JP4213906B2 - Method for producing conjugated linoleic acid - Google Patents
Method for producing conjugated linoleic acid Download PDFInfo
- Publication number
- JP4213906B2 JP4213906B2 JP2002110773A JP2002110773A JP4213906B2 JP 4213906 B2 JP4213906 B2 JP 4213906B2 JP 2002110773 A JP2002110773 A JP 2002110773A JP 2002110773 A JP2002110773 A JP 2002110773A JP 4213906 B2 JP4213906 B2 JP 4213906B2
- Authority
- JP
- Japan
- Prior art keywords
- linoleic acid
- lactobacillus
- conjugated
- conjugated linoleic
- acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- OYHQOLUKZRVURQ-IXWMQOLASA-N linoleic acid Natural products CCCCC\C=C/C\C=C\CCCCCCCC(O)=O OYHQOLUKZRVURQ-IXWMQOLASA-N 0.000 title claims description 65
- JBYXPOFIGCOSSB-GOJKSUSPSA-N 9-cis,11-trans-octadecadienoic acid Chemical compound CCCCCC\C=C\C=C/CCCCCCCC(O)=O JBYXPOFIGCOSSB-GOJKSUSPSA-N 0.000 title claims description 48
- 229940108924 conjugated linoleic acid Drugs 0.000 title claims description 39
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- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 78
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Description
【０００１】
【発明の属する技術分野】
本発明は、Lactobacillus oris、Lactobacillus pontis及びLactobacillus panis から選ばれる１種又は２種以上の乳酸菌を利用して特定の共役脂肪酸を効率よく製造する方法及びこの方法により得られた共役脂肪酸を含有する飲食品に関するものである。
【０００２】
【従来の技術】
共役脂肪酸は隣り合う炭素が単結合を挟んで二重結合を持つ脂肪酸であるが、とりわけ炭素数１８の脂肪酸分子内に共役ジエンを１個持つ共役リノール酸は、様々な生理活性を持つことが明らかにされている。
【０００３】
共役リノール酸を工業的に製造する方法としては、例えばリノール酸を含む油脂、あるいは遊離型のリノール酸をエチレングリコールなどの有機溶媒下で共役化するアルカリ共役化法が知られている。
【０００４】
アルカリ共役化法では、通常、不飽和脂肪酸と過剰のアルカリとを有機溶媒中で１５０℃以上に加熱する。この時、得られる共役脂肪酸は、二重結合の結合位置や配置が異なる混合物の形であり、例えば原料にリノール酸を用いた場合には、cis-9, trans-11型あるいはtrans-9, cis-11型、trans-10, cis-12型の共役リノール酸が得られ、この他にも幾つかの位置あるいは幾何異性体を含んでいる。
【０００５】
従って、上記共役脂肪酸の生理作用は、共役脂肪酸の混合物としての作用であり、特定の共役脂肪酸が製造できれば学術的にも産業的にも応用価値は高い。また、アルカリ共役化法では、環化およびその他の副反応も起こり、共役脂肪酸の収率が低下すること、精製が困難になることなどの問題点も指摘されている。
【０００６】
一方、微生物あるいはその酵素が共役脂肪酸を作ることが報告されている。例えば、ルーメン細菌が多価不飽和脂肪酸から共役脂肪酸を産生すること（Shorland F.B., et al., Nature , vol.175, p.1129, 1955）をはじめとして、Treponema（Yokoyama, et al., J. Bacteriology , vol.107,p519-527,1971）、Butyrivibrio fibrisolvens （Kepler C.R., et al., J. Biol. Chem., vol.242, p5686-92, 1967）、Propionibacterium freudenreichii（Jiang J., Doctoral thesis, Swedish Uni. Uppsala, 1998 ）、種々の肺の病原菌の一割強（Jack C.I.A., et al., Clinica Chlimica Acts., vol224, p139-4, 1994 ）などがリノール酸異性化活性を持ち、また、これらの微生物によって産生される共役リノール酸はcis-9、trans-11型異性体が主体であることも報告されている。
【０００７】
一方、反芻動物は自らの体内でcis-9,trans-11型共役リノール酸を産生していることから、乳製品や畜肉にはcis-9,trans-11型共役リノール酸が多く含有されており、従って、cis-9,trans-11型共役リノール酸は通常の食生活でヒトが摂取する機会の多い共役リノール酸であると考えられる。それゆえ、本異性体の生理効果については多くの研究が行われ、種々の癌に対する防御作用があることが明らかとなってきた（Ha, Y.L., et al., Cancer Research vol.50, p1097-1101, 1990, Ip C., et al., Cancer Research vol.51, p6118-6124 ）。
【０００８】
以上のように、微生物を用いれば、前述のアルカリ異性化法に比べ副産物が少なく、抗癌作用の期待されるcis-9,trans-11型共役リノール酸含量の高い生成物が得られることから、微生物、中でも食品への利用価値が高い乳酸菌を用いた共役リノール酸製造に関しては、精力的に研究が進められており、いくつかの乳酸菌がリノール酸から共役リノール酸への変換能力を持つこと（京大院農・松村賢司ら、1999年度日本農芸化学会、福岡）や、また発酵乳中で乳酸菌を用いて共役リノール酸を製造できること（Tung Y. Lin, et al., Food Chemistry vol.67, p1-5, 1999、Tung Y. Lin, et al., Food Chemistry vol.69, p27-31, 2000）などが報告されている。
【０００９】
しかしながら、上述した種々の微生物による共役化反応ではcis-9,trans-11型共役リノール酸以外に all trans型共役リノール酸もまた同時に副産してしまうことがわかってきた。
【００１０】
【発明が解決しようとする課題】
前述の通り、アルカリ共役化法は、高温における反応のため副反応が起こりやすく特定の共役脂肪酸を得ることが困難であり、その後の精製工程が煩雑であるなどの問題点があった。更に、微生物による変換反応によって得られる共役リノール酸では、all trans型共役リノール酸もまた同時に副産してしまい、cis-9, trans-11型共役リノール酸のみを選択的に得る方法については現在のところまだ報告されていない。
【００１１】
本発明は、生理活性が高いとされているcis-9,trans-11型共役リノール酸に代表される共役脂肪酸を選択的かつ高効率で製造する製造法を得ることを目的とする。
【００１２】
【課題を解決するための手段】
本発明に係る共役リノール酸の製造法では、ラクトバチルス・オリス (Lactobacillus oris) 及び／又はラクトバチルス・ポンティス (Lactobacillus pontis)の乳酸菌を用いて二重結合を少なくとも二つ以上有する不飽和脂肪酸を共役化する工程を含み、
