JP7365202B2 - Method for producing fats and oils with high diacylglycerol content - Google Patents

Description

The present invention relates to a method for producing fats and oils with high diacylglycerol content.

It is known that fats and oils containing diacylglycerol in high concentrations have physiological effects such as suppressing the increase in blood triglycerides (neutral fats) after meals and having a low tendency to accumulate in the body (for example, Patent Document 1) reference).
Diacylglycerol is generally produced by a glycerolysis reaction between glycerin and fat or oil, or an esterification reaction between glycerin and a fatty acid (see, for example, Patent Documents 2 to 5). These manufacturing methods are broadly divided into chemical methods using alkaline catalysts, etc., and enzymatic methods using enzymes such as lipase, but it is preferable to carry out the reaction under mild conditions using enzymes in terms of flavor etc. It is said that

Japanese Patent Application Publication No. 10-176181 Special Publication No. 6-65310 Special Publication No. 6-65311 Japanese Patent Application Publication No. 4-330289 Special Publication No. 2004-528843

When carrying out the esterification reaction between glycerin and fatty acids, it is a common practice to efficiently proceed with the reaction by creating a vacuum in the reaction system, removing generated water, and keeping the pressure below a certain level. For convenience, the vacuum conditions are generally kept constant during operation, and the desired quality can be obtained at a relatively high reaction rate.
However, when performing an esterification reaction between glycerin and fatty acids using an immobilized enzyme as a catalyst while maintaining a constant degree of vacuum, high vacuum conditions are used to improve diacylglycerol purity and reaction yield, which may reduce the durability of the enzyme. It was found that the results were not necessarily satisfactory, and there were problems from an industrial perspective.
Therefore, an object of the present invention is to provide an industrially advantageous method for producing highly purified diacylglycerol in high yield.

After intensive study in view of the above-mentioned problems, the inventors of the present invention found that by carrying out an enzyme esterification reaction under a predetermined pressure and under conditions where the water content of the oil phase and the pressure satisfy the formula (i) described later, It has been found that the esterification reaction can be promoted while suppressing the excessive decrease in the water content of the enzyme, suppressing the decrease in enzyme activity, and producing highly pure diacylglycerol in high yield.

That is, the present invention includes a step of esterifying glycerin and a fatty acid or a lower alkyl ester thereof under a pressure of 200 to 1200 Pa using an immobilized enzyme, and throughout the step, the oil phase moisture (x) The relationship between and pressure (y) is expressed by the following formula (i);
(i) 10000x+100≧y≧2500x-300
(However, x≦0.6, 200≦y≦1200)
(Here, x indicates oil phase moisture (mass%) and y indicates pressure (Pa).)
The present invention provides a method for producing fats and oils with a high diacylglycerol content, which satisfies the following.

According to the present invention, fats and oils with high diacylglycerol purity can be industrially advantageously produced in high yield.

The method for producing fats and oils with high diacylglycerol content of the present invention includes a step of esterifying glycerin and a fatty acid or a lower alkyl ester thereof under a pressure of 200 to 1200 Pa using an immobilized enzyme, and during this step, , the relationship between oil phase moisture (x) and pressure (y) is expressed by the following equation (i);
(i) 10000x+100≧y≧2500x-300
(However, x≦0.6, 200≦y≦1200)
(Here, x indicates oil phase moisture (mass%) and y indicates pressure (Pa).)
This is a manufacturing method that satisfies the following.
Here, the oil phase is a fatty acid, a lower alkyl ester thereof, and an oil or fat, and the "oil phase moisture" is moisture contained in the oil phase.
Furthermore, in this specification, "oil" is synonymous with "oil", and substances constituting fats and oils (oil) include not only triacylglycerol but also monoacylglycerol and diacylglycerol. That is, the fat (oil) contains one or more of monoacylglycerol, diacylglycerol, and triacylglycerol.

[Immobilized enzyme]
The immobilized enzyme used in the present invention is preferably an immobilized lipase, and the lipase is not particularly limited, and lipases derived from animals, plants, and microorganisms can be used. For example, Rhizopus spp., Aspergillus spp., Mucor spp., Rhizomucor spp., Pseudomonas spp., Geotrichum spp., Penicillium spp. ) genus, Candida genus Examples include lipases originating from.
The type of immobilized lipase is not particularly limited, and a lipase selective for positions 1 and 3 showing specificity for the sn-1 and sn-3 positions of glycerol, a non-position-specific (random type) lipase, etc. may be used. I can do it. Among these, 1,3-position selective lipase is preferred from the viewpoint of reactivity. Examples of commercially available immobilized 1,3 position selective lipases include Lipozyme RM IM (manufactured by Novozyme Japan).

