JPS61173791A - Method of hydrolyzing fat and oil with lipase - Google Patents

Abstract

PURPOSE:To keep high decomposition ratio of fats and oils, to maintain enzyme activity and to continue a reaction stably for a long period, by hydrolyzing fats and oils with lipase while keeping always glycerin concentration in a water phase existing in a reaction system in a specific range. CONSTITUTION:In hydrolyzing fats and oils with lipase, glycerin concentration in a water phase existing in a reaction system is kept always at 10-40wt% to carry out the reaction. After the hydrolysis is over, the reaction mixture is separated into an oil layer, an emulsion layer and a water layer, the emulsion layer is taken out, used again for the following fats and oils hydrolysis, the enzyme dissolved in the water phase is concentrated and recovered from the water layer by the use of an ultrafilter, and preferably it is reused for the ensuring fats and oils hydrolysis.

Description

Detailed Description of the Invention [Industrial Field of Application] The present invention proposes that in a fat decomposition reaction using a fat decomposing enzyme (lipase), if a certain amount of glycerin is maintained in the reaction system, a high degree of fat decomposition can be achieved. This invention relates to a novel enzyme reaction method that maintains the activity of the enzyme while maintaining the efficiency, enables reuse and reuse of the enzyme, and allows the reaction to continue stably for a long period of time.

[Conventional technology and problems]

Although the method of producing glycerin and fatty acids by decomposing fats and oils using lipase has been known for a long time, there are various obstacles to industrialization. The main reason for this is that lipase rapidly deactivates during the reaction and cannot be recovered and reused, so a large amount of expensive enzyme must be used. Therefore, the current situation is that there is no cost advantage compared to high-pressure decomposition methods, and the characteristics of enzymes cannot be utilized. Therefore, in recent years, various methods have been developed to stabilize enzymes and make it possible to recover and reuse them. Considerations have been made. For example, a method of stabilizing an enzyme by immobilizing it on a polysaccharide gel (Japanese Unexamined Patent Application Publication No. 1983-1989)
146284), or a method of adding a sialkylene ether in a monohydric or polyhydric alcohol polio (Japanese Patent Laid-open No. 173080/1983). However, each of these methods has major drawbacks when it comes to industrialization. For example, the method using immobilized lipase requires a large amount of immobilization carrier, and the cost required for this cannot be ignored. Furthermore, it is generally said that immobilization reduces the activity expression rate of the enzyme, and therefore a large amount of enzyme is often required. Alternatively, in the method of adding a polyoxyalkylene ether of a monohydric or polyhydric alcohol, since this additive is present in the reaction product, a step for removing it is necessarily necessary. Furthermore, if this additive cannot be recovered and reused, the cost required for the additive becomes large.

In this way, when considering industrialization, conventional enzyme stabilization methods
It has various disadvantages in terms of process and cost, and it is difficult to find a technology that surpasses the high-pressure decomposition method.

[Means for solving problems]

As a result of intensive studies aimed at overcoming these drawbacks, the present inventors have established a new fat and oil decomposition technology that allows stable reuse of enzymes and is advantageous in terms of cost.

In other words, glycerin, which is a product of fat and oil decomposition reaction, acts very effectively to stabilize lipase, and if the reaction is carried out under conditions where a certain amount or more of glycerin is always present in the reaction system, found that lipase can be stabilized and easily reused.

That is, in the hydrolysis reaction of fats and oils using a fat-degrading enzyme (lipase), an enzyme reaction method is characterized in that the reaction is carried out while always maintaining the glycerin concentration in the aqueous phase present in the reaction system at 10 to 40% by weight. It provides:

Glycerin contributes to the stabilization of enzymes if it is present in a certain amount or more, but on the other hand, if a large amount of glycerin is present, the fat and oil decomposition rate decreases, making it difficult to obtain the desired high fat and oil decomposition rate of 80 to 90% or more. It becomes. This is probably due to the fact that when a large amount of glycerin is present, a reverse reaction to the decomposition reaction expressed by the following reaction formula occurs, or due to a problem in reaction equilibrium.

