Method for producing fatty acids

Abstract

To provide a fatty acid production method enabling an enzyme amount to be reduced regardless of throwaway reaction of the enzyme, thereby enabling fatty acid to be produced with a simple facility.SOLUTION: There is provided a fatty acid production method by hydrolysis of fat, the hydrolysis includes a hydrolysis step which is repeatedly executed in an immobilized enzyme, and in an addition amount of the enzyme added in each hydrolysis step, the addition amount of the enzyme in the first hydrolysis step is most, and the addition amount of the enzyme in the second and following hydrolysis steps, is 0.5 mass times or lower of the addition amount of the first hydrolysis step.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for producing fatty acids. [Background technology]

[0002] Fatty acids are used as raw materials for food, cleaning agents, paints, pharmaceuticals, etc., and are produced by hydrolysis of fats and oils. There is a method of hydrolysis under high temperature and high pressure conditions, which is highly productive, but requires equipment that can withstand high pressure, and fatty acids that contain many double bonds are easily damaged. There is also a method of hydrolysis using enzymes, which is carried out at low temperatures and causes less damage to fatty acids, but the reaction rate is low, so multiple hydrolysis steps are required. In this case, the enzyme (lipase), which is a protein, is water-soluble and is lost with each reaction, so an immobilized enzyme is used and the enzyme is recovered and reused after each reaction (Patent Document 1). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2018-68166 A Summary of the Invention [Problem to be solved by the invention]

[0004] However, when using immobilized enzymes, the enzyme activity decreases during the enzyme immobilization process, so more enzyme is required. In addition, there is a problem that an enzyme recovery process (recovery device) or complex reaction equipment such as a column packed with immobilized enzymes is required.

[0005] Therefore, an object of the present invention is to provide a method for producing fatty acids, which can reduce the amount of enzyme used and produce fatty acids using simple equipment, even though the enzyme is used in the reaction and is disposable. [Means for solving the problem]

[0006] In order to achieve the above object, the present invention provides the following method for producing fatty acids.

[0007] [1] A method for producing fatty acids by hydrolysis of fats and oils, the hydrolysis including repeated hydrolysis steps using a non-immobilized enzyme, wherein the amount of the enzyme added in each hydrolysis step is the largest in the first hydrolysis step, and the amount of the enzyme added in the second or subsequent hydrolysis steps is 0.5 times or less by mass of the amount added in the first hydrolysis step. [2] A method for producing fatty acids, wherein the hydrolysis step is repeated three or more times, the first and second hydrolysis steps are performed with the addition of a non-immobilized enzyme, and the third and subsequent hydrolysis steps are performed without adding the enzyme. [3] The enzyme is a powdered lipase derived from Candida cylindracea; The first hydrolysis step is carried out by applying the enzyme to the vegetable oil (oil phase) at a concentration of 4×10 3 ~2×10 6 The amount is added so that the total amount is U / L. The second hydrolysis step is carried out by adding the enzyme in an amount of 0.2 to 0.5 times the amount of the enzyme added in the first hydrolysis step. A method for producing a fatty acid according to [1] or [2]. [4] The method for producing fatty acids according to any one of [1] to [3], wherein the concentration of free fatty acids in the first hydrolysis step is 85 mass% or more. [5] The method for producing a fatty acid according to any one of [1] to [4], wherein the oil or fat is linseed oil and the fatty acid is linseed fatty acid. [6] The method for producing fatty acids according to [5], wherein the linseed oil does not exhibit breakage in a break test (Reference 1.11-2013 of the Standard Method for the Analysis of Fats, Oils, and Related Materials). [7] The method for producing fatty acids according to any one of [1] to [6], wherein after the hydrolysis step, the oil phase is dried under reduced pressure at 80 to 110°C or less. Effect of the Invention

[0008] According to the present invention, fatty acids can be produced using a small amount of enzymes and with simple equipment and processes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The hydrolysis reaction of fats and oils using enzymes is a reversible reaction, and both glycerides and free fatty acids in fats and oils serve as substrates for the enzymes. Therefore, in order to increase the hydrolysis rate of fats and oils, the hydrolysis process is repeated, but even if the hydrolysis of fats and oils progresses, the amount of enzyme required in each hydrolysis process must be the same, or at least the ratio of the enzyme to the oil phase that can serve as a substrate must be the same. At this time, since the non-immobilized enzyme dissolves in the aqueous phase and is lost during oil-water separation during hydrolysis, the same amount of enzyme was added each time. However, the present inventors have found that in the hydrolysis of fats and oils using a non-immobilized enzyme, the reaction can proceed even if the ratio of the enzyme added to the oil phase is reduced in the repeated hydrolysis steps, and have completed the present invention. Based on this finding, the present inventors have completed the method for producing fatty acids according to the present invention.