Immobilization carriers include celite, diatomaceous earth, kaolinite, silica gel, molecular sieves, porous glass, activated carbon, calcium carbonate, inorganic carriers such as ceramics, ceramic powder, polyvinyl alcohol, polypropylene, chitosan, ion exchange resin, hydrophobic adsorption. Examples include organic polymers such as resins, chelate resins, and synthetic adsorption resins.
The shape of the immobilization carrier is not particularly limited, and examples thereof include particles, powder, granules, fibers, sponges, and the like.
Among these, ion exchange resins are preferred from the viewpoint of production efficiency, high affinity with fatty acids, and high water retention capacity. Further, among ion exchange resins, porous resins are preferable because they have a large surface area and can increase the amount of enzyme adsorption.

The particle diameter of the resin used as the immobilization 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-based, polystyrene-based, acrylamide-based, and divinylbenzene-based resins, and phenol-formaldehyde-based resins (eg, Duolite A-568 manufactured by Dow Chemical Company) are preferred from the standpoint of improving enzyme adsorption properties.
At this time, the amount of enzyme used is preferably 10 to 300% by weight, more preferably 20 to 250% by weight, and further preferably 30 to 200% by weight based on the weight of the carrier. During immobilization, the enzyme is put into a solution state, and it is preferable to adjust the pH of the buffer to 5 to 7 depending on the properties of the enzyme. The temperature during immobilization is preferably 0 to 60°C, more preferably 5 to 40°C.

In order to enhance the activity of the immobilized enzyme, a treatment may be performed to adsorb fat-soluble fatty acids or derivatives thereof onto the carrier before immobilizing the enzyme. 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, or ethanol, and then added to a carrier dispersed in water.
The fat-soluble fatty acids to be used include saturated or unsaturated, straight-chain or branched fatty acids having 8 to 18 carbon atoms, which may be substituted with hydroxyl groups. Specific examples include capric acid, lauric acid, myristic acid, oleic acid, linoleic acid, α-linolenic acid, and ricinoleic acid. Examples of the derivatives include esters of these fatty acids and monohydric or polyhydric alcohols, phospholipids, and derivatives obtained by adding ethylene oxide to these esters. Specific examples include methyl esters, ethyl esters, monoglycerides, diglycerides, ethylene oxide adducts thereof, polyglycerin esters, sorbitan esters, sucrose esters, etc. of the above-mentioned fatty acids. Two or more of these fat-soluble fatty acids or derivatives thereof may be used in combination.

[Fatty acid or its lower alkyl ester]
The fatty acid or lower alkyl ester thereof used in the present invention is preferably a linear or branched saturated or unsaturated fatty acid having 4 to 22 carbon atoms, preferably 8 to 18 carbon atoms, such as butyric acid, valeric acid, caproic acid. , enanthic acid, caprylic acid, pelargonic acid, capric acid, undecanoic acid, lauric acid, myristic acid, palmitic acid, zomaric acid, stearic acid, oleic acid, elaidic acid, linoleic acid, linolenic acid, arachidonic acid, gadolenic acid, arachine Acids such as behenic acid, erucic acid, eicosapentaenoic acid, docosahexaenoic acid, etc. can be used. Further, examples of lower alcohols that form esters with the fatty acids include those having 1 to 6 carbon atoms, such as methanol, ethanol, 1-propanol, 2-propanol, n-butanol, 2-butanol, and t-butanol. . Two or more types of these fatty acids or lower alkyl esters thereof can also be used in combination.

In the present invention, fatty acids obtained by hydrolyzing raw material fats and oils may be used.
Here, the raw material fat to be hydrolyzed may be either vegetable oil or animal fat. For example, soybean oil, rapeseed oil, safflower oil, rice oil, corn oil, sunflower oil, cottonseed oil, olive oil, sesame oil, peanut oil, pearl barley oil, wheat germ oil, perilla oil, linseed oil, perilla oil, sacha inchi oil, walnut oil. , kiwi seed oil, salvia seed oil, grape seed oil, macadamia nut oil, hazelnut oil, pumpkin seed oil, camellia oil, tea seed oil, borage oil, palm oil, palm olein, palm stearin, coconut oil, palm kernel oil, cacao. vegetable oils such as fat, monkey fat, shea butter, and algae oil; animal fats and oils such as fish oil, seal oil, lard, beef tallow, and butter fat; or their transesterified oils, hydrogenated oils, fractionated oils, etc. can be mentioned. These may be used alone or in combination of two or more.
The raw material oil is preferably an oil with a low content of 30% by mass or less of saturated fatty acids among the fatty acids constituting the oil.
Examples of methods for hydrolyzing fats and oils include high temperature and high pressure decomposition methods and enzymatic decomposition methods.
The high temperature and high pressure decomposition method is a method for obtaining fatty acids and glycerin by adding water to fats and oils and reacting at high temperature and high pressure conditions. Furthermore, the enzymatic decomposition method is a method in which fatty acids and glycerin are obtained by adding water to fats and oils and reacting at low temperatures using fat hydrolase as a catalyst.

