JP2008220236A - Method for producing middle-chain and long-chain fatty acid monoglyceride - Google Patents
Method for producing middle-chain and long-chain fatty acid monoglyceride Download PDFInfo
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Abstract
Description
本発明は、脂肪酸モノグリセリドの製造方法に関する。 The present invention relates to a method for producing a fatty acid monoglyceride.
脂肪酸モノグリセリドとは、グリセリン１分子と脂肪酸１分子とがエステル結合した物質であり、モノアシルグリセロールまたはグリセリン脂肪酸エステルともいわれる油脂の一種である。脂肪酸モノグリセリドは、乳化剤（食品添加物）として加工食品（マーガリン、チョコレート、アイスクリーム、パン、ケーキなど）に幅広く使用されている。さらに、中鎖脂肪酸のモノグリセリドは、抗菌活性を有することも知られている。 Fatty acid monoglyceride is a substance in which one molecule of glycerin and one molecule of fatty acid are ester-bonded, and is a kind of fats and oils also referred to as monoacylglycerol or glycerin fatty acid ester. Fatty acid monoglycerides are widely used in processed foods (margarine, chocolate, ice cream, bread, cakes, etc.) as emulsifiers (food additives). Furthermore, monoglycerides of medium chain fatty acids are also known to have antibacterial activity.
現在、脂肪酸モノグリセリドは、高温条件下（２２０−２５０℃）でアルカリ触媒を用いて、あるいは無触媒下で脂肪酸とグリセリンとをエステル化する方法；高温・高圧条件下で、油脂をグリセロリシスする方法などの化学法により工業的に製造されている。しかし、これらの化学法による脂肪酸モノグリセリドの製造は、多くのエネルギーを必要とし、アルカリ排水処理が必要となるなどの問題がある。さらに、高温高圧条件下の化学法による製造方法は、熱、酸素などに対して不安定な不飽和脂肪酸を用いる脂肪酸モノグリセリドの製造には、適用できないという問題もある。 Currently, fatty acid monoglycerides are a method of esterifying a fatty acid and glycerin under high temperature conditions (220-250 ° C.) with or without an alkali catalyst; a method of glycerolysis of fats and oils under high temperature and high pressure conditions, etc. It is manufactured industrially by the chemical method. However, the production of fatty acid monoglycerides by these chemical methods has problems such as requiring a lot of energy and requiring alkaline wastewater treatment. Furthermore, the production method by the chemical method under high temperature and high pressure conditions also has a problem that it cannot be applied to the production of fatty acid monoglyceride using unsaturated fatty acid which is unstable to heat, oxygen and the like.
そこで、穏和な条件下で反応可能な酵素を用いて、脂肪酸モノグリセリドを製造する方法の研究がなされている。例えば、特許文献１には、リパーゼを用いて、高度不飽和脂肪酸を含有する脂肪酸またはそのエステルとグリセリンとから、高度不飽和脂肪酸含有部分グリセリドを製造する方法が記載されている。しかし、この方法で得られるグリセリド（アシルグリセロールという場合もある）に含まれる脂肪酸モノグリセリドは、最大でも１９％であること、およびグリセリンが高度不飽和脂肪酸に対してモル比で３０倍以上の量が必要であることからも、この方法は、実用的な脂肪酸モノグリセリドの製造方法ではない。 Thus, research has been conducted on methods for producing fatty acid monoglycerides using enzymes capable of reacting under mild conditions. For example, Patent Document 1 describes a method for producing a highly unsaturated fatty acid-containing partial glyceride from a fatty acid containing a highly unsaturated fatty acid or an ester thereof and glycerin using lipase. However, the fatty acid monoglyceride contained in the glyceride (sometimes referred to as acylglycerol) obtained by this method is at most 19%, and the amount of glycerin is 30 times or more in molar ratio to the highly unsaturated fatty acid. In view of the necessity, this method is not a practical method for producing fatty acid monoglycerides.
特許文献２には、ペニシリウム属に属する微生物から得られたモノおよびジグリセリドリパーゼを用いて、Ｃ８：０からＣ１８：０の炭素数が偶数個の脂肪酸またはオレイン酸（Ｃ１８：１）とグリセリンとから、部分グリセリド（脂肪酸モノグリセリドの純度、＞９５％）を製造する方法が記載されている（ここで、Ｃｎ：ｍは、炭素数がｎであり、二重結合数がｍ個の直鎖脂肪酸を表す。ｍが０のときは、直鎖飽和脂肪酸である。以下、本明細書において同様である）。しかし、この方法では、グリセリンを大過剰（１０倍モル以上）用いる必要があること、および脂肪酸のエステル化率が最大でも７５％であることから、効率的な方法ではない。 In Patent Document 2, mono- and diglyceride lipases obtained from microorganisms belonging to the genus Penicillium are used, and fatty acids having an even number of carbon atoms from C8: 0 to C18: 0 or oleic acid (C18: 1) and glycerin. , A method for producing partial glycerides (purity of fatty acid monoglycerides,> 95%) is described (where Cn: m is a linear fatty acid having n carbon atoms and m double bonds). When m is 0, it is a straight-chain saturated fatty acid (the same applies hereinafter). However, this method is not an efficient method because it is necessary to use glycerin in a large excess (10-fold mol or more) and the esterification rate of the fatty acid is 75% at the maximum.
特許文献３には、ペニシリウム属に属する微生物から得られたモノおよびジグリセリドリパーゼを用いて、共役脂肪酸とグリセリンとから、部分グリセリド（脂肪酸モノグリセリドが主成分）を製造する方法が記載されている。さらに、特許文献４には、ペニシリウム属に属する微生物から得られたモノおよびジグリセリドリパーゼを用いて、ω−３系高度不飽和脂肪酸とグリセリンとから、部分グリセリド（脂肪酸モノグリセリドが主成分）を製造する方法が記載されている。 Patent Document 3 describes a method for producing a partial glyceride (a fatty acid monoglyceride is a main component) from a conjugated fatty acid and glycerin using mono- and diglyceride lipase obtained from a microorganism belonging to the genus Penicillium. Furthermore, in Patent Document 4, partial glycerides (fatty acid monoglycerides are the main components) are produced from ω-3 highly unsaturated fatty acids and glycerin using mono- and diglyceride lipases obtained from microorganisms belonging to the genus Penicillium. A method is described.
これらの特許文献３および４には、（ｉ）０〜２０℃でエステル化反応を行い、反応途中で減圧してエステル化率を高める方法（特許文献３の段落番号０００８に記載の第１の方法の前半）、（ｉｉ）常温で、反応開始時から減圧下で反応を行う方法（特許文献３の第１の方法の後半、特許文献４、および非特許文献１）、（ｉｉｉ）常温でエステル化反応を行った後（途中から減圧）、０〜２０℃でグリセロリシスを行う方法(特許文献３の第２の方法)、などを用いることにより、脂肪酸のエステル化率が９６％以上で、脂肪酸モノグリセリドを９０％以上含む生成物が得られることが記載されている。 In these Patent Documents 3 and 4, (i) a method of performing an esterification reaction at 0 to 20 ° C. and reducing the pressure during the reaction to increase the esterification rate (the first method described in Paragraph No. 0008 of Patent Document 3) The first half of the method), (ii) a method in which the reaction is performed at room temperature under reduced pressure from the start of the reaction (second half of the first method of Patent Document 3, Patent Document 4, and Non-Patent Document 1), (iii) at room temperature The esterification rate of fatty acid is 96% or more by using a method of performing glycerolysis at 0 to 20 ° C. (second method of Patent Document 3) after performing the esterification reaction (reduced pressure from the middle), It is described that a product containing 90% or more of fatty acid monoglycerides can be obtained.