[0010] The method for producing a fatty acid of the present invention will be described in detail below. In the embodiment of the present invention, A (numerical value) to B (numerical value) means A or more and B or less.

[0011] <Oils> In the method for producing fatty acids of the present invention, fats and oils are used as raw materials. Examples of fats and oils that can be used include vegetable oils and animal oils. For example, vegetable fats and oils such as linseed oil, perilla oil, soybean oil, rapeseed oil, safflower oil, rice oil, corn oil, sunflower oil, cottonseed oil, olive oil, sesame oil, grapeseed oil, peanut oil, pearl barley oil, wheat germ oil, perilla oil, chia seed oil, sacha inchi oil, walnut oil, kiwi seed oil, salvia seed oil, grape seed oil, macadamia nut oil, hazelnut oil, pumpkin seed oil, camellia oil, tea seed oil, borage oil, palm oil, palm olein, palm stearin, coconut oil, palm kernel oil, cacao butter, monkey fat, shea butter, and algae oil can be mentioned; animal fats and oils such as fish oil, lard, beef tallow, and butter fat; or transesterified oils, hydrogenated oils, or fractionated oils thereof can be mentioned. These oils and fats may be used alone or in combination of two or more. In particular, since the method for producing fatty acids of the present invention does not involve high-temperature treatment, it is preferable to use it for producing linseed fatty acids and perilla fatty acids that have a high linolenic acid content, such as linseed oil and perilla oil.

[0012] The oil can be either unrefined or refined, but oil that has been subjected to an adsorption process such as a decolorization process is preferred in terms of maintaining the reactivity of the enzyme. In addition, it is preferable that the linseed oil does not break in the break test (Reference 1.11-2013 of the Standard Methods for Analysis of Fats and Oils). The linseed oil that can be used is the one that is distributed as NB linseed oil.

[0013] <Enzyme> In the method for producing fatty acids of the present invention, an enzyme that is not immobilized is used. As the enzyme that is not immobilized, an enzyme-containing liquid containing culture medium components of the enzyme, or an enzyme-containing liquid obtained by drying can be used. In addition, an enzyme that has had culture medium components and the like removed, that is, an enzyme that is essentially composed of the enzyme itself, can be used. As the enzyme of the present invention, it is more preferable that the enzyme is cultured, the bacterial cells are removed, and the enzyme is powdered without being immobilized. The powdering can be performed by drying after culture, or by removing the bacterial cells and drying, or by dissolving and / or dispersing grain powder and / or sugar powder in an aqueous liquid containing the enzyme to obtain a lipase-containing aqueous liquid, which is then further dried to obtain a powder.

[0014] The enzyme is preferably a lipase. The lipase is not particularly limited, and lipase derived from an animal, a plant, or a microorganism can be used. Examples of the lipase include lipoprotein lipase, monoacylglycerol lipase, diacylglycerol lipase, triacylglycerol lipase, galactolipin lipase, and the like. Examples of lipases include triacylglycerol lipase and phospholipase. Among these, triacylglycerol lipase is preferred. Lipases are also used in transesterification, and some lipases have 1,3-selectivity. In general, the selectivity of lipases is derived from the reaction rate of the 1,3 and 2 positions of glycerol. However, since the reaction rate of hydrolysis is faster than that of transesterification, even lipases with 1,3-selectivity can hydrolyze fatty acids at the 2 position, so lipases regardless of selectivity can be used in the present invention.

