JPH03127992A - Production of fatty acid ester - Google Patents

Abstract

PURPOSE:To decrease the free fatty acid content in an ester after reaction by reacting a fatty acid with an alcohol in the presence of an immobilized lipase. CONSTITUTION:An ester is produced by reacting a fatty acid with an alcohol. The above process is carried out by reacting in the presence of an immobilized lipase having an anion-exchange group on the carrier surface and then in the presence of a lipase immobilized to a hydrophobic carrier free from anion exchange group on the carrier surface. The lipase used in the above process is an enzyme capable of bonding an acyl-enzyme (R-COO-E) or (R-COS-E) by bonding a fatty acid (R-COOH) with an enzyme (HO-E) or (HS-E) and any kind of enzyme such as phospholipase, lipoprotein lipase, protease, etc., can be used provided that the enzyme has the above function. The immobilized lipase can be prepared by contacting a lipase solution with an anion exchange material or a hydrophobic carrier.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention is directed to fixation in order to reduce unreacted fatty acids as much as possible when producing fatty acid esters, which are basic materials for the oil chemical industry, food industry, and pharmaceutical industry. The present invention relates to a method for producing fatty acid ester using chemical lipase.

[Prior art and its problems]

A method of producing a product with a high monoglyceride content by carrying out a reaction at a water content of 3 $ or less using a reaction solution containing a high acid side, glycerin, and lipase immobilized on an anion exchanger (Japanese Patent Publication No. 51593/1983) Although there is.

When using an immobilized lipase having a hydrophilic anion exchange group, it is difficult to control the water content of the reaction system, and the acid value after one reaction is high.

Ester synthesis of 0.2M (R,5)-1-phenylethanol and 0.3M or heptanoic acid in cyclohexane is carried out using lipase immobilized on bleaching clay containing octadecyltrichlorosilane (M, N
orin et al, Appl, Microbiol
, Biotechnol.

28.527 (1988)), but in ester synthesis using 3M heptanoic acid as a substrate, the synthesis rate was as high as the ester synthesis rate using 0.3M heptanoic acid as a substrate, despite the high substrate concentration. I couldn't get it.

[Problem that the invention seeks to solve]

When fatty acid esters are synthesized using immobilized lipases that have anion exchange groups on the surface of the carrier, hydrolysis is likely to occur because the immobilized lipases have parent wood groups, and a large amount of fatty acids remains in the esters, reducing their utility value. It was difficult to obtain certain esters. In addition, in order to use a high acid value part as a substrate, it is necessary to carry out the reaction using a high concentration of fatty acids of 15 to 50%, but the high concentration of fatty acids can cause free lipase,
and inhibited lipase bound to a hydrophobic carrier that does not have an anion exchange group on the carrier surface.

[Means for solving problems]

The present inventors have completed the present invention as a result of extensive research into a method for producing esters using immobilized lipase in order to reduce the free fatty acid content in the esters after reaction. That is, in the present invention, when producing an ester by reacting a substrate containing a fatty acid and an alcohol, the reaction is carried out in the presence of an immobilized lipase having an anion exchange group on the surface of the carrier, and then anion is added to the surface of the carrier. This is a method for producing a fatty acid ester, characterized in that the reaction is carried out in the presence of a lipase immobilized on a hydrophobic carrier in which no exchange group is present.

The reaction solution used in the present invention usually contains 15% or more of a fatty acid, and the fatty acid component used is a fatty acid or an oil containing a free fatty acid. In order to complete the esterification of fatty acids, it is preferable to react the alcohol with a molar concentration of at least four times the amount of fatty acids.The alcohol component includes not only monohydric alcohols but also polyhydric alcohols such as glycerol. . Fatty acids are R-COOH (R
: An aliphatic group) that serves as a substrate for ester synthesis or transesterification by lipase.

The lipase used in the present invention refers to fatty acids (R-COOI+
)To8 Element (No-E) It is also 1t, ()Is-E) An enzyme that produces force binding dithia shi/L' 9 M(R-Coo-E) or (R-CO5-E), Any enzyme having such a function, such as phospholipase, lipoprotein lipase, or protease, may be used. Heat-resistant Shutomonas lipase or Muc that has activity even when the water content is significantly reduced
More preferred are lipases of the genus Or (see FIG. 1 of Japanese Patent Publication No. 62-51593).

The immobilized lipase in which an anion exchange group is present on the surface of the carrier of the present invention is an immobilized lipase in which an anion exchange group to which lipase is not bound is present on the surface of the carrier to which lipase is bound. This immobilized lipase (Special Publication No. 63-1
It can be produced by a synthetic method such as that disclosed in Japanese Patent Publication No. 8476). That is, when a lipase solution with an ionic strength of 0.1 or less is brought into contact with an anion exchanger, lipase, which is generally an acidic protein, ionically bonds to the anion exchanger, and lipase proteins that have an affinity for hydrophobic groups bond with the anion exchanger. Forms a strong hydrophobic bond with hydrophobic carriers. Thereafter, by washing the unimmobilized lipase with water, the desired immobilized lipase can be easily obtained. In addition, if you want to fix it more firmly,
Treat with polyvalent reaction reagents such as geltaraldehyde.