As the oil-fat hydrolase, lipase is preferable, and there are no particular limitations, and the above-mentioned animal-derived, plant-derived, and microbial-derived lipases can be used. Among them, from the viewpoint of hydrolysis efficiency, it is preferable to use a so-called non-selective lipase that does not have positional or chain length selectivity, and it is more preferable to use a non-selective lipase produced by Candida cylindracea. is preferred. For example, there is lipase AY "Amano" 30SD-K (manufactured by Amano Enzyme Co., Ltd.).

The hydrolysis reaction can be carried out according to a conventional method.
The hydrolysis reaction may be controlled by the free fatty acid concentration expressed by the following formula (1), and may be terminated when a predetermined decomposition rate is reached.
Free fatty acid concentration (%) = acid value (AV) of hydrolyzed oil × (average fatty acid molecular weight of raw material oil/56.1/10) (1)
The free fatty acid concentration of the fatty acid obtained by hydrolyzing fats and oils is preferably 80% by mass or more, more preferably 84% by mass or more, further 88% by mass or more, and even more preferably 92% by mass or more, from the viewpoint of suppressing the transfer reaction of diacylglycerol. .
To obtain fatty acids from the reaction solution after hydrolysis, the fat/oil hydrolase and the aqueous phase may be separated from the oil phase by static separation, centrifugation, or the like.
In addition to fatty acids, hydrolyzed oils include unreacted fats and fats and partially hydrolyzed fats and oils, but they can be used as they are as fatty acids as raw materials for esterification reactions, and can be purified by distillation, wintering, etc. It may also be used after adjusting the fatty acid composition.

[Glycerin]
The glycerin used in the present invention preferably has a purity of 95% by mass or more from the viewpoint of esterification reactivity.

[Raw material preparation ratio]
The ratio of moles of fatty acid to moles of glycerin [FA/GLY] when carrying out the esterification reaction is determined by the point at which the composition of the reaction oil is optimal (the residual amount of fatty acids, etc. and glycerin in the reaction oil, and the amount of monoacylglycerol or 5.0 or less, further 4.0 or less, further 3.0 or less, further 2 It is preferable to set it to .5 or less, and from the viewpoint of improving the reaction rate and distillation residue ratio, it is preferable to set it to 0.5 or more, further 1.0 or more, further 1.2 or more, and even 1.5 or more. preferable.
The ratio of moles of fatty acid to moles of glycerin [FA/GLY] is expressed by the following formula (2).
FA/GLY = (moles of free fatty acids + moles of monoacylglycerols + moles of diacylglycerols x 2 + moles of triacylglycerols x 3) / (moles of glycerin + moles of monoacylglycerols + moles of diacylglycerols + moles of triacylglycerols) moles)...(2)

[Esterification reaction]
The present invention includes a step of esterifying glycerin and a fatty acid or a lower alkyl ester thereof under a pressure of 200 to 1200 Pa, and the relationship between oil phase moisture (x) and pressure (y) is expressed by the following formula (i):
(i) 10000x+100≧y≧2500x-300
(However, x≦0.6, 200≦y≦1200)
(Here, x indicates oil phase moisture (mass%) and y indicates pressure (Pa).)
Operate to satisfy. The esterification reaction progresses by removing reaction product water from the reaction system, but in the present invention, reaction product water is removed under reduced pressure. However, when using an immobilized enzyme as an esterification catalyst, if too much water is removed from the reaction system, the water content in the immobilized enzyme will decrease, resulting in a decrease in enzyme activity, resulting in high diacylglycerol purity. It was found that it became impossible to obtain fats and oils with a high reaction yield.
In the present invention, the enzymatic esterification reaction is carried out when the oil phase moisture in the reaction system is 0.6% by mass or less and at a pressure of 1200 Pa or less and a degree of vacuum of 200 Pa or more while adjusting the pressure with respect to the oil phase moisture. By promoting the esterification reaction, suppressing an excessive decrease in the amount of water in the immobilized enzyme, and suppressing a decrease in enzyme activity, fats and oils with high diacylglycerol purity can be obtained at a high reaction yield. Furthermore, as a result of being able to suppress a decrease in enzyme activity, the immobilized enzyme is also excellent in reusability. Note that when the oil phase water content of the reaction system exceeds 0.6% by mass, the pressure does not need to be 1200 Pa or less, but it is preferably 200 Pa or more.
Hereinafter, the step of esterifying glycerin and a fatty acid or a lower alkyl ester thereof under a pressure of 200 to 1200 Pa using an immobilized enzyme will also be referred to as an "esterification step."
In the present invention, the relationship between oil phase moisture (x) and pressure (y) during the esterification process satisfies the above formula (i), but the esterification reaction is promoted while suppressing the decrease in enzyme activity, and the diacyl From the point of view of obtaining fats and oils with high glycerol purity at a high reaction yield, the following formula (ii);
(ii) 10000x-100≧y≧2500x-175
(However, x≦0.55, 200≦y≦1200)
It is preferable to satisfy the following formula (iii);
(iii) 10000x-300≧y≧2500x-75
(However, x≦0.51, 200≦y≦1200)
It is more preferable to satisfy the following.

