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JP6289053B2 - Production method of sophorolipid - Google Patents

Production method of sophorolipid Download PDF

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JP6289053B2
JP6289053B2 JP2013241740A JP2013241740A JP6289053B2 JP 6289053 B2 JP6289053 B2 JP 6289053B2 JP 2013241740 A JP2013241740 A JP 2013241740A JP 2013241740 A JP2013241740 A JP 2013241740A JP 6289053 B2 JP6289053 B2 JP 6289053B2
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健一 四方
健一 四方
佐藤 仁
仁 佐藤
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Description

本発明は、ソフォロリピッドの製造方法に関する。   The present invention relates to a method for producing sophorolipid.

酵母等の微生物が生産する両親媒性物質はバイオサーファクタント(生物界面活性剤)と呼ばれ、環境適合性と機能性を兼ね備えた材料として、食品、化粧品、ライフサイエンス、環境・エネルギー分野等での応用が研究されている。
なかでもソフォロリピッドは、比較的安価な基質から得られるため、現在産業利用されている代表的なバイオサーファクタントの一つである。
Amphiphiles produced by microorganisms such as yeast are called biosurfactants (biosurfactants), and are materials that have both environmental compatibility and functionality in the fields of food, cosmetics, life science, environment and energy. Application has been studied.
Among them, sophorolipid is one of typical biosurfactants currently used in industry because it is obtained from a relatively inexpensive substrate.

ソフォロリピッドの製造方法として、例えば、カンジダ属酵母を植物油と遊離脂肪酸を混合した液体培地で培養し、ソフォロリピッドを発酵生産する方法(特許文献1)、カンジダ属酵母を植物油、脂肪酸等の代わりに6〜30個の炭素原子の鎖長をもつ2−アルカノールを含有する培地で培養し、非環状のソフォロリピッドを製造する方法(特許文献2)が知られている。また、ジアセチルラクトン型のソフォロリピッドを得るために、培養液への酸素供給量を制限して微生物を培養する方法(特許文献3)が知られている。   As a method for producing sophorolipid, for example, Candida yeast is cultured in a liquid medium in which vegetable oil and free fatty acid are mixed, and sophorolipid is fermented and produced (Patent Document 1). Instead, a method (Patent Document 2) for producing an acyclic sophorolipid by culturing in a medium containing 2-alkanol having a chain length of 6 to 30 carbon atoms is known. Moreover, in order to obtain a diacetyllactone type sophorolipid, a method of culturing a microorganism by limiting the amount of oxygen supplied to the culture solution (Patent Document 3) is known.

特開2002−45195号公報JP 2002-45195 A 特開平8−325287号公報JP-A-8-325287 国際公開第2010/050413号International Publication No. 2010/050413

しかしながら、従来技術の方法では、基質である炭素源(糖、油脂、脂肪酸誘導体等)からソフォロリピッドへの変換速度(発酵速度)が遅く、工業生産に適した生産性の向上が大きな課題と考えられた。
したがって、本発明は、変換速度が速く、効率よく基質からソフォロリピッドを製造することのできる方法を提供することに関する。
However, in the method of the prior art, the conversion rate (fermentation rate) from the carbon source (sugar, fat, fatty acid derivative, etc.) as a substrate to sophorolipid is slow, and improvement of productivity suitable for industrial production is a major issue. it was thought.
Therefore, the present invention relates to providing a method capable of producing sophorolipid from a substrate with high conversion rate and efficiency.

本発明者らが検討した結果、炭素源基質からソフォロリピッドへの変換速度を速めるには、発酵に用いられる微生物の数を増やし、酸素供給量を増加させることが有効と考えられた。しかし、酸素供給量が増え、培養液中の溶存酸素濃度が高まるとかえって微生物のソフォロリピッドの生産性に影響を及ぼすことが懸念された。そこで更に鋭意検討したところ、一定数の微生物に対し供給される酸素の供給速度を特定の範囲に制御することで、培養液中の溶存酸素濃度を制御することにより、変換速度が速まり、極めて効率よく基質からソフォロリピッドを製造できることを見出した。   As a result of the study by the present inventors, it was considered effective to increase the number of microorganisms used for fermentation and increase the oxygen supply amount in order to increase the conversion rate of the carbon source substrate to sophorolipid. However, there is a concern that the productivity of sophorolipids of microorganisms may be affected if the oxygen supply amount increases and the dissolved oxygen concentration in the culture solution increases. As a result of further intensive studies, the conversion rate was increased by controlling the dissolved oxygen concentration in the culture solution by controlling the supply rate of oxygen supplied to a certain number of microorganisms to a specific range. It was found that sophorolipid can be efficiently produced from a substrate.

すなわち、本発明は、ソフォロリピッドを生産する能力を有する微生物を好気的条件下、炭素源を含有する培養液中で培養し、ソフォロリピッドを得る工程を含むソフォロリピッドの製造方法であって、
前記培養液中のソフォロリピッドを生産する能力を有する微生物の濃度が波長660nmにおける吸光度として100〜300であり、且つ培養液に供給される酸素の供給速度が0.1〜0.5mol/L/hrである、製造方法を提供するものである。
That is, the present invention is a method for producing a sophorolipid comprising a step of culturing a microorganism having the ability to produce sophorolipid in a culture solution containing a carbon source under aerobic conditions to obtain the sophorolipid. There,
The concentration of microorganisms capable of producing sophorolipid in the culture solution is 100 to 300 as absorbance at a wavelength of 660 nm, and the supply rate of oxygen supplied to the culture solution is 0.1 to 0.5 mol / L. The production method is / hr.

本発明によれば、基質からソフォロリピッドへの変換速度を高めることができるので、ソフォロリピッドを効率よく製造することができる。   According to the present invention, since the conversion rate from a substrate to sophorolipid can be increased, sophorolipid can be produced efficiently.

