JP6990076B2 - Method for producing fatty acids - Google Patents

Description

The present invention relates to a method for producing fatty acids by an enzymatic decomposition method.

Fatty acids are widely used as intermediate raw materials for foods, additives for various other industrial products, and intermediate raw materials. Such fatty acids are generally produced by hydrolyzing vegetable oils such as rapeseed oil and soybean oil and animal oils such as beef tallow.
As a method for hydrolyzing fats and oils, there are a high-temperature high-pressure decomposition method and an enzymatic decomposition method (for example, Patent Documents 1 and 2). The enzymatic decomposition method uses a fat hydrolase such as lipase as a catalyst, adds water to the fat and oil, and reacts under relatively low temperature conditions, and has the advantage of not producing transunsaturated fatty acids.

When oils and fats are industrially hydrolyzed by the enzymatic decomposition method, an immobilized enzyme in which the enzyme is immobilized on an inorganic or organic immobilized carrier is used in order to use the enzyme efficiently, and this is used for repeated hydrolysis reactions. used.

Japanese Unexamined Patent Publication No. 2000-160188 Japanese Unexamined Patent Publication No. 2007-99959

However, if the hydrolysis reaction of fats and oils is carried out intermittently while reusing the immobilized enzyme, especially at long time intervals, the hydrolysis activity of the enzyme decreases during the reaction suspension period, and the productivity of fatty acids deteriorates. It turned out to be.
Therefore, the present invention suppresses a decrease in the hydrolysis activity of the enzyme during the reaction suspension period even if the immobilized enzyme is reused and the hydrolysis reaction of the fat and oil is carried out at intervals, and fatty acids are produced with high productivity. It seeks to provide a method that can be done.

The present inventor has investigated various causes for the decrease in the hydrolysis activity of the enzyme during the reaction suspension period, and found that the cause is that water remains attached to the immobilized enzyme after the hydrolysis reaction. I found. That is, in the hydrolysis reaction of fats and oils, a large amount of water is supplied into the reaction system in order to promote the equilibrium reaction toward the hydrolysis, so that a large amount of water adheres to the immobilized enzyme after the hydrolysis reaction. The longer this state was and the more water was attached, the lower the hydrolytic activity of the enzyme.
Therefore, as a result of further investigation, a treatment was performed in which the immobilized enzyme used in the hydrolysis reaction was brought into contact with the fat and oil, and the remaining water adhering to the immobilized enzyme was consumed to reduce the water activity of the immobilized enzyme to a predetermined value or less. It was found that if it is reduced, the decrease in the hydrolysis activity of the enzyme during the reaction rest period can be suppressed.

That is, the present invention describes the following steps (A) and (B):
(A) A step of contacting an oil-water mixture having a water content of 10% by mass or more with an immobilized enzyme to hydrolyze fats and oils to obtain fatty acids.
(B) A method for producing fatty acids, which comprises a step of bringing the immobilized enzyme used for the hydrolysis reaction of the fat and oil into contact with the fat and oil to reduce the water activity of the immobilized enzyme to 0.75 or less.

According to the present invention, since the decrease in the hydrolysis activity of the enzyme during the reaction suspension period can be suppressed, the fatty acids can be highly productive even if the hydrolase reaction of the fat and oil is carried out at intervals by reusing the immobilized enzyme. Obtainable.

The method for producing fatty acids of the present invention comprises (A) a step of contacting an oil-water mixture having a water content of 10% by mass or more with an immobilized enzyme to hydrolyze the fat and oil to obtain fatty acids, and (B) the fat and oil. It has a step of bringing the immobilized enzyme used in the hydrolysis reaction into contact with a fat or oil to reduce the water activity of the immobilized enzyme to 0.75 or less.
In the present specification, "fatty acids" may contain fats and oils in addition to fatty acids.
"Fat and oil" is synonymous with "oil", and the substances constituting the fat and oil (oil) include not only triglyceride (TAG) but also monoglyceride (MAG) and diglyceride (DAG). That is, the fat (oil) contains any one or more of monoglyceride, diglyceride and triglyceride.

