【発明の詳細な説明】（産業上の利用分野）本発明は、リン脂質を原料として酵素を用いて改質する
方法、詳しくは天然リン脂質の脱アシル化によりリゾリ
ン脂質を得る方法に関する。DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method of modifying phospholipids as raw materials using enzymes, and more particularly to a method of obtaining lysophospholipids by deacylation of natural phospholipids.
（従来の技術）リン脂質を脱アシル化する酵素はホスホリパーゼＡと特
定のリパーゼがある。(Prior Art) Enzymes that deacylate phospholipids include phospholipase A and specific lipases.
これらの酵素による脱アシル化は、いずれも最適ｐＨ範
囲、最適緩衝液、及びカルシウムイオンを要求する条件
で、水溶液とリン脂質溶液との乳化による界面反応で進
行する。従来、これらの条件を考慮した酵素分解方法と
して、次に示すような方法が知られている。Deacylation by these enzymes proceeds through an interfacial reaction by emulsification of an aqueous solution and a phospholipid solution under conditions that require an optimal pH range, an optimal buffer solution, and calcium ions. Conventionally, the following methods are known as enzymatic decomposition methods that take these conditions into consideration.
■腸内で脂質系の物質の吸収を促進する組成物による方
法（特開平１ −１７５９４３号）■リン脂質の酵素分
解方法（特開昭６４ − １６５９５号）■リン脂質の酵素分解方法（特開昭６３ − ４４８９
３号）■リン脂質の改質方法（特開昭６３ − ４２６９１号
）■乳化剤組成物の製造法（特開昭６３ − ３０２９
２９号）しかるに、これら従来の酵素分解方法では次の
様な問題が生じる。■の方法は、リン脂質またはリン脂
質と油脂の混合物に０．１４重量部の水を加えた乳化系
反応であり、生戒物の精製や脱水が難しい。しかも反応
時間が２４時間と長いにもかかわらず反応収率が７割と
低い。■と■の方法は、リン脂質またはリン脂質と油脂
の混合物に０．１〜１．０重量部の水を加えた乳化系反
応であり、生成物の精製や脱水が難しい。しかも反応収
率が８割と低い。■Method using a composition that promotes the absorption of lipid-based substances in the intestines (Japanese Patent Application Laid-open No. 1-175943) ■Method for enzymatic decomposition of phospholipids (Japanese Patent Laid-open No. 16595-1982) ■Method for enzymatic decomposition of phospholipids Kaisho 63-4489
No. 3) ■ Method for modifying phospholipids (JP-A No. 63-42691) ■ Method for producing emulsifier compositions (JP-A-63-3029)
(No. 29) However, these conventional enzymatic decomposition methods cause the following problems. Method (2) is an emulsification reaction in which 0.14 parts by weight of water is added to phospholipids or a mixture of phospholipids and fats and oils, and it is difficult to purify and dehydrate the raw materials. Moreover, despite the long reaction time of 24 hours, the reaction yield is as low as 70%. Methods (1) and (2) are emulsion-based reactions in which 0.1 to 1.0 parts by weight of water is added to phospholipids or a mixture of phospholipids and oils, and it is difficult to purify and dehydrate the products. Moreover, the reaction yield is as low as 80%.
■の方法は、緩衝液中にリン脂質を１０重量％分敗させ
た乳化系反応であり、生戒物の精製や脱水が難しい。し
かも反応収率は最適条件でも８割以下である。■の方法
は、リン脂質またはリン脂質と油脂の混合物にホスホリ
パーゼＡ２とリパーゼを添加し、反応時にｐＨ調整用の
水酸化ナトリウム溶液とカルシウム供給用の塩化カルシ
ウム溶液を使用する乳化系反応である。しかも反応収率
が低く生戒物の精製が難しい。Method (2) is an emulsification reaction in which 10% by weight of phospholipids are dissolved in a buffer solution, and it is difficult to purify and dehydrate raw materials. Moreover, the reaction yield is less than 80% even under optimal conditions. Method (2) is an emulsification reaction in which phospholipase A2 and lipase are added to phospholipids or a mixture of phospholipids and fats and oils, and a sodium hydroxide solution for pH adjustment and a calcium chloride solution for calcium supply are used during the reaction. Moreover, the reaction yield is low and it is difficult to purify the raw materials.
（発明が解決しようとする課Ｂ）本発明では、工業的に大量生産が可能な天然リン脂質又
は天然リン脂質と油脂の混合物を原料とした酵素分解に
よるリゾリン脂質の製造法を検討した．リゾリン脂質の製造法を工業的な生産方法に拡大するに
は、従来法では次のような三つの問題点がある。第一点
は、この反応はリン脂質を溶解した有機溶媒と水溶液と
の界面反応であり、単位容積当たりの反応面積を保持す
るため、反応中は激しい撹拌を続ける必要があり、それ
でも反応収率は８０〜９０％程度で、収率に限界がある
ことである。(Problem B to be Solved by the Invention) In the present invention, a method for producing lysophospholipids by enzymatic decomposition using natural phospholipids or a mixture of natural phospholipids and fats and oils as raw materials, which can be industrially mass-produced, was investigated. In order to expand the production method of lysophospholipids to an industrial production method, the following three problems exist in the conventional method. The first point is that this reaction is an interfacial reaction between an organic solvent in which phospholipids are dissolved and an aqueous solution, and in order to maintain the reaction area per unit volume, it is necessary to continue vigorous stirring during the reaction, but even so, the reaction yield is The yield is about 80 to 90%, and there is a limit to the yield.
第二点は、反応生底物のリゾリン脂質が強い親水性界面
活性を示すため、反応液からの脱水に際し、リゾリン脂
質が水中にξセルとして可溶化し、リゾリン脂質の収量
が低下することである。第三点は、リン脂質が含水系で
発泡性を示すため、反応液からの減圧脱水に際し、著し
い発泡現象が生じて脱水が面倒であることである。The second point is that the lysophospholipids in the reaction raw material exhibit strong hydrophilic surface activity, so when the reaction solution is dehydrated, the lysophospholipids are solubilized as ξ cells in water, reducing the yield of lysophospholipids. be. The third point is that since phospholipids are water-containing and exhibit foaming properties, a significant foaming phenomenon occurs during vacuum dehydration from the reaction solution, making dehydration troublesome.
