JPH049516B2 - - Google Patents

Description

[Detailed description of the invention]

This invention relates to a method of hydrolyzing fats and oils using lipase. Conventional methods for hydrolyzing fats and oils using lipase involve mixing and stirring an aqueous lipase solution and fats or oils, or reacting them in a semi-solid slurry state that is mixed and emulsified. This is a method of mixing with oils and fats that do not contain oil. In these methods, most of the lipase is adsorbed by fats and oils and their hydrolyzed fatty acids during the reaction, so even if the lipase aqueous solution is recovered by simple separation by layer separation, the lipase in the aqueous solution is Recovery of lipase is almost impossible because it contains less than 10% by weight of the amount of lipase originally used. The impossibility of recovering lipase is the biggest drawback of the lipase-based fat and oil hydrolysis method.
That is, in order to reduce the cost of lipase by reducing the amount of lipase used as much as possible, the reaction rate becomes extremely slow and the reaction takes several days to reach the goal, which is an industrially inappropriate method. Therefore, in order to industrialize this hydrolysis reaction, it is necessary to completely recover the lipase so that it can be recycled. This recycling makes it possible to reduce the cost of lipase and increase the rate of hydrolysis by increasing the amount of lipase used. For this purpose, methods of immobilizing enzymes are generally employed. However, conventional reactions using immobilized enzymes are mainly carried out in aqueous systems;
Most of the carriers on which enzymes are immobilized are hydrophilic, such as Cephadex and porous glass beads. Even if this enzyme immobilization technology using a hydrophilic carrier is applied to the above-mentioned lipase, since the substrate is made of water-insoluble fats and oils, the immobilized lipase cannot be sufficiently dispersed in the substrate, and hydration occurs naturally. Decomposition rate decreases. Furthermore, the substances involved in the hydrolysis reaction include oil and water, which do not dissolve in each other in addition to lipase, and it is necessary that there be sufficient contact between the oil and water and that lipase be present at the interface. It is said that. The fact that no technology has been developed that fully satisfies the complexity of this reaction system is the reason why an industrial method for hydrolyzing fats and oils using immobilized lipase has not been developed. As a result of intensive studies to overcome the above-mentioned two drawbacks, the inventors found that alkylation,
By using a phenylated or tritylated agarose gel and immobilizing lipase in this gel by adsorbing and entrapping it mainly through hydrophobic bonds, and bringing it into contact with oil and fat, the oil and fat can be absorbed without adding water to this contact system. The present invention was completed based on the discovery that the hydrolysis reaction can be achieved satisfactorily. That is, the present invention relates to a method for hydrolyzing fats and oils using lipase, which is characterized in that the fats and oils are hydrolyzed using lipase immobilized on an alkylated, phenylated or tritylated agarose gel. The method of this invention is carried out by, for example, a method of dispersing the above-mentioned immobilized lipase in fats and oils to hydrolyze the fats and oils, or a method of hydrolyzing the fats and oils by passing the fats and oils through a column filled with the above-mentioned immobilized lipase. , can be implemented. In the above invention, since the alkylated, phenylated or tritylated agarose gel with a water content of about 95% by weight is oil-water amphiphilic, the immobilization of lipase from the aqueous lipase solution is effectively carried out and the immobilized lipase is can. Since a large amount of water is present in this immobilized gel, there is no need to add water to the hydrolysis reaction system. Moreover, because this gel has been alkylated, phenylated, or tritylated, lipophilic groups have been introduced, so it is extremely well dispersed in and in contact with fats and oils, resulting in good results for the expression of lipase activity. bring. By bringing this immobilized lipase into direct contact with fats and oils, an interface between fats and oils-lipase-water is effectively formed. Another advantage of this invention is that because the immobilized lipase is tightly adsorbed to the gel, it does not migrate to the hydrolyzed fatty acids, unlike conventional non-immobilized systems. There is no need to remove lipase protein from fatty acids (e.g., fatty acid distillation step), and since the by-product glycerin is included in the gel, pure fatty acids can be obtained by simply separating the gel or centrifuging it. It is in being able to receive. The type of lipase used in this invention is not limited, but generally includes Candeida genus, Chromobacterium genus, Aspergillus genus, Penicillium genus,
It is possible to use lipases sourced from microorganisms such as Geotrichum and Rhizopus, lipases obtained from animal organs such as pancreas, and lipases obtained from plant seeds such as castor seeds. The lipase may be in the form of an aqueous solution or a powder, but in order to ensure efficient immobilization, it is better to have less foreign proteins other than the lipase. The agarose gel according to the present invention for immobilizing lipase is made by alkylating, phenylating or tritylating agarose to form a gel-like substance containing a large amount of water molecules by known means, and various types are already commercially available. has been done. Here, lipophilicity is imparted to the agarose gel by alkylation, phenylation, or tritylation, but from the viewpoint of both the dispersibility (accessibility) to the oily substrate and the immobilization rate of lipase due to this imparting of lipophilicity, , those alkylated with an alkyl group, particularly an alkyl group having 6 to 18 carbon atoms, are preferred. Furthermore, it is desirable that the alkyl group is an octyl group, a decyl group, a dodecyl group, or a mixed alkyl group thereof. Note that agarose gel is a polysaccharide gel made from natural agar. The means for immobilizing lipase on this agarose gel is arbitrary, and any known immobilization technique such as physical adsorption, cross-linking, or covalent bonding can be used. Considering the immobilization cost and reusability of the carrier gel, the physical adsorption method is the best. The reaction temperature for hydrolysis is generally 10 to 50°C, but the optimum temperature is 30 to 40°C. The reaction rate is better at higher temperatures as long as the activity of the enzyme is not lost, and if an appropriate thermostable lipase is used, higher temperature reactions are possible. Next, the present invention will be explained with reference to examples.
In the Examples below, refined olive oil and olive oil with a hydrolysis rate of 95.7% by weight were used as representative examples of fats and oils. The lipase-immobilized gel used was prepared by the following physical adsorption method. <Preparation of immobilized lipase> Thoroughly wash 100 ml of commercially available octyl, phenyl, or tritylated agarose gel, wash 4 times with 0.1 M phosphate buffer (PH = 7.0), and repeat centrifugation to obtain a concentration of 2400 units/ml. Contains lipase
In addition to 60 ml of 0.1M phosphate buffer (PH=7.0), lipase was impregnated into the gel while being mixed for 1 hour under ice cooling. After this was filtered off with suction, the water on the surface of the gel was absorbed with paper to obtain an immobilized lipase gel.
The immobilization rate of these lipases was 97-99%,
Activity was 1400-1425 units/ml gel. Example 1 Refined olive oil (saponification value: 192.4, acid value:
0.2, iodine value: 82) 25 ml of the above immobilized lipase octyl agarose gel was added to 50 g, and gentle stirring was continued at 35°C. After 7 hours, the hydrolysis rate of this oil reached 93.1% by weight. Example 2 Partially hydrolyzed olive oil with a hydrolysis rate of 95.7% by weight (saponification value: 200.1, acid value: 191.5, melting point 25°C)
25 ml of the same immobilized lipase gel used in Example 1 was added to 50 g, and the mixture was slowly stirred at 35°C. After 1.5 hours, the acid value was 198.3. The saponification value at that time was 202.0, so the hydrolysis rate was
It is 98.2% by weight. Example 3 The immobilized lipase gel was recovered by filtering the reaction product of Example 2, and this recovered gel was added to 50 g of the same partially hydrolyzed olive oil used in Example 2 and reacted in the same manner. That is, the same gel was repeatedly tested. As a result, the acid values shown in the following table were obtained, and it was confirmed that a hydrolysis rate of 98 to 99% by weight could be maintained even after repeating the test five times.