For this reason, the amount of glycerin present in the reaction system is determined as the concentration of glycerin in the aqueous phase of the reaction system.
If KtK is maintained at 40% by weight, preferably 15 to 30% by weight, and the fat decomposition reaction is carried out,
The present invention was completed based on the discovery that the enzyme is stabilized and can be easily recovered and reused, while the reaction can be carried out while maintaining the hydrolysis rate of fats and oils at a high level.

As a method of keeping the glycerin concentration within the above-mentioned range, when stirring and mixing the substrate fats and oils and water with lipase to react, a 1, for example, 15% by weight aqueous glycerin solution is used instead of water.

As the fat and oil decomposition reaction progresses, glycerin is produced and the glycerin concentration in the aqueous phase increases, so the amount of glycerin aqueous solution added should not be set so that the final glycerin concentration produced after the fat and oil is decomposed falls within the above range. must not. To explain in more detail by giving an example, for example, if a 15% by weight aqueous glycerin solution is used at the initial stage of the reaction and the glycerin concentration in the aqueous phase is maintained within 30% by weight after the reaction is completed,
It is necessary to control the supply ratio and rate of oil/glycerin aqueous solution by the following calculation.

Molecular weight of fats and oils = M, molecular weight of glycerin = 9'2°, amount of fats and oils = Substituting ma = 95%, x/y =
L54 The present invention is characterized in that the feed ratio of oil/fat/glycerin aqueous solution is controlled in this way, and the reaction is carried out by setting the glycerin concentration in the aqueous phase to always be within the above range during the entire reaction process. That is.

In this way, in the method of using glycerin as an enzyme stabilizer, compared to the method of adding polyoxyalkylene ether of monohydric or polyhydric alcohol, since glycerin is a reaction product, it is difficult to remove the reaction product from the end product. There is no need to remove this, which is very advantageous in terms of cost and process.

When reusing the stabilized enzyme by keeping the glycerin concentration in the system constant, the following method is used to recover the enzyme. That is, after the reaction is completed, the reaction system is separated into three layers by standing still or by means such as centrifugation. The three layers are, from the top, an oil layer, an emulsion layer, and an aqueous layer. 80-95% of the enzyme is in this emulsion layer, and 5-20% is in the aqueous layer.
include. Therefore, in order to reuse enzymes,
This emulsion layer is taken out and used for the next enzyme reaction.
In addition, the aqueous layer is treated with an ultra-transient membrane to concentrate and recover the dissolved enzyme, which is then used for the next enzyme reaction. Since it is stabilized by glycerin, the enzyme recovered by K in this way has almost no loss of activity and can be reused for the next reaction. Note that if the glycerin concentration is low, the fat and oil decomposition rate may decrease as the reaction is repeated. In such a case, it is preferable to supplement with a small amount of fresh surface lipase. The replenishment amount is preferably 5 to 20% of the initially added enzyme.

The ultratransient membrane used to recover enzymes from the aqueous layer is
There are no particular limitations on the material or shape as long as it does not allow enzymes to pass through, such as cellulose acetate membrane, polyacrylonitrile membrane, polysulfone membrane, polyamide membrane,
Any material such as a polyimide membrane can be used, and any shape can be used such as a tubular shape, a spiral shape, a hollow fiber shape, or a flat membrane shape. In addition, the molecular weight cutoff of the ultratransient membrane is 3000 to 20000.
It is preferable that the degree of According to the ultra-proper method, the aqueous glycerin solution passes through the ultra-filter membrane and the concentrated enzyme solution remains inside the membrane, so it can be reused for the next enzyme reaction.The concentration ratio of the enzyme solution is 5. The supplement is at least twice that amount, preferably 20 times or more.