本発明のソフォロリピッドの製造方法は、ソフォロリピッドを生産する能力を有する微生物を好気的条件下、炭素源を含有する培養液中で培養し、ソフォロリピッドを得る工程を含むものであり、前記培養液中のソフォロリピッドを生産する能力を有する微生物の濃度が波長660nmにおける吸光度として100〜300であり、且つ培養液に供給される酸素の供給速度が0.1〜0.5mol/L/hrである。   The method for producing sophorolipid of the present invention comprises a step of culturing a microorganism having the ability to produce sophorolipid in a culture solution containing a carbon source under aerobic conditions to obtain sophorolipid. And the concentration of the microorganism having the ability to produce sophorolipid in the culture solution is 100 to 300 as absorbance at a wavelength of 660 nm, and the supply rate of oxygen supplied to the culture solution is 0.1 to 0.5 mol / L / hr.

ソフォロリピッドは、ソフォロースとヒドロキシ脂肪酸とからなる糖脂質である。ソフォロースは、グルコースがβ−1,2結合した二糖類であり、ヒドロキシ脂肪酸は、ω位、或いはω−1位にヒドロキシ基を有する脂肪酸である。ヒドロキシ脂肪酸の脂肪酸部分は、特に限定されないが、炭素数6〜30の飽和又は不飽和脂肪酸が好ましい。また、ソフォロースは、ヒドロキシ基が一部アセチル化したものも含む。
ソフォロリピッドは、ヒドロキシ脂肪酸のカルボキシル基が遊離した酸型と分子内のソフォロースと結合したラクトン型に大別され、一般的に発酵生産では酸型とラクトン型の混合物として得られることが知られている。
Sophorolipid is a glycolipid composed of soforose and hydroxy fatty acid. Soforus is a disaccharide in which glucose is β-1,2 linked, and hydroxy fatty acid is a fatty acid having a hydroxy group at the ω-position or ω-1 position. The fatty acid portion of the hydroxy fatty acid is not particularly limited, but a saturated or unsaturated fatty acid having 6 to 30 carbon atoms is preferable. Soforus also includes those in which the hydroxy group is partially acetylated.
Sophorolipids are broadly divided into acid types in which the carboxyl group of hydroxy fatty acid is liberated and lactone types in combination with Sophorose in the molecule, and are generally known to be obtained as a mixture of acid and lactone types in fermentation production. ing.

本発明で用いられるソフォロリピッドを生産する能力を有する微生物は、基質からソフォロリピッドを生成し、菌体外に産出する能力を有する微生物であればよく、例えば、カンジダ属(Candida)に属する微生物が挙げられる。
カンジダ属に属する微生物としては、Candida bombicolaCandida bogoriensisCandida magnoliaeCandida gropengiesseriCandida apicola等が挙げられる。なかでも、ソフォロリピッド生産性の点から、Candida bombicolaが好ましい。
The microorganism having the ability to produce the sophorolipid used in the present invention may be any microorganism that has the ability to produce the sophorolipid from the substrate and to produce it outside the cell. For example, it belongs to the genus Candida. Examples include microorganisms.
Examples of the microorganisms belonging to the genus Candida, Candida bombicola, Candida bogoriensis, Candida magnoliae, Candida gropengiesseri, Candida apicola , and the like. Among these, Candida bombicola is preferable from the viewpoint of productivity of sophorolipid .

ソフォロリピッドを生産する能力を有する微生物は、該微生物が資化し得る炭素源を含むものであれば、従来公知の培地を用いて培養することができる。例えば、YM培地等の市販の液体培地を用いることができる。また、該微生物が資化し得る炭素源、窒素源、無機塩類、その他必要な栄養源等を培地の成分として適宜用いることができる。
炭素源としては、例えば、糖(グルコース、アラビノース、キシロース、マンノース、フラクトース、ガラクトース、シュークロース、マルトース、ラクトース、ソルビトール、マンニトール、イノシット、グリセリン、可溶性澱粉、廃糖蜜、転化糖等)、油脂類(大豆油、ナタネ油、サフラワー油、米油、コーン油、パーム油、ヒマワリ油、綿実油、オリーブ油、ゴマ油、落花生油、ハトムギ油、小麦胚芽油、シソ油、アマニ油、エゴマ油、サチャインチ油、クルミ油、キウイ種子油、サルビア種子油、ブドウ種子油、マカデミアナッツ油、ヘーゼルナッツ油、カボチャ種子油、椿油、茶実油、ボラージ油、パームオレイン、パームステアリン、やし油、パーム核油、カカオ脂、サル脂、シア脂、藻油等の植物性油脂;魚油、ラード、牛脂、バター脂等の動物性油脂;又はそれらのエステル交換油、水素添加油もしくは分別油;カプリン酸、ウンデカン酸、ラウリン酸、トリデカン酸、ミリスチン酸、ペンタデカン酸、パルミチン酸、マルガリン酸、ステアリン酸、ノナデカン酸、アラキジン酸、ベヘン酸、リグノセリン酸、リンデル酸、ツズ酸、ミリストレイン酸、パルミトレイン酸、ペトロセリン酸、オレイン酸、エライジン酸、バクセン酸、エルカ酸、ソルビン酸、リノール酸、リノエライジン酸、γ−リノレン酸、リノレン酸、アラキドン酸等の脂肪酸又はそのエステル等)が挙げられる。また、酢酸等の資化しうる有機酸、エタノール等のアルコール類等を用いてもよい。
窒素源としては、例えば、アンモニア、無機・有機アンモニウム塩、尿素、コーングルテンミール、大豆粉、酵母エキス、肉エキス、魚肉エキス、ポリペプトン、ペプトン、各種アミノ酸、ソイビーンミール等が挙げられる。
A microorganism having the ability to produce sophorolipid can be cultured using a conventionally known medium as long as it contains a carbon source that can be assimilated by the microorganism. For example, a commercially available liquid medium such as a YM medium can be used. Moreover, a carbon source, a nitrogen source, inorganic salts, other necessary nutrient sources, and the like that can be assimilated by the microorganism can be appropriately used as components of the medium.
Examples of the carbon source include sugars (glucose, arabinose, xylose, mannose, fructose, galactose, sucrose, maltose, lactose, sorbitol, mannitol, inosit, glycerin, soluble starch, waste molasses, invert sugar, etc.), fats and oils ( Soybean oil, rapeseed oil, safflower oil, rice oil, corn oil, palm oil, sunflower oil, cottonseed oil, olive oil, sesame oil, peanut oil, pearl barley oil, wheat germ oil, perilla oil, linseed oil, sesame oil, sacha inch oil, Walnut oil, kiwi seed oil, salvia seed oil, grape seed oil, macadamia nut oil, hazelnut oil, pumpkin seed oil, coconut oil, tea seed oil, borage oil, palm olein, palm stearin, palm oil, palm kernel oil, cocoa butter , Vegetable oils such as monkey fat, shea fat, algae oil; fish oil, lard, beef tallow Animal fats and oils such as butterfat; or transesterified oils, hydrogenated oils or fractionated oils thereof; capric acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid, margaric acid, stearic acid, nonadecane Acid, arachidic acid, behenic acid, lignoceric acid, lindelic acid, tuzuic acid, myristoleic acid, palmitoleic acid, petroceric acid, oleic acid, elaidic acid, vaccenic acid, erucic acid, sorbic acid, linoleic acid, linoelaidic acid, and fatty acids such as γ-linolenic acid, linolenic acid, and arachidonic acid, and esters thereof). Further, organic acids that can be assimilated such as acetic acid, alcohols such as ethanol, and the like may be used.
Examples of the nitrogen source include ammonia, inorganic / organic ammonium salts, urea, corn gluten meal, soybean flour, yeast extract, meat extract, fish extract, polypeptone, peptone, various amino acids, and soy bean meal.