また、従来法では水溶液としてｐＨ４Ｊｊｌ整用の緩衝
液やカルシウム供給用の塩類溶液を使用するため、次の
ような二つの問題点がある。第一点は、上記の水溶液に
用いる無機物質が反応生戒物に混入することである．反
応規模を拡大すると、この混入物の処理工程が必要とな
るが、現在、リン脂質中に混入する無機物質の効率良い
除去方法は確立されていない。第二点は、カルシウム塩
溶液を用いた大量の分解反応では、水に可溶でクロロホ
ルム／メタノール系溶媒に不溶な物質が、反応生戒物中
にしばしば出現することである。この物質はリゾリン脂
質と水に対する挙動が類似しているため、反応生戒物中
から除去することが難しい。Furthermore, in the conventional method, a buffer solution for adjusting the pH to 4Jjl or a salt solution for supplying calcium is used as the aqueous solution, so there are the following two problems. The first point is that the inorganic substances used in the aqueous solution mentioned above contaminate the reactants. Expanding the scale of the reaction requires a step to treat these contaminants, but at present no efficient method for removing inorganic substances mixed in phospholipids has been established. The second point is that in large-scale decomposition reactions using calcium salt solutions, substances that are soluble in water but insoluble in chloroform/methanol solvents often appear in the reactants. Since this substance behaves similarly to lysophospholipid and water, it is difficult to remove it from the reactant.
即ち、本発明の目的は、上記課題を解決し、天然リン脂
質又は天然リン脂質と油脂の混合物から、リゾリン脂質
を酵素分解方法を用いて収率良く製造し、しかも反応生
戒物の精製が容易な工業的製法を提供することである。That is, an object of the present invention is to solve the above-mentioned problems, to produce lysophospholipids in high yield from natural phospholipids or a mixture of natural phospholipids and fats and oils by using an enzymatic decomposition method, and to purify the reactive substances. The objective is to provide an easy industrial manufacturing method.
（課題を解決するための手段）本発明は、リン脂質を酵素を用いて改質する際に、天然
リン脂質又は天然リン脂質と油脂の混合物を原料とし、
ホスホリパーゼＡ！、ホスホリパーゼＡ２活性を有する
リパーゼおよびパンクレアチンから選ばれる一種以上の
酵素を用いて、非イオン性無極性有機溶媒に０．０１〜
２容量％の水を加えた反応液中で、加水分解反応させ、
リゾリン脂質を得ることを特徴とするリン脂質の酵素改
質方法である。(Means for Solving the Problems) The present invention uses natural phospholipids or a mixture of natural phospholipids and fats and oils as raw materials when modifying phospholipids using enzymes,
Phospholipase A! , a lipase having phospholipase A2 activity, and one or more enzymes selected from pancreatin, in a nonionic nonpolar organic solvent with a concentration of 0.01~
A hydrolysis reaction is carried out in a reaction solution to which 2% by volume of water is added,
A method for enzymatic modification of phospholipids characterized by obtaining lysophospholipids.
本発明において用いる天然リン脂質は、天然レシチンと
呼ばれる天然脂質原料から脱脂処理をしたり、また、そ
の脱脂処理物からホスファチジルコリンなどの特定リン
脂質を濃縮することによって得られる。天然リン脂質と
油脂の混合物には天然品と工業製品がある。天然品は食
品工業でレシチンと呼ばれる前述の天然レシチンが相当
する。The natural phospholipid used in the present invention can be obtained by defatting a natural lipid raw material called natural lecithin, or by concentrating specific phospholipids such as phosphatidylcholine from the defatted product. Mixtures of natural phospholipids and fats and oils include natural products and manufactured products. The natural product corresponds to the aforementioned natural lecithin, which is called lecithin in the food industry.
この天然レシチンには、動物性の卵黄レシチンや植物性
の大豆レシチン、菜種レシチン、サフラワーレシチン、
綿実レシチン、ひまわりレシチン等が挙げられる．一般
に天然レシチンはリン脂質を６０〜７０％含む油脂との
混合物である。工業製品には、ホスファチジルコリンを
３０〜９５％に濃縮する様な天然レシチン中のリン脂質
！戒を変えたＭｉ戒物を３０〜７０％含む油脂又は油脂
と脂肪酸との混合物がある。さらに、供給の面から大豆
レシチンと卵黄レシチンを起源とするものが有利である
。This natural lecithin includes animal egg yolk lecithin, vegetable soybean lecithin, rapeseed lecithin, safflower lecithin,
Examples include cottonseed lecithin and sunflower lecithin. Generally, natural lecithin is a mixture with fats and oils containing 60 to 70% phospholipids. Industrial products include phospholipids in natural lecithin that concentrate phosphatidylcholine to 30-95%! There are oils and fats or mixtures of oils and fatty acids containing 30 to 70% of Mi precepts with different precepts. Furthermore, from the viewpoint of supply, those originating from soybean lecithin and egg yolk lecithin are advantageous.
本発明に用いる酵素は、ホスホリパーゼＡ２、ホスホリ
パーゼＡ！活性を有するリパーゼ及びパンクレアチンか
ら選ばれる１種以上のものである．ホスホリパーゼＡ２
は、ヘビ毒由来、ハチ毒由来、細菌由来及び牛や豚の膵
臓由来の公知のものが何れも使用出来る。この中でもホ
スホリパーゼＡ２活性の力価、酵素の価格及び除去の簡
便さから豚膵臓由来のものが好ましい。ホスホリパーゼ
Ａ２活性を有するリパーゼは豚膵臓由来、及び微生物由
来の公知のものが何れも使用出来る。この中でもホスホ
リパーゼＡ２活性の高い微生物由来のリパーゼとしてア
スペルギルス・ニガー、ムコール・ミーハイ、リゾープ
ス・ジャパニカス、リゾーブス・ニベウスなどが好まし
い。特にホスホリパーゼＡ２活性の力価、及び酵素の価
格からアスベルギルス・ニガー由来のものが好ましい。The enzymes used in the present invention are phospholipase A2 and phospholipase A! One or more types selected from active lipase and pancreatin. Phospholipase A2
Any of the known ones derived from snake venom, bee venom, bacteria, and cow or pig pancreas can be used. Among these, those derived from pig pancreas are preferred because of the potency of phospholipase A2 activity, the cost of the enzyme, and the ease of removal. As the lipase having phospholipase A2 activity, any known lipase derived from pig pancreas or microorganism can be used. Among these, as lipases derived from microorganisms with high phospholipase A2 activity, Aspergillus niger, Mucor miehai, Rhizopus japanicus, Rhizopus niveus, etc. are preferred. Particularly preferred is one derived from Asbergillus niger in view of the potency of phospholipase A2 activity and the price of the enzyme.
