JPH0411193B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for ester group exchange reaction of oils and fats using lipase, specifically, a method for transesterification of oils and fats using lipase, which is characterized in that the reaction is carried out in the presence of lipase, water, chitosan and/or chitosan derivatives, a superabsorbent resin, and saccharides. This invention relates to a method for transesterification of oils and fats. In the case of a reaction system using an enzyme as a catalyst, the reaction is generally carried out in the presence of a large amount of water in order to fully demonstrate the activity of the enzyme present in the system.
However, in the case of the transesterification reaction of oils and fats using lipase, in a reaction system containing a relatively large amount of water, hydrolysis, which is an undesirable side reaction, is promoted, making it extremely difficult to obtain a preferable reaction composition. For these reasons, the transesterification reaction of oils and fats using conventionally known lipases is carried out in a reaction system in which the water content is extremely reduced. For example, in JP-A-55-71797 (Fuji Oil),
Water present in the reaction system is 0.18% by weight based on the substrate
The following methods are described. Also, JP-A-52-
Publication No. 104506 (Unilever Nahmrose Bennoteshape) describes a method in which the reaction is carried out in the presence of a small amount of water or in the presence of 0.2 to 1% by weight of water relative to the substrate. however,
In a reaction system with extremely low water content, such as in known methods, the enzyme is in a dead or sleeping state;
It cannot exist in a living state, and therefore the enzyme lipase is not activated sufficiently and a sufficient reaction rate or no reaction rate can be obtained or the reaction does not proceed at all. Furthermore, in a reaction system with extremely low water content,
Even if the amount of enzyme used is increased in order to obtain a sufficient reaction rate, not only is it not possible to further increase the reaction rate, but the added enzyme is largely inactivated, making it unsuitable for industrial use. In order to industrially utilize the transesterification reaction of fats and oils using lipase, it is necessary to technically solve these difficulties. The purpose of the present invention is to fully activate the lipase in the reaction system without concurrently causing hydrolysis, which is a preferable side reaction, in order to achieve industrial utilization of the transesterification reaction of oils and fats using lipase. The goal is to increase the reaction rate to a certain degree.
Furthermore, the present invention aims to prevent inactivation of lipase within the system, enable effective reuse of lipase, and improve the economic efficiency of the process. The present inventors investigated various enzyme reaction conditions for effectively exerting lipase activity in the transesterification reaction of fats and oils by lipase, and found that lipase, water, chitosan and/or chitosan derivatives, super absorbent resin, It was discovered that by conducting the reaction in the presence of sugars and sugars, the degree of side reactions can be suppressed and the reaction can proceed rapidly. Therefore, the object of the present invention could be achieved. That is, the present invention is a method for transesterifying fats and oils using lipase, which is characterized in that the reaction is carried out in a system in which lipase, water, chitosan and/or chitosan derivatives, a superabsorbent resin, and saccharides are present. The preferred amount of water in the reaction system is 1 to 30% by weight, more preferably 10 to 15% by weight based on the substrate.
Weight%. In addition, preferred chitosan and/or
Alternatively, the amount of the chitosan derivative is 0.005 to 5 g/g, more preferably 0.1 to 1 g/g, based on the amount of water (unit amount) in the system. Further, the preferable amount of the super absorbent resin is 0.001 to 3 g/g, more preferably 0.02 to 1 g/g, based on the water content (unit amount) in the system.
g/g. In addition, the preferable amount of sugars is 0.001 to 2 g/g based on the amount of water (unit amount) in the system;
More preferably, it is 0.01 to 1 g/g. In addition to the reaction conditions, the preferable amount of lipase enzyme (activity unit U) is 5 to 50% relative to the substrate (unit amount).
