JP5586855B2 - Method for producing monoacylglycerol-rich oil and fat - Google Patents

Description

  The present invention relates to a method for efficiently producing an oil and fat having a high monoacylglycerol content.

  Monoacylglycerol (hereinafter sometimes referred to as “monoglyceride” or “monoglyceride”) has been widely used as an emulsifier and antibacterial agent in the fields of foods, pharmaceuticals, cosmetics, and the like. Foods that focus on activity are attracting attention. In order to obtain this monoacylglycerol, a method of reacting fatty acid or fatty acid methyl and glycerin, or a method of chemically reacting fat and glycerin with a metal catalyst added at high temperature is used.

Examples of the method for producing monoacylglycerol using a fatty acid as a raw material include an esterification method, a transesterification method, and a glycerolysis method.
For example, a method for producing a mixture of monoglyceride and diglyceride in which fatty acid or a lower alkyl ester thereof and glycerol are reacted in the presence of partial glyceride lipase is known (Patent Document 1).
Also known is a method for producing monoglyceride (Patent Document 2) in which an excess of glycerin at a molar ratio of 3 times or more with respect to conjugated linoleic acid is used and reacted at 5 ° C. in the presence of Penicillium camembertii mono and diglyceride lipase. ing.

On the other hand, a method for producing monoglycerides (Patent Document 3) is known in which glycerin is used in a molar ratio of 30 times or more with respect to highly unsaturated fatty acid in the presence of lipase derived from Pseudomonas sp. Immobilized on calcium carbonate. ing.

  Therefore, a method for efficiently producing fats and oils containing a high amount of monoacylglycerol by reacting a fatty acid with glycerin is desired.

JP 61-181390 A Japanese Patent No. 3970669 Japanese Patent No. 3861941

  Among the prior arts, the chemical reaction using a metal catalyst is at a high temperature of 200 ° C. or higher. Therefore, when the raw material contains a large amount of unsaturated fatty acid, it tends to deteriorate, and it is difficult to completely remove the metal catalyst. From the point of view, its application has been a problem. Further, in the technique using the partial glyceride lipase (Patent Document 1), the monoacylglycerol concentration (monoacylglycerol / raw material) decreases with the progress of the reaction. Later, a purification step such as rectification is required, which is problematic in terms of production efficiency. Furthermore, in the technique using the mono- and diglyceride lipase (Patent Document 2), since the reaction is performed at a low temperature that is not the optimum temperature for the lipase, the reaction efficiency is not good. Moreover, in the technique (patent document 3) in which glycerin is used in a molar ratio of 30 times or more with respect to the fatty acid using lipase immobilized on calcium carbonate, the monoglyceride concentration is only 16.7%.

  An object of the present invention is to provide a method for efficiently producing monoacylglycerol using fatty acid and glycerin as raw materials without impairing lipase activity.

  When the present inventor reacts fatty acid or a lower alkyl ester thereof with glycerin using an immobilized partial glyceride lipase, which is an immobilized enzyme, the present invention surprisingly provides an enzyme immobilization carrier (hereinafter also referred to as an immobilization carrier). Immobilized partial glyceride lipase prepared by increasing the proportion of partial glyceride lipase used relative to the amount decreases the production efficiency of monoacylglycerol, whereas the proportion of partial glyceride lipase used relative to the amount of immobilized carrier It has been found that the production efficiency of monoacylglycerol increases when the amount is reduced. Furthermore, it has been found that the surface area of the immobilization carrier is involved in the production efficiency of monoacylglycerol, and the immobilization partial glyceride lipase prepared so that the esterification activity per unit area of the immobilization carrier falls within a specific range. As a result, it was found that high-accuracy monoacylglycerol can be produced at a high reaction rate when the reaction is carried out in the presence of.

That is, the present invention relates to a monoacylglycerol in which a fatty acid or a lower alkyl ester thereof and glycerin are reacted in the presence of an immobilized partial glyceride lipase having an esterification activity per unit surface area of the immobilized carrier of 100 to 140000 U / m ^2. The present invention provides a method for producing a high content fat.