However, since polyvalent reaction reagents are undesirable if mixed into foods, etc., after the reaction is completed, unreacted polyvalent reaction reagents are completely removed using a reducing agent, etc., and then thoroughly washed. Furthermore, in order to obtain an immobilized lipase with anion exchange groups present on the surface of the carrier, lipase is adsorbed onto a carrier into which as many ion exchange groups as possible are introduced. As the amount of lipase adsorbed increases, the amount of unadsorbed lipase gradually increases, but when the amount exceeds a certain level, it suddenly increases.

If the amount of liver adsorption is kept below that amount, the desired immobilized lipase can be obtained.

The lipase immobilized on a hydrophobic carrier having no anion exchange group on the carrier surface of the present invention refers to a lipase adsorbed on a hydrophobic carrier. The hydrophobic carrier is macroporous polystyrene adsorption resin Amberlite XAD-
8 (manufactured by ROHM & Haas), Diaion HP
-20, Sepabeads SP-205 (Mitsubishi Kaoru Co., Ltd. 1m) etc., macroporous phenolic resin, resin Duolite S-
761. Examples include Duolite S-587 (Diamond, manufactured by Shermrock), ceramic powder, glass powder, metal powder, etc., into which hydrophobic groups are introduced using a silane coupling agent. When a lipase solution is brought into contact with a hydrophobic carrier, the lipase protein that has an affinity for hydrophobic groups forms a strong hydrophobic bond with the hydrophobic carrier. Ultrasonic treatment and deaeration treatment to improve contact between lipase and hydrophobic carrier.

Immobilized pase with high activity can be obtained by treatment with a polar solvent. Thereafter, by washing the unimmobilized lipase with water or a salt solution, the desired immobilized lipase can be easily obtained. In addition, in order to fix it more firmly, it is treated with a polyvalent reaction reagent such as geltaraldehyde to adsorb it or create a bond between lipases. The post-treatment of the multivalent reaction reagent is the same as in the case of immobilization on an anion exchanger.

In order to produce fatty acid ester according to the present invention, first of all,
The reaction is carried out using immobilized lipase that has an anion exchange group on the surface of the carrier. In this case, the water content of the reaction solution is 2
It can be set in a wide moisture range below z. Next, a reaction is carried out using lipase immobilized on a hydrophobic carrier that does not have an anion exchange group on the carrier surface. This reduces unreacted fatty acids in the reaction product. Second
When carrying out the step reaction, it is preferable that the water content is as low as possible, and it is usually carried out at a water content of 11.1% or less. Moisture can be removed from the reaction solution by distillation under reduced pressure, by using a dehydrating agent such as a molecular sieve, or by using an inert dry gas such as nitrogen gas. The reaction may be carried out in either a fluidized bed reactor or a fixed bed reactor. FIG. 1 shows an explanatory cross-sectional view of a countercurrent fluidized bed reactor.

This reactor is described in Japanese Patent Application No. 62-2550587. In this reactor, lipase immobilized on a hydrophobic carrier with no anion exchange group on the carrier surface is present in the upper stirring tank, and lipase immobilized on a hydrophobic carrier with no anion exchange group on the carrier surface is present in the lower stirring tank. presence of chemical lipase. Substrate containing fatty acids is supplied from the middle lower stage, alcohol is supplied from the middle upper stage, unreacted alcohol is collected from the lower end, and ester continuously flows out from the upper end. Since the unreacted alcohol contains water produced by the reaction, it is dehydrated by distillation under reduced pressure, dry nitrogen, etc., and then supplied again as alcohol from the middle upper stage.

[Effect]

Substrates such as high acid particles containing 15-30% fatty acids can be prepared by reacting them with alcohol in the presence of an immobilized lipase that has anion exchange groups on the surface of the carrier.
Fatty acid concentration can be reduced to 15% or less. The above-mentioned immobilized lipase is stabilized by binding to a large amount of carrier, and at the same time, due to the microenvironmental effect of the anion exchange group, protons around the lipase are eliminated and the pH is maintained near the optimum pH. (See Japanese Patent Publication No. 62-51593). Further, as shown in Example 1 of the present application, if the water content is 2z or less, the glyceride synthesis reaction proceeds. Therefore, even if some water is generated by the condensation reaction of a highly concentrated fatty acid-containing substrate, the reaction is not hindered. The present inventors previously disclosed that it is good to react at a water content in the range of 0.5 to 3.0%, but in the present invention, which aims to remove unreacted fatty acids, the water content is reduced in the pre-reaction. It has been found that good results can be obtained by keeping the water content below 2% and keeping the water content below 1.1% in the post-reaction.

