JPS6251593B2 - - Google Patents

Description

[Detailed description of the invention]

(a) Industrial application field The present invention uses a reaction solution containing a high acid value oil, glycerin, and immobilized lipase in order to utilize high acid value oil with a high free fatty acid content in fats and oils.
This invention relates to a method for producing monoglyceride-containing products that are widely used as emulsifiers in foods and cosmetics, or as compounding agents for plastics and spindle oils. (b) Conventional technology At present, 85-90% of oil resources are used as edible oils, but when rice bran oil, palm oil, etc. , preventive measures (stabilizing) are being taken (Ogiji Shimizu, Jun Kurokawa, Yushi, Vol. 37, No. 2, P67 (1984)). Oil extraction factories are spread out to different production areas, and heat treatment is carried out immediately after harvesting, but rice bran oil from countries with delayed collection methods becomes a high-acid oil with a high amount of free fatty acids, making it difficult to use it for human consumption. It transforms into something that cannot be done. This high acid value oil is only used as a raw material for soaps and the like, and no attempt has been made to synthesize monoglycerides that can be used as emulsifiers for foods and the like. Monoglycerides are currently produced by chemical methods that require long periods of time at high temperatures (VR Kaufman and N. Garti,
JAOCS, Vol. 59, P471 (1982)).
In addition, biochemical methods include limited degradation using lipase (Japanese Patent Publication No. 17525-1983), ester synthesis reaction of lipase (Japanese Patent Publication No. 7754-1983, 23535-1983), and transesterification reaction of lipase (Japanese Patent Publication No. 59-1989).
118094), but these all use soluble enzymes or enzyme dry powders, and are unsuitable for constructing a reactor suitable for continuous reactions, and are not suitable for production in high acid value oils. (Kosugi, Suzuki, Abstracts of the 1986 Japanese Society of Fermentation Engineering)
P206 (1983)). Furthermore, no knowledge has been obtained regarding a reaction system that simultaneously uses a lipase transesterification reaction and an ester synthesis reaction. (c) Problems to be Solved by the Invention In addition to triglycerides and fatty acids, high acid value oils contain a certain amount of glycerin, diglycerides, and monoglycerides. In order to obtain a product with a high monoglyceride content, the objective is to explore conditions for effectively utilizing the ester decomposition, ester synthesis, and transesterification effects of lipase. (d) Means for Solving the Problems The inventors of the present invention have conducted intensive research into a manufacturing method for obtaining monoglyceride-rich products in high yields in order to utilize high-acid-value oils in a high-yield manner. If a reaction solution containing lipase immobilized on an anion exchanger is used and the reaction is carried out at a water content of 3.0% by weight or less, the immobilization is such that lipase activity is not easily inhibited even in a reaction system containing a large amount of free fatty acids. It has been discovered that a reaction can be carried out using lipase, and that monoglyceride-rich products can be continuously produced in high yield by simultaneously using the ester synthesis and transesterification abilities of lipase. That is, the present invention uses a reaction solution containing high acid value oil, glycerin, and immobilized lipase to reduce the water content.
This is a method for producing a product with a high monoglyceride content, which is characterized by carrying out the reaction at a concentration of 3.0% by weight or less. The high acid value oil used in the present invention refers to an oil or fat raw material with a high free fatty acid content in the oil or fat, and an oil or fat with an acid value of 30 or more. Average molecular weight of free fatty acids is 270~
In the case of 290, an acid value of 30 or more refers to an oil or fat raw material containing about 15% or more of free fatty acids. This high acid value oil is obtained by decomposing triglycerides during storage, and contains fatty acids and triglycerides as main components, as well as fat-soluble vitamins such as glycerin, diglyceride, monoglyceride, and tocopherol. Also, depending on the degree of purification, it may contain wax, phospholipids, sterols, etc.
Since high acid value oil is a source of fatty acids and glycerin that constitute monoglycerides, any oil that contains fatty acids and glycerin as constituent units in a form that is susceptible to lipase action can be used instead. Glycerin used in the present invention is a skeleton for binding fatty acids to form monoglyceride, and also serves as a solvent for substances involved in the reaction. High acid value oils contain some glycerin, which needs to be added to promote the transesterification reaction. Adding an organic solvent such as hexane to the reaction solution reduces the amount of glycerin used and facilitates separation of unreacted glycerin after the reaction. Lipase used in the present invention is an enzyme produced by microorganisms and higher animals and plants, and is a biopolymer that acts on ester bonds to catalyze ester decomposition, transesterification, or ester synthesis reactions. When used as an industrial catalyst, lipases produced by bacteria of the genus Pseudomonas or Chromobacterium, or filamentous fungi of the genus Mucor or Rhizopus can be cited, since they can be produced in large quantities. When a large amount of α-monoglyceride is to be produced, the monoglyceride content will be increased if a Rhizopus filamentous fungus having specificity for the α-position of glyceride is used. The immobilized lipase used in the present invention may be prepared by any of the reported enzyme immobilization methods such as covalent feeding method, ionic bonding method, hydrophobic bonding method, entrapping immobilization method, etc., but preferably , a lipase is bonded to an anion exchanger and reinforced with a divalent reaction reagent such as glutaraldehyde. By this immobilization treatment, the enzyme protein is bonded to the carrier at multiple points, so that it is less likely to be desorbed even in the presence of a surfactant or an organic solvent, and its stability against chemical treatment and heat treatment is increased. Furthermore, the optimal pH of immobilized lipase shifts to the acidic side, making it possible to remove the effects of fatty acid inhibition from the microenvironment near the active center of the enzyme. (Unexamined Japanese Patent Publication No. 1983-
