JP4714377B2 - Purification method of sophorose lipid - Google Patents

Description

（ここで、各構造式において、Ｒ₁およびＲ₂はそれぞれ、独立して、水素またはアセチル基であり、ｎは、一般に、１１〜２０の整数であり、より通常には、１３〜１７、さらにより通常には１４〜１６である）。
【００１８】
ＳＬは、カンジダ属酵母を培養することにより生成され得る。培養生成されたＳＬは、複数分子種の混合物であって、通常、ラクトン型を５０％以上含む。従って、本明細書中では、ソホロースリピッド（ＳＬ）とは、単一の物質のみならず、複数分子種の混合物をも意味する。
【００１９】
培養に使用される酵母としては、カンジダ ボンビコラ（Ｃａｎｄｉｄａ ｂｏｍｂｉｃｏｌａ）、カンジダ アピコラ（Ｃ．ａｐｉｃｏｌａ）、カンジダ ペトロフィラム（Ｃ．ｐｅｔｒｏｐｈｉｌｕｍ）、およびカンジダ ボゴリエンシス（Ｃ．ｂｏｇｏｒｉｅｎｓｉｓ）が挙げられる。これらは、保存機関から分譲された菌株またはその継代培養であり得る。
【００２０】
ＳＬは、上記のような菌株を、適当な液体培地を用いて培養することにより、培養液中に蓄積される。
【００２１】
使用され得る液体培地の組成について、以下に説明する。
【００２２】
炭素源としては、糖類および油性基質が用いられ得る。糖類としては、グルコース、フルクトース、ガラクトースなどの単糖類、およびスクロース、マルトースなどの二糖類が挙げられる。好ましくは、グルコースが使用される。培養初発濃度は、３０〜１５０ｇ／Ｌ、好ましくは９０〜１２０ｇ／Ｌである。油性基質としては、培養によるＳＬ産生に使用され得ることが報告されているいずれの油性基質もが使用可能であり、このような油性基質として、植物性油脂（Ｚｈｏｕら、Ｊ．Ａｍ．Ｏｉｌ．Ｃｈｅｍ．Ｓｏｃ．、６９：８９−９１（１９９２）など）、動物性油脂（Ｄｅｓｈｐａｎｄｅら、Ｂｉｏｒｅｓｏｕｒｃｅ Ｔｅｃｈｎｏｌ．，５４：１４３−１５０（１９９５））、脂肪酸（Ａｓｍｅｒら、Ｊ．Ａｍ．Ｏｉｌ．Ｃｈｅｍ．Ｓｏｃ．，６５：１４６０−１４６６（１９８８）など）、脂肪酸エステル（Ｄａｖｉｌａら Ａｐｐｌ．Ｍｉｃｒｏｂｉｏｌ．Ｂｉｏｔｅｃｈｎｏｌ．３８：６−１１（１９９２））、ｎ−アルカン（Ｔｕｌｌｏｃｈら、Ｃａｎ．Ｊ．Ｃｈｅｍ．，４６：３３３７−３３５１（１９６８）などが報告されている。好ましくは、植物性油脂または脂肪酸、あるいは植物油脂を原料とする脂肪酸エステルが使用され得る。植物性油脂として、通常、食用植物油が使用され得る。使用される植物油としては、例えば、ダイズ油、ナタネ油、綿実油、ヒマワリ油、カポック油、ゴマ油、コーン油、コメ油、落花生油、ベニバナ油、オリープ油、アマニ油、キリ油、ヒマシ油、パーム油、パーム核油、ヤシ油など、またはこれらの混合物が挙げられる。ダイズ油、ナタネ油、ヒマワリ油、ベニバナ油、またはこれらの混合物が好ましい。このような脂肪酸の炭素数は、６〜２４、好ましくは、１２〜２０の範囲であり、さらにそれぞれの分子内に０〜３箇所の不飽和結合を含んでもよい。用いられる脂肪酸としては、カプロン酸、カプリル酸、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、ベへン酸、およびリグノセリン酸のような飽和脂肪酸、ならびにトウハク酸、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、リノール酸、γ−リノレン酸、およびリノレン酸のような不飽和脂肪酸、ならびにこれらの混合物が挙げられる。脂肪酸エステルとしては、前出の脂肪酸のエステルが挙げられる。培養の開始時に添加され得る油性基質の濃度は、５０〜２００ｇ／Ｌ、好ましくは１００〜１５０ｇ／Ｌの範囲である。連続的に供給される場合、上記濃度に相当する量の油性基質が、培養期間中に均等速度で培養系に添加される。
【００２３】
窒素源としては、２〜５ｇ／Ｌの酵母エキス、０．５〜２ｇ／Ｌの尿素などが添加され得るが、これらに限定されない。
【００２４】
さらに、酵母の生育に必要な各種有機物および無機塩類（リン酸塩、マグネシウム塩を含む）が適当量で添加され得る。
【００２５】
培養形態は、上記のような液体培地を用いたバッチ培養、または培養系に油性基質を連続添加する流加培養であり得、通気攪拌することが望ましい。培養ｐＨ条件は、開始時にｐＨ約３．５〜約６．０であり、好ましくは、約４．０〜約５．５である。通常、培養中、ｐＨは、外部から調節されない。培養温度は、上記菌株が、ソホロースリピッド産生して生育し得るのに適当な温度であり、通常、２０〜３５℃であり、好ましくは２８〜３０℃である。５Ｌ〜５ｋＬ容量の培養の場合、通気攪拌の速度は、０．１〜２．０ｖｖｍ、１００〜６００ｒｐｍであり得る。攪拌速度に関して、培養槽が大きくなるにつれて、同じ溶存酸素条件を作るために必要とされる攪拌速度は遅くなる。このような攪拌は、培養液中に十分な溶存酸素条件を提供するために行われ得、従って、酵母の培養に適切な溶存酸素条件を与えるような、いずれの攪拌速度も望ましい。
【００２６】
培地中の糖類と油性基質とは、培養過程で同時に利用され、時間の経過と共に濃度が低下する。糖類が先に完全消費される。ＳＬの生成は、培地中の油性基質が全て消費されると停止する。培地に残留している油性基質は、培養液を湯浴中で加熱後、遠心分離することで油層となって分離するため、ＳＬの生成の終点を、培養中の培養液を一部、分取して上記操作を行い、油層の消失を確認することによって、容易に知ることができる。従って、ＳＬの生成を、培地に残留している油性基質の量を測定することによりモニタリングすることにより、最大収量のＳＬを含有する培養液を滅菌処理して菌体を殺菌した後、本精製方法に供し得る。
