Oil and fat manufacturing method

Abstract

The present invention addresses the problem of providing an effective new means for efficiently reducing the amount of diacylglycerol in a fat / oil (by conversion into triacylglycerol). Disclosed is a method for producing a fat / oil, including a step in which a lipase having a ratio of transesterification activity to hydrolysis activity of 0.007 or higher is caused to act on a diacylglycerol-containing fat / oil.

Description

[Technical Field]

[0001] The present invention relates to a method for producing fats and oils, and more particularly to a method for producing fats and oils using lipase. [Background technology]

[0002] Fats and oils such as palm oil have been converted into high-quality fats and oils by removing diacylglycerol or converting it to triacylglycerol (see, for example, Patent Document 1). Furthermore, fats and oils with high added value have been produced by modifying the fatty acid composition of triacylglycerol using a transesterification reaction of lipase (see, for example, Patent Documents 2 and 3). Generally, two-stage processes are required to remove diacylglycerol / convert it to triacylglycerol and to modify the fatty acid composition of triacylglycerol.

[0003] The above-mentioned Patent Document 1 discloses that the amount of diacylglycerol in a diacylglycerol-containing fat or oil is reduced and the fatty acid composition of triacylglycerol is changed using a lipase derived from the genus Pseudomonas or Rhizopus. Patent Document 1 also discloses that the yield of triacylglycerol decreases when the water content of the fat or oil on which the enzyme is reacted is 110 ppm or more.

[0004] Meanwhile, Patent Document 4 discloses a lipase derived from Penicillium sp. that can be used for producing and modifying fats and oils. This lipase acts on monoglycerides (monoacylglycerols) and diglycerides (diacylglycerols), but is not at all active on triglycerides (triacylglycerols). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Publication No. 64-2588 [Patent Document 2] International Publication No. 2012 / 077614 Brochure [Patent Document 3] International Publication No. 2019 / 155789 Brochure [Patent Document 4] Japanese Patent Application Publication No. 2-174676 Summary of the Invention [Problem to be solved by the invention]

[0006] Under the above circumstances, an object of the present invention is to provide a new and effective means for efficiently reducing diacylglycerol in fats and oils (by converting it to triacylglycerol). [Means for solving the problem]

[0007] In order to solve the above problems, we have conducted extensive research focusing on the "ratio of hydrolysis activity to transesterification activity" of the enzymes used, and as a result, we have discovered a method for efficiently and effectively modifying fats and oils (in other words, a method for producing modified fats and oils). Based on this result, the following invention is provided. [1] A method for producing fats and oils, comprising a step of allowing a lipase having a ratio of transesterification activity to hydrolysis activity of 0.007 or more to act on a diacylglycerol-containing fat and oil. [2] The method for producing an oil or fat according to [1], wherein the water content of the diacylglycerol-containing oil or fat is 110 ppm or more. [3] The manufacturing method according to [1] or [2], wherein the ratio is 0.01 or more. [4] The method according to any one of [1] to [3], wherein the lipase is an immobilized lipase. [5] The method according to any one of [2] to [4], wherein the water content is 1000 ppm or less. [6] A method for producing fats and oils, comprising a step of allowing a lipase having a ratio of transesterification activity to hydrolysis activity of 0.001 or more to act on a diacylglycerol-containing fat and oil having a water content of less than 110 ppm. DETAILED DESCRIPTION OF THE INVENTION

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

[0009] Lipases typically exhibit hydrolysis activity when the reaction system is aqueous, and transesterification activity when the reaction system is oil-based. Furthermore, transesterification activity can be improved by treating lipases with immobilization or other treatments. Conventionally, when performing transesterification or ester synthesis reactions in oil systems, it has been common to determine the enzyme to be used based solely on transesterification activity. However, since substrates (oils and fats, etc.) contain trace amounts of water, it is possible that hydrolysis activity also affects the reaction efficiency, etc. Based on this concept, the researchers discovered that the ratio between hydrolysis activity and transesterification activity is important for the efficiency of ester synthesis reactions in oil systems, leading to the completion of the present invention. The ratio between hydrolysis activity and transesterification activity can be calculated as follows. Hydrolysis activity and transesterification activity can be measured using the method described below.