179091). Examples of immobilized carriers for lipase include synthetic resins, polysaccharides, ceramics, etc.
Since the monoglyceride yield may vary depending on the carrier, it is necessary to select an appropriate carrier. The water content of the reaction solution used in the present invention is 3.0% by weight.
The following is required: The water content in the present invention is determined by the weight ratio of the water content in the reaction solution and the water content attached to the immobilized enzyme, which is obtained by subtracting the dry weight of the immobilized enzyme from the total weight of the reaction solution. When the water content exceeds 3.0% by weight, hydrolysis occurs and the yield decreases. 0.5-3.0% by weight
Near this point, the free fatty acid content after reaction is at its lowest.
In a state of severe dehydration of 0.5% by weight or less, even the water needed to express the enzymatic activity of lipase is taken away from the lipase protein, so the lipase action is not exerted and the yield decreases. The reaction conditions used in the present invention are around the optimum pH and temperature of the immobilized lipase used, except that the water content is set within the above-mentioned range. The reaction method used in the present invention is a batch method in which immobilized lipase is shaken together with a reaction solution, and a continuous method using a reaction tank filled with immobilized lipase in a column or a reaction tank in which lipase is immobilized on a membrane surface. It is carried out in a regular manner. In a continuous reaction, unreacted glycerin can be separated, subjected to dehydration treatment, and then subjected to the reaction again. Glycerin after the reaction contains water generated during the synthesis reaction. On a small scale, glycerin is dehydrated by passing it through a dehydrating agent column such as a molecular sieve, but on a large scale, glycerin is dehydrated by taking advantage of the difference in boiling point between glycerin and water. Also, the acid value is 30
By mixing and reacting the following low acid value oils, it is possible to suppress the ester synthesis reaction that produces water after the reaction and save energy for dehydration. (e) Action Lipase catalyzes ester decomposition, ester synthesis, and transesterification reactions. The present invention is based on the discovery of conditions for exhibiting ester synthesis and transesterification ability by using immobilized lipase and adjusting the water content in the reaction solution. If the water content in the reaction solution is set to a point that satisfies two requirements: the amount necessary to express the activity of the lipase protein and the water content that does not cause hydrolytic action, the hydrolytic ability can be increased. It has been found that it is possible to exhibit ester synthesis and transesterification abilities. The ester synthesis effect is observed as a decrease in the acid value of the oil in the reaction solution. Since ester synthesis is the reverse reaction of hydrolysis, water is produced along with glyceride after the reaction. This water is mostly concentrated into hydrophilic glycerin. Dehydrating glycerin is easy because its boiling point is 290°C, which is significantly different from the boiling point of water. Separation of monoglyceride-rich substances and glycerin is carried out by membrane separation that takes advantage of the difference between monoglyceride-rich substances which are hydrophobic and glycerin which is hydrophilic, and separation that takes advantage of the fact that glycerin has a heavy specific gravity of 1.27. . The transesterification effect is observed as triglyceride decreases and monoglyceride and diglyceride increase during the reaction. If the reaction time is prolonged, the produced monoglyceride may convert to diglyceride, so it is necessary to control the reaction time depending on the purpose. Transesterification reactions do not produce water, and hydrolysis reactions consume water. In the present invention, the reaction is carried out in a system that suppresses hydrolysis as much as possible, so hydrolysis is slight. However, if a low acid value oil is mixed into the reaction system, the water content in the reaction solution can be controlled, and the glycerin layer can be dehydrated. can save energy. (f) Examples The present invention will be specifically explained using examples. Example 1 1 g of macroporous anion exchange resin (Dowex MWA-1, manufactured by Dow Chemicel) and 1185 units of various enzymes were suspended in 1 ml of 1/15M pine quill vain buffer (PH5) and shaken overnight at 10°C. .
An additional 1 ml of 1/15M pine irvain buffer was added, followed by 80 μ of a 25% glutaraldehyde solution. After shaking at 10°C for 10 minutes, 0.2 ml of 20% sodium hydrogen sulfate solution was added, followed by shaking at 10°C for 10 minutes to remove excess glutaraldehyde. The obtained immobilized lipase was thoroughly washed with water and then dried on a suction funnel. The lipase activity measurement method is
It was carried out using a modified method of Nord et al. (Yamada et al., Nichino Kagaku Vol. 36, p. 860 (1960)), and the amount of enzyme that reacted at 40℃ to 60℃ at pH 7 and released 1 microequivalent of acid per minute was 1 unit. And so. Rice bran oil with a high acid value (high acid crude oil) is heated to 80°C to completely dissolve the wax, then cooled with water to 20°C and aged for 1 hour. N-hexane cooled to 20°C was added so that the oil concentration in the mixture was 70%, and the mixture was filtered by suction. Next, n-hexane was distilled off from the liquid to obtain a dewaxed high acid value oil. The acid value of the high acid value oil was 73.6. 0.5 g of immobilized lipase, 1 g of glycerin, and 1 g of high acid value oil were placed in a 100 ml Erlenmeyer flask, sealed with a silicone rubber stopper, and reacted for 120 hours in a constant temperature bath at 40°C or 60°C with shaking 133 times per minute. . After adding a 1:1 mixture of ethanol and benzene to separate the immobilized enzyme, the acid value of the solution was measured. Furthermore, the water content of high acid value oil measured by the Karl-Futscher method is 0.133%, and the water content of glycerin is 0.555%.
Therefore, even including the water adhering to the immobilized lipase, the water content in the reaction solution was 3.0% by weight or less.