【００２７】
また、精製したＳＬを製品への配合において利用するために、精製の過程における油性基質の残留はできるだけ少ないことが望ましい。従って、本精製方法に用いる培養液は、炭素源として添加した油性基質の残留が、ｎ−ヘキサン抽出物として、培養終了液の重量基準で０．５％以下であることが望ましい。炭素源として添加した油性基質の残留は、好ましくは０．２％以下、さらにより好ましくは０．１％以下である。残留する油性基質の測定は、以下の手順で行われ得る。培養液を密栓容器に取り、この培養液と実質的に等量のｎ−ヘキサンを添加して激しく振り混ぜた後、遠心分離（例えば、１，０００×ｇで１０分間）し、分離したヘキサン層を回収する。この工程は３回以上繰り返され得る。ヘキサン層を回収して、ヘキサン溶媒を揮発除去し、残留物の重量を測定する。
【００２８】
上記菌株を培養する場合、培養の進行と共に培養液が酸性化する傾向がある。従って、培養を終了する時点で、培養液は酸性化されている。この培地のｐＨの範囲は、５．５未満であり得、しばしば、４．０以下、より頻繁には、３．５以下であり、一方、しばしば下限が２．０、より頻繁には、２．５であり得る。培養終了時点での培養液のｐＨは、培養に使用した培地の組成、培養条件などに依存し得る。本精製方法においては、代表的には、出発培養液として、ｐＨが２．０〜３．５である培養液が使用され得る。従って、通常、含水グルコース８０〜１２０ｇ、酵母エキス１〜５ｇ、リン酸一カリウム５〜１５ｇ、硫酸マグネシウム７水和物５〜１５ｇ、塩化ナトリウム０．５〜２ｇ、ダイズ油８０〜１２０ｇ、尿素０．５〜２ｇ（量は全て培地１ｋｇ当たりの量である）の組成の培地を用いて、温度約３０℃、通気攪拌０．５〜１．０ｖｖｍ、２５０〜６００ｒｐｍ、初発ｐＨ４．０〜５．０、培養期間４〜１２日間で、上記菌株が培養され得る。
【００２９】
酸性化した培養液中において、生成したＳＬは分散状態で存在するが、攪拌を停止すれば、このＳＬは、粘稠性の含水物として速やかに沈殿する性質を持つ。すなわち、培養の進行に伴って酸性化した培養液中では、ＳＬは、難溶性または不溶性であり、静置状態では沈殿している。これは、酸型ＳＬのカルボキシル基が酸性下で非解離型になっていること、ならびにラクトン型ＳＬがもともと疎水的な分子構造を持っているためと考えられる。
【００３０】
本精製方法では、上述のような酸性化した培養液を、アルカリを添加することによって中和し、上述のような不溶化ＳＬを可溶化する。好ましくは、培養液を高温（例えば、８０〜９５℃）に加熱して殺菌して培養を終了し、室温まで冷却して静置（例えば、室温下で半日〜１日間）し、生じた上澄を取り除き、そして取り除いた上澄と実質的に等しい量の水（例えば、工業用水または井水）を添加し、これを精製のために用いる。中和の条件は、ｐＨ５．５以上、より好ましくはｐＨ６．０〜７．５の範囲である。アルカリは、中和のために添加されるものであればいずれでもよく、このようなアルカリとしては、水酸化ナトリウム、水酸化カリウム、炭酸ナトリウム、炭酸アンモニウム、リン酸ナトリウムなどが挙げられる。好ましくは、水酸化ナトリウムである。通常、４８％水酸化ナトリウムが使用され得る。上記アルカリを、培養液のｐＨが上記範囲になるようにモニタリングしながら添加する。好ましくは、上記アルカリを攪拌しながら添加する。ラクトン型ＳＬは中性下ではなお難水溶性であると考えられるが（Ｉｎｏｕｅら、Ａｇｒｉｃ．Ｂｉｏｌ．Ｃｈｅｍ．，４４：２２２１−２２２３（１９８０））、本工程によって、ラクトン型ＳＬを含むＳＬ成分全体が、中性下において可溶化あるいは安定した分散状態になる。
【００３１】
本精製方法では、次いで、上記工程により得られた、中和状態の溶液を固液分離し、溶液中の固形分を除去する。この固液分離のための技術としては、濾過または遠心分離が挙げられる。濾過は、そのままでも可能であるし、水を加えて粘度を下げることで、濾過速度をより速くすることもできる。本工程で使用され得る濾過は、加圧濾過（例えば、板枠型圧濾過器を用いる）、減圧濾過（例えば、回転円筒真空濾過器を用いる）が好ましい。ラボスケールでは、ブフナロートなどを用いる減圧濾過もまた可能である。濾過は、当該分野で一般に使用されている装置を用いて実施され得る。遠心分離は、当該分野で一般に使用されている装置を用いて実施され得る。例えば、連続遠心分離機による連続的な遠心分離が使用され得る。連続遠心分離機としては、かご型遠心分離機、ディスク型遠心分離機などが挙げられる。連続遠心分離の場合、連続的に液を流し込むことにより、分離に供され得る。例えば、約２ｋＬ／時で流し込まれ得る。バッチ型の遠心分離の場合、例えば、１０００×ｇで１５分間で実施され得る。上記のような濾過または遠心分離によって、固形分と、可溶化した状態のＳＬを含む溶液とを容易に分離することができる。この固形分には、菌体および、培養におけるＳＬの生成において生じた不純物が含まれ得る。
【００３２】
次いで、固形分を除去したＳＬ溶液を、酸を添加することにより酸性化し、ＳＬを再び不溶化する。添加する酸としては、無機酸（例えば、塩酸、フッ化水素酸、硫酸、硝酸、リン酸など）、および有機酸（例えば、酢酸、シュウ酸、酒石酸、コハク酸、マレイン酸、フマル酸、グルクロン酸、クエン酸、リンゴ酸、アスコルビン酸、安息香酸、タンニン酸、アルギン酸、ポリグルタミン酸、ナフタレンスルホン酸、ナフタレンジスルホン酸、ポリガラクツロン酸など）が挙げられる。なお、有機物と反応することのない酸が好ましい。塩酸および硫酸が好ましい。例えば、５％〜３６％の塩酸、２０〜９８％の硫酸が用いられ得る。酸性化の条件は、ｐＨ５．５以下、より好ましくはｐＨ４．０以下、より好ましくはｐＨ３．５以下、さらにより好ましくはｐＨ３．０以下であって、下限がｐＨ２．０である範囲である。通常、ｐＨ２．５〜４．０の範囲が使用され得る。上記酸を、培養液のｐＨが上記範囲になるようにモニタリングしながら添加する。好ましくは、上記酸を攪拌しながら添加する。