[Step (A)]
This step is a step of hydrolyzing fats and oils by contacting an oil-water mixture having a water content of 10% by mass or more with an immobilized enzyme to obtain fatty acids. In the present specification, the "oil-water mixture" is a mixture of fat and water to be hydrolyzed, but it may be in a phase-separated state or in an emulsified state.
The fats and oils to be hydrolyzed may be either vegetable fats or oils or animal fats and oils. For example, soybean oil, rapeseed oil, safflower oil, rice oil, corn oil, sunflower oil, cottonseed oil, olive oil, sesame oil, peanut oil, honeybee oil, wheat germ oil, perilla oil, flaxseed oil, sesame oil, chia seed oil, sacha inch oil. , Walnut oil, kiwi seed oil, salvia seed oil, grape seed oil, macadamia nut oil, hazelnut oil, pumpkin seed oil, camellia oil, brown seed oil, borage oil, palm oil, palm olein, palm stea, palm oil, palm kernel Vegetable fats and oils such as oils, cacao fats, monkey fats, shea fats and algae oils; animal fats and oils such as fish oils, lard, beef fats and butter fats; or their ester exchange oils, hydrogenated oils or fractionated oils. Can be mentioned. These fats and oils may be used alone or in combination of two or more.

The fatty acids constituting the fats and oils to be hydrolyzed are not particularly limited and may be either saturated fatty acids or unsaturated fatty acids, but 60 to 100% by mass of the fatty acids constituting the fats and oils are unsaturated fatty acids. It is preferable, more preferably 70 to 99% by mass, further 75 to 97% by mass, and further preferably 80 to 95% by mass as unsaturated fatty acids in terms of appearance and industrial productivity of fats and oils. The unsaturated fatty acid has 14 to 24 carbon atoms, and more preferably 16 to 22 carbon atoms from the viewpoint of physiological effects. The amount of fatty acid in the present specification is a free fatty acid equivalent.

Further, among the fatty acids constituting the fats and oils to be hydrolyzed, the content of saturated fatty acids is preferably 30% by mass or less, more preferably 20% by mass or less, and further 15 in terms of suppressing crystal precipitation at low temperatures. It is more preferably mass% or less, more preferably 10 mass% or less. Further, in terms of the industrial productivity of fats and oils, it is preferably 0.5% by mass or more. As the saturated fatty acid, those having 14 to 24 carbon atoms and further 16 to 22 carbon atoms are preferable.

It is preferable that the fats and oils are extracted from the plants or animals that are the raw materials thereof, and then the solids other than the oils are removed by filtration, centrifugation or the like. Then, it is preferable to add and mix water and, in some cases, an acid, and then degumming by separating the gum component by centrifugation or the like. Further, it is preferable that the fats and oils are deoxidized by adding and mixing an alkali, washing with water and dehydrating. Further, it is preferable that the fats and oils are decolorized by contacting them with an adsorbent such as activated clay and then separating the adsorbent by filtration or the like. Further, deodorization is a process of removing unpleasant odors and taste substances to obtain refined oil having good flavor, hue and storage stability, and removes odorous components while blowing steam under high temperature and reduced pressure conditions. It is preferable to perform steam deodorization. These processes are preferably performed in the above order, but the order may be changed. In addition to this, the fat and oil may be wintered to separate the solid content at a low temperature in order to remove the wax content.

The water may be distilled water, ion-exchanged water, degassed water, tap water, well water or the like. Other water-soluble components such as glycerin may be mixed. If necessary, a buffer solution having a pH of 3 to 9 may be used so that the stability of the enzyme can be maintained.

The water content of the oil-water mixture is 10% by mass or more, but from the viewpoint of industrial productivity, it is preferably 10 to 85% by mass, more preferably 15 to 75% by mass, and further preferably 20 to 65% by mass. In the present invention, the water in the oil-water mixture includes water contained in the fat and oil added into the reaction system in addition to the water added into the reaction system.
As a method of controlling the water content of the oil-water mixture, (1) a method of measuring the water content of the reaction component in advance by the Karl Fischer method or the like and controlling the total water content, and (2) completely controlling the reaction component. There is a method of dehydrating and adding a predetermined amount of water later.

The immobilized enzyme is an immobilized oil / fat hydrolase in which an oil / fat hydrolase is immobilized on a carrier.
Lipase is preferable as the fat hydrolase. The lipase is not particularly limited, and a lipase derived from an animal, a plant, or a microorganism can be used. For example, the genus Rhizopus, the genus Aspergillus, the genus Mucor, the genus Rhizomucor, the genus Pseudomonas, the genus Geotrichum, the genus Geotrichum, the genus Penicilium. Examples include lipases of origin.
Among them, from the viewpoint of hydrolysis efficiency, it is preferable to use a so-called non-selective lipase having no position / chain length selectivity, and further, a non-selective lipase produced by Candida cylindracea is used. Is preferable.