  According to the method for producing a monoacylglycerol-rich oil and fat of the present invention, highly pure monoacylglycerol can be obtained efficiently.

  The immobilized partial glyceride lipase used in the present invention is obtained by immobilizing a partial glyceride lipase on an immobilization carrier. In the present invention, the esterification activity per unit surface area of the immobilized carrier (hereinafter referred to as “A value”) is determined by immobilizing lipase on a carrier whose average particle diameter is known and measuring the esterification activity. The specific surface area per unit mass of the carrier determined from the mass of the immobilized partial glyceride lipase used in the above and the average particle size of the carrier is determined according to the following formula (1).

A [U / m ² ] = Esterification activity of immobilized lipase [U] / (Immobilized carrier specific surface area [m ² / g] × Immobilized lipase mass [g]) Formula (1)

In the present invention, the esterification activity [U] of the immobilized lipase in the formula (1) is a mixture of fatty acid and glycerin in a total of 100 g and fatty acid / glycerin in a molar ratio of 0.6. The fatty acid concentration after adding 5 g on a dry mass basis and stirring for 60 minutes at 40 ° C. and 400 Pa is measured and defined as μmol number of fatty acid decreased per minute. Here, in the immobilized lipase, the mass of the lipase relative to the mass of the immobilized carrier is extremely small, and therefore the mass of the immobilized carrier is approximately equal to the mass of the immobilized lipase. In the present invention, the A value measured as described above is in the range of 100 to 140000 U / m ² . This A value is preferably 200 to 70000 U / m ² , more preferably 500 to 30000 U / m ² , particularly from the viewpoint of efficiently obtaining a monoacylglycerol-rich oil and fat without improving the reaction rate and by-producing diacylglycerol. 1500 to 10000 U / m ² is preferred.

  The esterification activity of the immobilized partial glyceride lipase used in the present invention is preferably 5 to 8000 U, more preferably 10 to 4000 U, particularly 50 to 2000 U, and most preferably 200 to 1200 U, from the viewpoint of efficiently obtaining a monoacylglycerol-rich oil.

The specific surface area of the immobilization carrier used for the immobilization partial glyceride lipase used in the present invention is the surface area per 1 g of the immobilization carrier, and 0.0027 to 0.536 m from the viewpoint of efficiently obtaining fats and oils with high monoacylglycerol content. ² / g, further 0.0045~0.268m ² / g, especially 0.0077~0.179m ² / g, especially 0.0107~0.134m ² / g are preferred. The specific surface area can be calculated by classifying the immobilized carrier with a sieve to obtain an average particle size, assuming that the shape of the carrier is spherical and the true specific gravity is 1.12 g / cm ³ .

The partial glyceride lipase used in the present invention is a lipase that hydrolyzes monoacylglycerol and diacylglycerol partial glycerides but hardly hydrolyzes triacylglycerol.
Examples of partial glyceride lipase include monoglyceride lipase or diglyceride lipase derived from animal organs such as rat small intestine and porcine adipose tissue; monoglyceride lipase derived from Bacillus sp. 2000), Pseudomonas sp. LP7315-derived monoglyceride lipase (Journal of Bioscience and Bioengineering, 91 (1), 27-32, 2001), Penicillium silopium-derived lipase (J. Biochem, 87 (1), 205) -211, 1980), Penicillium camembertii U-150 lipase (J. Fermentation and Bioengineering, 72 (3), 162-167, 1991) and the like. Examples of commercially available products include “monoglyceride lipase (MGLPII)” (Asahi Kasei Corporation), “Lipase G“ Amano ”50” (Amano Enzyme).

  The immobilization carrier used in the present invention is an inorganic carrier such as celite, diatomaceous earth, kaolinite, silica gel, molecular sieves, porous glass, activated carbon, calcium carbonate, ceramics; cellulose powder, polyvinyl alcohol, polypropylene, chitosan, ion exchange Examples thereof include organic polymer carriers such as resins, hydrophobic adsorption resins, chelate resins, and synthetic adsorption resins. In particular, ion exchange resins are preferable in terms of water retention. The shape of the immobilization carrier is not particularly limited, and examples thereof include particles, powders, granules, fibers, and sponges.