【００３３】
次いで、再不溶化したＳＬを沈降させ、上澄を取り除く。再不溶化したＳＬの沈降は、静置あるいは遠心分離により生じ得る。この静置は、通常、室温下で半日〜２日間、行われ得る。遠心分離は、数十〜数百×ｇで１０分から１時間で実施され得る。沈降物と上澄との分離は、デカンテーションなどで可能である。したがって、高濃度のＳＬを比較的容易に含水物として得ることができる。上記により得られたＳＬ生成物は、酸型ＳＬおよびラクトン型ＳＬの混合物に加えて水分を含む、含水物である。この水分は、ＳＬを高温または減圧下に置いたとき失われ得る。
【００３４】
上記ＳＬ含水物中に存在し得る、ＳＬ以外の不純物をさらに取り除くために、上記ＳＬ含水物に水を添加して攪拌し、次いで、例えば、静置または遠心分離を行うことによりＳＬを沈降させ、例えば、デカンテーションにより上澄を除去し、ＳＬを回収し得る。例えば、上記ＳＬ含水物に、例えば、蒸留水（または脱イオン水または工業用水もしくは井水）がＳＬ含水物と実質的に等しい量で添加され、５０〜２５０ｒｐｍで攪拌される。次いで、ＳＬを沈降させるために、例えば、室温で半日〜２日間、静置され得る。あるいは、ＳＬを沈降させるために、例えば、室温で５００×ｇで約３０分の遠心分離に供され得る。上澄の除去のためには、デカンテーションを行い得る。上記の手順は、必要に応じて、繰り返され得る。これにより、このＳＬ含水物に含まれ得る、培地中の、あるいは精製過程で添加したアルカリ、酸などに由来する少量の混入物（例えば、水溶性（無機）塩類、低分子および高分子の水溶性有機物など）が、ＳＬをほとんど損失することなく除去され得る。
【００３５】
本発明の精製方法により得られたＳＬ含水物は、そのまま原料として、製品に配合され得る。ＳＬ含水物はまた、種々の誘導体の出発物質として利用され得る。ＳＬは、ラクトン型と酸型に分離することなく混合物の形態で十分に利用され得る。ソホロースリピッドは、小麦製品の品質改良において利用されることが公知である（特開昭６１−２０５４４９号公報）。ソホロースリピッド誘導体は、化粧品の湿潤剤（油化学、３６、７４８−７５３（１９８７））、およびゲル化剤（特公平７−１７６６８号公報）として利用されている。
【００３６】
本発明の精製方法で得られたＳＬ含水物のメタノール溶液、および中和処理前のＳＬ含水物をメタノールに溶解した後フィルター濾過で不溶物を除いたものを、ＨＰＬＣで分析すると、基本的に同一のクロマトグラムが得られる。これは、本精製方法における工程において、酸型ＳＬだけでなく、本来難水溶性のラクトン型ＳＬもまた、可溶化および再不溶化沈殿で回収され、特定のＳＬ成分が大量に分解および損失することはないことを示している。
【００３７】
本発明の精製方法で使用される化学薬品は、基本的にアルカリおよび酸のみであり、必要な設備は、処理槽と、固液分離に必要な装置（例えば、連続遠心分離機あるいは菌体除去に使われ得る一般的な濾過装置）程度である。また、本発明の精製方法は、有機溶媒を使用しない。
【００３８】
従って、本発明により、培養液からのＳＬ分離精製に必要なコストを低減するだけでなく、環境および作業者への負荷を軽減することで、ＳＬは、より安価にかつより安全に市場に提供され得る。
【００３９】
以下、本発明を実施例により説明するが、本発明はこれに限定されない。本実施例で使用した材料、試薬、装置などは、他に特定のない限り、商業的な供給源から入手可能である。使用され得る試薬は、工業的に利用可能な等級の試薬である。
【００４０】
【実施例】
（実施例１）
２Ｌ容量の三角フラスコに２０ｇ／Ｌ含水グルコース、１０ｇ／Ｌポリペプトンおよび５ｇ／Ｌ酵母エキスからなる液体培地８００ｍｌ（ｐＨ４．６）を調製し、通気性のある蓋をして蒸気滅菌した。ここにカンジダ ボンビコラ ＡＴＣＣ２２２１４株１白金耳を植菌し、スターラーで攪拌しながら３０℃にて５０時間培養した。これを前培養液とする。３０Ｌ容量の培養槽（ファーメンテーター）に、１００ｇ／Ｌ含水グルコース、１００ｇ／Ｌダイズ油、２．５ｇ／Ｌ酵母エキス、１０ｇ／Ｌリン酸一カリウム、１０．２５ｇ／Ｌ硫酸マグネシウム７水和物、１ｇ／Ｌ塩化ナトリウムおよび１ｇ／Ｌ尿素からなる液体培地１８ｋｇ（ほぼ１８Ｌ）を調製し、塩酸を使用してｐＨを４．５に調整してから蒸気滅菌した。ただし不溶物の生成を避けるため尿素は別溶液として蒸気滅菌し、冷却後に混合した。混合後の培地のｐＨは４．６であった。１時間以上通気攪拌を行ってから、この培地に、前述の前培養液８００ｍｌを添加した。これを、３０℃で１０日間、通気攪拌培養（１．０ｖｖｍ、２５０ｒｐｍ）した。
【００４１】
ここで、培地由来の残留油脂量を調べた。培養液を密栓容器に取り、この培養液と等量のｎ−ヘキサンを添加して激しく振り混ぜた後、１，０００×ｇで１０分間遠心分離し、分離したヘキサン層を回収した。この工程を３回繰り返した。ヘキサン層を回収して、ヘキサン溶媒を６０〜８０℃程度の湯浴上にて揮発除去し、残留物の重量を測定した。培地由来の残留油脂は、ｎ−ヘキサン抽出物として、上記培養液の重量の約０．１％であった。
【００４２】
次いで、培養液を９０℃で１５分処理し、培養終了液を得た。この培養終了液は、ｐＨ２．７であった。この培養終了液は、室温で半日〜１日間の静置により、下から順に、液状の褐色沈殿物層、主に菌体と思われる乳白色の固形物層、上澄の３層に分離した。よく混合均一化した培養終了液を一部分取し、分取した培養終了液と等容量のｎ−ヘキサンでこの培養終了液を洗浄した後、２倍容量の酢酸エチルで３回抽出し、溶媒および水分を加熱減圧下で除去した。この結果、この培養終了液は、１４％のＳＬを含有すると見積もられた。