The ester synthesis reaction using lipase is mediated by an acyl enzyme (R-COO=E) in a solvent, and then the active enzyme (H
This is a repetition of the reaction resulting in O-E). At this time, when decomposing the acyl enzyme, hydrolysis occurs, or ester synthesis or In the present invention, after the ester synthesis reaction, by using lipase immobilized on a hydrophobic carrier that does not have an anion exchange group on the carrier surface, unreacted Fatty acids and partial esters can be reduced.

Lipase immobilized on a hydrophobic carrier that does not have an anion exchange group on the carrier surface has a low affinity for water and has an affinity for alcohols and partial esters such as diglyceride. This product effectively exhibits its function under conditions of low moisture content.

Furthermore, the effects of the present invention can be further improved by using, as the carrier, a lipase immobilized thereon, such as pancreatic lipase or lipase of the genus Mucor, which exhibits activity even in systems with extremely low water content.

〔Example〕

Next, the present invention will be explained in more detail with reference to Examples.

Example 1 Macroporous anion exchange resin (DOWEX NW
A-1. (manufactured by Dow Chemical Company) 1g, Shutomonas mephiti force, Variant lipolyte force (Feikoken Bacteria 5
02) was desalted and concentrated from River 1185 units.
- Adsorbed. This adsorbed material was dried on a glass filter while being filtered under suction. Assuming that the molecular weight of the protein of this lipase purified to a specific activity of 2200 units/mg protein is 17500, the number of molecules of 1185 units is calculated as follows.

Lipase number = (1185/2200) X to-3/
12,500 units 4.3 X 10-8 Comparing this number of moles with the exchange capacity of DOWEX NWA-1, 4.2X 10-'/(4,3X 10-") = 9.8
X 104 or 9,8X1 per molecule of lipase
0' anion exchange groups are present on the hydrophobic polystyrene support. On the other hand, the number of acidic amino acids involved in ionic bonds on the 17,500 protein is estimated to be 10 or less.

As shown from the above calculation examples, this immobilization method yielded an immobilized lipase in which an anion exchange group was present on the surface of the carrier. The lipase activity measurement method is carried out using a modified method by Nord et al. (White Layering Vol. 36, p. 860 (1960)). was taken as 1 unit.

Macroporous adsorption resin (Amberlite XAD-8
) Ig, Shutomonas mephiti, Variant,
Two components were adsorbed by dispersing and dissolving lipase from Lipolyty Riki (Feikoken Bokuyori No. 520) using ultrasonic waves. This adsorbed material was dried on a glass filter while being suction-filtered to obtain lipase immobilized on a hydrophobic carrier with no anion exchange group on the carrier surface.

High acid value dtI was obtained by dewaxing high acid value rice bran oil (high acid crude oil). Since the acid value of the high acid value oil was 75.0, the free fatty acid content was 36% when calculated from the fatty acid average molecular weight of the unbran oil. A low acid formulation was prepared by adding 10% oleic acid to refined rice bran oil. The acid value of low acid details is 21.9
Met.

DOWEX NW is the dry weight of immobilized lipase that makes the amount of enzyme at the time of preparation of immobilized lipase 400 units.
0.314 g of A-1 immobilized lipase and 0.407 g of Amberlite XAD-8 were used in the following reaction.

First, 1g of high acid value oil, 0.314g of Dowex N
WA-1 immobilized lipase, various amounts of glycerin at 60%
The results of reaction treatment at ℃ for 120 hours are shown in Table 1.

The amount of glycerin used in the reaction in Table 1 shows the ratio of the number of moles of glycerin to the number of moles of fatty acid in the high acid value oil. After the reaction was completed, the acid value was measured after stopping the reaction with benzene/ethanol (1:1) and removing the immobilized lipase by filtration. The water content in the table was determined using the Karl Fischer method and the drying method. It has been found that the acid value reducing ability is high when the water content is 2% or less, preferably 1.84% or less, and the glycerin content is 4 times or more that of the fatty acid.

Next, the low acid content of Ig, DOWEX NWA-1 immobilized lipase, and glycerin were reacted at 60° C. for 12 hours, and the results are shown in Table 2.

In Table 2, the amount of glycerin is shown as the ratio of the number of moles of glycerin to the number of moles in the low acid value oil. DOWEX MIIA-
When using 1 immobilized lipase, there was only a slight decrease in the acid value of the low acid part.

Next, 1 g of high acid value oil, Amberlite XAD-8 immobilized lipase, and glycerin were reacted at 60° C. for 120 hours, and the results are shown in Table 3.

