JP5997479B2 - Method for synthesizing sugar fatty acid esters by enzymatic method - Google Patents

Description

  The present invention relates to a method for producing a sugar fatty acid ester useful in various fields. More specifically, the present invention relates to a method for producing a sugar fatty acid ester under mild conditions with high reaction efficiency and low fatty acid degradation, and further, the ester of a sugar fatty acid monoester, diester, triester, etc. Among them, the present invention relates to a method for controlling the yield of a specific ester.

  Sucrose fatty acid monoester retains the function as an emulsifier and is widely used in food additives, surfactants, chemical products and the like. A fatty acid monoester of sucrose, which is a typical disaccharide, is known to have a chemical method based on a transesterification reaction between a lower alcohol ester of a fatty acid and sucrose (for example, Japanese Patent No. 3002922). In addition, acylation of trehalose with fatty acid chloride or fatty acid anhydride (Japanese Patent Publication No. 62-50478) is also known. According to these chemical methods, many diesters and triesters are produced in addition to the monoester, and a mixture of esters is obtained. In addition, these chemical methods use a solvent with a bad odor such as pyridine, require energy by reacting at high temperature / high pressure or high temperature / reduced pressure, are liable to cause side reactions, and products are easily discolored. There are problems such as.

Many methods for esterifying monosaccharides, disaccharides, and cyclodextrins with fatty acids using an enzyme (lipase) have been reported. In Japanese Patent Publication No. 3-35912, J. Am. Oil Chem. Soc. 61, 1761-1765 (1984), Japanese Patent Publication No. 7-94483, and Japanese Patent Laid-Open No. 7-115983, monosaccharides, disaccharides, When lipase derived from Candida cylindracea (current name: Candida rugosa ) is added and reacted under normal pressure or reduced pressure, sugar fatty acid monoester Can be synthesized, however, a large amount of diesters and triesters can be produced simultaneously with the monoester, and a mixture of esters can be obtained.

In JP-A-3-76593, sugar fatty acid monoester can be synthesized with high purity by reacting glucose in the presence of tert-butanol, immobilized Candida antarctica lipase, and molecular sieve. However, this method can be applied to monosaccharides such as glucose and fructose, but when it is applied to disaccharides such as sucrose, the reaction efficiency is low due to the low solubility of the sugar in the solvent (the present application). See Examples). This tendency to decrease the reactivity is said to be more pronounced with long chain fatty acids such as stearic acid and oleic acid, compared to medium chain fatty acids such as lauric acid. In order to increase the efficiency of these reactions, pyridine or the like that increases the solubility of long-chain fatty acids must be used as a solvent, but pyridine has drawbacks such as a bad odor (Japanese Patent Laid-Open No. 3-168091).

  Various improved methods have been reported for synthesizing sugar fatty acid monoesters. In JP-A-5-76783, JP-A-9-271387 and the like, a fatty acid monoester is efficiently obtained by reacting glucose having a reducing end methylated as a substrate and using lipase under normal pressure or reduced pressure. Although it can be synthesized, this method has disadvantages such as not being applied to disaccharides, and the removal of the reducing end methyl group also removes fatty acid esters. Sucrose is ketalized (JP-A-8-217783), sucrose is acetalized (JP-A-8-266296 and JP2010-233566), etc. Esters have been synthesized, but complicated operations such as derivatization of sugar ketals and acetals and removal of intramolecular ketals and acetals of products are required.

In J. Ferment. Bioeng. 78, 70-73 (1994), glycerin or trimethylpropane is bound to disaccharides such as maltose, and fatty acids such as oleic acid are ester-linked using lipase derived from Candida cylindracea. I am letting. According to this method, in addition to the monoester, a large amount of diester and triester are produced, and a mixture of esters is obtained.

  Japanese Patent Application Laid-Open No. 61-191610 reports a tablet containing a sucrose fatty acid ester containing 90% or more of a monoester and rapidly dissolving in gastric juice. JP-A-2000-256386 reports a method for purifying sucrose fatty acid monoesters in a simple and high yield from sucrose fatty acid ester mixtures such as monoesters and diesters and triesters of sucrose and fatty acids. Has been. Thus, the usefulness of obtaining a specific ester in a high yield is also pointed out.

Japanese Patent No. 3002922 Japanese Patent Publication No.62-50478 Japanese Patent Publication No. 3-35912 Japanese Patent Publication No. 7-94483 Japanese Patent Laid-Open No. 7-115983 Japanese Unexamined Patent Publication No. 3-76593 Japanese Patent Laid-Open No. 3-168091 Japanese Patent Laid-Open No. 5-176783 JP 9-271387 A JP-A-8-217783 JP-A-8-266296 JP 2010-233566 JP 61-191610 A JP 2000-256386 A

J. Am. Oil Chem. Soc. 61, 1761-1765 (1984) J. Ferment. Bioeng. 78, 70-73 (1994)

  Since sugar fatty acid esters are useful in various fields, it is desired to produce them efficiently. Therefore, there is a need for a method for producing a sugar fatty acid ester under mild conditions with high reaction efficiency and low fatty acid degradation.

  Further, depending on the application field of the sugar fatty acid ester, for example, a high purity sugar fatty acid monoester may be required. In the above method, a mixture of a plurality of types of esters such as monoester, diesel, triester and the like can be obtained, but any of the esters cannot be synthesized efficiently.

  Therefore, when synthesizing a sugar fatty acid ester in which a fatty acid is bound to a cyclic sugar such as a cyclosaccharide or a mixture of disaccharides or trisaccharide or more oligosaccharides or a mixture of monosaccharides such as glucose or fructose, the fatty acids bound to the sugar It is required to establish a method for synthesizing sugar fatty acid esters that can control the number of these, that is, the purity of any of monoesters, diesters, triesters, and the like.

  The present invention has been made in view of the above circumstances, and a hydroxyl group of a cyclic sugar such as a disaccharide or a mixture of trisaccharide or more oligosaccharides or cyclodextrin is modified in advance with a hydroxy-terminated (poly) oxyalkylene group, Providing a method for selectively producing, for example, sugar fatty acid monoesters, by mixing a hydroxy-terminated (poly) oxyalkylene group-modified saccharide with a fatty acid and reacting with an enzyme (lipase) that can react under mild conditions The task is to do.

  In order to achieve the above object, the present invention employs the following configuration.

  Disaccharides (such as maltose, sucrose, and lactose) or trisaccharide or higher oligosaccharide mixtures or cyclic sugars such as cyclodextrins, such as alkylene oxides such as propylene oxide and butylene oxide, and ethylene chlorohydrin at mild temperatures Etherification is carried out by the action of an etherifying agent such as alkylene chlorohydrone, and at least one hydroxyl group mainly possessed by the saccharide is modified with a hydroxy-terminated (poly) oxyalkylene group.

  This reaction is a mild reaction that does not use a bad odor solvent such as pyridine and other volatile and flammable solvents. By modifying the hydroxyl group of the sugar with a hydroxy-terminated (poly) oxyalkylene group, it is considered that the solubility of the sugar in an organic solvent is increased and the lipase is likely to act. Moreover, the hydroxyl group bonded to the pyranose ring of the sugar is sterically bulky to enter the active center of the lipase, resulting in a low lipase reactivity. However, by adding a linker such as a (poly) oxyalkylene group to the hydroxyl group, the steric bulk of the terminal hydroxyl group bonded to the linker is reduced, and the hydroxyl group can easily enter the active center of the lipase. The lipase reactivity is thought to increase.

Next, a fatty acid, an organic solvent (tert-butanol or acetone), and a lipase (fixed) are added to a cyclic sugar such as a cyclosaccharide or a mixture of disaccharides or trisaccharide or more oligosaccharides modified with a hydroxy-terminated (poly) oxyalkylene group. Candida antarctica lipase, immobilized Rhizomucor miehei lipase, immobilized Thermomyces lanuginosa lipase, and immobilized Alcaligenes sp. Lipase, free form ( Candida rugosa ) lipase, etc.) allows the sugar fatty acid monoester to be efficiently produced and the monoester purity relative to the total ester to be 90% or higher.

That is, the present invention is a method for synthesizing a sugar fatty acid ester,
(1) an etherification step of etherification with an etherifying agent to modify at least one hydroxyl group of a saccharide with a hydroxy-terminated (poly) oxyalkylene group; and (2) the hydroxy-terminated (poly) oxyalkylene group There is provided a synthesis method including an enzyme esterification reaction step of esterifying at least one hydroxyl group of the above and a carboxyl group of a fatty acid by dehydration condensation using an enzyme.

  In order to modify with a hydroxy-terminated (poly) oxyalkylene group in the synthesis method of the present invention, at least one hydroxyl group of a saccharide that is etherified with an etherifying agent may be a hydroxyl group at the 6-position of the saccharide.