【００４３】
培養終了液１５ｋｇを室温で半日〜１日間静置し、生じた上澄を除去した後、工業用水または井水を、除去した上澄に等しい量で添加した。得られた培養終了液に攪拌しながら４８重量％の水酸化ナトリウム溶液を徐々に加え、ｐＨ６．５とし、ＳＬを可溶化した。連続遠心分離機（ウェストファリア（ウェストファリアセパレーターＡＧ製））を用いて２ｋＬ／時で処理することにより、この乳白色の固形物を沈殿させ、上澄を回収した。回収した上澄に攪拌しながら７８重量％の硫酸を徐々に加え、ｐＨ３．０とし、ＳＬを再不溶化した。室温で２日間静置後、デカンテーションにより上澄を可能な限り除去し、ＳＬ含水物３．９７ｋｇを得た。
【００４４】
このＳＬ含水物は、室温で１日以上静置すると、濁りのない液状の褐色沈殿物と若干量の上澄とに分離し、これら二層の界面において固形物はほとんど認められなかった。よく混合均一化したものの一部分取を分析した結果、得られたＳＬ含水物は、１０５℃乾燥による重量の減少から５３％の水分を含むと見積もられた。
【００４５】
次いで、上述の手順に従って、培地由来の残留油脂量を調べた。培地由来の残留油脂は、ｎ−ヘキサン抽出物として、ＳＬ含水物の乾燥重量に対し０．５％含んでいた。
【００４６】
次いで、水溶性塩類の混入を調べた。ビーカーおよび濾紙の重量を正確に測定しておいた。濾紙は１０５℃で１時間以上乾燥した後、デシケーター内で放冷したものを使用した。ビーカーにＳＬ含水物約１０ｇを正確に量り取り、エタノールを試料水分の２０倍重量以上で添加して湯浴上で加熱溶解した。この溶液を上述の濾紙を用いて濾過した。ビーカー内の不溶物を、熱エタノールを用いて２〜３回洗い出すことにより、完全に濾紙上に移した。濾紙を１０５℃の乾燥機で１時間乾燥し、デシケーターで放冷後、重量を正確に測定した。次式から、エタノール不溶分を算出した：エタノール不溶分（重量％）＝濾紙上残留物重量（ｇ）／［ＳＬ含水物重量（ｇ）×（１００−水分量（％））］×１０，０００。このＳＬ含水物における水溶性塩類の混入は、エタノール不溶分として２．３％であった。
【００４７】
また、このＳＬ含水物は、上述した方法（ｎ−ヘキサン洗浄−酢酸エチル抽出−溶媒・水分除去）を用いて、４８％のＳＬを含有すると見積もられた。
【００４８】
（実施例２）
実施例１で得られたＳＬ含水物に等重量（３．９ｋｇ）の蒸留水を添加し、２５０ｒｐｍで３０分、攪拌した。次いで、これを、室温で半日間、静置した。デカンテーションを行うことにより、上澄を除去した。この手順を、３回、繰り返した。
【００４９】
実施例１と同様に、このＳＬ含水物の組成について分析した。得られたＳＬ含水物は、５１％の水分を含むと見積もられた。培地由来の残留油脂を、ｎ−ヘキサン抽出物として、ＳＬ含水物の乾燥重量に対し０．５％含んでいた。また水溶性塩類の混入は、エタノール不溶分として０．１％であった。
【００５０】
また、このＳＬ含水物は、実施例１の方法（ｎ−ヘキサン洗浄−酢酸エチル抽出−溶媒・水分除去）を用いて、５０％のＳＬを含有すると見積もられた。
【００５１】
【発明の効果】
従って、本発明により、有機溶媒を用いることなく、培養終了液から液体および固形成分を除去し、高濃度のＳＬ含水物を得る方法が提供される。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for purifying sophorose lipids produced in culture.
[0002]
[Prior art]
Biosurfactants, which are surfactants produced by microorganisms, exhibit performance equivalent to that of chemically synthesized surfactants, are highly biodegradable and have low toxicity, and are expected to be applied in various industrial fields. Yes.
[0003]
Among them, sophorose lipids produced by Candida yeasts such as Candida bombicola and C. apicola are also relatively inexpensive. There is great expectation for wider commercial use because it is produced from raw materials and has high productivity.
[0004]
SL is accumulated in the culture medium by subjecting the strain to aeration and agitation culture using an appropriate liquid medium. Glucose and fats and oils are frequently used as the carbon source. Usually, 100 g / L or more of sophorose lipid can be obtained as a dry product by batch culture for about one week. This is remarkably high compared to the productivity of other biosurfactants, which is often several g / L.