前記二重結合を少なくとも二つ以上有する不飽和脂肪酸がリノール酸であり、前記共役化する工程によって、 cis-9,trans-11 型の共役リノール酸を得ることにより、上述の課題を解決するものである。
【００１４】
本発明においては、二重結合を少なくとも二つ以上有する不飽和脂肪酸を含有する培地に、ラクトバチルス・オリス (Lactobacillus oris) 及び／又はラクトバチルス・ポンティス (Lactobacillus pontis)の乳酸菌を接種して培養することが好ましい。このための培地として、乳培地を用いることが好適である。
【００１５】
本発明は更に、上述の本発明による方法によって得られた共役脂肪酸を含有する飲食品を提供することができる。
【００１６】
【発明の実施の形態】
本発明による方法においては、ラクトバチルス・オリス (Lactobacillus oris) 及び／又はラクトバチルス・ポンティス (Lactobacillus pontis)の乳酸菌を用いて二重結合を少なくとも二つ以上有する不飽和脂肪酸を共役化する工程を含み、
前記二重結合を少なくとも二つ以上有する不飽和脂肪酸がリノール酸であり、前記共役化する工程によって、 cis-9,trans-11 型の共役リノール酸を得るものであるため、生理活性が高いとされているcis-9,trans-11型共役リノール酸に代表される共役脂肪酸を選択的かつ高効率で製造することができる。
【００１７】
本発明による共役脂肪酸の製造方法で用いられるラクトバチルス・オリス（La ctobacillus oris）は人の唾液から分離された乳酸菌である（Farrow J.A.E. et al., Int. J. Syst. Bacteriol.vol.38, p116, 1988）。菌株としては、例えば、Lactobacillus oris NCD02160(ATCC49062), NCD02161, NCD02162, NCD02163, NCD02164等が挙げられ、特に、NCD02160(ATCC49062)が好ましい。
【００１８】
また、ラクトバチルス・ポンティス（Lactobacillus pontis）は、ライ麦パン種から分離された乳酸菌であり（Vogel,R.F., et al., Int. J. Syst. Bacteriol., vol.44, p223-229, 1994）、菌株としては、例えば、Lactobacillus pontis ATCC51518, ATCC51519等が挙げられ、特にATCC51519を用いることが好ましい。
【００２０】
本発明において共役脂肪酸を製造するためにラクトバチルス・オリス (Lactobacillus oris) 及び／又はラクトバチルス・ポンティス (Lactobacillus pontis) の乳酸菌を用いて不飽和脂肪酸を共役化する処理としては、原料の不飽和脂肪酸を含有する増殖培地中で乳酸菌を培養して直接共役脂肪酸を生成させる方法、或いは何らかの方法で乳酸菌を培養して集菌し洗浄した菌体（洗浄菌体）を原料の不飽和脂肪酸を含有する溶液に加えて反応させることにより共役脂肪酸を生成させる方法等何れの方法を用いることも可能である。更に、不飽和脂肪酸を含有する溶液中では、乳酸菌の生菌のみならず、死菌体、菌体抽出液などを利用して反応を行うことも可能である。
【００２１】
ラクトバチルス・オリス (Lactobacillus oris) 及び／又はラクトバチルス・ポンティス (Lactobacillus pontis) の乳酸菌を培養するための培地としては、乳酸菌の増殖用に通常用いられる培地や乳を含む培地を用いることが出来るが、特に乳を用いた発酵乳中で不飽和脂肪酸を処理することが好ましく、これにより乳中に含まれる蛋白成分などの影響によって原料油類の均質化が比較的容易となる。なお、本明細書中において乳とは、牛乳・山羊乳などの獣乳の生乳、脱脂粉乳、全脂粉乳、生クリーム、あるいは豆乳・アーモンド乳・ココナッツミルク等の植物乳の各種乳蛋白質含有物を指す。
【００２２】
また、不飽和脂肪酸を含有する溶液中でラクトバチルス・オリス (Lactobacillus oris) 及び／又はラクトバチルス・ポンティス (Lactobacillus pontis) の乳酸菌の洗浄菌体或いは菌体末、死菌体、菌体抽出液などを利用して反応を行う場合、洗浄菌体や菌体末、死菌体、菌体抽出液の調製は常法に従えば良い。例えば、洗浄菌体は、生理食塩水やバッファーなどで培養菌体を洗浄して得ることができ、菌体末は凍結乾燥や噴霧乾燥などの乾燥技術により得ることができる。
【００２３】
死菌体を得る方法としては、細胞壁溶解酵素を作用させる方法の他に、低浸透圧で処理する方法、凍結融解する方法、高圧処理する方法、破砕処理する方法、加熱処理する方法等がある。中でも、菌体を加熱処理し自己融解させる方法は、コストをかけずに大量の菌体を処理できる点で好ましい。菌体抽出液は、上記のようにして得た洗浄菌体、菌体末、死菌体などに適切な溶媒を添加した後、遠心分離上清として得ることができる。
【００２４】
本発明において、原料として用いられる、二重結合を少なくとも二つ以上有する不飽和脂肪酸は特に限定されず、例えばリノレン酸、アラキドン酸、エイコサペンタエン酸、ドコサヘキサエン酸などいずれも好適に使用し得る。特に、リノール酸を原料として共役リノール酸を生成させた場合、生理活性の報告されている特定の異性体が特異的かつ効率よく生成し、他の異性体（副産物）の量は極微量となるため好ましい。
【００２５】
また、上記不飽和脂肪酸は、塩またはエステルなどを形成していても良く、塩としては、ナトリウム塩、カリウム塩などのアルカリ金属塩、カルシウム塩、マグネシウム塩などのアルカリ土類金属塩、アンモニンム塩等が挙げられ、エステルとしてはメチルエステル、エチルエステルのほか上記脂肪酸を含むその他の脂質類（リン脂質、糖脂質等）、モノグリセライド、ジグリセライド、トリグリセライドなども用いることも可能である。また、天然油脂も原料として用いることができ、例えばリノール酸を分子内に多く含むものとしてはサフラワー油、綿実油、大豆油、ヒマワリ種子油、トウモロコシ油、落花生油、米ヌカ油、アマニ油、カカオ脂などの植物由来の天然油脂などが、リノレン酸を分子内に多く含むものとしてはアマニ油、ナタネ油などが、多価不飽和脂肪酸を多く含むものとしてはイワシ油、ニシン油、タラ肝油などの動物由来の天然油脂などが挙げられ、さらにこれらのリパーゼ分解物なども原料として用いることが可能である。
【００２６】
ラクトバチルス・オリス (Lactobacillus oris) 及びラクトバチルス・ポンティス (Lactobacillus pontis) から選ばれる１種又は２種以上の乳酸菌を用いて共役脂肪酸を製造するための共役化処理法としては例えば以下の(a)〜(e)を挙げることができる。
(a) 乳酸菌を増殖培地や発酵乳中で培養して発酵生産の形で不飽和脂肪酸を共役化する方法
(b) 不飽和脂肪酸を乳酸菌の洗浄菌体で共役化する方法
(c) 乳酸菌菌末を用いて不飽和脂肪酸を共役化する方法
(d) 乳酸菌死菌体を用いて不飽和脂肪酸を共役化する方法
(e) 乳酸菌菌体抽出液を用いて不飽和脂肪酸を共役化する方法
【００２７】
以下、処理法(a)〜(e)について詳述する。
(a) 乳酸菌を増殖培地や乳培地中で培養して発酵生産の形で不飽和脂肪酸を共役化する方法：
増殖培地あるいは発酵乳中で共役化を行う際は、基本操作は一般的に行われている手法で行えば良いが、スターターとしては以下のものを用いるのが好ましい。
【００２８】
まず菌体調製において変換能を効率的に誘導するため、文献（京都大学大学院農学研究科・大村依子ら、平成11年度日本生物工学会要旨集、p191）を参考にしてリノール酸などの不飽和脂肪酸をMRS（LACTOBACILLI MRS BROTH、DIFCO社製）培地などの乳酸菌用増殖培地に添加した。この濃度は０〜0.2%で、特に0.05〜0.1%が好ましい。この際の培養は、ｐＨ3.5〜5.5で停止することが好ましく、特に、4.5〜5.5で停止することが好ましい。ｐＨ3.5 以下まで培養すると菌の過増殖により共役化能が低下してしまう場合もあり、ｐＨ5.5 以上では共役化を行うのに充分な菌体量が得られないためである。