  As sugars that can be used in the synthesis method of the present invention, disaccharides selected from the group consisting of maltose, sucrose, lactose, cellobiose, trehalose, xylobiose, and melibiose; ~ 10-linked fructooligosaccharides, or oligosaccharides in which one or more monosaccharides are mixed and bonded; or a cyclic sugar selected from the group consisting of cyclodextrin, cyclofructan, and cyclic tetrasaccharide, or of these cyclic sugars Examples thereof include branched cyclic sugars in which one molecule of the above oligosaccharide is further bonded to one molecule.

  In the synthesis method of the present invention, examples of the etherifying agent include chlorohydrin selected from unsubstituted or ethylene oxide substituted with 1 to 4 alkyl groups having 1 to 3 carbon atoms, or ethylene chlorohydrin.

  In the etherification step of the present invention, ethylene oxide, propylene oxide, butylene oxide (1,2-butylene oxide, 2,3-butylene oxide) may be used as ethylene oxide which is unsubstituted or substituted with 1 to 4 alkyl groups having 1 to 3 carbon atoms. Butylene oxide, isobutylene oxide), pentylene oxide (2-propylethylene oxide, 1-methyl-2-ethylethylene oxide, 1-methyl-2,2-dimethyl-ethylene oxide, 2-ethyl-2-methyl-ethylene oxide, 2-isopropyl C 2-5 alkylene oxides such as ethylene oxide are preferred, and ethylene chlorohydrin is preferred as the chlorohydrin.

  As fatty acids that can be used in the synthesis method of the present invention, butyric acid, hexanoic acid, octanoic acid (caprylic acid), nonanoic acid, decanoic acid, dodecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid (palmitic acid), octadecanoic acid ( Stearic acid), icosanoic acid, docosanoic acid, 2-ethylhexanoic acid, 3,5,5-trimethylhexanoic acid, isostearic acid, 9-tetradecenoic acid, 2-hexadecenoic acid, 9-hexadecenoic acid, 9-octadecenoic acid ( Oleic acid), 9,12-octadecanedienic acid (linoleic acid), 6,9,12-octadecanetrienoic acid (α-linolenic acid), γ-linolenic acid, arachidonic acid, dihomo-γ-linolenic acid, eicosapentaenoic acid , Aliphatic carboxylic acid having 4 to 22 carbon atoms selected from the group consisting of docosapentaenoic acid and docosahexaenoic acid; or benzenecarboxylic acid Benzoic acid), 3-phenyl-2-propenoic acid (cinnamic acid), hydroxycinnamic acid, 4-methoxycinnamic acid, 3,4-dihydroxycinnamic acid (caffeic acid), α-cyano-4-hydroxy An aromatic carboxylic acid selected from the group consisting of cinnamic acid, sinapinic acid; or combinations thereof.

Enzymes that can be used in the present invention include animal pancreatic lipase; seed lipase; or Candida antarctica fraction A lipase (CALA) or fraction B lipase (CALB), Candida rugosa ( Candida rugosa). ) Origin, Geotrichum candidum origin, Pseudomonas sp. Origin, Pseudomonas aeruginosa origin, Burkholderia cepacia origin, Alcaligenes sp. Serratia marcescens (Serratia marcescens) derived from Pseudomonas fluorescens (Pseudomonas fluorescens) derived from Rhizomucor miehei (Rhizomucor miehei) origin, derived from Rhizopus oryzae (Rhizopus oryzae), thermo Mrs. Ranuginoza (Thermomyces lanug inosa ), Fusarium heterosporum , Penicillium camembertii , Aspergillus niger , or closely related lipase .

  The present invention further provides a saccharide wherein at least one hydroxyl group of a saccharide is modified with a hydroxy-terminated (poly) oxyalkylene group as a precursor of a saccharide fatty acid ester suitable for the method for synthesizing a saccharide fatty acid ester according to the present invention, and the hydroxy A sugar fatty acid ester of a terminal-type (poly) oxyalkylene group-modified saccharide and a fatty acid is also provided.

  The present invention provides a composition comprising a sugar fatty acid ester having an ester bond between at least one hydroxyl group of a hydroxy-terminated (poly) oxyalkylene group that modifies at least one hydroxyl group of a saccharide and a carboxyl group of a fatty acid.

  In the composition of the present invention, at least one hydroxyl group of a saccharide modified with a hydroxy-terminated (poly) oxyalkylene group may be a hydroxyl group at the 6-position of the saccharide.

  In the composition of the present invention, the sugar fatty acid ester has the general formula (1):

｛式中、Ｓは、糖類の少なくとも１つの水酸基を除いた残基を示し、ｍは１〜ｐの整数を示し、かつ、ｐは前記糖類が有する水酸基の最大個数を示し、ｍ個のＸは、それぞれ独立して、一般式（２）：

{In the formula, S represents a residue obtained by removing at least one hydroxyl group of a saccharide, m represents an integer of 1 to p, and p represents the maximum number of hydroxyl groups of the saccharide, and m X Are each independently a general formula (2):

（式中、Ｒ^１、Ｒ^２、Ｒ^３およびＲ^４は、独立して、Ｈまたは炭素数１〜３のアルキル基を示し、ｎは１〜６の整数を示す。）で表されるヒドロキシ終端型（ポリ）オキシアルキレン基；または一般式（３）：

(Wherein R ¹ , R ² , R ³ and R ⁴ independently represent H or an alkyl group having 1 to 3 carbon atoms, and n represents an integer of 1 to 6). Terminal (poly) oxyalkylene group; or general formula (3):

［Ｒ^５は炭素数４〜２２の脂肪酸からカルボキシル基を除いた残基を示し、Ｌは、存在しないか、または、独立して、一般式（４）：

[R ⁵ represents a residue obtained by removing a carboxyl group from a fatty acid having 4 to 22 carbon atoms, and L is absent or, independently, represented by the general formula (4):

（式中、Ｒ^１、Ｒ^２、Ｒ^３およびＲ^４は、独立して、Ｈまたは炭素数１〜３のアルキル基を示し、ｎは１〜６の整数を示す。）で表される（ポリ）オキシアルキレン基のリンカーを示す。］で表される基である。ただし、少なくとも１つのＸは、一般式（３）で表される基であり、かつ、少なくとも１つのＬは前記（ポリ）オキシアルキレン基のリンカーである。｝で表される。ここで、（ポリ）オキシアルキレン基は、オキシアルキレン基およびポリオキシアルキレン基を含むことを意図している。

(Wherein R ¹ , R ² , R ³ and R ⁴ independently represent H or an alkyl group having 1 to 3 carbon atoms, and n represents an integer of 1 to 6) ( The linker of a poly) oxyalkylene group is shown. ]. However, at least one X is a group represented by the general formula (3), and at least one L is a linker of the (poly) oxyalkylene group. }. Here, the (poly) oxyalkylene group is intended to include an oxyalkylene group and a polyoxyalkylene group.

R ¹ , R ² , R ³ and R ⁴ in the general formulas (2) and (4) are independently H, a methyl group, an ethyl group, a propyl group, or an isopropyl group.

  In the composition of the present invention, as the saccharide, a disaccharide selected from the group consisting of maltose, sucrose, lactose, cellobiose, trehalose, xylobiose, melibiose; glucooligosaccharide having 3 to 10 glucose bound thereto, and 3 to 10 fructose Single-linked fructooligosaccharides, or oligosaccharides bonded by mixing one or more types of monosaccharides; or cyclic sugars selected from the group consisting of cyclodextrins, cyclofructans, and cyclic tetrasaccharides, or one molecule of these cyclic sugars Furthermore, branched cyclic saccharides in which one molecule of the above oligosaccharide is bonded.

  In the general formula (1), p is the maximum number of hydroxyl groups that the saccharide has. Specifically, disaccharides selected from the group consisting of maltose, sucrose, lactose, cellobiose, trehalose, xylobiose, melibiose; and gluco-oligosaccharides with 3-10 glucose bound, fructo-oligos with 3-10 fructose bound The maximum number of hydroxyl groups in an oligosaccharide to which one or more types of sugars or monosaccharides are bonded is p = 3q + 2 (q is glucose, fructose to be bonded, or one or more types are mixed and bonded. The number of monosaccharides is shown.) For example, the maximum number p of hydroxyl groups possessed by disaccharides such as maltose is 8, and the maximum number p possessed by trisaccharides such as maltotriose is 11. The maximum number of hydroxyl groups of a cyclic sugar selected from the group consisting of cyclodextrin, cyclofructan, and cyclic tetrasaccharide is represented by p = 3r (r is the number of sugars contained in the cyclic sugar). For example, the maximum number p of hydroxyl groups possessed by α-cyclodextrin is 18. The maximum number of hydroxyl groups in the branched cyclic saccharide in which one molecule of the oligosaccharide is further bonded to one molecule of the cyclic saccharide is p = 3 (q + r) (q is glucose to be bound, fructose, or one or a plurality of types mixed) And r represents the number of saccharides contained in the cyclic saccharide).