パンクレアチンは、牛や豚の膵臓由来のものが使用出来
る。特に豚膵臓由来のパンクレアチンは酵素の価格が非
常に安価であるので好ましい。Pancreatin can be derived from cow or pig pancreas. In particular, pancreatin derived from pig pancreas is preferred because the enzyme is very inexpensive.
本発明における加水分解は、例えば、天然リン脂質又は
天然リン脂質と油脂の混合物の１部を、後述する有機溶
媒１０〜５００部に溶解し、これに酵素を加えて行われ
る。酵素量は酵素の種類、純度、及び力価によって変化
する。また、酵素量は天然リン脂質と油脂の混合物では
、リン脂質の含量によって変化する。ホスホリパーゼＡ
２やホスホリパーゼＡｔ活性を有するリパーゼの場合、
リン脂質１ｇに１ ． ０００〜１００，０００ユニン
トを添加する。Hydrolysis in the present invention is carried out, for example, by dissolving a part of a natural phospholipid or a mixture of a natural phospholipid and an oil in 10 to 500 parts of an organic solvent described below, and adding an enzyme to the solution. The amount of enzyme varies depending on the type, purity, and potency of the enzyme. Furthermore, in a mixture of natural phospholipids and fats and oils, the amount of enzyme changes depending on the content of phospholipids. Phospholipase A
2 or a lipase with phospholipase At activity,
1 g of phospholipid. 000 to 100,000 units.
従って、使用する酵素のユニット数によって添加重量は
変化する．パンクレアチンの場合、リン脂質１ｇに０．
５〜２．０ｇを添加する。Therefore, the weight added will vary depending on the number of enzyme units used. In the case of pancreatin, 1g of phospholipid contains 0.
Add 5-2.0g.
反応温度は、１０℃から使用する有機溶媒の沸点までの
範囲で任意に選択できるが、反応効率を考えると、室温
から５０℃までの温度が望ましい。また、この方法は酵
素の熱安定性が良く、例えば豚膵臓由来のホスホリパー
ゼＡ２は７０℃でも活性を示す．反応時間は酵素量や反
応温度によって異なるが、通常１〜１０時間程度である
。また、従来の大量の水を使用する界面反応においては
、撹拌速度が反応進行率に大きな影響を与えたが、本発
明では通常６０〜１５０ｒｐｍの低速撹拌で充分に反応
が進行する．また、従来の酵素を用いるリン脂質の加水
分解反応では、反応進行に大量の水、ｐｔ＋調整用の緩
衝液、及びカルシウム塩溶液の添加が必要であった。し
かし本発明では、これらの溶液を必要としないため、反
応生戒物中に無機物や大量の水が共存せず、反応終了後
の精製も容易である。The reaction temperature can be arbitrarily selected within the range from 10°C to the boiling point of the organic solvent used, but in consideration of reaction efficiency, a temperature from room temperature to 50°C is desirable. In addition, this method provides good thermostability of the enzyme; for example, phospholipase A2 derived from pig pancreas shows activity even at 70°C. The reaction time varies depending on the amount of enzyme and reaction temperature, but is usually about 1 to 10 hours. In addition, in conventional interfacial reactions using large amounts of water, the stirring speed had a large effect on the rate of reaction progress, but in the present invention, the reaction progresses sufficiently with low-speed stirring of usually 60 to 150 rpm. Furthermore, in the conventional hydrolysis reaction of phospholipids using enzymes, it was necessary to add a large amount of water, a buffer for adjusting pt+, and a calcium salt solution for the reaction to proceed. However, in the present invention, since these solutions are not required, inorganic substances and large amounts of water do not coexist in the reactants, and purification after the reaction is completed is easy.
本発明における酵素反応は加水分解反応であるため、加
水分解に必要な最少量の水の添加が望ましい。水は有機
溶媒中に０．０１〜２容量％含まれるように添加する。Since the enzyme reaction in the present invention is a hydrolysis reaction, it is desirable to add the minimum amount of water necessary for hydrolysis. Water is added in an amount of 0.01 to 2% by volume in the organic solvent.
この範囲の水量では、反応終了後の溶媒による再沈、乾
燥濃縮、濾過剤処理等の精製手段により容易に脱水され
、特別な脱水工程を必要としない。水の添加が０．０１
容量％未満では充分に加水分解が行われず、２容量％を
超えると、水除去に伴う反応液の発泡現象、反応生成物
の損失等を生じ、また、余分な脱水工程が必要になり、
コストと時間がかかってしまう。If the amount of water is within this range, it can be easily dehydrated by purification means such as reprecipitation with a solvent, dry concentration, and treatment with a filtering agent after the reaction, and no special dehydration step is required. Addition of water is 0.01
If it is less than 2% by volume, sufficient hydrolysis will not occur, and if it exceeds 2% by volume, foaming of the reaction solution due to water removal, loss of reaction products, etc. will occur, and an extra dehydration step will be required.
It costs money and time.
