Method of manufacturing oils and fats

Abstract

This invention provides a novel and effective method for efficiently reducing diacylglycerol in oils and fats (by converting it to triacylglycerol). [Solution] A method for producing oil and fat comprising the step of reacting a diacylglycerol-containing oil or fat with a lipase having a ratio of transesterification activity to hydrolysis activity of 0.007 or more and 0.5 or less, wherein the lipase is an immobilized lipase, the water content of the diacylglycerol-containing oil or fat is 110 ppm or more, the value of the hydrolysis activity is the value obtained by multiplying the value of the hydrolysis activity (u / g) of the lipase before immobilization treatment by the amount of lipase used for immobilization (g) and dividing by the amount of immobilized lipase (g), and the value of the transesterification activity is the value of the transesterification activity of the immobilized lipase.

Description

Technical Field

[0001] The present invention relates to a method for producing fats and oils. More specifically, it relates to a method for producing fats and oils using lipase.

Background Art

[0002] Regarding fats and oils such as palm oil, conversion into high-quality fats and oils is carried out by removing diacylglycerol or converting it into triacylglycerol (for example, see Patent Document 1). In addition, modification is carried out by changing the fatty acid composition of triacylglycerol using the ester exchange reaction of lipase to produce fats and oils with high added value (for example, see Patent Documents 2 and 3). Generally, when performing both removal of diacylglycerol / conversion into triacylglycerol and modification of the fatty acid composition of triacylglycerol, a two-step process is required.

[0003] Patent Document 1 cited above discloses reducing the amount of diacylglycerol in diacylglycerol-containing fats and oils and changing the fatty acid composition of triacylglycerol using lipase derived from the genus Pseudomonas or the genus Rhizopus. Further, Patent Document 1 shows that when the water content of the fat and oil on which the enzyme acts is 110 ppm or more, the yield of triacylglycerol decreases.

[0004] On the other hand, as a lipase that can be used for the production and modification of fats and oils, Patent Document 4 discloses a lipase derived from Penicillium sp. The lipase is said to act on monoglyceride (monoacylglycerol) and diglyceride (diacylglycerol), but not at all on triglyceride (triacylglycerol).

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

[0006] Against this background, the present invention aims to provide a novel means that is effective in efficiently reducing diacylglycerol in oils and fats (by converting it to triacylglycerol). [Means for solving the problem]

[0007] In order to solve the above problems, we conducted extensive research focusing on the "ratio of hydrolysis activity to transesterification activity" of the enzyme used, and as a result, we discovered an efficient and effective method for modifying oils and fats (in other words, a method for producing modified oils and fats). Based on these results, the following invention is provided. [1] A method for producing oils and fats, comprising the step of reacting a diacylglycerol-containing oil or fat with a lipase having a ratio of transesterification activity to hydrolysis activity of 0.007 or more. [2] The method for producing oil and fat according to [1], wherein the water content of the diacylglycerol-containing oil and fat is 110 ppm or more. [3] The manufacturing method according to [1] or [2], wherein the ratio is 0.01 or greater. [4] The manufacturing method according to any one of [1] to [3], wherein the lipase is an immobilized lipase. [5] The manufacturing method according to any one of [2] to [4], wherein the water content is 1000 ppm or less. [6] A method for producing oils and fats, comprising the step of reacting a lipase having a ratio of transesterification activity to hydrolysis activity of 0.001 or more with a diacylglycerol-containing oil or fat having a water content of less than 110 ppm. [Modes for carrying out the invention]

[0008] This invention relates to a method for producing oils and fats using lipase. The production method of this invention is characterized by a step of acting a lipase having a ratio of transesterification activity to hydrolysis activity, i.e., "transesterification activity / hydrolysis activity" of 0.007 or more, on a diacylglycerol-containing oil or fat, preferably a step of acting on a diacylglycerol-containing oil or fat with a water content of 110 ppm or more, or a step of acting a lipase having a ratio of 0.001 or more on a diacylglycerol-containing oil or fat with a water content of less than 110 ppm. In this step, the diacylglycerol in the diacylglycerol-containing oil or fat reacts with free fatty acids or free fatty acid esters by the action of the lipase to produce triacylglycerol. As a result, the oil or fat is modified. In other words, modified oil or fat is obtained.

