JP2002218993A - Method for producing glyceroglycolipid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new method for the production of a large amount of glyceroglycolipid at a low cost by using only two kinds of natural raw materials comprising an oil-and-fat and a carbohydrate in contrast with conventional method for producing glyceroglycolipid with lipase necessitating three kinds of raw materials comprising glycerol, sugar and fatty acid or fatty acid derivative, etc. SOLUTION: The method for the production of glyceroglycolipid contains the 1st step to decompose a glyceride in an oil-and-fat raw material into glycerol and a fatty acid with lipase, the 2nd step to treat the glycerol with a glycosyltransferase and transfer a sugar from the carbohydrate raw material to form a sugar glycerol and the 3rd step to add the fatty acid produced by the 1st step to the sugar glycerol with a lipase the same as or different from the lipase used in the 1st step and obtain the objective glyceroglycolipid.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing glyceroglycolipids by an enzymatic reaction.

[0002]

2. Description of the Related Art Glyceroglycolipids are glycolipids mainly found in plants and microorganisms, and are particularly present in many living organisms as membrane components. Recent studies have shown that Kaffir lime
Glyceroglycolipids extracted from ( Citrus hystrix ) (the sugar moiety is galactose; the fatty acid moiety is α-linolenic acid or α-linolenic acid and palmitic acid) are found to have a carcinogenesis promotion inhibitory effect (J. Agric. Food Chem. .,
1995, 43, 2779-2783), and its physiological functions have attracted attention.

[0003] Industrially, emulsifiers and surfactants having high safety and useful physiological functions can be expected to be applied to cosmetics, pharmaceuticals, foods and the like.

In order to obtain such glyceroglycolipids,
There is a method of extracting from plants and microorganisms containing it, and various materials such as spinach, pumpkin, diatoms and chlorella have been studied as raw materials, but in each case only about 0.1% of the raw material can be obtained, Has become a problem.

[0005] Enzymatic synthesis methods have also been proposed (JP-A-5-168489, JP-A-6-62885, JP-A-7-31493). In the methods disclosed in these patents, three kinds of substances such as glycerol and sugar, and fatty acids or fatty acid derivatives are always synthesized as raw materials. However, there is no example of enzymatically synthesizing glyceroglycolipids using only two kinds of raw materials, fats and oils and saccharides.

[0006]

SUMMARY OF THE INVENTION The present invention provides a novel method for synthesizing glyceroglycolipids which can be produced inexpensively and in large quantities by using only two kinds of natural raw materials, fats and oils and saccharides. Is what you do.

[0007]

According to the process for producing glyceroglycolipid according to the present invention, glyceride in a fat or oil raw material is decomposed into glycerol and a fatty acid by a lipase in a first step, and a glycerol is transferred to the glycerol in a second step. The sugars are transferred from the saccharide raw material to sugar glycerol, and the water content of the reaction system is reduced to 5% or less, and then the fatty acid produced in the first step is transferred to the sugar glycerol by lipase as a third step. Characterized by the enzyme reaction of

[0008] Since both the glycerol portion and the fatty acid portion of glyceride (mainly triglyceride) in the fat and oil raw material are used, glyceroglycolipid is synthesized from only two kinds of substrates, ie, the fat and oil raw material and the saccharide raw material as a sugar donor. be able to.

[0009]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The oils and fats according to the present invention include linseed oil, perilla oil, perilla oil, safflower oil, sunflower oil,
Soybean oil, corn oil, peanut oil, cottonseed oil, sesame oil,
In addition to vegetable oils such as rice oil, rapeseed oil, olive oil, palm oil, palm kernel oil, and coconut oil, fish oils and animal fats are also included.

Since the fatty acids constituting the glyceride in each fat and oil are various, the composition of the fatty acid portion of the synthesized glyceroglycolipid can be changed by changing the fat and oil used.

In the present invention, lipase is used in two steps, a first step of decomposing a fat material to produce fatty acids and glycerol, and a third step of adding fatty acids to sugar glycerol. In the first step, it is necessary to release the fatty acid attached to the 2-position of glycerol. Therefore, it is desirable to use a lipase having no regiospecificity. However, since the fatty acid in glyceride undergoes intramolecular transfer during the reaction, 1, The objective can be achieved even by using a lipase having 3-specificity. In the reaction of the third step, lipase can be used regardless of the presence or absence of regiospecificity. Therefore, in the third step, the lipase used in the first step can be used as it is, the same lipase can be newly added, or another type of lipase can be used.

The lipase used in such a reaction includes:
Mucor miehei , Aspergillus
Niger (Aspergillus niger), Candida Ann talc Atlantica (Candida antarctica), Rhizopus oryzae (Rhi
It can be used regardless of its origin, such as those derived from microorganisms such as zopus oryzae ), those derived from plants, and those derived from animals. Further, these lipases do not necessarily have to be isolated and purified, and crude enzymes and commercially available enzyme preparations containing these enzymes can also be used. Further, these lipases can be used by being immobilized on various carriers.

The carbohydrate raw material according to the second step of the present invention includes:
Any sucrose, lactose, maltose, starch and the like can be used as long as the sugar can be transferred to glycerol by a glycosyltransferase. As the glycosyltransferase, those suitable for the sugar to be transferred are prepared from microorganisms and used. A commercially available enzyme preparation can also be used.

For example, Rahne Aquatilis ( Rahne
lla aquatilis ) Fructosyl glycerol can be produced by transferring fructose from sucrose to glycerol using levan sucrose prepared from JCM1683 strain. Further, commercially available β-1,4-galactosidase (Bacillus circulans ( Bacillus cirrus)
culans ), by transferring galactose from lactose to glycerol using Daiwa Kasei Co., Ltd.)
Galactosyl glycerol can be made.