Although enzymes can be stably recovered and reused by the methods described above, in the method according to the present invention, enzymes are not deactivated even in reactions at high temperatures and can be easily recovered and reused. Enzymes are generally very unstable to heat. This is also the case with lipases, where a gradual decrease in activity is observed at temperatures exceeding 40°C, and therefore, efforts are being made to develop heat-resistant lipases in various fields. However, it has been found that even if such a heat-stable lipase is not used, and even if a normal lipase, which is easily deactivated at high temperatures, is used, the method of the present invention can suppress the deactivation of the enzyme and allow stable reuse. . That is, the method according to the present invention has a high fat and oil decomposition rate of 80 to 90% even at high temperatures of 45 to SO ℃, and enzymes can also be recovered and reused. Oils and fats that are solid at room temperature, such as palm oil, can be efficiently and easily subjected to KW elementary decomposition. Furthermore, if the reaction can be carried out at a high temperature, the rate of decomposition of fats and oils will be increased, and therefore a large amount of fats and oils can be decomposed within a certain amount of time compared to reactions at low temperatures.

The type of lipase used in the present invention is not particularly limited, but generally includes Candida, Chromobacterium, Aspergillus, Penicillium, Mucor, Geotrichum, Rhizopus, and Arthrobacter.
Lipases sourced from microorganisms such as Hycomyces spp.
Lipase obtained from animal organs such as pancreas, lipase obtained from plant pollen such as castor seeds, etc. can be used.

In addition, these lipase-purified enzyme preparations are not
An unpurified product can also be used, and its purity is not particularly limited.

In addition, the oils and fats used in the present invention include soybean oil, palm oil,
Vegetable oils and fats such as coconut oil, olive oil, linseed oil, castor oil, and tung oil, and animal oils and fats such as beef tallow, lard, and fish oil are exemplified.

As described above, the present invention utilizes the characteristics of lipase to enable recovery and reuse of barse while maintaining a high decomposition rate, and also enables reaction at high temperatures and recovery and reuse. This is a fat and oil decomposition reaction method that has many advantages.

〔Example〕

Examples of the present invention will be described below, but the present invention is not limited to these examples.
Add 200 t of glycerin aqueous solution, and add 0.11 (0.05% of soybean oil) of lipase (320,000 units/f) produced by Cantype Cylindrace.
The reaction was carried out at 30°C (with constant stirring).

After 24 hours, the reaction solution was centrifuged and separated into three layers. Centrifugation was performed at 8000 rpm for 5 minutes.

160 tons was collected from the upper layer (oil phase) and the acid value and saponification value were measured. Acid value = 174, saponification value = 195
was obtained, and the hydrolysis rate was calculated using the formula below, which was 89
．． It was 2%.

The enzyme in the lower layer (aqueous phase) was recovered using a flat membrane type ultra-transient membrane having a molecular weight cutoff of 20,000 and made of polyacrylonitrile.

Processing is carried out at a pressure of 1 b/s2 - 1170 t of lower layer liquid is
It was concentrated to 2F. The glycerin concentration of the membrane permeate is 2.
It was 9% by weight.

A total of 82 tons of the emulsion layer separated in this way and the ultra-superconcentrated lower layer concentrate were added again to 200 f of soybean oil.
Then, a second reaction was carried out by adding 200 tons of 18% by weight aqueous glycerin solution (the reason why the 18% by weight glycerin aqueous solution was used was based on the glycerin concentration in the collected emulsion layer and the aqueous phase of the membrane concentrate (42 tons in total). is 29% by weight, this is to adjust the glycerin concentration of the entire aqueous phase to 20% by weight). The reaction was allowed to proceed for 24 hours while stirring at 30°C. The decomposition rate of fats and oils was 90.1%, and the glycerin concentration in the aqueous layer was 29% by weight.

After 24 hours, using the enzyme recovered by centrifugation and membrane separation in the same manner as above, fresh soybean oil 200%
A third reaction was carried out in the same manner as with t and 200f of a 18% by weight aqueous glycerin solution.

When the comb-reaction reaction was carried out in this way, the oil and fat hydrolysis rates shown in Table 1 were obtained, and the enzyme was not deactivated at all.
The original enzyme could be used as is, and a high decomposition rate could always be maintained. The results are shown in Table 1.Table 1 Example-2 Olive oil was used as the fat, and 200f of olive oil was mixed with 1
Add 5 weight polyglycerin aqueous solution 10 (It, Example-
0.1 f of the same lipase used in 1 was added and reacted at 30°C for 24 hours with stirring.