In the present invention, the hydroxy-terminated (poly) oxyalkylene group represented by the general formula (2) is a group having a hydroxyl group formed at the terminal as a result of hydrogenation of the terminal of the (poly) oxyalkylene group. means. When n is 1, the hydroxy-terminated (poly) oxyalkylene group represented by the general formula (2) can also be referred to as a “hydroxyalkyl group”. In any case, it has a hydroxyl group at the terminal.
The modification with a hydroxy-terminated (poly) oxyalkylene group in which the hydroxyl group of the saccharide is represented by the general formula (2) can also be referred to as “hydroxyalkylation” when n is 1.

  In the general formula (3), when L is not present, the fatty acid is directly ester-bonded to the hydroxyl group of the saccharide, and when L is present, the fatty acid and saccharide are not directly ester-bonded and are a (poly) oxyalkylene group. Bond through a linker.

  In the composition of the present invention, as the fatty acid, butyric acid, hexanoic acid, octanoic acid (caprylic acid), nonanoic acid, decanoic acid, dodecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid (palmitic acid), octadecanoic acid (stearic acid) ), Icosanoic acid, docosanoic acid, 2-ethylhexanoic acid, 3,5,5-trimethylhexanoic acid, isostearic acid, 9-tetradecenoic acid, 2-hexadecenoic acid, 9-hexadecenoic acid, 9-octadecenoic acid (oleic acid) ), 9,12-octadecanedienic acid (linoleic acid), 6,9,12-octadecanetrienoic acid (α-linolenic acid), γ-linolenic acid, arachidonic acid, dihomo-γ-linolenic acid, eicosapentaenoic acid, docosa An aliphatic carboxylic acid having 4 to 22 carbon atoms selected from the group consisting of pentaenoic acid and docosahexaenoic acid; or benzenecarboxylic acid (benzoic acid) ), 3-phenyl-2-propenoic acid (cinnamic acid), hydroxycinnamic acid, 4-methoxycinnamic acid, 3,4-dihydroxycinnamic acid (caffeic acid), α-cyano-4-hydroxycinnamic acid An aromatic carboxylic acid selected from the group consisting of acid, sinapinic acid; or combinations thereof.

  In the sugar fatty acid ester of the present invention, the hydroxyl group of the sugar has four states. That is, a hydroxyl group modified with a hydroxy-terminated (poly) oxyalkylene group, a hydroxyl group esterified with a fatty acid, a hydroxyl group esterified with a fatty acid ester via a linker of a (poly) oxyalkylene group, and an unmodified hydroxyl group.

Hereinafter, a reaction scheme for obtaining a hydroxy-terminated (poly) oxyalkylene group-modified saccharide and a sugar fatty acid ester of oleic acid from maltose, which is a disaccharide, is exemplified.
This sugar fatty acid ester is a specific example in which the hydroxyl group at the 6-position of maltose is modified with a hydroxypropyl group, and the hydroxyl group of the hydroxypropyl group is enzymatically esterified with a fatty acid.

  First, maltose (G2), which is a disaccharide, is hydroxypropylated using, for example, propylene oxide. This figure shows a state in which the hydroxyl group at the 6-position of any one of the two linked monosaccharide molecules (reducing terminal or non-reducing terminal) is modified with a hydroxypropyl group depending on the reaction conditions. The hydroxypropylated maltose is then esterified with oleic acid using lipase. In this figure, a state in which esterification has occurred specifically at the hydroxyl group of the hydroxypropyl group is shown.

  The esterification reaction with fatty acids by lipase in organic solvents is greatly improved by modifying cyclic sugars such as cyclosaccharides with disaccharides or trisaccharide or higher oligosaccharide mixtures or cyclodextrins. In addition, sugar fatty acid monoesters can be synthesized efficiently.

  Monosaccharides that are the smallest units that constitute cyclic sugars such as disaccharides, trisaccharide or higher oligosaccharide mixtures, and cyclodextrins are not particularly limited. However, tetrasaccharides such as erythrose and threose (tetraose), ribose, arabinose, xylose, Preferable examples include pentoses such as lyxose, hexoses such as allose, altrose, glucose, mannose, gulose, idose, galactose, and talose. Among these, glucose and fructose are more preferable.

  Preferred examples of the disaccharide include sucrose (sucrose), maltose (maltose), lactose (lactose), cellobiose, trehalose, xylobiose, melibiose and the like.

  As oligosaccharides, one or more types of monosaccharides mixed with 3-10 glucoses such as maltotriose, maltotetraose and maltopentaose, 3-10 fructose-linked fructose Examples of the oligosaccharides bonded to each other include oligosaccharides other than these.

Examples of cyclic sugars include cyclodextrin, cyclofructan, cyclic tetrasaccharide and the like. Examples of the cyclodextrin include α-, β-, and γ-cyclodextrin.
A branched cyclic saccharide in which the oligosaccharide is further bonded to these cyclic saccharides may be used.

  Disaccharide, oligosaccharide mixture of trisaccharide or more, cyclic sugar such as cyclodextrin, and substance that modifies hydroxyl group of branched cyclic sugar with hydroxy-terminated (poly) oxyalkylene group include alkylene such as ethylene oxide, propylene oxide, butylene oxide Examples include oxides and etherifying agents such as alkylene chlorohydrins such as ethylene chlorohydrin, but the reaction is easy, and hydroxypropylated cyclodextrins are already used as cosmetics, pharmaceuticals, deodorant sprays, and softeners. From the viewpoint of being used, propylene oxide is particularly preferable.

  The fatty acid (carboxylic acid) of the acyl donor that esterifies the hydroxyl group of the sugar is not particularly limited as long as it does not impair the effects of the present invention, and is an aliphatic carboxylic acid having 4 to 22 carbon atoms, or aromatic A carboxylic acid is preferred.

  Examples of the aliphatic carboxylic acid having 4 to 22 carbon atoms (fatty acid) include butyric acid, hexanoic acid, octanoic acid (caprylic acid), nonanoic acid, decanoic acid, dodecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid (palmitic acid) , Linear saturated fatty acids such as octadecanoic acid (stearic acid), icosanoic acid, docosanoic acid, branched saturated fatty acids such as 2-ethylhexanoic acid, 3,5,5-trimethylhexanoic acid, isostearic acid, 9-tetradecenoic acid, 2-hexadecenoic acid, 9-hexadecenoic acid, 9-octadecenoic acid (oleic acid), 9,12-octadecanedienic acid (linoleic acid), 6,9,12-octadecanetrienoic acid (α-linolenic acid), γ-linolenic acid And unsaturated fatty acids such as acid, arachidonic acid, dihomo-γ-linolenic acid, eicosapentaenoic acid, docosapentaenoic acid, and docosahexaenoic acid. Among these, from the viewpoint of reaction efficiency, a linear saturated aliphatic carboxylic acid having 6 to 16 carbon atoms is more preferable, and caprylic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, and α-linolenic acid are particularly preferable.

  Examples of the aromatic carboxylic acid include benzenecarboxylic acid (benzoic acid), 3-phenyl-2-propenoic acid (cinnamic acid), hydroxycinnamic acid, 4-methoxycinnamic acid, and 3,4-dihydroxycinnamic acid. (Caffeic acid), α-cyano-4-hydroxycinnamic acid, sinapinic acid and the like.

  These fatty acids and carboxylic acids may be used alone or in combination of two or more.

  The esterification reaction using an enzyme such as lipase may be a reaction in an organic solvent, a reaction in an aqueous solution, or a reaction in the absence of a solvent. From the viewpoint of reaction efficiency, the reaction in an organic solvent may be performed. preferable. The organic solvent to be used is preferably a solvent having no primary hydroxyl group or secondary hydroxyl group. For example, ketones such as acetone, tert-butanol, tert-amyl alcohol, acetonitrile, tertiary alcohols, and nitriles may be used. Acetone and tert-butanol are preferable, and a solvent in which the above organic solvent is appropriately mixed may be used. Further, an organic solvent such as pyridine, dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, N-methyl-2-pyrrolidinone dipropyl sulfoxide may be mixed and reacted.

  The enzyme that esterifies the hydroxyl group of the saccharide and the carboxyl group of the fatty acid is not particularly limited as long as the enzyme has a catalytic activity to ester bond the hydroxyl group and the carboxyl group. For example, a hydrolase such as lipase, esterase, or protease is preferable. And lipase is a more preferred enzyme.