[Table] Example 4 Immobilized lipase trityl agarose gel described above
100 ml was mixed with 100 g of glass beads having an average diameter of 1.5 mm, and the mixture was packed into a 100 cm long column. While maintaining this column at 35° C., 200 g of the same refined olive oil as in Example 1 was injected from the top using a liquid feed pump. As a result of repeating this process by pumping the distillate from the lower part into the upper part, the hydrolysis rate of this oil reached 92.3% by weight after 7 hours. Example 5 Immobilized lipase phenyl agarose gel as described above
Using 25ml, the other operations were the same as in Example 1.
Refined olive oil was subjected to a hydrolysis reaction under the following conditions. In this example, a hydrolysis rate of 92.0% by weight was obtained after 7 hours.

Claims (1)

[Scope of Claims] 1. A method for hydrolyzing fats and oils using lipase, which comprises hydrolyzing fats and oils using lipase immobilized on an alkylated, phenylated or tritylated agarose gel. 2. A method for hydrolyzing fats and oils using lipase according to claim 1, which comprises dispersing immobilized lipase in fats and oils to hydrolyze the fats and oils. 3. A method for hydrolyzing fats and oils using lipase according to claim 1, wherein the fats and oils are passed through a column filled with immobilized lipase for hydrolysis. 4 Alkylated agarose gel has 6 or more carbon atoms
A method for hydrolyzing fats and oils using a lipase according to any one of claims 1 to 3, which has 18 alkyl groups.