The immobilized carrier includes inorganic carriers such as Celite, diatomaceous earth, kaolinite, silica gel, molecular sieves, porous glass, activated carbon, calcium carbonate, and ceramics, ceramic powder, polyvinyl alcohol, polypropylene, chitosan, ion exchange resin, and hydrophobic adsorption. Examples thereof include organic polymers such as resins, chelate resins, and synthetic adsorption resins. Of these, an ion exchange resin is preferable because of its high water retention capacity. Further, among the ion exchange resins, the porous one is preferable from the viewpoint that the adsorption amount of the enzyme can be increased by having a large surface area.

The particle size of the resin used as the immobilized carrier is preferably 50 to 2000 μm, more preferably 100 to 1000 μm. The pore diameter is preferably 10 to 150 nm, more preferably 10 to 100 nm. Examples of the material include phenol formaldehyde, polystyrene, acrylamide, divinylbenzene and the like, and a phenol formaldehyde resin (for example, Duolite A-568 manufactured by Rohman and Haas) is preferable from the viewpoint of improving enzyme adsorption.
At this time, the amount of the oil-and-fat hydrolase used is preferably 0.1 to 300% by mass, further 0.5 to 200% by mass, and further preferably 1 to 150% by mass with respect to the carrier mass from the viewpoint of industrial productivity. At the time of immobilization, the enzyme is put into a solution state, and it is preferable to adjust the pH to 3 to 7 with a buffer. The temperature at the time of immobilization is preferably 0 to 60 ° C, more preferably 3 to 40 ° C.

In order to enhance the activity of the immobilized enzyme, a treatment of adsorbing a fat-soluble fatty acid or a derivative thereof on a carrier may be performed in advance before immobilizing the fat-and-fat hydrolase. Examples of the method for performing the treatment include a method in which a fat-soluble fatty acid or a derivative thereof is once dispersed and dissolved in an organic solvent such as chloroform, hexane and ethanol, and then added to a carrier dispersed in water.
Examples of the fat-soluble fatty acid used include saturated or unsaturated, linear or branched-chain fatty acids having 8 to 18 carbon atoms, which may be substituted with hydroxyl groups. Specific examples thereof include capric acid, lauric acid, myristic acid, oleic acid, linoleic acid, α-linolenic acid, and ricinoleic acid. Examples of the derivative include an ester of these fatty acids and a monohydric or polyhydric alcohol, a phospholipid, and a derivative obtained by adding ethylene oxide to these esters. Specific examples thereof include methyl esters, ethyl esters, monoglycerides, diglycerides of the above fatty acids, ethylene oxide adducts thereof, polyglycerin esters, sorbitan esters, sucrose esters and the like. Two or more of these fat-soluble fatty acids or derivatives thereof may be used in combination.

The hydrolyzing activity of the fat hydrolase has a titer of 20 U / g (dry mass) or more, further 100 to 10,000 U / g (dry mass), and further 500 to 5000 U / g (dry mass) measured by the method described later. It is preferably in the range.

The amount of the immobilized enzyme used (dry mass) can be appropriately determined in consideration of the activity of the enzyme, but it is industrially used in an amount of 1 to 20% by mass and further 2 to 15% by mass with respect to the fat and oil. It is preferable from the viewpoint of productivity.

Means for contacting the oil-water mixture having a water content of 10% by mass or more with the immobilized enzyme include immersion, stirring, and passing the liquid through a column filled with the immobilized enzyme by a pump or the like. In the case of stirring, although it depends on the diameter of the reaction vessel, 10 to 1000 r / min is preferable, and 20 to 1000 r / min is preferable from the viewpoint that the immobilized enzyme does not settle, crushing is suppressed, and the hydrolysis reaction proceeds efficiently. 700 r / min, more preferably 30 to 500 r / min.

In the present invention, the hydrolysis of fats and oils can be carried out by a batch type, continuous type or semi-continuous type hydrolysis reaction device. The oil and fat and water may be supplied into the hydrolysis reactor by either a parallel flow type or a countercurrent type. As the fat and oil and water supplied to the hydrolysis reaction apparatus, fat and oil and water that have been degassed or deoxidized in advance may be used, if necessary.

