producing BC under culture conditions in which KLa, which is an index of oxygen supply capacity, shows a high value in a culture solution containing BC, and have completed the present invention. It was.
That is, until now, it has been reported that the presence of an antifoaming agent generally lowers KLa, but by using a horizontal culture apparatus, in the culture system of BC, the presence of the antifoaming agent conversely causes KLa. It has been found that the value of can be increased and the production rate of BC can be improved. This is because in the normal culture solution of cellulose-producing bacteria for producing BC, the fluidity of the culture solution changes greatly because fine bubbles remain in the solution for a long time, and a new bubble solution generated by aeration. Although the dispersion into the inside is hindered and the KLa is lowered, the concentration of the antifoaming agent has been conventionally used (the foam rises due to the foaming phenomenon from the gas-liquid interface to the gas phase side, and finally the liquid is discharged to the outside with the ventilation. It is considered that the addition of more than the concentration that can suppress the phenomenon in which a part is generated promotes the renewal of the interface of fine bubbles in the liquid phase, and as a result, KLa increases.
[0006]
[Means for Solving the Problems]
That is, the present invention uses a horizontal culture apparatus comprising a stirring blade on the horizontal axis in the tank, and uses a simulated solution containing 2% by weight of bacterial cellulose and having a plastic viscosity of 15 to 20 poise. This relates to a method for producing a cellulosic substance by culturing a cellulose-producing bacterium for at least a certain period of time under a culture condition in which an oxygen transfer capacity coefficient (KLa) measured in the above is about 50 to 100 / hr.
As one of the specific embodiments of the present invention, there is a method of culturing in the presence of an antifoaming agent using a horizontal culture apparatus comprising a stirring blade on a horizontal axis in a tank. As the antifoaming agent, conventionally known antifoaming agents such as polypropylene glycol (PPG), silicon, esters, alcohols and fatty acids and derivatives thereof can be used. Although depending on the type, it is preferably 0.001% by weight or more, more preferably 0.005% by weight or more.
As another specific embodiment, there is a method of culturing under the condition that the bubble ratio is 10% or less by using a horizontal culture apparatus having a stirring blade on the horizontal axis in the tank. The bubble ratio can be calculated by the following method.
Bubble ratio calculation method :
Assuming that the apparent specific gravity of the simulated liquid containing air bubbles is A and the true specific gravity of the simulated liquid is B,

[0007]
Conventionally known various forms such as a portal blade or a turbine blade provided in the horizontal culture apparatus can be used.
For example, as a turbine blade, in addition to a general Rushton turbine, a paddle type without a disk, a type having a disk directly above a paddle type, a curved paddle type, a cover type, a marine impeller type, or the like can be used.
In the culture apparatus of the present invention, the same type and different types of blades such as a portal blade and a turbine blade can be provided on the same shaft or separate shafts in the same tank.
The horizontal axis can be used tilted up to about 80 °.
KLa is an important and general index of apparatus performance in culture, but varies depending on the properties of the liquid used as a control, the shape of the stirring blade, and the rotational speed. The present inventors set up a simulated solution close to the intended bacterial / cellulose solution (culture solution of cellulose-producing bacteria), and evaluate it with KLa as a factor directly related to the improvement of productivity. be able to. If it measures with this evaluation system, arbitrary conditions can be selected for the shape and rotation speed of a stirring blade.
[0008]
Method for measuring KLa :
A simulated liquid containing 2% by weight of bacterial cellulose and having a plastic viscosity of 15 to 20 poise is put on 1/3 volume of a 2 liter glass jar fermenter (horizontal culture device), and defoamed appropriately. Measured with the agent added. The simulated oxygen solution in which the dissolved oxygen concentration was saturated by 0 to 10% by ventilating nitrogen while rotating the stirring blades was then aerated with air having an oxygen partial pressure of 20 to 21%, thereby increasing the dissolved oxygen concentration Is measured using a dissolved oxygen electrode.