[Table] In Table 1, it can be seen that the acid value of high acid value oil decreases when the reaction is performed using a lipase from a higher animal, plant, or microorganism. In particular, when lipases from bacteria of the genus Pseudomonas, bacteria of the genus Chromobacterium, filamentous fungi of the genus Rhizopus, and filamentous fungi of the genus Mucor were used, the degree of decrease in acid value was high. For Chromobacterium bacteria, 1185 units of lipase was immobilized at 60°C.
The reaction was carried out at 60°C and 40°C. The degree of reduction in acid value was greater when the reaction was carried out at 60°C, which is close to the optimum temperature. Example 2 Rhizopus niveus filamentous fungus lipase (manufactured by Amano Pharmaceutical Co., Ltd.) was immobilized in the same manner as in Example 1, and the acid value of the reaction solution after reaction at 40°C was measured. In addition, the determination of glycerides and fatty acids in the reaction solution after reaction with high acid value oil was performed using synchrography (IATROSKYAN TH).
-10 type, made by Yatron).

Table 2 shows that the triglyceride and fatty acid contents decrease and the monoglyceride and diglyceride contents increase with the start of the reaction. After the start of the reaction, the acid value also decreases significantly, indicating that ester synthesis is occurring, but transesterification is also observed at the same time. 72
After that time, the acid value does not decrease and the ester synthesis reaction is considered to have reached equilibrium, but the produced monoglyceride continues to be transesterified into diglyceride, so in order to increase the monoglyceride yield, it is necessary to carry out the reaction for about 72 hours. It turns out that time is desirable. Table 3 shows the analysis results of the reaction products obtained by immobilizing enzymes of various origins on Dowex MWA-1 in an experiment similar to the above.

[Table] Using the lipase of Pseudomonas mephiteica (Feikoken Bacteria No. 520), monoglyceride equivalent to that of Rhizopus niveus or Mucor diapanicus (manufactured by Amano Pharmaceutical) can be obtained even at a reaction temperature of 60°C. I understand. Example 3 Using Chromobacterium viscosum lipase (manufactured by Toyo Jozo), Example 1 was applied to various carriers.
It was immobilized using the same method. Table 4 shows the acid values after the reaction after reacting at 60° C. for 5 days in the same manner as in Example 1.

[Table] When using spherosil DEA or DEAE cellulofine immobilized on a carrier, the acid value decreased significantly, and the composition of the reaction product was analyzed and the result was as shown in Table 5.

[Table] It can be seen that when spherosil, which is a macroporous silica bead, or cellulofine, which is a cellulose bead, more than 30% of monoglyceride is produced even when Chromobacterium lipase is used. Example 4 Pseudomonas mephiteica
Lipase (No. 520) was immobilized on Dowex MWA-1 in the same manner as in Example 1. immobilized lipase
0.05 to 10 g of glycerin was added to 0.5 g of high acid value oil and reacted in the same manner as in Example 1, and the acid value after the reaction was measured. The water content in the glycerin and high acid value oil was determined by the Karl Fusscher method, and the water content in the immobilized lipase was determined from the weight loss after heating at 110°C, and the water content in the reaction solution was calculated.