It is 10,000 U/g, more preferably 50 to 1,000 U/g. However, the activity unit U of the enzyme is the fatty acid equivalent to 0.06 ml of 0.05N sodium hydroxide aqueous solution when the enzyme is added to 5 ml of olive oil emulsion and 4 ml of 0.1M phosphate buffer and reacted at 37°C for 30 minutes. Each generation was considered as one activity unit (U). Furthermore, the reaction rate can be further increased by the presence of 0.05 to 5.0% by weight of surfactant relative to the substrate. As the surfactant, one or two or more selected from the group consisting of sucrose fatty acid ester, sorbitan fatty acid ester, propylene glycol fatty acid ester, polyglycerin fatty acid ester, lecithin, and monoglyceride can be effectively used. In particular, sucrose fatty acid esters with HLB of about 1 to 8 are effective. The above-mentioned substrate refers to fats and oils or fats and oils and fatty acids, and when glycerin, monoglyceride, diglyceride, alcohol ester of fatty acid, etc. are present in the system, these substances are also included in the above-mentioned substrate. As the lipase used in the present invention, Rhizopus type, Aspergillus type, Candida type, Mucor type, pancreatic lipase, etc. can be used, and many of these lipases are available as commercial products. When specifically transesterifying fatty acid groups at the 1 and 3 positions of glycerides, lipases having properties suitable for the purpose, such as Rhizopus deremer, Rhizopus japonicus, Mucor japonicus, etc., may be used. The above-mentioned fats and oils used in the present invention include general vegetable or animal fats or processed fats, or
These mixed fats and oils include, for example, soybean oil,
These include cottonseed oil, rapeseed oil, olive oil, corn oil, coconut oil, safflower oil, beef tallow, lard, and fish oil. Furthermore, glycerides with specific compositions that are raw materials for cocoa butter substitutes, namely 1,3-distearo-2-oleoglyceride, 1-palmito-2oleo-3-stearoglyceride, and 1,3-dipalmito-2-oleoglyceride. When the object of the transesterification reaction is oil or fat containing a large amount of oleic acid at the 2-position of the glyceride, such as olive oil, camellia oil, sasanya oil, palm oil, monkey fat, iritzpe butter, kokum butter, shea butter, Maua butter, furwara butter, Borneo tallow butter, or fractionated fats and oils thereof can be used. Furthermore, as the fatty acid, linear saturated or unsaturated fatty acids having 2 to 22 carbon atoms can be used. for example,
Palmitic acid, stearic acid, oleic acid, etc. can be used. Alcohol esters of fatty acids include esters of the above fatty acids and linear saturated monohydric alcohols having 1 to 6 carbon atoms, such as methyl palmitate, ethyl palmitate, methyl stearate, and ethyl stearate. can. Chitosan ((1→4)-2-
Amino-2-deoxy-β-D-glucan) is a deacetylated product of chitin ((1→4)-2-acetamido-2-deoxy-β-D-glucan). Furthermore, depending on the deacetylation conditions for chitin, partially deacetylated products having different deacetylation rates can be produced, and these can also be used. The deacetylation rate is preferably 30% or more, more preferably 70% or more. However, partially deacetylated products randomly deacetylated in a homogeneous reaction system with a deacetylation rate of 40 to 60%, more preferably 45 to 55% can be effectively used. The chitosan derivatives used in the present invention include acids,
Acid anhydrides, salts, aldehydes, epoxy compounds, etc., or reactants with these derivatives,
These partial reactants are also included. Such derivatives include N-acyl chitosan, N-mixed acyl chitosan, N-partial acyl chitosan, N,O-
Acyl chitosan, N-allylidene chitosan, N-
Examples include alkylidene chitosan and chitosan salt. The superabsorbent resin used in the present invention includes water-absorbing polyurethane resin, polyhydroxyethyl methacrylate, polyacrylic acid resin, starch-acrylic acid graft polymer (graft polymerization of acrylic acid on starch, neutralization, and starch-acrylonitrile graft polymer (starch is graft-polymerized with acrylonitrile on starch using ceric salt or radiation, hydrolyzed, purified, and dried), starch is cross-linked with monochloroacetic acid Carboxymethylated and crosslinked with formalin, cellulose-acrylonitrile graft polymer, cellulose carboxymethylated with monochloroacetic acid and crosslinked with formalin,
Alternatively, self-crosslinked products obtained by hydrolyzing vinyl alcohol and acrylic acid copolymers or vinyl acetate and methyl methacrylate copolymers, polyvinyl alcohol intermolecularly crosslinked with dialdehyde or radiation, crosslinked polyethylene oxide, etc. It will be done. These super absorbent resins may be used alone or in combination of two or more. Among these superabsorbent resins, those obtained by hydrolyzing and self-crosslinking starch-acrylic acid graft polymers, vinyl alcohol and acrylic acid copolymers, or vinyl acetate and methyl methacrylate copolymers are preferably used. sell. The former is the product name Sunwet IM- manufactured by Sanyo Chemical Industries, Ltd.