[0005]
However, the price difference compared with surfactants by chemical synthesis is still obvious, and it is a factor that limits commercial use to those with high cost performance (Kozaric, surfactant science series vol. 48, Biosurfactants pp. 378-382 (1993)). One of the factors raising the production cost is the separation and purification process from the culture end solution.
[0006]
When the medium is acidified as the culture progresses, the produced SL exists in a dispersed state, but if the stirring is stopped, this SL has the property of quickly precipitating as a viscous hydrate. Therefore, although it can be easily separated from other liquid components by standing or centrifuging the culture end solution, in this method, solid components such as bacterial cells are directly above the SL layer or mixed in the layer. Therefore, removal by filtration is required. However, as described above, since SL is viscous, it is difficult to permeate the filter medium, and even if a filter aid is used, it is not so improved.
[0007]
Another method is an extraction method using an organic solvent. In this method, the culture end solution is washed with normal hexane (also referred to as n-hexane) to remove unreacted residual fats and oils, and then mixed with ethyl acetate to dissolve SL and separate from the aqueous phase. Is volatilized and removed by heating under reduced pressure. In this method, solid components such as cells are removed, but it is necessary to use an organic solvent having a volume several times that of the culture solution. In order to remove and recover the organic solvent from the SL hydrate obtained here, or to treat the waste liquid containing the organic solvent, dedicated equipment and energy are required, leading to an increase in production cost. Furthermore, the use of an organic solvent is generally not preferable from the viewpoint of impact on the environment and impact on the health of workers.
[0008]
Therefore, there is a need for a simple purification method that can remove solid components such as cells without using an organic solvent. According to such a purification method, SL is provided to the market at a lower cost and with a smaller environmental load, which can be extremely advantageous from an industrial viewpoint.
[0009]
[Problems to be solved by the invention]
Therefore, this invention provides the method of removing a liquid and a solid component from a culture completion liquid, and using the organic solvent, and obtaining a highly concentrated SL hydrate.
[0010]
[Means for Solving the Problems]
When the present inventors have neutralized the culture-finished solution containing sophorose lipid that is insoluble under acidic conditions by adding alkali while stirring, the entire SL component is solubilized or stably dispersed. As a result, the present invention has been completed.
[0011]
The present invention provides a method for purifying sophorose lipid, which includes the following steps. That is, the method for purifying the sophorose lipid of the present invention comprises:
Neutralizing an acidic culture solution containing insoluble sophorose lipid obtained by culturing to solubilize the sophorose lipid;
A step of subjecting the culture solution to solid-liquid separation to remove solids in the culture solution;
Acidifying the culture and resolubilizing the sophorose lipid; and
The step of allowing the sophorose lipid to settle and removing the supernatant
And thereby a high concentration of sophorose lipid hydrate can be obtained.
[0012]
In one embodiment, the purification method further includes a step of washing the sophorose lipid hydrate with water one or more times, and a step of removing the obtained supernatant.
[0013]
In another embodiment, the purification method uses a culture solution having a pH of 2.0 to 3.5 at the end of the culture.
[0014]
In still another embodiment, the purification method described above is a culture solution in which the residue of an oily substrate added as a carbon source at the end of the culture is 0.2% or less based on the weight of the culture end solution as an n-hexane extract. Is used.
[0015]
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a simple method for separating and purifying a high-concentration sophorose lipid-containing product containing almost no contaminating solids from a culture solution containing sophorose lipid, and without using an organic solvent. provide.
[0016]
Sophorose lipid (SL) is a glycolipid composed of sophorose or sophorose having a partially acetylated hydroxyl group and a hydroxy fatty acid. Sophorose is a sugar composed of two molecules of glucose linked by β1 → 2. Hydroxy fatty acid is a fatty acid having a hydroxyl group. Sophorose lipids are also broadly divided into acid forms in which the carboxyl groups of hydroxy fatty acids are liberated and lactone forms in which intramolecular sophorose is bound. Each structural formula is shown below (right side, acid type; left side, lactone type):
[0017]
[Chemical 1]

(Here, in each structural formula, R ₁ And R ₂ Are each independently hydrogen or an acetyl group, and n is generally an integer from 11 to 20, more usually from 13 to 17, and even more usually from 14 to 16.)
[0018]
SL can be produced by culturing Candida yeast. Cultured SL is a mixture of multiple molecular species and usually contains 50% or more of lactone type. Therefore, in this specification, sophorose lipid (SL) means not only a single substance but also a mixture of plural molecular species.
[0019]
Examples of yeast used in the culture include Candida bombicola, Candida apicola, C. petrophyllum, and C. bogoriensis. These can be strains or their subcultures that have been distributed from a preservation agency.
[0020]
SL is accumulated in a culture solution by culturing the above-described strain using an appropriate liquid medium.
[0021]
The composition of the liquid medium that can be used is described below.
[0022]
As the carbon source, sugars and oily substrates can be used. Examples of the saccharide include monosaccharides such as glucose, fructose, and galactose, and disaccharides such as sucrose and maltose. Preferably glucose is used. The initial culture concentration is 30 to 150 g / L, preferably 90 to 120 g / L. As the oily substrate, any oily substrate that has been reported to be used for SL production by culture can be used, and as such an oily substrate, vegetable oils (Zhou et al., J. Am. Oil. Chem. Soc., 69: 89-91 (1992), etc.), animal fats and oils (Deshpande et al., Bioresource Technol., 54: 143-150 (1995)), fatty acids (Asmer et al., J. Am. Oil. Chem. Soc., 65: 1460-1466 (1988)), fatty acid esters (Davila et al. Appl. Microbiol. Biotechnol. 38: 6-11 (1992)), n-alkanes (Tulloch et al., Can. J. Chem., 46). : 3337-3351 (1968) Preferably, vegetable oils or fatty acids, or fatty acid esters derived from vegetable oils and fats can be used, and edible vegetable oils can be usually used as vegetable oils and fats. Rapeseed oil, cottonseed oil, sunflower oil, kapok oil, sesame oil, corn oil, rice oil, peanut oil, safflower oil, oil oil, flaxseed oil, gill oil, castor oil, palm oil, palm kernel oil, coconut oil, etc., or Preferred are soybean oil, rapeseed oil, sunflower oil, safflower oil, or mixtures thereof, such fatty acids having a carbon number in the range of 6-24, preferably 12-20, In addition, each molecule may contain 0 to 3 unsaturated bonds, such as caproic acid, caprylic acid, capric acid, urine. Saturated fatty acids such as ndecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid, margaric acid, stearic acid, nonadecanoic acid, arachidic acid, behenic acid, and lignoceric acid, as well as tosuccinic acid, lindelic acid , Unsaturated fatty acids such as, tuzuic acid, myristoleic acid, palmitoleic acid, petrothelic acid, oleic acid, elaidic acid, vaccenic acid, erucic acid, linoleic acid, γ-linolenic acid, and linolenic acid, and mixtures thereof Fatty acid esters include those of the fatty acids described above The concentration of the oily substrate that can be added at the start of the culture is in the range of 50 to 200 g / L, preferably 100 to 150 g / L. The amount of oily substrate corresponding to the above concentration is It is added to the culture system in equal speed.