[0009] Lipases typically exhibit hydrolytic activity in aqueous reaction systems and transesterification activity in oil-based reaction systems. Furthermore, transesterification activity can be improved by immobilization or other treatments applied to lipases. Conventionally, when performing transesterification or esterification reactions in oil-based systems, it was common to select an enzyme based solely on its transesterification activity. However, since substrates (such as oils and fats) contain trace amounts of water, it is possible that hydrolytic activity also influences reaction efficiency. Based on this idea, investigations revealed that the ratio of hydrolytic activity to transesterification activity is important for the efficiency of esterification reactions in oil-based systems, leading to the completion of this invention. The ratio of hydrolytic activity to transesterification activity can be calculated as follows. Note that hydrolytic activity and transesterification activity can be measured by the methods described later.

[0010] For hydrolysis activity, first, the hydrolysis activity of the lipase before immobilization is measured. Then, the hydrolysis activity value per gram of immobilized lipase is calculated from the amount of lipase used for immobilization. For example, if the hydrolysis activity of the lipase before immobilization is 10,000 u / g, and 1 g of this lipase (equivalent to 1 g in terms of activity) is used for immobilization, resulting in 4 g of immobilized lipase, then the hydrolysis activity value used to calculate the ratio with transesterification activity is 10,000 u / g × 1 g ÷ 4 g = 2,500 u / g. On the other hand, for transesterification activity, the lipase after immobilization is used. That is, the transesterification activity of the immobilized lipase is measured using the method described below, and the measurement result (transesterification activity value of the immobilized lipase) is used to calculate the ratio with hydrolysis activity. Note that the above is the calculation method for immobilized lipase, but the same method can be used to calculate each activity for lipase treated by methods other than immobilization. Furthermore, if the lipase is untreated, its measured values ​​(hydrolysis activity value, transesterification activity value) can be used directly in the ratio calculation.

[0011] The ratio of transesterification activity to hydrolysis activity of lipase is not particularly limited as long as the diacylglycerol in the diacylglycerol-containing oil is reduced by the esterification reaction. An example of the above ratio is preferably 0.007 or higher when lipase is applied to a diacylglycerol-containing oil with a water content of 110 ppm or more. From the viewpoint of further reducing the diacylglycerol in the diacylglycerol-containing oil, the ratio in the above example is preferably 0.01 or higher, more preferably 0.02 or higher, even more preferably 0.03 or higher, even more preferably 0.05 or higher, and even more preferably 0.07 or higher. There is no particular upper limit to the ratio, but examples include 0.5 or less, 0.3 or less, or 0.1 or less.

[0012] Another example of the above ratio is when lipase is applied to a diacylglycerol-containing oil with a water content of less than 110 ppm, where the ratio is preferably 0.001 or higher. From the viewpoint of further reducing the diacylglycerol in the diacylglycerol-containing oil, the ratio in this example is preferably 0.002 or higher, more preferably 0.003 or higher. There is no particular upper limit to the ratio, but examples include 0.5 or less, 0.3 or less, 0.1 or less, 0.05 or less, 0.01 or less, or 0.005 or less.

[0013] As long as the ratio of transesterification activity to hydrolysis activity satisfies the above conditions, the origin of the lipase is not particularly limited. For example, lipases derived from the genera Rhizopus, Penicillium, Burkholderia, Aspergillus, Candida, Pseudomonas, Mucor, Thermomyces, or Geotrichum can be used. Preferably, lipases derived from the genera Rhizopus, Penicillium, or Burkholderia are used. An example of a lipase derived from the genus Rhizopus is the lipase produced by Rhizopusoryzae (specifically, lipase DF (Amano Enzyme Co.)), an example of a lipase derived from the genus Penicillium is the lipase produced by Penicillium camembertii (specifically, lipase G (Amano Enzyme Co.)), and an example of a lipase derived from the genus Burkholderia is the lipase produced by Burkholderia cepacia (specifically, lipase PS (Amano Enzyme Co.)).