しかし、（ｉ）または（ｉｉｉ）の方法では反応温度を低温にしなくてはならず、多くのエネルギーを必要とし、さらに、反応液が固化するため大容量の反応の場合は、反応液の内部からの減圧脱水の効率が悪いなどの問題があり、実用的ではない。特に、（ｉｉｉ）の方法では、反応に１週間を要するため実用的ではない。一方で、（ｉｉ）の方法（３０℃、５ｍｍＨｇの減圧下で脱水）は、反応系が液状であることから、工業的な大容量の反応に適する。しかし、（ｉ）、（ｉｉ）、および（ｉｉｉ）のいずれの方法も、原料が共役脂肪酸およびω−３系高度不飽和脂肪酸に限定されており、その他の脂肪酸を用いた場合の脂肪酸モノグリセリドの製造方法については、開示されていない。すなわち、特許文献３および４に記載の方法によっては、限られた脂肪酸のみしか適用できない。後述の比較例に記載するように、この方法によってＣ１４：０などの脂肪酸を用いて脂肪酸モノグリセリドの製造を試みたが、脂肪酸のエステル化率が低く、純度の高い脂肪酸モノグリセリドを得ることができない。 However, in the method (i) or (iii), the reaction temperature must be lowered, and a large amount of energy is required. Further, since the reaction liquid is solidified, There is a problem that the efficiency of vacuum dehydration from is poor, and it is not practical. In particular, the method (iii) is not practical because the reaction takes one week. On the other hand, the method (ii) (dehydration at 30 ° C. under a reduced pressure of 5 mmHg) is suitable for industrial large-capacity reactions because the reaction system is liquid. However, in any of the methods (i), (ii), and (iii), the raw materials are limited to conjugated fatty acids and ω-3 polyunsaturated fatty acids, and fatty acid monoglycerides when other fatty acids are used. The manufacturing method is not disclosed. That is, only limited fatty acids can be applied by the methods described in Patent Documents 3 and 4. As will be described later in Comparative Examples, production of fatty acid monoglycerides using fatty acids such as C14: 0 was attempted by this method, but fatty acid monoglycerides with low purity and low purity cannot be obtained.
非特許文献２には、ペニシリウム属に属する微生物から得られたリパーゼを用いて、脂肪酸モノグリセリドの融点以下（すなわち、固形の反応系）、常圧下で反応させることにより、Ｃ１０：０、Ｃ１２：０、およびＣ１４：０の脂肪酸、ならびにオレイン酸（Ｃ１８：１）、リノール酸（Ｃ１８：２）、およびリノレン酸（Ｃ１８：３）とグリセリンとから、脂肪酸モノグリセリド（グリセリドに対する脂肪酸モノグリセリドの純度、９８％以上）を製造する方法が開示されている。しかし、この方法では、いずれの脂肪酸においても、エステル化率が最大でも８７％であること、オレイン酸、リノール酸、およびリノレン酸のモノグリセリドは、室温以下の低温で反応させなければならず、反応に多くのエネルギーを必要とするなどの問題がある。 Non-Patent Document 2 discloses that C10: 0 and C12: 0 are obtained by reacting under normal pressure with a lipase obtained from a microorganism belonging to the genus Penicillium below the melting point of fatty acid monoglyceride (that is, a solid reaction system). , And C14: 0 fatty acids, and oleic acid (C18: 1), linoleic acid (C18: 2), and linolenic acid (C18: 3) and glycerin to fatty acid monoglycerides (purity of fatty acid monoglycerides relative to glycerides, 98% A method for producing the above is disclosed. However, in this method, in any fatty acid, the esterification rate is at most 87%, and monoglycerides of oleic acid, linoleic acid, and linolenic acid must be reacted at a low temperature below room temperature. However, there are problems such as requiring a lot of energy.
さらに、酵素法を用いた脂肪酸モノグリセリドの製造方法においては、いずれも原料である脂肪酸の炭素数は偶数であり、炭素数が奇数の脂肪酸（Ｃ９：０、Ｃ１５：０など）を原料とする酵素法を用いた脂肪酸モノグリセリドの製造方法は、未だ報告されていない。
本発明の目的は、脂肪酸（特に、Ｃ９：０からＣ１８：０の直鎖飽和脂肪酸、オレイン酸、リノール酸、およびリノレン酸の不飽和脂肪酸）を含む部分グリセリド、特に純度の高い脂肪酸モノグリセリドを、効率的に製造し得る方法を提供することにある。 The object of the present invention is to provide a partial glyceride containing fatty acids (especially C9: 0 to C18: 0 linear saturated fatty acids, unsaturated fatty acids of oleic acid, linoleic acid, and linolenic acid), particularly fatty acid monoglycerides of high purity. It is to provide a method that can be efficiently manufactured.
本発明者らは上記課題を解決するために鋭意研究を重ねた結果、リパーゼを用いた脂肪酸とグリセリンとのエステル化反応において、反応系中の水分量が重要であることを見出したことに基づき、本発明を完成した。 Based on the finding that the amount of water in the reaction system is important in the esterification reaction between a fatty acid and glycerin using lipase as a result of intensive studies to solve the above problems. The present invention has been completed.
本発明は、リパーゼを用いるエステル化反応によって脂肪酸とグリセロールとから脂肪酸モノグリセリドを製造する方法を提供し、該方法は、該脂肪酸のエステル化率が６０％に達した以降のエステル化反応を、水分含量０．３〜２．５質量％に制御して行う工程を包含する。 The present invention provides a method for producing a fatty acid monoglyceride from a fatty acid and glycerol by an esterification reaction using a lipase, and the method comprises subjecting the esterification reaction after the esterification rate of the fatty acid to 60% to moisture to The process includes controlling the content to 0.3 to 2.5% by mass.
１つの実施態様では、上記脂肪酸のエステル化率が６０％に達した以降のエステル化反応を、得られる脂肪酸モノグリセリドの融点以下に制御して行う工程をさらに包含する。 In one embodiment, the method further includes a step of controlling the esterification reaction after the esterification rate of the fatty acid reaches 60% to be equal to or lower than the melting point of the obtained fatty acid monoglyceride.
１つの実施態様では、上記脂肪酸は、Ｃ９：０からＣ１８：０の直鎖飽和脂肪酸、オレイン酸、リノール酸、およびリノレン酸からなる群より選択される少なくとも１種の脂肪酸である。 In one embodiment, the fatty acid is at least one fatty acid selected from the group consisting of C9: 0 to C18: 0 linear saturated fatty acids, oleic acid, linoleic acid, and linolenic acid.
１つの実施態様では、上記リパーゼは、モノグリセリドリパーゼまたはモノおよびジグリセリドリパーゼである。 In one embodiment, the lipase is a monoglyceride lipase or a mono and diglyceride lipase.
本発明によれば、脂肪酸のエステル化率が６０％に達した以降のエステル化反応を、水分含量０．３〜２．５質量％に制御することによって、穏和な条件で反応可能なリパーゼを用いて、脂肪酸モノグリセリド、特にＣ９：０からＣ１８：０の直鎖飽和脂肪酸、オレイン酸、リノール酸、およびリノレン酸の脂肪酸モノグリセリドを効率よく、かつ高純度で製造する方法が提供される。 According to the present invention, a lipase capable of reacting under mild conditions is controlled by controlling the esterification reaction after the fatty acid esterification rate reaches 60% to a water content of 0.3 to 2.5% by mass. The present invention provides a method for producing fatty acid monoglycerides, particularly fatty acid monoglycerides of C9: 0 to C18: 0 linear saturated fatty acids, oleic acid, linoleic acid, and linolenic acid efficiently and with high purity.
従来、反応系中の水分量が重要であることは分かっておらず、それを適切に保つための反応条件も明確に解明されていなかったため、限られた脂肪酸モノグリセリドのみしか得ることができなかった。しかし、反応系中の水分量が重要であるという知見に基づいてなされた本発明の製造方法により、従来技術の問題が解消され、幅広い脂肪酸モノグリセリドを実用的に合成し得る。 Previously, it was not known that the amount of water in the reaction system was important, and the reaction conditions for maintaining it appropriately were not clearly elucidated, so only limited fatty acid monoglycerides could be obtained. . However, the production method of the present invention based on the knowledge that the amount of water in the reaction system is important solves the problems of the prior art, and a wide range of fatty acid monoglycerides can be synthesized practically.
（リパーゼ）
本発明の製造方法において触媒として使用されるリパーゼは、グリセリド類を基質として認識するものであれば限定されない。例えば、モノグリセリドリパーゼ、モノおよびジグリセリドリパーゼ、トリグリセリドリパーゼ、クチナーゼ、エステラーゼなどが挙げられる。これらの中でもリパーゼが好ましく、特に、脂肪酸トリグリセリドを基質としてほとんど認識せず、脂肪酸モノグリセリドおよび／または脂肪酸ジグリセリドを基質として認識するリパーゼが好ましい。このようなリパーゼとして、モノグリセリドリパーゼ、モノおよびジグリセリドリパーゼなどが挙げられる。以下、リパーゼについて説明する。
(Lipase)
The lipase used as a catalyst in the production method of the present invention is not limited as long as it recognizes glycerides as a substrate. Examples include monoglyceride lipase, mono and diglyceride lipase, triglyceride lipase, cutinase, esterase and the like. Among these, lipases are preferable, and in particular, lipases that hardly recognize fatty acid triglycerides as substrates and recognize fatty acid monoglycerides and / or fatty acid diglycerides as substrates are preferable. Such lipases include monoglyceride lipase, mono and diglyceride lipase and the like. Hereinafter, lipase will be described.