【００２９】
以上のようにして培養した培養液をスターターとして用いるのが好ましく、スターター接種量は発酵乳調製用培地の 0.5〜８％程度が好ましく、特に１〜４％程度が好ましい。原料は培地中に0.02〜0.8 ％程度が好ましく、特に0.1〜0.2％が好ましい。培養温度は２０〜４０℃程度、好ましくは２８〜３７℃である。
【００３０】
より効率良く目的とする共役脂肪酸を得るためには培養はｐＨ3.5〜5.5で停止することが好ましく、特にｐＨ4.5〜5.5で停止することが好ましい。また、中和培養でも同様に目的とする共役脂肪酸を得ることが出来る。
【００３１】
(b) 不飽和脂肪酸を乳酸菌の洗浄菌体で共役化する方法：
処理法(a) のスターター調製時と同様の培養条件によって培養した菌体を生理食塩水にて洗浄したものを回収し、緩衝液に懸濁させたものを洗浄菌体として用いるのが好ましく、この菌体を懸濁させる緩衝液および洗浄菌体反応は適度なｐＨを維持できる条件の水溶液を用いて行う。例えば0.1〜1.0Ｍのリン酸緩衝液が好ましく、ｐＨは5.0〜7.5であるが、好ましくは6.0〜7.0である。菌体懸濁液の菌体濃度は、湿重で0.025〜0.25％であり、好ましくは0.025〜0.1 ％である。
【００３２】
原料は、添加時の原料を牛血清アルブミン等のバッファーとの混合液として用いることが原料を均一に混ぜるためには、好ましい。おり原料濃度は反応溶液中に0.1〜4.0％程度添加することが好ましく、特に0.3〜1.0％が好ましい。また牛血清アルブミンの混合比率は原料に対して５分の１程度が好ましい。洗浄菌体による変換反応の際の温度は２０〜５２℃だが、好ましくは３２〜３７℃である。変換生成の適正な時間は０〜９６時間であり、好ましくは２４〜７２時間である。なお、ｐＨを中和しながら培養した菌体も同様に用いることが出来る。
【００３３】
(c) 乳酸菌菌末を用いて不飽和脂肪酸を共役化する方法：
乳酸菌菌末は、例えば処理法(a) のスターター調製時の培養条件と同様の方法にて得た乳酸菌菌体を乾燥処理により粉末化することにより得ることが出来る。乾燥処理方法としては凍結乾燥、噴霧乾燥などを利用することができる。尚、菌末調製後の変換反応は、処理法(b) の洗浄菌体反応の反応条件と同様に行えば良い。
【００３４】
(d) 乳酸菌死菌体を用いて不飽和脂肪酸を共役化する方法：
乳酸菌死菌体は、例えば処理法(a) スターター調製時の培養条件と同様の方法にて得た乳酸菌菌体の細胞壁を破壊することにより得ることができる。細胞壁破壊は、細胞壁破壊酵素で処理する方法や、菌体を溶媒に懸濁させて低浸透圧で処理する方法、凍結融解する方法、高圧処理する方法、破砕処理する方法、加熱処理する方法などを利用することができる。細胞壁破壊液調製後の変換反応は、処理法(b) の洗浄菌体反応の反応条件と同様に行えば良い。
【００３５】
(e) 乳酸菌菌体抽出液を用いて不飽和脂肪酸を共役化する方法：
乳酸菌菌体抽出液としては、例えば処理法(b)〜(d)と同様の方法にて得た乳酸菌洗浄菌体、菌体末、死菌体などを適切な溶媒で抽出し、遠心分離など方法により残渣を除去して得ることができる。尚、菌体抽出液調製後の反応は、処理法(b)の洗浄菌体反応の反応条件と同様に行えば良い。
【００３６】
上記処理法(a)〜(e)等、ラクトバチルス・オリス (Lactobacillus oris) 及びラクトバチルス・ポンティス (Lactobacillus pontis) から選ばれる１種又は２種以上の乳酸菌の何れかにより得られる反応液または培養液の脂肪酸組成は、ガスクロマトグラフィー分析により内部標準物質と各脂肪酸の面積値の比率をもとに各脂肪酸量を算出できる。なお、例えば、共役リノール酸を対象とする場合には、ガスクロマトグラフィーは以下の表１に示す条件で行えばよい。
【００３７】
【表１】
本発明により得られる共役脂肪酸は、医薬品、食品、化粧品等の形態で投与することができる。例えば共役リノール酸の生理効果を訴求する医薬品や栄養補助食品等の形態で用いる場合であれば、カプセル剤、顆粒剤、錠剤、散剤等の固形製剤、或いはシロップ剤等の液状製剤として経口投与することができる。また、経口投与剤でなくとも、注射剤、皮膚外用剤、直腸投与剤等非経口形態で投与することも可能である。
【００３９】
各製剤の製造時には、乳糖、澱粉、結晶セルロース、乳酸カルシウム、メタケイ酸アルミン酸マグネシウム、無水ケイ酸等の賦形剤、白糖、ヒドロキシプロピルセルロース、ポリビニルピロリドン等の結合剤、カルボキシメチルセルロース、カルボキシメチルセルロースカルシウム等の崩壊剤、ステアリン酸マグネシウム、タルク、モノグリセリド、蔗糖脂肪酸エステル等の滑沢剤や、その他、医薬・食品等として許容され得る成分を適宜使用すればよい。
【００４０】
また、同様の生理効果を期待して一般食品形態（「明らか食品」の形態）で用いる場合には、本発明の方法により得られた共役脂肪酸をそのまま或いは適宜精製処理したものを油脂、錠菓、発酵乳、飴、調味料、ふりかけ等の飲食品に添加し、常法を用いて製造すればよい。発酵食品として用いる場合には、発酵原料中の二重結合を二つ以上有する脂肪酸を添加し、発酵菌により発酵（培養）させ、製造することができる。特にリノール酸を含む乳を発酵した発酵乳とすれば、前記のとおりcis-9, trans-11型共役リノール酸を特異的かつ多量に含有させることが容易であるため好ましい。
【００４１】
なお、発酵乳とは、乳等省令により定められている発酵乳、乳製品乳酸菌飲料等の生菌含有タイプの飲料や殺菌処理の施された発酵乳を含有する乳性飲料、更には、ケフィア等のことである。発酵に際しては、その他の菌、例えばラクトバチルス・カゼイ、ラクトバチルス・アシドフィルス、ラクトバチルス・ガッセリ、ラクトバチルス・ゼアエ、ラクトバチルス・ジョンソニー、ラクトバチルス・デルブルッキィ サブスピーシーズ ブルガリカス、ラクトバチルス・デルブルッキィ サブスピーシーズ デルブルッキィ等のラクトバチルス属細菌やストレプトコッカス・サーモフィルス等のストレプトコッカス属細菌、ラクトコッカス・ラクチス サブスピーシーズ．ラクチス、ラクトコッカス・ラクチス サブスピーシーズ．クレモリス、ラクトコッカス・プランタラム、ラクトコッカス・ラフィノラクチス等のラクトコッカス属細菌、ロイコノストック・メセンテロイテス、ロイコノストック・ラクチス等のロイコノストック属細菌、エンテロコッカス・フェカーリス、エンテロコッカス・フェシウム等のエンテロコッカス属細菌等を使用できる。
【００４２】
また、ビフィドバクテリウム・ブレーベ、ビフィドバクテリウム・ビフィダム、ビフィドバクテリウム・ロンガム、ビフィドバクテリウム・アニマーリス等のビフィドバクテリウム属細菌や酵母その他の微生物を使用しても良い。これらは、１種または２種以上を組み合わせて使用することができる。
【００４３】
また、これらの食品には、その他の食品素材、すなわち、各種糖質や乳化剤、増粘剤、甘味料、酸味料、果汁等を適宜配合してもよい。具体的には、蔗糖、異性化糖、グルコース、フラクトース、パラチノース、トレハロース、ラクトース、キシロース等の糖類、ソルビトール、キシリトール、エリスリトール、ラクチトール、パラチニット、還元水飴、還元麦芽糖水飴等の糖アルコール、ショ糖脂肪酸エステル、グリセリン脂肪酸エステル、レシチン等の乳化剤、カラギーナン、グァーガム、キサンタンガム、ペクチン、ローカストビーンガム等の増粘（安定）剤、が挙げられる。この他にも、ビタミンＡ、ビタミンＢ類等の各種ビタミン類やカルシウム、鉄、マンガン、亜鉛等のミネラル類を配合しても、優れた風味の耐光性発酵乳を得ることができる。
【００４４】
これら医薬品、食品等の形態での使用に際しては、本発明の方法により得られた共役脂肪酸を適宜配合することができる。また、共役脂肪酸の生理効果を訴求する場合であれば、その効果を得られかつ過剰摂取等の問題が生じない程度の量、１０ｍｇ〜１０００ｍｇ／日程度の摂取が見込まれる量を適宜配合しておけばよい。
【００４５】
【実施例】
以下、実施例により本発明を具体的に説明するが、本発明はこれら実施例になんら制約されるものではない。
【００４６】
実施例１：共役脂肪酸を産生する乳酸菌のスクリーニング