The lipase is not particularly limited as long as it does not impair the effects of the present invention, and may be any of animal origin, microorganism origin, or plant origin. For example, lipase derived from pancreas of animals such as pigs and humans, lipase derived from seeds such as wheat germ, fraction A lipase (CALA) or fraction B derived from Candida antarctica (also referred to as Psudozyma antarctica ) lipase (CALB), Candida Rugoza (Candida rugosa) derived from: (. Pseudomonas sp) (formerly known as Candida cylindracea (Candida cylindracea)), Jiotorikamu-Kanjidamu (Geotrichum candidum) derived from Pseudomonas derived from the genus Pseudomonas Aeruginoza (Pseudomonas aeruginosa) origin, Burkholderia cepacia (Burkholderia cepacia) derived from: (. Alcaligenes sp) (formerly known as Pseudomonas cepacia (Pseudomonas cepacia)), Alcaligenes derived from the genus, Serratia marcescens (Serratia marcescens) derived from Pseudomonas fluorescens (Ps eudomonas fluorescens) derived from Rhizomucor miehei (Rhizomucor miehei) derived from Rhizopus oryzae (Rhizopus oryzae) derived from (formerly known as: Rhizopus delemar (Rhizopus delemar)), Thermo Mrs. Ranuginoza (Thermomyces lanuginosa) (formerly Humicola Ranuginoza (Humicola lanuginosa )), Fusarium heterosporum , Penicillium camembertii , Aspergillus niger , or closely related lipases. it can. Among these, in particular, the fraction B lipase derived from Candida antarctica (CALB), the lipase derived from Rhizomucor miehei , the lipase derived from Thermomyces lanuginosa , and the genus Alcaligenes sp. .) Derived lipase is a preferred enzyme.

  The enzyme may be used as a free lipase or may be immobilized on a carrier, but the immobilized enzyme can be easily recovered from the reaction solution when the enzyme is immobilized. The enzyme can be easily reused.

  Enzyme immobilization methods include physicochemical adsorption or binding to an inert carrier such as Celite, or binding to an ion exchange resin, in a gel lattice made of a polymer such as polyacrylamide. A known method such as a comprehensive method including an enzyme can be applied.

  As the enzyme, one kind of enzyme may be used alone, or a plurality of enzymes may be used in combination.

  The amount of the enzyme to be used is not particularly limited as long as the effects of the present invention are not impaired. Usually, 10 to 100,000 units (U), more preferably 1,000 to 50,000 U, is added to 1 g of sugar as a reaction raw material. Is preferred. Here, 1U is the amount of enzyme that liberates 1 micromole of fatty acid per minute in the hydrolysis reaction of fats and oils such as olive oil.

  The temperature at which the enzyme reaction is performed may be any temperature as long as the enzyme does not lose its activity, but is preferably 0 to 80 ° C, more preferably 20 to 60 ° C.

  The reaction time for performing the esterification reaction is not particularly limited as long as it is a reaction time for obtaining a sugar fatty acid ester as a product. Although depending on the reaction temperature, the amount of enzyme, and the base mass, the esterification reaction can be sufficiently carried out usually in about 2 to 120 hours.

  The molar ratio of the raw material sugar to fatty acid may be any as long as the product sugar fatty acid ester can be obtained, but preferably fatty acid / sugar = 0.5 to 20, more preferably fatty acid / sugar = 1 to 6 .

  The amount of sugar and fatty acid dissolved in the organic solvent may be any as long as the product sugar fatty acid ester can be obtained. Preferably, the total amount of sugar and fatty acid is 1 to 300 mg per 1 mL of solvent. Preferably it is 4-100 mg.

  It is sufficient to add only an organic solvent, sugar, fatty acid, and enzyme to the reaction system and react while shaking or stirring, but in order to increase the reaction efficiency, the reaction is carried out while removing the water produced during esterification. desirable. In order to remove the generated water, a method using a known dehydrating agent such as molecular sieve, which performs the reaction under reduced pressure, can be employed.

  The sugar fatty acid ester synthesized by the above method can be quantified and analyzed by the following method.

<Analytical conditions for disaccharide fatty acid monoesters and diesters by HPLC>
After recovering the supernatant from the reaction solution, 100 μL of this supernatant and 100 μL of methanol were mixed, and the supernatant was recovered by centrifugation. The precipitate obtained by this centrifugation was washed with 200 μL of methanol and centrifuged, and the supernatant was collected and mixed with the previous supernatant. After removing the solvent under reduced pressure, the residue was dissolved in 400 μL of acetone: acetonitrile: TFA = 33: 67: 0.05 (volume ratio). 40 μL of this was loaded onto Cosmo Seal 5C18-MS (Nacalai Tesque, 4.6 × 250 mm, 40 ° C.), and the disaccharide fatty acid monoester and diester were analyzed. The elution solvent at the time of this analysis was acetone: acetonitrile: TFA = 33: 67: 0.05 (volume ratio), the flow rate was 0.5 mL / min, and a differential refractometer was used as a detector.

  The product (ester of oleic acid and hydroxypropyl maltose (HP-G2)) produced by the reaction described in the examples below is fractionated and purified under the above-mentioned HPLC conditions, and a calibration curve is prepared. The amount of the product was calculated from the calibration curve, and the conversion rate to the product (monoester and diester) relative to the substrate was determined. Here, for example, when the conversion rate to a monoester is 50% and the conversion rate to a diester is 5%, when there are 100 molecules of HP-G2 as a raw material, 50 molecules of HP-G2 are converted into a monoester. , Which means that 5 molecules of HP-G2 were each converted to diester.

<Measurement of esterification rate by saponification decomposition method>
Mixtures of tri- or higher oligosaccharides and cyclic sugars such as cyclodextrins were analyzed in this way. After removing the solvent contained in 0.6 mL of the reaction solution under reduced pressure, the residue was suspended in 0.6 mL of hexane, centrifuged, and the solvent layer containing unreacted fatty acid was removed. (At this time, there was no other than unreacted fatty acid in the solvent layer.) After washing the precipitate with 0.2 mL of hexane, the precipitate obtained by centrifugation was dissolved in 0.6 mL of isopropanol. After removing molecular sieves or enzyme-derived insoluble matter by centrifugation, 2 micromolar internal standard (palmitic acid) was added, and this solution was divided equally into 0.3 mL portions. One of them was subjected to GC analysis without treatment, and the other was subjected to GC analysis after saponification decomposition.

  Saponification decomposition was performed as follows. To 0.3 mL of the isopropanol solution in which the sample was dissolved, 1.5 mL of 0.1 M NaOH / 92% ethanol solution was added and heated at 65 ° C. for 10 minutes. After cooling to room temperature, 2.5 mL of water was added, and 2M hydrochloric acid was added until acidic. After extracting the fatty acid with 2 mL hexane, the hexane layer was recovered. After removing the solvent with an evaporator, 0.5 mL of toluene / hexane / methanol (4: 1: 1, v / v / v) solution was added, and fatty acid was methyl esterified with 2M trimethylsilyldiazomethane diethyl ether solution, followed by GC analysis. .

  For GC analysis without treatment, remove 0.3 mL of the isopropanol solution in which the sample was dissolved, add 0.5 mL of a toluene / hexane / methanol solution (4: 1: 1, v / v / v), and trimethylsilyl. After fatty acid was methyl esterified with diazomethane, GC analysis was performed. Calculate the relative amount of oleic acid with respect to the internal standard when saponified and decomposed, and the relative amount of oleic acid with respect to the internal standard when not saponified and decomposed, and the difference between them was defined as the amount of oleic acid bound to the sugar. . The esterification rate of oleic acid per sugar molecule was calculated from the amount of oleic acid relative to the internal standard and the amount of substrate in the reaction system. Here, for example, in hydroxypropylated β-cyclodextrin, if there are 100 molecules of hydroxypropylated β-cyclodextrin as a raw material, 50 molecules of hydroxypropylated β-cyclodextrin become monoester, 5 molecules of hydroxypropylated When each β-cyclodextrin is converted to a diester, 50 + 5x2 = 60 molecules of fatty acid will be quantified because the saponification decomposition method only quantifies the amount of bound fatty acid. . Since it is defined by the esterification rate of oleic acid per sugar molecule, the esterification rate is 60%. After this saponification decomposition analysis, if the ratio of monoester to diester is analyzed to 10: 1 by MS analysis etc., conversion rate from 60% esterification to conversion rate from hydroxypropylated β-cyclodextrin to monoester Can be calculated as 50% and the conversion rate to diester is 5%.

(Production Example 1) Preparation of hydroxypropylated maltose (HP-G2) using propylene oxide
20 g of water, 0.3 g of sodium hydroxide, 30 g of maltose (G2), and 9 g of propylene oxide were added and reacted overnight at 42 ° C. with shaking. After the reaction, 1.2 M HCl was added to adjust the pH to 5, and then unreacted propylene oxide and water were removed under reduced pressure to prepare crude purified HP-G2.

This crude purified HP-G2 was analyzed by MALDI / TOF-MS (AXIMA Confidence, Shimadzu Corporation) using 2,5-dihydroxybenzoic acid (DHBA) as a matrix. As a result, the (HP) ₁ -G2 content was 51.3 mol%. there were. The analysis results are shown in Table 1.

Where G2 is maltose, (HP) ₁ -G2 is a substance in which one of the maltose hydroxyl groups is hydroxypropylated, and (HP) ₂ -G2 is a substance in which _two maltose hydroxyl groups are hydroxypropylated , (HP) ₃ -G2 means a substance obtained by hydroxypropylation of three of the maltose hydroxyl groups. HP-G2 means a mixture of (HP) ₁ -G2, (HP) ₂ -G2 and (HP) ₃ -G2, or includes all the hydroxypropylated hydroxyl groups without specifying the number. This is used to express it. The same applies to other hydroxyalkylated saccharides.