The reaction temperature of hydrolysis can be determined by the characteristics of the enzyme, but from the viewpoint of improving the reaction rate and suppressing the inactivation of the enzyme, it is 0 to 80 ° C, further 10 to 70 ° C, and further 20 to 60. ° C is preferred.

The hydrolysis reaction is controlled by the concentration of free fatty acid in the obtained hydrolyzed oil, and can be terminated when a predetermined fatty acid concentration is reached. In the present invention, the hydrolysis reaction of fats and oils is carried out in the range of free fatty acid concentration of 85% by mass or more, further 86 to 98% by mass, and further 91 to 97% by mass from the viewpoint of increasing the free fatty acid concentration and yield. Is preferable.
The free fatty acid concentration is expressed by the following formula from the acid value and fatty acid composition of the oil phase.
Free fatty acid concentration (% by mass) = xxy / 56.11 / 10
(X = acid value of oil phase [mgKOH / g], y = average molecular weight obtained from fatty acid composition)
The acid value of the oil phase can be measured by "acid value (23.1-1996)" in "Standard Oil and Fat Analysis Test Method 2003" edited by the Japan Oil Chemists' Society.

The reaction time of hydrolysis can be appropriately determined in consideration of the amount of immobilized enzyme used and the enzyme activity, but from the viewpoint of industrial productivity, it is 1 to 24 hours, further 2 to 12 hours, and further 2. 5 to 6 hours, more preferably 3 to 4.5 hours.

The hydrolysis reaction is preferably carried out in the presence of an inert gas such as nitrogen so that contact with air is avoided as much as possible.

After the hydrolysis reaction, in order to obtain fatty acids as an oil phase from the reaction solution, the immobilized enzyme and the aqueous phase may be separated from the oil phase by static separation, centrifugation or the like.
The oil phase may be used as it is as fatty acids, or the free fatty acid fraction may be fractionated by a fractionating means such as chromatography, molecular distillation, liquid liquid partitioning, crystal fractionation, deoxidation method or the like.
After separation and recovery, the immobilized enzyme used in the hydrolysis reaction is allowed to act again on the oil-water mixture having a water content of 10% by mass or more and reused for hydrolysis of fats and oils.

[Step (B)]
This step is a step of bringing the immobilized enzyme used for the hydrolysis reaction of the fat and oil into contact with the fat and oil to reduce the water activity of the immobilized enzyme to 0.75 or less.
After the hydrolysis reaction of the fat and oil, the oil and water are separated by filtration or an inert gas blow, and the water content of the recovered immobilized enzyme is usually 15% by mass or more, and the water activity is usually 0.9 or more. be.
In this way, the immobilized enzyme after the hydrolysis reaction of the fat and oil is in a state where a large amount of water is attached, but by contacting the immobilized enzyme with the fat and oil, it remains attached to the immobilized enzyme in the hydrolysis of the fat and oil. The water activity of the immobilized enzyme can be reduced to 0.75 or less by consuming the water. Therefore, the decrease in the hydrolytic activity of the enzyme during the reaction rest period is suppressed.

The water activity of the immobilized enzyme after contact with the fat is 0.75 or less, but from the viewpoint of suppressing the decrease in the hydrolysis activity of the enzyme, it is 0.1 to 0.75, and further 0.2 to 0.65. Further, 0.3 to 0.6 is preferable. The water activity of the immobilized enzyme refers to a value obtained according to the method described in [Analytical Method] (iii) described later.

In this step, the fat and oil is not particularly limited, and may be any of the above-mentioned vegetable fats and oils, animal fats and oils, and the like.
The amount of the fat or oil used in contact with the immobilized enzyme used in the hydrolysis reaction of the fat or oil may be such that the water activity of the immobilized enzyme can be reduced to 0.75 or less. From the viewpoint of water activity reduction efficiency and industrial productivity, the amount of fats and oils used is 1000% by mass or more, further 1250 to 5000% by mass, and further 1500 to 4000% by mass with respect to the initial water content in the reaction system. Further, it is preferably 1750 to 3000% by mass. Here, the initial water content in the reaction system is contained in the fat and oil added into the reaction system in addition to the water remaining attached to the immobilized enzyme used for the hydrolysis reaction of the fat and oil before the step (B). Include water. When the hydrolysis of fats and oils is carried out by a hydrolysis reaction device equipped with an enzyme tower (reaction tower) filled with an immobilized enzyme and a substrate supply tower for supplying fats and oils and water to the enzyme tower, after the hydrolysis reaction, Since water remains in the substrate supply tower and in the supply path between the substrate supply tower and the enzyme tower, the initial water content in the reaction system is the water remaining in the hydrolysis reaction device before the step (B). Also includes. Before step (B), estimate the residual water content in the reaction system such as the residual water content of the immobilized enzyme used for the hydrolysis reaction of the fat and oil, supply the fat and oil accordingly, and contact the immobilized enzyme. By doing so, the water activity of the immobilized enzyme can be reduced to 0.75 or less.