300, the latter manufactured by Sumitomo Chemical Co., Ltd.
The product name Sumikagel S-50 is available as a commercial product. The saccharides used in the present invention include monosaccharides, oligosaccharides and polysaccharides. Monosaccharides include glycerose, threolose, erythrose, ribose, xylose, arabinose, lyxose, glucose, mannose, allose, altrose, gyulose, indose, galactose, talose, dioxyacetone, ketothreose, erythroketopentose, fructose, tagatose, Among these, arabinose, ribose, galactose, and glucose were particularly effective. Examples of oligosaccharides include maltose, cellobiose, trehalose,
Examples include gentiobiose, isomaltose, lactose, sucrose, melibiose, raffinose, gentianose, stachyose, and among these, trehalose, raffinose, lactose, and sucrose were particularly effective. Examples of polysaccharides include xylan, alaban, starch, dextrin, cellulose, glycogen, dextran, inulin, levan, galactan, and cyclodextrin, among which dextrin, cellulose, glycogen, dextran, and cyclodextrin are particularly effective. It was hot. These saccharides may be used alone or in combination of two or more. In the present invention, various methods can be used to make lipase, water, chitosan and/or chitosan derivatives exist in the system, but a preferred method is to first bring lipase and water into sufficient contact with each other, and then A method of preparing lipase by adding chitosan and/or a chitosan derivative to the lipase, mixing well and bringing the lipase into sufficient contact with the lipase, or by bringing the lipase into sufficient contact with the chitosan and/or the chitosan derivative, and then adding water therein.
Alternatively, chitosan is brought into sufficient contact with an aqueous solution of acid and/or salt, and lipase is added therein, mixed thoroughly, and if necessary, water is added. There is a method of preparation by adding chitosan and/or a chitosan derivative, water, and lipase in an inert solvent and mixing thoroughly. The prepared enzyme-containing composition may be dispersed and added to a system having a substrate. Furthermore, various methods can be used for adding the superabsorbent resin and saccharide, and preferably, at any preparation stage in the method for preparing the enzyme-containing composition,
A method is used in which the superabsorbent resin and saccharide are added and thoroughly mixed. For example, after thoroughly mixing sugars and enzymes, chitosan and/or chitosan derivatives are added, thoroughly mixed, a superabsorbent resin is further added, and water is further added and thoroughly mixed and brought into contact to form an enzyme-containing composition. There are various methods of adjustment, and the enzyme-containing composition thus adjusted can be dispersed and added to a system containing a substrate to carry out the reaction. The transesterification reaction of the present invention can be carried out in a batch stirring reaction, a continuous flow reaction, etc., which are reaction systems in which the enzyme-containing composition is dispersed. Alternatively, the reaction can be carried out in a fixed enzyme reactor in which the enzyme-containing composition is packed into a column and the substrate solution is passed through the column. The transesterification reaction of the present invention is carried out at a reaction temperature of 20 to 50°C.
It is preferable to carry out the reaction at a temperature of 0.degree. C., and the desired composition can be obtained in about 3 to 50 hours, although the reaction time varies depending on the system. For example, if a mixture of high melting point oil and fatty acid is used as a reaction substrate, the reaction temperature may result in a non-uniform system, but in such cases, an organic solvent inert to lipase The reaction substrate can be dissolved in a homogeneous system to carry out the reaction. This type of organic solvent includes n-hexane, industrial hexane, petroleum ether, etc.