The particle diameter of the carrier is preferably 10 to 2000 μm, more preferably 20 to 1200 μm, particularly preferably 30 to 700 μm, particularly preferably 40 to 500 μm, and the pore diameter is preferably 10 to 150 nm, more preferably 10 to 100 nm.
As the ion exchange resin, a porous anion exchange resin is preferable. Examples of the material for the resin include phenol formaldehyde, polystyrene, acrylamide, and divinylbenzene. In particular, a phenol formaldehyde-based resin (trade name: Duolite A-568 manufactured by Rohm and Hass) is preferable from the viewpoint of improving lipase adsorption.

  In the present invention, it is preferable to treat the immobilization carrier with a fat-soluble fatty acid or a derivative thereof as a pretreatment for immobilization of the enzyme in order to obtain an adsorption state that exhibits high activity. These may be used singly, but a further effect is exhibited by combining two or more. As a method for contacting these fat-soluble fatty acids or derivatives thereof and the immobilization carrier, these may be added as they are in water or an organic solvent, but in order to improve dispersibility, fat-soluble fatty acids or derivatives thereof are added to the organic solvent. Once dispersed / dissolved, it may be added to an immobilized carrier dispersed in water. Examples of the organic solvent include chloroform, hexane, ethanol, and the like. The ratio of the fat-soluble fatty acid or derivative thereof to the immobilization carrier is 0.01 to 1 part by mass, particularly 0.05 to 0.5 mass, with respect to 1 part by mass (dry mass) of the immobilization carrier. Part is preferred. The contact temperature is preferably 0 to 80 ° C, particularly preferably 20 to 60 ° C. The contact time may be about 5 minutes to 5 hours. After this treatment, the immobilized carrier is recovered by filtration, but may be dried at this time. The drying temperature is preferably room temperature to 80 ° C., and may be dried under reduced pressure.

  Examples of the fat-soluble fatty acid used for the pretreatment of the immobilization carrier include C4-C24 linear or branched, saturated or unsaturated fatty acids that may be substituted with hydroxyl groups. Preferred fat-soluble fatty acids include fatty acids having 8 to 18 carbon atoms, for example, linear saturated fatty acids such as capric acid, lauric acid and myristic acid, unsaturated fatty acids such as oleic acid and linoleic acid, hydroxy fatty acids such as ricinoleic acid, Examples thereof include branched fatty acids such as isostearic acid. Examples of the fat-soluble fatty acid derivative include esters of a fatty acid having 8 to 18 carbon atoms and a compound having a hydroxyl group, monohydric alcohol ester, polyhydric alcohol ester, phospholipid, or ethylene oxide added to these esters. Examples include derivatives. Examples of monohydric alcohol esters include methyl esters and ethyl esters, and examples of polyhydric alcohol esters include monoacylglycerol, diacylglycerol, and derivatives thereof, or polyglycerin fatty acid esters, sorbitan fatty acid esters, and sucrose fatty acid esters. It is done. These fat-soluble fatty acids or derivatives thereof are desirably liquid at room temperature in the process. Moreover, as these fat-soluble fatty acids or derivatives thereof, a mixture of the above fatty acids, for example, naturally-derived fatty acids such as soybean fatty acids can be used.

  The immobilized partial glyceride lipase of the present invention can be prepared by a general method for producing an immobilized enzyme. For example, Duolite A-568 (manufactured by Rohm & Hass) is used as an immobilization carrier. Sodium hydroxide aqueous solution is added to the immobilization carrier and stirred for alkali treatment, buffer solution is added and stirred for pH adjustment, ethanol is substituted, and fatty acid is added and stirred for pretreatment with a fat-soluble fatty acid or derivative thereof. After the lipase was added and fixed while stirring, the method was obtained by further replacing the oil by adding soybean fatty acid, or by adding a partial glyceride lipase solution to the fixed carrier and stirring the mixture. The partial glyceride lipase is adsorbed and immobilized on the immobilized carrier, and then the insoluble carrier is filtered from the partial glyceride lipase solution. How is washed with-exchange water or buffer and the like. The enzyme may be purified as necessary, or may be used by dispersing or dissolving in advance in an aqueous medium such as water or a buffer solution.