リパーゼは、微生物、動物、および植物由来のいずれでもよいが、好ましくはペニシリウム（Penicillium）属、シュードモナス（Pseudomonas）属、バークホルデリア（Burkholderia）属、アルカリゲネス（Alcaligenes）属、スタフィロコッカス（Staphylococcus）属、バシラス（Bacillus）属、キャンディダ（Candida）属、ゲオトリカム（Geotrichum）属、リゾプス（Rhizopus）属、リゾムコール（Rhizomucor）属、ムコール（Mucor）属、（アスペルギルス）Aspergillus属、（サーモミセス）Thermomyces属（以前の名称はフミコーラ（Humicola）属）、シュードチマ（Pseudozyma）属などの微生物が生産するリパーゼ、豚膵臓由来のリパーゼなどが挙げられる。より好ましくは、ペニシリウム（Penicillium）属およびバシラス（Bacillus）属が生産するリパーゼである。これらのリパーゼは一般に市販されており、容易に入手可能である。 The lipase may be derived from microorganisms, animals, and plants, but preferably the genus Penicillium, Pseudomonas, Burkholderia, Alcaligenes, Staphylococcus Genus, Bacillus genus, Candida genus, Geotrichum genus, Rhizopus genus, Rhizomucor genus, Mucor genus, (Aspergillus) Aspergillus genus, Thermomyces Examples include lipases produced by microorganisms such as the genus (formerly Humicola) and Pseudozyma, and lipases derived from porcine pancreas. More preferably, it is a lipase produced by the genus Penicillium and the genus Bacillus. These lipases are generally commercially available and are readily available.
リパーゼは、精製（粗精製および部分精製を含む）されたものを用いてもよい。さらに、遊離型のまま使用してもよく、あるいはイオン交換樹脂、多孔性樹脂、セラミックス、炭酸カルシウムなどの担体に固定して使用してもよい。遊離型のリパーゼを用いる場合は、一旦酵素を水に溶かしてから反応系に添加してもよく、反応系にリパーゼと水とを別々に添加してもよい。固定化されたリパーゼを使用する場合は、反応系への水の添加は任意である。 The lipase may be purified (including crude and partial purification). Furthermore, it may be used as it is, or may be used by being fixed to a carrier such as an ion exchange resin, a porous resin, ceramics, or calcium carbonate. When using a free lipase, the enzyme may be once dissolved in water and then added to the reaction system, or the lipase and water may be added separately to the reaction system. When an immobilized lipase is used, addition of water to the reaction system is optional.
本発明の製造方法に使用されるリパーゼの量は、反応温度、反応時間、圧力（減圧度）、脂肪酸の種類などにより適宜設定され得、好ましくは反応混合液１ｇ当たり１単位（Ｕ）〜１００００Ｕ、より好ましくは５Ｕ〜１０００Ｕである。酵素活性の１Ｕとは、リパーゼの場合、オリーブ油の加水分解において１分間に１μモルの脂肪酸を遊離する酵素量をいう。モノグリセリドリパーゼ、あるいはモノおよびジグリセリドリパーゼの場合はモノオレイン（グリセリンモノオレエイト）の加水分解において１分間に１μモルの脂肪酸を遊離する酵素量である。 The amount of lipase used in the production method of the present invention can be appropriately set depending on the reaction temperature, reaction time, pressure (degree of reduced pressure), type of fatty acid, etc., preferably 1 unit (U) to 10000 U per 1 g of reaction mixture. More preferably, it is 5U-1000U. In the case of lipase, 1 U of enzyme activity refers to the amount of enzyme that liberates 1 μmol of fatty acid per minute in the hydrolysis of olive oil. In the case of monoglyceride lipase or mono- and diglyceride lipase, the amount of enzyme that liberates 1 μmol of fatty acid per minute in hydrolysis of monoolein (glycerin monooleate).
（グリセリン）
本発明の製造方法に使用されるグリセリンの量は、特に限定されない。通常、遊離脂肪酸１モル量に対して、好ましくは１〜１０倍モル量、より好ましくは１．５〜３倍モル量である。
(Glycerin)
The amount of glycerin used in the production method of the present invention is not particularly limited. Usually, it is preferably 1 to 10 times the molar amount, more preferably 1.5 to 3 times the molar amount with respect to 1 mol of the free fatty acid.
（脂肪酸）
本発明の製造方法で使用される脂肪酸は、遊離型、金属塩型、およびエステル型のいずれの形態でもよい。本発明においては、エステル化反応が進行しやすい点で、遊離型の脂肪酸（遊離脂肪酸）が好ましい。本発明の製造方法で使用される脂肪酸は、特に限定されず、炭素数が偶数の脂肪酸でも奇数の脂肪酸でもよい。好ましくはＣ９：０からＣ１８：０の直鎖飽和脂肪酸、オレイン酸（Ｃ１８：１）、リノール酸（Ｃ１８：２）、リノレン酸（Ｃ１８：３）などが挙げられる。
(fatty acid)
The fatty acid used in the production method of the present invention may be in a free form, a metal salt form, or an ester form. In the present invention, a free fatty acid (free fatty acid) is preferable in that the esterification reaction easily proceeds. The fatty acid used in the production method of the present invention is not particularly limited, and may be an even fatty acid or an odd fatty acid. Preferably, C9: 0 to C18: 0 linear saturated fatty acid, oleic acid (C18: 1), linoleic acid (C18: 2), linolenic acid (C18: 3) and the like can be mentioned.
（脂肪酸モノグリセリドの製造方法）
本発明は、リパーゼを用いるエステル化反応によって脂肪酸とグリセロールとから脂肪酸モノグリセリドを製造する方法であって、該脂肪酸のエステル化率が６０％に達した以降のエステル化反応を、水分含量０．３〜２．５質量％に制御して行う工程を包含する。
(Method for producing fatty acid monoglyceride)
The present invention is a method for producing a fatty acid monoglyceride from a fatty acid and glycerol by an esterification reaction using lipase, and the esterification reaction after the esterification rate of the fatty acid reaches 60% The process of carrying out by controlling to -2.5 mass% is included.
リパーゼを用いた脂肪酸とグリセリンとのエステル化反応は、脂肪酸のエステル化率によって、反応前期と反応後期とに区分される。本明細書において、脂肪酸のエステル化率とは、原料とする遊離脂肪酸に対するグリセリンと反応した遊離脂肪酸の割合をいう。エステル化率は、反応系中の遊離脂肪酸量（酸価）をアルカリによる滴定で測定し、以下の式で求められる。 The esterification reaction between a fatty acid and glycerin using lipase is divided into an early reaction period and a late reaction stage depending on the esterification rate of the fatty acid. In the present specification, the esterification rate of fatty acid refers to the ratio of free fatty acid reacted with glycerin to free fatty acid used as a raw material. The esterification rate is obtained by the following equation by measuring the amount of free fatty acid (acid value) in the reaction system by titration with alkali.
｛（反応前の酸価−反応後の酸価）／（反応前の酸価）｝×１００ {(Acid value before reaction−acid value after reaction) / (acid value before reaction)} × 100
本発明においては、脂肪酸のエステル化率が６０％に達する前を反応前期、そして達した以降を反応後期とする。脂肪酸のエステル化率は、サンプリングすることによって経時的に測定することができる。 In the present invention, the time before the esterification rate of the fatty acid reaches 60% is defined as the first reaction period, and the time after the esterification ratio is reached as the second reaction period. The esterification rate of the fatty acid can be measured over time by sampling.
まず、反応前期では、酵素の活性が失われない量の水分が反応系中に存在すれば、脂肪酸とグリセリンとから脂肪酸モノグリセリドが優先的に合成される。したがって、反応前期では、水分量はあまり重要ではなく、適宜設定し得る。例えば、反応開始時（原料仕込み時）には、反応系中に、好ましくは脂肪酸とグリセリンとの合計質量に対して０．３〜２０質量％、より好ましくは１〜１０質量％の割合で水を添加し、そして反応前期にわたって反応系中の水分量を、好ましくは０．１〜２０質量％、より好ましくは０．３〜５質量％に制御すればよい。 First, in the first reaction period, fatty acid monoglyceride is preferentially synthesized from fatty acid and glycerin if an amount of water that does not lose the enzyme activity is present in the reaction system. Therefore, in the first reaction period, the water content is not so important and can be set as appropriate. For example, at the start of the reaction (at the time of raw material charging), water is preferably added in the reaction system at a ratio of 0.3 to 20% by mass, more preferably 1 to 10% by mass with respect to the total mass of fatty acid and glycerin. And the water content in the reaction system is preferably controlled to 0.1 to 20% by mass, more preferably 0.3 to 5% by mass over the first half of the reaction.