[0014] The above lipases may be used individually or in combination of multiple types. When the lipase is applied to a diacylglycerol-containing oil with a water content of 110 ppm or more, among the above lipases, lipases derived from the genus Rhizopus (preferably Rhizopus oryzae) or the genus Penicillium (preferably Penicillium camembertii) are preferred, and more preferably lipases derived from the genus Rhizopus (preferably Rhizopus oryzae) are preferred. When lipase is applied to diacylglycerol-containing oils with a water content of less than 110 ppm, among the above lipases, lipases derived from the genus Rhizopus (preferably Rhizopus oryzae) or the genus Burkhorrideria (preferably Burkhorrideria cepacia) are preferred, and more preferably lipases derived from the genus Burkhorrideria (preferably Burkhorrideria cepacia).

[0015] In one aspect of the present invention, lipase immobilized on a carrier (immobilized lipase) is used. Immobilized lipase can be prepared according to conventional methods. Alternatively, commercially available immobilized lipase can be used. Furthermore, treatments such as immobilization can be used to change the ratio of hydrolytic activity to transesterification activity.

[0016] Examples of oils and fats on which lipase can act include vegetable oils such as soybean oil, rapeseed oil, rice oil, corn oil, sunflower oil, cottonseed oil, peanut oil, safflower oil, olive oil, palm oil, palm soft oil, fractionated palm oil, palm kernel oil, coconut oil, and cocoa butter; animal oils such as fish oil, lard, beef tallow, and milk fat; and fractionated oils of these, hydrogenated oils, and synthetic oils such as trilaurin, triolein, and tripalmitin.

[0017] If necessary, fatty acids such as myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, and γ-linolenic acid or their esters (hereinafter, the expression “fatty acids etc.” is used as an expression encompassing fatty acids and fatty acid esters) may be added alone or in combination of two or more kinds. Also, these fatty acids may originally be contained in the oil or fat. The addition amount or content of the fatty acids etc. is, for example, 1 to 1000 parts by weight, preferably 1.5 to 100 parts by weight, more preferably 2 to 30 parts by weight, still more preferably 2.5 to 10 parts by weight, 3 to 7.5 parts by weight, or 3.5 to 5 parts by weight per 100 parts by weight of the oil or fat.

[0018] To carry out an ester synthesis reaction using lipase, for example, lipase may be added to a diacylglycerol-containing oil or fat and reacted. Specifically, the water content (water concentration; hereinafter, simply referred to as “water content”) of the reaction system (including the raw material oil (oil or fat, or a mixture of oil or fat and fatty acids etc.) and the enzyme) at the start of the reaction is not particularly limited and is adjusted to less than 110 ppm (for example, 10 ppm or more and less than 110 ppm, preferably 40 to 105 ppm, more preferably 60 to 100 ppm, still more preferably 80 to 97 ppm) or 110 ppm or more (for example, 110 to 1000 ppm, preferably 150 to 800 ppm, more preferably 200 ppm to 500 ppm, still more preferably 200 ppm to 400 ppm, even more preferably 220 to 300 ppm), and then lipase is added and reacted. The higher the water content of the reaction system, the more likely a hydrolysis reaction occurs rather than an ester synthesis reaction. Therefore, when the water content of the reaction system is high, it is preferable that the ratio of the hydrolysis activity to the transesterification activity of the lipase is high.

[0019] For example, the reaction is carried out under the conditions of 30 to 100 °C, preferably 35 to 80 °C, for a predetermined time (for example, 1 hour to 48 hours). To promote the reaction, stirring during the reaction is advisable. In addition to batch-type reactions, continuous reactions using a column, a flow cell, etc. can be employed. For reactions using immobilized lipase, a batch stirred tank reactor, a flow-through stirred tank reactor, a packed bed reactor, a fluidized bed reactor, etc. can be utilized.

[0020] The addition amount of lipase is not particularly limited as long as the desired reaction proceeds. For example, 10 to 100 parts by weight may be added per 100 parts by weight of the raw material oil.

[0021] As the ester synthesis reaction proceeds, water is generated along with the production of triacylglycerol. Therefore, it is preferable to remove the water from the reaction system. For example, water can be removed by distillation under reduced pressure, using a dehydrating agent such as molecular sieves, or using a dry inert gas such as nitrogen gas.