  The amount of the partial glyceride lipase used at this time is such that lipase G “Amano” 50 (50000 u / g or more) is used as the partial glyceride lipase with respect to 100 parts by mass of the immobilization carrier from the viewpoint of efficiently obtaining a fat having a high monoacylglycerol content. When used, it is preferably 0.1 to 500 parts by mass, more preferably 1 to 200 parts by mass, particularly 5 to 100 parts by mass, and particularly preferably 10 to 80 parts by mass. The number of u referred to here is 1 unit [u], which is the amount of enzyme that causes an increase of 1 μmol of fatty acid per minute by the LV emulsification method. In the LV emulsification method, lipase is allowed to act on an emulsion of vinyl laurate (pH 5.6, 30 minutes at 40 ° C.), the reaction is stopped with an ethanol / acetone mixed solvent, and the generated fatty acid is neutralized with sodium hydroxide. Thereafter, the remaining sodium hydroxide is titrated with hydrochloric acid to quantify the generated fatty acid to determine the u number.

  The immobilization temperature of the partial glyceride lipase may be any temperature that does not cause enzyme inactivation, and is preferably 0 to 60 ° C, particularly preferably 5 to 30 ° C. The pH of the aqueous enzyme solution used for immobilization may be in a range that does not cause denaturation of the enzyme, and is preferably pH 3-9. In particular, in order to obtain the maximum activity when using a lipase whose optimum pH is acidic, the pH is preferably 4 to 6. Moreover, as a buffer solution used for the enzyme aqueous solution, a general acetate buffer solution, phosphate buffer solution, Tris-HCl buffer solution, or the like can be used.

  The fatty acid used as a raw material for producing the monoacylglycerol of the present invention is preferably a saturated or unsaturated fatty acid having 4 to 22 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, zomarinic acid, stearic acid, oleic acid, elaidic acid, linoleic acid, linolenic acid, arachidonic acid, gadrenic acid, arachic acid, behenic acid, erucic acid, eicosapentaene An acid, docosahexaenoic acid, etc. can be used. Moreover, as a lower alcohol which forms the said fatty acid and ester, a C1-C3 lower alcohol is preferable. Examples of the lower alcohol having 1 to 3 carbon atoms include methanol, ethanol, 1-propanol, 2-propanol and the like. Two or more of these fatty acids or lower alkyl esters thereof (hereinafter simply referred to as “fatty acids etc.”) 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 esterification reaction of fatty acid or the like and glycerin in the presence of the immobilized partial glyceride lipase may be performed by bringing the immobilized partial glyceride lipase and the raw material (fatty acid or the like and glycerin) into contact with each other. For example, a column filled with the immobilized enzyme may be passed with a pump or the like. In the case of stirring, 30 to 1000 r / min is preferable, and 100 to 700 r / min is more preferable from the viewpoint of efficiently obtaining a high monoacylglycerol-containing fat.

  The immobilized partial glyceride lipase used in the present invention is capable of setting the esterification activity [U] as follows according to the particle size range of the immobilized carrier from the viewpoint of efficiently obtaining a monoacylglycerol-rich oil and fat. preferable. That is, when the particle size of the immobilized carrier is 20 to 100 μm, the esterification activity is preferably 50 to 8000 U, more preferably 200 to 4000 U, and particularly preferably 500 to 2000 U. When the particle size of the immobilized carrier is 100 to 400 μm, The esterification activity is preferably 10 to 4000 U, more preferably 50 to 2000 U, and particularly preferably 200 to 1200 U. When the particle size of the immobilized carrier is 400 to 1200 μm, the esterification activity is 5 to 2000 U, further 10 to 1200 U, particularly 50 to It is preferable that it is 800U.