Next, 4 g of crude purified HP-G2 was loaded onto an 830 mL activated carbon column, and G2, (HP) ₁ -G2 (HP) with a gradient of 0% ethanol (1.5 L) / 50% ethanol (1.5 L) ) ₂ -G2 and the like were eluted. Fractions mainly composed of (HP) ₁ -G2 were collected, and 1.1 g of purified HP-G2 was obtained by deethanol and lyophilization using an evaporator.

Table 1 shows the results of analyzing this purified HP-G2 by MALDI / TOF-MS using DHBA as a matrix. As a result, the (HP) ₁ -G2 content was 94.4 mol%. Moreover, when purified HP-G2 was analyzed by ion trap LC-MS (Finnigan LCQ ^DECA , Thermo Quest), a sodium ion addition molecule [M + Na ⁺ ] with m / z = 423.3 was observed. It was confirmed that one site was a hydroxypropylated material.

(Example 1) Effect of saccharide solubility due to hydroxypropylation of hydroxyl group An appropriate amount of maltose (G2) or purified HP-G2 was added to 1 mL of an organic solvent (tert-butanol, acetone) and heated to 50 ° C. Stir for 1 hour. The supernatant was collected by centrifugation, and after removing the solvent, the weight of the residue was measured. This value was defined as the solubility (mg / mL) per mL of the organic solvent. The results are shown in Table 2.

(Example 2) Esterification of purified HP-G2 and oleic acid using lipase
25 mg of substrate (G2, and purified HP-G2 prepared in Production Example 1), 100 mg of oleic acid (Tokyo Kasei, purity of about 90%), 5 mL of tert-butanol (Wako Pure Chemical Industries), 100 mg Candida antarctica lipase (Novozym 435, Novozymes Japan) and 500 mg of molecular sieve 3A (Kanto Chemical) were mixed and reacted at 50 ° C. with shaking. Samples are taken after 1, 3 and 5 days and the content of the product (G2 or purified HP-G2 oleic acid monoesters and diesters) is determined by HPLC and converted to monoesters and diesters for G2 and purified HP-G2 The rate was calculated. The results are shown in Table 3.

  It was found that the conversion rate when purified HP-G2 was used as a substrate was 31 times higher on the first day of the reaction and 7.7 times higher on the fifth day of the reaction later than when G2 was used as the substrate. Therefore, it was found that hydroxypropylated maltose oleate can be efficiently synthesized by modifying the hydroxyl group of maltose, which is a disaccharide, with a hydroxypropyl group using a lipase reaction.

  Reasons for efficient synthesis are as follows: (1) Lipase, which is extremely difficult to act on solid substrates, has been improved by increasing the solubility of sugar in organic solvents by hydroxyalkylating sugar. 2) Since a linker such as a (poly) oxyalkylene group is present at the hydroxyl group of the sugar, the hydroxyl group of the hydroxyalkyl group can easily enter the active center of the lipase, and the reactivity of the lipase is increased.

  The reaction for modifying the hydroxyl group of maltose with a hydroxypropyl group is a reaction under simple and mild conditions, and does not use a reagent having a bad odor such as pyridine. The subsequent esterification reaction using lipase has a higher reaction efficiency than the conventional method (reaction using maltose that is not hydroxypropylated) and is mild under the conditions, and there is a risk of fatty acid degradation. Therefore, this synthesis method is a novel and excellent synthesis method.

Example 3 LC / MS Analysis of (HP) ₁ -G2 Oleic Acid Monoester The monoester and diester synthesized in Example 2 were fractionated and purified by HPLC and analyzed by LC-MS. As a result, monoesters and diesters were detected as sodium-added molecules [M + Na ⁺ ] with m / z = 687.4 and m / z = 951.5, respectively. Collision-induced cleavage with sodium adduct molecules as parent ion peaks showed that in monoester, [M-OCOR-H + Na ⁺ ] fragment ion m / z = 405 (R = CH ₃ (CH ₂ ) ₇ CH = CH (CH ₂ ) ₇ ) and [M-OCH ₂ CH (CH ₃ ) OCOR-H + Na ⁺ ] fragment ions m / z = 347 were detected. On the other hand, in the diester, [M-OCOR-H + Na +] in the fragment ion m / z = 669, and _{[M-OCH 2 CH (CH} 3) OCOR-H + Na +] in the fragment ion m / z = 611 was detected respectively. From the above, it was confirmed that they were hydroxypropylated maltose oleic acid monoester and hydroxypropylated maltose oleic acid diester in which one or two oleic acids were ester-bonded to (HP) ₁ -G2. As a result of MALDI / TOF-MS analysis, no triester was detected.

(Example 4) Effect of disaccharide type on esterification of hydroxypropylated disaccharide and fatty acid using lipase Also in disaccharides other than maltose, disaccharide fatty acid ester is efficiently obtained by hydroxypropylating the hydroxyl group. It was investigated whether it could be synthesized. By the same method as in Production Example 1, crude purified HP-sucrose and crude purified HP-lactose were prepared from sucrose (sucrose) or lactose. These were analyzed for the composition ratio of the product by MALDI / TOF-MS using DHBA as a matrix. As a result, in crude purified HP-sucrose, unreacted sucrose was 5.4 mol%, one hydroxyl group was hydroxypropylated (HP) ₁ -sucrose 28.1 mol%, and two hydroxyl groups were hydroxypropyl (HP) ₂ -sucrose was 36.5 mol%, 3 hydroxyl groups were hydroxypropylated (HP) ₃ -sucrose was 23.6 mol%, and 4 hydroxyl groups were hydroxypropylated (HP ₄ ) Sucrose was 6.4 mol%. In the crude purified HP-lactose, unreacted lactose was 38.7 mol%, one hydroxyl group was hydroxypropylated (HP) ₁ -lactose was 49.3 mol%, and two hydroxyl groups were hydroxypropylated (HP) ₂ -Lactose was 9.3 mol%, and (HP) ₃ -Lactose in which three hydroxyl groups were hydroxypropylated was 2.7 mol%.

  25 mg substrate (sucrose, crude HP-sucrose, lactose, crude HP-lactose, maltose (G2), and crude HP-G2), 100 mg oleic acid, 5 mL tert-butanol, 100 mg Novozym 435 and 500 mg of molecular sieve 3A were mixed and reacted at 50 ° C. with shaking. Sampling was performed after 1, 3 and 5 days, and the content of the product (oleic acid monoester) was measured by HPLC, and the conversion ratio of the raw material to the monoester was calculated. The results are shown in Table 4.

  As a result, it was found that hydroxypropylated disaccharide fatty acid esters can be efficiently synthesized using lipase by hydroxypropylating not only maltose but also the hydroxyl group of disaccharides such as sucrose and lactose. It was found that it can be applied to sugar.

(Example 5) Effect of fatty acid type on esterification of purified HP-G2 and fatty acid using lipase
Mix 25 mg purified HP-G2, 100 mg fatty acid (caprylic acid, palmitic acid, linoleic acid, Tokyo Chemical), 5 mL tert-butanol, 100 mg Novozym 435, and 500 mg molecular sieve 3A And reacted at 50 ° C. with shaking. 1, 3, and 5 days after sampling, the content of the product (various fatty acid monoesters of purified HP-G2) was measured by HPLC, and the conversion rate of the purified HP-G2 into a monoester was calculated. The results are shown in Table 5.

  As a result, not only liquid oleic acid (C18: 1) with 18 carbon atoms and one double bond, but also linoleic acid (C18: 2) with more double bonds than oleic acid (2), Similar to oleic acid, palmitic acid (C16: 0), which has 16 carbon atoms and no double bonds, is solid at room temperature, and caprylic acid (C8: 0), which has a short carbon number (8) and has no double bonds, is the same as oleic acid. It was found that hydroxypropylated maltose fatty acid monoester can be efficiently synthesized using lipase, and that this technology can be applied to a wide range of fatty acids.

(Example 6) Effect of solvent type on esterification of purified HP-G2 and oleic acid using lipase
Mix 25 mg purified HP-G2, 100 mg oleic acid, 5 mL solvent (hexane, acetonitrile, acetone), 100 mg Novozym 435, and 500 mg molecular sieve 3A and react at 50 ° C with shaking I let you. Samples were taken after 1, 3 and 5 days, and the content of the product (oleic acid monoesters and diesters of purified HP-G2) was measured by HPLC, and the conversion ratio of the purified HP-G2 to monoesters and diesters was calculated. The results are shown in Table 6. The reaction in tert-butanol in Example 2 is also shown.

  As a result, it was found that the esterification reaction proceeds efficiently not only in tert-butanol but also in acetonitrile and acetone. In particular, the esterification rate on the first day in acetone was found to be greater than the esterification rate on the first day in tert-butanol.