KLa is calculated from the above equation (1). For simplicity, the dissolved oxygen concentration is measured every 5 to 30 seconds, and the dissolved oxygen concentration DO1 at time t1 and the dissolved oxygen concentration DO2 at time t2 are as follows. Calculate KLa by the equation.
((DO2-DO1) / (t2-t1)) / (C ^* -(DO1 + DO2) / 2), unit (/ hr) (where C ^* is the dissolved oxygen concentration in equilibrium with the oxygen partial pressure of bubbles)
[0009]
In the present specification, “culture conditions under which KLa is about 50 to 100 / hr” is a numerical value obtained by the aforementioned measurement system defined in the present specification.
Depending on the type of bacterial cells, the composition of the culture medium, the type of the culture apparatus, and the like, it is possible for a trader to appropriately select which period and how long the culture is controlled under the specific apparatus conditions. Since the viscosity of the medium increases with the growth of the cells, it is usually constant at least when the cellulose concentration in the culture becomes 5 g / L or higher, or when the production rate becomes 0.2 g / L / hr. It is preferable to achieve a sufficient range of oxygen supply to the culture system by achieving KLa in the specific range for the period. It is also possible to provide this specific device condition intermittently during the culture period.
By the method of the present invention, a very high production rate of biocellulose can be obtained.
[0010]
DETAILED DESCRIPTION OF THE INVENTION
In carrying out the method of the present invention, in addition to the culture method and the culture operation method described above, it is described in Japanese Patent Application No. 6-192287 “between the culture device and the separation device such as the flotation separation device and the edge filter”. A method for producing a cellulosic material in which a culture solution containing cells is circulated, wherein the cellulosic material as a product is separated from the cells and the culture solution in the separation device. And a method for producing a cellulosic material by culturing a cellulose-producing bacterium, and withdrawing the culture solution from the culture system and the amount of the withdrawal. By continuously supplying an approximately equal volume of a new culture solution, the concentration of the cellulosic substance in the culture solution during culture is, for example, 10 g / L or less, or the oxygen consumption rate is 15 μmol / L. It may take the above method, "which is characterized by maintaining such can be kept above hr low.
[0011]
Cellulose-producing bacteria used in the present invention include, for example, Acetobacter xylinum subsp. Sucrofermentans , Acetobacter xylinum ATCC 23768, represented by BPR2001 strain, Acetobacter xylinum ATCC 23769, Acetobacter pasturianus ( A. pasteurianus ) ATCC 10245, Acetobacter xylinum ATCC 14851, Acetobacter xylinum ATCC 11142, Acetobacter xylinum ATCC 10821 and other acetic acid bacteria, Agrobacterium genus, Rhizobium genus, Sarsina, Pseudomonas, Achromobacter, Alkaligenes, Aerobacter, Azotobacter and Zoo These are various mutant strains created by subjecting the genus Glare and those to mutation treatment by a known method using NTG (nitrosoguanidine) or the like.
The BPR2001 strain was deposited on February 24, 1993 at the Patent Microbiology Depositary Center of the Biotechnology Institute of Industrial Technology, Ministry of International Trade and Industry (accession number FERM P-13466), and then on February 7, 1994. Has been transferred to the deposit under the Budapest Treaty concerning the international recognition of deposits under patent procedure (accession number FERM BP-4545).
[0012]
Chemical mutagenesis methods using mutagens such as NTG include, for example, Bio Factors, Vol. L, p.297-302 (1988) and J. Gen. Microbiol, Vol. 135, p.2917-2929. (1989) etc. Therefore, those skilled in the art can obtain mutant strains used in the present invention based on these known methods. The mutant strain used in the present invention can also be obtained by other mutation methods such as irradiation.
In the composition of the medium used in the production method of the present invention, sucrose, glucose, fructose, mannitol, sorbitol, galactose, maltose, erythritol, glycerin, ethylene glycol, ethanol, etc. are used alone or in combination as a carbon source. be able to. Furthermore, starch hydrolyzate, citrus molasses, beet molasses, beet juice, sugarcane juice, citrus juice and other fruit juices containing these can be used in addition to sucrose. As the nitrogen source, organic or inorganic nitrogen sources such as ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate, nitrates and urea can be used, or Bact-Peptone, Bact-Soytone, Yeast-Extract, beans Nitrogen-containing natural nutrient sources such as rich may be used. Add amino acids, vitamins, fatty acids, nucleic acids, 2,7,9-tricarboxy-1H pyrrolo [2,3,5] -quinoline-4,5-dione, sulfite pulp waste liquor, lignin sulfonic acid, etc. as organic micronutrients May be.