After 24 hours, centrifugation and ultra-transient membrane separation were performed under the same conditions as in Example 1. The hydrolysis rate of the oil phase was 88.2%. Moreover, the glycerin concentration of the aqueous phase was 33% by weight.

A total of 73 furnaces of concentrate from the emulsion layer and membrane were recovered, to which were added 200 tons of fresh olive oil and 100 tons of an 8 weight glycerin aqueous solution. The glycerin concentration in the aqueous phase of the system is 15% by weight.

After reacting for 24 hours at a temperature of sO<0>C, centrifugation and ultra-transient membrane separation were performed in the same manner. The separated emulsion layer and ultra-filter concentrate were used for the third reaction.

When the reaction was repeated in this manner, the oil and fat hydrolysis rates shown in Table 2 were obtained.

Table 2 Example-3 200 tons of 10% by weight glycerin aqueous solution in 200 tons of soybean oil
f, and to this add 0.1f of the lipase used in Example-1.
was added and reacted at 30°C for 24 hours. After 24 hours, centrifugation and ultra-transient membrane separation were performed under the same conditions as in Example-1. The hydrolysis rate of fats and oils was 92.5%, and the glycerin concentration in the aqueous phase was 19% by weight.

In the same manner as in Example-1, the emulsion layer and membrane concentrate were collected, and 200 r of fresh soybean oil and 200 r of an 8% by weight aqueous glycerin solution were added (to this,
The glycerin concentration in the aqueous phase in the reaction system was 10% by weight), and the reaction was similarly carried out for 24 hours. After 24 hours, the enzyme was recovered by centrifugation and membrane separation in the same manner, and soybean oil and soybean oil were added to it again.
A third reaction was carried out by adding an aqueous glycerin solution. After the reaction was repeated in this manner without any reaction, the hydrolysis rate of the oil and fat was measured, and the results shown in Table 3 were obtained.

Table 3 As shown in Table 3, when the initial concentration of the glycerin aqueous solution was set to 10% by weight, the hydrolysis rate of fats and oils decreased as the number of reactions increased.

Example 4 In Example 3, a decrease in the decomposition rate was observed with each number of reactions, so here the reaction was repeated by adding fresh enzyme little by little to the recovered enzyme solution. That is, the reaction was carried out under the same conditions as in Example 3, and fresh lipase o, o1 was added to the enzyme recovery solution obtained by centrifugation and membrane separation.
r (10% of the amount of charged enzyme) was added, and a second reaction was performed. Similarly, during the third reaction, fresh lipase 0.01F was added and the reaction was repeated.

In this way, the oil and fat hydrolysis rates shown in Table 4 were obtained, assuming that the reaction was repeated.

Table 4 As shown in Table 4, when the initial concentration of the glycerin aqueous solution is low, the activity is reduced, so by repeatedly adding small amounts of the enzyme, it was possible to prevent dryness.0 Also, when the glycerin concentration is low The decomposition rate reached after 24 hours is higher when the glycerin concentration is higher.
and added the same lipase O used in Example-1.
, a t (0.4% based on beef tallow) and heated to 45°C.
The mixture was reacted with stirring for 24 hours. Since the viscosity of beef tallow decreases significantly at 45° C., stirring was possible with a small stirring power. After 24 hours, the reaction solution was centrifuged and separated into three layers. During centrifugation, the hydrolysis rate of the oil phase in the upper layer was measured to be 90.2% using a device capable of keeping the liquid valve at 45° C. at all times. The glycerin concentration in the lower aqueous phase was 29% by weight.

For the lower aqueous phase, the enzyme was concentrated using an ultrafiltration membrane. Here, as an ultra-filter membrane, the molecular weight cutoff is 1
Using 5000 polysulfone membrane, pressure 1go/cab
The operation was performed in 2.