In the present invention, in the esterification step, when the oil phase moisture is 0.6% by mass or less, the pressure remains constant as long as the relationship between the oil phase moisture (x) and the pressure (y) satisfies formula (i). However, by promoting the esterification reaction and suppressing the decrease in enzyme activity by suppressing an excessive decrease in the amount of water in the immobilized enzyme, and further suppressing the transfer reaction, the purity of diacylglycerol can be improved. In order to obtain fats and oils with a high reaction yield, it is preferable to carry out the esterification reaction while changing the pressure, and it is more preferable to change the pressure so that it decreases as the water content of the oil phase decreases.
The pressure is preferably changed from a low degree of vacuum to a high degree of vacuum. Specifically, the esterification reaction may be carried out under a pressure of 200 to 1200 Pa, and the degree of vacuum may be adjusted to fall within the range of formula (i) based on the water content of the oil phase. Further, the degree of vacuum may be changed continuously so as to fall within the range of the above formula (i), or may be changed stepwise. However, since the water content is high in the early stage of the reaction, if the water content in the oil phase is more than 0.6% by mass, the reaction may be carried out at a pressure of more than 1200 Pa. Afterwards, as the reaction progresses, the water content in the oil phase increases. When the pressure becomes 0.6% by mass or less, the pressure is adjusted to 1200 Pa or less. For example, when the water content in the oil phase reaches 0.3% by mass, it is preferable to increase the degree of vacuum and adjust the pressure to less than 1200 Pa and 450 Pa or more, more preferably less than 1200 Pa and 575 Pa or more. , more preferably less than 1200 Pa and 675 Pa or more. When the reaction further progresses and the water content in the oil phase becomes 0.2% by mass or less, it is preferable to increase the degree of vacuum and adjust the pressure to a range of less than 1200 Pa and 200 Pa or more, and less than 1200 Pa and 325 Pa or more. It is more preferable to adjust the pressure between 1,200 Pa and 425 Pa or more. When the reaction further progresses and the water content in the oil phase becomes 0.1% by mass or less, it is preferable to further increase the degree of vacuum and adjust the pressure between 1100 Pa and 200 Pa or more, and 900 Pa and 200 Pa or more. It is more preferable to adjust the pressure between 700 Pa and 200 Pa or more.
In addition, if you understand the relationship between the change in oil phase moisture with respect to pressure and reaction time in the first esterification reaction, when using the immobilized enzyme repeatedly, the change in oil phase moisture with respect to pressure will change over reaction time. Therefore, moisture measurement can be omitted.

In addition, in the esterification reaction of the present invention, from the viewpoint of obtaining fats and oils with high diacylglycerol purity at a high reaction yield, in addition to reducing the pressure, as a method for removing the water produced by the reaction, for example, an absorbent such as zeolite or molecular sieves can be used. Methods such as the use of gas, ventilation of dry inert gas into the reaction tank, etc. may be used in combination.

In the esterification step, the oil phase moisture x is 0.25% by mass or more, preferably 0.3 to 0.6% by mass, and the esterification step is carried out for 20 minutes or more, preferably 60 The esterification reaction is carried out for at least 1 minute, and then the esterification reaction is carried out while removing the reaction product water from the reaction system to lower the oil phase moisture, so that the oil phase moisture x is less than 0.25% by mass, preferably 0. It is preferable to carry out the esterification reaction at .2% by mass or less for 15 minutes or more, preferably 60 minutes or more.

The amount of immobilized enzyme used in the esterification reaction can be determined appropriately taking into account the activity of the enzyme, but from the viewpoint of improving the reaction rate, the amount of immobilized enzyme used in the esterification reaction is On the other hand, 1 to 30% by mass, more preferably 2 to 20% by mass.

