JPS6253152B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention uses a reactor (bioreactor) that uses a separation membrane in the fat and oil decomposition reaction using lipase, thereby maintaining enzyme activity while maintaining a high fat and oil decomposition rate, and separating the produced glycerin and fatty acids. The present invention also relates to an enzyme reaction method characterized in that the enzyme reaction is carried out simultaneously with the decomposition reaction. Although methods for producing glycerin and fatty acids by decomposing fats and oils using lipases have been known for a long time, there are various obstacles to industrialization. The main reason for this is that fat-degrading enzymes rapidly deactivate during the reaction and cannot be recovered and reused, so large amounts of expensive enzymes must be used. Therefore, the current situation is that it does not offer cost advantages compared to high-pressure decomposition methods, and the characteristics of enzymatic decomposition cannot be utilized. When investigating the causes of lipase inactivation, inhibitors,
It can be said that heat and water are the main factors. Particularly focusing on water, lipase decomposition reaction is a hydrolysis reaction, so although water is required for the reaction, lipase has the contradictory property of deactivating as the amount of water increases. ing. Therefore, it is easy to imagine that one of the factors to prevent deactivation is to supply only the water necessary for the reaction and not to supply excess water. However, as shown in Table 1 below, in the method of reacting by mixing and stirring water, oil, and enzymes, if the amount of water is extremely reduced, the decomposition rate of fats and oils will decrease, resulting in a sufficient decomposition reaction. You will not be able to make it happen.

