JP2012034622A - Method for producing fat and oil with high diacylglycerol content - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing fat and oil with high diacylglycerol content by esterification with a fatty acid, and effectively producing the fat and oil containing high amount of highly pure diacylglycerol in an industrially advantageous condition.SOLUTION: The method for producing the fat and oil with high diacylglycerol content includes: reacting glycerine and the fatty acid, or a lower alkyl ester thereof using an immobilized lipase in which a 1,3-selective lipase is immobilized on an immobilized carrier having an average particle size of less than 300 μm.

Description

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

It is known that fats and oils containing diacylglycerol at a high concentration have a physiological action such as suppressing an increase in blood triglycerides (neutral fat) after meals and having less accumulation in the body (for example, Patent Document 1). reference).
The production of diacylglycerol is generally carried out by a glycerolysis reaction between glycerin and fats and oils or an esterification reaction between glycerin and a fatty acid (see, for example, Patent Documents 2 to 4). These production methods are roughly classified into a chemical method using an alkali catalyst or the like and an enzyme method using an enzyme such as lipase, but it is preferable in terms of flavor and the like to perform the reaction under mild conditions using the enzyme. It is said.

JP-A-10-176181 Japanese Examined Patent Publication No. 6-65310 Japanese Patent Publication No. 6-65311 JP-A-4-330289 Special table 2004-528843 gazette

Conventionally, when performing a reaction using an enzyme, an immobilized enzyme in which the enzyme is immobilized on an inorganic or organic immobilization carrier is used in order to efficiently use the enzyme. Usually, these immobilization carriers are used in the state of particles that are somewhat large due to their handling properties (see, for example, Patent Document 5).
However, since what kind of pore distribution and particle size is preferable depends on the reaction system to be applied, it is still unclear what type of immobilization support is suitable for diacylglycerol production. Not. For this reason, it cannot be said that the existing immobilized enzyme is necessarily satisfied in terms of balance between production efficiency and quality and cost.

In order to improve the reaction rate of the esterification reaction, it is conceivable to increase the lipase usage ratio relative to the immobilization carrier and increase the lipase activity, but it has been found that the purity of diacylglycerol decreases. Moreover, it is not desirable to increase the amount of lipase used from the viewpoint of industrial productivity.
Accordingly, an object of the present invention is to provide a method for producing a fat and oil having a high content of diacylglycerol by esterifying a fatty acid with glycerin and efficiently producing the fat and fat having a high content of diacylglycerol under industrially advantageous conditions. It is in.

  The inventor has made various studies on the production method of diacylglycerol. If lipase is adsorbed on an immobilization carrier having a specific particle size, the esterification activity is high even with a small adsorption rate, and the reaction rate is improved. Surprisingly, the inventors have found that the selectivity of diacylglycerol is enhanced, and that a fat with a high content of diacylglycerol can be obtained efficiently.

  That is, the present invention has a high content of diacylglycerol in which glycerin is reacted with a fatty acid or a lower alkyl ester thereof using an immobilized lipase in which a 1,3-position selective lipase is immobilized on an immobilized carrier having an average particle size of less than 300 μm. The manufacturing method of fats and oils is provided.

  According to the method of the present invention, the reaction rate can be increased without reducing the purity of diacylglycerol, so that fats and oils with a high diacylglycerol content can be obtained efficiently. Moreover, since the usage-amount of lipase can be reduced, it is industrially advantageous.

Examples of the immobilization carrier used in the production method of the present invention include celite, diatomaceous earth, kaolinite, silica gel, molecular sieves, porous glass, activated carbon, calcium carbonate, ceramics and other inorganic carriers, ceramic powder, polyvinyl alcohol, Examples thereof include organic polymers such as polypropylene, chitosan, ion exchange resin, hydrophobic adsorption resin, chelate resin, and synthetic adsorption resin. Of these, ion exchange resins are preferred from the viewpoints of production efficiency, affinity with fatty acids, and water retention.
The shape of the immobilization carrier is not particularly limited, and examples thereof include particles, powders, granules, fibers, and sponges.