培養液中の初発の炭素源の含有量は、微生物の増殖の点から、1〜30%(w/v)、更に5〜20%(w/v)が好ましい。また、窒素源の含有量は1〜10%(w/v)が好ましい。また、培養様式によって、培養液中の濃度が前記範囲となるように培養期間中に連続的又は断続的に添加してもよい。   The content of the initial carbon source in the culture solution is preferably 1 to 30% (w / v), more preferably 5 to 20% (w / v) from the viewpoint of the growth of microorganisms. Further, the content of the nitrogen source is preferably 1 to 10% (w / v). Moreover, you may add continuously or intermittently during a culture | cultivation period so that the density | concentration in a culture solution may become the said range by a culture | cultivation mode.

培養方法は、好気的条件下であればよく、通気攪拌培養、振盪培養等の一般的な方法を適用することができる。
培養温度は、使用する微生物の増殖に悪影響を与えない範囲であれば特に制限されないが、通常、20〜33℃が好ましく、28〜30℃がより好ましい。このとき培養液の初発pH(30℃)は2〜7が好ましく、3〜6がより好ましい。
The culture method may be an aerobic condition, and general methods such as aeration stirring culture and shaking culture can be applied.
The culture temperature is not particularly limited as long as it does not adversely affect the growth of microorganisms to be used, but it is usually preferably 20 to 33 ° C, more preferably 28 to 30 ° C. At this time, the initial pH (30 ° C.) of the culture solution is preferably 2 to 7, and more preferably 3 to 6.

培養液のpHを調整する緩衝剤としては、例えば、炭酸、酢酸、クエン酸、フマル酸、リンゴ酸、乳酸、グルコン酸、酒石酸等の有機酸塩、リン酸、塩酸、硫酸等の無機塩、水酸化ナトリウム等の水酸化物、アンモニア又はアンモニア水等が挙げられ、これらを単独又は2種以上組み合わせて用いることができる。   Examples of the buffer for adjusting the pH of the culture solution include organic acid salts such as carbonic acid, acetic acid, citric acid, fumaric acid, malic acid, lactic acid, gluconic acid and tartaric acid, inorganic salts such as phosphoric acid, hydrochloric acid and sulfuric acid, Examples thereof include hydroxides such as sodium hydroxide, ammonia or aqueous ammonia, and these can be used alone or in combination of two or more.

ソフォロリピッドを生産する能力を有する微生物を用いてソフォロリピッドを生産する場合、一般的には、先ず前培養を行って菌体を活性化させ、次いで、これを本培養の培養液に接種して培養を行い、ソフォロリピッドを生産することが好ましい。前培養及び本培養の期間は、適宜設定できるがそれぞれ10〜100時間が好ましく、20〜60時間がより好ましい。
本発明においては、ソフォロリピッドを生産する際、培養液中のソフォロリピッドを生産する能力を有する微生物の濃度は、波長660nmにおける吸光度として100〜300である。該濃度(OD660)は、ソフォロリピッドの生産性、液物性の点から、120以上が好ましく、また、200以下、更に160以下が好ましい。また、該濃度(OD660)は、100〜200がより好ましく、さらに好ましくは120〜160である。
When producing sophorolipids using microorganisms capable of producing sophorolipids, in general, preculture is first performed to activate the cells, and then this is inoculated into the culture medium of the main culture. Then, it is preferable to produce sophorolipid by culturing. Although the period of pre-culture and main culture can be set as appropriate, it is preferably 10 to 100 hours and more preferably 20 to 60 hours.
In the present invention, when producing sophorolipid, the concentration of the microorganism having the ability to produce sophorolipid in the culture solution is 100 to 300 as absorbance at a wavelength of 660 nm. The concentration (OD660) is preferably 120 or more, more preferably 200 or less, and even more preferably 160 or less, from the viewpoint of productivity of the sophorolipid and liquid physical properties. The concentration (OD660) is more preferably 100 to 200, and still more preferably 120 to 160.

また、ソフォロリピッドを生産する際、培養液に供給される酸素の供給速度は0.1〜0.5mol/L/hrである。
酸素供給速度は、培養系において空気中の酸素が液相の微生物に移動する速度として次式で示される。
In addition, when producing sophorolipid, the supply rate of oxygen supplied to the culture solution is 0.1 to 0.5 mol / L / hr.
The oxygen supply rate is expressed by the following equation as the rate at which oxygen in the air moves to the liquid phase microorganism in the culture system.