As a means for bringing the immobilized enzyme used for the hydrolysis reaction of the fat and oil into contact with the fat and oil, the same means as described above, for example, dipping, stirring, passing a liquid through a column filled with the immobilized enzyme by a pump or the like, etc. Can be mentioned.
The contact temperature between the immobilized enzyme and the fat and oil may be adjusted to the enzyme characteristics without consuming the residual water in the reaction system and reducing the water activity of the immobilized enzyme and by not inactivating the enzyme. The range of the hydrolysis reaction temperature of the fat and oil is preferable.
The contact between the immobilized enzyme and the fat and oil can be terminated when the concentration of free fatty acid in the obtained partially decomposed oil reaches an equilibrium state. For example, the free fatty acid concentration is 70% by mass or less, further 5 to 60% by mass, further 10 to 50% by mass, and further 15 to 40% by mass. The obtained partially decomposed oil can be used as a raw material for the subsequent hydrolysis reaction.

After the hydrolysis reaction, the longer the state in which a large amount of water adheres to the immobilized enzyme, the more easily the hydrolysis activity of the enzyme decreases. It is preferable to carry out the reaction within 120 hours after the completion of the hydrolysis reaction.

In the present invention, before the immobilized enzyme used for the hydrolysis reaction of the fat and oil is brought into contact with the fat and oil, an inert gas blow that supplies an inert gas such as nitrogen to the immobilized enzyme is performed in advance, and the immobilized enzyme is as much as possible. It is preferable to reduce the amount of water adhering to the water.

The immobilized enzyme after the step (B) can be reused for the subsequent hydrolysis reaction.
In the present invention, after an interval of 24 hours or more, 72 hours or more, and 120 hours or more after the completion of the hydrolysis reaction, the immobilized enzyme is reused to start the hydrolysis reaction of fats and oils. In addition, the effect of the present invention is more effectively exhibited.
The residual activity rate of the immobilized enzyme when the immobilized enzyme is reused to initiate the hydrolysis reaction of fats and oils is preferably high from the viewpoint of fatty acid productivity, more preferably 80% or more, and further 85 to 85. 99%, more preferably 90-98%. The residual activity rate of the immobilized enzyme refers to a value obtained according to the method described in [Analytical method] (vi) described later.
The number of times the immobilized enzyme is reused varies depending on the enzyme activity, but from the viewpoint of efficient use of the enzyme, it is preferably once or more, further 2 times or more, further 5 times or more, and further 10 times or more. ..

[Analysis method]
(I) Measurement of acid value (AV) The acid value (AV) was measured according to "Acid value (23.1-1996)" in "Standard Oil and Fat Analysis Test Method 2003" edited by Japan Oil Chemists' Society.

(Ii) Calculation of free fatty acid concentration The free fatty acid concentration of the fatty acid obtained by hydrolyzing fats and oils was determined by the following formula (1). The average fatty acid molecular weight of flaxseed oil was 280.
Free fatty acid concentration [% by mass] = acid value of hydrolyzed oil (AV) / average molecular weight of fatty acid of flaxseed oil / 56.11 / 10 ... (1)

(Iii) Measurement of water activity The water activity of the immobilized enzyme was measured using a water activity precision measuring device LabMaster-aw.