１００mMリン酸緩衝液（ｐＨ6.5）１ｍL中にリノール酸５０mgとBSA １０mgを溶解し、あらかじめリノール酸−BSA−複合体溶液を調製した。１５ｍLの0.07％リノール酸含有MRS（LACTOBACILLI MRS BROTH、DIFCO社製）培地に各菌を接種し、２８℃、１２０ｒｐｍ、２０時間振盪培養した。得られた培養液はｐＨ 4.7であった。
【００４７】
この培養液を遠心分離して菌体を回収し生理食塩水で２回洗浄して洗浄菌体を得た。この洗浄菌体にリノール酸−BSA−複合体溶液を100μL、100ｍＭリン酸緩衝液（ｐＨ6.5）を0.9ｍL添加し、酸素吸収・炭酸ガス発生剤（Anaero Pack、三菱ガス化学(株)社製）にて嫌気状態に保った酸素不透過性のビニール袋内で３７℃、１２０ｒｐｍで２４時間反応した。
【００４８】
得られた反応液に内部標準物質（HEPTADECANOIC ACID）を1mg添加後Bligh-Dyer 法にて抽出し、メチルエステル化（４％塩酸メタノール溶液で３０分間室温静置）後、ガスクロマトグラフィーの分析に供し脂肪酸分析を行った。
【００４９】
【表２】
共役リノール酸のピークは標準品（リノール油脂製 CLA８０）のリテンションタイムを基準に判断し、基質として添加したリノール酸を１００とした時の相対値を算出した。その結果、表２に示したようにLactobacillus orisで共役リノール酸の産生が認められた。このLactobacillus oris NCD02160は、ATCC49062としてアメリカン・タイプ・カルチャー・コレクション（ＡＴＣＣ）に登録されている。
【００５１】
実施例２：共役脂肪酸の異性体の同定
実施例１でLactobacillus orisによって産生された共役リノール酸の異性体を調べた。図１はLactobacillus orisによって産生された共役リノール酸の異性体のガスクロマトグラムのチャートである。図１に示したようにLactobacillus orisによって産生された共役リノール酸の全てがcis-9,trans-11型共役リノール酸であった。以上の結果から、数ある乳酸菌の中でLactobacillus orisが選択的、且つ効率的にcis-9,trans-11型共役リノール酸を産生することが明らかとなった。
【００５２】
実施例３：発酵乳中での共役脂肪酸の産生
１００mMリン酸緩衝液（ｐＨ6.5）１ｍL中にリノール酸５０mgとBSA １０mgを溶解し、あらかじめリノール酸−BSA −複合体溶液を調製した。このリノール酸−BSA−複合体溶液２００μLをMRS（LACTOBACILLI MRS BROTH、DIFCO社製）培地１５ｍL に添加後、Lactobacillus orisを接種し２８℃、１２０ｒｐｍ、２０時間振盪培養し、ｐＨ４．７の培養液を調製した。
【００５３】
１５ｍL 容試験管（キャップ付き）に５ｍL 分注した１０％スキムミルク（グルコース１％、大豆ペプチド0.1％添加）培地に、リノール酸−BSA−複合体溶液を１００μL、先に調製した各菌株の培養液を２００μL添加し、キャップを閉めた。この培地を２８℃、１２０ｒｐｍで４８時間培養しｐＨ4.6 の発酵乳を調製し、実施例１と同様に脂肪酸分析を行った。
【００５４】
その結果、添加したリノール酸の２．０％が共役リノール酸に変換されていた。産生された共役リノール酸の全てがcis-9、trans-11型共役リノール酸であり、実施例２と同様の結果が得られた。以上の結果から、発酵乳中でもLactobacillus orisが選択的、且つ効率的にcis-9,trans-11型共役リノール酸を産生することが明らかとなった。
【００５５】
実施例４：Lactobacillus oris 死菌体による共役脂肪酸の産生
１００mMリン酸緩衝液（ｐＨ6.5）１ｍL中にリノール酸５０mgと BSA１０mgを溶解し、あらかじめリノール酸−BSA −複合体溶液を調製した。このリノール酸−BSA−複合体溶液２００μLをMRS（LACTOBACILLI MRS BROTH、DIFCO社製）培地１５ｍL に添加後、Lactobacillus orisを接種し２８℃、１２０ｒｐｍ、２０時間振盪培養し、ｐＨ4.7 の培養液を調製した。
【００５６】
この培養液0.5ｍLを、0.3%glc加ＩＬＳ培地15mLに接種し、３７℃、１２０rpmで１８時間（ｐＨ5.6まで）培養し、遠心分離して菌体を回収し0.2Ｍグリシン緩衝液（ｐＨ10.6）で２回洗浄し、洗浄後菌体を 6.7％蔗糖−５０mMトリスアミノメタン−１mＭ EDTA溶液２ｍLに溶解した。この液に lysozyme溶液(10mg/mL in 25mＭ トリスアミノメタン ,ｐＨ8.0、生化学工業(株)社製)0.8ｍLおよびN-Acetylmuramidase水溶液（1mg/mL 、生化学工業(株)社製）0.15ｍLを添加し、３７℃、１２０ｒｐｍで３０分反応した。反応液をセントリプレップ１０（amicon社製）に移し濃縮操作を行うことによりLactobacillus oris死菌体懸濁液を約0.6ｍL
得た。
【００５７】
この死菌体懸濁液にリノール酸−BSA−複合体溶液を１００μL、１００mMリン酸緩衝液（ｐＨ6.5）を0.9ｍL添加し、酸素吸収・炭酸ガス発生剤（Anaero Pack、三菱ガス化学(株)(株)社製）にて嫌気状態に保った酸素不透過性のビニール袋内で３７℃、１２０ｒｐｍで２４時間反応した。
【００５８】
得られた反応液について実施例１と同様に脂肪酸分析を行った結果、添加したリノール酸の１．６％が共役リノール酸に変換されていた。産生された共役リノール酸の全てがcis-9,trans-11型共役リノール酸であり、洗浄菌体および発酵乳中と同様の結果が得られた。以上の結果から、細胞壁を破壊した死菌体でもLactobacillus orisが選択的、且つ効率的にcis-9,trans-11型共役リノール酸を産生することが明らかとなった。
【００５９】
実施例５：Lactobacillus pontisによる共役脂肪酸の産生
１００mMリン酸緩衝液（ｐＨ6.5）１ｍL中にリノール酸５０mgと BSA１０mgを溶解し、あらかじめリノール酸−BSA −複合体溶液を調製した。このリノール酸−BSA−複合体溶液２００μLをMRS（LACTOBACILLI MRS BROTH、DIFCO社製）培地１５ｍL に添加後、Lactobacillus pontis （ATCC51518）を接種し２８℃、１２０ｒｐｍ、４８時間振盪培養し、ｐＨ4.9 の培養液を調製した。
【００６０】
１５ｍL 容試験管（キャップ付き）に５ｍL 分注した１０％スキムミルク生地に、リノール酸−BSA −複合体溶液を１００μL 、先きに調整した各菌株の培養液を４００μL 添加し、キャップを閉めた。この培地を２８℃、１２０ｒｐｍで４８時間培養し、ｐＨ5.5の発酵乳を調整し、実施例１と同様に脂肪酸分析を行った。
【００６１】
その結果、添加したリノール酸の０．５％が共役リノール酸に変換されていた。産生された共役リノール酸の全てがcis-9,trans-11型共役リノール酸であった。以上の結果から、Lactobacillus pontisが選択的、且つ効率的にcis-9,trans-11型共役リノール酸を産生することが明らかとなった。
【００６２】
【発明の効果】
以上説明した通り、本発明の共役不飽和脂肪酸の製造方法によればcis-9, trans-11型共役不飽和脂肪酸を選択的に高効率で製造できるという効果がある。
【図面の簡単な説明】
【図１】 Lactobacillus orisによって産生された共役リノール酸の異性体のガスクロマトグラムのチャートである。[0001]
BACKGROUND OF THE INVENTION
The present inventionLactobacillus oris,Lactobacillus pontisas well asLactobacillus panis The present invention relates to a method for efficiently producing a specific conjugated fatty acid using one or two or more lactic acid bacteria selected from the above and a food or drink containing the conjugated fatty acid obtained by this method.