[0023]
As a nitrogen source, 2-5 g / L yeast extract, 0.5-2 g / L urea, etc. may be added, but are not limited thereto.
[0024]
Furthermore, various organic substances and inorganic salts (including phosphates and magnesium salts) necessary for yeast growth can be added in appropriate amounts.
[0025]
The culture form may be batch culture using a liquid medium as described above, or fed-batch culture in which an oily substrate is continuously added to the culture system, and is preferably aerated and agitated. The culture pH condition is about 3.5 to about 6.0 at the start, preferably about 4.0 to about 5.5. Usually, during culture, pH is not adjusted from the outside. The culture temperature is an appropriate temperature at which the strain can produce and grow a sophorose lipid, and is usually 20 to 35 ° C, and preferably 28 to 30 ° C. In the case of culturing in a volume of 5 L to 5 kL, the speed of aeration and stirring can be 0.1 to 2.0 vvm and 100 to 600 rpm. Regarding the agitation rate, the agitation rate required to produce the same dissolved oxygen conditions becomes slower as the culture vessel becomes larger. Such agitation can be performed to provide sufficient dissolved oxygen conditions in the culture, and therefore any agitation rate that provides suitable dissolved oxygen conditions for yeast culture is desirable.
[0026]
The saccharide and the oily substrate in the medium are simultaneously used in the culturing process, and the concentration decreases with time. The sugar is completely consumed first. SL production stops when all the oily substrate in the medium is consumed. The oily substrate remaining in the culture medium is separated into an oil layer by heating the culture solution in a hot water bath and then centrifuging it. By taking the above operation and confirming the disappearance of the oil layer, it can be easily known. Therefore, the production of SL is monitored by measuring the amount of oily substrate remaining in the culture medium, so that the culture medium containing the maximum yield of SL is sterilized to sterilize the cells, and then purified. Can be subjected to a method.
[0027]
Further, in order to use the refined SL in blending into a product, it is desirable that the oily substrate remains as little as possible during the purification process. Therefore, in the culture solution used in this purification method, the residue of the oily substrate added as a carbon source is desirably 0.5% or less based on the weight of the culture end solution as an n-hexane extract. The residual oily substrate added as a carbon source is preferably 0.2% or less, and more preferably 0.1% or less. The residual oily substrate can be measured by the following procedure. Take the culture solution in a sealed container, add substantially the same amount of n-hexane as this culture solution, shake vigorously, and then centrifuge (for example, 1,000 × g for 10 minutes) to separate the hexane. Collect the layer. This process can be repeated three or more times. The hexane layer is collected, the hexane solvent is removed by volatilization, and the weight of the residue is measured.
[0028]
When culturing the above strain, the culture solution tends to be acidified as the culture progresses. Therefore, the culture solution is acidified when the culture is completed. The pH range of this medium can be less than 5.5, often 4.0 or less, more often 3.5 or less, while often the lower limit is 2.0, more often 2 .5. The pH of the culture solution at the end of the culture can depend on the composition of the medium used for the culture, the culture conditions, and the like. In this purification method, typically, a culture solution having a pH of 2.0 to 3.5 can be used as a starting culture solution. Therefore, normally, hydrous glucose 80-120 g, yeast extract 1-5 g, monopotassium phosphate 5-15 g, magnesium sulfate heptahydrate 5-15 g, sodium chloride 0.5-2 g, soybean oil 80-120 g, urea 0 Using a medium having a composition of 0.5 to 2 g (all amounts are per kg of medium), the temperature is about 30 ° C., aeration and stirring is 0.5 to 1.0 vvm, 250 to 600 rpm, and the initial pH is 4.0 to 5. 0, the strain can be cultured in a culture period of 4 to 12 days.
[0029]
In the acidified culture broth, the produced SL exists in a dispersed state, but when stirring is stopped, this SL has the property of quickly precipitating as a viscous hydrate. That is, SL is hardly soluble or insoluble in a culture solution acidified as the culture progresses, and precipitates in a stationary state. This is presumably because the carboxyl group of the acid type SL is non-dissociated under acidic conditions and that the lactone type SL originally has a hydrophobic molecular structure.
[0030]
In this purification method, the acidified culture solution as described above is neutralized by adding an alkali to solubilize the insolubilized SL as described above. Preferably, the culture solution is heated to high temperature (for example, 80 to 95 ° C.) to be sterilized to terminate the culture, cooled to room temperature, and allowed to stand (for example, half a day to one day at room temperature). The supernatant is removed and an amount of water (eg, industrial or well water) substantially equal to the removed supernatant is added and used for purification. The neutralization condition is pH 5.5 or more, more preferably pH 6.0 to 7.5. Any alkali may be used as long as it is added for neutralization. Examples of such an alkali include sodium hydroxide, potassium hydroxide, sodium carbonate, ammonium carbonate, and sodium phosphate. Sodium hydroxide is preferable. Usually 48% sodium hydroxide can be used. The alkali is added while monitoring so that the pH of the culture solution is in the above range. Preferably, the alkali is added with stirring. Lactone-type SL is still considered to be poorly water-soluble under neutrality (Inoue et al., Agric. Biol. Chem., 44: 2221-2223 (1980)). The whole is solubilized or stably dispersed under neutral conditions.
