JPH0430837B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a method and apparatus for producing triglycerides by causing lipase to act on a substrate containing monoglycerides and/or diglycerides and fatty acids. [Conventional technology] The main component of edible oils and fats is triglyceride (TG).
In addition, it contains free fatty acids (FFA), monoglycerides (MG), and diglycerides (DG), which are hydrolysis products of TG. These products are produced by the hydrolysis of the main component TG by enzymes contained in the raw materials or external factors during the oil and fat extraction operation from the collection of raw materials. Among these hydrolysis products, FFA and MG can be removed in the purification step. For example, it is possible to separate and remove a significant proportion of FFA and MG from TG by methods such as alkali purification or distillation/deacidification. On the other hand, DG has physical and chemical properties such as boiling point.
Since it is similar to TG, it is difficult to separate it from each other, and a method for separating DG has not been established. The presence of DG in TG forms a eutectic mixture.
Problems arise such as a decrease in SFI (solid fat content index), interference with crystal nucleation, and difficulty in tempering operations in the thiokolate production process. As described above, when DG is contained in TG, its uses are limited, so a method is needed to effectively reduce the DG content in fats and oils. It is widely known that lipase acts on fats and oils to cause hydrolysis, ester synthesis, and transesterification reactions. Among these, hydrolysis and synthesis of fatty acid esters are reversible reactions as shown in the following formula. DG + FFA → ← TG + H ₂ O (1) From equation (1), it is clear that the reaction proceeds to the hydrolysis side in a system with a lot of water, and to the TG synthesis side in a system with a little water. It is advantageous to carry out the above reaction while removing water, but in any case, lipase serves as a catalyst that promotes the hydrolysis reaction or synthesis reaction. Japanese Patent Publication No. 63-12599 relates to an esterification method in which a lipolytic enzyme having transesterification activity acts on a substrate containing partial glycerides and free fats or esters of their lower alcohols in a system that discharges water or water and lower alcohols. Vacuum distillation and the use of absorbents such as zeolite and silica gel have been shown to be a method for discharging water or water and lower alcohols from the system. The amount of water in the reaction system is stated to be approximately 0.18% or less. JP-A-60-203196 relates to a transesterification method using lipase that performs an ester synthesis reaction following a hydrolysis reaction of oils and fats. It is described that water is removed by venting into the reaction system and then exhausting it outside the reaction system to remove the water in the reaction system along with it. JP-A No. 62-19090 discloses that glycerin and C ₄ to C ₂₂ saturated (unsaturated) fatty acids have specific properties without substantially adding water and while removing water by-produced by the reaction. This method concerns the production of diglycerides by the action of lipase produced by mutant strains of Candida and Cylindrace.The method for dehydrating the reaction system is to use an absorbent or to vent dry air or inert gas into the reaction tank. It is shown that the water content of the reaction system is reduced to 0.1% or less by stirring and exhausting the system to remove water. As mentioned above, in the conventional method, the pressure inside the reaction system is reduced or dry gas is passed through the reaction system to remove moisture in the system by entraining the gas. Merely reducing the pressure is inefficient in removing water, and if you try to remove the maximum amount of water using drying gas, it is necessary to introduce a large amount of gas, and the accompanying stirring action causes significant depletion of the immobilized enzyme, which can be counterproductive. However, there was a drawback in that water could not be removed to the maximum extent with a gas amount that did not deplete the immobilized enzyme, making it impossible to obtain highly concentrated triglycerides. [Problems to be Solved by the Invention] The present invention is capable of removing water in the reaction system to the utmost extent when performing an ester synthesis reaction by causing lipase to act on monoglyceride and/or diglyceride and fatty acid, and producing a highly concentrated triglyceride. An object of the present invention is to provide a method and apparatus capable of efficiently manufacturing. [Means for Solving the Problems] The method for producing highly concentrated triglycerides according to the present invention comprises an immobilized lipase presence region comprising a fixed bed filled with lipase immobilized on a carrier and an immobilized lipase not filled with immobilized lipase. A substrate containing monoglyceride and/or diglyceride and fatty acids is supplied to a reaction tank in which a lipase-free region is installed, and gas is introduced only into the immobilized lipase-free region and then discharged, and the substrate is immobilized. Ester synthesis is performed by bringing the substrate and immobilized lipase into contact in the region where immobilized lipase exists, and the reaction product obtained by ester synthesis is moved to the region where immobilized lipase is not present and brought into contact with dry gas to combine the reaction product and the immobilized lipase. Water is removed from the reaction product by discharging the gas after contact to the outside of the reaction tank, and the reaction product from which water has been removed by contact with the dry gas is circulated to the area where the immobilized lipase is present and esterified again. It consists of performing synthesis. The substrate in the method for producing high concentration triglycerides of the present invention may be any substrate as long as it contains at least one of monoglycerides and diglycerides in addition to fatty acids. In addition to TG obtained by transesterification of oils and fats, MG, DG,
It can also be applied to products containing by-products such as FFA.