[0002]
[Prior art]
Conjugated fatty acids are fatty acids in which adjacent carbons have a double bond with a single bond sandwiched between them. In particular, conjugated linoleic acid having one conjugated diene in a fatty acid molecule having 18 carbon atoms can have various physiological activities. It has been revealed.
[0003]
As a method for industrially producing conjugated linoleic acid, for example, an oil / fat containing linoleic acid or an alkali conjugation method in which free linoleic acid is conjugated in an organic solvent such as ethylene glycol is known.
[0004]
In the alkali conjugation method, an unsaturated fatty acid and excess alkali are usually heated to 150 ° C. or higher in an organic solvent. At this time, the conjugated fatty acid obtained is in the form of a mixture in which the position and arrangement of double bonds are different. For example, when linoleic acid is used as a raw material, cis-9, trans-11 type or trans-9, Conjugated linoleic acid of cis-11 type, trans-10 type, cis-12 type is obtained, and also contains several position or geometric isomers.
[0005]
Therefore, the physiological action of the conjugated fatty acid is an action as a mixture of conjugated fatty acids, and if the specific conjugated fatty acid can be produced, the application value is high both academically and industrially. Further, in the alkali conjugation method, cyclization and other side reactions also occur, and problems such as a decrease in the yield of conjugated fatty acid and difficulty in purification have been pointed out.
[0006]
On the other hand, it has been reported that microorganisms or their enzymes produce conjugated fatty acids. For example, rumen bacteria produce conjugated fatty acids from polyunsaturated fatty acids (Shorland F.B., et al., Nature, vol.175, p.1129, 1955),Treponema(Yokoyama, et al., J. Bacteriology, vol.107, p519-527,1971),Butyrivibrio fibrisolvens (Kepler C.R., et al., J. Biol. Chem., Vol.242, p5686-92, 1967),Propionibacterium freudenreichii(Jiang J., Doctoral thesis, Swedish Uni. Uppsala, 1998) and more than 10% of various lung pathogens (Jack CIA, et al., Clinica Chlimica Acts., Vol224, p139-4, 1994) It has been reported that conjugated linoleic acid having isomerization activity and produced by these microorganisms is mainly composed of cis-9 and trans-11 isomers.
[0007]
On the other hand, ruminants produce cis-9, trans-11 conjugated linoleic acid in their bodies, so dairy products and livestock meat contain a lot of cis-9, trans-11 conjugated linoleic acid. Therefore, it is considered that cis-9, trans-11 type conjugated linoleic acid is a conjugated linoleic acid that is frequently consumed by humans in a normal diet. Therefore, many studies have been conducted on the physiological effects of this isomer, and it has been revealed that it has protective effects against various cancers (Ha, YL, et al., Cancer Research vol.50, p1097- 1101, 1990, Ip C., et al., Cancer Research vol. 51, p6118-6124).
[0008]
As described above, when microorganisms are used, there are fewer by-products than the alkali isomerization method described above, and a product with a high content of cis-9, trans-11 conjugated linoleic acid, which is expected to have anticancer activity, can be obtained. Conjugated linoleic acid production using microorganisms, especially lactic acid bacteria that have high utility value for food, has been energetically studied, and some lactic acid bacteria have the ability to convert linoleic acid to conjugated linoleic acid (Kyoto University Agriculture, Matsumura Kenji et al., 1999 Agricultural Chemical Society of Japan, Fukuoka) and the ability to produce conjugated linoleic acid using lactic acid bacteria in fermented milk (Tung Y. Lin, et al., Food Chemistry vol.67, p1-5, 1999, Tung Y. Lin, et al., Food Chemistry vol.69, p27-31, 2000).
[0009]
However, it has been found that in the conjugation reaction by various microorganisms described above, all trans conjugated linoleic acid is also produced as a by-product in addition to cis-9, trans-11 conjugated linoleic acid.
[0010]
[Problems to be solved by the invention]
As described above, the alkali conjugation method has a problem that a side reaction is likely to occur due to the reaction at a high temperature and it is difficult to obtain a specific conjugated fatty acid, and the subsequent purification process is complicated. Furthermore, in conjugated linoleic acid obtained by a conversion reaction by microorganisms, all trans conjugated linoleic acid is also produced as a by-product at the same time, and there is currently no method for selectively obtaining only cis-9, trans-11 type conjugated linoleic acid. Not yet reported.
[0011]
An object of the present invention is to obtain a production method for selectively and efficiently producing a conjugated fatty acid represented by cis-9, trans-11 type conjugated linoleic acid, which is considered to have high physiological activity.
[0012]
[Means for Solving the Problems]
According to the present inventionConjugated linoleic acidIn the manufacturing method ofLactobacillus oris (Lactobacillus oris) And / or Lactobacillus pontis (Lactobacillus pontis)A step of conjugating an unsaturated fatty acid having at least two double bonds usingSee
The unsaturated fatty acid having at least two double bonds is linoleic acid, and by the conjugating step, cis-9, trans-11 To obtain the type of conjugated linoleic acidThus, the above-described problems are solved.
[0014]
In the present invention, a medium containing an unsaturated fatty acid having at least two double bonds,Lactobacillus oris (Lactobacillus oris) And / or Lactobacillus pontis (Lactobacillus pontis)It is preferable to inoculate and culture lactic acid bacteria. A milk medium is preferably used as the medium for this purpose.
[0015]
The present invention further provides a food or drink containing a conjugated fatty acid obtained by the above-described method according to the present invention.Can provide.
[0016]
DETAILED DESCRIPTION OF THE INVENTION
In the method according to the invention,Lactobacillus oris (Lactobacillus oris) And / or Lactobacillus pontis (Lactobacillus pontis)A step of conjugating an unsaturated fatty acid having at least two double bonds usingSee
The unsaturated fatty acid having at least two double bonds is linoleic acid, and by the conjugating step, cis-9, trans-11 To obtain the type of conjugated linoleic acidTherefore, a conjugated fatty acid represented by cis-9, trans-11 type conjugated linoleic acid, which is considered to have high physiological activity, can be selectively and highly efficiently produced.
[0017]
Lactobacillus oris used in the method for producing a conjugated fatty acid according to the present invention (La ctobacillus oris) Is a lactic acid bacterium isolated from human saliva (Farrow J.A.E. et al., Int. J. Syst. Bacteriol. Vol.38, p116, 1988). As a strain, for example,Lactobacillus oris NCD02160 (ATCC49062), NCD02161, NCD02162, NCD02163, NCD02164 and the like are mentioned, and NCD02160 (ATCC49062) is particularly preferable.
[0018]
Also, Lactobacillus pontis (Lactobacillus pontis) Is a lactic acid bacterium isolated from rye bread seeds (Vogel, R.F., et al., Int. J. Syst. Bacteriol., Vol.44, p223-229, 1994).Lactobacillus pontis ATCC51518, ATCC51519, etc. are mentioned, and it is particularly preferable to use ATCC51519.