  As the ion exchange resin, a porous anion exchange resin is preferable. Since such a porous carrier has a large surface area, a larger amount of enzyme adsorbed can be obtained. Examples of the material of the anion exchange resin include phenol formaldehyde series, polystyrene series, acrylamide series, divinylbenzene series, and the like, and phenol formaldehyde series resins (for example, Rohm and Hass Co., Ltd.) are particularly effective in achieving the effects of the present invention. Duolite A-568) is preferred.

  The average particle size of the immobilization carrier is less than 300 μm, but preferably 120 μm to less than 300 μm, more preferably 140 μm to 280 μm, and particularly preferably 150 μm to 260 μm from the viewpoint of improving the reaction rate and the purity of diacylglycerol.

The immobilization carrier having an average particle size of less than 300 μm is not particularly limited, but an immobilization carrier having a larger particle size can be obtained by cutting, crushing, or the like. At this time, since a fine powdery carrier is also generated, it is preferable to separate by sieving. The particle diameter range of the immobilization carrier is preferably 75 μm to 500 μm, more preferably 106 μm to 300 μm, and particularly preferably 150 μm to 250 μm from the same point as described above.
In the present invention, the average particle diameter means an average value obtained by a laser diffraction scattering method in accordance with a volume standard, and the particle diameter range means a width of a JIS standard sieve opening value.

The specific surface area of the immobilization carrier is a surface area per immobilizing carrier 1g, although 0.018 m ² / g or more, from the viewpoint of diacylglycerol purity improvement, 0.018m ² /g~0.054m ² / g by weight, more 0.019m ² /g~0.049m ² / g, especially 0.020m ² /g~0.045m ² / g are preferred. This specific surface area can be calculated by calculating the average particle diameter of the immobilized carrier, assuming that the shape of the carrier is spherical, and the true specific gravity is 1.12 g / cm ³ (Dulite A-568 manufactured by Rohm and Hass). .

  The immobilized lipase used in the present invention is obtained by immobilizing a 1,3-position selective lipase on the above-described immobilized carrier. The mass ratio of the lipase to the immobilization carrier is preferably 0.25 to 2.5, more preferably 0.5 to 1.5, and particularly preferably 0.5 to 1 from the viewpoint of production efficiency and diacylglycerol purity. In addition, the mass ratio of the lipase with respect to the immobilization carrier is a dry mass value of the lipase with respect to the dry mass of the immobilization carrier at the time of charging.

  The esterification activity of the immobilized lipase is preferably 3000 to 12000 U, more preferably 4500 to 11000 U, and particularly preferably 6000 to 9000 U from the viewpoint of production efficiency. Here, the esterification activity (U) of the immobilized lipase is defined as the number of moles of fatty acid decreased per minute when the fatty acid concentration was measured by the method described in Examples below.

  The 1,3-position selective lipase is a lipase that specifically acts on the 1,3-positions of glycerides, and commercially available lipases derived from microorganisms as well as those derived from animals and plants can also be used. Examples of the lipase derived from microorganisms include genus Rhizopus, Aspergillus, Mucor, Rhizomucor, Pseudomonas, Getrichemi, and Geotrichem. And those of origin such as the genus Penicillium and Candida.

  When the lipase is immobilized on the immobilization carrier, the lipase may be directly adsorbed on the immobilization carrier. It may be used after being treated with a derivative. Examples of the fat-soluble fatty acid to be used include saturated or unsaturated, linear or branched fatty acids having 8 to 18 carbon atoms, which may be substituted with a hydroxyl group. Specific examples include capric acid, lauric acid, myristylic acid, oleic acid, linoleic acid, α-linolenic acid, ricinoleic acid, isostearic acid and the like. Examples of the derivatives include esters of these fatty acids with mono- 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, and sucrose esters of the above fatty acids. Two or more of these fat-soluble fatty acids or derivatives thereof may be used in combination.