[0010] Regarding hydrolytic activity, first, the hydrolytic activity of the lipase before immobilization is measured. Then, the hydrolytic activity per 1 g of immobilized lipase is calculated from the amount of lipase used in the immobilization. For example, if the hydrolytic 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, the hydrolytic activity used to calculate the ratio to the transesterification activity is 10,000 u / g × 1 g ÷ 4 g = 2,500 u / g. Meanwhile, 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 of the immobilized lipase) is used to calculate the ratio to the hydrolytic activity. Note that the above calculation method is for immobilized lipase, but the respective activities can also be calculated in a similar manner for lipases treated by methods other than immobilization. In addition, in the case of untreated lipase, the measured values ​​(hydrolysis activity value, transesterification activity value) may be directly used in calculating the ratio.

[0011] The ratio of transesterification activity to hydrolysis activity of lipase is not particularly limited as long as it can reduce diacylglycerol in diacylglycerol-containing fats and oils by ester synthesis reaction. An example of the ratio is preferably 0.007 or more when lipase is applied to diacylglycerol-containing fats and oils having a water content of 110 ppm or more. From the viewpoint of further reducing diacylglycerol in diacylglycerol-containing fats and oils, the ratio in this example is preferably 0.01 or more, more preferably 0.02 or more, even more preferably 0.03 or more, even more preferably 0.05 or more, and even more preferably 0.07 or more. The upper limit of this ratio is not particularly limited, but examples include 0.5 or less, 0.3 or less, or 0.1 or less.

[0012] Another example of the ratio is preferably 0.001 or more when lipase is applied to diacylglycerol-containing fats and oils having a water content of less than 110 ppm. From the viewpoint of further reducing diacylglycerol in diacylglycerol-containing fats and oils, the ratio in this example is preferably 0.002 or more, more preferably 0.003 or more. The upper limit of the ratio is not particularly limited, but examples include 0.5 or less, 0.3 or less, 0.1 or less, 0.05 or less, 0.01 or less, and 0.005 or less.

[0013] The origin of the lipase is not particularly limited as long as the ratio of transesterification activity to hydrolysis activity satisfies the above-mentioned condition. For example, lipases derived from the genus Rhizopus, Penicillium, Burkholderia, Aspergillus, Candida, Pseudomonas, Mucor, Thermomyces, or Geotrichum can be used. Preferably, lipases derived from the genus Rhizopus, Penicillium, or Burkholderia are used. An example of a lipase derived from the genus Rhizopus is a lipase produced by Rhizopus oryzae (a specific example is Lipase DF (Amano Enzyme Co., Ltd.)), an example of a lipase derived from the genus Penicillium is a lipase produced by Penicillium camenbertii (a specific example is Lipase G (Amano Enzyme Co., Ltd.)), and an example of a lipase derived from the genus Burkholderia is a lipase produced by Burkholderia cepacia (a specific example is Lipase PS (Amano Enzyme Co., Ltd.)).

[0014] The lipases may be used singly or in combination. When the lipase is to be applied to a diacylglycerol-containing fat or oil having a water content of 110 ppm or more, among the above lipases, preferred are lipases derived from the genus Rhizopus (preferably Rhizopus oryzae) or Penicillium (preferably Penicillium camembertii), more preferably lipases derived from the genus Rhizopus (preferably Rhizopus oryzae). When a lipase is allowed to act on a diacylglycerol-containing fat or oil having a water content of less than 110 ppm, among the above-mentioned lipases, a lipase derived from the genus Rhizopus (preferably Rhizopus oryzae) or the genus Burkholderia (preferably Burkholderia cepacia) is preferred, and a lipase derived from the genus Burkholderia (preferably Burkholderia cepacia) is more preferred.

[0015] In one embodiment of the present invention, a lipase immobilized on a carrier (immobilized lipase) is used. The immobilized lipase can be prepared by a conventional method. Commercially available immobilized lipases can also be used. Furthermore, treatments such as immobilization can be used to change the ratio of hydrolysis activity to transesterification activity.

[0016] Examples of fats and oils on which lipase can be applied include vegetable fats and oils such as soybean oil, rapeseed oil, rice oil, corn oil, sunflower oil, cottonseed oil, peanut oil, safflower oil, olive oil, palm oil, soft palm oil, fractionated palm oil, palm kernel oil, coconut oil, and cocoa butter; animal fats and oils such as fish oil, lard, beef tallow, and milk fat; and fractionated oils thereof, hardened oils, and synthetic fats and 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 term "fatty acids, etc." is used to encompass fatty acids and fatty acid esters) may be added to the fat or oil, either singly or in combination of two or more. These fatty acids may also be those originally contained in the fat or oil. The amount or content of the fatty acids, etc. added is, for example, 1 to 1,000 parts by weight, preferably 1.5 to 100 parts by weight, more preferably 2 to 30 parts by weight, and even 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 fat or oil.