脂肪酸のエステル化率が６０％に達した以降、すなわち反応後期では、反応系中の水分含量を０．３〜２．５質量％に制御する。水分含量は、カールフィッシャー水分計を用いて経時的に測定すればよい。この反応系中の水分含量は、特に重要である。脂肪酸のエステル化率が６０％を超えると、脂肪酸モノグリセリドが基質として認識され脂肪酸ジグリセリドへの変換反応が進行し始めるので、脂肪酸のエステル化率が６０％に達した以降の水分含量を制御する。反応後期においては、反応系中の水分含量が０．３質量％よりも低い場合、エステル化反応はほとんど進行せず、原料である脂肪酸が未反応のまま残り効率が悪くなる。一方、反応系中の水分含量が２．５質量％よりも高い場合、エステル化反応は効率よく進行するものの、同時に脂肪酸モノグリセリドから脂肪酸ジグリセリドへの変換反応も盛んに進むため、脂肪酸ジグリセリドが副産物として反応系内に大量に蓄積し、純度の高い脂肪酸モノグリセリドが得られない。これは、水分量により酵素の基質特異性が変化するためと推定される。すなわち、反応系中の水分含量が０．３質量％よりも少ない場合、リパーゼが脂肪酸およびグリセリンを基質として認識しなくなり、グリセリンと脂肪酸とのエステル化反応が進行し難くなると推定される。一方、反応系中の水分含量が２．５質量％よりも多い場合、リパーゼが脂肪酸および脂肪酸モノグリセリドも基質と認識し、脂肪酸モノグリセリドから脂肪酸ジグリセリドの反応が進行すると推定される。 After the esterification rate of the fatty acid reaches 60%, that is, in the later stage of the reaction, the water content in the reaction system is controlled to 0.3 to 2.5% by mass. The moisture content may be measured over time using a Karl Fischer moisture meter. The water content in the reaction system is particularly important. If the esterification rate of the fatty acid exceeds 60%, the fatty acid monoglyceride is recognized as a substrate and the conversion reaction into the fatty acid diglyceride starts to proceed, so that the water content after the fatty acid esterification rate reaches 60% is controlled. In the later stage of the reaction, when the water content in the reaction system is lower than 0.3% by mass, the esterification reaction hardly proceeds and the raw fatty acid remains unreacted and the efficiency is deteriorated. On the other hand, when the water content in the reaction system is higher than 2.5% by mass, the esterification reaction proceeds efficiently, but at the same time, the conversion reaction from fatty acid monoglyceride to fatty acid diglyceride also actively proceeds. A large amount of fatty acid monoglyceride accumulates in the reaction system and cannot be obtained. This is presumably because the substrate specificity of the enzyme changes depending on the amount of water. That is, when the water content in the reaction system is less than 0.3% by mass, it is presumed that the lipase does not recognize fatty acid and glycerin as substrates, and the esterification reaction between glycerin and fatty acid is difficult to proceed. On the other hand, when the water content in the reaction system is more than 2.5% by mass, lipase recognizes fatty acids and fatty acid monoglycerides as substrates, and it is estimated that the reaction of fatty acid diglycerides proceeds from fatty acid monoglycerides.
したがって、反応後期の水分含量は、酵素の活性が失われず、かつ脂肪酸モノグリセリドから脂肪酸ジグリセリドへの変換反応が抑制される水分含量である０．３〜２．５質量％に制御する必要がある。さらにこの反応後期では、酵素の活性が失われない水分含量(０．３質量％以上)の範囲内で、水分量が少なくすれば少なくするほど脂肪酸モノグリセリドの純度が向上するので、水分含量を上記範囲内で極力少なく維持することが好ましい。 Therefore, it is necessary to control the water content in the late reaction to 0.3 to 2.5% by mass, which is the water content that does not lose the activity of the enzyme and suppresses the conversion reaction from fatty acid monoglyceride to fatty acid diglyceride. Furthermore, in the latter stage of the reaction, the purity of the fatty acid monoglyceride is improved as the water content is reduced within the range of the water content (0.3% by mass or more) in which the enzyme activity is not lost. It is preferable to keep it as small as possible within the range.
至適な水分含量は、用いる脂肪酸によって多少異なる。例えば、Ｃ９：０の脂肪酸は、他の脂肪酸（Ｃ１０：０〜Ｃ１８：０、Ｃ１８：１、Ｃ１８：２、およびＣ１８：３）に比べて、水分含量を上記範囲内でやや高く（１質量％以上）設定することが好ましい。 The optimum moisture content varies somewhat depending on the fatty acid used. For example, the C9: 0 fatty acid has a slightly higher moisture content within the above range (1 mass) than other fatty acids (C10: 0 to C18: 0, C18: 1, C18: 2, and C18: 3). % Or more) is preferably set.
このように、反応系中の水分含量が０．３〜２．５質量％の範囲内である場合、エステル化反応は効率よく進行し、かつ脂肪酸モノグリセリドから脂肪酸ジグリセリドの変換反応が抑制されて純度の高い脂肪酸モノグリセリドが得られる。 Thus, when the water content in the reaction system is within the range of 0.3 to 2.5% by mass, the esterification reaction proceeds efficiently, and the conversion reaction from fatty acid monoglyceride to fatty acid diglyceride is suppressed, and the purity is increased. High fatty acid monoglycerides.
上記水分含量に制御するための反応条件（反応温度および圧力）は、反応系中の水分含量を考慮して適宜設定され得る。反応温度は、好ましくは−２０〜１００℃、より好ましくは０〜８０℃、さらに好ましくは２０〜７０℃である。反応系中の圧力は、好ましくは０．０１〜１００ｍｍＨｇ、より好ましくは１〜３０ｍｍＨｇである。 Reaction conditions (reaction temperature and pressure) for controlling the water content can be appropriately set in consideration of the water content in the reaction system. Reaction temperature becomes like this. Preferably it is -20-100 degreeC, More preferably, it is 0-80 degreeC, More preferably, it is 20-70 degreeC. The pressure in the reaction system is preferably 0.01 to 100 mmHg, more preferably 1 to 30 mmHg.
上記水分含量を保つために、モレキュラーシーブなどの脱水剤を反応系中に添加してもよい。あるいは、水分含量が０．３質量％より少なくなった場合には、反応途中で反応系中に水を添加して、０．３〜２．５質量％の範囲内となるようにしてもよい（例えば、水分含量が０．２質量％まで低下した場合に、水分含量が１質量％となるように水を添加するなど）。さらに、これらの水分含量制御方法を組み合わせてもよい。 In order to maintain the water content, a dehydrating agent such as molecular sieve may be added to the reaction system. Alternatively, when the water content is less than 0.3% by mass, water may be added to the reaction system during the reaction so that the water content is within the range of 0.3 to 2.5% by mass. (For example, when the water content is reduced to 0.2% by mass, water is added so that the water content becomes 1% by mass). Furthermore, these water content control methods may be combined.
エステル化反応は、静置反応でもよいし、各種の撹拌法、振盪法、超音波法、窒素などの吹き込み法、ポンプなどによる循環混合法、弁またはピストンを用いる混合法などにより、あるいはこれらの方法の組合せにより、反応液をよく混合しながら行ってもよい。 The esterification reaction may be a stationary reaction, various stirring methods, shaking methods, ultrasonic methods, nitrogen blowing methods, circulating mixing methods using pumps, mixing methods using valves or pistons, etc. Depending on the combination of methods, the reaction solution may be mixed well.
反応混合液から脂肪酸モノグリセリドを単離・精製する方法としては、任意の単離・精製方法を採用し得る。単離・精製方法としては、例えば、脱酸、水洗、蒸留、溶媒抽出、イオン交換クロマトグラフィー、膜分離など、およびこれらの方法の組合せが挙げられる。 Any isolation / purification method may be employed as a method for isolating / purifying the fatty acid monoglyceride from the reaction mixture. Examples of the isolation / purification method include deoxidation, water washing, distillation, solvent extraction, ion exchange chromatography, membrane separation, and combinations of these methods.