  As a method for contacting these fat-soluble fatty acids or derivatives thereof with a carrier, these may be added directly to a carrier in water or an organic solvent, but in order to improve dispersibility, a fat-soluble fatty acid or derivative thereof is added to an organic solvent. Once dispersed and dissolved, it may be added to a carrier dispersed in water. Examples of the organic solvent include chloroform, hexane, acetone, ethanol, and the like. The amount of the fat-soluble fatty acid or derivative thereof used is preferably 1 to 1000 parts, more preferably 10 to 500 parts, with respect to 100 parts by mass (hereinafter simply referred to as parts) of the immobilized carrier. The contact temperature is preferably 0 to 80 ° C., more preferably 20 to 60 ° C., and the contact time is preferably about 5 minutes to 5 hours. The carrier after this treatment is collected by filtration, but may be dried. The drying temperature is preferably 0 to 80 ° C., and drying under reduced pressure may be performed.

  The temperature at which the lipase is immobilized can be determined depending on the properties of the lipase, but is preferably a temperature at which the lipase is not deactivated, that is, 0 to 80 ° C., more preferably 20 to 60 ° C. Moreover, the pH of the lipase solution used at the time of immobilization may be in a range where denaturation of the lipase does not occur and can be determined by the characteristics of the lipase as well as the temperature, but is preferably pH 3-9. In order to maintain this pH, a buffer solution is used. Examples of the buffer solution include an acetate buffer solution, a phosphate buffer solution, and a Tris-HCl buffer solution. The lipase concentration in the lipase solution is preferably not more than the saturation solubility of lipase and sufficient from the viewpoint of immobilization efficiency. Moreover, the lipase solution can also use the supernatant which removed the insoluble part by centrifugation as needed, and what was refine | purified by ultrafiltration etc.

  In the present invention, after the lipase is immobilized on the immobilization carrier, it is preferably dehydrated without contacting with fat-soluble fatty acid, fatty acid triglyceride, fatty acid partial glyceride or the like. The fat-soluble fatty acid is preferably a vegetable liquid oil such as rapeseed oil, soybean oil or sunflower oil, or a fatty acid produced from fish oil such as sardine oil, tuna oil or bonito oil. The fatty acid, fatty acid triglyceride or fatty acid partial glyceride used is preferably selected as an oil phase substrate in an actual esterification reaction using an immobilized lipase prepared by the method of the present invention.

  In the method of the present invention, the fatty acid used in the esterification reaction or a lower alkyl ester thereof (hereinafter also referred to as a fatty acid) is linear or branched C4-22, preferably C8-18 saturated or unsaturated. Fatty acids are preferred, such as butyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, undecanoic acid, lauric acid, myristic acid, palmitic acid, zomarinic acid, stearic acid, oleic acid, elaidic acid, Linoleic acid, linolenic acid, arachidonic acid, gadrenic acid, arachidic acid, behenic acid, erucic acid, eicosapentaenoic acid, docosahexaenoic acid, and the like can be used. Examples of lower alcohols that form esters with 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 of these fatty acids or their lower alkyl esters can be used in combination. A mixture of the above fatty acids, for example, naturally derived fatty acids such as soybean fatty acid can also be used.

  The raw material charge ratio when the esterification reaction is performed is such that the ratio R of the number of moles of fatty acid groups to the number of moles of glycerin groups R (R = fatty acid etc. (mol) / glycerin (mol)) is 0.5 to 5.0, Further, 1.0 to 4.0, particularly 1.2 to 3.0, and particularly 1.5 to 2.5 is the point at which the composition of the reaction oil is optimized (fatty acid in the reaction oil and glycerin The residual amount and the production amount of monoacylglycerol or triacylglycerol are suppressed, and the distillation load is reduced and the content of diacylglycerol is high, which is preferable from the viewpoint of increasing production efficiency.

  The method of reacting glycerin with a fatty acid or the like using an immobilized lipase is not particularly limited, and the immobilized lipase and the raw material (such as glycerin and a fatty acid) may be brought into contact with each other. Examples thereof include a method of passing a solution through a column filled with immobilized lipase with a pump or the like. In the case of stirring, 10 to 1000 r / min is preferable, 50 to 700 r / min, particularly 100 to 600 r / min is preferable from the viewpoint of production efficiency and suppression of crushing of the immobilized lipase.