In addition, DG after pre-purification to remove MG and FFA
Fatty acids may be added to the content. Alternatively, a triglyceride with a low concentration obtained by an esterification reaction of glycerin and fatty acid may be used. Enzymes used in the present invention include Rhizopus, Aspergillus, Penicillum,
Examples include lipases derived from the genus Candida, Pseudomonas, Mucor, and Geotricum. It is preferable to use lipase immobilized on a carrier because this allows the contact surface between the substrate and lipase to be expanded. Examples of carriers include ion exchange resins, styrene/divinylbenzene copolymers,
Polymers such as polymethyl methacrylate and photocurable resins, and inorganic materials such as zeolite, diatomaceous earth, perlite, kaolin, silica, and alumina can be used. Particularly preferred is a porous carrier having macroscopic pores, with an average pore diameter of 600 Å or more, preferably 800 Å or more. On the other hand, if the average pore diameter is too high, the specific surface area will decrease and the expression of lipase activity will tend to deteriorate.
500000 Å or less is preferable. As a method for immobilizing lipase, it is preferable to immobilize lipase on a carrier by physical adsorption. The reaction temperature when performing the ester synthesis reaction by allowing the immobilized lipase to act on the aforementioned substrate is as follows:
Depending on the lipase used, a temperature in the range of 20 to 75°C is preferred. The amount of dry gas vented to the immobilized lipase-free area of the reaction tank depends on the volume ratio of the substrates present in the immobilized lipase-free area and the immobilized lipase-free area, temperature, and other operating conditions. It depends, but in order to obtain a high concentration of triglyceride, 5
~20 vvm [aeration amount per minute (/min)/capacity of fats and oils in the immobilized lipase-free area in the reaction tank ()], preferably 7 to 20 vvm, more preferably 10 to 20 vvm. The amount of ventilation
If it is less than 5 vvm, the water content in the reaction system cannot be sufficiently lowered, or it takes a long time to reach a predetermined water content, making it impossible to effectively obtain a high concentration of triglyceride. On the other hand, even if the aeration rate is 20 vvm or more, it is not expected to improve the triglyceride concentration much, and if the aeration rate is too high, problems such as scattering of reaction products will occur, resulting in a decrease in the yield of reaction products and difficulty in recovery. At the aeration rate within the above range, which would otherwise require additional operations, the drying gas and the reaction product can effectively contact each other, and the ester synthesis reaction can be carried out satisfactorily. The dry gas may be any gas that is not reactive towards fats and oils, such as nitrogen gas, argon gas, helium gas, and the like. The gas discharged from the immobilized lipase-free region of the reaction tank is cooled to separate moisture, and can be circulated to the reaction tank again and reused as dry gas. A reaction tank having a structure in which an immobilized lipase-present region and an immobilized lipase-free region where immobilized lipase is not filled are installed, and gas is introduced only into the immobilized lipase-free region and then discharged. As a specific example, as shown in Fig. 1, an inner cylinder 3 having a lower height is inserted into an outer cylinder 2 of an upright cylindrical reaction tank 1, and is installed so as to have a space above and below. , a packed bed 4 of immobilized lipase is provided between the outer cylinder 2 and the inner cylinder 3, a gas inlet 6 is provided directly below the inner cylinder 3, and a gas outlet 7 is provided at the top of the tank. Symbol 5 is a mixture of substrate and reaction product,
Symbol 8 is a heating jacket. The part marked with symbol 4 is the area where immobilized lipase exists, the bottom of the reaction tank, and the inner cylinder 3.