この反応系は、反応開始から終了まで液状を保っている系であるため、終始、反応液の混合が容易である。さらに、反応終了時に反応槽からの反応液の抜き出しを容易に行い得るので、工業的にも好ましい。しかし、本発明の製造方法においては、上記脂肪酸のエステル化率が６０％に達した以降のエステル化反応を、得られる脂肪酸モノグリセリドの融点以下に制御して行う工程を包含してもよい。脂肪酸のエステル化率が６０％に達した以降のエステル化反応を、得られる脂肪酸モノグリセリドの融点以下に制御して行うことによって、副産物である脂肪酸ジグリセリドの含量を１質量％以下に抑えることができる。すなわち、反応前期には、反応液の撹拌が容易である液状を保つ温度で、適切な水分量を保ちながら減圧下で反応を行い、反応後期に入った時に、反応により得られた脂肪酸モノグリセリドの融点以下の温度（反応系が固化する温度）まで冷却して反応を行う。このように温度制御を行うと、脂肪酸モノグリセリドから脂肪酸ジグリセリドへの変換反応がほとんど起こらないため、副産物である脂肪酸ジグリセリドの含量が１質量％以下になる。 Since this reaction system is a system that maintains a liquid state from the start to the end of the reaction, it is easy to mix the reaction liquid throughout. Furthermore, since the reaction liquid can be easily extracted from the reaction tank at the end of the reaction, it is industrially preferable. However, the production method of the present invention may include a step in which the esterification reaction after the esterification rate of the fatty acid reaches 60% is controlled below the melting point of the obtained fatty acid monoglyceride. By controlling the esterification reaction after the fatty acid esterification rate reaches 60% to below the melting point of the fatty acid monoglyceride obtained, the content of fatty acid diglyceride as a by-product can be suppressed to 1% by mass or less. . That is, in the first stage of the reaction, the reaction is carried out under reduced pressure while maintaining an appropriate amount of water at a temperature at which the reaction liquid can be easily stirred, and when the latter stage of the reaction is started, The reaction is carried out by cooling to a temperature below the melting point (temperature at which the reaction system solidifies). When temperature control is performed in this manner, since the conversion reaction from fatty acid monoglyceride to fatty acid diglyceride hardly occurs, the content of fatty acid diglyceride as a by-product becomes 1% by mass or less.
反応後期に反応系を固化させる製造方法においては、反応終了後に反応系の温度を上昇させて溶解させる工程を追加することにより、反応槽からの反応液の抜き出しを容易に行い得るので、工業的にも好ましい。さらに、反応系中の脂肪酸ジグリセリドの含量が１質量％以下であるため、脂肪酸モノグリセリドを容易に単離・精製することができる。一方、反応開始時から脂肪酸モノグリセリドの融点以下の温度（反応系が固化する温度）、減圧下で反応を行った場合、脂肪酸、グリセリン、酵素、および水が十分に混合されず、エステル化反応は、ほとんど進行しない（後述の実施例２を参照）。 In the manufacturing method in which the reaction system is solidified in the late stage of the reaction, the reaction solution can be easily extracted from the reaction tank by adding a step of increasing the temperature of the reaction system after the reaction is completed, so Also preferred. Furthermore, since the content of the fatty acid diglyceride in the reaction system is 1% by mass or less, the fatty acid monoglyceride can be easily isolated and purified. On the other hand, when the reaction is carried out at a temperature below the melting point of the fatty acid monoglyceride from the start of the reaction (temperature at which the reaction system solidifies) under reduced pressure, the fatty acid, glycerin, enzyme, and water are not sufficiently mixed, Little progress (see Example 2 below).
これに対し、反応後期に反応系を固化させる製造方法では、反応前期の反応系は液状であるため、脂肪酸、グリセリン、酵素、および水が十分に混合されているので、反応系が固化しても、エステル化反応が効率よく進行する。さらに、この方法における脂肪酸のエステル化率は、反応開始時から脂肪酸モノグリセリドの融点以下の温度かつ常圧下で反応を行った場合（例えば、非特許文献２を参照）よりも高くなる。 On the other hand, in the production method in which the reaction system is solidified in the late stage of the reaction, since the reaction system in the early stage of the reaction is liquid, the fatty acid, glycerin, enzyme, and water are sufficiently mixed. Also, the esterification reaction proceeds efficiently. Furthermore, the esterification rate of the fatty acid in this method is higher than when the reaction is carried out at the temperature below the melting point of the fatty acid monoglyceride and under normal pressure from the start of the reaction (see, for example, Non-Patent Document 2).
本発明の製造方法によれば、脂肪酸のエステル化率が好ましくは９５％以上、より好ましくは９８％以上となり効率よく脂肪酸モノグリセリドを得ることができ、この脂肪酸モノグリセリドの純度は、好ましくは純度９０％以上、より好ましくは９５％以上であり得る。 According to the production method of the present invention, the fatty acid monoglyceride can be efficiently obtained with an esterification rate of fatty acid of preferably 95% or more, more preferably 98% or more. The purity of this fatty acid monoglyceride is preferably 90% purity. More preferably, it may be 95% or more.
以下、本発明の脂肪酸モノグリセリド製造方法を実施例に基づいて説明するが、これらはあくまで一例にすぎず、また水分含量を制御するための反応温度、圧力（減圧度）などの条件は、反応容器の形状、大きさ、反応液の容量、混合方法、脂肪酸の種類などによって異なるものであり、本発明は、本実施例に限定されるものではない。 Hereinafter, although the fatty acid monoglyceride manufacturing method of this invention is demonstrated based on an Example, these are only examples, and conditions, such as reaction temperature and pressure (decompression degree) for controlling a water content, are reaction containers. However, the present invention is not limited to this example.
（実施例１：酵素法による脂肪酸モノグリセリドの合成に及ぼす圧力（減圧度）の影響）
約３０ｍＬのバイアル瓶（内径２．８ｃｍおよび高さ５ｃｍ）に、グリセリンと遊離脂肪酸（Ｃ１８：１）との混合液（遊離脂肪酸／グリセリン＝１／２（モル比））１０ｇ、０．１ｇの水、および反応系１ｇに対して２００Ｕのペニシリウム・カマンベルティ（Penicillium camembertii）由来モノおよびジグリセリドリパーゼ（リパーゼＧ；天野エンザイム株式会社製）を添加した。次いで、反応温度を３０℃に保ちながらマグネチックスターラーで撹拌し、表１に記載の圧力条件下（２ｍｍＨｇ、５ｍｍＨｇ、１５ｍｍＨｇ、および７６０ｍｍＨｇ）で、それぞれ４８時間反応を行った。反応開始から７時間後および４８時間後（反応終了後）の脂肪酸のエステル化率および水分量（カールフィッシャー水分計（株式会社ダイアインスツルメンツ製）で測定）、ならびに反応液中の組成を測定した。反応液中の組成の測定は、イアトロスキャンＭＫ−６（株式会社三菱化学ヤトロン製）を用いるＴＬＣ−ＦＩＤ法で行った。ＴＬＣの展開溶媒として、トルエン／クロロホルム／酢酸＝５０／２０／０．７（容量比）の混合溶媒を使用した。結果を表１に示す。
(Example 1: Effect of pressure (decompression degree) on synthesis of fatty acid monoglyceride by enzymatic method)
About 30 mL vial (inner diameter 2.8 cm and
これとは別に、Ｃ１０：０の脂肪酸（反応温度４０℃；圧力１ｍｍＨｇ、５ｍｍＨｇ、１５ｍｍＨｇ、および７６０ｍｍＨｇ）およびＣ１５：０の脂肪酸（反応温度５０℃；圧力４ｍｍＨｇ、１０ｍｍＨｇ、２０ｍｍＨｇ、および５０ｍｍＨｇ）を用いて、上記Ｃ１８：１の脂肪酸を用いた場合と同様の反応を行った。結果を表１に示す。
Separately, C10: 0 fatty acids (
表１に示すように、Ｃ１８：１の脂肪酸は、脂肪酸のエステル化率が６０％に達した以降（反応開始から７時間より以降）の水分含量を、０．３１〜２．２質量％に制御（反応温度が３０℃で圧力が２〜５ｍｍＨｇ）した場合（実験Ａ１およびＡ２）、Ｃ１０：０の脂肪酸は、０．３１〜１．８質量％に制御（反応温度が４０℃で圧力が５ｍｍＨｇ）した場合（実験Ｂ２）、そしてＣ１５：０の脂肪酸は、０．９２〜１．６質量％に制御（反応温度が５０℃で圧力が１０ｍｍＨｇ）した場合（実験Ｃ２）、エステル化率が高く、かつ脂肪酸モノグリセリドの含量も高いことがわかる。いずれの脂肪酸の場合も、脂肪酸のエステル化率が６０％に達した以降の水分含量を、０．３〜２．５質量％に制御することにより、エステル化率が高く、脂肪酸モノグリセリドが高収率で得られることがわかる（表１に評価を○で示す）。 As shown in Table 1, the C18: 1 fatty acid has a water content of 0.31 to 2.2% by mass after the esterification rate of the fatty acid reaches 60% (after 7 hours from the start of the reaction). When controlled (reaction temperature is 30 ° C. and pressure is 2 to 5 mmHg) (experiment A1 and A2), C10: 0 fatty acid is controlled to 0.31 to 1.8% by mass (reaction temperature is 40 ° C. and pressure is 5 mmHg) (experiment B2), and C15: 0 fatty acids were controlled to 0.92 to 1.6 mass% (reaction temperature was 50 ° C. and pressure was 10 mmHg) (experiment C2). It is high and the content of fatty acid monoglyceride is also high. In any fatty acid, by controlling the water content after the fatty acid esterification rate reaches 60% to 0.3 to 2.5% by mass, the esterification rate is high and the fatty acid monoglyceride has high yield. It turns out that it is obtained by rate (indicated by ○ in Table 1).
