JPS62278987A - Recovery of enzymatic reaction product - Google Patents

Abstract

PURPOSE:To improve the recovery of enzymatic reaction product and shorten the recovery time in the recovery of a reaction product from an enzymatic reaction system, by adjusting the pH of the system after the completion of the enzymatic reaction. CONSTITUTION:An enzyme such as lipase is reacted to a substrate composed of one or more kinds of oil or fat such as fish oil, lard, olive oil, synthetic oil or fat, etc. The reaction time can be remarkably shortened and the yield can be improved by utilizing a buffer solution and adding metallic ion to the reaction system. After the completion of the hydrolysis, the pH of the system is adjusted to 2-5 and the reaction product is recovered e.g. by settling, centrifugal separation, etc. A hydrolyzed oil or fat can be produced in high yield in a short time by this process. The product can be obtained in a short time in high yield by adding an insoluble organic solvent such as hexane to the system after pH adjustment, extracting the product and recovering the solvent layer or by recovering the solvent layer by centrifugal separation and distilling out the solvent.

Description

[Detailed Description of the Invention] 3. Detailed Description of the Invention "Field of Industrial Application" The present invention relates to a method for recovering an enzymatic reaction product produced by allowing an enzyme to act on a substrate. The present invention relates to a method for recovering an enzymatic reaction product, which comprises adjusting.

"Prior Art and Problems" The presence of water is essential in an enzymatic reaction using fats and oils as a substrate and lipase as an enzyme, that is, hydrolysis reactions of fats and oils using lipase. Generally, the reaction is carried out by mixing and stirring or by emulsifying and leaving the emulsion to stand still. However, when using ion-exchanged water or distilled water, the expression of lipase activity is low, so the reaction is carried out using a buffer solution. In addition, a lipase activator such as calcium chloride is added. It is customary to obtain sufficient lipase activity by coexisting pressure ions and carry out the hydrolysis reaction.

In order to recover the target fat/oil hydrolysis reaction product after the hydrolysis reaction is completed, conventional methods include leaving the reaction solution still and recovering the oil layer, or using water-insoluble organic solvents such as hexane, petroleum ether, and petroleum hexane. Methods for recovering oil and fat hydrolysis reaction products by means of centrifugation, centrifugation, and the like are carried out singly or in combination.

In the hydrolysis reaction using lipase, new calcium citrate is produced, for example, when calcium chloride is added to Mcllvaine buffer (made with disodium hydrogen phosphate and citric acid). The oil and fat hydrolysis reaction products are adsorbed and/or incorporated into the salts produced. For this reason, conventional methods for recovering oil and fat hydrolysis reaction products have drawbacks such as prolonged standing time, decreased capacity and extended time in centrifugation, and extended time due to decreased filtration rate in filtration separation. There is. Furthermore, in the conventional method, oil and fat hydrolysis reaction products adsorbed and/or incorporated into newly generated salts such as carnoum citrate are not recovered. In other words, this causes a decrease in the recovery of oil and fat hydrolysis reaction products. This, together with the fact that it takes a long time to separate and recover the oil and fat hydrolysis reaction products, is a major disadvantage in industrial use.

"Means for Solving the Problems" The object of the present invention is to obtain fat and oil hydrolysis reaction products by enzymes, particularly lipases, in a short period of time and in high yield.

While investigating the recovery of fat and oil hydrolysis reaction products using lipase, the present inventors discovered that salt compounds such as calcium citrate, which cause a decrease in recovery rate and an increase in time, caused the pH of the reaction system to decrease. It was discovered that the ionic bonds were broken and disappeared through adjustment, leading to the present invention. We have also found that the amount of adsorption of lipase on reaction products such as fatty acids can be reduced by adjusting the pH of the reaction system.

That is, the present invention hydrolyzes 7S fats and oils by allowing an enzyme such as lipase to act on the whole hydrolysis reaction system or a part of the hydrolysis reaction system before recovering the oil and fat hydrolysis reaction product from the reaction system. The content of the present invention is a method for recovering a fat and oil hydrolysis reaction product, which comprises performing a recovery operation of the oil and fat hydrolysis reaction product after adjusting the pH of the oil and fat hydrolysis reaction product.

In the method for recovering oil and fat hydrolysis reaction products of the present invention, the substrate oils and fats 4 are not particularly limited, but include brown oil, lard, beef tallow, soybean oil,
Animal and vegetable oils such as palm oil and olive oil, oils and fats extracted from microorganisms, synthetic oils, and the like can be used. In addition, in the present invention, lipases that originate from microorganisms such as Diotrichum lotus and Aspargillus, lipases that originate from animals such as pancreas, and lipases that originate from plants such as castor seeds can be used. It can be applied to hydrolysis reactions that can occur. The type of lipase is also not particularly limited. Furthermore, the present invention is applicable to hydrolysis reactions by a static method and hydrolysis reactions by a stirring method.