[0015] The source of the lipase is not particularly limited and may be bacteria, yeast, filamentous fungi, actinomycetes, etc. The sources of lipases include, for example, Alcaligenes sp., Pseudomonas sp., Arthrobacter sp., Staphylococcus sp., Torulopsis sp., Escherichia sp., Mycotorula sp., Propionibacterum sp., Chromobacterum sp., Xanthomonas sp., Lactobacillus sp., Clostridium sp., Candida sp., Geotrichum sp., Sacchromycopsis sp., and the like. Preferred examples of the genus include those of the genus Nocardia, Fusarium, Aspergillus, Rhizomucor, Mucor, Thermomyces, Rhizopus, Penicillium, Phycomyces, Puccinia, Bacillus, and Streptomyces. Among them, the non-selective lipase produced by Candida cylindracea is preferred because of its high hydrolytic reactivity.

[0016] The lipase-containing composition of the present invention may consist of lipase alone, or may contain, in addition to lipase, any of the components, auxiliaries, etc. used in the medium for lipase.

[0017] The lipase-containing composition of the present invention may be in the form of a liquid or powder (powdered lipase). The powdered lipase may be used in the form of a lipase powder preparation, which can be obtained, for example, by dissolving and / or dispersing lipase and grain powder and / or sugar powder in an aqueous liquid to obtain a lipase-containing aqueous liquid, and then drying and powdering the resulting liquid. As the lipase, a commercially available lipase can be used, for example, Lipase OF (powdered lipase manufactured by Meito Sangyo Co., Ltd.). Examples of the grain powder and / or sugar powder include soybean powder such as full fat soybean powder and defatted soybean powder, wheat flour, rice flour, and dextrin. The grain powder and / or sugar powder is suitably, for example, 250 to 1000% by mass, preferably 500 to 1000% by mass, more preferably 500 to 750% by mass, relative to the lipase.

[0018] In the present invention, the amount of enzyme refers to the hydrolysis titer of the enzyme. The hydrolysis titer of the enzyme is expressed as the product of the hydrolysis titer (U / g) and the mass of the enzyme (g). For example, when the hydrolysis titer is about 4×10 5 The enzyme amount (titer) of 1 kg of the aforementioned Lipase OF, which is known to be U / g, is approximately 4 × 10 5 U / g×1000g=approx. 4×10 8 The hydrolytic titer (U / g) is the activity of an enzyme that hydrolyzes fats and oils, and can be measured, for example, using JIS 0601-1995 "Method for measuring activity of industrial lipase."

[0019] <Hydrolysis process> The method for producing fatty acids of the present invention includes the following hydrolysis step of the first embodiment or the hydrolysis step of the second embodiment, in which the above-mentioned fats and oils are hydrolyzed using the above-mentioned enzymes.

[0020] In the hydrolysis step, water is added to the oil phase, hydrolysis is carried out with an enzyme, and then the water is separated. The repeated hydrolysis step is one in which this step is repeated two or more times. The repeated hydrolysis step is preferably carried out two to five times, and more preferably three to four times.

[0021] (Amount of enzyme added in the hydrolysis step in the first embodiment) The hydrolysis step in the first embodiment includes hydrolysis steps repeatedly performed using a non-immobilized enzyme, and the amount of the enzyme added in each hydrolysis step is the largest in the first hydrolysis step, and the amount of the enzyme added in the second and subsequent hydrolysis steps is 0.5 times or less by mass of the amount added in the first hydrolysis step. Preferably, the second hydrolysis step is performed by adding 0.2 to 0.5 times by mass of the enzyme added in the first hydrolysis step.

[0022] The amount of enzyme added was 4 x 10 3 ~2×10 6 U / L (4×10 for 1 L of oil phase) 3 ~2×10 6 U) is preferably added, and 2×10 4 ~2×10 5 U / L (2×10 for 1 L of oil phase) 4 ~2×10 5 U) is more preferably added, and 3.2 × 10 4 ~8×10 4 U / L (3.2×10 for 1 L of oil phase) 4 ~8×10 4 For example, it is more preferable to add about 4 × 10 U of the enzyme derived from Candida cylindracea. 5 In the case of a powdered lipase having an activity of 10 U / g, the enzyme is preferably added in an amount of 0.01 to 5.0 kg / kL, more preferably 0.05 to 0.5 kg / kL, and even more preferably 0.08 to 0.2 kg / kL relative to the vegetable oil (oil phase) in the first hydrolysis step.