しかし、エステル化率が６０％に達した以降（表１では、反応開始から７時間より以降）も、水分含量が２．５質量％を越える場合（実験Ａ３、Ａ４、Ｂ３、Ｂ４、およびＣ３）、脂肪酸ジグリセリドの生成量が増加して脂肪酸モノグリセリド生成効率が低下する傾向にある。なお、実験Ｃ４については、反応開始から７時間目のエステル化率は５７％であり、水分含量は３．２質量％であった。さらに、反応を進めて、エステル化率が６０％に達した時点の水分含量を測定すると３．０質量％であったため、脂肪酸ジグリセリドの生成量が増加した。したがって、脂肪酸モノグリセリド生成効率と反応液中の水分量との関連性が示唆された。実験Ｂ１およびＣ１においては、反応開始時から水分含量が少なく、酵素が脂肪酸およびグリセリンを基質として認識しなくなり、エステル化反応は、ほとんど進行しなかったと考えられる。 However, even after the esterification rate reaches 60% (in Table 1, after 7 hours from the start of the reaction), when the water content exceeds 2.5% by mass (experiments A3, A4, B3, B4, and C3 ), The production amount of fatty acid diglyceride tends to increase and the production efficiency of fatty acid monoglyceride tends to decrease. For Experiment C4, the esterification rate 7 hours after the start of the reaction was 57%, and the water content was 3.2% by mass. Further, when the reaction was advanced and the water content at the time when the esterification rate reached 60% was measured, it was 3.0% by mass, so the amount of fatty acid diglyceride produced increased. Therefore, the relationship between the fatty acid monoglyceride production efficiency and the amount of water in the reaction solution was suggested. In Experiments B1 and C1, the water content is low from the start of the reaction, the enzyme does not recognize fatty acid and glycerin as substrates, and the esterification reaction is considered to have hardly proceeded.
（実施例２：酵素法による脂肪酸モノグリセリドの合成に及ぼす温度の影響）
約３０ｍＬのバイアル瓶に、グリセリンと遊離脂肪酸（Ｃ１０：０）との混合液（遊離脂肪酸／グリセリン＝１／２（モル比））１０ｇ、０．１ｇの水、および反応系１ｇに対して２００ＵのリパーゼＧを添加した。次いで、反応温度を表２に記載の温度（３０℃、４０℃、および５０℃）に保ちながらマグネチックスターラーで撹拌し、１〜３ｍｍＨｇの圧力条件下で、それぞれ４８時間反応を行った。反応開始から７時間後および４８時間後（反応終了後）の脂肪酸のエステル化率および水分量、ならびに反応液中の組成を測定した。結果を表２に示す。
(Example 2: Effect of temperature on synthesis of fatty acid monoglyceride by enzymatic method)
In a 30-mL vial, 10 g of a mixed solution of glycerin and free fatty acid (C10: 0) (free fatty acid / glycerin = 1/2 (molar ratio)), 0.1 g of water, and 200 g for 1 g of the reaction system Of lipase G was added. Subsequently, it stirred with the magnetic stirrer, maintaining reaction temperature at the temperature (30 degreeC, 40 degreeC, and 50 degreeC) of Table 2, and it reacted for 48 hours, respectively under the pressure conditions of 1-3 mmHg. The fatty acid esterification rate and water content, and the composition in the reaction solution were measured 7 hours and 48 hours after the start of the reaction (after completion of the reaction). The results are shown in Table 2.
これとは別に、Ｃ１８：１の脂肪酸（圧力１〜３ｍｍＨｇ；反応温度２０℃、３０℃、４０℃、および５０℃）、Ｃ１４：０の脂肪酸（圧力７〜１２ｍｍＨｇ；反応温度３０℃、４０℃、５０℃、および６０℃）、および亜麻仁油脂肪酸（圧力１〜３ｍｍＨｇ；反応温度１０℃、２０℃、３０℃、４０℃、および５０℃）を用いて、上記Ｃ１０：０の脂肪酸を用いた場合と同様の反応を行った。結果を表２に示す。なお、亜麻仁油脂肪酸（亜麻仁油由来脂肪酸）とは、亜麻仁油をケン化分解して得られる遊離脂肪酸である。亜麻仁油脂肪酸は、Ｃ１６：０を４．９質量％、Ｃ１８：０を３．３質量％、Ｃ１８：１を１７．０質量％、Ｃ１８：２を１６．２質量％、Ｃ１８：３を５７．４質量％、およびその他の脂肪酸を１．２質量％の割合で含む。
Separately, C18: 1 fatty acids (pressure 1 to 3 mmHg;
表２に示すように、Ｃ１０：０の脂肪酸は、脂肪酸のエステル化率が６０％に達した以降（反応開始から７時間より以降）の水分含量を、０．３１〜１．０質量％に制御（反応温度が３０℃で圧力が１〜３ｍｍＨｇ）した場合（実験Ｄ２）、Ｃ１８：１の脂肪酸は、０．３２〜２．３質量％に制御（反応温度が２０〜３０℃で圧力が１〜３ｍｍＨｇ）した場合（実験Ｅ１およびＥ２）、Ｃ１４：０の脂肪酸は、１．４〜２．５質量％に制御（反応温度が５０℃で圧力が７〜１２ｍｍＨｇ）した場合（実験Ｆ３）、亜麻仁油脂肪酸は、０．３１〜２．０質量％に制御（反応温度が１０〜３０℃で圧力が１〜３ｍｍＨｇ）した場合（実験Ｇ１〜Ｇ３）、エステル化率が高く、かつ脂肪酸モノグリセリドの含量も高いことがわかる。いずれの脂肪酸の場合も、脂肪酸のエステル化率が６０％に達した以降の水分含量を、０．３〜２．５質量％に制御することにより、エステル化率が高く、脂肪酸モノグリセリドが高収率で得られることがわかる（表２に評価を○で示す）。 As shown in Table 2, the C10: 0 fatty acid has a moisture content of 0.31 to 1.0 mass% after the esterification rate of the fatty acid reaches 60% (after 7 hours from the start of the reaction). When controlled (reaction temperature is 30 ° C. and pressure is 1 to 3 mmHg) (Experiment D2), C18: 1 fatty acid is controlled to 0.32 to 2.3 mass% (reaction temperature is 20 to 30 ° C. and pressure is 1 to 3 mmHg) (experiment E1 and E2), C14: 0 fatty acid is controlled to 1.4 to 2.5% by mass (reaction temperature is 50 ° C. and pressure is 7 to 12 mmHg) (experiment F3) When the linseed oil fatty acid is controlled to 0.31 to 2.0% by mass (reaction temperature is 10 to 30 ° C. and pressure is 1 to 3 mmHg) (experiment G1 to G3), the esterification rate is high and the fatty acid monoglyceride It can be seen that the content of is also high. In any fatty acid, by controlling the water content after the fatty acid esterification rate reaches 60% to 0.3 to 2.5% by mass, the esterification rate is high and the fatty acid monoglyceride has high yield. It can be seen that it is obtained at a rate (indicated by a circle in Table 2).