[0020]
In order to produce conjugated fatty acids in the present inventionLactobacillus oris (Lactobacillus oris) And / or Lactobacillus pontis (Lactobacillus pontis) As a process for conjugating unsaturated fatty acids using lactic acid bacteria, a method in which lactic acid bacteria are directly cultivated in a growth medium containing unsaturated fatty acids as a raw material to produce conjugated fatty acids directly, or lactic acid bacteria are cultured by some method. Any method such as a method of producing conjugated fatty acid by adding and reacting the collected and washed cells (washed cells) to a solution containing the unsaturated fatty acid as a raw material can be used. Furthermore, in a solution containing an unsaturated fatty acid, it is possible to carry out the reaction using not only viable bacteria of lactic acid bacteria but also dead cell bodies, cell body extract, and the like.
[0021]
Lactobacillus oris (Lactobacillus oris) And / or Lactobacillus pontis (Lactobacillus pontis) As a medium for culturing lactic acid bacteria, a medium usually used for the growth of lactic acid bacteria or a medium containing milk can be used, but it is particularly preferable to treat unsaturated fatty acids in fermented milk using milk. As a result, the homogenization of the raw oils becomes relatively easy due to the influence of protein components contained in the milk. In this specification, milk means raw milk of animal milk such as cow milk and goat milk, skim milk powder, whole milk powder, fresh cream, or various milk protein-containing substances of vegetable milk such as soy milk, almond milk and coconut milk. Point to.
[0022]
Also, in solutions containing unsaturated fatty acidsLactobacillus oris (Lactobacillus oris) And / or Lactobacillus pontis (Lactobacillus pontis) When the reaction is carried out using washed microbial cells or microbial cells, dead cells, or microbial cell extracts of lactic acid bacteria, preparation of washed microbial cells, microbial cells, dead cells, or microbial cell extracts is a conventional method. You can follow. For example, the washed cells can be obtained by washing the cultured cells with physiological saline or a buffer, and the cell powder can be obtained by a drying technique such as freeze drying or spray drying.
[0023]
As a method for obtaining dead cells, in addition to the method of allowing cell wall lytic enzyme to act, there are a method of treating at low osmotic pressure, a method of freezing and thawing, a method of treating with high pressure, a method of crushing, a method of heat treating, etc. . Especially, the method of heat-processing and self-melting a microbial cell is preferable at the point which can process a lot of microbial cells without incurring cost. The bacterial cell extract can be obtained as a centrifugal supernatant after adding a suitable solvent to the washed bacterial cells, bacterial cells, dead cells and the like obtained as described above.
[0024]
In the present invention, the unsaturated fatty acid having at least two double bonds used as a raw material is not particularly limited, and any of linolenic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid and the like can be suitably used. In particular, when conjugated linoleic acid is produced using linoleic acid as a raw material, specific isomers that have been reported to have physiological activity are produced specifically and efficiently, and the amount of other isomers (by-products) is extremely small. Therefore, it is preferable.
[0025]
In addition, the unsaturated fatty acid may form a salt or an ester, and examples of the salt include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, and ammonium salt. Examples of esters that can be used include methyl esters, ethyl esters, and other lipids containing the above fatty acids (phospholipids, glycolipids, etc.), monoglycerides, diglycerides, triglycerides, and the like. Natural oils and fats can also be used as a raw material. For example, those containing a large amount of linoleic acid in the molecule include safflower oil, cottonseed oil, soybean oil, sunflower seed oil, corn oil, peanut oil, rice bran oil, linseed oil, Plant-derived natural fats such as cacao butter contain linolenic acid in the molecule, linseed oil, rapeseed oil, etc., and those containing many polyunsaturated fatty acids include sardine oil, herring oil, cod liver oil Natural fats and oils derived from animals such as these, and these lipase degradation products can also be used as raw materials.
[0026]
Lactobacillus oris (Lactobacillus oris) And Lactobacillus pontis (Lactobacillus pontis) Examples of the conjugation treatment method for producing a conjugated fatty acid using one or more lactic acid bacteria selected from (a) to (e) include the following (a) to (e).
(a) Method of conjugating unsaturated fatty acids in the form of fermentation production by culturing lactic acid bacteria in growth medium or fermented milk
(b) A method of conjugating unsaturated fatty acids with lactic acid bacteria washed cells
(c) Method of conjugating unsaturated fatty acids using lactic acid bacteria powder
(d) A method for conjugating unsaturated fatty acids using dead lactic acid bacteria
(e) Method for conjugating unsaturated fatty acids using lactic acid bacteria cell extract
[0027]
Hereinafter, the processing methods (a) to (e) will be described in detail.
(a) Method of conjugating unsaturated fatty acids in the form of fermentation production by culturing lactic acid bacteria in growth medium or milk medium:
When conjugation is performed in a growth medium or fermented milk, the basic operation may be performed by a commonly performed technique, but it is preferable to use the following as a starter.
[0028]
First, in order to efficiently induce the conversion ability in cell preparation, unsaturated materials such as linoleic acid were referred to the literature (Yoshiko Omura, Kyoto University Graduate School of Agricultural Science, 1999, Biomedical Society of Japan, 1999). Fatty acids were added to a growth medium for lactic acid bacteria such as MRS (LACTOBACILLI MRS BROTH, manufactured by DIFCO) medium. This concentration is 0 to 0.2%, particularly preferably 0.05 to 0.1%. In this case, the culture is preferably stopped at pH 3.5 to 5.5, particularly preferably 4.5 to 5.5. If the culture is carried out to pH 3.5 or less, the conjugation ability may be reduced due to the overgrowth of the bacteria, and if the pH is 5.5 or more, a sufficient amount of cells for conjugation cannot be obtained.
[0029]
The culture medium cultured as described above is preferably used as a starter, and the starter inoculation amount is preferably about 0.5 to 8%, particularly preferably about 1 to 4% of the medium for preparing fermented milk. The raw material is preferably about 0.02 to 0.8% in the medium, particularly preferably 0.1 to 0.2%. The culture temperature is about 20 to 40 ° C, preferably 28 to 37 ° C.
[0030]
In order to obtain the target conjugated fatty acid more efficiently, the culture is preferably stopped at pH 3.5 to 5.5, particularly preferably at pH 4.5 to 5.5. Similarly, the target conjugated fatty acid can be obtained by neutralization culture.
[0031]
(b) Method of conjugating unsaturated fatty acids with lactic acid bacteria wash cells:
It is preferable to collect the cells cultured under the same culture conditions as in the starter preparation of the treatment method (a), recover the cells washed with physiological saline, and use the cells suspended in the buffer as the washed cells. The buffer solution for suspending the cells and the washing cell reaction are carried out using an aqueous solution under conditions capable of maintaining an appropriate pH. For example, a 0.1 to 1.0 M phosphate buffer is preferred, and the pH is 5.0 to 7.5, preferably 6.0 to 7.0. The cell concentration of the cell suspension is 0.025 to 0.25% by wet weight, and preferably 0.025 to 0.1%.
[0032]
In order to uniformly mix the raw materials, it is preferable to use the raw materials at the time of addition as a mixed solution with a buffer such as bovine serum albumin. The concentration of the starting material is preferably added to the reaction solution by about 0.1 to 4.0%, particularly preferably 0.3 to 1.0%. The mixing ratio of bovine serum albumin is preferably about 1/5 of the raw material. The temperature during the conversion reaction with the washed cells is 20 to 52 ° C, preferably 32 to 37 ° C. The proper time for generating the conversion is 0 to 96 hours, preferably 24 to 72 hours. In addition, the microbial cell cultured while neutralizing pH can be used similarly.
[0033]
(c) Method for conjugating unsaturated fatty acids using lactic acid bacteria powder:
Lactic acid bacteria powder can be obtained, for example, by pulverizing lactic acid bacteria cells obtained by the same method as the culture conditions for preparing the starter in the treatment method (a) by drying treatment. As the drying method, freeze drying, spray drying, or the like can be used. The conversion reaction after the preparation of the bacterial powder may be performed in the same manner as the reaction conditions for the washing cell reaction in the treatment method (b).