Table 3: The moisture range showing the ability to reduce the acid value was narrow, from 0.90% to 1.03%, and when the moisture content became 0.84% or less, the ability to reduce the acid value decreased. This is thought to be because the reaction did not proceed well due to the lack of water required for the lipase reaction.

Next, 1 g of low acid value oil, Amberlite XAD-8 immobilized lipase, and glycerin were reacted at 60° C. for 120 hours, and the results are shown in Table 4.

Table 4 shows that using Amberlite XAD-8 and reducing the water content to 1.1z or less, preferably 1.08% or less, the acid value of the low acid value oil was further reduced.

Example 2 1 g of DOWEX NWA-1 immobilized lipase Ig or Amberlite XAD-8 immobilized lipase prepared in the same manner as in Example 1, 1 g of glycerin, and 1 g of high acid value oil were shaken 133 times at 60°C. After 120 hours of reaction, one reaction solution was removed by filtration and the acid value of the filtrate was measured.
．． 6. 56.1 for those using Amberlight XAD-8
It turns out that the former is better for reacting with high acid value oil first.

Next, 1 g of DOWEX NWA-1 immobilized lipase or 1 g of Amberlite XAD-8 immobilized lipase, glycerin Ig, and 1 g of low acid value oil were reacted at 60° C. for 120 hours. After the reaction, the acid value was 17. for those using DOWEX NWA-1 immobilized lipase. O, Amberlight XA
The value was 11.0 when D-8 immobilized lipase was used, and the latter had a higher ability to reduce the acid value of oils and fats that had a somewhat low acid value. However, even in this case, at the initial stage of the reaction when the reaction time was 6 hours and 12 hours, DOWEX N
The decrease in acid value was greater when WA-1 immobilized lipase was used.

Example 3 In an Actor as shown in Figure 1, 14.5 g of Amberlite XAD-8 immobilized lipase prepared in the same manner as in Example 1 was placed in the two upper stirring tanks, and 14.5 g of Amberlite XAD-8 immobilized lipase was placed in the two lower stirring tanks. Same as Example 1! l! ! Manufactured DOWEX AwA-1
Immobilized lipase was present. Water-soluble product collection port (9
) was blocked, and glycerin was flowed through the water-soluble substrate supply port (3) at a rate of 11 mQ/hr, and high acid value oil was flowed through the oily substrate supply port (7) at a rate of 5.5 m12/hr. The high acid value oil was a 1:1 mixture of oleic acid and rice bran oil and had an acid value of 152.1.

After filling the reactor with the substrate, start stirring, bring the reaction temperature to 60 °C, and increase the glycerin inflow 1−/h.
r, high acid value oil inflow rate is 1 mQ/hr, and when water-soluble products are collected from the water-soluble product recovery port (9) at 1n+Q/hr, the oily product is collected from the oily product recovery port (1) at approximately 1 mQ/hr.
It eluted continuously in hr. Table 5 shows the changes over time in the acid value of the oily product and the water content in the water-soluble product.

From the upper part of Table 5, it was found that the operation to reduce the acid value from 152.1 to about 173 could be performed continuously.

In addition, the water content in the supplied glycerin is 0.46 L%, and the water content in the recovered water-soluble product is 2 to 3 L%, so the water content near Amberlite XAD-8 immobilized lipase is 1.1 L%. % or less, it was expected that the water content near the DOWEX NWA-1 immobilized lipase would be 2z or less.

〔Effect of the invention〕

It has been reported that surfactants such as monoglycerides and sugar esters can be synthesized by lipase. In this case, since the alkaline purification method cannot be used to remove unreacted fatty acids, a synthesis method that reduces unreacted fatty acids as much as possible is required.

There have been attempts to produce low acid value oils from high acid value oils using lipase, but it is necessary to produce oil with a triglyceride content that is less susceptible to hydrolysis and has a faster crystallization rate. The present invention provides a solution to these technical needs.

[Brief explanation of the drawing]

FIG. 1 is an explanatory cross-sectional view of the reactor used in this example. 1... Oily product collection port 2... Static tank at upper end 3... Water-soluble substrate supply port 4... Stirring blade 5... Static tank 6... Stirring tank 7... Oily product Substrate supply port 8... Lower end static tank 9... Water-soluble product collection port 10... Stirring shaft 11... Sieve plate 12... Stirring shaft cover

Claims (1)

[Claims] (1) When producing an ester by reacting a fatty acid with an alcohol, the reaction is carried out in the presence of an immobilized lipase that has an anion exchange group on the surface of the carrier, followed by a hydrophobic hydrophobic acid that has no anion exchange group on the surface of the carrier. 1. A method for producing a fatty acid ester, comprising further carrying out the reaction in the presence of a lipase immobilized on a carrier.