しかし、脂肪酸のエステル化率が６０％に達した以降（反応開始から７時間より以降）の水分含量が０．３質量％未満の場合（実験Ｄ３、Ｄ４、Ｅ３、Ｅ４、Ｆ４、Ｇ４、およびＧ５）、エステル化率が低下し、脂肪酸モノグリセリドの収率も低いことがわかる。すなわち、用いる脂肪酸によって多少異なるが、反応温度を高温にすると、水分が減少しやすいためにエステル化率が低下する。一方、飽和脂肪酸（Ｃ１０：０およびＣ１４：０）を用いた実験Ｄ１、Ｆ１、およびＦ２のように、反応温度を低温にすると、反応開始時から反応液が固化して撹拌効率が低下し、反応が進まなかった。 However, when the water content after the esterification rate of fatty acid reaches 60% (after 7 hours from the start of the reaction) is less than 0.3% by mass (experiments D3, D4, E3, E4, F4, G4, and G5), it can be seen that the esterification rate decreases and the yield of fatty acid monoglycerides is also low. That is, although somewhat different depending on the fatty acid to be used, when the reaction temperature is increased, the water content tends to decrease and the esterification rate decreases. On the other hand, as in Experiments D1, F1, and F2 using saturated fatty acids (C10: 0 and C14: 0), when the reaction temperature is lowered, the reaction solution is solidified from the start of the reaction and the stirring efficiency is lowered. The reaction did not progress.
実施例１および２より、約３０ｍＬのバイアル瓶を用いたリパーゼＧによる効率的な脂肪酸モノグリセリドの合成反応は、反応後期（脂肪酸のエステル化率が６０％に達した以降）の水分含量に依存していることが分かった。その関係を図１に示す。図１の白部領域の反応条件（反応温度および圧力）の場合、効率よくかつ高収率で脂肪酸モノグリセリドが得られる。脱水条件が厳しい斜線域（白部領域よりも右下部）では、水分含量が低すぎることにより反応率の低下を招く。一方、脱水条件が緩やかな点領域（白部領域よりも左上部）では、水分含量が高すぎることにより脂肪酸モノグリセリドから脂肪酸ジグリセリドへの変換反応が起こる。なお、反応開始時から固化する場合は、撹拌されにくいために反応率が低下する。 From Examples 1 and 2, the efficient synthesis reaction of fatty acid monoglyceride by lipase G using about 30 mL vial depends on the water content in the late stage of reaction (after the fatty acid esterification rate reaches 60%). I found out. The relationship is shown in FIG. In the case of the reaction conditions (reaction temperature and pressure) in the white area in FIG. 1, fatty acid monoglycerides can be obtained efficiently and in high yield. In the shaded area where the dehydration conditions are severe (lower right area than the white area), the water content is too low, leading to a decrease in the reaction rate. On the other hand, in the point region where the dehydration conditions are mild (upper left part of the white part region), the conversion of fatty acid monoglyceride to fatty acid diglyceride occurs because the water content is too high. When solidifying from the start of the reaction, the reaction rate decreases because stirring is difficult.
この白部領域の反応条件下の場合、反応容器、撹拌、反応液量、脂肪酸の種類などによって、多少の誤差はあるが、ほぼ反応系中の水分含量が０．３〜２．５質量％に制御され得る。反応後期（エステル化率が約６０％に達した以降（実施例１および２の場合は、反応開始から７時間より以降））における水分量が０．３質量％よりも小さくなると酵素はその活性を失うため、反応後期の水分量を０．３質量％以上に保っておく必要がある。反応後期（エステル化率が約６０％に達した以降（実施例１および２の場合は、反応開始から７時間より以降））の水分量を２．５質量％以下、さらに反応終了時（エステル化率が約９５％に達した時、実施例１および２の場合は、反応開始から４８時間後）の水分量を１．５質量％以下に制御することによって、脂肪酸ジグリセリド含量を１０質量％以下にすることができる。さらに、この反応後期の水分含量が０．３質量％に近ければ近いほど、脂肪酸ジグリセリド含量がより少なくなる。 Under the reaction conditions in the white area, there are some errors depending on the reaction vessel, stirring, the amount of the reaction solution, the type of fatty acid, etc., but the water content in the reaction system is almost 0.3 to 2.5% by mass. Can be controlled. If the amount of water in the late stage of the reaction (after the esterification rate reaches about 60% (in the case of Examples 1 and 2 after 7 hours from the start of the reaction)) becomes less than 0.3% by mass, the enzyme is activated. Therefore, it is necessary to keep the water content in the late reaction at 0.3% by mass or more. The water content in the latter stage of the reaction (after the esterification rate reaches about 60% (in the case of Examples 1 and 2, after 7 hours from the start of the reaction)) is 2.5% by mass or less, and at the end of the reaction (ester When the conversion rate reached about 95%, in the case of Examples 1 and 2, the water content of the fatty acid diglyceride content was 10% by mass by controlling the water content in the case of 48 hours after the reaction start) to 1.5% by mass or less. It can be: Furthermore, the closer the water content in this late reaction is to 0.3% by weight, the lower the fatty acid diglyceride content.
（実施例３：各種脂肪酸モノグリセリドの合成）
約３０ｍＬのバイアル瓶に、グリセリンと遊離脂肪酸（Ｃ９：０）との混合液（遊離脂肪酸／グリセリン＝１／２（モル比））１０ｇ、０．１ｇの水、および反応系１ｇに対して２００ＵのリパーゼＧを添加した。次いで、反応温度を３０〜６３℃の範囲および圧力を２〜２３ｍｍＨｇの範囲で種々変更し、マグネチックスターラーで撹拌して４８時間反応を行った（実験Ｈ）。
(Example 3: Synthesis of various fatty acid monoglycerides)
In a 30-mL vial, 10 g of a mixed solution of glycerin and free fatty acid (C9: 0) (free fatty acid / glycerin = 1/2 (molar ratio)), 0.1 g of water, and 200 g for 1 g of the reaction system Of lipase G was added. Next, the reaction temperature was variously changed within the range of 30 to 63 ° C. and the pressure within the range of 2 to 23 mmHg, and the reaction was carried out for 48 hours while stirring with a magnetic stirrer (Experiment H).
Ｃ１０：０からＣ１７：０の直鎖飽和脂肪酸、Ｃ１８：１、Ｃ１８：２、およびＣ１８：３の脂肪酸についても、上記Ｃ９：０を用いた場合と同様の反応を行った（実験Ｉ〜Ｓ）。各種脂肪酸のモノグリセリドの合成に適した反応温度および圧力、その反応温度および圧力における反応開始から７時間後および４８時間後（反応終了後）の脂肪酸のエステル化率および水分量、および反応液中の組成を表３に示す。 For C10: 0 to C17: 0 linear saturated fatty acids, C18: 1, C18: 2, and C18: 3 fatty acids, the same reaction as in the case of using C9: 0 was performed (Experiments I to S). ). Reaction temperature and pressure suitable for the synthesis of monoglycerides of various fatty acids, esterification rate and water content of fatty acid at 7 hours and 48 hours (after completion of the reaction) at the reaction temperature and pressure, and in the reaction solution The composition is shown in Table 3.
いずれの脂肪酸の場合も（実験Ｈ〜Ｓ）、反応後期の水分量を０．３〜２．５質量％とすることによって、高収率で脂肪酸モノグリセリドが得られた。また、脂肪酸の種類によって、適した水分含量が多少異なり、例えばＣ９：０の脂肪酸は、他の脂肪酸よりも水分含量がやや多い傾向にあることがわかる。 In the case of any fatty acid (experiments H to S), fatty acid monoglycerides were obtained in a high yield by setting the water content in the late stage of the reaction to 0.3 to 2.5 mass%. In addition, the suitable water content varies somewhat depending on the type of fatty acid. For example, it can be seen that a C9: 0 fatty acid tends to have a slightly higher water content than other fatty acids.
なお、これらの反応では反応液は固化せずに液状であるため、実用的な製造方法である。 In these reactions, the reaction solution is liquid without being solidified, and is a practical production method.