[Table] Therefore, in a reaction where water, oil, and enzymes are mixed and stirred to perform a decomposition reaction, in order to ensure a high decomposition rate,
The reaction has no choice but to provide more water than is necessary for the reaction, sacrificing recovery and reuse of the enzyme. As a result of intensive studies to overcome these drawbacks, the present inventors have developed a system that can maintain a high decomposition rate, prevent enzyme deactivation, and simultaneously separate the reaction products glycerin and fatty acids. discovered a temporary reaction method. The reaction method according to the present invention uses a separation membrane called a reverse osmosis membrane, an ultrafiltration membrane, an ion exchange membrane, or a dialysis membrane, and an oil phase in which lipase is suspended on one side of the membrane. It is characterized in that an aqueous phase is present on the opposite side, and the enzyme, oil, and water are brought into contact with each other through a membrane to perform a hydrolysis reaction of fats and oils. If only an oil phase in which enzymes are suspended is present on one side of the membrane and an aqueous phase on the other side, water will enter the oil phase as the decomposition reaction progresses, and as mentioned above, the amount of water will increase and the enzyme will become inactive. However, by applying some pressure to the oil phase side, water can be prevented from entering the oil phase, and enzyme deactivation can be prevented. However, on the other hand, if the pressure applied to the oil phase side is too high, the decomposition rate decreases. Therefore, it is preferable to maintain the pressure applied to the oil phase at the minimum pressure necessary to prevent water from penetrating into the oil phase, and this varies depending on the type of oil used, temperature, etc. Generally the pressure is 0.01
It is necessary to maintain the pressure at Kg/cm ² or higher, and the optimum pressure varies depending on the shape and size of the membrane reactor, but
Desirably 0.05-5Kg/cm ² , more preferably 0.05
~0.5Kg/ ^cm2 . The reaction mechanism of the membrane reactor of the present invention is that water penetrates through the membrane to the membrane surface on the oil phase side, contacts enzymes on this membrane surface, and an oil and fat decomposition reaction occurs. Ru. Since the fat and oil decomposition reaction occurs on the membrane surface in this way, a very high decomposition rate can be obtained, and since almost no water is mixed into the oil phase, it is thought that deactivation of enzymes can be prevented. Details are unknown. In the fat and oil decomposition reaction method of the present invention, the glycerin produced in the decomposition reaction is released into the water phase and almost no longer exists in the oil phase. As a result, at the end of the decomposition reaction, the oil phase becomes a fatty acid solution in which enzymes are suspended, and the aqueous phase becomes glycerin water, making it possible to separate glycerin and fatty acids at the same time as the reaction. Furthermore, since the fat and oil decomposition reaction is determined by the contact time between water, oil, and enzyme, the rate of fat and oil decomposition can be increased by increasing the membrane area. Therefore, by increasing the membrane area, it is possible to further shorten the reaction time, which conventionally required 20 to 30 hours. The lipase used in the present invention may be produced by microorganisms as long as it has a strong ability to decompose fats and oils, or may be obtained from animal organs, plant seeds, etc., and in particular, the source thereof may be specified. isn't it. Furthermore, these lipases can be used not only as purified enzyme preparations but also as unpurified products, and there are no particular limitations on their purity. As the separation membrane used in the present invention, as mentioned above, semipermeable membranes or porous membranes such as reverse osmosis membranes, ultrafiltration membranes, ion exchange membranes, and dialysis membranes are used, especially if they do not allow enzymes to pass through. Although there are no restrictions on the material, it is desirable to use a material that has a high affinity for water. There are no particular limitations on the form of the membrane, and any form of membrane can be used, such as a flat membrane, hollow fiber membrane, or spiral membrane. The membrane reactor in which the membranes are set can be a flat membrane in which many membranes are set to form a multi-chamber structure, or a hollow fiber membrane in which many hollow fiber membranes can be set. A reactor that is more compact and allows for a larger membrane area is desirable, but there are no particular restrictions on the shape of the reactor. In carrying out the present invention, the oil phase in which the enzyme is suspended and water may be left standing on both sides of the membrane, or may be passed through with a pump or the like. When left standing, it is necessary to apply pressure to the oil phase side using a hydraulic machine or gas pressure, but when pumping liquid, it is sufficient to use pump pressure, and pumping liquid is preferable from an industrial perspective. . In addition, in the case of pump liquid passage, oil and water can be circulated through the membrane, respectively, and thereby the membrane area can be reduced. From this point of view as well, pumping is considered preferable. In the reaction method of the present invention, the enzyme is suspended in the fatty acid solution that comes out of the membrane reactor after the reaction is completed.
This is separated, collected and reused. For this separation, filtration, centrifugation, separation using an ultrafiltration membrane, etc. are useful. However, in this case, in order to prevent enzyme deactivation, a method is desired that does not generate heat, can be separated in a short time, and has less enzyme loss. The fats and oils used in the method of the present invention include soybean oil, palm oil, coconut oil, olive oil, linseed oil,
Vegetable oils such as castor oil, beef tallow, pork tallow,
Examples include animal fats and oils such as fish oil. Among these fats and oils, in the case of liquid oils that are liquid at room temperature, the above-mentioned pumping is possible, but in the case of solid fats that are solid at room temperature, this is not possible. Therefore, in the case of these solid fats, it is preferable to carry out the reaction by heating to a temperature above the melting point, or to add an organic solvent that is compatible with the solid fat to impart fluidity and carry out the decomposition reaction. However, in the reaction under heating, extreme heating is not possible because the enzyme is largely inactivated by heat, and it is preferable to maintain the temperature at the lowest temperature at which the oil has fluidity. Also, in the case of adding an organic solvent, it is preferable to add an organic solvent that does not easily lose enzyme activity in the minimum amount that provides fluidity. Examples of organic solvents that do not easily deactivate lipase include hexane and isooctane.
It is not limited to this. Although the reaction method according to the present invention uses a membrane, it does not involve immobilizing the enzyme on the membrane.