[0018] To carry out an ester synthesis reaction using lipase, for example, lipase may be added to a diacylglycerol-containing fat or oil and then reacted. Specifically, the water content (water concentration; hereinafter simply referred to as "water content") of the reaction system (including the raw material oil (fat or oil, or a mixture of fat and oil with fatty acids, etc.) and the enzyme) at the start of the reaction is not particularly limited, and is adjusted to less than 110 ppm (e.g., 10 ppm or more but less than 110 ppm, preferably 40 to 105 ppm, more preferably 60 to 100 ppm, and even more preferably 80 to 97 ppm) or 110 ppm or more (e.g., 110 to 1000 ppm, preferably 150 to 800 ppm, more preferably 200 ppm to 500 ppm, even more preferably 200 ppm to 400 ppm, and even more preferably 220 to 300 ppm), and then lipase is added and the reaction is carried out. The higher the water content of the reaction system, the more likely a hydrolysis reaction occurs 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 at a temperature of 30 to 100°C, preferably 35 to 80°C, for a predetermined time (for example, 1 to 48 hours). To promote the reaction, stirring is preferably carried out during the reaction. In addition to a batch reaction, a continuous reaction using a column, a fluidized bed, or the like can also be employed. For reactions using immobilized lipase, a batch-type stirred tank reactor, a flow-through stirred tank reactor, a packed bed reactor, a fluidized bed reactor, or the like can be used.

[0020] The amount of lipase to be added is not particularly limited as long as the desired reaction proceeds, and may be, for example, 10 to 100 parts by weight 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, and it is therefore preferable to remove the water from the reaction system, for example, by distillation under reduced pressure, by using a dehydrating agent such as a molecular sieve, or by using a dry inert gas such as nitrogen gas. [Example]

[0022] The diacylglycerol (DG) reducing effect of various lipases with different ratios of transesterification activity to hydrolysis activity was investigated (Test Examples 1 and 2). The hydrolysis activity and transesterification activity were measured by the following methods. (Hydrolysis activity measurement method) Hydrolytic activity was measured using Lipase Kit S (SB Biosciences, Inc.). A color-developing stock solution was prepared by adding 2.4 mL of the pH 7.0 buffer solution provided with the kit and 22 mL of purified water to the color-developing reagent provided with the kit. 250 μL of the pH 7.0 buffer solution provided with the kit and 2000 μL of purified water were added to 250 μL of the color-developing stock solution to prepare a color-developing solution. 1 mL of the color-developing solution and 50 μL of a diluted solution prepared by diluting the enzyme solution to an appropriate concentration were placed in a test tube and incubated at 37°C for 5 minutes. 100 μL of the substrate solution provided with the kit was added, and the reaction was stopped by adding 2 mL of acetone. After the reaction was stopped, the supernatant was collected and its absorbance was measured at 412 nm. Using the sample containing the substrate solution after the addition of acetone as a blank, the hydrolytic activity (U / g) was calculated using the following formula: Hydrolysis activity (U / g)=(A412sample-A412blank)×20×n (where A412sample is the absorbance at 412 nm of the sample supernatant, A412blank is the absorbance at 412 nm of the blank, 20 is the coefficient, and n is the dilution factor of the enzyme solution.) (Method for measuring transesterification activity) 5 mL of tricaprylin (Wako Pure Chemical Industries, Ltd.) and 6 mL of methyl laurate (Wako Pure Chemical Industries, Ltd.) were added to a 50 mL disposable centrifuge tube and preheated at 30 ± 1°C for 10 minutes. 0.1 g of enzyme sample was added, the tube was capped and mixed by inversion, then the tube was placed on a rotator and the enzyme reaction was carried out at 30°C and 50 rpm for 30 minutes. 30 μL of the reaction solution was dissolved in 1 mL of hexane to prepare a sample for gas chromatography. The area value of methyl caprylate produced as a result of the enzyme reaction (transesterification) was determined by gas chromatography analysis (column: DB-1HT (Agilent J&W, 5 m × 0.25 mm, df 0.1 μm), 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), and the transesterification activity was calculated using the following formula. Interesterification activity (u / g) = A / a × 34 × 1 / 30 × 1 / 0.1 × 11 A: Area value of methyl caprylate sample a: The slope calculated from the calibration curve (area value of methyl octanoate = a × methyl octanoate concentration) created from the area value and methyl octanoate concentration (mmol / L) when 1 mmol / L to 7.5 mmol / L of methyl octanoate is analyzed by gas chromatography under the above conditions. 34: Dilution ratio when the reaction solution is diluted with hexane * (1 mL + 30 μL) / 30 μL ≒ 34 1 / 30: Conversion factor for reaction time per minute 1 / 0.1: Conversion factor per 1g 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, Burkholderia acepacia-derived lipase) was prepared according to standard methods, and its hydrolytic activity and transesterification activity were measured. The hydrolytic activity and transesterification activity of each lipase are shown in Table 1.

