JPH047680B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Background of the Invention It is known that higher fatty acid esters of sugars and sugar alcohols can be chemically synthesized by transesterification of fatty acid esters with sugars or sugar alcohols. Sugar fatty acid esters are produced in large quantities by this method. Since all of these chemical synthesis methods involve a heating step, they have drawbacks such as coloring of the product due to heating and the formation of complex mixtures.

In order to avoid these drawbacks, a biochemical synthesis method for fatty acid esters using free fatty acids and sugars or sugar alcohols as substrates and incubation in the presence of lipase has recently been proposed, and a patent application is currently being filed by the present applicants. . Since the incubation is carried out in an aqueous medium such as water or a buffer and under stirring, the substrate concentration cannot be too high. For this reason, energy costs are required to recover the product after the reaction is completed, and as is well known, the esterification reaction between acid and alcohol is a reversible reaction, so a low substrate concentration is disadvantageous for the esterification rate. be.

DISCLOSURE OF THE INVENTION The present invention provides a method for synthesizing fatty acid esters by incubating fatty acids and sugars or sugar alcohols as substrates in the presence of lipase. It relates to a method characterized by incubating in a dissolvable amount of an aqueous medium under reduced pressure while gradually removing moisture, and after the final moisture content becomes 5% or less, the incubation is continued by standing at normal pressure.

In this enzymatic reaction, it is only necessary to incubate with stirring at a relatively high water content during the initial period of the reaction. Therefore, at the beginning of the reaction, incubation is started in the presence of sufficient water to dissolve the sugar or sugar alcohol, and then the water is gradually removed under reduced pressure while the incubation is carried out with stirring. The reaction can be continued simply by standing under normal pressure. Therefore, the esterification rate is improved as a whole, and the concentration of the target product at the end of the reaction is high.

Detailed Description Sugars that can be used in the present invention include glucose,
Monosaccharides such as fructose, ribose, arabinose, mannose, galactose, xylol, sucrose, maltose, lactose, cellobiose,
Disaccharides such as trehalose and palatinose, trisaccharides such as maltotriose, raffinose, cellotriose and mannotriose, tetrasaccharides such as cellotetrose and stachyose, dextrin, cyclodextrin, mannan, fructan, galactan, xylan, alaban, cellulose, There are cellulose derivatives (CMC, hydroxypropylcellulose, methylcellulose), etc.

Examples of sugar alcohols include sorbitol, sorbitan, arabitol, xylitol, mannitol, dulcitol, maltitol, lactitol-paratitol, and the like.

The fatty acid is preferably a saturated or unsaturated fatty acid having 8 to 22 carbon atoms. Examples include caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid,
Saturated fatty acids such as behenic acid, caproleic acid, lindelic acid, myristoleic acid, palmitoleic acid, oleic acid, cadreic acid, erucic acid, decadienoic acid, linoleic acid, hiratic acid, linolenic acid,
Unsaturated fatty acids such as eicosatrienoic acid, docosatrienoic acid, hexadecatetraenoic acid, stearidonic acid, arachidonic acid, docosatetraenoic acid, eicosapentaenoic acid, sardine acid, and sabinic acid, iproric acid, yarapinoleic acid, ricinoleic acid, There are hydroxy fatty acids such as feronic acid.

As is well known, there are lipases derived from microorganisms and those derived from animals, both of which can be used. As for those derived from microorganisms,
Aspergillus niger (Amano Pharmaceutical, Lipase AP
-6), Mucor genus (Amano Pharmaceutical, lipase
MAP-10), Mucor miehei (manufactured by Novo Industries, lipase SP-225), Pseudomonas
(Amano Pharmaceutical, Lipase P), Rhizopus
japonicus (Osaka Seiken Co., Ltd., Lipase Cyken "100"), Rhizopus delemor (Tanabe Seiyaku Co., Ltd., Talipase), Candida cylindracea (Meito Sangyo Co., Ltd., Lipase MY), etc. Examples of animal origin include pancreatin derived from pig pancreas.