[Table] Since the water content in glycerin was 0.555%, it can be seen that increasing the amount of glycerin added decreases the water content in the reaction solution. The acid value decreased maximum when the amount of glycerin added was 1 g. As the amount of glycerin added increases, the water content in the reaction solution increases, indicating that the hydrolysis action of lipase cannot be suppressed. It is also understood that if the amount of glycerin added is significantly increased, the moisture needed to express lipase activity is also taken away from the lipase protein, so that the transesterification and ester synthesis effects become ineffective. FIG. 1 shows the results of experiments similar to those described above performed using Rhizopus niveus and Mucor japanicus lipases (manufactured by Amano Seiyaku) immobilized on Dowex. The water content of the reaction solution at which the acid value decreases maximum varies depending on the lipase, but in any case, the hydrolytic action of lipase can be suppressed by reducing the water content to 3.0% by weight or less. FIG. 2 shows the results of an experiment similar to that described above in which Chromobacterium viscosum lipase (manufactured by Toyo Jozo Co., Ltd.) was immobilized on various carriers in the same manner as in Example 1. Although the water content of the reaction solution at which the acid value decreases maximum varies depending on the immobilized carrier, it is shown that in any case, if the water content is 3.0% by weight or less, the hydrolytic action of lipase can be suppressed. Example 5 Lipase of Pseudomonas fluorescens biotype (Feikoken Bibori No. 5495) was immobilized on Dowex MWA-1 in the same manner as in Example 1. 0.5g of immobilized lipase, n per 1g of high acid value oil
- Added 0.429g of hexane to create 70% micellar, which is often used in the essential oil process. And glycerin 0.4~
1.4 g was added and the reaction was carried out in the same manner as in Example 1 at 60°C for 120 hours. For comparison, a sample without n-hexane was reacted in the same manner, and the acid value after the reaction was measured.

[Table] Table 7 shows that when hexane is added to the reaction solution, the degree of acid value decrease increases and the amount of glycerin added increases.
This shows that the acid value after the reaction is the lowest at 0.6 g, whereas if no hexane is added, the acid value after the reaction will not be the lowest unless 1.0 g of glycerin is added. Example 6 1 g of refined rice bran oil (manufactured by Yuwax Yakuhin), 1 g of glycerin, and 0.5 g of immobilized lipase obtained by immobilizing Rhizopus niveus lipase (manufactured by Amano Pharmaceutical) on Dowex MWA-1 in the same manner as in Example 1 were heated at 40°C. at 72
The reaction was carried out in the same manner as in Example 1. In addition, 0.5 g of refined rice bran oil and 0.5 g of high acid value oil were mixed to make a mixed oil and reacted in the same manner. Refined rice bran oil was mostly triglyceride and its acid value was 0.0. On the other hand, the acid value of the mixed oil was 33.2. Table 8 shows the analysis results of those reaction products.

[Table] From Table 8, it can be seen that the acid value of refined rice bran oil increases slightly, which indicates that the hydrolyzing function of lipase is exerted even under the reaction conditions of the present invention. The production of monoglycerides from refined rice bran oil is thought to be mainly accomplished by transesterification. Since the acid value of the mixed oil has decreased, it is thought that monoglycerides are synthesized through ester synthesis and transesterification. The fact that the amount of monoglyceride synthesized is greater in the mixed oil than in the refined oil indicates that the present invention, which uses both the transesterification tree function and the ester synthesis function, is superior. (G) Effects The present invention uses a high-acid-value oil with a high amount of free fatty acids in its fats and oils, which is an inexpensive raw material that is not edible and can only be used as a raw material for soap, etc. Since it is possible to synthesize monoglycerin-rich products, it contributes to the advanced utilization of high acid value oils. Since the reaction proceeds at room temperature and pressure, it is energy-saving, and since the physiologically active substances such as tocopherols in fats and oils are not denatured, it is also possible to effectively utilize their antioxidant and physiologically active functions. Since immobilized enzymes are used, continuous operation is possible, and glycerin can be easily reused. Moreover, the glycerin separated from the reaction product is
Since it contains a high concentration of monoglyceride, it can also be used as an emulsifier by simply removing fatty acids using alkali purification or the like. In addition, after monoglycerides are collected using molecular distillation or the like, other glycerides and fatty acids can be reacted again and used for monoglyceride synthesis, which brings great benefits to the oil chemical industry.

[Brief explanation of the drawing]

FIG. 1 shows the water content of reaction solutions using immobilized lipases of the present invention and the degree of decrease in acid value after the reaction. FIG. 2 shows the water content of the reaction solution and the degree of decrease in the acid value after the reaction when immobilized on various carriers were used.

Claims (1)

[Claims] 1. A method for producing a product with a high monoglyceride content, which comprises carrying out a reaction at a water content of 3.0% by weight or less using a reaction solution containing a high acid value oil, glycerin, and immobilized lipase.