[0024] (2) Preparation of substrate oil An oil (TG content 89.4 wt%, DG content 6.7 wt%, the remainder mainly fatty acids) made by mixing refined palm oil with glycerol dioleate, palmitic acid, and oleic acid was dried under reduced pressure at 80°C for more than 12 hours until the moisture content was 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 12 hours or more, and 20 g of substrate oil were added to an Erlenmeyer flask and shaken at 60°C and 160 rpm to react. After 22 hours, the reaction solution was analyzed by GC to confirm changes in the composition of the substrate oil. The residual DG rate was calculated from the DG ratio before and after the reaction.

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

[0027] When the water content was 95 ppm, the DG reduction effect was confirmed even when using a lipase 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) were used as enzyme samples.

[0029] (2) Preparation of substrate oil Crude palm oil (hereinafter referred to as CPO) (TG content 92.5 wt%, DG content 4.8 wt%, the remainder mainly fatty acids) was dried under reduced pressure at 80°C for 2 hours or more 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 12 hours or more, 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 analyzed by GC to confirm the change in the composition of the substrate oil.

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

[0032] At a water content of 228 ppm, the DG reduction effect was not confirmed with lipase (LPS-IM) with a low ratio of hydrolysis activity to transesterification activity, but the DG reduction effect was confirmed 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 activity amount for the DG reduction effect.

[0033] The fatty acid composition of each sample after reaction is shown in the table below. [Table 3] The "theoretical value" is the composition ratio calculated based on the abundance ratio of free fatty acids and DG in the base oil when no interesterification reaction occurs and only the synthesis reaction of free fatty acids and DG in the base oil occurs.

[0034] It was confirmed that in Sample 3, an ester synthesis reaction occurred along with an ester exchange reaction (Tables 2 and 3). On the other hand, it was confirmed that in Sample 4, an ester synthesis reaction occurred (Table 2), but an ester exchange reaction did not (Table 3). It was also confirmed that in Sample 5, an ester synthesis reaction did not occur (Table 2), but an ester exchange reaction did occur (Table 3). These results demonstrate that an ester synthesis reaction occurs when the ratio of hydrolysis activity to ester exchange activity is high, regardless of whether or not there is ester exchange reaction ability. [Industrial Applicability]

[0035] The present invention is useful for modifying and improving the physical properties of fats and oils or processed fat products (e.g., shortening, margarine). For example, the present invention can be applied to improve spreadability, emulsion stability, solid fat content (SFC), solidification, selective concentration of specific fatty acids, and production of low-trans fats and oils or processed fat products containing low-trans fats and oils. The fats and oils obtained by applying the present invention or processed fat products containing the same exhibit improved physical properties and are highly useful industrially.

[0036] The present invention is not limited to the above-described embodiments and examples. Various modifications within the scope of the claims and within the scope that can be easily conceived by a person skilled in the art are also included in the present invention. The contents of papers, published patent applications, patent publications, and other publications explicitly stated in this specification are incorporated herein by reference in their entirety.

Claims

1. A method for producing a diacylglycerol-containing oil or fat, comprising the step of allowing a lipase having a ratio of transesterification activity to hydrolysis activity of 0.007 or more to act on the diacylglycerol-containing oil or fat, the lipase is a lipase derived from the genus Penicillium and / or a lipase derived from the genus Burkholderia, and is an immobilized lipase; The water content of the diacylglycerol-containing oil or fat is 110 ppm or more, the hydrolytic activity value is a value obtained by multiplying the hydrolytic activity (u / g) of the lipase before immobilization by the amount (g) of the lipase used for immobilization and dividing the product by the amount (g) of the immobilized lipase; The method for producing fats and oils, wherein the transesterification activity value is the transesterification activity value of the immobilized lipase.

2. The method of claim 1 , wherein the ratio is 0.5 or less.

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

4. The method according to any one of claims 1 to 3, wherein the water content is 1000 ppm or less.

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