[0013]
When using an auxotrophic mutant that requires an amino acid or the like for growth, it is necessary to supplement the required nutrients. Examples of inorganic salts include phosphates, magnesium salts, calcium salts, iron salts, manganese salts, cobalt salts, molybdates, red blood salts, chelate metals, and the like.
Furthermore, the above-mentioned cellulose production promoting factor can be appropriately added to the medium.
For example, when acetic acid bacteria are used as production bacteria, the pH of the culture is controlled to 3 to 7, preferably around 5. The culture temperature is 10 to 40 ° C, preferably 25 to 35 ° C. The oxygen concentration supplied to the culture apparatus may be 1 to 100%, preferably 21 to 80%. Those skilled in the art can appropriately select the composition ratio of each component in these media and the inoculation of bacterial cells to the media according to the culture method.
[0014]
The BC produced by the method of the present invention may be recovered as it is, and further subjected to a treatment for removing impurities other than the cellulosic material containing the cells contained in this material.
In order to remove impurities, washing with water, pressure dehydration, dilute acid washing, alkali washing, treatment with bleach such as sodium hypochlorite and hydrogen peroxide, treatment with lytic enzymes such as lysozyme, sodium lauryl sulfate, Impurities can be almost completely removed from the cellulosic substance by treatment with a surfactant such as deoxycholic acid, heat washing in the range from room temperature to 200 ° C. alone or in combination.
The cellulosic material referred to in the present invention thus obtained includes cellulose, a heteropolysaccharide having cellulose as a main chain, and a glucan such as β-1,3, β-1,2, and the like. Constituent components other than cellulose in the case of heteropolysaccharides are hexoses such as mannose, fructose, galactose, xylose, arabinose, rhamnose, glucuronic acid, pentoses, and organic acids.
In addition, these polysaccharides may be a single substance, or two or more polysaccharides may be mixed by hydrogen bonding or the like.
[0015]
【Example】
The following examples illustrate the invention in more detail.
Example 1
Using the horizontal culture apparatus shown in FIG. 1, the required power is changed in the range of about 40 to 170 kg · cm / sec · L using the above-mentioned KLa measurement system, and the KLa value is defoamed accordingly. It was measured how it changed with and without the agent. The result is shown in FIG. It can be seen that when the required power is relatively high, the effect of adding an antifoaming agent becomes particularly significant.
Moreover, the influence which the density | concentration of an antifoamer has on the value of KLa was measured by the same system. The result is shown in FIG. It can be seen that the effect of adding an antifoaming agent is particularly remarkable when the rotational speed is relatively high.
Furthermore, the influence which the density | concentration of an antifoamer has on the bubble ratio was measured by the same system (rotation speed: 200 rpm). The result is shown in FIG.
[Brief description of the drawings]
FIG. 1 shows a horizontal culture apparatus used in a KLa measurement system.
FIG. 2 shows changes in the value of KLa accompanying changes in required power.
FIG. 3 shows the effect of antifoam concentration on the value of KLa.
FIG. 4 shows the influence of the concentration of an antifoaming agent on the bubble ratio and the value of KLa.

Claims (1)

Oxygen transfer capacity coefficient measured using a simulated culture solution containing 2% by weight of bacterial cellulose and having a plastic viscosity of 15 to 20 poises using a horizontal culture apparatus equipped with a stirring blade on the horizontal axis in the tank Cellulose-producing substances are produced by culturing cellulose-producing bacteria in the presence of an antifoaming agent having a concentration of 0.001% by weight or more under a culture condition in which (KLa) is about 50 to 100 / hr for at least a certain period. how to.

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