本発明に用いる非イオン性無極性有機溶媒は、エーテル
、炭化水素、エステルの群から選ばれるが、具体例とし
ては、例えば、エーテルとしてジエチルエーテル、イソ
プロビルエーテル、ジオキサン、テトラヒド口フラン等
、炭化水素としてｎ−へキサン、ｎ−へブタン、石油エ
ーテル、シクロヘキサン等、エステルとして酢酸メチル
、酢酸エチル等が挙げられる。これらの有機溶媒は非イ
オン性無極性溶媒であり、酵素はこれら溶媒との接触で
は活性が低下しない。また基質である天然リン脂質又は
天然リン脂質と油脂の混合物全体を溶解することが出来
、酵素及び少量含まれる水と撹拌することにより、微小
界面を形威し、反応を進行させることが出来る。この溶
媒中で反応物のリゾリン脂質は微少の水層に移行し、恰
もこの水は相間移動触媒の役割を果たすことが出来る。The nonionic nonpolar organic solvent used in the present invention is selected from the group of ethers, hydrocarbons, and esters. Specific examples include ethers such as diethyl ether, isopropyl ether, dioxane, tetrahydrofuran, etc. Examples of hydrogen include n-hexane, n-hebutane, petroleum ether, and cyclohexane, and examples of ester include methyl acetate and ethyl acetate. These organic solvents are nonionic, nonpolar solvents, and the activity of enzymes does not decrease when they come into contact with these solvents. In addition, it is possible to dissolve the entire substrate of natural phospholipid or a mixture of natural phospholipid and oil, and by stirring it with the enzyme and a small amount of water, it is possible to form a micro-interface and allow the reaction to proceed. In this solvent, the reactant lysophospholipid migrates to a minute water layer, and this water can function as a phase transfer catalyst.
また、この加水分解条件では、使用する酵素のリパーゼ
活性が発現しないため、リン脂質と共存する油脂はわず
かしか分解を受けない。一方、油脂以外に脂肪酸が共存
してもリン脂質の加水分解率に大きな影響を与えない。Furthermore, under these hydrolysis conditions, the lipase activity of the enzyme used is not expressed, so the fats and oils coexisting with phospholipids are only slightly degraded. On the other hand, the coexistence of fatty acids in addition to fats and oils does not significantly affect the hydrolysis rate of phospholipids.
前述以外の有機溶媒、例えば、ハロゲン化炭化水素やア
ブロテックな非イオン性極性溶媒では、酵素の活性が発
揮出来ない．また、ジクロロエタン、クロロホルム等の
ハロゲン化炭化水素中では酵素が失活する。さらに、メ
タノール、エタノール等は酵素の阻害剤であり、アセト
ン、アセトニトリル、ジメチルスルホキシド、ジメチル
ホルムアミド等の非イオン性極性溶媒は、蛋白質である
酵素を一部溶解し、酵素の構造を変化させ、活性を失わ
せる恐れがある。Organic solvents other than those mentioned above, such as halogenated hydrocarbons and nonionic polar solvents such as Abrotech, cannot exhibit enzyme activity. Furthermore, the enzyme is inactivated in halogenated hydrocarbons such as dichloroethane and chloroform. Furthermore, methanol, ethanol, etc. are enzyme inhibitors, and nonionic polar solvents such as acetone, acetonitrile, dimethyl sulfoxide, dimethyl formamide, etc. partially dissolve the protein enzyme, change the structure of the enzyme, and activate the enzyme. There is a risk of losing.
反応終了後は、エーテル洗浄とアセトンによる再結晶、
乾燥濃縮、濾過助剤による脱酵素等の公知の精製手段に
より容易に目的物が得られる。一般的には分解率は４０
％以上、特に反応溶媒と酵素の選択により、天然リン脂
質単独又は天然リン脂質と油脂の混合物でも分解率は９
０％以上となる。After the reaction is complete, wash with ether and recrystallize with acetone.
The desired product can be easily obtained by known purification methods such as dry concentration and deenzyme removal using a filter aid. Generally, the decomposition rate is 40
% or more, depending on the selection of the reaction solvent and enzyme, the decomposition rate of natural phospholipids alone or a mixture of natural phospholipids and fats and oils can be reduced to 9.
It will be 0% or more.
従来のホスホリパーゼＡ２を使用する界面反応では、ホ
スホリパーゼＡ，の窒素末端の疎水性部分が基質の界面
と結合して加水分解が進行する。In the conventional interfacial reaction using phospholipase A2, the hydrophobic portion of the nitrogen terminal of phospholipase A binds to the interface of the substrate, and hydrolysis proceeds.
一方、本発明においては、基質と酵素の疎水性部分が有
機溶媒に溶解し均一系に近い反応で進行し、その際、酵
素の活性は有機溶媒中でも保持される。また、微量に加
えられた水は有機溶媒中に分散される．（発明の効果）本発明によれば、下記のような効果が得られる。On the other hand, in the present invention, the substrate and the hydrophobic portion of the enzyme are dissolved in an organic solvent, and the reaction proceeds in a nearly homogeneous system, and at this time, the activity of the enzyme is maintained even in the organic solvent. Also, a small amount of water added is dispersed in the organic solvent. (Effects of the Invention) According to the present invention, the following effects can be obtained.
（１） 工業的に入手が可能な天然リン脂質原料と安
価な酵素を用い、リゾリン脂質が大量に生産出来る．（２）温和な反応条件で高収率で目的化学物が得られる
．（３） 反応液中に無機物を添加しないため、反応生
成物の精製が容易である。(1) Lysophospholipids can be produced in large quantities using industrially available natural phospholipid raw materials and inexpensive enzymes. (2) The target chemical can be obtained in high yield under mild reaction conditions. (3) Since no inorganic substances are added to the reaction solution, the reaction product can be easily purified.
（４）水添加量が微量なため、水除去に伴う反応液の発
泡現象、反応生戒物の損失、エネルギーの大量消費が防
止出来る。(4) Since the amount of water added is small, it is possible to prevent foaming of the reaction solution, loss of reactants, and large consumption of energy due to water removal.
《５）本発明で得られるリゾリン脂質は、高濃度のマグ
ネシウムやカルシウム等のイオン存在下でも強いＯ／Ｗ
型乳化を保持できる強親水性乳化剤として食品工業に応
用される。<5) The lysophospholipid obtained by the present invention has strong O/W properties even in the presence of high concentrations of ions such as magnesium and calcium.
It is applied in the food industry as a strong hydrophilic emulsifier that can maintain type emulsification.
以上の効果により、本発明はリゾリン脂質の工業的製造
法として極めて好適である。Due to the above effects, the present invention is extremely suitable as an industrial method for producing lysophospholipids.