  In this reaction, the reaction molar ratio R [R = fatty acid etc. (mol) / glycerin (mol)] of fatty acid etc. is preferably 0.2 to 1.5, more preferably 0.3 to 1.35, in particular. It is preferable that it is 0.4-1.2. Moreover, it is preferable to add 0.1-50 mass parts of said fixed partial glyceride lipase with respect to 100 mass parts of raw materials which totaled fatty acid etc. and glycerin, and also 0.5-40 mass parts, especially 2.5-. 30 parts by mass, particularly 5 to 25 parts by mass is preferable from the viewpoint of improving the reaction rate and efficiently obtaining a high monoacylglycerol-containing fat.

  The reaction temperature of the esterification reaction is preferably 0 to 100 ° C., more preferably 10 to 80 ° C., and particularly preferably 20 to 70 ° C. from the viewpoint of improving the reaction rate and suppressing the deactivation of the enzyme. The reaction time is preferably 1 to 200 hours, more preferably 2 to 150 hours, from the viewpoint of industrial productivity. Moreover, pressure may be adjusted as appropriate, and the pressure condition is preferably 10 to 10,000 Pa, more preferably 100 to 1000 Pa, from the viewpoint of efficiently obtaining a high monoacylglycerol-containing fat.

  Moreover, it is preferable to carry out while removing the reaction product water out of the reaction system from the viewpoint of efficiently obtaining a monoacylglycerol-rich oil and fat. For example, the water produced in the esterification reaction is carried out under substantially anhydrous conditions except for the water contained in the lipase preparation, and the reaction product water is reduced in pressure; the use of an absorbent such as zeolite or molecular sieves; It is removed from the system by a method such as ventilation of a dry inert gas.

  According to the method of the present invention, a monoacylglycerol-rich oil and fat can be produced with as high a reaction rate as possible while hardly producing triacylglycerol and suppressing production of diacylglycerol, using glycerin and fatty acid as raw materials. it can. The resulting monoacylglycerol-rich oil / fat preferably has a monoacylglycerol concentration of 40% by mass or more, more preferably 50% by mass or more, further 60% by mass or more, particularly 70% by mass or more, and particularly 75% by mass. The above is preferable. The monoacylglycerol concentration can be determined by monoacylglycerol / (monoacylglycerol + diacylglycerol + triacylglycerol) × 100 (mass%). In addition, the yield of the monoacylglycerol-containing oil after the esterification reaction is preferably 30% by mass or more from the viewpoint of allowing the esterification reaction to proceed to lower the fatty acid concentration and increase the monoacylglycerol content. Further, it is preferably 40% by mass or more, particularly 50% by mass or more, and particularly preferably 60% by mass or more. Here, the yield of monoacylglycerol-containing fats and oils can be determined by the sum of mass% of monoacylglycerol, diacylglycerol and triacylglycerol in the esterification reaction oil.

  Hereinafter, the present invention will be described in more detail with reference to examples.

[Analysis method]
(I) Measurement of Acid Value According to “Acid Value (2.3.1-1996)” of “Standard Oil Analysis Test Method” edited by Japan Oil Chemistry Association, the free fatty acid contained in 1 g of the sample A value obtained by calculating the number of mg of potassium hydroxide required for summing.

(Ii) Measurement of fatty acid composition Obtained by preparing fatty acid methyl ester according to “Preparation Method of Fatty Acid Methyl Ester (2.4.1.2-1996)” in “Standard Analysis Method for Fats and Oils” edited by Japan Oil Chemistry Association. The samples were measured according to American Oil Chemists. 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 formula (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 [mg KOH / g], y = average molecular weight determined from fatty acid composition)

(Iv) Measurement of glyceride composition About 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.