In the same manner as in Example 1, 200 p of shredded beef tallow and 200 f of an 18 wt % aqueous glycerin solution were added to a total of 851 of the emulsion layer and ultrafilter concentrate separated by centrifugation, and 45 The second reaction was carried out for 24 hours at ℃. After the completion of the second reaction, the third reaction was carried out in the same manner as in Example 1, and the reactions were repeated sequentially. As a result, the hydrolysis rate of beef tallow was As shown in Table 5, there was no decrease at all even when the reaction was repeated 5 times.

Thus, it was found that even at a high temperature of 45°C, the enzyme was not deactivated at all in this reaction method and could be reused.

Comparative Example-1 200 fl of soybean oil, 200 fl of water, and 0.1 t of the same lipase used in Example-1 were mixed with stirring and reacted at 30° C. for 24 hours. The reaction solution was separated into three layers by centrifugation under the same conditions as in Example-1.

The hydrolysis rate of soybean oil in the oil phase was 93.0%. Furthermore, the glycerin concentration in the aqueous phase was 9.7%.

The aqueous phase was subjected to ultra-transient membrane treatment in the same manner as in Example-1 to recover the enzyme, which was used together with the emulsion layer in the next reaction. That is, 200v of soybean oil and 200v of water were added to the recovered enzyme solution, and a second reaction was carried out at 30°C for 24 hours. When the mixture was recycled in this manner without any reaction, the results shown in Table 6 were obtained for the soybean oil hydrolysis rate.

Table 6 As shown, in the method of simply reacting water and fat without glycerin, the enzyme activity decreases significantly as the reaction is repeated.

Comparative Example-2 200t of soybean oil, 50% glycerin aqueous solution 2001
The same lipase 0.1f as used in Example-1 was mixed with stirring and reacted at 30°C for 24 hours. After the reaction, centrifugation was performed under the same conditions as in Example-1.

The hydrolysis rate of soybean oil in the oil phase was 66%.

On the other hand, the glycerin concentration in the aqueous phase was 57% by weight.

When an aqueous glycerin solution of as much as 50% by weight is used in this way, the hydrolysis rate of fats and oils is greatly reduced.

Comparative Example-3 200 t of beef tallow, 200 F of water, and the same lipase O, at (0.4% of beef tallow) used in Example-1 were mixed with stirring and reacted at 45° C. for 24 hours. After the reaction, centrifugation was performed under the same conditions as in Example 5 to separate into three layers. The hydrolysis rate of beef tallow in the oil phase was 91.0%, and the glycerin concentration in the aqueous phase was 9.4% by weight. The enzyme was recovered by membrane treatment and used together with the emulsion layer in the next reaction. That is, 200 vol of beef tallow and 200 f of water were added to the recovered enzyme solution, and a second reaction was carried out at 45° C. for 24 hours. When the reaction was repeated in this way, the following results were obtained.

Table 7 As shown above, in a system in which a certain amount of glycerin is not present in a high-temperature reaction at 45°C, the enzyme activity decreases markedly as the reaction recycles.

Patent Application No. 13903/1983 dated March 1, 1985, Patent Application No. 60-13903 (2) Title of Invention: Process for Hydrolyzing Oils and Fats by Lipase 3, Patent Application No. 13903 (Departed from Kaoru Furuya, Procedural Ordinance for the Applicant) Related Patent Applicant (091) Kao Soap Co., Ltd. 4, Agent Nakai Building, 1-3 Nihonbashi Yokoyama-cho, Chuo-ku, Tokyo Column 6 of the detailed description of the invention in the specification, Contents of the amendment (11 Specification, page 19, line 7) Corrected “reaction solution” to “after reaction”

Claims (1)

[Scope of Claims] 1. In the hydrolysis reaction of fats and oils using lipolytic enzymes (lipases), the reaction is carried out while always maintaining the glycerin concentration in the aqueous phase present in the reaction system at 10 to 40% by weight. Enzyme reaction method. 2. After the hydrolysis reaction is completed, the mixture is divided into three layers: an oil layer, an emulsion layer, and an aqueous layer. This emulsion layer is taken out and used again for the next fat and oil decomposition reaction. Concentrate and recover the enzyme dissolved in the
The method according to claim 1, characterized in that this is also used again in the next fat and oil decomposition reaction.