The inside of the area becomes an area where no immobilized lipase exists. When dry gas is introduced into the inner cylinder 3 from the gas inlet 6, the mixture of substrates and reaction products is dehydrated while being stirred and moves up the inner cylinder 3, and the gas containing moisture is discharged from the gas outlet 7 into the system. Exhausted outside. The mixture of substrates and reaction products that has risen up the inner cylinder along with the gas overflows the upper edge of the inner cylinder and reaches the packed bed 4 of immobilized lipase provided between the inner cylinder and the outer cylinder.
The ester synthesis reaction takes place. The substrate and reaction product that have passed through the packed bed 4 of immobilized lipase are introduced into the inner cylinder again and are dehydrated by contacting with the dried gas. The space above and below the inner cylinder 3 serves as a circulation path for substrates and reaction products. In this type of reaction tank, as the dry gas rises in the inner cylinder, the reaction product moves from the area where immobilized lipase is present to the area where there is no immobilized lipase due to the draft action, and the area where there is no immobilized lipase. This has the advantage that the circulation of the dehydrated reaction product from the immobilized lipase to the area where the immobilized lipase is present is carried out automatically. In case of continuous operation, a substrate supply port and a reaction product discharge port are additionally provided. Contrary to the diagram, a packed bed of immobilized lipase is provided in the inner cylinder, and dry gas is introduced between the inner cylinder and the outer cylinder, so that the inner cylinder side becomes the reaction zone and the inner cylinder and the outer cylinder The space between them may be used as a dehydration area. Further, as shown in FIG. 2, a region (packed layer) 4' in which lipase immobilized on a carrier exists is provided in the lower half of the cylindrical container constituting the reaction tank 1, and the upper half without the packed layer is provided. The dry gas inlet 6 is located at the lower part of the immobilized lipase-free area 9, the gas outlet 7 is located at the upper part, and an appropriate position between the dry gas inlet and the gas outlet in the upper half (to form a liquid level) is provided. A liquid circulation pipe 10 may be provided from the lower half of the carrier to the lower part of the packed layer 4' of lipase immobilized on the carrier. The substrate introduced from below the immobilized lipase existing region 4' ascends the immobilized lipase packed bed 4' and undergoes an ester synthesis reaction. The mixture of substrate and reaction product leaving the immobilized lipase packed bed 4' dehydrates and rises while contacting the dry gas from the dry gas inlet 6 in the immobilized lipase-free area 9, and contains water. The gas is exhausted to the outside of the system from the gas exhaust port 7. After contacting the gas, the mixture of substrate and reaction product is again introduced into the immobilized lipase packed bed 4' through the liquid circulation pipe 10 and subjected to the ester synthesis reaction again. When circulating the liquid, a liquid draft method by blowing dry gas may be used, but a forced circulation reaction tank using a pump or the like may also be used. Such a reaction vessel used in the present invention is
Area where reaction is the main activity (immobilized lipase presence area) and area where dehydration is the main activity (immobilized lipase absence area)
Since these are provided separately, the immobilized lipase is not affected by the stirring action of the blown gas, and its depletion is significantly suppressed. The shape of the immobilized lipase can be granular, film, honeycomb, or the like. If the immobilized lipase is too fine, the flow of the substrate and reaction products will be slow, and if it is too coarse, the specific surface area will decrease and the expression of lipase activity will be impaired.
The particle size when using granular immobilized lipase is
0.5-5 mm is preferred. The volume ratio of the area where immobilized lipase is present and the area where immobilized lipase is not present is preferably in the range of about 5:1 to 1:5. The present invention will be specifically explained below using Examples. Example 1 Diatomaceous earth (specific surface area: 0.8 m ² /g, pore volume:
1.08 cc/g, average pore size: 97200 Å) was molded into a particle size of 2 to 3 mm using a silica-based binder to prepare 10 parts by weight of carrier, and to this was added 0.3 parts by weight of lipase originating from the genus Pseudomonas. 40 parts by impregnation with a solution of 10 parts by weight of water.