(V) Measurement of dry weight ratio of immobilized enzyme Washed 3 times 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, and then left at 70 ° C. for 15 hours. Thus, the solvent was removed, and the mass of the immobilized enzyme alone was weighed (b parts by mass), and the mass ratio was determined from the following formula with respect to 100 parts by mass of the immobilized carrier.
Immobilized enzyme dry weight ratio = b / a × 100 (%)
(A: immobilized enzyme mass with oil and water attached, b: immobilized enzyme mass)

[Preparation of immobilized lipase 1]
50 g of Duolite A-568 (Rohm & Hass, average particle size of 480 μm, the same applies hereinafter) was stirred in 500 mL of 0.1N sodium hydroxide aqueous solution for 1 hour. Then, it was washed with 500 mL of distilled water for 1 hour, and the pH was equilibrated with 500 mL of 500 mM acetate buffer (pH 5) for 2 hours. Thereafter, the pH was equilibrated twice with 500 mL of 50 mM acetate buffer (pH 5) for 2 hours each. Thereafter, filtration was performed and the carrier was recovered, followed by ethanol replacement with 250 mL of ethanol for 30 minutes. After filtration, 250 mL of ethanol containing 50 g of soybean fatty acid was added, and the soybean fatty acid was adsorbed on the carrier for 30 minutes. Thereafter, the carrier was recovered by filtration, and then washed four times with 500 mL of 50 mM acetate buffer (pH 5), ethanol was removed, and the carrier was recovered by filtration. Thereafter, 50 g of lipase G “Amano” 50 (Amano Enzyme Co., Ltd., hereinafter the same), which is a partial glyceride lipase, was brought into contact with a lipase solution dissolved in 400 mL of 50 mM acetate buffer (pH 5) for immobilization. . Furthermore, the immobilized lipase was recovered by filtration, and washed with 500 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, 500 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 immobilized lipase 1.

[Preparation of immobilized lipase 2-4]
The immobilized lipase 2 to 4 were obtained in the same manner as the immobilized lipase 1 except that the amount of lipase G “Amano” 50 used was 5, 25, and 100 g.

[Preparation of immobilized lipase 5]
Duolite A-568 500 g was ground and classified with a sieve of 63 μm and 38 μm, and treated in the same manner as immobilized lipase 1 except that an immobilized carrier having an average particle diameter of 51 μm was used to obtain immobilized lipase 5. It was.

  Duolite A-568 500 g was ground and classified with a sieve of 250 μm and 150 μm, and treated in the same manner as immobilized lipase 1 except that an immobilized carrier having an average particle size of 200 μm was used to obtain immobilized lipase 6. It was.

[Preparation of immobilized lipase 7 to 10]
The lipase was treated in the same manner as the immobilized lipase 1 except that the lipase was lipase A-10FG (Nagase ChemteX Corp.), which is a 1,3-position selective lipase, and the amount used was 5, 25, 50, 100 g. Immobilized lipases 7 to 10 were obtained.

[Preparation of immobilized lipase 11]
20 g of Duolite A-568 was stirred in 200 mL of 0.1N aqueous sodium hydroxide for 1 hour. Then, it was washed with 200 mL of distilled water for 1 hour, and equilibrated with 200 mL of 500 mM phosphate buffer (pH 7) for 2 hours. Thereafter, the pH was equilibrated twice with 200 mL of 50 mM phosphate buffer (pH 7) for 2 hours each. Thereafter, filtration was performed and the carrier was recovered, followed by ethanol replacement with 100 mL of ethanol for 30 minutes. After filtration, 100 mL of ethanol containing 20 g of soybean fatty acid was added, and the 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 phosphate buffer (pH 7), ethanol was removed, and the carrier was recovered by filtration. Thereafter, 20 g of lipase AY “Amano” 30G (Amano Enzyme Co., Ltd.), a non-selective lipase, was contacted with a lipase solution dissolved in 400 mL of 50 mM phosphate buffer (pH 7) for 4 hours for immobilization. Furthermore, the immobilized lipase was recovered by filtration, and washed with 100 mL of 50 mM phosphate buffer (pH 7) to remove non-immobilized lipase and protein. All the above operations were performed at a temperature of 20 ° C. Thereafter, 100 g of deodorized soybean oil was added and stirred at a temperature of 40 ° C. for 10 hours, followed by filtration to separate from the deodorized soybean oil, whereby immobilized lipase 11 was obtained.