（実施例）以下、実施例に基づき本発明を具体的に説明する。尚、
各例中、％は重量基準である．実施例１ナス型フラスコ中に、天然大豆レシチン（辻製油製、商
品名ＳＯＹ−ＳＬＰペースト、リン脂質６０％、脂肪酸
１４％、油脂分２６％）１０ｇ，エチルエーテル１，０
００ ｄ、及びレシターゼＩＯＬ（豚膵臓由来のホスホ
リパーゼＡ２、ノボインダストリー製、商品名） ９０
，０００ユニットを添加し、撹拌子で緩やかに撹拌しな
がら蒸留水ｌ１Ｒ１を加えた．室温で９時間放置した。(Examples) Hereinafter, the present invention will be specifically described based on Examples. still,
In each example, percentages are by weight. Example 1 In an eggplant-shaped flask, 10 g of natural soybean lecithin (manufactured by Tsuji Oil Co., Ltd., trade name SOY-SLP paste, 60% phospholipid, 14% fatty acid, 26% fat and oil), 1.0 g of ethyl ether
00 d, and recitase IOL (phospholipase A2 derived from porcine pancreas, manufactured by Novo Industries, trade name) 90
,000 units were added, and distilled water l1R1 was added while stirring gently with a stirrer. It was left at room temperature for 9 hours.
経時後、エチルエーテルをデカンテーションで除去し、
新しいエチルエーテル３００一を加えて撹拌し、再度、
デカンテーションでエチルエーテルを除去した．さらに
冷アセトン３００一で２回、撹拌洗浄とデカンテーショ
ンを繰り返し、粗生戒物を乾燥濃縮した。このｍ縮吻を
クロロホルム・メタノール同量混液３００−に溶解シ、
この溶液中に濾過助剤（ダイカライト・オリエント社製
、商品名ダイカライト・バーライト）３ｇを添加し、撹
拌後、濾過して、その母液から溶媒を留去した後、乾燥
して２．９ｇ （収率４８％）の黄色の軟らかいロウ状
物を得た。After time, ethyl ether was removed by decantation.
Add fresh ethyl ether 300 and stir again.
Ethyl ether was removed by decantation. Further, stirring and washing with 300 g of cold acetone and decantation were repeated twice to dry and concentrate the crude raw material. Dissolve this proboscis in a mixture of equal amounts of chloroform and methanol,
3 g of a filter aid (manufactured by Dicalite Orient Co., Ltd., trade name: Dicalite Barite) was added to this solution, stirred, filtered, and the solvent was distilled off from the mother liquor, followed by drying. 9 g (yield 48%) of a yellow soft waxy material was obtained.
ロウ状物の分析値は下記の通りであった。The analysis values of the waxy substance were as follows.
■ ＴＬＣ（薄層クロマトグラフィー）メルク社製Ｔ
Ｌ Ｃ （Ｐｌａｔｅｓ Ｓｉｌｉｃａ Ｇｅｌ ６０
）、２０Ｘ２０Ｃｌｌ，厚さ０．２５Ｂ，展開液：クロロホルム／メタノール／水６５／２５／４
（ｖ／ｖ／ｖ）Ｒｆ値：０．１０、０．３９、０．５１、０．５９、０
．６５発色剤１’，？’−ジクロロフルオレソセン試薬全スポット橙色、Ｒｆ０．１０と０．３９が強く呈色デ
ィトマーレスター試薬全スポット青色、Ｒｆ０．１０が強く呈色■ＴＬＣ−Ｆ
ＩＤ （イヤトロスキャン法）展開液：ＴＬＣと同様薄層棒：クロマロソド−ｓｎリゾリン脂質群 ９２％その他
８％実施例２ナス型フラスコ中に天然卵黄レシチン（キューピー社製
、商品名卵黄レシチンＰ Ｌ−１００、リン脂質９０％
、脂肪酸１０％）１０ｇＳｎ−ヘキサン５００ ｄ、お
よびリパーゼＡ「アマノ」６ （アスペルギルス・ニガ
ー由来のリパーゼ、天野製薬製、商品名）３６０，００
０ユニットを添加し、撹拌子で緩やかに撹拌しながら蒸
留水１）Ｒｌを加えた。４５℃で１０時間反応させた。■ TLC (Thin Layer Chromatography) Merck T
L C (Plates Silica Gel 60
), 20X20Cll, thickness 0.25B, developing solution: chloroform/methanol/water 65/25/4
(v/v/v) Rf value: 0.10, 0.39, 0.51, 0.59, 0
．． 65 color former 1',? '-Dichlorofluorescene reagent all spots orange, Rf0.10 and 0.39 strongly colored Ditmar-Lester reagent all spots blue, Rf0.10 strongly colored ■TLC-F
ID (Iyatroscan method) Developing solution: Same as TLC Thin layer bar: Chromarosodo-sn Lysophospholipid group 92% Others
8% Example 2 Natural egg yolk lecithin (manufactured by Kewpie Co., Ltd., trade name Egg yolk lecithin PL-100, 90% phospholipid) in an eggplant-shaped flask
, fatty acid 10%) 10 g Sn-hexane 500 d, and lipase A "Amano" 6 (lipase derived from Aspergillus niger, manufactured by Amano Pharmaceutical Co., Ltd., trade name) 360,00
0 units were added, and distilled water 1) Rl was added while gently stirring with a stirrer. The reaction was carried out at 45°C for 10 hours.
後の処理は実施例１に従い、５．８ｇ （収率６４％）
の黄色の軟らかいロウ状物を得た。The subsequent treatment was carried out according to Example 1, and 5.8 g (yield 64%)
A yellow soft waxy substance was obtained.
ロウ状物の分析値は下記の通りであった。The analysis values of the waxy substance were as follows.
■ＴＬＣ実施例１と同じ条件Ｒｆ値： ０．１３と０．２４に強＜ 、０．３６に弱
い発色が認められた。■TLC Same conditions as Example 1 Rf value: strong color development was observed at 0.13 and 0.24, and weak color development was observed at 0.36.