It can be used in an amount of 0.1 to 5 times (by weight). After completion of the transesterification reaction, if an organic solvent is used, the organic solvent is removed by distillation to obtain a reaction oil. Fatty acids, monoglycerides, diglycerides, added surfactants, etc. in the reaction oil should be removed by conventional chemical purification methods such as alkali neutralization, and physical purification methods such as steam distillation, vacuum distillation, and chromatography. I can do it. The preferred amount of water added to the reaction system as part of the enzyme-containing composition is 1 to
It is 30% by weight. Moisture content of substrate is 1% by weight
If it is less than that, sufficient reaction rate cannot be obtained,
Further, if the water content is 30% by weight or more based on the substrate, even if the water content is further increased, a faster reaction rate cannot be obtained. Further, a more preferable water content of the reaction system is 10 to 15% by weight based on the substrate. After the reaction, the enzyme-containing composition can be recovered by washing separately with a solvent. The activity of the enzyme-containing composition decreases when it is reused, but the activity is restored by adding and mixing water. However, if the amount of water in the enzyme-containing composition is relatively large, the activity is less likely to decrease and it can be reused more often. As a result of various studies from this point of view, it was determined that a more preferable amount of water added to the reaction system as part of the enzyme composition is about 10 to 15% by weight based on the substrate. The effects of the present invention are that it is possible to suppress the ester hydrolysis reaction, which is an undesirable side reaction, and to increase the rate of the transesterification reaction. If preferable conditions are selected, a reaction rate approximately 10 to 20 times faster than that of conventionally known methods can be obtained. When using enzymes industrially, increasing the reaction rate not only dramatically shortens the operating time of the reactor and provides an efficient and highly productive process, but also The residence time of the composition in the reactor can be shortened,
Therefore, there is an advantage that deactivation of the enzyme or change in the shape of the enzyme-containing composition due to causes such as stress caused by stirring in the reactor and changes in surface physical properties can be reduced. In addition, another effect of the present invention is that the enzyme is stabilized by using chitosan and/or a chitosan derivative, so there is very little deactivation of the enzyme, and the enzyme-containing composition recovered after the reaction is effective. This makes it possible to reuse the material and improve the economic efficiency of the process. The enzyme-containing composition according to the present invention can be reused 10 times or more, and can dramatically improve the economic efficiency of the process. According to the transesterification method for fats and oils according to the present invention, a cocoa butter substitute can be efficiently produced from inexpensive raw materials by using a regioselective lipase. Furthermore, under specific conditions, a triglyceride composition similar to cacao butter can be obtained, making this a very useful method industrially. Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited to these Examples. Example 1 4 g of chitosan (manufactured by Kyowa Yushi Kogyo Co., Ltd., trade name Flownatsu N), 5 g of ion-exchanged water, 0.2%
After thoroughly mixing and contacting 2 g of acetic acid aqueous solution and 2.4 g of lipase derived from Rhizopus delemar (lipase activity 3500 U/g per 1 g), 2 g of dextrin and a super absorbent resin (manufactured by Sanyo Kasei Co., Ltd., trade name: Sunwet) were brought into contact. After preparing an enzyme-containing composition by adding and mixing 4 g of IM-300), the enzyme-containing composition was mixed with 34.3 g of palm central oil, 25.7 g of stearic acid, and n-hexane.
The reaction was carried out at a reaction temperature of 40°C and a stirring speed of 300 rpm (1
(time). A small amount of the reaction mixture was separated over time and the triglyceride composition and acid value according to carbon number were measured by heating gas chromatography, and the results shown in Table 1 were obtained.

[Table] After separating the enzyme-containing composition from the reaction mixture by decantation and thoroughly washing with n-hexane, the enzyme-containing composition was dispersed and added to the same substrate mixed solution as the first time. The reaction was repeated under these conditions. When the triglyceride composition and acid value according to carbon number of each reaction mixture were measured, the results shown in Table 2 were obtained.