  The amount (dry mass) of the immobilized lipase used for the esterification reaction can be appropriately determined in consideration of the activity of the lipase, but it is 0.1 to 50 parts with respect to 100 parts of the raw material obtained by totaling fatty acid and the like and glycerin. Further, 0.5 to 30 parts, particularly 1.0 to 15 parts are preferred.

  The reaction temperature for the esterification reaction is not particularly limited, but 20 to 80 ° C., particularly 30 to 70 ° C. is preferable in terms of reactivity. The reaction time is preferably within 10 hours from the viewpoint of industrial productivity, more preferably 0.1 to 5 hours, particularly preferably 0.2 to 2.5 hours, and particularly preferably 0.3 to 1 hour.

  In addition, it is preferable to perform dehydration under reduced pressure during the esterification reaction from the viewpoint of increasing the diacylglycerol content of the reaction oil. The pressure is preferably 10 to 10,000 Pa, and particularly preferably 100 to 1000 Pa.

  In the present invention, the purity of diacylglycerol as a reaction product is preferably 80% or more, more preferably 85 to 99.5%, further 90 to 99%, and particularly 95 to 98%. From the viewpoint of industrial productivity. Here, the diacylglycerol purity is [diacylglycerol / (diacylglycerol + triacylglycerol) × 100].

  In the present invention, the diacylglycerol + triacylglycerol content [% by mass] in the reaction product is preferably 60% or more, more preferably 60 to 99%, further 65 to 98%, particularly 70 to 97%. It is preferable from the viewpoint of physiological effects and industrial productivity.

  The oil containing fat with high diacylglycerol obtained by the esterification reaction can be made into a product by performing post-treatment. The post-treatment is preferably performed by deoxidation (removing unreacted fatty acid and by-product monoacylglycerol), acid treatment, water washing, and deodorization. The deacidification step refers to a step of removing unreacted fatty acid and by-produced monoacylglycerol from the reaction product by distilling the diacylglycerol-rich oil and fat obtained by the esterification reaction under reduced pressure. The acid treatment step refers to a step of adding and mixing a chelating agent such as citric acid to the deoxidized oil and further dehydrating under reduced pressure as necessary. Further, the obtained acid-treated oil may be subjected to a decoloring step by contact with an adsorbent from the viewpoint of further improving the hue and flavor. The water washing step refers to a step of performing an operation of adding water to the acid-treated oil and vigorously stirring to perform oil-water separation. Washing with water is preferably repeated a plurality of times (for example, 3 times) to obtain washing oil. The deodorizing step refers to a step of subjecting the washing oil to steam distillation under reduced pressure. Deodorization includes batch type, continuous type, semi-continuous type, etc. In addition to a method of performing a thin film deodorization device or a tray type deodorization device alone, a deodorization treatment using these thin film deodorization devices and a tray type deodorization device were used. You may carry out in combination with a deodorizing process.

[Analysis method]
(i) Measurement of acid value In accordance with “Standard value of fats and oils analysis test method” edited by Japan Oil Chemistry Association, 2003, “acid value (2.3.1-1996)”, free fatty acids contained in 1 g of sample A value obtained by calculating the number of mg of potassium hydroxide required for summing.

(ii) Measurement of fatty acid composition The fatty acid composition was measured according to the "Preparation Method of Fatty Acid Methyl Esters (2.4.1.2-1996)" edited by the Japan Oil Chemists' Society, "Standard Oil Analysis Test Method" 2003 edition. Fatty acid methyl esters were prepared and the resulting samples were obtained from American Oil Chemists. It was measured by Society Official Method Ce 1f-96 (GLC method).