[0034]
(d) Method of conjugating unsaturated fatty acids using dead lactic acid bacteria:
Lactic acid bacteria dead cells can be obtained, for example, by destroying the cell walls of the lactic acid bacteria cells obtained by the same method as the treatment conditions (a) culture conditions at the starter preparation. Cell wall destruction is treated with cell wall disrupting enzyme, microbial cells are suspended in a solvent and treated at low osmotic pressure, freeze-thaw method, high-pressure treatment method, crushing treatment method, heat treatment method, etc. Can be used. The conversion reaction after preparation of the cell wall disrupting solution may be performed in the same manner as the reaction conditions for the washing cell reaction in the treatment method (b).
[0035]
(e) Method of conjugating unsaturated fatty acid using lactic acid bacteria cell extract:
As the lactic acid bacteria cell extract, for example, lactic acid bacteria washed cells, cell powder, dead cells and the like obtained by the same method as the treatment methods (b) to (d) are extracted with an appropriate solvent, centrifuged, etc. It can be obtained by removing the residue by a method. The reaction after the preparation of the bacterial cell extract may be performed in the same manner as the reaction conditions for the washing bacterial cell reaction in the treatment method (b).
[0036]
The above processing methods (a) to (e), etc.Lactobacillus oris (Lactobacillus oris) And Lactobacillus pontis (Lactobacillus pontis) The fatty acid composition of the reaction solution or culture solution obtained from either one or two or more lactic acid bacteria selected from the following is the amount of each fatty acid based on the ratio of the area value of the internal standard substance and each fatty acid by gas chromatography analysis Can be calculated. For example, when conjugated linoleic acid is targeted, gas chromatography may be performed under the conditions shown in Table 1 below.
[0037]
[Table 1]
The conjugated fatty acid obtained by the present invention can be administered in the form of pharmaceuticals, foods, cosmetics and the like. For example, if it is used in the form of a pharmaceutical or nutritional supplement that promotes the physiological effect of conjugated linoleic acid, it is orally administered as a solid preparation such as a capsule, granule, tablet, powder, or a liquid preparation such as a syrup. be able to. Moreover, even if it is not an oral administration agent, it can also administer in parenteral forms, such as an injection, a skin external preparation, and a rectal administration agent.
[0039]
When manufacturing each preparation, excipients such as lactose, starch, crystalline cellulose, calcium lactate, magnesium aluminate metasilicate, and anhydrous silicic acid, binders such as sucrose, hydroxypropylcellulose, polyvinylpyrrolidone, carboxymethylcellulose, carboxymethylcellulose calcium Etc., lubricants such as magnesium stearate, talc, monoglyceride and sucrose fatty acid ester, and other components acceptable as pharmaceuticals and foods may be used as appropriate.
[0040]
In addition, when the same physiological effect is expected and used in the form of a general food (in the form of “obvious food”), the conjugated fatty acid obtained by the method of the present invention is used as it is or after appropriate purification treatment as a fat or tablet. It can be added to foods and drinks such as fermented milk, rice cake, seasonings and sprinkles, and manufactured using conventional methods. When used as a fermented food, it can be produced by adding a fatty acid having two or more double bonds in the fermentation raw material and fermenting (culturing) it with fermenting bacteria. In particular, fermented milk obtained by fermenting milk containing linoleic acid is preferable because it can easily contain a specific and large amount of cis-9, trans-11 conjugated linoleic acid as described above.
[0041]
Fermented milk refers to fermented milk specified by a ministerial ordinance, milk-containing beverages such as dairy lactic acid bacteria beverages, dairy drinks containing sterilized fermented milk, and kefir Etc. During fermentation, other fungi such as Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus gasseri, Lactobacillus zeae, Lactobacillus johnsonii, Lactobacillus delbruchy subspecies Bulgaricus, Lactobacillus delbruxy subspecies Lactobacillus genus bacteria such as Delbrukki, Streptococcus bacterium such as Streptococcus thermophilus, Lactococcus lactis subspecies. Lactis, Lactococcus lactis Subspecies. Lactococcus bacteria such as Cremolis, Lactococcus plantarum, Lactococcus raffinoractis, Leuconostoc bacterium such as Leuconostoc mesenterotetes, Leuconostoc lactis, Enterococcus faecalis, Enterococcus faecium Bacteria etc. can be used.
[0042]
Further, Bifidobacterium bacteria such as Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium animaris, yeast and other microorganisms may be used. These can be used alone or in combination of two or more.
[0043]
In addition, other food materials, that is, various sugars, emulsifiers, thickeners, sweeteners, acidulants, fruit juices, and the like may be appropriately mixed with these foods. Specifically, sugars such as sucrose, isomerized sugar, glucose, fructose, palatinose, trehalose, lactose, xylose, sorbitol, xylitol, erythritol, lactitol, palatinit, reduced starch syrup, reduced maltose starch syrup, sucrose fatty acid Examples thereof include emulsifiers such as esters, glycerin fatty acid esters, and lecithin, and thickening (stabilizing) agents such as carrageenan, guar gum, xanthan gum, pectin, and locust bean gum. In addition to this, even when various vitamins such as vitamin A and vitamin B and minerals such as calcium, iron, manganese, and zinc are blended, an excellent flavor of light-resistant fermented milk can be obtained.
[0044]
When used in the form of these pharmaceuticals and foods, the conjugated fatty acid obtained by the method of the present invention can be appropriately blended. In addition, when appealing the physiological effect of conjugated fatty acids, an appropriate amount is added so that the effect can be obtained and an amount such as overdose is not caused, and an amount of intake of about 10 mg to 1000 mg / day is expected. Just keep it.
[0045]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not restrict | limited to these Examples at all.
[0046]
Example 1: Screening for lactic acid bacteria producing conjugated fatty acids
A linoleic acid-BSA-complex solution was prepared in advance by dissolving 50 mg of linoleic acid and 10 mg of BSA in 1 mL of 100 mM phosphate buffer (pH 6.5). Each bacterium was inoculated into 15 mL of 0.07% linoleic acid-containing MRS (LACTOBACILLI MRS BROTH, manufactured by DIFCO) medium, and cultured with shaking at 28 ° C., 120 rpm for 20 hours. The obtained culture broth was pH 4.7.
[0047]
The culture broth was centrifuged to collect the cells and washed twice with physiological saline to obtain washed cells. 100 μL of linoleic acid-BSA-complex solution and 0.9 mL of 100 mM phosphate buffer (pH 6.5) are added to the washed cells, and an oxygen absorption / carbon dioxide generator (Anaero Pack, Mitsubishi Gas Chemical Co., Ltd.) The reaction was carried out for 24 hours at 37 ° C. and 120 rpm in an oxygen-impermeable plastic bag kept in an anaerobic state.
[0048]
1 mg of internal standard substance (HEPTADECANOIC ACID) was added to the resulting reaction solution, extracted by the Bligh-Dyer method, methyl esterified (4% hydrochloric acid in methanol for 30 minutes at room temperature), and analyzed by gas chromatography. The fatty acid analysis was performed.
[0049]
[Table 2]
The peak of conjugated linoleic acid was judged on the basis of the retention time of a standard product (CLA80 manufactured by Linoleum Fats and Oils), and the relative value when the linoleic acid added as a substrate was taken as 100 was calculated. As a result, as shown in Table 2.Lactobacillus orisProduction of conjugated linoleic acid was observed. thisLactobacillus oris NCD02160 is registered with the American Type Culture Collection (ATCC) as ATCC49062.
[0051]
Example 2: Identification of isomers of conjugated fatty acids
In Example 1Lactobacillus orisThe isomers of conjugated linoleic acid produced by were investigated. Figure 1Lactobacillus oris2 is a gas chromatogram chart of an isomer of conjugated linoleic acid produced by As shown in Figure 1Lactobacillus orisAll of the conjugated linoleic acid produced by cis-9, trans-11 type conjugated linoleic acid. From the above results, it was revealed that Lactobacillus oris produces cis-9, trans-11 conjugated linoleic acid selectively and efficiently among a number of lactic acid bacteria.