■ＴＬＣ−ＦＩＤ （イヤトロスキャン法）実施例ｌと
同じ条件リゾリン脂質群 ４０％ホスファチ
ジルコリン ４２％ホスファチジルエタノ
ールアミン １０％その他
８％実施例３ナス型フラスコ中に、天然大豆リン脂質（ナツターマン
社製、商品名ＳＯＹ−ＰＣ９５、リン脂質１００％）１
ｇ、酢酸エチル５０−、及び豚膵臓起源のパンクレアチ
ン（アメリカ薬局方、力価等量×１、シグマ社製）Ｉｇ
を添加し、撹拌子で緩やかに撹拌しながら蒸留水０．２
−を加えた。５０℃で４時間放置した．後の処理は実施
例１に従い０．６ｇ（収率６０％）の黄色の軟らかいロ
ウ状物を得た。■TLC-FID (Iyatroscan method) Same conditions as Example 1 Lysophospholipid group 40% phosphatidylcholine 42% phosphatidylethanolamine 10% others
8% Example 3 In an eggplant-shaped flask, 1 natural soybean phospholipid (manufactured by Natsuterman, trade name SOY-PC95, 100% phospholipid)
g, ethyl acetate 50-, and pancreatin derived from pig pancreas (American Pharmacopoeia, titer equivalent x 1, manufactured by Sigma) Ig
Add 0.2 of distilled water while stirring gently with a stirrer.
- was added. It was left at 50°C for 4 hours. The subsequent treatment was carried out in accordance with Example 1 to obtain 0.6 g (yield 60%) of a yellow soft waxy substance.
ロウ状物の分析値は下記の通りであった。The analysis values of the waxy substance were as follows.
■ＴＬＣ実施例１と同じ条件Ｒｆ値：Ｏ．＋３と０．２８に強い発色が認められた。■TLCSame conditions as Example 1Rf value: O. Strong color development was observed at +3 and 0.28.
■ＴＬＣ−ＦＩＤ （イヤトロスキャン法）実施例１と
同じ条件ホスファチジルコリン ５５％リゾホスフ
ァチジルコリン ４５％実施例４ナス型フラスコ中に、天然卵黄レシチン（キューピー社
製、商品名卵黄レシチンＰＬ−６０、リン脂［６０％、
油脂３５％）１０ｇ、ジオキサン５００ ｍｌ、及びレ
シターゼ１０Ｌ（豚膵臓由来のホスホリパーゼＡ，、商
品名、ノボインダストリー社製）４５．０００ユニット
を添加し、撹拌子で緩やかに撹拌しながら蒸留水１）Ｉ
１ｌを加えた。２５℃で９時問おいた。後の処理は実施
例１に従い、２．９ｇ （収率４９％）の黄色のロウ状
物を得た。■TLC-FID (Iyatroscan method) Same conditions as Example 1 Phosphatidylcholine 55% Lysophosphatidylcholine 45% Example 4 In an eggplant-shaped flask, put natural egg yolk lecithin (manufactured by Kewpie Co., Ltd., brand name Egg Yolk Lecithin PL-60, phospholipid). [60%,
Add 10 g of fat and oil (35%), 500 ml of dioxane, and 45,000 units of recitase (phospholipase A derived from pig pancreas, trade name, manufactured by Novo Industries), and add 10 g of distilled water while gently stirring with a stir bar. I
Added 1 liter. It was kept at 25°C for 9 hours. The subsequent treatment was carried out in accordance with Example 1 to obtain 2.9 g (yield 49%) of a yellow waxy substance.
ロウ状物の分析値は下記の通りであった。The analysis values of the waxy substance were as follows.
■ＴＬＣ実施例１と同じ条件Ｒｆ値：０．１５に強＜ 、０．２６と０．３８に弱い
発色が認められた。■TLC Same conditions as in Example 1 Rf value: strong color development was observed at 0.15 and weak color development at 0.26 and 0.38.
■ＴＬＣ−ＦＩＤ （イヤトロスキャン法）実施例１と
同じ条件リゾリン脂質群 ８５％ホスファ
チジルコリン ４％ホスファチジルエタノ
ールアミン ３％その他
６％実施例５ナス型フラスコ中に、工業製品の大豆レシチン（ルーカ
スマイヤー社製、商品名工ピクロン１３５Ｆ，リン脂質
５５％、脂肪酸１６％、油脂２９％）２ｇ、石油エーテ
ル５０＋ｄ及びリパーゼＦ「アマノ」 （商品名、リゾ
ブス・ジャパニカス由来のリパーゼ、天野製薬製） １
００，０００ユニットを添加し、撹拌子で緩やかに撹拌
しながら蒸留水０．１−を加えた。４０℃で３時間放置
した。後の処理は実施例１に従い、０．８ｇ （収率７
３％）の黄色のロウ状物を得た。■TLC-FID (Iyatroscan method) Same conditions as Example 1 Lysophospholipid group 85% phosphatidylcholine 4% phosphatidylethanolamine 3% others
6% Example 5 In an eggplant-shaped flask, 2 g of industrial product soybean lecithin (manufactured by Lukasmeyer, trade name Picron 135F, 55% phospholipids, 16% fatty acids, 29% fats and oils), petroleum ether 50+d, and lipase F "Amano ” (Product name: Lipase derived from Rhizobus japanicus, manufactured by Amano Pharmaceutical) 1
00,000 units were added, and 0.1 - of distilled water was added while gently stirring with a stirrer. It was left at 40°C for 3 hours. The subsequent treatment was carried out in accordance with Example 1, and 0.8 g (yield 7
3%) of a yellow waxy substance was obtained.
ロウ状物の分析値は下記の通りであった。The analysis values of the waxy substance were as follows.
■ＴＬＣ実施例１と同じ条件Ｒｆ値７ ０．１３と０．２９に強＜ 、０．３６に弱
い発色が認められた。■TLC Same conditions as Example 1 Rf value 7 Strong color development was observed at 0.13 and 0.29, and weak color development was observed at 0.36.