(iii) Measurement of fatty acid concentration Using the acid value and fatty acid composition measured by the above method, the fatty acid concentration was determined according to the following equation (1) according to the knowledge of fat and oil products (Shoshobo Co., Ltd.).
Fatty acid concentration (mass%) = xxy / 56.1 / 10 (1)
(X = acid value [mgKOH / g], y = average molecular weight determined from fatty acid composition)

(iv) Measurement of esterification activity of immobilized lipase 4 g of immobilized lipase was added to a total of the raw material fatty acid and glycerin on a dry mass basis in a 200 ml four-necked flask equipped with a crescent blade. Raw material fatty acids shown in Table 1 below were added, and glycerin was added while stirring at 50 ° C. and 400 r / min. The total of the raw material fatty acid and glycerin was 80 g, and the fatty acid / glycerin molar ratio was 2.0. Next, the reaction was carried out under reduced pressure (400 Pa) with a vacuum pump. The reaction solution after 30 minutes was sampled, and the esterification activity of the immobilized lipase was calculated from the value of the fatty acid concentration. The esterification activity [U] of the immobilized lipase was defined as the number of μmoles of fatty acid decreased per minute.

(v) Measurement of glyceride composition Approximately 3 g of a sample of the reaction finished oil was collected in a test tube capable of being centrifuged, and centrifuged at 3000 r / min for 10 minutes to remove the precipitated glycerin. Next, about 10 mg of the upper layer and 0.5 mL of a trimethylsilylating agent (“silylating agent TH”, manufactured by Kanto Chemical) were added to a glass sample bottle, sealed, and heated at 70 ° C. for 15 minutes. To this, 1.5 mL of water and 1.5 mL of hexane were added and shaken. After standing, the upper layer was subjected to gas chromatography (GLC) to analyze the glyceride composition.

(vi) Measurement of dry mass ratio of immobilized lipase After washing 3 times each alternately with 10 parts by mass of hexane and acetone with respect to a part by mass of immobilized enzyme a to which oil and moisture are adhered, leave at 70 ° C. for 15 hours. Thus, the solvent was removed, and the mass of only the immobilized enzyme was weighed (b part by mass), and the mass ratio with respect to 100 parts by mass (dry mass) of the immobilized carrier was determined from the following formula (2).
Dry mass ratio of immobilized lipase = b / a × 100 (%) (2)
(A: mass of immobilized lipase with oil and water attached, b: mass of immobilized lipase)

(vii) Amount of lipase adsorbed per surface area The amount of lipase adsorbed per surface area is determined by the specific surface area [m ² / g] of the immobilized carrier and the mass ratio of lipase to the immobilized carrier (in the formula, “lipase / immobilized carrier mass ratio”). From the following formula (3).
Lipase adsorption amount per surface area [g / m ² ] = (Lipase / immobilized carrier mass ratio) / immobilized carrier specific surface area [m ² / g] (3)

(viii) Measurement of average particle diameter LS 13 320 (manufactured by BECKMAN COULTER) was used and measured by a laser diffraction scattering method.

[Raw fatty acid]
The fatty acid used as a reaction raw material was obtained by hydrolyzing fats and oils with a high-pressure hydrothermal decomposition apparatus. The water was 50 parts by mass with respect to 100 parts by mass of soybean oil, and treated with high-pressure hot water (5 MPa, 240 ° C., average residence time 4 h). Next, after cooling, the mixture was centrifuged, and the fatty acid layer was dehydrated under reduced pressure at a temperature of 70 ° C. and a vacuum degree of 400 Pa for 30 minutes to obtain soybean fatty acid. Table 1 shows the glyceride composition. Here, FFA is a free fatty acid, GLY is glycerin, MAG is monoacylglycerol, DAG is diacylglycerol, and TAG is triacylglycerol (the same applies hereinafter).

(Preparation of immobilization carrier)
500 g of Duolite A-568 (Rohm & Hass, average particle size of 549 μm, hereinafter the same) was ground, dry classified with 250 μm and 150 μm sieves specified in JIS-Z8801, and further wet-classified with water. Next, it was dried at 70 ° C. to obtain an immobilized carrier having an average particle diameter of 235 μm.