(Example 7) Effect of molar ratio on esterification of purified HP-G2 and oleic acid using lipase
25 mg substrate (G2 or purified HP-G2), 100 mg oleic acid (substrate: oleic acid = 1: 6 (molar ratio)) or 16.4 mg oleic acid (substrate: oleic acid = 1: 1 (molar ratio) )), 5 mL acetone, 100 mg Novozym 435, and 500 mg molecular sieve 3A were mixed and reacted at 50 ° C. with shaking. Sampling after 1 and 2 days, measuring the content of the product (G2 or purified HP-G2 oleic acid monoester and diester) by HPLC and calculating the conversion rate of monoester and diester to G2 or purified HP-G2 did. The results are shown in Table 7.

  As a result, similar to the reaction in tert-butanol in Example 2, in the reaction in acetone in the presence of an excess amount of oleic acid, purified HP-G2 had a higher esterification rate than G2. On the other hand, when oleic acid was present in excess, a large amount of diester was present. However, when the amount of oleic acid is reduced to the substrate: oleic acid = 1: 1 (molar ratio), the monoester: diester = 60: 4 (molar ratio) in the reaction on the first day of purified HP-G2 Therefore, it was found that by using equimolar amounts of oleic acid with respect to the substrate, it was possible to synthesize monoesters selectively (the molar ratio of monoesters to the total of ester bodies was 90% or more). Further, even when equimolar amounts of oleic acid were used in acetone relative to the substrate, purified HP-G2 was about 6 times higher in esterification than G2. As a result of MALDI / TOF-MS analysis, no triester was detected at any of the molar ratios of 1: 1 and 1: 6. Thus, it was found that the production ratio of monoesters and diesters can be controlled by considering the balance of the substrates (saccharides and fatty acids).

(Example 8) Effect of amount of molecular sieve 3A on esterification of purified HP-G2 and oleic acid using lipase
25 mg purified HP-G2, 16.4 mg oleic acid (substrate: oleic acid = 1: 1 (molar ratio)), 5 mL acetone, 100 mg Novozym 435, and molecular sieve 3A (0, 100, 300, 500 mg) was mixed and reacted at 50 ° C. with shaking. Sampling was conducted after 1 and 2 days, and the content of the product (oleic acid monoester and diester of purified HP-G2) was measured by HPLC, and the conversion ratio of monoester and diester to purified HP-G2 was calculated. The results are shown in Table 8.

  As a result, although the molecular sieve that removes the water generated during the esterification reaction does not exist in the reaction system, the reaction is found to be more reactive. The product content was highest when the molecular sieve amount was 300 mg.

(Example 9) Effect of base mass on esterification of purified HP-G2 and oleic acid using lipase
25, 75, 150, 300 mg purified HP-G2, 16.4, 49.2, 98.4, 196.8 mg oleic acid (substrate: oleic acid = 1: 1 (molar ratio)), 5 mL acetone, 100 mg Novozym 435 , And 300 mg of molecular sieve 3A were mixed and reacted at 50 ° C. with shaking. Samples were taken after 1, 2 and 3 days, the content of the product (purified HP-G2 oleic acid monoester and diester) was measured by HPLC, and the amount of monoester and diester produced in the 5 mL reaction system was calculated. . The results are shown in Table 9.

  As a result, when 25 mg or 75 mg of substrate is used, monoester can be synthesized with a purity of 90% or more (monoester / (monoester + diester)), but when there are more substrates, The total amount of product increased and the diester ratio also increased. Moreover, it turned out that the production amount ratio of a monoester and a diester can be controlled by controlling the base mass.

(Example 10) Effect of lipase type on esterification of purified HP-G2 and oleic acid
25 mg purified HP-G2, 16.4 or 100 mg oleic acid (substrate: oleic acid = 1: 1 (molar ratio) or 1: 6), 5 mL tert-butanol or acetone, 100 mg immobilized lipase ( Novozym 435, Lipozym TLIM (from Thermomyces lanuginosa , Novozymes Japan), Lipozym RMIM (from Rhizomucor miehei , Novozymes Japan), ion exchange resin Duolite A568 (Rhom and Haas) Ltd., importer Sumika Chemtex) to immobilized lipase-QLM (. Alcaligenes (Alkaligenes sp) Meito Sangyo)) or 100 mg of free lipase (lipase-QLM, lipase-oF ( Candida Rugoza (Candida rugosa ), name sugar industry)), and 300 mg of molecular sieve 3A were mixed and reacted at 50 ° C. with shaking. Sampling was conducted after 1 to 5 days, and the composition of the product (purified HP-G2 oleic acid monoester) was measured by HPLC, and the conversion rate of the purified HP-G2 into a monoester was calculated. The results are shown in Table 10.

  As a result, not only the immobilized Novozym 435 but also the immobilized Lipozym TLIM, Lipozym RMIM, Lipase-QLM, non-immobilized free Lipase-QLM, and Lipase-OF were found to have significant products.

(Production Example 2) Preparation of hydroxyethylated maltose (HE-G2) using ethylene chlorohydrin
Add 300 g of water, 6 g of sodium hydroxide, 300 g of maltose (G2), and 90 g of ethylene chlorohydrin, and with stirring at 38 ° C, 24% NaOH to keep the pH below 11.8 The solution was reacted overnight while dropping the solution as appropriate. After the reaction, 1.2 M HCl was added to adjust the pH to 5, then subjected to activated carbon column chromatography and eluted with 30% ethanol to obtain a crude purified HE-G2 solution. The liquid was concentrated by evaporation and dried to obtain crude purified HE-G2.

When the composition ratio of the product was analyzed by MALDI / TOF-MS using DHBA as a matrix, unreacted G2 was 5.6 mol%, and one hydroxyl group was hydroxyethylated (HE) ₁ -G2 52.8 mol%, (HE) ₂ -G2 hydroxy-ethylated at _two hydroxyl groups was 35.5 mol%, and hydroxy-hydroxylated three (HE) ₃ -G2 was 6.1 mol% .

(Example 11)
Mix 25 mg crude HE-G2, 16.4 mg oleic acid (substrate: oleic acid = 1: 1 (molar ratio)), 5 mL acetone, 100 mg Novozym 435, and 300 mg molecular sieve 3A. 1 and 2 days after reaction at 50 ° C. with shaking, the content of the product (oleic acid monoester and diester of crude HE-G2) was determined by HPLC, and the monoester for crude HE-G2 And the conversion rate to diester was calculated. The results are shown in Table 11.

As a result, it was found that the reactivity of lipase was improved not only by modifying the hydroxyl group of maltose with a hydroxypropyl group but also with a hydroxyethyl group. In addition, when the product after the esterification reaction was analyzed by MALDI / TOF-MS, the monoesters of (HE) ₁ -G2 and (HE) ₂ -G2 were m / z = 673.2 and 717.2 sodium, respectively. It was detected as an additional molecule [M + Na ⁺ ]. The triester sodium-added molecule [M + Na ⁺ ] was not detected.

(Production Example 3) Production of oligosaccharide mixture having three or more sugars An oligosaccharide mixture having three or more sugars was produced by the following method. In accordance with the method described in JP-A-9-143191, tapioca starch was decomposed with α-amylase to obtain a maltooligosaccharide having a reducing sugar of 25. Thereafter, the maltooligosaccharide was adjusted to a 40% aqueous solution, 4% of fresh yeast (manufactured by Oriental Yeast Co., Ltd.) was added to the maltooligosaccharide, and the mixture was stirred at 35 ° C. for 2 days. Thereafter, purification with diatomaceous earth, activated carbon, ion exchange resin, and spray drying were performed to obtain a final product. As a result, 0.7 wt% for G2 or less, 18.1 wt% for maltotriose (G3), 19.9 wt% for maltotetraose (G4), 28.4 wt% for maltopentaose (G5), and maltohexaose (G6) An oligosaccharide mixture containing 14.3 wt% and 18.6 wt% of maltoheptaose (G7) or more (an average of 4.6 glucose bonds) was obtained.

(Example 12) Hydroxypropylation of trisaccharide or higher oligosaccharide mixture using lipase and esterification of oleic acid Maltoose was changed to the oligosaccharide mixture (No.3L-P) produced in Production Example 3, The reaction was carried out in the same manner as in Production Example 1 to prepare a crudely purified hydroxypropylated trisaccharide or higher oligosaccharide mixture (crude purified HP-No. 3L-P). As a result of analysis by MALDI / TOF-MS, the number of hydroxypropyl groups bonded per molecule was 1.62 for G3, 1.76 for G4, 2.03 for G5, and 2.17 for G6.

  25 mg of No.3L-P (oligosaccharide mixture produced in Production Example 3) or crude purified HP-No.3L-P (crudely purified hydroxypropylated trisaccharide or higher oligosaccharide mixture), 100 mg oleic acid , 5 mL of acetone or tert-butanol, 100 mg of Novozym 435, and 300 mg of molecular sieve 3A were mixed and reacted at 50 ° C. for 3 days with shaking. Esterification rate of oleic acid to HP-No.3L-P (a crudely purified hydroxypropylated trisaccharide or higher oligosaccharide mixture) of 1 molecule (average 4.6 glucose binds) by saponification decomposition method ( = (Number of moles of oleic acid released by saponification decomposition) / (number of moles of crudely purified HP-No.3L-P (a mixture of crudely purified hydroxypropylated trisaccharide or more oligosaccharides)) x 100) . The results are shown in Table 12.