（実施例４：反応途中で固化させる方法によるＣ１０：０脂肪酸モノグリセリドの合成）
約３０ｍＬのバイアル瓶に、グリセリンとＣ１０：０の脂肪酸との混合液（Ｃ１０：０の脂肪酸／グリセリン＝１／２（モル比））１０ｇ、０．１ｇの水、および反応系１ｇに対して２００ＵのリパーゼＧを添加した。次いで、反応温度を３０℃に保ちながらマグネチックスターラーで撹拌し、２ｍｍＨｇの減圧条件下で反応を行った。次いで、脂肪酸のエステル化率が６０％に達した７時間後に反応温度を２０℃に下げ、水分含量を０．３〜２．５質量％に制御した。反応液が固化した時点で撹拌を停止し、さらに減圧下で反応を継続した。反応開始時から４８時間後のエステル化率は９８％であり、脂肪酸モノグリセリド含量は９８質量％、脂肪酸ジグリセリド含量はＴＬＣ−ＦＩＤ法では検出限界以下（１質量％以下）、そして水分量は２．５質量％であった。このように、水分含量を０．３〜２．５質量％に制御し、かつ反応後期の反応温度を下げて反応溶液を固化させることによって、副産物である脂肪酸ジグリセリド含量を１質量％以下に減らすことができる。
(Example 4: Synthesis of C10: 0 fatty acid monoglyceride by a method of solidifying during the reaction)
In about 30 mL vial, 10 g of mixed liquid of glycerin and C10: 0 fatty acid (C10: 0 fatty acid / glycerin = 1/2 (molar ratio)), 0.1 g of water, and 1 g of reaction system 200 U of lipase G was added. Subsequently, it stirred with the magnetic stirrer, keeping reaction temperature at 30 degreeC, and it reacted on 2 mmHg pressure reduction conditions. Subsequently, 7 hours after the esterification rate of the fatty acid reached 60%, the reaction temperature was lowered to 20 ° C., and the water content was controlled to 0.3 to 2.5% by mass. Stirring was stopped when the reaction solution solidified, and the reaction was further continued under reduced pressure. The esterification rate 48 hours after the start of the reaction was 98%, the fatty acid monoglyceride content was 98% by mass, the fatty acid diglyceride content was below the detection limit (1% by mass or less) in the TLC-FID method, and the water content was 2. It was 5 mass%. Thus, by controlling the water content to 0.3 to 2.5% by mass and lowering the reaction temperature in the late stage of the reaction to solidify the reaction solution, the content of fatty acid diglyceride as a by-product is reduced to 1% by mass or less. be able to.
Ｃ１０：０の脂肪酸を用いて、反応温度を下げずに反応を行った場合、反応後期の水分含量を０．３７〜１．５質量％に制御すると、脂肪酸モノグリセリドが効率よく得られる（表３の実験Ｉを参照）。このように、副産物である脂肪酸ジグリセリド含量をできるだけ少なくするには、水分含量が０．３質量％未満にならない範囲で、できる限り水分含量を低く保たなければならず、水分含量の抑制を厳密に行う必要がある。しかし、反応後期の反応温度を下げた場合、水分含量の制御を厳密に行わなくても、０．３〜２．５質量％の範囲内であれば、副産物である脂肪酸ジグリセリド含量を１質量％以下に抑えることができ、より効率よく脂肪酸モノエステルが得られることがわかる。したがって、反応後期の反応温度を、得られる脂肪酸モノエステルの融点以下、すなわち、反応溶液が固化する温度まで下げて反応を行うと、水分含量が０．３〜２．５質量％の範囲内であれば、脂肪酸の種類によって厳密に制御しなくてもよいことがわかる。 When the reaction is carried out using C10: 0 fatty acids without lowering the reaction temperature, fatty acid monoglycerides can be efficiently obtained by controlling the water content in the latter stage of the reaction to 0.37 to 1.5% by mass (Table 3). See Experiment I). Thus, in order to reduce the content of fatty acid diglyceride as a by-product as much as possible, the water content must be kept as low as possible within the range where the water content does not become less than 0.3% by mass, and the suppression of the water content is strictly limited. Need to be done. However, when the reaction temperature in the later stage of the reaction is lowered, the content of fatty acid diglyceride as a by-product is 1% by mass within the range of 0.3 to 2.5% by mass without strictly controlling the water content. It can be suppressed to the following, and it can be seen that the fatty acid monoester can be obtained more efficiently. Therefore, when the reaction is carried out by lowering the reaction temperature in the latter stage of the reaction to a temperature equal to or lower than the melting point of the obtained fatty acid monoester, that is, the temperature at which the reaction solution solidifies, the water content is within the range of 0.3 to 2.5% by mass. If it exists, it turns out that it is not necessary to control strictly according to the kind of fatty acid.
（比較例１：従来技術の酵素法（特許文献３）による脂肪酸モノグリセリドの合成）
約３０ｍＬのバイアル瓶に、グリセリンと遊離脂肪酸（Ｃ１２：０）との混合液（遊離脂肪酸／グリセリン＝１／２（モル比））１０ｇ、０．２ｇの水、および反応系１ｇに対して２００ＵのリパーゼＧを添加した。次いで、マグネチックスターラーで撹拌し、４ｍｍＨｇの減圧条件下で、水分含量を考慮することなく４８時間反応を行った。この時、反応系をよく撹拌するために、反応温度を遊離脂肪酸が溶解する温度（４５℃）に設定した。反応後の脂肪酸のエステル化率および反応液中の組成を測定した。結果を表４に示す。
(Comparative Example 1: Synthesis of fatty acid monoglyceride by enzyme method of prior art (Patent Document 3))
In a 30-mL vial, 10 g of a mixed solution of glycerin and free fatty acid (C12: 0) (free fatty acid / glycerin = 1/2 (molar ratio)), 0.2 g of water, and 200 g for 1 g of the reaction system Of lipase G was added. Next, the mixture was stirred with a magnetic stirrer and reacted for 48 hours under reduced pressure of 4 mmHg without considering the water content. At this time, in order to sufficiently stir the reaction system, the reaction temperature was set to a temperature (45 ° C.) at which the free fatty acid was dissolved. The esterification rate of the fatty acid after the reaction and the composition in the reaction solution were measured. The results are shown in Table 4.
これとは別に、Ｃ１４：０の脂肪酸（反応温度３０℃および５０℃；圧力５ｍｍＨｇ）およびＣ１５：０の脂肪酸（反応温度５０℃；圧力４ｍｍＨｇ）を用いて、上記Ｃ１２：０の脂肪酸を用いた場合と同様の反応を行った。結果を表４に示す。 Separately, fatty acids of C14: 0 (reaction temperatures of 30 ° C. and 50 ° C .; pressure of 5 mmHg) and fatty acids of C15: 0 (reaction temperature of 50 ° C .; pressure of 4 mmHg) were used. The same reaction as in the case was performed. The results are shown in Table 4.
Ｃ１２：０、Ｃ１４：０、およびＣ１５：０の脂肪酸は融点が高いため、反応系中で脂肪酸が溶解する温度で反応させたが、従来技術の酵素法と同じ方法では、エステル化は、ほとんど進行しなかった（エステル化率、＜２６％）。さらに、Ｃ１４：０の脂肪酸の反応温度を３０℃に下げてもエステル化は、ほとんど進行しなかった。 Since fatty acids of C12: 0, C14: 0, and C15: 0 have a high melting point, they were reacted at a temperature at which the fatty acid was dissolved in the reaction system. No progress (esterification rate, <26%). Furthermore, esterification hardly proceeded even when the reaction temperature of the C14: 0 fatty acid was lowered to 30 ° C.
本発明によれば、Ｃ９：０からＣ１８：０の直鎖飽和脂肪酸、オレイン酸、リノール酸、およびリノレン酸の脂肪酸モノグリセリドを効率よく、かつ高純度で製造する方法が提供される。したがって、本発明の製造方法によって得られた脂肪酸モノグリセリドは、乳化剤などの食品添加物として有用であり、さらに、中鎖脂肪酸のモノグリセリドは、抗菌活性を有することから、抗菌剤などとしても期待され得る。 ADVANTAGE OF THE INVENTION According to this invention, the method of manufacturing the fatty acid monoglyceride of C9: 0 to C18: 0 linear saturated fatty acid, oleic acid, linoleic acid, and linolenic acid efficiently and with high purity is provided. Therefore, the fatty acid monoglyceride obtained by the production method of the present invention is useful as a food additive such as an emulsifier. Furthermore, since the monoglyceride of a medium chain fatty acid has antibacterial activity, it can be expected as an antibacterial agent or the like. .
Claims (4)
該脂肪酸のエステル化率が６０％に達した以降のエステル化反応を、水分含量０．３〜２．５質量％に制御して行う工程を包含する、脂肪酸モノグリセリドの製造方法。 A method for producing a fatty acid monoglyceride from a fatty acid and glycerol by an esterification reaction using lipase,
A method for producing a fatty acid monoglyceride, comprising a step of performing an esterification reaction after the esterification rate of the fatty acid reaches 60% by controlling the water content to 0.3 to 2.5% by mass.
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| JP2010183873A (en) * | 2009-02-12 | 2010-08-26 | Kao Corp | Method for producing fat and oil highly containing monoacylglycerol |
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