Figure 0006289053
Figure 0006289053

dC/dt:酸素供給速度(mol/L/hr)
*:培養液中の飽和溶存酸素濃度(mol/L)
C:培養液中の溶存酸素濃度(mol/L)
kLa:酸素物質移動容量係数(hr-1
酸素供給速度の算出方法の詳細は実施例に記載した。
斯かる酸素供給速度は、ソフォロリピッドの生産性の点から、0.1mol/L/hr以上であるが、更に0.15mol/L/hr以上であるのが好ましく、また、ソフォロリピッドの変換効率の点から、0.5mol/L/hr以下であり、更に0.3mol/L/hr以下であるのが好ましい。また、酸素供給速度は、0.1〜0.3mol/L/hr、更に0.15〜0.3mol/L/hrであるのが好ましい。
dC / dt: oxygen supply rate (mol / L / hr)
C * : Saturated dissolved oxygen concentration (mol / L) in the culture solution
C: dissolved oxygen concentration (mol / L) in the culture solution
kLa: oxygen mass transfer capacity coefficient (hr −1 )
Details of the method for calculating the oxygen supply rate are described in the Examples.
Such an oxygen supply rate is 0.1 mol / L / hr or more from the viewpoint of productivity of the sophorolipid, but is more preferably 0.15 mol / L / hr or more. From the viewpoint of conversion efficiency, it is preferably 0.5 mol / L / hr or less, and more preferably 0.3 mol / L / hr or less. The oxygen supply rate is preferably 0.1 to 0.3 mol / L / hr, more preferably 0.15 to 0.3 mol / L / hr.

酸素供給速度は、通気酸素濃度、通気速度(通気量)、撹拌回転数、圧力等によって調整することができる。
本発明において、通気酸素濃度は、供給される空気中の酸素濃度であり、酸素供給速度の点から、21%(体積比率、以下同じ)より高い濃度とするのが好ましく、更に30%以上、より好ましくは40%以上とするのが好ましい。上限としてはソフォロリピッドの変換効率の点から、90%、より好ましくは85%とするのが好ましい。なお、通気酸素濃度は培養挙動に合わせて変化させることが好ましい。
また、通気速度(通気量)は、泡量の点から0.2〜1vvmが好ましく、撹拌回転数は、撹拌翼径が0.5m以下では200〜1000r/minが好ましく、撹拌翼径が0.5m以上では50〜200r/minが好ましい。圧力は常圧から微加圧の条件が好ましく、加圧条件としては0〜0.1MPaの範囲が好ましい。
The oxygen supply rate can be adjusted by the aeration oxygen concentration, the aeration rate (aeration amount), the number of stirring revolutions, the pressure, and the like.
In the present invention, the aeration oxygen concentration is the oxygen concentration in the supplied air, and is preferably higher than 21% (volume ratio, the same shall apply hereinafter) from the viewpoint of the oxygen supply rate, and more preferably 30% or more, More preferably, it is 40% or more. The upper limit is preferably 90%, more preferably 85%, from the viewpoint of sophorolipid conversion efficiency. The aeration oxygen concentration is preferably changed according to the culture behavior.
The aeration rate (aeration rate) is preferably 0.2 to 1 vvm from the viewpoint of the amount of foam, and the stirring rotation speed is preferably 200 to 1000 r / min when the stirring blade diameter is 0.5 m or less, and the stirring blade diameter is 0. When it is 0.5 m or more, 50 to 200 r / min is preferable. The pressure is preferably from normal pressure to slightly pressurized, and the pressurized condition is preferably in the range of 0 to 0.1 MPa.

このような酸素の供給により、培養液中の溶存酸素濃度は制御される。
ソフォロリピッドを生産する際、培養液(30℃)中の溶存酸素濃度は、0.5ppm以上、更に1ppm以上であるのが好ましく、より好ましくは2ppm以上である。また、ソフォロリピッドの変換効率の点から、10ppm以下、更に7ppm以下、より好ましくは3.5ppm以下であるのが好ましい。また、溶存酸素濃度は、0.5〜10ppm、更に1〜7ppmが好ましく、より好ましくは2〜3.5ppmである。
By supplying such oxygen, the dissolved oxygen concentration in the culture solution is controlled.
When producing sophorolipid, the dissolved oxygen concentration in the culture solution (30 ° C.) is preferably 0.5 ppm or more, more preferably 1 ppm or more, and more preferably 2 ppm or more. Further, from the viewpoint of the conversion efficiency of sophorolipid, it is preferably 10 ppm or less, more preferably 7 ppm or less, more preferably 3.5 ppm or less. The dissolved oxygen concentration is preferably 0.5 to 10 ppm, more preferably 1 to 7 ppm, and more preferably 2 to 3.5 ppm.

このような培養により、培養液中にソフォロリピッドが蓄積するので、培養終了後、適当な分離・精製手段により培養液からソフォロリピッドを採取することができる。
例えば、酢酸エチル等を用いた溶剤抽出後、分別沈殿、液液分配、カラムクロマトグラフ及び高速液体クロマトグラフ等を単独或いは組み合わせて用いることによりソフォロリピッドを取得することができる。
As a result of such culture, sophorolipid accumulates in the culture solution, and therefore, after completion of the culture, the sophorolipid can be collected from the culture solution by an appropriate separation / purification means.
For example, sophorolipid can be obtained by solvent extraction using ethyl acetate or the like and then using fractional precipitation, liquid-liquid distribution, column chromatograph, high performance liquid chromatograph or the like alone or in combination.