The enzyme is suspended in the oil phase. Therefore, deactivation of the enzyme during immobilization, which occurs with immobilized enzymes, does not occur. Furthermore, enzyme deactivation in the enzyme separation step after the membrane reaction is extremely rare, and the enzyme can be easily recovered and reused while maintaining high enzyme activity. As described above, the present invention utilizes the characteristics of lipase to enable its recovery and reuse while maintaining a high decomposition rate, as well as to simultaneously separate fatty acids and glycene. This reaction method has many advantages such as being able to shorten the time. The present invention will be described below with reference to Examples, but the present invention is not limited to these Examples. Example 1 A membrane reactor having a multi-chamber structure was prepared by setting 10 cellulose acetate membranes (DRS-10, manufactured by Daicel Chemical Industries, Ltd.) with an area of 0.02 m ² . FIG. 1 is a schematic cross-sectional view of an example of such a multi-chamber structured membrane reactor. As shown in FIG. A filling material 2 consisting of a plastic net is placed in each chamber, and water is circulated and supplied from a water tank 5 to every other chamber 11 through lines 9 and 9' by a pump 7. 1. Oil (enzyme suspended) is pumped from the oil tank 6 into the chamber adjacent to the pump 8.
It is designed to be circulated and supplied through a 0,10' line. 3 indicates the outer frame. 300g of soybean oil and lipase (Meito Sangyo Co., Ltd.)
Oil in which 0.15 g of OF) was suspended was placed in oil tank 6, and pumped into the chamber on one side of each membrane of the membrane reactor. Similarly, 300 g of water was placed in the water tank 5, and the water was pumped into the chambers on the opposite side of each membrane. The pump flow rates for the oil phase and water phase were changed, and the pump flow rates were determined so that a pressure of 0.2 Kg/cm ² was applied to the oil phase side, and the oil and water phases were circulated at a temperature of 30°C. In addition, as the viscosity and density of the liquid changed as the reaction progressed, the pump flow rate had to be constantly adjusted to maintain a pressure of 0.2 Kg/cm ² . The reaction was carried out in this manner, and after 24 hours, a hydrolysis rate of 95% was obtained. on the other hand,
After 24 hours, the glycerin concentration in the oil phase was 0.1%, and the water concentration was also 0.1%. The glycerin concentration in the water that passed through the other side of the membrane was 9%. While such a high decomposition rate was obtained, glycerin and fatty acids were already separated at the end of the membrane reaction. Note that when only the pressure was increased to 0.5 Kg/cm ² under the same conditions, the decomposition rate decreased to 90% after 24 hours. Therefore, the pressure is preferably maintained near the minimum pressure necessary to prevent water from penetrating into the oil phase, and is appropriately selected according to the shape, size, etc. of the membrane reactor. Example 2 Same conditions as Example 1, 300 g of soybean oil, water
300g, enzyme 0.15g, temperature 30℃, pressure 0.2Kg/cm ² ,
After 24 hours of reaction in a membrane area of 0.2 m ² , the fatty acid solution was centrifuged to separate the enzyme. Centrifugation at 25℃
I did it in 10 minutes at 8000rpm. The enzyme thus obtained was suspended again in 300 g of fresh soybean oil, and a second reaction was carried out under the same conditions. A hydrolysis rate of 89% was obtained after 24 hours. The mixture was collected again in the same manner and a third reaction was performed. After 24 hours, the hydrolysis rate was 83%. In this way, the enzyme activity decreased by just 6% per reaction, and the enzyme could be recovered and reused with very little deactivation. Example 3 In Example 2, there was 6% inactivation in each reaction, but to compensate for this, an enzyme recovery and reuse experiment was conducted in a system in which 6% fresh enzyme was added. . That is, the same conditions as Example 1 (pressure 0.2 Kg/cm ² )
After reacting for 24 hours, the enzyme was separated using the separation method described in Example 2, and 0.009 g of fresh enzyme, equivalent to 6% of the initial addition amount, was added to the separated enzyme. The decomposition reaction was carried out again under the same conditions as in 1. A decomposition rate of 95% was obtained after 24 hours. Since the enzyme can be recovered and reused with a very low degree of deactivation, it was found that a high decomposition rate of 95% could be maintained by supplementing only a few percent of the deactivated amount for each batch. On the other hand, a method in which water, oil, and enzymes are simply stirred and mixed to decompose fats and oils, but the enzymes are not recovered;
0.15g of lipase was required for each application. Therefore, the method of the present invention requires only about 1/20 of the amount used. Example 4 The number of membranes in the membrane reactor as shown in FIG. 1 as in Example 1 was increased to 40, and the same conditions as in Example 1 were carried out.
i.e. 300g oil, 300g water, 0.15g enzyme, 0.2 pressure
When the reaction was carried out at a rate of Kg/cm ² and a temperature of 30°C, an increase in the decomposition rate was observed as shown in Figure 2. A is membrane 10
(0.2 m ² ), and B indicates the case of 40 membranes (0.8 m ² ). That is, it took 24 hours with 10 membranes (0.2 m ² ) to reach the maximum decomposition rate of 95%, but it became possible with 40 membranes (0.8 m ² ) in 9 hours. It was found that by increasing the membrane area in this way, the reaction time could be significantly shortened, which would have great advantages in industrialization. Example 5 In Example 1, a flat membrane was used, but in this example, a hollow fiber membrane was used instead of the flat membrane, and a decomposition reaction was carried out by passing an aqueous phase inside the hollow fiber membrane and an oil phase outside. I made it happen. 0.15 g of enzyme was suspended in 300 g of olive oil, this was placed in a container, and the liquid was pumped to the outside of the hollow fiber membrane. On the other hand, 300 g of water was placed in a container and pumped through the inside of the hollow fiber membrane. 0.3 on the oil phase side
A pressure of Kg/cm ² was applied. The hollow fiber membrane is manufactured by Asahi Kasei Corporation.
Ultrafiltration membrane ACL-1010 (polyacrylonitrile) was used. The area of this membrane is 0.2 m ² . 30
After reacting at ℃ for 24 hours, a decomposition rate of 95% was obtained, and glycerin and fatty acids were separated in the same manner as in Example 1.

[Brief explanation of the drawing]

FIG. 1 is a schematic cross-sectional view of an example of an apparatus used in the method of the present invention, and FIG. 2 is a graph showing an example of the relationship between reaction time (horizontal axis) and decomposition rate (vertical axis). 1... Membrane, 2... Filling, 3... Outer frame, 4...
Gasket.

Claims (1)

[Claims] 1. An oil phase in which lipase is suspended is present on either side of a separation membrane, and an aqueous phase is present on the other side, and the enzyme, oil, and water are brought into contact with each other across the membrane. A reaction method characterized by carrying out a hydrolysis reaction of fats and oils. 2. The reaction method according to claim 1, characterized in that a pressure necessary to prevent water from entering the oil phase side is applied to the oil phase side. 3. The reaction method according to claim 2, wherein the pressure is 0.01 Kg/cm ² or more.