■ＴＬＣ−ＦＩＤ（イヤトロスキャン法）実施例１と同
じ条件リゾリン脂質群 ４２％ホスファチ
ジルコリン ４４％ホスファチジルエタノ
ールアミン １０％その他
４％実施例６ナス型フラスコ中に、天然大豆リン脂質（ユニ旦−ル社
製、商品名ボレソクＰＣ８０＋、リン脂質９０％）Ｉｇ
、酢酸エチル１０１Ｒｌ、及びレシターゼ１０Ｌ（豚膵
臓由来のホスホリパーゼＡ２、商品名、ノボインダスト
リー社製〉の脱水粉末８， ０００ユニットを添加し、
撹拌子で緩やかに撹拌しながら蒸留水０．０１ｄを加え
た。５５℃で３時問おいた。後の処理は実施例１に従い
、０．５ｇ （収率５６％）の黄色のロウ状物を得た。■TLC-FID (Iatroscan method) Same conditions as Example 1 Lysophospholipid group 42% phosphatidylcholine 44% phosphatidylethanolamine 10% others
4% Example 6 In an eggplant-shaped flask, natural soybean phospholipid (manufactured by Unitan, trade name Boresoku PC80+, phospholipid 90%) Ig
, ethyl acetate 101Rl, and 8,000 units of dehydrated powder of lecitase 10L (phospholipase A2 derived from pig pancreas, trade name, manufactured by Novo Industries) were added,
0.01 d of distilled water was added while stirring gently with a stirrer. It was incubated at 55°C for 3 hours. The subsequent treatment was carried out in accordance with Example 1 to obtain 0.5 g (yield 56%) of a yellow waxy substance.
ロウ状物の分析値は下記の通りであった。The analysis values of the waxy substance were as follows.
■ＴＬＣ実施例１と同じ条件Ｒｆ値：０．Ｉ２に強＜ 、０．２４に弱い発色が認め
られた。■TLC Same conditions as Example 1 Rf value: 0. Strong color development was observed in I2 and weak color development was observed in 0.24.
■ＴＬＣ−ＦＩＤ （イヤトロスキャン法）実施例１と
同じ条件ホスファチジルコリン ７％リゾホスファ
チジルコリン ９３％以上の結果から、収率は
原料リン脂質の加水分解率に反比例することがわかる。(2) TLC-FID (Iatroscan method) Same conditions as Example 1 Phosphatidylcholine 7% Lysophosphatidylcholine The results of 93% or more indicate that the yield is inversely proportional to the hydrolysis rate of the raw material phospholipid.
加水分解率が低いと、反応生戒物中に未分解のリン脂質
が存在する。また、本法は、リン脂質単独で反応を進行
させ、リン脂質に油脂や脂肪酸が大量に混入しても反応
を進行させることができた。後者の場合、原料中のリン
脂質含量に相対する酵素量の添加が必要であった。When the hydrolysis rate is low, undegraded phospholipids are present in the reactant. In addition, in this method, the reaction was able to proceed with the phospholipid alone, and even when the phospholipid was mixed with a large amount of fats and oils and fatty acids, the reaction was able to proceed. In the latter case, it was necessary to add an amount of enzyme relative to the phospholipid content in the raw material.
比較例１ナス型フラスコ中に、天然大豆レシチン（辻製油製、商
品名ＳＯＹ−ＳＬＰペースト、リン脂質６０％、脂肪酸
１４％、油脂分２６％）１０ｇ、メタノール１ ， ０
００ ＋ｄ、及びレシターゼ１０Ｌ（豚膵臓由来のホス
ホリパーゼＡ２、商品名、ノボインダストリ一社製）
９０．０００ユニットを添加し、撹拌子で緩やかに撹拌
しながら、蒸留水１−を加えた。室温で１０時間放置し
た．後の処理は実施例１に従い５．０ｇ（収率８３％）
の黄色の軟らかいロウ状物を得た。Comparative Example 1 In an eggplant-shaped flask, 10 g of natural soybean lecithin (manufactured by Tsuji Oil Co., Ltd., trade name SOY-SLP paste, 60% phospholipid, 14% fatty acid, 26% fat and oil), 1.0 g of methanol.
00 +d, and recitase 10L (phospholipase A2 derived from pig pancreas, trade name, manufactured by Novo Industries Co., Ltd.)
90,000 units were added, and 1-1 of distilled water was added while gently stirring with a stirrer. It was left at room temperature for 10 hours. The subsequent treatment was carried out in accordance with Example 1, 5.0 g (yield: 83%)
A yellow soft waxy substance was obtained.
ロウ状物の分析値は下記の通りであった。The analysis values of the waxy substance were as follows.
■ＴＬＣ実施例１と同じ条件Ｒｆ値： ０．３８と０．６４に強＜ 、０．１２に弱
い発色が認められた。■TLC Same conditions as Example 1 Rf value: Strong color development was observed at 0.38 and 0.64, and weak color development was observed at 0.12.
■ＴＬＣ−ＦＩＤ （イヤトロスキャン法）実施例１と
同じ条件ホスファチジルエタノールアくン ２０％ホスファチ
ジルコリン及びその他 ７５％リゾリン脂質群
５％以上の結果より、本法の酵素によ
る天然リン脂質の加水分解に関し、非イオン性無極性溶
媒を使用しない条件では分解率が低下することが判明し
た。■TLC-FID (Iyatroscan method) Same conditions as Example 1 Phosphatidylethanolamine 20% Phosphatidylcholine and others 75% Lysophospholipid group
From the results of 5% or more, it was found that with regard to the hydrolysis of natural phospholipids by the enzyme of this method, the decomposition rate is reduced under conditions where a nonionic nonpolar solvent is not used.
比較例２ナス型フラスコ中に、天然卵黄レシチン（キューピー社
製、商品名卵黄レシチンＰＬ−１００、リン脂質９０％
、脂肪酸１０％）１０ｇ，ｎ−ヘキサン５０〇一、及び
リパーゼＢサッポロ（シュードモナス・フレイジ由来の
リパーゼ、商品名、サッポロビール製） ３６０．ＱＯ
Ｏユニントを添加し、撹拌子で緩やかに撹拌しながら蒸
留水０．５−を加えた。３７℃で２４時間反応させた．
後の処理は実施例ｌに従い、７．５ｇ （収率８３％）
の黄色の軟らかいロウ状物を得た。Comparative Example 2 In an eggplant-shaped flask, natural egg yolk lecithin (manufactured by Kewpie Co., Ltd., trade name Egg Yolk Lecithin PL-100, 90% phospholipid) was added.
, fatty acid 10%) 10g, n-hexane 501, and Lipase B Sapporo (lipase derived from Pseudomonas phage, trade name, manufactured by Sapporo Breweries) 360. QO
0 unit was added, and 0.5-liter of distilled water was added while stirring gently with a stirrer. The reaction was carried out at 37°C for 24 hours.
The subsequent treatment was carried out according to Example 1, and 7.5 g (yield 83%)
A yellow soft waxy substance was obtained.