本発明によれば、0.004〜0.01mol/L/hr、好ましくは0.005〜0.009、より好ましくは0.006〜0.008の変換速度で基質からソフォロリピッドを製造することができる。なお、変換速度とは、培養液1Lに含まれるソフォロリピッド生産量を発酵1時間あたりの量で示した値である。
ソフォロリピッド生産量の測定方法は実施例に記載した。
According to the present invention, sophorolipid is produced from a substrate at a conversion rate of 0.004 to 0.01 mol / L / hr, preferably 0.005 to 0.009, more preferably 0.006 to 0.008. be able to. The conversion rate is a value indicating the amount of sophorolipid produced in 1 L of the culture broth as an amount per hour of fermentation.
The method for measuring sophorolipid production was described in the examples.

本発明により得られるソフォロリピッドは、界面活性剤、化粧品基材、各種中間体の原料等としての利用が期待される。   The sophorolipid obtained by the present invention is expected to be used as a surfactant, a cosmetic base, a raw material for various intermediates, and the like.

上述した実施形態に関し、本発明はさらに以下の製造方法を開示する。   In relation to the above-described embodiment, the present invention further discloses the following manufacturing method.

<1>ソフォロリピッドを生産する能力を有する微生物を好気的条件下、炭素源を含有する培養液中で培養し、ソフォロリピッドを得る工程を含むソフォロリピッドの製造方法であって、
前記培養液中のソフォロリピッドを生産する能力を有する微生物の濃度が波長660nmにおける吸光度として100〜300であり、且つ培養液に供給される酸素の供給速度が0.1〜0.5mol/L/hrである、製造方法。
<1> A method for producing a sophorolipid comprising a step of culturing a microorganism having an ability to produce a sophorolipid in a culture solution containing a carbon source under an aerobic condition to obtain the sophorolipid,
The concentration of microorganisms capable of producing sophorolipid in the culture solution is 100 to 300 as absorbance at a wavelength of 660 nm, and the supply rate of oxygen supplied to the culture solution is 0.1 to 0.5 mol / L. / Hr, the production method.

<2>ソフォロリピッドを生産する能力を有する微生物が、好ましくはカンジダ属(Candida)に属する微生物であり、より好ましくはCandida bombicolaCandida bogoriensisCandida magnoliaeCandida gropengiesseri、又はCandida apicolaであり、更に好ましくはCandida bombicolaである<1>に記載のソフォロリピッドの製造方法。
<3>炭素源が、好ましくは糖と油脂類、より好ましくはグルコースと脂肪酸又はその塩である<1>又は<2>に記載のソフォロリピッドの製造方法。
<4>培養液中のソフォロリピッドを生産する能力を有する微生物の濃度が波長660nmにおける吸光度として、好ましくは120以上であり、また、好ましくは200以下、より好ましくは160以下であり、また、好ましくは100〜200、より好ましくは120〜160である<1>〜<3>のいずれか1に記載のソフォロリピッドの製造方法。
<5>培養液に供給される酸素の供給速度が、好ましくは0.15mol/L/hr以上であり、また、好ましくは0.3mol/L/hr以下であり、また、好ましくは0.1〜0.3mol/L/hr、より好ましくは0.15〜0.3mol/L/hrである<1>〜<4>のいずれか1に記載のソフォロリピッドの製造方法。
<6>培養液に酸素濃度が好ましくは21%超、より好ましくは30%以上、更に好ましくは40%以上であり、また、好ましくは90%以下、より好ましくは85%以下であり、また、好ましくは20%超90%以下、より好ましくは30%以上90%以下、更に好ましくは40%以上85%以下である空気を通気させる<1>〜<5>のいずれか1に記載のソフォロリピッドの製造方法。
<7>培養時の通気速度(通気量)が好ましくは0.2〜1vvmであり、撹拌回転数が、撹拌翼径0.5m以下では好ましくは200〜1000r/min、撹拌翼径0.5m以上では好ましくは50〜200r/minであり、加圧条件が好ましくは0〜0.1MPaである<1>〜<6>のいずれか1に記載のソフォロリピッドの製造方法。
<8>培養時の温度が、好ましくは20〜33℃、より好ましくは28〜30℃であり、培養液の初発pH(30℃)が、好ましくは2〜7、より好ましくは3〜6である<1>〜<7>のいずれか1に記載のソフォロリピッドの製造方法。
<9>培養液中の30℃における溶存酸素濃度が、好ましくは0.5ppm以上、より好ましくは1ppm以上、更に好ましくは2ppm以上であり、また、好ましくは10ppm以下、より好ましくは7ppm以下、更に好ましくは3.5ppm以下であり、また、好ましくは0.5〜10ppm、より好ましくは1〜7ppm、更に好ましくは2〜3.5ppmである<1>〜<8>のいずれか1に記載のソフォロリピッドの製造方法。
<10>ソフォロリピッドを、好ましくは0.004〜0.01mol/L/hr、より好ましくは0.005〜0.009、更に好ましくは0.006〜0.008の変換速度で得る<1>〜<9>のいずれか1に記載のソフォロリピッドの製造方法。
<2> The microorganism having the ability to produce sophorolipid is preferably a microorganism belonging to the genus Candida , more preferably Candida bombicola , Candida bogoriensis , Candida majoriand , Candida group , Candida group , Candida group , or Candida group The method for producing a sophorolipid according to <1>, which is preferably Candida bombicola .
<3> The method for producing a sophorolipid according to <1> or <2>, wherein the carbon source is preferably sugar and fats and oils, more preferably glucose and fatty acid or a salt thereof.
<4> The concentration of the microorganism having the ability to produce sophorolipid in the culture solution is preferably 120 or more, preferably 200 or less, more preferably 160 or less, as the absorbance at a wavelength of 660 nm. The method for producing a sophorolipid according to any one of <1> to <3>, which is preferably 100 to 200, more preferably 120 to 160.
<5> The supply rate of oxygen supplied to the culture solution is preferably 0.15 mol / L / hr or more, preferably 0.3 mol / L / hr or less, and preferably 0.1 The manufacturing method of the sophorolipid as described in any one of <1>-<4> which is -0.3 mol / L / hr, More preferably, it is 0.15-0.3 mol / L / hr.
<6> The oxygen concentration in the culture medium is preferably more than 21%, more preferably 30% or more, still more preferably 40% or more, preferably 90% or less, more preferably 85% or less, The sophoro according to any one of <1> to <5>, wherein the air is preferably more than 20% and 90% or less, more preferably 30% or more and 90% or less, and still more preferably 40% or more and 85% or less. Manufacturing method of lipid.
<7> The aeration rate (aeration amount) during the culture is preferably 0.2 to 1 vvm, and the stirring rotation speed is preferably 200 to 1000 r / min when the stirring blade diameter is 0.5 m or less, and the stirring blade diameter is 0.5 m. The method for producing a sophorolipid according to any one of <1> to <6>, wherein the pressure is preferably 50 to 200 r / min and the pressurizing condition is preferably 0 to 0.1 MPa.
<8> The temperature during culture is preferably 20 to 33 ° C., more preferably 28 to 30 ° C., and the initial pH (30 ° C.) of the culture solution is preferably 2 to 7, more preferably 3 to 6. The method for producing a sophorolipid according to any one of <1> to <7>.
<9> The dissolved oxygen concentration in the culture medium at 30 ° C. is preferably 0.5 ppm or more, more preferably 1 ppm or more, still more preferably 2 ppm or more, preferably 10 ppm or less, more preferably 7 ppm or less, Preferably it is 3.5 ppm or less, Preferably it is 0.5-10 ppm, More preferably, it is 1-7 ppm, More preferably, it is 2-3.5 ppm, Any 1 of <1>-<8> A method for producing sophorolipid.
<10> A sophorolipid is obtained at a conversion rate of preferably 0.004 to 0.01 mol / L / hr, more preferably 0.005 to 0.009, still more preferably 0.006 to 0.008 <1 The manufacturing method of the sophorolipid as described in any one of>-<9>.