[0052]
Example 3: Production of conjugated fatty acids in fermented milk
In 1 mL of 100 mM phosphate buffer (pH 6.5), 50 mg of linoleic acid and 10 mg of BSA were dissolved to prepare a linoleic acid-BSA-complex solution in advance. After adding 200 μL of this linoleic acid-BSA-complex solution to 15 mL of MRS (LACTOBACILLI MRS BROTH, manufactured by DIFCO) medium,Lactobacillus orisAnd cultured with shaking at 28 ° C. and 120 rpm for 20 hours to prepare a culture solution having a pH of 4.7.
[0053]
100 μL of linoleic acid-BSA-complex solution in 10% skim milk (glucose 1%, soybean peptide 0.1% added) medium dispensed 5 mL into a 15 mL test tube (with cap) Was added and the cap was closed. This medium was cultured at 28 ° C. and 120 rpm for 48 hours to prepare fermented milk having a pH of 4.6, and fatty acid analysis was performed in the same manner as in Example 1.
[0054]
As a result, 2.0% of the added linoleic acid was converted to conjugated linoleic acid. All of the produced conjugated linoleic acid was cis-9, trans-11 type conjugated linoleic acid, and the same results as in Example 2 were obtained. From the above results, even in fermented milkLactobacillus orisWas found to produce cis-9, trans-11 conjugated linoleic acid selectively and efficiently.
[0055]
Example 4:Lactobacillus orisProduction of conjugated fatty acids by dead cells
50 mg of linoleic acid and 10 mg of BSA were dissolved in 1 mL of 100 mM phosphate buffer (pH 6.5) to prepare a linoleic acid-BSA-complex solution in advance. After adding 200 μL of this linoleic acid-BSA-complex solution to 15 mL of MRS (LACTOBACILLI MRS BROTH, manufactured by DIFCO) medium,Lactobacillus orisAnd cultured with shaking at 28 ° C. and 120 rpm for 20 hours to prepare a culture solution having a pH of 4.7.
[0056]
0.5 mL of this culture solution is inoculated into 15 mL of 0.3% glc-added ILS medium, cultured at 37 ° C. and 120 rpm for 18 hours (up to pH 5.6), centrifuged, and the cells are collected and 0.2 M glycine buffer (pH 10). Washed twice in .6), and after washing, the cells were dissolved in 2 mL of a 6.7% sucrose-50 mM trisaminomethane-1 mM EDTA solution. To this solution, lysozyme solution (10 mg / mL in 25 mM Trisaminomethane, pH 8.0, manufactured by Seikagaku Corporation) 0.8 mL and N-Acetylmuramidase aqueous solution (1 mg / mL, manufactured by Seikagaku Corporation) 0.15 mL was added and reacted at 37 ° C. and 120 rpm for 30 minutes. By transferring the reaction solution to Centriprep 10 (manufactured by amicon) and performing a concentration operationLactobacillus orisAbout 0.6 mL of dead cell suspension
Obtained.
[0057]
To this dead cell suspension, 100 μL of linoleic acid-BSA complex solution and 0.9 mL of 100 mM phosphate buffer (pH 6.5) were added, and an oxygen absorption / carbon dioxide generator (Anaero Pack, Mitsubishi Gas Chemical ( The reaction was carried out for 24 hours at 37 ° C. and 120 rpm in an oxygen-impermeable plastic bag kept in an anaerobic state.
[0058]
As a result of conducting a fatty acid analysis in the same manner as in Example 1 for the obtained reaction solution, 1.6% of the added linoleic acid was converted to conjugated linoleic acid. All of the produced conjugated linoleic acid was cis-9, trans-11 type conjugated linoleic acid, and the same results were obtained as in washed cells and fermented milk. From the above results, even dead cells that destroyed the cell wallLactobacillus orisWas found to produce cis-9, trans-11 conjugated linoleic acid selectively and efficiently.
[0059]
Example 5:Lactobacillus pontisProduction of conjugated fatty acids
50 mg of linoleic acid and 10 mg of BSA were dissolved in 1 mL of 100 mM phosphate buffer (pH 6.5) to prepare a linoleic acid-BSA-complex solution in advance. After adding 200 μL of this linoleic acid-BSA-complex solution to 15 mL of MRS (LACTOBACILLI MRS BROTH, manufactured by DIFCO) medium,Lactobacillus pontis (ATCC51518) was inoculated and cultured with shaking at 28 ° C. and 120 rpm for 48 hours to prepare a culture solution having a pH of 4.9.
[0060]
100 μL of the linoleic acid-BSA-complex solution and 400 μL of the previously prepared culture solution of each strain were added to 10% skim milk dough dispensed 5 mL into a 15 mL test tube (with cap), and the cap was closed. This medium was cultured at 28 ° C. and 120 rpm for 48 hours to prepare fermented milk having a pH of 5.5, and fatty acid analysis was performed in the same manner as in Example 1.
[0061]
As a result, 0.5% of the added linoleic acid wasConvert to conjugated linoleic acidIt had been. All of the conjugated linoleic acid produced was cis-9, trans-11 type conjugated linoleic acid. From the above results,Lactobacillus pontisWas found to produce cis-9, trans-11 conjugated linoleic acid selectively and efficiently.
[0062]
【The invention's effect】
As described above, according to the method for producing a conjugated unsaturated fatty acid of the present invention, there is an effect that a cis-9, trans-11 type conjugated unsaturated fatty acid can be selectively produced with high efficiency.
[Brief description of the drawings]
FIG. 1 is a gas chromatogram chart of isomers of conjugated linoleic acid produced by Lactobacillus oris.
Claims (4)
前記二重結合を少なくとも二つ以上有する不飽和脂肪酸がリノール酸であり、前記共役化する工程によって、 cis-9,trans-11 型の共役リノール酸を得ることを特徴とする共役リノール酸の製造法。 Look including the step of conjugating an unsaturated fatty acid having at least two double bonds with the lactic acid bacteria of Lactobacillus oris (Lactobacillus oris) and / or Lactobacillus Pontis (Lactobacillus pontis),
Production of conjugated linoleic acid , wherein the unsaturated fatty acid having at least two double bonds is linoleic acid, and conjugated linoleic acid of cis-9, trans-11 type is obtained by the conjugating step Law.
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| JP2002110773A JP4213906B2 (en) | 2002-04-12 | 2002-04-12 | Method for producing conjugated linoleic acid |
| DE60329672T DE60329672D1 (en) | 2002-04-12 | 2003-04-11 | PROCESS FOR PRODUCING CONJUGATED FATTY ACID AND FOOD / BEVERAGES PRODUCED BY THIS METHOD |
| EP03746461A EP1500706B1 (en) | 2002-04-12 | 2003-04-11 | Process for producing conjugated fatty acid and food/drink obtained by the process |
| US10/511,230 US20050208195A1 (en) | 2002-04-12 | 2003-04-11 | Process for producing conjugated fatty acid and food/drink obtained by the process |
| KR1020047016341A KR100928890B1 (en) | 2002-04-12 | 2003-04-11 | Method for producing conjugated fatty acid and food and drink obtained by the method |
| PCT/JP2003/004633 WO2003087385A1 (en) | 2002-04-12 | 2003-04-11 | Process for producing conjugated fatty acid and food/drink obtained by the process |
| AU2003236111A AU2003236111A1 (en) | 2002-04-12 | 2003-04-11 | Process for producing conjugated fatty acid and food/drink obtained by the process |
| CA2481894A CA2481894C (en) | 2002-04-12 | 2003-04-11 | Process for producing conjugated fatty acid and food/drink produced by the process |
| AT03746461T ATE445707T1 (en) | 2002-04-12 | 2003-04-11 | METHOD FOR PRODUCING CONJUGATE FATTY ACID AND FOODS/BEVERAGES PRODUCED BY THIS METHOD |
| US12/082,917 US20080227165A1 (en) | 2002-04-12 | 2008-04-15 | Process for producing conjugated fatty acid and food/drink produced by the process |
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