The presence of water is essential in the hydrolysis reaction of fats and oils using lipase, and ion-exchanged water or a buffer solution is used. The present invention exhibits a particularly remarkable effect when a slow III/EI water is used as water. And the effect of increasing the yield is significant. When metal ions are added using a 1ilii solution, for example, Manoquilpuain (
The effect is particularly great when chloride is added to the Mcllvaine buffer.

In the hydrolysis reaction of fats and oils using lipase, after the reaction is completed, three layers are usually formed: an oil layer containing residual substrate fats, partial glycerides, and fatty acids, an emulsion layer, and an aqueous layer containing lipase and glycerin. In the present invention, the pl-! After adjusting, the oil and fat hydrolysis reaction product can be recovered in a high yield and in a short time by applying conventional methods such as standing still and/or centrifugation.

As the acid that can be used in the present invention, commonly used acids such as hydrochloric acid and sulfuric acid can be used in combination with 1α or N2.

In the present invention, even if the pH is adjusted to the alkaline side, there is almost no effect, or even if there is, the effect is small. After the completion of the hydrolysis reaction, the pH is preferably 6.5 or less, more preferably 2.5 or less.
By collecting the separated oil layer by allowing it to stand after adjusting it to 0 to 5.0, or by collecting it in combination with centrifugation, oils and fats can be removed from the reaction system in a higher yield and in a shorter time than with conventional methods. A hydrolysis reaction product can be obtained. In addition, after adjusting the pH3Pl, a water-insoluble organic solvent such as hexane is added, and the oil and fat hydrolysis reaction product is extracted by stirring for a short time, and then the solvent layer is collected by standing still, or by centrifugation, etc. By collecting the solvent layers and distilling off the solvent, an oil/fat hydrolysis reaction product can be obtained from the reaction system in a higher yield and in a shorter time than in conventional methods.

Is it water-insoluble from the reuse of lipase? As the tj medium, hexane, petroleum ether, petroleum hexane, etc., which do not inactivate the enzyme, are preferable.

The present invention is characterized by adjusting the end of the hydrolysis reaction (7&pt); however, when using lipase dissolved in ion-exchanged water or a buffer solution, the aqueous layer is removed from the reaction system prior to pH adjustment. By adding acids to a portion of the lipase, it is possible to reuse the liquid other than the lipase without affecting the lipase.Also, when using immobilized lipase that has been immobilized by a known method, etc. If there is, it can be reused by removing it from the reaction system together with the aqueous layer (and/or separating the immobilized lipase by filtration, etc.). It is advantageous in terms of cost.

The hydrolysis reaction of fats and oils by lipase has a faster reaction rate at higher temperatures, but is generally 20 to 50°C, and the optimum temperature varies depending on the origin of the lipase, but is usually 30 to 40°C. If a thermostable lipase is used, the reaction can be carried out at higher temperatures. However, there is no temperature limit for increasing the recovery rate and shortening the time by carrying out the present invention, and effective effects are shown at the optimal temperature of the lipase used in the hydrolysis reaction of fats and oils.

In a method for recovering fat and oil hydrolysis reaction products that does not use an organic solvent, the oil and fat hydrolysis reaction products are completely melted and the recovery rate is higher at high temperatures. However, regarding the temperature when recovering the fat and oil hydrolysis reaction product, it is not necessary to be significantly higher than the melting point of the fat and oil hydrolysis reaction product;
In terms of energy, the temperature is preferably higher than the melting point of the fat/oil hydrolysis reaction product to 1 degree plus the melting point. In a method for recovering fat and oil hydrolysis reaction products using a water-insoluble solvent such as hexane, it is necessary to perform the recovery at a temperature below the boiling point of the organic solvent used.

Above, we have described in detail the method for recovering hydrolysis reaction products in high yield and in a short time when hydrolyzing fats and oils using lipase, but the present invention can also be widely used to recover enzymatic reaction products other than lipase. Needless to say, it is.

"Examples" The present invention will be described in more detail below with reference to Examples and Comparative Examples, but the present invention is not limited by these in any way.

In the following Examples and Comparative Examples 1, olive oil was used as the substrate fat, and lipase derived from Candida cylindracea (trade name: 7 Libarase MYj (manufactured by Meito Sangyo)) was used as the lipase. The buffer used was a Mcllvaine buffer with a pH of 7 and an oxygen concentration. The hydrolysis reaction was carried out at 40°C.

Example 1 20 g of olive oil, buffer'/11150 ml, 0.
300m of 2M calcium chloride aqueous solution 5mff with a stopper
The mixture was placed in an Erlenmeyer flask and maintained at the reaction temperature while stirring with a magnetic stirrer. After confirming that the reaction temperature had been reached, 5 ml of lipase solution was added and a hydrolysis reaction was carried out for 60 minutes. Add lipase solution to 5g of lipase powder at pH
The mixture was prepared by adding 100 m7 of Mc11vaineNl buffer solution, keeping it at 40°C, and performing I'A PI' using a magnetic cooler for 30 minutes. After the hydrolysis reaction was completed, the reaction system Using a pH meter, apply 6N until the pH value reaches 12.5.
Hydrochloric acid was added. 100ml of hexane after pH adjustment! was added and surface 1↑ was performed for 3 minutes.