以下の実施例及び比較例において、酸素濃度の「%」は空気中の体積比率を示し、それ以外の「%」は「%(w/v))」を意味する。   In the following examples and comparative examples, “%” of the oxygen concentration indicates a volume ratio in the air, and other “%” means “% (w / v))”.

〔菌体濃度の測定〕
培養液を200倍に希釈し1cm角の石英ガラス製セルに注入し、分光光度計UV―1800(島津製作所製)を用いて、660nmの吸光度を測定し、希釈率からOD660を算出した。
[Measurement of bacterial cell concentration]
The culture broth was diluted 200 times, poured into a 1 cm square quartz glass cell, the absorbance at 660 nm was measured using a spectrophotometer UV-1800 (manufactured by Shimadzu Corporation), and OD660 was calculated from the dilution rate.

〔酸素供給速度の算出〕
酸素供給速度は以下の式により算出した。
[Calculation of oxygen supply rate]
The oxygen supply rate was calculated by the following formula.

Figure 0006289053
Figure 0006289053

(式中、C*は培養液中の飽和溶存酸素濃度、Cは培養液中の溶存酸素濃度である。kLaは酸素物質移動容量係数である。)
培養液中の飽和溶存酸素濃度と溶存酸素濃度は溶存酸素濃度センサー(エイブル製)を用いて測定を行った。
酸素物質移動容量係数(kLa)は、ガッシングアウト法により算出した。具体的には、培養液に窒素を通気し溶存酸素濃度を0ppmにした後に、窒素に換えて空気を通気し、その溶存酸素濃度変化からkLaの算出を行った。
(In the formula, C * is the saturated dissolved oxygen concentration in the culture solution, C is the dissolved oxygen concentration in the culture solution, and kLa is the oxygen mass transfer capacity coefficient.)
The saturated dissolved oxygen concentration and dissolved oxygen concentration in the culture solution were measured using a dissolved oxygen concentration sensor (manufactured by Able).
The oxygen mass transfer capacity coefficient (kLa) was calculated by the gassing-out method. Specifically, nitrogen was aerated through the culture solution to make the dissolved oxygen concentration 0 ppm, then air was aerated instead of nitrogen, and kLa was calculated from the dissolved oxygen concentration change.

〔ソフォロリピッドの分析〕
培養液2mLをサンプリングし、ヘキサン1mLで2回洗浄した後、酢酸エチル1mLで2回抽出し回収を行った。その後、溶媒を留去し、酢酸エチル画分の回収を行った。回収した酢酸エチル画分0.01gに対して、内部標準としてドデカンとメタノール1mL、濃塩酸0.03mLを添加し、100℃温浴バスにて2時間メタノール交換を行った。冷却後、飽和食塩水1mL、ヘキサン1.5mLを添加し混合を行い、静置後にヘキサン相を回収した。得られたヘキサン相の溶媒を留去し、シリル化剤TMSI−Hを0.2mL添加し70℃で20分シリル化を行った後、水1.5mL、ヘキサン1.5mLを添加し混合を行い、静置後にヘキサン相を回収し、GC分析に供した。
[Analysis of Sophorolipid]
2 mL of the culture solution was sampled, washed twice with 1 mL of hexane, and then extracted twice with 1 mL of ethyl acetate for recovery. Thereafter, the solvent was distilled off, and the ethyl acetate fraction was collected. To 0.01 g of the collected ethyl acetate fraction, dodecane, 1 mL of methanol, and 0.03 mL of concentrated hydrochloric acid were added as internal standards, and methanol was exchanged in a 100 ° C. warm bath for 2 hours. After cooling, 1 mL of saturated saline and 1.5 mL of hexane were added and mixed, and the hexane phase was recovered after standing. The solvent of the obtained hexane phase was distilled off, 0.2 mL of silylating agent TMSI-H was added and silylation was performed at 70 ° C. for 20 minutes, and then 1.5 mL of water and 1.5 mL of hexane were added and mixed. After standing, the hexane phase was recovered and subjected to GC analysis.