(Iv) Measurement of dry mass ratio of immobilized enzyme After washing with hexane and acetone 10 times by mass alternately with hexane and acetone 10 times by mass with respect to a mass part of the immobilized enzyme to which oil and water are attached, leave it at 70 ° C. for 15 hours. By doing so, the solvent was removed and the mass of the immobilized enzyme alone was weighed (b part by mass). The dry mass ratio of the immobilized enzyme was determined by the following formula (2).
Dry mass ratio of immobilized enzyme = b / a × 100 [mass%] ... (2)
(A: mass of immobilized enzyme with oil and water attached, b: mass of immobilized enzyme)

(V) Measurement of hydrolysis activity (expression activity) Add 5 g of immobilized enzyme and 50 g of rapeseed oil to a 100 mL three-necked flask, and stir with a crescent blade (width 50 mm x height 20 mm) at 430 r / min in a water bath. Was heated to 40 ° C. 30 g of distilled water was added thereto to start the reaction. Sampling was performed at the time of passage, oil and water were separated by centrifugation (3000 r / min, 1 minute), and then the acid value of the oil phase was measured. The time required for the acid value to reach 175 mgKOH / g was determined, and the hydrolysis activity was determined from the following formula (3).
Hydrolytic activity [U / g (dry mass)]
= (Acid value 175 arrival time [hr] /523.12) ^ (-1 / 1.0919)
× Canola oil [g] / (immobilized enzyme [g] × dry mass ratio [-]) ・ ・ ・ ・ ・ (3)

(Vi) Calculation of residual activity rate The residual activity rate of the immobilized enzyme was determined by the following formula (4).
Residual activity rate [%] = Hydrolytic activity after storage [U / g (dry mass)] / Hydrolytic activity after step (B) [U / g (dry mass)] × 100 ... (4)

[Preparation of immobilized enzyme]
Anion exchange resin Duolite A-568 (manufactured by Rohman and Haas, 96% by mass of particles having a particle size distribution of 150 to 850 μm) is crushed and classified, and 1 part by mass of a resin having 97% by mass of particles having a particle size of 150 to 425 μm. Was stirred in 10 parts by mass of N / 10 NaOH solution for 1 hour. After filtration, the mixture was washed with 10 parts by mass of ion-exchanged water, and pH was equilibrated with 10 parts by mass of 500 mM phosphate buffer (pH 7). After filtration, pH equilibration was performed twice for 2 hours with 10 parts by mass of 50 mM phosphate buffer (pH 7). After that, filtration was performed, the carrier was recovered, and then ethanol substitution was performed with 4 parts by mass of ethanol for 30 minutes. After filtration, 4.22 parts by mass of ethanol containing 0.58 parts by mass of rapeseed fatty acid was added, and soybean fatty acid was adsorbed on the carrier for 30 minutes. After recovering the carrier by filtration, the carrier was washed 4 times with 5 parts by mass of 50 mM phosphate buffer (pH 7) for 30 minutes each, and the carrier was recovered by filtration.
Next, 0.029 parts by mass of a commercially available lipase AY "Amano" (manufactured by Amano Enzyme) derived from the genus Candida cylindracea was contacted with an enzyme solution dissolved in 18 parts by mass of 50 mM phosphate buffer (pH 7) for 2 hours. , Immobilization was performed. After filtration, the immobilized enzyme was recovered and washed with 5 parts by mass of 50 mM phosphate buffer (pH 7) to remove the non-immobilized enzyme and protein. All of the above operations were performed at 20 ° C. Then, 4 parts by mass of rapeseed oil was added, and the mixture was stirred at 40 ° C. for 2 hours. After filtration, 4 parts by mass of rapeseed oil was added and stirred while reducing the pressure at 40 ° C., and when the degree of vacuum reached 750 Pa-abs, the pressure was returned to normal pressure, and then the mixture was filtered and separated from fats and oils to obtain an immobilized enzyme.
The hydrolytic activity of the immobilized enzyme was 3621 U / g (dry mass).

[Step (A)]
87.8 g of the immobilized enzyme was weighed by dry weight and charged into a 5 L volumetric flask. 2000 g of flaxseed oil and 1200 g of distilled water were added thereto. The water content of the oil-water mixture was 37.5% by mass. The hydrolysis reaction was carried out at 40 ° C. with stirring under a nitrogen stream. When the free fatty acid concentration reached 93% by mass, the oil and water were separated by centrifugation to obtain fatty acids as an oil phase, and the immobilized enzyme was recovered by filtration.
The water content of the recovered immobilized enzyme was 16.5% by mass, the water activity was 0.94, and the hydrolysis activity was 3585 U / g (dry mass).

[Comparative Example 1]
The immobilized enzyme recovered in the step (A) was stored at 50 ° C. for 136 hours, and then the hydrolysis activity was measured. The hydrolysis activity was 2549 U / g (dry mass), and the residual activity rate was 71.1%.