In the first step and the second step, it is desirable to use water as a reaction solvent, but in the third step, the water content in the reaction system needs to be 5% or less. Therefore, after completion of the second step, freeze-drying or heat treatment is performed to reduce the water content in the reaction system to 5% or less.

When lyophilization is performed to remove water,
Efficient water removal cannot be achieved if the oil phase is mainly composed of fatty acid lipase hydrolyzate composed mainly of fatty acids. Therefore, it is preferable to separate the oil phase before freezing and freeze-dry only the aqueous phase. After lyophilization, the oil phases are combined again and the reaction of the third step is performed.

In the reaction of the third step, an organic solvent can be used as a reaction solvent. Any organic solvent can be used as long as it maintains a liquid state at the reaction temperature and stably maintains the lipase activity.

Specific examples of the organic solvent include aliphatic hydrocarbons such as n-heptane, n-pentane, n-hexane, petroleum ether and isooctane; aromatic hydrocarbons such as benzene, toluene and xylene; acetone, methyl Ketones such as isobutyl ketone; nitrogen-containing solvents such as acetonitrile, 2-nitropropane, pyridine and dimethylformamide; ether solvents such as dimethyl ether, diethyl ether, tetrahydrofuran and dioxane; halogens such as carbon tetrachloride, chloroform and methylene chloride Sulfoxide solvents such as dimethyl sulfoxide; and alcohols such as ethanol, t-butyl alcohol, t-amyl alcohol, diacetone alcohol, 3-methyl-3-pentenol, and propylene glycol.
These can be used in combination of two or more kinds.

[0019]

EXAMPLES Next, the present invention will be described specifically with reference to examples, but the present invention is not limited to the examples.

[0020]

[Example 1] [Synthesis of glyceroglycolipid from linseed oil and lactose] <First step> Lipase MY ( Candida cylindra) was added to 40 ml of pure water containing 12.8 g of lactose.
cea ) (from Meito Sangyo Co., Ltd.) (250 mg) and linseed oil (50 g) were added, and reacted at 40 ° C. for 24 hours to obtain a linseed oil decomposition product. When analyzed by high-performance liquid chromatography and thin-layer chromatography, linseed oil was mostly decomposed into glycerol and fatty acids, but a part was present as monoglyceride or diglyceride. <Second Step> In the reaction solution of the first step, as a β-galactosidase, Cryptococcus laurentii variety Lorenti OKN-4 ( Cryptococcus laurentii var. La .
urentii OKN-4; FERM P-7629) strain immobilized with calcium alginate 34.25 g of immobilized cells (β-galactosidase activity 19 units) and 1.75 g of commercially available baker's yeast were added.
The reaction was performed at 0 ° C. for 120 hours. Analysis by high performance liquid chromatography revealed that 3.38 g of galactosylglycerol had been formed. <Third Step> A part of the reaction solution of the second step (containing 0.25 g of galactosylglycerol) was freeze-dried to remove water. 25 ml of acetone, lipase PLG (from Alcaligenes ), celite immobilized enzyme, Meito industry
0.2 g) and 1 g of Molecular Sieves 3A / 16 were added and reacted at 40 ° C. for 12 days. When the reaction solution was analyzed by high performance liquid chromatography and thin layer chromatography, formation of glyceroglycolipid was confirmed, and the amounts of the formation were 0.03 g as a monoester and 0.15 g as a diester.

[0021]

Example 2 [Reaction when oil phase was separated from reaction liquid and only aqueous phase was freeze-dried] In the second step of Example 1,
During centrifugation for removing baker's yeast and immobilized enzyme, an oil phase and an aqueous phase of the reaction solution were also separated. When only the aqueous phase was freeze-dried and then combined with the oil phase again and subjected to the lipase reaction in the third step, the amount of glyceroglycolipid produced was 0.08 g as a monoester and 0.34 g as a diester.

[0022]

According to the present invention, various glyceroglycolipids can be efficiently produced at low cost from two kinds of raw materials, fats and oils and saccharides. The glyceroglycolipids obtained by the method of the present invention are suitable for use as emulsifiers and surfactants in fields requiring safety, such as cosmetics, pharmaceuticals, and foods. Depending on the composition of the constituent fatty acids, various physiological functions can be expected.

Continued on the front page (72) Inventor Shiro Hino 4-9-11, Toyosu, Koto-ku, Tokyo Inside Nisshin Sugar Co., Ltd. Product Development Department (72) Inventor Kazunori Moriya 4-9-11, Toyosu, Koto-ku, Tokyo Nisshin Sugar Refining Co., Ltd. Product Development Department (72) Inventor Osamu Ozawa 4-9-1-11 Toyosu, Koto-ku, Tokyo Nisshin Sugar Co., Ltd. Product Development Department F-term (reference) 4B064 CA21 CA33 CB03 CB26 CC03 CD22 DA16 DA20 4D077 AB08 AB11 AB12 AC01 CA01 CA16 DC36X

Claims (2)

[Claims] A first step of decomposing glyceride in a raw material of fats and oils into glycerol and a fatty acid with a lipase; , 5% of water of reaction system
After the following, a glyceroglycolipid characterized by comprising a third step of obtaining the glyceroglycolipid by adding the fatty acid produced in the first step to the sugar glycerol by the same or another type of lipase as the lipase Manufacturing method. 2. Before starting the reaction of the third step, an aqueous phase and an oil phase are separated, and the water in the aqueous phase is removed to reduce the water in the reaction system by 5%.
The method for producing a glyceroglycolipid according to claim 1, wherein after the reaction is performed, a fatty acid addition reaction with a lipase is performed again together with the oil phase.