After stirring, the reaction solution was transferred to a separating funnel, and the aqueous layer was removed. Furthermore, the remaining solution was centrifuged at 300 QrpmlO minutes to obtain a hexane layer. Hexane in the obtained hexane layer was distilled off under reduced pressure at 40°C, and oil and fat hydrolysis reaction products were recovered. The results are shown in Table 2.

Incidentally, the recovery rate was expressed by the following formula.

Recovery rate (%) = Example 2 The hydrolysis reaction of fats and oils was carried out according to the method of Example 1. pl (adjustment is carried out for the entire reaction system, and the pH is adjusted to 4.
, 5. Next, the oil and fat hydrolysis reaction products were collected in the same manner as in Example 1, and the recovery rate was determined. The results are shown in Table 2.

Example 3 The hydrolysis reaction of fats and oils was carried out according to the method of Example 1. After one reaction was completed, the reaction solution was transferred to a dispersion funnel and the aqueous layer was removed. Next, 3N hydrochloric acid was added to a portion of the remaining reaction system to adjust the pH to 4.5. After pH adjustment, hexane 100
ml was added to a separatory funnel and the separatory funnel was shaken for 30 seconds. Next, a hexane layer was obtained by centrifugation according to the method of Example 1. Hexane was distilled off under reduced pressure at 40°C to collect the oil and fat hydrolysis reaction product, and the recovery rate was determined. The results are shown in Table 2.

Using the removed lipase-containing aqueous layer (buffer/& layer), the hydrolysis reaction of fats and oils was carried out again according to the method of Example 1. However, prior to the reaction, a 0.16% buffer solution of lipase was added to bring the total volume of the aqueous layer to 155 ml. The fat and oil hydrolysis reaction product was collected as described above.

A differential refractometer was used as a detector, and glyceride and fatty acid compositions of oil and fat hydrolysis reaction products were analyzed by high performance liquid chromatography using a column for gel permeation chromatography.

The results are shown in Table 1 (% by weight).

Table 1 As is clear from the results in Table 1, it was possible to reuse the lipase.

Example 4 In pH adjustment W, pH was adjusted using 6N sodium hydroxide.
The hydrolysis reaction of fats and oils was carried out in accordance with the method of Example 1 except that the concentration was adjusted to 9.0, and the products of the hydrolysis reaction of fats and oils were collected. The results are shown in Table 2.

Example 5 The hydrolysis reaction of fats and oils, pH adjustment, and addition of hegisan were carried out according to the method of Example 1. After adding hexane, the mixture was transferred to a separating funnel and allowed to stand for 2 hours. After standing still, the aqueous layer and emulsion layer were removed to obtain only the hexane layer. 40 hexane
The oil and fat hydrolysis products were recovered by distillation under reduced pressure at °C, and the recovery rate was determined.

The results are shown in Table 2.

Comparative Example 1 The hydrolysis reaction of fats and oils was carried out according to the method of Example 1. After the reaction was completed, 100 m# of hexane was added and stirred for 3 minutes. Hereinafter, the fat and oil hydrolysis reaction product was collected according to the method of Example 1, and the recovery rate was determined. The results are shown in Table 2.

Comparative Example 2 The water decomposition reaction of fats and oils was carried out according to the method of Example 1. After the reaction was completed, 100ml of hexane was added to the tatara and stirred for 3 minutes. After completing the PA stirring, transfer to a separating funnel and add Example 5.
The mixture was allowed to stand for 2 hours according to the method described in . Hereinafter, the fat and oil hydrolysis reaction product was collected according to the method of Example 5, and the recovery rate was determined. The results are shown in Table 2. Compared to Example 5, the recovery rate is low.

Comparative Example 3 Buffer solution 155mi! The hydrolysis reaction of fats and oils was carried out according to the method of Example 1, except that 0.2 M chloride water/8 solution was not added. After the reaction was completed, the fat and oil hydrolysis reaction product was collected according to the method of Comparative Example 2, and the recovery rate was determined. The results are shown in Table 2.

Table 2 "Actions/Effects" According to the method for recovering enzymatic reaction products of the present invention, remarkable effects can be obtained in increasing the recovery rate and shortening the recovery time.

Claims (1)

[Scope of Claims] 1. A method for recovering an enzymatic reaction product, which comprises adjusting the pH after the enzymatic reaction is completed when the enzymatic reaction product is recovered from an enzymatic reaction system in which an enzyme acts on a substrate. 2. The recovery method according to claim 1, wherein the substrate is one or more types of fats and oils. 3. The recovery method according to claim 1 or 2, wherein the enzyme is lipase. 4. Adjusting the pH to acidic Claims 1 and 2
Collection method described in Section 3 or Section 3. 5. The recovery method according to claim 1, 2, 3, or 4, wherein the pH is adjusted to 2.0 to 5.0.