[Example 1]
[Step (B)]
To a 500 mL four-necked flask, 20 g of the immobilized enzyme recovered in the step (A) was weighed by dry weight, and 279 g of flaxseed oil was added. The amount of flaxseed oil used was 2892% by mass of the initial water content in the reaction system. The mixture was stirred at 40 ° C., the immobilized enzyme and flaxseed oil were brought into contact with each other until the free fatty acid concentration became equilibrium, and then filtered to recover the immobilized enzyme.
The recovered immobilized enzyme had a water activity of 0.46 and a hydrolysis activity of 3641 U / g (dry mass).

The immobilized enzyme recovered in the step (B) was stored at 50 ° C. for 136 hours, and then the hydrolysis activity was measured. The hydrolysis activity was 3383 U / g (dry mass), and the residual activity rate was 92.9%.

[Example 2]
[Step (B)]
To a 500 mL four-necked flask, 20 g of the immobilized enzyme recovered in the step (A) was weighed by dry weight, and 172 g of flaxseed oil was added. The amount of flaxseed oil used was 1786% by mass of the initial water content in the reaction system. The mixture was stirred at 40 ° C., the immobilized enzyme and flaxseed oil were brought into contact with each other until the free fatty acid concentration became equilibrium, and then filtered to recover the immobilized enzyme. The recovered immobilized enzyme had a water activity of 0.62 and a hydrolysis activity of 3550 U / g (dry mass).

The immobilized enzyme recovered in the step (B) was stored at 50 ° C. for 136 hours, and then the hydrolysis activity was measured. The hydrolysis activity was 3096 U / g (dry mass), and the residual activity rate was 87.2%.

[Example 3]
[Step (B)]
To a 500 mL four-necked flask, 20 g of the immobilized enzyme recovered in the step (A) was weighed by dry weight, and 118 g of flaxseed oil was added. The amount of flaxseed oil used was 1225% by mass of the initial water content in the reaction system. The mixture was stirred at 40 ° C., the immobilized enzyme and flaxseed oil were brought into contact with each other until the free fatty acid concentration became equilibrium, and then filtered to recover the immobilized enzyme. The recovered immobilized enzyme had a water activity of 0.71 and a hydrolysis activity of 3564 U / g (dry mass).

The immobilized enzyme recovered in the step (B) was stored at 50 ° C. for 136 hours, and then the hydrolysis activity was measured. The hydrolysis activity was 2984 U / g (dry mass), and the residual activity rate was 83.7%.

The results of Examples and Comparative Examples are shown in Table 1.

As is clear from Table 1, where the hydrolase activity of the enzyme decreases during the reaction rest period (Comparative Example 1), as in Examples 1 to 3, the immobilized enzyme after use and the fat and oil are contacted and fixed. By reducing the water activity of the enzyme to a predetermined value or less, the decrease in the hydrolase activity of the enzyme was suppressed. As a result, in the hydrolysis reaction of fats and oils that reuse the immobilized enzyme, fatty acids can be obtained with good productivity even if there is an interval until the next reaction.

Claims (4)

Next steps (A) , (B) and (C) :
(A) A step of contacting an oil-water mixture having a water content of 10% by mass or more with an immobilized enzyme to hydrolyze fats and oils to obtain fatty acids.
(B) The immobilized enzyme used for the hydrolysis reaction of the fat and oil is brought into contact with the fat and oil of 1000% by mass or more with respect to the initial water content in the reaction system, and the water activity of the immobilized enzyme is reduced to 0.75 or less. The process of lowering,
(C) After the step (B), a step of hydrolyzing fats and oils using an immobilized enzyme having a water activity reduced to 0.75 or less to obtain fatty acids.
A method for producing fatty acids, including. The method for producing a fatty acid according to claim 1, wherein the amount of the fat and oil used in the step (B) is 1250 to 5000% by mass with respect to the initial water content in the reaction system. The method for producing a fatty acid according to claim 1 or 2, further comprising a step of blowing an inert gas against the immobilized enzyme used for the hydrolysis reaction of fats and oils before the step (B). The method for producing a fatty acid according to any one of claims 1 to 3, wherein in the step (B), the fat or oil is brought into contact with the immobilized enzyme within 120 hours after the completion of the hydrolysis reaction of the fat or oil.