[Table] Comparative Example 1 After thoroughly mixing 10 g of chitosan (Fronac N) and 0.5% acetic acid aqueous solution, 25%
Glutaraldehyde solution was added gradually to form a gel. The gelled product was thoroughly washed with ion-exchanged water, pulverized, and air-dried. The air-dried material was further completely dried in a dryer at 100°C. 5 g of the dried product, 8 g of ion-exchanged water, and lipase derived from Lirhizopus derema (lipase activity per 1 g)
After thoroughly mixing and contacting 4.8 g of 3500 U/g) to prepare an enzyme-containing composition, the enzyme-containing composition was dispersed and added to a substrate mixed solution consisting of 34.3 g of palm central oil, 25.7 g of stearic acid, and 100 g of n-hexane. ,
The reaction was carried out at a reaction temperature of 40° C. and a stirring speed of 200 rpm (first time). The triglyceride composition and acid value of the reaction mixture according to carbon number were measured over time in the same manner as in Example 1, and the results shown in Table 3 were obtained. In the case of this example, the hydrolysis reaction could not be suppressed as compared to the case of Example 1.

[Table] In the same manner as in Example 1, the reaction was repeated under the same conditions as the first time using the enzyme-containing composition recovered from the reaction mixture. The reaction time required for the C ₅₀ triglyceride to reach approximately 35% was 4 hours for the second reaction, 5 hours for the third reaction,
The fourth time was 7 hours, the fifth time was 10 hours, and the sixth time was 15 hours. The reaction mixtures from the first to sixth reactions were mixed, n-hexane was removed, and celite filtration was performed. 100 g of this reaction mixture and 400 g of industrial hexane were mixed and dissolved, and the mixture was cooled to 8°C under stirring conditions. After that, it was separated into a crystal part and a liquid part. The yield of the crystalline portion was 41% and its acid value was 170.2, and the acid value of the liquid portion was 34.4. This liquid portion was subjected to Florisil carrier chromatography and eluted with industrial hexane. The eluted section consisted mostly of glycerides, and the fatty acids were adsorbed to the carrier and were not eluted. The solvent was removed from the elution section, and the solid fat index (SFI) was measured, and the results shown in Table 4 were obtained. Table 4 also shows the amount of cocoa butter measured at the same time.
SFI was also shown.

[Table] As is clear from the results shown in Table 4, it can be seen that triglycerides having physical properties similar to cocoa butter were produced. Comparative Example 2 Chitin (manufactured by Tokyo Kasei Kogyo Co., Ltd.) was added to T.
The deacetylation rate was determined by homogeneous deacetylation by the method of Sannan (Makromol Chem 177 , 3589 (1976) and Makromol Chem 178 , 3197 (1977)).
48% random deacetylation was obtained. 2 g of the deacetylated product, 4 g of water, and lipase derived from Rhizopus delema (lipase activity per 1 g: 3500 U/g)
After sufficiently mixing and contacting 2.4 g of the enzyme-containing composition, 1.0 g of a super absorbent resin (Sunwet IM-300) was added and mixed to prepare an enzyme-containing composition. 25.7g and n
- It was dispersed and added to a substrate mixed solution consisting of 100 g of hexane, and the reaction was carried out for 8 hours at a reaction temperature of 42° C. and a stirring speed of 300 rpm (first time). Analysis of the reaction mixture revealed an acid value of 99.5, _C48 ; 8.5%, _C50 ;
They were 34.6%, C ₅₂ ; 38.0%, and C ₅₄ 18.9%. In the case of this example, the ester group exchange rate was poorer than in Example 1. In the same manner as in Example 1, the reaction was repeated using the enzyme-containing composition recovered from the reaction mixture under the same conditions as the first time. The reaction time required for the C ₅₀ triglyceride to reach approximately 35% was 7 hours for the second reaction, 10 hours for the third reaction, 25 hours for the fourth reaction, and 35 hours for the fifth reaction.
It was time. Comparative Example 3 1 g of chitosan (Fronac N), 1 g of ion-exchanged water, 0.8 g of 0.2% acetic acid aqueous solution, and lipase derived from Rhizopus derema (lipase activity per 1 g)
After thoroughly mixing and contacting 2.4 g of 3500U/g), 0.4 g of a super absorbent resin (Sunwet IM-300) was added and mixed to prepare an enzyme-containing composition. g, stearic acid
Add dispersed to a mixed substrate solution consisting of 25.7g and 100g of n-hexane, reaction temperature 40℃, stirring speed
The reaction was carried out at 300 rpm (first time). The triglyceride composition and acid value of the reaction mixture according to carbon number were measured over time in the same manner as in Example 1, and the results shown in Table 5 were obtained. In the case of this example, the ester group exchange rate was poorer than in Example 1.