The molar ratio of sugar or sugar alcohol to fatty acid in the starting reaction mixture is preferably in the range of 1:3 to 3:1. The initial reaction mixture is prepared by adding substrate and lipase to water or buffer. The amount of water at this time is sufficient as long as the sugar or sugar alcohol in the substrate can be completely dissolved, and the presence of excess water is uneconomical. The amount varies depending on the water solubility of the sugar or sugar alcohol used, but is generally 30% of the total mixture.
~15%.

The amount of enzyme added depends on the titer, but in the case of the commercially available products shown, it is generally 0.1 to 10% of the total weight of the substrate.
It is.

The optimum pH of lipase is generally in the range of 4.0 to 8.0, preferably 5.0 to 7.0.

The starting reaction mixture prepared as described above is incubated under reduced pressure with gradual removal of water to a final moisture content of less than 5%, preferably less than 2%.

The degree of depressurization is related to the dehydration time to reach the specified final moisture content, so it cannot be defined unconditionally, but in order to reach the specified moisture concentration in a few hours, it must be 100 to 1 Torr.
A degree of vacuum is required.

The incubation temperature depends on the heat resistance of the enzyme, but is generally in the range of 30°C to 50°C, with the optimum temperature being around 37°C. However, in the case of thermostable enzymes,
Temperatures may exceed 40°C. This temperature remains the same during the dehydration stage under reduced pressure and the subsequent incubation stage.

The reaction equipment used is preferably a rotary evaporator, for example, so that the contents can be mixed uniformly in the dehydration step under reduced pressure.

The operation involves first dissolving the sugar or sugar alcohol in the required amount of water or buffer, adding the enzyme and dissolving it, then adding the fatty acid on top of this, and using a rotary evaporator for several hours under reduced pressure. Incubate with gradual dehydration. When the moisture content has decreased to 5% or less, preferably 2% or less, return to normal pressure and leave it in a thermostatic oven for 1 to 4 days, the reaction will proceed further and the desired fatty acid ester will be produced at a high concentration and yield. It can be obtained with

After the reaction is complete, for example, remove oil-soluble components (fatty acids, fatty acid esters) from the reaction mixture in chloroform,
The desired fatty acid ester can be isolated by a conventional method by extraction with a solvent such as tetrahydrofuran.

Examples of the present invention are shown below.

Example 1 Put 9.1 g of sorbitol into a 300 ml eggplant flask and dissolve in 5 g of M/15 phosphate buffer (PH7).
Add 0.5 g of lipase derived from Candida cylindracea (Lipase MY, manufactured by Meito Sangyo) and dissolve it. Furthermore, 14.1 g of oleic acid was added, and the mixture was stirred at 37°C using a rotary evaporator. The degree of pressure reduction at this time was approximately 20 Torr, and dehydration was performed to a moisture content of 2% over a period of 5 hours.

The ester synthesis rate at this time was 10% based on fatty acids.

Next, the reaction mixture, which had been dehydrated under reduced pressure, was placed in a thermostat and left at 37°C for 3 days. After the completion of the reaction, the ester synthesis rate was 87% based on fatty acids.

Example 2 Put 19g of sucrose in a 300ml eggplant-shaped flask,
Dissolve in 10g of M/15 phosphate buffer (PH7). 1.0 g of lipase derived from Rhizopus japonicus (Lipase Saiken "100" manufactured by Osaka Seiken Co., Ltd.) is added to this and dissolved. Add 8g of caprylic acid to this,
Rotate and stir in a rotary evaporator at 40°C. The degree of pressure reduction at this time was approximately 10 Torr, and dehydration to 1% moisture took 8 hours.

The ester synthesis rate at this time was 5% based on fatty acids.

The reaction mixture was then placed in a thermostat and left at 40°C for 4 days. After completion of the reaction, the ester synthesis rate was 89% based on fatty acids.

Claims (1)

[Claims] 1. In a method for synthesizing fatty acid esters by incubating fatty acids and sugars or sugar alcohols as substrates in the presence of lipase, the substrate and lipase are mixed in an aqueous medium in an amount that allows the sugars or sugar alcohols to be substantially completely dissolved. During,
A method for producing a fatty acid ester, which comprises incubating under reduced pressure while gradually removing moisture, and after the final moisture content reaches 5% or less, leaving it to stand at normal pressure and continuing incubation.