[0031]
Next, in this purification method, the neutralized solution obtained by the above step is subjected to solid-liquid separation, and the solid content in the solution is removed. Examples of the technique for solid-liquid separation include filtration or centrifugation. The filtration can be performed as it is, or the filtration rate can be increased by adding water to lower the viscosity. The filtration that can be used in this step is preferably pressure filtration (for example, using a plate frame type pressure filter) or vacuum filtration (for example, using a rotating cylindrical vacuum filter). At the lab scale, vacuum filtration using a buchner funnel or the like is also possible. Filtration can be performed using equipment commonly used in the art. Centrifugation can be performed using equipment commonly used in the art. For example, continuous centrifugation with a continuous centrifuge can be used. Examples of the continuous centrifuge include a cage centrifuge and a disk centrifuge. In the case of continuous centrifugation, it can be subjected to separation by pouring the liquid continuously. For example, it can be poured at about 2 kL / hour. In the case of batch-type centrifugation, for example, it can be performed at 1000 × g for 15 minutes. By filtration or centrifugation as described above, a solid content and a solution containing SL in a solubilized state can be easily separated. This solid content may include bacterial cells and impurities generated in the production of SL in culture.
[0032]
Next, the SL solution from which the solid content has been removed is acidified by adding an acid, and the SL is insolubilized again. Examples of acids to be added include inorganic acids (for example, hydrochloric acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid), and organic acids (for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, glucuron). Acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, etc.). In addition, the acid which does not react with organic substance is preferable. Hydrochloric acid and sulfuric acid are preferred. For example, 5% to 36% hydrochloric acid, 20 to 98% sulfuric acid can be used. The conditions for acidification are pH 5.5 or less, more preferably pH 4.0 or less, more preferably pH 3.5 or less, even more preferably pH 3.0 or less, and the lower limit is a range of pH 2.0. Usually a pH range of 2.5 to 4.0 can be used. The acid is added while monitoring so that the pH of the culture solution is in the above range. Preferably, the acid is added with stirring.
[0033]
The reinsolubilized SL is then allowed to settle and the supernatant is removed. Sedimentation of the re-insolubilized SL can occur by standing or centrifugation. This standing can usually be performed for half a day to two days at room temperature. Centrifugation can be carried out at several tens to several hundreds × g for 10 minutes to 1 hour. The sediment and the supernatant can be separated by decantation or the like. Therefore, a high concentration of SL can be obtained as a water-containing material relatively easily. The SL product obtained as described above is a hydrous material containing moisture in addition to a mixture of acid type SL and lactone type SL. This moisture can be lost when the SL is placed at high temperature or under reduced pressure.
[0034]
In order to further remove impurities other than SL that may be present in the SL hydrate, water is added to the SL hydrate and stirred, and then the SL is allowed to settle by, for example, standing or centrifuging. For example, the supernatant can be removed by decantation and the SL can be recovered. For example, for example, distilled water (or deionized water or industrial water or well water) is added to the SL water-containing material in an amount substantially equal to that of the SL water-containing material, and stirred at 50 to 250 rpm. It can then be allowed to stand, for example, at room temperature for half a day to 2 days in order to precipitate SL. Alternatively, it can be subjected, for example, to centrifugation at 500 xg for about 30 minutes at room temperature to precipitate SL. Decantation can be performed to remove the supernatant. The above procedure can be repeated as necessary. Thereby, a small amount of contaminants (for example, water-soluble (inorganic) salts, low-molecular and high-molecular water-soluble substances) that can be contained in the SL water-containing material, or are derived from alkali, acid, or the like added in the medium or added during the purification Can be removed with little loss of SL.
[0035]
The SL water-containing product obtained by the purification method of the present invention can be blended into a product as it is as a raw material. SL hydrate can also be utilized as a starting material for various derivatives. SL can be fully utilized in the form of a mixture without separation into lactone and acid forms. Sophorose lipids are known to be used in improving the quality of wheat products (Japanese Patent Application Laid-Open No. 61-205449). Sophorose lipid derivatives are used as cosmetic wetting agents (Oil Chemical, 36, 748-753 (1987)) and gelling agents (Japanese Patent Publication No. 7-17668).
[0036]
When the methanol solution of the SL hydrate obtained by the purification method of the present invention and the SL hydrate prior to the neutralization treatment were dissolved in methanol and the insolubles were removed by filter filtration, The same chromatogram is obtained. This is because not only the acid type SL but also the inherently poorly water-soluble lactone type SL is recovered by solubilization and re-insolubilization precipitation in the process of this purification method, and specific SL components are decomposed and lost in large quantities. Indicates that there is no.
[0037]
The chemicals used in the purification method of the present invention are basically only alkalis and acids, and the necessary equipment is a processing tank and equipment required for solid-liquid separation (for example, continuous centrifuge or cell removal) It is about a general filtration device) that can be used. Moreover, the purification method of the present invention does not use an organic solvent.
[0038]
Therefore, the present invention not only reduces the cost required for the separation and purification of SL from the culture solution, but also reduces the burden on the environment and workers, thereby providing SL to the market more inexpensively and more safely. Can be done.
[0039]
EXAMPLES Hereinafter, although an Example demonstrates this invention, this invention is not limited to this. Materials, reagents, equipment, etc. used in this example are available from commercial sources unless specified otherwise. The reagents that can be used are industrially available grade reagents.
[0040]
【Example】
Example 1
In a 2 L Erlenmeyer flask, 800 ml (pH 4.6) of a liquid medium composed of 20 g / L water-containing glucose, 10 g / L polypeptone and 5 g / L yeast extract was prepared, and steam-sterilized with a breathable lid. Here, Candida bombycola ATCC 22214 strain 1 platinum ear was inoculated and cultured at 30 ° C. for 50 hours while stirring with a stirrer. This is used as a preculture solution. In a 30 L culture tank (fermentor), 100 g / L hydrous glucose, 100 g / L soybean oil, 2.5 g / L yeast extract, 10 g / L monopotassium phosphate, 10.25 g / L magnesium sulfate heptahydrate 18 kg (approximately 18 L) of a liquid medium comprising 1 g / L sodium chloride and 1 g / L urea was prepared, and the pH was adjusted to 4.5 using hydrochloric acid, followed by steam sterilization. However, urea was steam sterilized as a separate solution to avoid the formation of insoluble matter, and mixed after cooling. The pH of the medium after mixing was 4.6. After aeration and agitation for 1 hour or longer, 800 ml of the above-mentioned preculture solution was added to this medium. This was cultured with aeration and agitation (1.0 vvm, 250 rpm) at 30 ° C. for 10 days.