[Raw fatty acid]
The fatty acid used as a reaction raw material was obtained by hydrolyzing fats and oils with an oil-water countercurrent type high-pressure hydrothermal cracker. Soybean oil was continuously fed from the lower side of the high-pressure hydrothermal decomposition apparatus, and water was continuously fed from the upper side of the apparatus. The liquid feeding amount was 50 parts by mass of water with respect to 100 parts by mass of the raw material fats and oils. At this time, the average residence time (hr) in the decomposition tower (column volume (m ³ ) / (flow rate of raw material oil (m ³ / hr) + flow rate of water (m ³ / hr))) was about 4 hr. . In the apparatus, the raw oil and fat was heated with high-pressure hot water (5.0 MPa, 240 ° C.). The reaction solution was collected from a sampling port in the middle of the oil-water counter-flow type high-pressure hydrothermal decomposition apparatus, sealed with nitrogen, and cooled to 25 ° C. in a light-shielded state. Thereafter, the mixture was centrifuged (5,000 g, 30 minutes), and after removing the aqueous layer, 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 a raw fatty acid X. In addition, the raw material fatty acid X was operated at a heater temperature of 230 ° C., a pressure of 2 Pa, and a flow rate of 150 ml / hr using a wipe film evaporator (Shinko Pantech 2-03 type, inner diameter 5 cm, heat transfer area 0.03 m ² ). Distillation was performed under conditions to obtain a raw material fatty acid Y. Each glyceride composition is shown in Table 1. Here, FFA is a free fatty acid, GLY is glycerin, MAG is monoacylglycerol, DAG is diacylglycerol, and TAG is triacylglycerol (the same applies hereinafter).

[Esterification activity of immobilized lipase]
A 300 ml four-necked flask with a crescent blade set was charged with 5 g of immobilized lipase based on 100 g of the raw fatty acid shown in Table 1 and the esterified raw material on a dry mass basis, at a temperature of 40 ° C., a stirring of 400 rpm, and a pressure of 400 Pa. Dehydrated under reduced pressure for minutes. Then, it wash | cleaned 3 times with raw material fatty acid. Thereafter, raw material fatty acid was added, and then glycerin was added to start the reaction, and the pressure was reduced with a vacuum pump. The esterification reaction was carried out at a temperature of 40 ° C., a stirring of 400 rpm, and a pressure of 400 Pa. The total of raw material fatty acid and glycerin was 100 g, and the molar ratio of FFA / GLY was 0.6. The reaction solution after 60 minutes was sampled, and the esterification activity was calculated from the value of the fatty acid concentration. The average molecular weight calculated from the fatty acid composition of each raw fatty acid was 279 for both raw fatty acids X and Y. Table 2 shows the preparation conditions and physical property values of the prepared immobilized lipase.

[Esterification reaction 1-18]
A 300 ml four-necked flask set with crescent moon blades was charged with 100 g of either X or Y raw fatty acid and immobilized lipase shown in Table 3 in an amount shown in Table 3 on a dry mass basis, temperature 40 ° C., stirring 400 rpm And dehydration under reduced pressure at 400 Pa for 30 minutes. Then, it wash | cleaned 3 times with raw material fatty acid. Thereafter, the starting fatty acid was added, and then the reaction was started by adding glycerin while stirring, and the pressure was reduced with a vacuum pump. The esterification reaction was performed at a temperature of 40 ° C., with stirring at 400 rpm, and a pressure of 400 Pa. The total of the raw fatty acid and glycerin was 100 g, and the molar ratio of FFA / GLY shown in Table 3 was used. The reaction solution was sampled at the reaction time shown in Table 3 to determine the glyceride composition.

[Esterification reaction 19-24]
Raw material fatty acid (X) and glycerin shown in Table 3 were added to a 300 ml four-necked flask with a crescent blade set, and stirring was started, and the amount of powdered lipase G shown in Table 4 was added. The esterification reaction was carried out at a temperature of 40 ° C., stirring at 400 rpm, and the pressure shown in Table 4. In the case of reduced pressure, a vacuum pump was used. The total of the raw material fatty acid and glycerin was 100 g, and the molar ratio of FFA / GLY shown in Table 4 was used. The reaction solution was sampled at the reaction time shown in Table 4 to determine the glyceride composition.