ロウ状物の分析値は下記の通りであった。The analysis values of the waxy substance were as follows.
■ＴＬＣ実施例１と同じ条件Ｒｆ値７ ０．３３と０．６２に強＜ 、０．１２に弱
い発色が認められた。■TLC Same conditions as Example 1 Rf value 7 Strong color development was observed at 0.33 and 0.62, and weak color development was observed at 0.12.
■ＴＬＣ−ＦＩＤ （イヤトロスキャン法）実施例１と
同じ条件ホスファチジルエタノールア≧ン １８％ホスファチ
ジルコリン及びその他 ７６％リゾリン脂質群
６％以上の結果より、本法の酵素によ
る天然リン脂質の加水分解に関し、ホスホリパーゼＡ２
活性を有しない酵素を使用する条件では分解率が低下す
ることが判明した。■TLC-FID (Iatroscan method) Same conditions as Example 1 Phosphatidylethanolone 18% phosphatidylcholine and others 76% lysophospholipid group
From the results of 6% or more, it was found that phospholipase A2
It was found that the decomposition rate decreased under conditions where an inactive enzyme was used.
比較例３実施例１の反応条件のうち、蒸留水の量を５０一に変え
た。エーテル層と水層が分離するので、全体を均一に保
つため、撹拌子で激しく撹拌し、乳化状態にした。反応
終了後、減圧下に溶媒を留去し、さらに連続的に脱水を
試みたが、反応液に発泡現象が認められた。そのため、
ベンゼン５０−を加えての減圧蒸留を３回繰り返して脱
水した。この蒸留残査を冷アセトン２００−で２回、撹
拌とデカンテーションを行い、粗生或物を得た。この粗
生戒物をクロロホルム／メタノール同量混液に溶解した
が、この溶媒に不溶な物質が出現した。この物質は、脱
水前の反応溶媒には認められなかった。Comparative Example 3 Among the reaction conditions of Example 1, the amount of distilled water was changed to 50-1. Since the ether layer and the aqueous layer were separated, in order to keep the whole mixture homogeneous, they were vigorously stirred with a stirring bar to form an emulsified state. After the reaction was completed, the solvent was distilled off under reduced pressure and further dehydration was attempted continuously, but a bubbling phenomenon was observed in the reaction solution. Therefore,
Dehydration was carried out by repeating vacuum distillation three times with addition of 50% of benzene. This distillation residue was stirred and decanted twice with 200 g of cold acetone to obtain a crude product. This crude raw material was dissolved in a mixture of equal amounts of chloroform and methanol, but a substance insoluble in this solvent appeared. This substance was not found in the reaction solvent before dehydration.
以上のように従来法では厳しい反応条件が要求され、さ
らに反応生底物の脱水や精製に特別な工程が必要である
。As described above, the conventional method requires severe reaction conditions and also requires special steps for dehydration and purification of the reaction raw material.
比較例４実施例４の反応条件のうち、レシターゼＩＯＬは脱水し
た乾燥品を使用し、蒸留水は無添加で、反応を行った。Comparative Example 4 Under the reaction conditions of Example 4, the reaction was carried out using a dehydrated dry recitase IOL and no distilled water was added.
後の処理は実施例１に従い５．１ｇ （収率８５％）の
黄色のロウ状物を得た。The subsequent treatment was carried out in accordance with Example 1 to obtain 5.1 g (yield: 85%) of a yellow waxy substance.
ロウ状物の分析値は下記の通りであった。The analysis values of the waxy substance were as follows.
■ＴＬＣ実施例１と同じ条件Ｒｆ値： ０．１５に弱＜ 、０．２５と０．３５に強
い発色が認められた。■TLC Same conditions as Example 1 Rf value: weak color development was observed at 0.15, and strong color development was observed at 0.25 and 0.35.
■ＴＬＣ−ＦＩＤ （イヤトロスキャン法）実施例ｌと
同じ条件ＬＰＬ群 １４％ホスファチジル
コリン ７ｌ％ホスファチジルエタノール
アミン １０％その他
５％以上の結果より、蒸留水を全《添加しない条件では
分解率が低下することが判明した。■TLC-FID (Iyatroscan method) Same conditions as Example 1 LPL group 14% phosphatidylcholine 7l% phosphatidylethanolamine 10% others
From the results of 5% or more, it was found that the decomposition rate decreased under conditions where no distilled water was added at all.
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP23295689AJPH0398590A (en) | 1989-09-11 | 1989-09-11 | Method for modifying phospholipid with enzyme |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP23295689AJPH0398590A (en) | 1989-09-11 | 1989-09-11 | Method for modifying phospholipid with enzyme |
| Publication Number | Publication Date |
|---|---|
| JPH0398590Atrue JPH0398590A (en) | 1991-04-24 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP23295689APendingJPH0398590A (en) | 1989-09-11 | 1989-09-11 | Method for modifying phospholipid with enzyme |
| Country | Link |
|---|---|
| JP (1) | JPH0398590A (en) |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5378623A (en)* | 1992-06-16 | 1995-01-03 | Sankyo Company, Limited | Phospholipase A1, process for its preparation and the use thereof |
| JP2009148244A (en)* | 2007-11-29 | 2009-07-09 | Gunma Prefecture | Method for producing lysophosphatidylethanolamine |
| JP2025105190A (en)* | 2023-12-28 | 2025-07-10 | 財團法人工業技術研究院 | Waste treatment system and method for converting waste to energy |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5378623A (en)* | 1992-06-16 | 1995-01-03 | Sankyo Company, Limited | Phospholipase A1, process for its preparation and the use thereof |
| US5521080A (en)* | 1992-06-16 | 1996-05-28 | Sankyo Company, Limited | Phospholipase A1, process for its preparation |
| US5538874A (en)* | 1992-06-16 | 1996-07-23 | Sankyo Company, Limited | Phospholipase A1, process for its preparation and the use thereof |
| JP2009148244A (en)* | 2007-11-29 | 2009-07-09 | Gunma Prefecture | Method for producing lysophosphatidylethanolamine |
| JP2025105190A (en)* | 2023-12-28 | 2025-07-10 | 財團法人工業技術研究院 | Waste treatment system and method for converting waste to energy |
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