[0041]
Here, the amount of residual fat derived from the medium was examined. The culture broth was placed in a sealed container, n-hexane of the same volume as this culture broth was added and shaken vigorously, and then centrifuged at 1,000 × g for 10 minutes to recover the separated hexane layer. This process was repeated three times. The hexane layer was recovered, and the hexane solvent was removed by volatilization on a hot water bath at about 60 to 80 ° C., and the weight of the residue was measured. The residual fat derived from the medium was about 0.1% of the weight of the culture solution as an n-hexane extract.
[0042]
Next, the culture solution was treated at 90 ° C. for 15 minutes to obtain a culture completion solution. The culture end solution was pH 2.7. This culture-finished solution was separated from the bottom into a liquid brown precipitate layer, a milky white solid layer that seems to be mainly microbial cells, and three layers of supernatant by standing at room temperature for half a day to 1 day. A portion of the culture end solution that has been well mixed and homogenized is taken, and this culture end solution is washed with an equal volume of n-hexane to the separated culture end solution, and then extracted three times with twice the volume of ethyl acetate. Water was removed under heating and reduced pressure. As a result, this culture completion solution was estimated to contain 14% SL.
[0043]
After 15 kg of the culture end solution was allowed to stand at room temperature for half a day to 1 day and the resulting supernatant was removed, industrial water or well water was added in an amount equal to the removed supernatant. While stirring, the 48% by weight sodium hydroxide solution was gradually added to the obtained culture end solution to adjust the pH to 6.5 to solubilize SL. This milky white solid was precipitated by processing at 2 kL / hour using a continuous centrifuge (Westphalia (manufactured by Westphalia Separator AG)), and the supernatant was collected. While stirring the collected supernatant, 78% by weight of sulfuric acid was gradually added to adjust the pH to 3.0, thereby resolubilizing SL. After standing at room temperature for 2 days, the supernatant was removed as much as possible by decantation to obtain 3.97 kg of SL hydrate.
[0044]
When this SL hydrous material was allowed to stand at room temperature for 1 day or longer, it separated into a liquid brown precipitate without turbidity and a slight amount of supernatant, and almost no solid matter was observed at the interface between these two layers. As a result of analyzing a part of the well-mixed material, it was estimated that the obtained SL water-containing product contained 53% of water due to the weight loss caused by drying at 105 ° C.
[0045]
Next, the amount of residual fat derived from the medium was examined according to the above-described procedure. The residual fat derived from the medium contained 0.5% as an n-hexane extract with respect to the dry weight of the SL water-containing product.
[0046]
Next, contamination of water-soluble salts was examined. The weight of the beaker and filter paper was accurately measured. The filter paper used was dried at 105 ° C. for 1 hour or longer and then allowed to cool in a desiccator. About 10 g of SL water-containing material was accurately weighed into a beaker, ethanol was added at a weight 20 times or more of the sample moisture, and heated and dissolved in a hot water bath. This solution was filtered using the filter paper described above. The insoluble matter in the beaker was completely transferred onto the filter paper by washing it out 2-3 times with hot ethanol. The filter paper was dried with a dryer at 105 ° C. for 1 hour, allowed to cool with a desiccator, and then the weight was accurately measured. The ethanol-insoluble content was calculated from the following formula: ethanol-insoluble content (% by weight) = residue weight on filter paper (g) / [SL water content (g) × (100−water content (%))] × 10, 000. The mixture of water-soluble salts in this SL water-containing product was 2.3% as an ethanol-insoluble matter.
[0047]
Further, this SL hydrate was estimated to contain 48% of SL using the method described above (n-hexane washing-ethyl acetate extraction-solvent / water removal).
[0048]
(Example 2)
An equal weight (3.9 kg) of distilled water was added to the SL water-containing product obtained in Example 1, and the mixture was stirred at 250 rpm for 30 minutes. This was then left at room temperature for half a day. The supernatant was removed by decantation. This procedure was repeated three times.
[0049]
In the same manner as in Example 1, the composition of the SL water-containing product was analyzed. The resulting SL hydrate was estimated to contain 51% moisture. The residual fat derived from the medium was contained as an n-hexane extract in an amount of 0.5% based on the dry weight of the SL water-containing product. Moreover, contamination with water-soluble salts was 0.1% as ethanol-insoluble matter.
[0050]
Further, this SL hydrate was estimated to contain 50% of SL using the method of Example 1 (n-hexane washing-ethyl acetate extraction-solvent / water removal).
[0051]
【The invention's effect】
Therefore, the present invention provides a method for removing a liquid and a solid component from a culture end solution without using an organic solvent to obtain a high-concentration SL hydrate.

Claims (4)

A method for purifying sophorose lipids, comprising:
Neutralizing an acidic culture solution containing insoluble sophorose lipid obtained by culturing to solubilize the sophorose lipid;
A step of subjecting the culture solution to solid-liquid separation to remove solids in the culture solution;
Acidifying the medium and resolubilizing the sophorose lipid; and precipitating the sophorose lipid and removing the supernatant, thereby providing a high concentration of sophorose lipid hydrate. ,
Purification method.   The purification method according to claim 1, further comprising a step of washing the sophorose lipid hydrate with water at least once and a step of removing the obtained supernatant.   The purification method according to claim 1 or 2, wherein a culture solution having a pH of 2.0 to 3.5 at the end of the culture is used. At the end of culture, the residual oily substrate was added as a carbon source, n- as hexane extract, have use the culture solution is not more than 0.2% by weight of the culture broth, any one of claims 1 to 3 The purification method according to one item.