  As a result, it was found that the esterification rate was significantly increased by hydroxypropylating No. 3L-P.

When the reaction solution was analyzed by MALDI / TOF-MS using DHBA as a matrix, the oligosaccharide oleate monoester was detected as a sodium ion addition molecule [M + Na ⁺ ] as shown in Table 13 and produced. Confirmed. On the other hand, no sodium ion addition molecule in the diester form of hydroxypropylated oligosaccharide was detected.

(Example 13) Esterification of hydroxypropylated α-cyclodextrin and oleic acid using lipase Esterification of α-cyclodextrin (α-CD) in which 6 glucoses were bound cyclically was carried out. A reaction was carried out in the same manner as in Production Example 1 except that maltose was changed to α-CD to prepare crude purified hydroxypropylated α-CD (crude purified HP-α-CD). When the composition ratio of the product was analyzed by MALDI / TOF-MS using DHBA as a matrix, it was found that 8.1 hydroxyl groups per molecule of α-CD were hydroxypropylated.

  Mix 25 mg α-CD or crude HP-α-CD, 100 mg oleic acid, 5 mL tert-butanol, 100 mg Novozym 435, and 300 mg molecular sieve 3A at 50 ° C with shaking For 3 days. Esterification rate of oleic acid to one molecule of crude purified HP-α-CD (= (moles of oleic acid liberated by saponification)) / crude purification (moles of HP-α-CD) ) X 100). The results are shown in Table 14.

  As a result, it was found that the esterification rate was significantly increased by hydroxypropylating α-CD.

In addition, after removing oleic acid contained in the sample after esterification of crude purified HP-α-CD with oleic acid, analysis by MALDI / TOF-MS revealed that hydroxypropyl groups having 6 to 10 hydroxypropyl groups bonded thereto. The monoesters of isomerized α-CD were detected as sodium ion addition molecules [M + Na ⁺ ] at m / z = 1607.7, 1665.3, 1723.6, 1781.5, and 1839.6, respectively. On the other hand, the sodium ion addition molecule [M + Na ⁺ ] of the hydroxyesterated α-CD diester was not detected. Further, when the sample after esterifying α-CD with oleic acid was similarly analyzed by MALDI / TOF-MS, the peak of the ester was not confirmed. Therefore, the esterification rate with respect to α-CD in Table 14 is estimated to be 2.5% because of the background level value of the esterification rate measurement by the saponification decomposition method. From these results, it can be estimated that the conversion ratio of the crude purified HP-α-CD to the monoester form is 11.8-2.5 = 9.3%, and the conversion ratio to the diester form is 0%.

(Example 14) Esterification of hydroxypropylated β-cyclodextrin and oleic acid using lipase Esterification of β-cyclodextrin (β-CD) in which seven glucoses were bound cyclically was carried out. A reaction was carried out in the same manner as in Production Example 1 except that maltose was changed to β-CD to prepare crude purified hydroxypropylated β-CD1 (crude purified HP-β-CD1). When the composition ratio of the product was analyzed by MALDI / TOF-MS using DHBA as a matrix, it was found that 5.9 hydroxyl groups per molecule of β-CD were hydroxypropylated.

  In the same reaction as in Production Example 1, the amount of propylene oxide used was increased 1.5 times, and the hydroxypropylation reaction was extended to 2 days. As a result, crude purified hydroxypropylated β-CD2 (crude purified HP-β-CD2) in which 9.8 hydroxyl groups were hydroxypropylated per molecule of β-CD was prepared.

  25 mg β-CD or crude HP-β-CD1 or crude HP-β-CD2, 100 mg oleic acid, 5 mL tert-butanol or acetone, 100 mg Novozym 435, and 300 mg molecular sieve 3A was mixed and reacted at 50 ° C. for 3 or 10 days with shaking. Esterification rate of oleic acid to one molecule of crude purified HP-β-CD (= (number of moles of oleic acid liberated by saponification)) / crude purification (number of moles of HP-β-CD) ) X 100). The results are shown in Table 15.

  As a result, it was found that esterification rate was significantly increased by hydroxypropylating β-CD. The esterification rate was higher when the number of hydroxypropyl groups bonded to β-CD was increased, and the esterification rate was higher in the reaction in acetone than in tert-butanol.

Further, after removing oleic acid contained in the sample after esterification of crude purified HP-β-CD2 with oleic acid in acetone for 3 days, analysis by MALDI / TOF-MS revealed that the hydroxypropyl group was 8 to 12 Single-linked hydroxypropylated β-CD monoesters were detected as sodium-added molecules [M + Na ⁺ ] at m / z = 1885.9, 1943.8, 2001.8, 2059.8, 2117.8, respectively. On the other hand, sodium adduct molecular diester of hydroxypropylated ^{β-CD [M + Na +} ] is not detected, also sodium adduct molecules of mono- and diester in the sample after reaction of β-CD [M ⁺ Na + ] Was not detected. From these results, it can be estimated that the conversion ratio of the crude purified HP-β-CD to the monoester form is 33.4.2-1 = 31.3%, and the conversion ratio to the diester form is 0%.

(Example 15) Esterification of hydroxybutylated β-cyclodextrin and oleic acid using lipase Maltose was changed to β-CD, propylene oxide was changed to butylene oxide, the amount of NaOH added was doubled, and the reaction time was increased. A reaction was carried out in the same manner as in Production Example 1 except that the period was changed to 2 days to prepare crude purified hydroxybutylated β-CD (crude purified HB-β-CD). When the composition ratio of the product was analyzed by MALDI / TOF-MS using DHBA as a matrix, it was found that 4.6 hydroxyl groups per molecule of β-CD were hydroxybutylated.

  Mix 25 mg β-CD or crude HB-β-CD, 100 mg oleic acid, 5 mL tert-butanol, 100 mg Novozym 435, and 300 mg molecular sieve 3A at 50 ° C with shaking For 3 days. Esterification rate of oleic acid to one molecule of crude purified HB-β-CD (= (number of moles of oleic acid released by saponification)) / crude purification (number of moles of HB-β-CD) ) X 100). The results are shown in Table 16.

  As a result, it was found that esterification rate was increased by hydroxybutylation of β-CD. Thus, it was confirmed that not only hydroxyethylation and hydroxypropylation, but also β-CD subjected to hydroxybutylation became a substrate for esterification.

Example 16: Determination of critical micelle concentration of hydroxypropylated maltose oleate and hydroxypropylated β-CD oleate
250 mg crude HP-G2 (prepared in Production Example 1), 164 mg oleic acid (substrate: oleic acid = 1: 1 (molar ratio)), 50 mL acetone, 1 g Novozym 435, and 3 g Of molecular sieves 3A were mixed and reacted at 50 ° C. for 2 days with shaking. The same reaction was performed 4 times. After the reaction, all solvent layers were collected, insolubles were removed by centrifugation, and then the solvent was removed with an evaporator. Unreacted oleic acid was removed by silica gel chromatography (elution solvent: hexane: ethyl acetate = 80: 20 (vol / vol)). As a result, 0.7 g of product was obtained. As a result of analyzing this product by MALDI / TOF-MS, the peak intensity ratio of unreacted crude purified HP-G2: monoester: diester was 3.5: 80.3: 16.2.

After preparing 25 mL of a 10 ⁻² mol / L solution of the product, this was appropriately diluted with distilled water to prepare a solution of 10 ⁻⁶ to 10 ⁻² mol / L. The surface tension of these diluted solutions was measured using an automatic surface tension meter CBVP-A3 type (Kyowa Interface Science Co., Ltd.). As a result, the critical micelle concentration was 8.7 × 10 ⁻⁶ mol / L. Similarly, the critical micelle concentration of hydroxypropylated β-CD oleate (reacted product in acetone in Table 15 for 10 days with hexane washed to remove unreacted oleic acid) was 1.3 x 10 ^-5 mol / L. These values are at the same level as the critical micelle concentration of conventional sucrose fatty acid monoesters. Therefore, the product composed mainly of hydroxypropylated maltose oleic acid monoester has the same function as sucrose fatty acid monoester, that is, has a surface activity, and is used as a food additive, a surfactant, It can be widely used in the field of products.

Example 17 Inclusion of Hydroxypropylated β-CD Oleate with Tryptophan Methyl
3 mg tryptophan methyl (3 hydrogen atoms in the methyl group are replaced by deuterium, Trp-Me), 0.8 mg β-CD or HP-β-CD or HP-β-CD oleate (in Table 15 in acetone) The reaction mixture was washed with hexane for 10 days to remove unreacted oleic acid), and 0.05 mL of water was mixed.