Examples

[0022] The diacylglycerol (DG) reduction effects of various lipases with different ratios of transesterification activity to hydrolysis activity were examined (Test Examples 1 and 2). The hydrolysis activity and transesterification activity were measured by the following methods. (Method for measuring hydrolysis activity) The hydrolysis activity was measured using a lipase kit S (SB Bioscience Co., Ltd.). A color-developing stock solution was prepared by adding 2.4 mL of a pH 7.0-adjusted buffer solution attached to the kit and 22 mL of purified water to the color-developing agent attached to the kit. A color-developing solution was prepared by adding 250 μL of the pH 7.0-adjusted buffer solution attached to the kit and 2000 μL of purified water to 250 μL of the color-developing stock solution. 1 mL of the color-developing solution and 50 μL of a diluted solution obtained by diluting the enzyme solution to an appropriate concentration were placed in a test tube, incubated at 37 °C for 5 minutes, then 100 μL of the substrate solution attached to the kit was added, and after reacting at 37 °C for 15 minutes, 2 mL of acetone was added to stop the reaction. The supernatant of the sample after the reaction was stopped was collected, and the absorbance was measured at 412 nm. Using the sample with the substrate solution added after adding acetone as a blank, the hydrolysis activity (U / g) was calculated from the following formula. Hydrolysis activity (U / g) = (A412sample - A412blank) × 20 × n (However, A412sample is the absorbance at 412 nm of the supernatant of the sample, A412blank is the absorbance at 412 nm of the blank, 20 is a coefficient, and n is the dilution factor of the enzyme solution) (Method for measuring transesterification activity) 5 mL of tricaprylin (manufactured by Wako Pure Chemical Industries, Ltd.) and 6 mL of methyl laurate (manufactured by Wako Pure Chemical Industries, Ltd.) were added to a 50 mL disposable centrifuge tube, and the tube was preheated at 30 ± 1 °C for 10 minutes. 0.1 g of the enzyme sample was added, the tube was capped, and mixed by inversion. The tube was then placed on a rotator and the enzymatic reaction was carried out at 30 °C and 50 rpm for 30 minutes. 30 μL of the resulting solution was dissolved in 1 mL of hexane and used as a sample for gas chromatography. Gas chromatography analysis (column: DB-1HT (Agilent J&W, 5m x 0.25mm, df 0.1μm), temperature conditions: 50°C, held for 1 minute, then heated to 370°C at a rate of 40°C / min, detector: FID, carrier gas: helium) was used to determine the area value of methyl capryphosphate produced as a result of the enzymatic reaction (transesterification), and the transesterification activity was calculated using the following formula. Transesterification activity (u / g) = A / a × 34 × 1 / 30 × 1 / 0.1 × 11 A: Area value of methyl capryphosphate in the sample a: The slope calculated from a calibration curve created from the area values ​​and methyl octanoate concentration (mmol / L) when methyl octanoate in concentrations of 1 mmol / L to 7.5 mmol / L was subjected to gas chromatography under the above conditions (area value for methyl octanoate = a × methyl octanoate concentration). 34: Dilution ratio when the reaction solution is diluted with hexane *(1mL + 30μL) / 30μL ≈ 34 1 / 30: Conversion factor to reaction time per minute 1 / 0.1: Conversion factor per gram of sample 11: Reaction volume (mL) * Tricaprylin 5mL + Methyl laurate 6mL

[0023] <Test Example 1> 1. Method (1) Preparation of enzyme samples Each lipase (Rhizopusoryzae-derived lipase and Burkholderiacepacia-derived lipase) was prepared according to conventional methods, and its hydrolytic and transesterification activities were measured. The hydrolytic and transesterification activities of each lipase are shown in Table 1.

[0024] (2) Preparation of substrate oils and fats A mixture of refined palm oil, glycerol dioleate, palmitic acid, and oleic acid (TG content 89.4% by weight, DG content 6.7% by weight, the remainder being mainly fatty acids) was dried under reduced pressure at 80°C for 12 hours or more until the moisture content reached 95 ppm.

[0025] (3) Reaction 0.2 g of enzyme sample, 10 g of molecular sieve 3A (dried under reduced pressure at 180°C for more than 12 hours), and 20 g of substrate oil were added to an Erlenmeyer flask and reacted by shaking at 60°C and 160 rpm. GC analysis was performed on the reaction solution after 22 hours to confirm the change in the composition of the substrate oil. The residual DG percentage was calculated from the DG ratio before and after the reaction.