〔GC分析条件〕
カラム:DB−1−HT/14m×250μm×0.1μm
カラム条件:流量0.7ml/分 60℃1分→10℃/分→300℃10分
インジェクション:スプリット50:1/280℃
検出:FID 280℃ H2:30ml/分、Air:400ml/分
[GC analysis conditions]
Column: DB-1-HT / 14 m × 250 μm × 0.1 μm
Column conditions: Flow rate 0.7 ml / min 60 ° C. 1 min → 10 ° C./min→300° C. 10 min Injection: Split 50: 1/280 ° C.
Detection: FID 280 ° C. H2: 30 ml / min, Air: 400 ml / min

〔培養方法〕
1.プレート培養
Candida bombicola NBRC10243株を用いた。
グルコース1%、酵母エキス1%、トリプトン1%、寒天1.5%を含む寒天培地のシャーレに種菌を1白金耳植菌し、温度30℃で2日間培養を行った。
[Culture method]
1. Plate culture
Candida bombicola NBRC10243 strain was used.
One platinum ear inoculum was inoculated in a petri dish of an agar medium containing 1% glucose, 1% yeast extract, 1% tryptone and 1.5% agar, and cultured at 30 ° C. for 2 days.

2.前培養
グルコース1%、酵母エキス1%、トリプトン1%を含む培養液100mLを坂口フラスコに入れ、121℃、20分高温で滅菌を行った。冷却後、プレートから1白金耳、植菌を行い、温度30℃、撹拌回転数120r/minの条件にて2日間撹拌培養を行った。
2. Preculture 100 mL of a culture solution containing 1% glucose, 1% yeast extract, and 1% tryptone was placed in a Sakaguchi flask and sterilized at 121 ° C. for 20 minutes at a high temperature. After cooling, 1 platinum loop and inoculation were performed from the plate, and stirring culture was performed for 2 days under the conditions of a temperature of 30 ° C. and a stirring rotation speed of 120 r / min.

3.本培養
パルミチン酸エチル10%、グルコース10%、酵母エキス8%、尿素1.2%を含む培養液をpH5.0に調整し、1.2Lにメスアップした。培養液を全容2Lのジャーファーメンターにて滅菌後に、前培養液24mL植菌し、温度30℃、撹拌回転数(撹拌翼径0.5m以下)600r/min、通気速度(通気量)0.6L/分(0.5vvm)の条件にて撹拌培養を行った。
3. Main culture A culture solution containing 10% ethyl palmitate, 10% glucose, 8% yeast extract, and 1.2% urea was adjusted to pH 5.0 and made up to 1.2 L. The culture solution was sterilized with a 2 L jar fermenter, inoculated with 24 mL of the preculture solution, temperature 30 ° C., stirring speed (stirring blade diameter 0.5 m or less) 600 r / min, aeration rate (aeration rate) 0. Stirring culture was performed under the condition of 6 L / min (0.5 vvm).

実施例1
培養開始から24時間後、通気酸素濃度を21〜85%に高め、更に36時間撹拌培養を行った。この時の菌体濃度(OD660nm)は150、培養液中の溶存酸素濃度は3ppm、酸素供給速度は0.172mol/L/hrであった。
培養後、GC分析よりソフォロリピッド変換速度を求めたところ0.0070mol/L/hrであった。
Example 1
After 24 hours from the start of the culture, the aeration oxygen concentration was increased to 21 to 85%, and the stirring culture was further performed for 36 hours. At this time, the bacterial cell concentration (OD 660 nm) was 150, the dissolved oxygen concentration in the culture solution was 3 ppm, and the oxygen supply rate was 0.172 mol / L / hr.
After culture, the sophorolipid conversion rate was determined by GC analysis and found to be 0.0070 mol / L / hr.

実施例2
通気酸素濃度を40%に高めた以外は実施例1と同様に培養を行った。菌体濃度(OD660nm)は154、培養液中の溶存酸素濃度は4ppm、酸素供給速度は0.213mol/L/hrであった。
培養後、GC分析よりソフォロリピッド変換速度を求めたところ0.0070mol/L/hrであった。
Example 2
Culture was performed in the same manner as in Example 1 except that the aeration oxygen concentration was increased to 40%. The bacterial cell concentration (OD660 nm) was 154, the dissolved oxygen concentration in the culture solution was 4 ppm, and the oxygen supply rate was 0.213 mol / L / hr.
After culture, the sophorolipid conversion rate was determined by GC analysis and found to be 0.0070 mol / L / hr.

実施例3
通気酸素濃度を50%に高めた以外は実施例1と同様に培養を行った。菌体濃度(OD660nm)は153、培養液中の溶存酸素濃度は9ppm、酸素供給速度は0.266mol/L/hrであった。
培養後、GC分析よりソフォロリピッド変換速度を求めたところ0.0066mol/L/hrであった。
Example 3
Culturing was performed in the same manner as in Example 1 except that the aeration oxygen concentration was increased to 50%. The bacterial cell concentration (OD660 nm) was 153, the dissolved oxygen concentration in the culture solution was 9 ppm, and the oxygen supply rate was 0.266 mol / L / hr.
After culturing, the sophorolipid conversion rate was determined by GC analysis and found to be 0.0066 mol / L / hr.

比較例1
本培養の培養液の組成を酵母エキス8%から酵母エキス2%に変更し、全培養期間を通じて通気酸素濃度を21%のままとした以外は実施例1と同様に培養を行った。
培養開始から48時間後の菌体濃度(OD660nm)は30、培養液中の溶存酸素濃度は4ppm、酸素供給速度は0.043mol/L/hrであった。
培養後、GC分析よりソフォロリピッド変換速度を求めたところ0.0016mol/L/hrであった。
Comparative Example 1
The culture was performed in the same manner as in Example 1 except that the composition of the culture solution of the main culture was changed from 8% yeast extract to 2% yeast extract and the aeration oxygen concentration was kept at 21% throughout the entire culture period.
The bacterial cell concentration (OD660 nm) 48 hours after the start of the culture was 30, the dissolved oxygen concentration in the culture solution was 4 ppm, and the oxygen supply rate was 0.043 mol / L / hr.
After culturing, the sophorolipid conversion rate was determined by GC analysis and found to be 0.0016 mol / L / hr.