Examples of methods for contacting the immobilized enzyme with the raw material (glycerin and fatty acid or lower alkyl ester thereof) include immersion, stirring, and passing the solution through a column packed with the immobilized enzyme using a pump or the like. When stirring, the stirring speed is preferably 10 to 1000 r/min, more preferably 50 to 700 r/min, and even more preferably 100 to 600 r/min, from the viewpoint of production efficiency and inhibition of enzyme crushing.

The reaction temperature of the esterification reaction is preferably 20 to 80°C, more preferably 30 to 70°C, from the viewpoint of reactivity. In addition, from the viewpoint of suppressing the transfer reaction to triacylglycerol and industrial productivity, the reaction time is preferably within 10 hours, more preferably 0.1 to 8 hours, and even more preferably 0.5 to 5 hours.

The water content of the immobilized enzyme in the reaction system is determined by setting the water content of the oil phase to more than 0.6% by mass in order to promote the esterification reaction while suppressing a decrease in enzyme activity and obtain a high yield of high-purity diacylglycerol. When the water content is preferably 2.5 to 10% by mass, more preferably 3.0 to 8% by mass, and when the oil phase water content is 0.6% by mass or less, it is 1.5 to 7% by mass. It is preferably 2.0 to 6% by mass, more preferably 2.0 to 6% by mass. In addition, the water content of the immobilized enzyme at the end of the esterification reaction is 1.0 to 3.0% by mass, since the reaction yield of high-purity diacylglycerol can be increased when the immobilized enzyme is reused. Further, 1.2 to 2.5% by mass is preferable.

In addition to the esterification step, the present invention includes a step of esterifying glycerin and a fatty acid or a lower alkyl ester thereof under a pressure of more than 1200 Pa in consideration of equipment load and industrial efficiency. Good too.

In this way, fats and oils with high diacylglycerol purity can be obtained with a high reaction yield. In addition, the immobilized enzyme used in the esterification reaction maintains high enzyme activity, so it can be reused in subsequent esterification reactions. Although the number of times the immobilized enzyme is reused in the subsequent esterification reaction varies depending on the enzyme activity, it is preferably one or more times, further two or more times, further five or more times, and further still ten times or more.
In the diacylglycerol-rich oil and fat of the present invention, the purity of diacylglycerol is preferably 80% by mass or more, more preferably 85 to 99.5%, further 90 to 99%, and even 90 to 98%. Preferable from the viewpoint of effectiveness and industrial productivity. Here, the diacylglycerol purity is [diacylglycerol/(diacylglycerol+triacylglycerol)×100].

Furthermore, in the diacylglycerol-rich oil and fat of the present invention, the content of diacylglycerol + triacylglycerol is preferably 70% by mass or more, more preferably 70 to 99% by mass, further preferably 75 to 98% by mass, and even more preferably 77% by mass. The content is preferably 97% by mass from the viewpoint of physiological effects and industrial productivity.

The diacylglycerol-rich fat and oil obtained by the esterification reaction can be used in the same manner as general edible fats and oils by performing a purification step if necessary.

In the following examples, "%" means "% by mass".

[Sampling method]
The vacuum line in the flask was shut off, the pressure was returned to normal pressure with nitrogen, and sampling was performed. Thereafter, about 3 g of a sample of the reaction product was collected in a test tube capable of centrifugation, and centrifuged at 3000 r/min for 5 minutes. The oil phase (upper layer) from which the precipitated catalyst was removed was analyzed.
[Analysis method]
(i) Measurement of oil phase water content The water content of the oil phase (upper layer) obtained in the above [sampling method] was measured using Karl Fischer AQ-300 (manufactured by Hiranuma Sangyo).

(ii) Measurement of acid value The oil phase (upper layer) obtained by the above [sampling method] was measured according to the "Acid value (2.3.1-1996)" in the "Standard oil and fat analysis test method" 2003 edition edited by the Japan Oil Chemists' Association. ”.
The free fatty acid concentration of the oil phase (upper layer) was determined using the following equation (1). The fatty acid average molecular weight of linseed oil was 280.
Free fatty acid concentration (%) = acid value (AV) of oil phase (upper layer) x (average fatty acid molecular weight of linseed oil/56.1/10) (1)

(iii) Measurement of glyceride composition In a glass sample bottle, add approximately 10 mg of the oil phase (upper layer) obtained in the above [sampling method] and 0.5 mL of a trimethylsilylating agent ("Silylating agent TH", manufactured by Kanto Kagaku). The mixture was then sealed and heated at 70°C for 15 minutes. 1.5 mL of water and 1.5 mL of hexane were added to this, and the mixture was shaken. After standing still, the oil phase (upper layer) was subjected to gas chromatography (GLC) to analyze the glyceride composition. The sum of monoacylglycerol + diacylglycerol + triacylglycerol was determined using the following formula (3), and the respective concentrations were determined by GC analysis.
Monoacylglycerol + diacylglycerol + triacylglycerol = 100 - free fatty acid concentration (%) (3)