[Table] In the same manner as in Example 1, the reaction was repeated under the same conditions as the first time using the enzyme-containing composition recovered from the reaction mixture, and the results shown in Table 6 were obtained.

[Table] Dispersed and added.
Comparative Example 4 23.23 g of chitosan and 43.43 g of water were mixed well, and then lipase derived from Rhizopus delema (1 g
Add 112g of lipase activity (3500U/g)
After thoroughly mixing to prepare an enzyme-containing composition, the enzyme-containing composition was dispersed and added to a substrate mixed solution consisting of 400 g of palm central oil, 300 g of stearic acid, and 1200 g of industrial hexane, and the reaction temperature was 40°C and the stirring speed was
The reaction was carried out at 300 rpm (first time). The triglyceride composition and acid value of the reaction mixture according to carbon number were measured over time in the same manner as in Example 1, and the results shown in Table 7 were obtained. In the case of this example, the hydrolysis reaction could not be suppressed as compared to the case of Example 1.

[Table] In the same manner as in Example 1, the enzyme-containing composition was recovered from the reaction mixture and reused five times. Table 8 shows changes over time in triglyceride composition and acid value according to carbon number of the reaction mixture in the fifth reaction.

[Table] Comparative Example 5 After sufficiently mixing and contacting 4 g of chitosan with 5 g of ion-exchanged water, 2 g of 0.2% acetic acid aqueous solution, and 2.4 g of lipase derived from Rhizopus delema (lipase activity 3500 U/g per 1 g), ethyl cellulose (Wako Pure Chemical Industries, Ltd. An enzyme-containing composition was prepared by adding and mixing 2 g of polyethylene glycol (with a degree of ethoxylation of about 49%) manufactured by Co., Ltd. and 4 g of polyethylene glycol (molecular weight: 6000), and a reaction was carried out in the same manner as in Example 1 for 8 hours. The reaction product is significantly hydrolyzed and its acid value is
Since the yield was as high as 147.5, the yield of the target triglyceride was extremely low.

Claims (1)

[Scope of Claims] 1. A method for transesterification of oils and fats using lipase, characterized in that the reaction is carried out in a system in which lipase, water, chitosan and/or chitosan derivatives, a superabsorbent resin, and saccharides are present. 2. The method for transesterifying fats and oils using lipase according to claim 1, wherein the reaction is carried out at a water content of 1 to 30% by weight of the substrate in the reaction system. 3 The amount of chitosan and/or chitosan derivative is 0.005 to 0.005 to the amount of water (unit amount) in the system.
3. The method for transesterification of oils and fats using lipase according to claim 1 or 2, wherein the reaction is carried out at a rate of 5 g/g. 4. The lipase according to any one of claims 1 to 3, characterized in that the amount of super absorbent resin is 0.001 to 3 g/g relative to the amount of water (unit amount) in the system. A method for transesterification of oils and fats. 5. Fats and oils produced by lipase according to any one of claims 1 to 4, characterized in that the reaction is carried out in an amount of saccharides of 0.001 to 2 g/g relative to the amount of water (unit amount) in the system. Transesterification reaction method. 6. Ester groups of fats and oils by the lipase according to any one of claims 1 to 5, characterized in that the enzyme amount of the lipase is 5 to 10,000 U/g with respect to the substrate (unit amount). Exchange reaction method. 7. Chitosan and/or chitosan derivatives are deacetylated products of chitin and have a deacetylation rate of 30% or more. Method for transesterification of oils and fats. 8 Chitosan and/or chitosan derivative is a random deacetylated product of chitin,
7. The method for transesterification of oils and fats using lipase according to any one of claims 1 to 6, characterized in that the deacetylation rate is 40 to 60%. 9 The chitosan derivative is N-acyl chitosan, N
- One or more selected from the group consisting of mixed acyl chitosan, N,O-acyl chitosan, N-allylidene chitosan, N-alkylidene chitosan, chitosan salt, and partial reactants thereof. A method for transesterifying fats and oils using a lipase according to any one of claims 1 to 8.