比較例2
本培養の培養液の組成を酵母エキス8%から酵母エキス4%に変更し、全培養期間を通じて通気酸素濃度を21%のままとした以外は実施例1と同様に培養を行った。
培養開始から48時間後の菌体濃度(OD660nm)は100、培養液中の溶存酸素濃度は0ppm、酸素供給速度は0.043mol/L/hrであった。
培養後、GC分析よりソフォロリピッド変換速度を求めたところ0.0029mol/L/hrであった。
Comparative Example 2
The culture was performed in the same manner as in Example 1 except that the composition of the culture solution for main culture was changed from 8% yeast extract to 4% yeast extract and the aeration oxygen concentration was kept at 21% throughout the entire culture period.
The bacterial cell concentration (OD660 nm) 48 hours after the start of the culture was 100, the dissolved oxygen concentration in the culture solution was 0 ppm, and the oxygen supply rate was 0.043 mol / L / hr.
After culturing, the sophorolipid conversion rate was determined by GC analysis and found to be 0.0029 mol / L / hr.

比較例3
全培養期間を通じて通気酸素濃度を21%のままとした以外は実施例1と同様に培養を行った。培養開始から48時間後の菌体濃度(OD660nm)は120、培養液中の溶存酸素濃度は0ppm、酸素供給速度は0.043mol/L/hrであった。
培養後、GC分析よりソフォロリピッド変換速度を求めたところ0.0026mol/L/hrであった。
Comparative Example 3
The culture was performed in the same manner as in Example 1 except that the aeration oxygen concentration was kept at 21% throughout the entire culture period. The cell concentration (OD660 nm) after 48 hours from the start of the culture was 120, the dissolved oxygen concentration in the culture solution was 0 ppm, and the oxygen supply rate was 0.043 mol / L / hr.
After culturing, the sophorolipid conversion rate was determined by GC analysis and found to be 0.0026 mol / L / hr.

比較例4
本培養の撹拌回転数を600r/minから800r/minにした以外は実施例1と同様に培養を行った。
培養開始から24時間後、通気酸素濃度を95%に高め、更に36時間撹拌培養を行った。この時の菌体濃度(OD660nm)は145、培養液中の溶存酸素濃度は25ppm、酸素供給速度は0.597mol/L/hrであった。
培養後、GC分析よりソフォロリピッド変換速度を求めたところ0.0011mol/L/hrであった。
各実施例及び比較例の条件と結果を表1に示す。
Comparative Example 4
The culture was performed in the same manner as in Example 1 except that the stirring rotation speed of the main culture was changed from 600 r / min to 800 r / min.
After 24 hours from the start of the culture, the aeration oxygen concentration was increased to 95%, and the stirring culture was further performed for 36 hours. At this time, the bacterial cell concentration (OD660 nm) was 145, the dissolved oxygen concentration in the culture solution was 25 ppm, and the oxygen supply rate was 0.597 mol / L / hr.
After culturing, the sophorolipid conversion rate was determined by GC analysis and found to be 0.0011 mol / L / hr.
Table 1 shows the conditions and results of each Example and Comparative Example.

Figure 0006289053
Figure 0006289053

表1に示すように、比較例1では菌数が少なく、比較例2、3では酸素供給速度が小さく溶存酸素濃度が低くなり、比較例4では酸素供給速度が大きく溶存酸素濃度が高くなり、これらの条件ではソフォロリピッドを効率良く得ることができなかった。これに対し、実施例に示す様に、高菌体濃度下、酸素供給速度を特定の範囲に制御し、培養液中の溶存酸素濃度を制御する条件でのみ、基質からソフォロリピッドへの変換速度が速くなり、ソフォロリピッドを効率良く得ることを達成した。   As shown in Table 1, in Comparative Example 1, the number of bacteria is small, in Comparative Examples 2 and 3, the oxygen supply rate is small and the dissolved oxygen concentration is low, and in Comparative Example 4, the oxygen supply rate is large and the dissolved oxygen concentration is high. Under these conditions, sophorolipid could not be obtained efficiently. On the other hand, as shown in the examples, the substrate is converted to sophorolipid only under the condition that the oxygen supply rate is controlled to a specific range under a high bacterial cell concentration and the dissolved oxygen concentration in the culture solution is controlled. The speed was increased, and sophorolipid was obtained efficiently.

Claims (2)

ソフォロリピッドを生産する能力を有するカンジダ属に属する微生物を好気的条件下、炭素源として糖及び油脂類を含有する培養液中で培養し、ソフォロリピッドを得る工程を含むソフォロリピッドの製造方法であって、
前記培養液中のソフォロリピッドを生産する能力を有するカンジダ属に属する微生物の濃度が波長660nmにおける吸光度として100〜300であり、培養液に供給される酸素の供給速度が0.1〜0.5mol/L/hrであり、且つ培養液中の30℃における溶存酸素濃度が0.5〜10ppmである、製造方法。
A method comprising culturing a microorganism belonging to the genus Candida having the ability to produce sophorolipid under aerobic conditions in a culture solution containing sugar and fats and oils as a carbon source to obtain the sophorolipid. A manufacturing method comprising:
The concentration of the microorganism belonging to the genus Candida having an ability to produce source Foro lipid in the culture medium is 100 to 300 as absorbance at a wavelength of 660 nm, the feed rate of oxygen supplied to the culture solution is 0.1 to 0 .5mol / L / hr der is, and the dissolved oxygen concentration at 30 ° C. in culture is 0.5 to 10, the manufacturing method.
培養液に酸素濃度が21%超90%以下の空気を通気させる請求項1記載のソフォロリピッドの製造方法。   The method for producing sophorolipid according to claim 1, wherein air having an oxygen concentration of more than 21% and 90% or less is aerated in the culture solution.
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