(iv) Calculation of reaction yield and diacylglycerol (DAG) purity From the glyceride composition after 5 hours of reaction, the reaction yield and DAG purity were calculated using the following formulas (4) and (5).
Reaction yield (%) = diacylglycerol + triacylglycerol (4)
DAG purity (%) = diacylglycerol/diacylglycerol yield x 100 (5)

(v) Measurement of dry mass ratio of immobilized enzyme After washing 3 times each with hexane and acetone in an amount of 10 times the mass of the immobilized enzyme a mass part with oil and moisture attached, leave it at 70°C for 15 hours. By doing so, the solvent was removed, and the mass of only the immobilized enzyme was weighed (b parts by mass). The dry mass ratio of the immobilized enzyme was determined using the following equation (6).
Dry mass ratio of immobilized enzyme = b/a (-) (6)
(a: mass of immobilized enzyme with oil and moisture attached, b: mass of immobilized enzyme)

(vi) Measurement of water content of immobilized enzyme The water content of the immobilized enzyme with oil and water attached was measured using Karl Fischer AQ-300 (manufactured by Hiranuma Sangyo). The water content of the immobilized enzyme was determined using the following equation (7).
Moisture of immobilized enzyme = (ab-b x (1-c))/c (%) (7)
(a: Water content (%) of the immobilized enzyme with oil and water attached, b: Water content (%) of the oil phase (upper layer), c: Dry mass ratio of the immobilized enzyme (-))

[Raw fatty acid used in esterification reaction]
20 kg of decolorized linseed oil and 12 kg of distilled water were placed in a 40 L jacket heated stirring tank, and the mixture was stirred at a temperature of 40° C. and 100 r/min. Thereafter, 200 g of lipase AY "Amano" 30SD (manufactured by Amano Enzyme) was applied to initiate a hydrolysis reaction. After 6 hours, stirring was stopped, static separation was performed, and the aqueous phase was extracted. Thereafter, while stirring at 100 r/min, 12 kg of distilled water was charged, and 200 g of lipase AY "Amano" 30SD (manufactured by Amano Enzyme) was applied to start the hydrolysis reaction again. After 18 hours, the entire volume was centrifuged to separate the oil phase. The operation of adding 60 wt % of distilled water to the oil phase, mixing and centrifuging was repeated twice, followed by dehydration under reduced pressure at 70° C. to obtain a hydrolyzed oil. This was used as a linseed fatty acid in the following esterification reaction. Table 1 shows the glyceride composition of flaxseed fatty acids.

[Esterification reaction]
Example 1
The total amount of flaxseed fatty acids and glycerin was 500 g, the molar ratio of fatty acid/glycerin was 2.0, and the amount of Lipozyme RM IM (manufactured by Novozymes Japan Co., Ltd., water content 2.0%) was 25 g on a dry mass basis. Lipozyme RM IM and linseed fatty acid were placed in a 1000 mL four-necked flask equipped with a crescent blade, and while stirring at 50° C. and 400 r/min, glycerin was added to start the reaction. Immediately, the pressure was reduced to 1,800 Pa using a vacuum pump, and the water content was measured 30 and 60 minutes later, and the pressure was adjusted to 1,200 Pa based on the analytical values. Thereafter, the moisture content was measured every 30 minutes, and the pressure was adjusted based on the moisture content. Specifically, the pressure was adjusted to 800 Pa 120 minutes after the start of the reaction, the pressure was adjusted to 400 Pa after 180 minutes, and the pressure was adjusted to 240 Pa 240 minutes after the start of the reaction. The reaction was terminated after 300 minutes, and the glyceride composition, acid value, and water content of the immobilized enzyme were also measured.

Example 2
The total amount of flaxseed fatty acids and glycerin shown in Table 1 was 3000 g, the molar ratio of fatty acid/glycerin was 2.0, and the amount of Lipozyme RM IM was 150 g based on dry mass. Lipozyme RM IM and linseed fatty acid were placed in a 5000 mL four-necked flask equipped with a crescent blade, and while stirring at 50° C. and 400 r/min, glycerin was added to start the reaction. Thereafter, the analysis was conducted under the same pressure adjustment conditions as in Example 1.