  From Tables 3 and 4, esterification reactions 3 to 5 and 9 to 15 using an immobilized partial glyceride lipase having an A value defined in the present invention all had high MAG concentrations, but the lipase species and the A value. The MAG concentration could not be increased in those not satisfying any one or more requirements for immobilization.

(I) Influence of lipase species By comparing esterification reactions 1 to 3 and 16 to 18, in the esterification reaction using immobilized lipase, the one in which lipase G, which is a partial glyceride lipase, is immobilized is selected in the 1,3-position The MAG concentration was the highest as compared with the immobilized lipase lipase and non-selective lipase lipase AY. Moreover, those using lipase could not increase the MAG concentration even when the A value was within the range defined by the present invention (esterification reactions 16 to 18).

(II) Effect of A value From the comparison of esterification reactions 3 to 15, in the esterification reaction performed using immobilized partial glyceride lipase within the range of the A value defined in the present invention, using immobilized partial glyceride lipase. In both cases, the MAG concentration increased, but when the A value exceeded the upper limit, the MAG concentration could not be increased, and the same was true even if the enzyme concentration was decreased (esterification reaction). 6-8). Further, the higher the A value, the faster the reaction rate, but at the same time, DAG tended to be by-produced (esterification reactions 8, 9 and 13). Further, when the feed ratio of the esterification reaction raw material is set to FFA / GLY = 0.2, the MAG concentration becomes higher, but the amount of glycerin in the reaction system increases, which is disadvantageous in terms of yield (esterification reaction 3 In addition, the glyceride composition of the reaction end oil shown in Tables 3 and 4 was obtained by sampling and centrifuging the reaction end oil and centrifuging the unreacted glycerin as described in the above “Measurement of glyceride composition”. Since it is measured after removal, only the amount of glycerin dissolved in the reaction-finished oil is measured, so the greater the amount of MAG having high affinity with glycerin in the reaction-finished oil, the more glycerin is actually In fact, it is the esterification reaction number 4 that “there is a lot of glycerin in the reaction system”, and 62% of glycerin in 100 g of the charged raw material. Against it, glycerin was precipitated by centrifugation removal was 52%.).
Further, if the carrier particle size is made smaller and the surface area of the immobilized carrier is made larger, it is possible to achieve both shortening of the reaction time and higher concentration of MAG, and the effect is particularly high when the carrier particle size is 200 μm or less. (Esterification reactions 10 and 11). Furthermore, when an immobilized partial glyceride lipase having a low A value was used at a high concentration, it was possible to achieve both a shortening of the reaction time and a high concentration of MAG (esterification reactions 12 and 13). At this time, even if the feed ratio of the esterification reaction raw material is set to FFA / GLY = 1.0, both high yield and high MAG concentration can be achieved (comparison between esterification reaction 14 and 15), and the fatty acid concentration of the raw fatty acid is high. The MAG concentration became higher (comparison between esterification reactions 12 and 14).

(III) Effect of lipase immobilization In the esterification reactions 19 to 24 carried out using non-immobilized powdered partial glyceride lipase, high MAG concentration and high yield were obtained under both reduced pressure and normal pressure conditions. I was unable to achieve both.

Claims (3)

A process for producing fats and oils with a high monoacylglycerol content in which fatty acid or a lower alkyl ester thereof and glycerin are reacted in the presence of an immobilized partial glyceride lipase having an esterification activity per unit surface area of the immobilized carrier of 200 to 10,000 U / m ² .   2. The mono of claim 1, wherein the reaction molar ratio R [R = fatty acid or its lower alkyl ester (mol) / glycerin (mol)] of fatty acid or its lower alkyl ester and glycerin is 0.2 to 1.5. A method for producing an acylglycerol-rich oil and fat.   The method for producing a fat and oil having a high monoacylglycerol content according to claim 1 or 2, wherein the immobilization carrier is an ion exchange resin.