[Preparation of immobilized lipase 1]
10 g of an immobilizing carrier having an average particle diameter of 235 μm was stirred in 100 mL of a 0.1N sodium hydroxide aqueous solution for 1 hour. After filtration, the mixture was stirred and washed with 100 mL of distilled water for 1 hour, filtered, and equilibrated with 100 mL of 500 mM acetate buffer (pH 5) for 2 hours. After filtration, the pH was equilibrated with 100 mL of 50 mM acetate buffer (pH 5) twice for 2 hours. Thereafter, filtration was performed and the carrier was recovered, followed by ethanol replacement with 50 mL of ethanol for 30 minutes. Thereafter, filtration was performed, 50 mL of ethanol containing 10 g of soybean fatty acid was added, and soybean fatty acid was adsorbed on the carrier for 30 minutes. Thereafter, the carrier was recovered by filtration, and then washed four times with 100 mL of 50 mM acetate buffer (pH 5), ethanol was removed, and the carrier was recovered by filtration. Thereafter, 5 g of lipase (Lipase A-10FG, manufactured by Nagase ChemteX) was contacted with a lipase solution dissolved in 100 mL of 50 mM acetate buffer (pH 5) for 2 hours for immobilization. Furthermore, the immobilized lipase was recovered by filtration, and washed with 100 mL of 50 mM acetate buffer (pH 5) to remove non-immobilized lipase and protein. All the above operations were performed at a temperature of 20 ° C. Thereafter, 100 g of soybean fatty acid was added, and the mixture was stirred at a temperature of 40 ° C. and depressurized until reaching a pressure of 400 Pa, and dehydrated to obtain an immobilized lipase 1.

[Preparation of immobilized lipases 2 and 3]
The immobilized lipase 2 and 3 were obtained in the same manner as the immobilized lipase 1 except that the amount of lipase used was 10 g and 15 g, respectively.

[Preparation of immobilized lipase 4-6]
The immobilized carrier was Duolite A-568 (average particle size 549 μm), and the amount of lipase used was 5 g, 10 g, and 15 g, respectively. Got.
The physical property values of each immobilized lipase are shown in Table 2.

[Esterification reaction]
The total amount of soybean fatty acid and glycerin shown in Table 1 was 80 g, the molar ratio of fatty acid / glycerin was 2.0, and the immobilized lipase was a charge amount based on 4 g of dry mass. Immobilized lipase and soybean fatty acid were placed in a 200 ml four-necked flask with a crescent blade set, glycerin was added while stirring at 50 ° C. and 400 r / min, and the reaction was carried out by reducing the pressure (400 Pa) with a vacuum pump. . Sampling was performed after returning to normal pressure every 30 minutes, and the glyceride composition was determined. From the glyceride composition, the reaction time to reach a DAG yield of 75% and the DAG purity at the 75% DAG yield were determined. The results are shown in Table 2.

As a result of comparison between Comparative Examples 1 to 3, the lipase immobilized on the immobilization carrier increased in reaction rate, but TAG was by-produced and the DAG selectivity decreased.
On the other hand, as a result of comparison between Examples 1 to 3 and Comparative Examples 1 to 3, those in which lipase was immobilized using an immobilized carrier having an average particle size of 235 μm had high esterification activity and had a DAG yield of 75. It was found that the reaction rate was improved even when the amount of lipase used per immobilization support was small. Furthermore, it was found that by-production of TAG was suppressed and DAG selectivity was increased.

Claims (4)

  A method for producing an oil and fat with a high content of diacylglycerol, wherein glycerin and a fatty acid or a lower alkyl ester thereof are reacted using an immobilized lipase in which a 1,3-position selective lipase is immobilized on an immobilized carrier having an average particle size of less than 300 μm.   The method for producing a fat and oil with a high content of diacylglycerol according to claim 1, wherein the average particle size of the immobilization carrier is less than 120 to 300 µm.   The method for producing a fat and oil having a high diacylglycerol content according to claim 1 or 2, wherein the immobilization carrier is an ion exchange resin.   The method for producing a high diacylglycerol-containing fat according to any one of claims 1 to 3, wherein the mass ratio of the 1,3-position selective lipase to the immobilized carrier of the immobilized lipase is 0.25 to 2.5.