Example 3
The total amount of flaxseed fatty acids and glycerin shown in Table 1 was 500 g, the molar ratio of fatty acid/glycerin was 2.0, and the amount of Lipozyme RM IM was 25 g based on dry mass. Lipozyme RM IM and linseed fatty acid were placed in a 1000 mL four-necked flask equipped with a crescent blade, and while stirring at 50° C. and 400 r/min, glycerin was added to start the reaction. Immediately, the pressure was reduced to 800 Pa using a vacuum pump. The pressure was kept constant and moisture was measured every 30 minutes. The reaction was terminated after 300 minutes, and the glyceride composition, acid value, and water content of the immobilized enzyme were also measured.

Example 4
The dry mass ratio of Lipozyme RM IM after use in Example 1 was measured. The dry mass ratio was 0.466. 43.7 g of Lipozyme RM IM with reaction oil attached (20.4 g of Lipozyme RM IM, 23.3 g of reaction oil) was charged into a 1000 mL four-necked flask equipped with a crescent blade. Next, it was washed three times with 200 g of linseed fatty acids shown in Table 1 to replace the oil adhering to Lipozyme RM IM with linseed fatty acids.
The total amount of flaxseed fatty acids and glycerin shown in Table 1 was 407.4 g, and the molar ratio of fatty acid/glycerin was 2.0. The flaxseed fatty acid is the total of the flaxseed fatty acid attached to Lipozyme RM IM and the newly charged flaxseed fatty acid. Furthermore, the mass ratio of Lipozyme RM IM to the total fatty acid/glycerin was 5%, which is the same as in Example 1.
Lipozyme RM IM and linseed fatty acid were placed in a 1000 mL four-necked flask, and while stirring at 50° C. and 400 r/min, glycerin was added to start the reaction. Thereafter, the analysis was conducted under the same pressure adjustment conditions as in Example 1.

Comparative example 1
The total amount of flaxseed fatty acids and glycerin was 50 g, the molar ratio of fatty acid/glycerin was 2.0, and the amount of Lipozyme RM IM was 2.5 g based on dry mass. Lipozyme RM IM and linseed fatty acid were placed in a 200 mL four-necked flask equipped with a crescent blade, and while stirring at 50° C. and 400 r/min, glycerin was added to start the reaction. Immediately, the pressure was reduced to 110 Pa using a vacuum pump. The pressure was kept constant and moisture was measured every 30 minutes. The reaction was terminated after 300 minutes, and the glyceride composition, acid value, and water content of the immobilized enzyme were also measured.

Comparative example 2
The total amount of flaxseed fatty acids and glycerin was 500 g, the molar ratio of fatty acid/glycerin was 2.0, and the amount of Lipozyme RM IM was 25 g on a dry mass basis. Lipozyme RM IM and linseed fatty acid were placed in a 1000 mL four-necked flask equipped with a crescent blade, and while stirring at 50° C. and 400 r/min, glycerin was added to start the reaction. Immediately, the pressure was reduced to 1200 Pa using a vacuum pump. The pressure was kept constant and moisture was measured every 30 minutes. After that, the same analysis as in Comparative Example 1 was performed.

Comparative example 3
The total amount of flaxseed fatty acids and glycerin was 500 g, the molar ratio of fatty acid/glycerin was 2.0, and the amount of Lipozyme RM IM was 25 g on a dry mass basis. Lipozyme RM IM and linseed fatty acid were placed in a 1000 mL four-necked flask equipped with a crescent blade, and while stirring at 50° C. and 400 r/min, glycerin was added to start the reaction. Immediately, the pressure was reduced to 1500 Pa using a vacuum pump. The pressure was kept constant and moisture was measured every 30 minutes. After that, the same analysis as in Comparative Example 1 was performed.

Comparative example 4
The dry mass ratio of Lipozyme RM IM after use in Comparative Example 1 was measured. The dry mass ratio was 0.480. A 200 mL four-necked flask equipped with a crescent blade was charged with 2 g of Lipozyme RM IM to which reaction oil was attached (0.96 g of Lipozyme RM IM, 1.04 g of reaction oil). Next, it was washed three times with 50 g of linseed fatty acids shown in Table 1 to replace the oil adhering to Lipozyme RM IM with linseed fatty acids.
The total amount of linseed fatty acids and glycerin shown in Table 1 was 19.2 g, and the molar ratio of fatty acid/glycerin was 2.0. The flaxseed fatty acid is the total of the flaxseed fatty acid attached to Lipozyme RM IM and the newly charged flaxseed fatty acid. Furthermore, the ratio of Lipozyme RM IM to the total fatty acid/glycerin was 5%, which is the same as in Example 1.
Lipozyme RM IM and linseed fatty acid were placed in a 200 mL four-necked flask, and while stirring at 50° C. and 400 r/min, glycerin was added to start the reaction. Immediately, the pressure was reduced to 240 Pa using a vacuum pump. The pressure was kept constant and moisture was measured every 30 minutes. After that, the same analysis as in Comparative Example 1 was performed.