Dry at ℃, 1mmHg, 4 hours to reduce moisture to 0.1
% or less of the immobilized enzyme was prepared. 30 g of the above immobilized enzyme was filled between the inner tube and the outer tube of a reaction tank, in which an inner tube with an outer diameter of 10 mm, an inner diameter of 7 mm, and a length of 180 mm was installed in an outer tube with an inner diameter of 20 mm and a height of 200 mm with a hot water jacket. In order to prevent the immobilized enzyme from moving, 20 mesh wire meshes made of stainless steel were provided above and below the immobilized enzyme filling tank to fix the immobilized enzyme, thereby forming an immobilized lipase presence area. (See Figure 1) 30 g of crude palm olein with a composition consisting of 87.0% by weight of triglyceride (TG), 7.3% by weight of diglyceride (DG), 0.3% by weight of monoglyceride (MG), and 5.4% by weight of free fatty acid (FFA) was used as a substrate. The reactor was placed in the reactor and nitrogen gas was blown into it from the bottom of the inner tube to stir the substrate and dehydrate the reaction product. When hot water at 60°C was passed through the jacket and the reaction was carried out for 24 hours with a nitrogen gas flow rate of 10 vvm, the concentration of triglyceride (TG) in the substrate reached 95% by weight. In addition, the concentrations of DG, MG, and FFA are
2.6% by weight, 0.1% by weight, 2.4% by weight, and the water content in the reaction system was 10 ppm or less. Example 2 An inner tube with an outer diameter of 45 mm, an inner diameter of 44 mm, and a height of 200 mm was attached to a jacketed reaction tank with an inner diameter of 80 mm and a height of 800 mm, and 200 g of the same immobilized enzyme used in Example 1 was added to the inner tube. The space between the outer tubes was filled with 900 ml (810 g) of the cooled palm olein, and dry nitrogen gas of 10 vvm was introduced at 60°C in the same manner as in Example 1, and the TG concentration reached 95% by weight in 24 hours. . The water content in the reaction system was 10 ppm or less. In addition, when the reaction was carried out under the condition of a nitrogen gas flow rate of 5 vvm, the TG concentration was 93% by weight in 24 hours, and the TG concentration was 10 vvm.
It was lower than at the time. The water content in the reaction system was 19 ppm or less. Example 3 100 g of the same immobilized enzyme used in Example 1 was filled between the inner tube and outer tube of the reaction tank used in Example 2, and the same cooled enzyme used in Example 1 was charged.
Use 1000ml (900g) of palm olein and change the amount of nitrogen gas fed into the reaction tank to 2, 5, 7, 10 or
The ester synthesis reaction was carried out at a temperature of 60° C. with various speeds of 14 vvm. The triglyceride concentration after 24 hours is shown in FIG. In FIG. 3, the horizontal axis shows gas flow velocity (vvm), and the vertical axis shows triglyceride concentration (wt%). From FIG. 3, it can be seen that a gas velocity of 5 to 20 vvm, particularly 7 to 20 vvm, is suitable for obtaining a high triglyceride concentration. Example 4 An immobilized lipase presence region filled with 100 g of the same immobilized enzyme used in Example 1 was provided between the inner tube and outer tube of the same reaction tank as used in Example 2,
1000 ml (900 g) of the same cooled palm olein used in Example 1 was charged, and dried nitrogen gas was introduced at a gas flow rate of 10 vvm to perform stirring, liquid circulation, and dehydration, and a synthesis reaction was performed at a temperature of 60°C. . The reaction was carried out at a rate of 41.7 ml/h (corresponding to a residence time of 24 hours) while continuously introducing the substrate and withdrawing the reaction product. The triglyceride concentration in the reaction product continuously extracted from the reaction tank was periodically analyzed and measured, and it stabilized after 48 hours. The triglyceride concentration in the reaction product at that time was 93.3
The water content in the reaction system was 19 ppm by weight. When the reaction was further continued, the results shown in Table 1 were obtained. Comparative Example 1 In the reaction tank used in Example 4, the same amount was dried in the same manner as in Example 4 except that the fixed bed of the immobilized enzyme was not provided and the immobilized enzyme was dispersed in the substrate to form a fluidized bed. Gas was introduced to carry out the ester synthesis reaction. It stabilizes 48 hours after starting the reaction, and the triglyceride concentration in the reaction product at that time is
The water content in the reaction system was 93.5% by weight and 19 ppm. When the reaction was further continued, the results shown in Table 1 were obtained.