These were analyzed by ion trap LC-MS (Finnigan LCQ ^DECA , Thermo Quest). In β-CD, sodium ion addition molecule [M + Na ⁺ ] of inclusion of β-CD and Trp-Me hydrogen ion adduct molecules [M ⁺ H +] is in the HP-beta-CD, hydrogen ion adduct molecules of HP-beta-CD and inclusion of ^{Trp-Me [M + H +} ] is, HP-beta In -CD oleate, the hydrogen ion addition molecule [M + H ⁺ ] of the inclusion product of HP-β-CD oleate and Trp-Me was confirmed.

  α-, β-, and γ-cyclodextrins contain various functional foods such as vitamin A and coenzyme Q10 in the molecule, various antibacterial agents such as iodine, various food flavors such as wasabi, and various pharmaceuticals such as prostaglandins. It is known to be included, and has functions such as substance stabilization, sustained release, and solubilization. By esterifying a fatty acid to cyclodextrin, it becomes amphiphilic, that is, has a function as a surfactant. Therefore, the substance obtained in this example can be expected as a new material having both the inclusion function and the surface activity function of the functional substance.

  According to the present invention, a hydroxyl group such as a disaccharide or a mixture of oligosaccharides of three or more sugars or a cyclic sugar such as cyclodextrin is previously modified with a hydroxy-terminated (poly) oxyalkylene group, and a hydroxy-terminated (poly) oxyalkylene group is obtained. The modified sugar and fatty acid are mixed, and the enzyme (lipase) that can react under mild conditions is reacted to control the purity of the sugar fatty acid ester. Can do.

Claims (13)

A method for synthesizing sugar fatty acid esters,
(1) an etherification step of etherification with an etherifying agent to modify at least one hydroxyl group of a saccharide with a hydroxy-terminated (poly) oxyalkylene group; and (2) the hydroxy-terminated (poly) oxyalkylene group A synthetic method comprising an enzymatic esterification reaction step of esterifying at least one hydroxyl group of the above and a carboxyl group of a fatty acid by dehydration condensation using lipase .   The synthesis method according to claim 1, wherein at least one hydroxyl group of the saccharide is a hydroxyl group at the 6-position of the saccharide.   The saccharide is a disaccharide selected from the group consisting of maltose, sucrose, lactose, cellobiose, trehalose, xylobiose, melibiose; glucooligosaccharide having 3 to 10 glucose bound thereto, fructo oligosaccharide having 3 to 10 fructose bound thereto, or An oligosaccharide in which one or more monosaccharides are mixed and bonded; or a cyclic sugar selected from the group consisting of cyclodextrin, cyclofructan and cyclic tetrasaccharide, or one molecule of these cyclic sugars and one oligosaccharide The synthesis method according to claim 1, which is a branched cyclic sugar to which molecules are bound.   The synthesis method according to claim 1, wherein the etherifying agent is ethylene oxide or ethylene chlorohydrin which is unsubstituted or substituted with 1 to 4 alkyl groups having 1 to 3 carbon atoms.   The etherifying agent is ethylene oxide, propylene oxide, butylene oxide (1,2-butylene oxide, 2,3-butylene oxide, isobutylene oxide), pentylene oxide (2-propylethylene oxide, 1-methyl-2-ethylethylene oxide, The synthesis method according to claim 1, which is selected from the group consisting of 1-methyl-2,2-dimethyl-ethylene oxide, 2-ethyl-2-methyl-ethylene oxide, 2-isopropylethylene oxide) and ethylene chlorohydrin.   The fatty acid is butyric acid, hexanoic acid, octanoic acid (caprylic acid), nonanoic acid, decanoic acid, dodecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid (palmitic acid), octadecanoic acid (stearic acid), icosanoic acid, docosanoic acid , 2-ethylhexanoic acid, 3,5,5-trimethylhexanoic acid, isostearic acid, 9-tetradecenoic acid, 2-hexadecenoic acid, 9-hexadecenoic acid, 9-octadecenoic acid (oleic acid), 9,12-octadecane From dienoic acid (linoleic acid), 6,9,12-octadecanetrienoic acid (α-linolenic acid), γ-linolenic acid, arachidonic acid, dihomo-γ-linolenic acid, eicosapentaenoic acid, docosapentaenoic acid, docosahexaenoic acid An aliphatic carboxylic acid having 4 to 22 carbon atoms selected from the group consisting of: benzenecarboxylic acid (benzoic acid), 3-phenyl-2-propenoic acid ( Cinnamic acid), hydroxycinnamic acid, 4-methoxycinnamic acid, 3,4-dihydroxycinnamic acid (caffeic acid), α-cyano-4-hydroxycinnamic acid, sinapinic acid The method of claim 1, which is an aromatic carboxylic acid; or a combination thereof. The lipase is an animal pancreatic lipase; a wheat germ lipase; or a Candida antarctica fraction A lipase (CALA) or a fraction B lipase (CALB), Candida rugosa, Geotricum lipase Derived from Geotrichum candidum, derived from Pseudomonas sp., Derived from Pseudomonas aeruginosa, derived from Burkholderia cepacia, derived from Alcaligenes sp., Serratia marcescens marcescens), Pseudomonas fluorescens, Rhizomucor miehei, Rhizopus oryzae, Thermomyces lanuginosa, Fusarium heteroporus The lipase of claim 1, selected from the group consisting of lipase derived from rum (Fusarium heterosporum), derived from Penicillium camembertii, derived from Aspergillus niger, or a closely related microorganism. Method. A composition comprising a sugar fatty acid monoester or a sugar fatty acid diester having one or two ester bonds of a hydroxyl group of a hydroxy-terminated (poly) oxyalkylene group that modifies at least one hydroxyl group of a saccharide and a carboxyl group of a fatty acid And the sugar fatty acid ester has the general formula (1):

{In the formula, S represents a residue obtained by removing at least one hydroxyl group of a saccharide, m represents an integer of 1 to p, and p represents the maximum number of hydroxyl groups of the saccharide, and m X Are each independently a general formula (2):

(Wherein R ¹ , R ² , R ³ and R ⁴ independently represent H or an alkyl group having 1 to 3 carbon atoms, and n represents an integer of 1 to 6). Terminal (poly) oxyalkylene group; or general formula (3):

[R ⁵ represents a residue obtained by removing a carboxyl group from a fatty acid having 4 to 22 carbon atoms, and L is absent or, independently, represented by the general formula (4):

(Wherein R ¹ , R ² , R ³ and R ⁴ independently represent H or an alkyl group having 1 to 3 carbon atoms, and n represents an integer of 1 to 6) ( The linker of a poly) oxyalkylene group is shown. ]. However, at least one X is a group represented by the general formula (3), and at least one L is a linker of the (poly) oxyalkylene group. } The composition represented by these.   The composition according to claim 8, wherein at least one hydroxyl group of the saccharide is a hydroxyl group at the 6-position of the saccharide.   The saccharide is a disaccharide selected from the group consisting of maltose, sucrose, lactose, cellobiose, trehalose, xylobiose, melibiose; glucooligosaccharide having 3 to 10 glucose bound thereto, fructo oligosaccharide having 3 to 10 fructose bound thereto, or An oligosaccharide in which one or more monosaccharides are mixed and bonded; or a cyclic sugar selected from the group consisting of cyclodextrin, cyclofructan and cyclic tetrasaccharide, or one molecule of these cyclic sugars and one oligosaccharide The composition according to claim 8 or 9, which is a branched cyclic sugar to which molecules are bound.   The fatty acid is butyric acid, hexanoic acid, octanoic acid (caprylic acid), nonanoic acid, decanoic acid, dodecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid (palmitic acid), octadecanoic acid (stearic acid), icosanoic acid, docosanoic acid , 2-ethylhexanoic acid, 3,5,5-trimethylhexanoic acid, isostearic acid, 9-tetradecenoic acid, 2-hexadecenoic acid, 9-hexadecenoic acid, 9-octadecenoic acid (oleic acid), 9,12-octadecane From dienoic acid (linoleic acid), 6,9,12-octadecanetrienoic acid (α-linolenic acid), γ-linolenic acid, arachidonic acid, dihomo-γ-linolenic acid, eicosapentaenoic acid, docosapentaenoic acid, docosahexaenoic acid An aliphatic carboxylic acid having 4 to 22 carbon atoms selected from the group consisting of: benzenecarboxylic acid (benzoic acid), 3-phenyl-2-propenoic acid ( Cinnamic acid), hydroxycinnamic acid, 4-methoxycinnamic acid, 3,4-dihydroxycinnamic acid (caffeic acid), α-cyano-4-hydroxycinnamic acid, sinapinic acid The composition according to any one of claims 8 to 10, which is an aromatic carboxylic acid; or a combination thereof. The composition according to any one of claims 8 to 11, wherein the sugar fatty acid ester has a diester / monoester molar ratio of 0 to 0.5. The composition according to any one of claims 8 to 12, wherein the sugar fatty acid ester has a diester / monoester molar ratio of 0 to 0.2.