Table 2 shows the reaction conditions, reaction products, and water content analysis values of the immobilized enzyme in Examples and Comparative Examples.

Further, Table 3 shows the moisture value and pressure conditions of the oil phase (upper layer) measured over time of the reaction.

As is clear from Tables 2 and 3, in the esterification reaction using immobilized lipase, the esterification is carried out under a vacuum of 1200 Pa or less and 200 Pa or more so that the oil phase moisture and pressure satisfy formula (i). It was confirmed that by proceeding with the reaction, a reaction oil with high diacylglycerol purity could be obtained in good yield (Examples 1 to 4). In Examples 1 to 3, the oil phase moisture was maintained at 0.25 to 0.7% for 20 minutes or more, and the oil phase moisture was maintained at less than 0.25% for 20 minutes or more, and the immobilized enzyme at the end of the esterification reaction was The moisture content was 3.0% or less.
Furthermore, as shown in Example 4, even when the immobilized lipase was used repeatedly, the diacylglycerol purity and reaction yield of the resulting reaction oil were high. The water content of the immobilized enzyme at the end of the reaction was 3.0% or less.
On the other hand, in Comparative Example 1, in which the esterification reaction was carried out in a vacuum at a pressure of less than 200 Pa, the diacylglycerol purity and reaction yield of the obtained reaction oil were able to be increased, but when the immobilized lipase was repeatedly used, As shown in Comparative Example 4, it became clear that the reaction yield was significantly reduced. The water content of the immobilized enzyme at the end of the reaction in Comparative Example 4 was 3.4%.
Further, as in Comparative Examples 2 and 3, it was revealed that when the pressure exceeds 1200 Pa in a vacuum degree, the resulting reaction oil had a high diacylglycerol purity but a low reaction yield. In Comparative Examples 2 and 3, the water content of the oil phase was less than 0.25% and was not maintained for more than 20 minutes, and the water content of the immobilized enzyme at the end of the reaction was 3.0% or more.

Claims (8)

It includes a step of esterifying glycerin and a fatty acid or its lower alkyl ester under a pressure of 200 to 1200 Pa using an immobilized enzyme, and throughout the step, the oil phase moisture (x) and pressure (y) are The relationship is the following formula (i);
(i) 10000x+100≧y≧2500x-300
(However, x≦0.6, 200≦y≦1200)
(Here, x indicates oil phase moisture (mass%) and y indicates pressure (Pa).)
A method for producing a diacylglycerol-rich fat or oil , which satisfies the following and changes the pressure so that it decreases as the water content of the oil phase decreases . In the step, the esterification reaction is carried out for 20 minutes or more at an oil phase moisture x of 0.25% by mass or more, and then the esterification reaction is carried out while removing the reaction product water from the reaction system to lower the oil phase moisture. The method for producing a diacylglycerol-rich fat or oil according to claim 1, wherein the esterification reaction is carried out for 15 minutes or more at an oil phase water content x of less than 0.25% by mass. Further comprising carrying out an esterification reaction at a pressure of more than 1200 Pa when the oil phase moisture is more than 0.6% by mass, and adjusting the pressure to 1200 Pa or less when the oil phase moisture becomes 0.6% by mass or less. The method for producing a diacylglycerol-rich fat or oil according to claim 1 or 2. High diacylglycerol content according to any one of claims 1 to 3, wherein in the step, the esterification reaction is carried out by adjusting the pressure to less than 1200 Pa and 450 Pa or more when the water content of the oil phase becomes 0.3% by mass or less. Method for producing fats and oils. The diacylglycerol-rich fat or oil according to any one of claims 1 to 4, wherein in the step, the esterification reaction is carried out by adjusting the pressure to less than 1200 Pa and 200 Pa or more when the water content of the oil phase becomes 0.2% by mass or less. manufacturing method. High diacylglycerol content according to any one of claims 1 to 5, wherein in the step, the esterification reaction is carried out by adjusting the pressure to 1100 Pa or less and 200 Pa or more when the water content of the oil phase becomes 0.1% by mass or less. Method for producing fats and oils. The method for producing a diacylglycerol-rich fat or oil according to any one of claims 1 to 6 , wherein the immobilized enzyme is an immobilized 1,3-position selective lipase. The method for producing a diacylglycerol-rich fat or oil according to any one of claims 1 to 7 , wherein the diacylglycerol purity of the diacylglycerol-rich fat or oil is 80% by mass or more.