[Table] In the case of Comparative Example 1, the results were similar to those of Example 4 in the short term, but in the long term operation, it was found that the leakage of the immobilized enzyme was severe and there was a difference in the life span of the immobilized enzyme. That is, in Comparative Example 1, which uses a fluidized bed format in which immobilized enzyme is dispersed in a substrate, 45% of the immobilized enzyme leaks out after 20 days of continuous operation, resulting in a decrease in activity and a decrease in triglyceride concentration. Although some leakage of the immobilized enzyme was observed in the case of fixed bed method No. 4, it was confirmed that stable activity was expressed for 40 days and a high triglyceride concentration was maintained. In addition, even in the fixed bed method, some leakage of immobilized enzyme is observed initially because the powder is attached to the raw material particles during the manufacturing process of the carrier. This is presumed to be due to the powdered immobilized enzyme leaking out together with the reaction product as the reaction time increased. In the case of a fixed bed, the amount of immobilized enzyme leaked out was only 7% after 20 days of continuous operation, but this amount corresponds to the amount of powder adhering to the carrier during the initial production of the immobilized enzyme carrier. In addition, in the case of a fluidized bed, not only does the powdered immobilized enzyme leak out quickly, but the granular immobilized enzymes collide with each other and become powdered, resulting in a greater amount of immobilized enzyme leaking than in the fixed bed method. It is presumed that the increase in the activity caused a rapid decline in activity. [Effects of the invention] Since water can be effectively removed from the reaction system and the water content can be maintained at a low level, high concentration triglyceride can be obtained from diglyceride and/or monoglyceride and fatty acids by the action of lipase, and moreover, it can be operated continuously for a long time. However, the activity does not decrease. Furthermore, the device of the present invention has a simple structure, prevents loss of immobilized lipase, and enables continuous operation for a long time.

[Brief explanation of drawings]

Figure 1 is an explanatory diagram of the fixed bed type reaction tank used in the examples, Figure 2 is an explanatory diagram of another type of reaction tank suitable for carrying out the method of the present invention, and Figure 3 is an illustration of the nitrogen supply to the reaction tank. This is a diagram showing the triglyceride concentration after 24 hours when the ester synthesis reaction was carried out by changing the amount of gas fed. The horizontal axis shows the gas flow rate (vvm) and the vertical axis shows the triglyceride concentration (wt%).

Claims (1)

[Scope of Claims] 1. An immobilized lipase-existing region consisting of a fixed bed filled with lipase immobilized on a carrier and an immobilized lipase-free region not filled with immobilized lipase are installed inside, and the immobilized lipase is A substrate containing a monoglyceride and/or diglyceride and a fatty acid is supplied to a reaction tank having a structure in which gas is introduced only into a lipase-free region and then discharged, and the substrate and immobilized lipase are brought into contact in an immobilized lipase-present region. By carrying out ester synthesis, moving the reaction product obtained from the ester synthesis to an area where no immobilized lipase exists and contacting it with dry gas, and discharging the gas after contacting with the reaction product to the outside of the reaction tank. Production of a highly concentrated triglyceride characterized by removing water from a reaction product and circulating the water-removed reaction product through contact with a dry gas to an area where immobilized lipase exists to perform ester synthesis again. Law. 2 Inside the outer cylinder constituting the reaction tank, an inner cylinder whose height is lower than the outer cylinder is installed with a space above and below it, and either between the outer cylinder and the inner cylinder or inside the inner cylinder. A highly concentrated product having a packed layer of lipase immobilized on a carrier on one side, a dry gas inlet on the side not having the packed layer of immobilized lipase, and a gas outlet on the top. Triglyceride production equipment. 3 A cylindrical container constituting the reaction tank has a packed layer of lipase immobilized on a carrier in the lower half, a dry gas inlet in the lower part of the upper half without the packed layer, and a gas outlet in the upper part. and a liquid circulation pipe is provided from a position between the dry gas inlet and the gas outlet in the upper half to the lower part of the packed bed of lipase immobilized on the carrier in the lower half. Highly concentrated triglyceride manufacturing equipment.