[0026] 2.Results The DG reduction effect of each sample (with a water content of 95 ppm in the reaction system at the start of the reaction) is shown in the table below. [Table 1]

[0027] When the water content was 95 ppm, a DG reduction effect was confirmed even when using lipases with a low ratio of hydrolysis activity to transesterification activity.

[0028] <Test Example 2> 1. Method (1) Enzyme sample Commercially available immobilized enzymes (LDF-IM, LGS-IM, LPS-IM, all manufactured by Amano Enzyme Co., Ltd.) were used as enzyme samples.

[0029] (2) Preparation of substrate oils and fats Crude palm oil (hereinafter referred to as CPO) (TG content 92.5% by weight, DG content 4.8% by weight, remainder mainly fatty acids) was dried under reduced pressure at 80°C for more than 2 hours to adjust the moisture content to 228 ppm.

[0030] (3) Reaction 1 g of enzyme sample, 10 g of molecular sieve 3A (dried under reduced pressure at 180°C for more than 12 hours), and 20 g of substrate oil were added to an Erlenmeyer flask and reacted by shaking at 60°C and 160 rpm. After 24 hours, the reaction solution was subjected to GC analysis to confirm the change in the composition of the substrate oil.

[0031] 2.Results The DG reduction effect of each sample (with a reaction system water content of 228 ppm at the start of the reaction) is shown in the table below. [Table 2]

[0032] At a water content of 228 ppm, no DG reduction effect was observed with lipases with a low ratio of hydrolysis activity to transesterification activity (LPS-IM), but a DG reduction effect was observed with other lipases with a high ratio of hydrolysis activity to transesterification activity. Furthermore, it was suggested that the ratio of hydrolysis activity to transesterification activity is more important than the amount of activity for DG reduction.

[0033] The fatty acid composition of each sample after reaction is shown in the table below. [Table 3] The "theoretical value" is calculated based on the relative abundance of free fatty acids and DG in the substrate oil, assuming that no transesterification reaction occurs and only the synthesis reaction between free fatty acids and DG in the substrate oil takes place.

[0034] In sample 3, both esterification and transesterification reactions were confirmed to occur (Tables 2 and 3). On the other hand, in sample 4, esterification reactions occurred (Table 2), but transesterification reactions did not (Table 3). Furthermore, in sample 5, esterification reactions did not occur (Table 2), but transesterification reactions did occur (Table 3). These results indicate that esterification reactions occur when the ratio of hydrolysis activity to transesterification activity is high, regardless of the presence or absence of transesterification activity. [Industrial applicability]

[0035] The present invention is useful for modifying and improving the physical properties of oils and fats or processed oil and fat products (e.g., shortening, margarine). For example, the present invention can be applied to improve spreadability, improve emulsion stability, optimize solid fat content (SFC), improve solidification properties, selectively concentrate specific fatty acids, and produce low trans fatty acid oils or processed oils. Oils and fats obtained by applying the present invention or processed oil and fat products containing them show improved physical properties and have high industrial value.

[0036] This invention is not limited in any way to the descriptions of embodiments and examples of the invention described above. Various modifications are also included in this invention, provided that they do not depart from the scope of the claims and are easily conceivable by those skilled in the art. The contents of papers, published patent gazettes, and other documents explicitly mentioned herein shall be cited in their entirety.

Claims

1. The process includes a step of reacting a diacylglycerol-containing oil or fat with a lipase having a ratio of transesterification activity to hydrolysis activity of 0.007 or more and 0.5 or less. The aforementioned lipase is an immobilized lipase, The water content of the aforementioned diacylglycerol-containing oil is 110 ppm or more. The hydrolysis activity value is obtained by multiplying the hydrolysis activity (u / g) of the lipase before immobilization treatment by the amount of lipase used for immobilization (g), and then dividing by the amount of immobilized lipase (g). A method for producing oils and fats, wherein the transesterification activity value is the same as the transesterification activity value of the immobilized lipase.

2. The manufacturing method according to claim 1, wherein the ratio is 0.01 or more.

3. The manufacturing method according to claim 1 or 2, wherein the water content is 1000 ppm or less.

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