[0023] (Amount of enzyme added in the hydrolysis step in the second embodiment) The hydrolysis step in the second embodiment includes a hydrolysis step repeated three or more times, in which the first and second hydrolysis steps are carried out by adding a non-immobilized enzyme, and the third and subsequent hydrolysis steps are carried out without adding the enzyme. As in the hydrolysis step in the first embodiment, the hydrolysis step in the second embodiment is preferably carried out such that the amount of the enzyme added in each hydrolysis step is the largest in the first hydrolysis step, and the amount of the enzyme added in the second and subsequent hydrolysis steps is 0.5 times or less by mass of the amount added in the first hydrolysis step. More preferably, the second hydrolysis step is carried out by adding 0.2 to 0.5 times by mass of the enzyme added in the first hydrolysis step.

[0024] The amount of enzyme added was 4 x 10 3 ~2×10 6 U / L (4×10 for 1 L of oil phase) 3 ~2×10 6 U) is preferably added, and 2×10 4 ~2×10 5 U / L (2×10 for 1 L of oil phase) 4 ~2×10 5 U) is more preferably added, and 3.2 × 10 4 ~8×10 4 U / L (3.2×10 for 1 L of oil phase) 4 ~8×10 4 For example, it is more preferable to add about 4 × 10 U of the enzyme derived from Candida cylindracea. 5 In the case of a powdered lipase having an activity of 10 U / g, the enzyme is preferably added in an amount of 0.01 to 5.0 kg / kL, more preferably 0.05 to 0.5 kg / kL, and even more preferably 0.08 to 0.2 kg / kL relative to the vegetable oil (oil phase) in the first hydrolysis step.

[0025] (Water content in hydrolysis process) In each hydrolysis, the amount of water relative to the oil phase (oils and fats, or a mixture of free fatty acids and oils and fats) is sufficient if it is in excess of the glycerides to be hydrolyzed. The amount of water relative to the oil phase (oils and fats, or a mixture of free fatty acids and oils and fats) is preferably 0.1 to 10 times by volume, more preferably 0.1 to 2 times by volume, even more preferably 0.2 to 1 times by volume, and particularly preferably 0.2 to 0.5 times by volume.

[0026] (Reaction temperature and time of hydrolysis process) Each hydrolysis reaction can be carried out without any problem as long as the temperature range is such that the enzyme is not inactivated and the oil phase (oil or mixture of free fatty acid and oil) is in a liquid state while stirring so that the oil phase (oil or mixture of free fatty acid and oil) and the aqueous phase are mixed. Generally, enzymes tend to become more active as the temperature increases, but since enzymes are proteins, they are denatured at high temperatures and their activity decreases. For example, the reaction is preferably carried out at 5 to 90°C, more preferably at 20 to 60°C, even more preferably at 20 to 40°C, and most preferably at 30 to 35°C. The reaction time of the hydrolysis reaction is affected by the amount of enzyme and the reaction time. The more the amount of enzyme, the shorter the reaction equilibrium is reached, and the higher the reaction temperature, the shorter the reaction equilibrium is reached, so the reaction temperature is appropriately selected. For example, in the case of powdered lipase, the enzyme is added to the vegetable oil (oil phase) in an amount of 0.01 to 5.0 kg / kL in the first hydrolysis step, and the reaction is preferably carried out for 3 to 20 hours at 20 to 40° C., more preferably for 10 to 20 hours.

[0027] In the first hydrolysis step, the free fatty acid concentration is preferably 85% by mass or more, and more preferably 90 to 98% by mass. The free fatty acid concentration (mass%) can be calculated by [neutralization value (or acid value) of the product]÷[theoretical acid value obtained by hydrolysis of fats and oils]×100.

[0028] (Separation of the aqueous phase after the hydrolysis reaction) After each hydrolysis reaction, the aqueous phase is separated. The aqueous phase may be separated using a centrifuge, or by leaving the mixture in a reaction tank and then discharging the lower aqueous layer from the tank.

[0029] <Processes other than the hydrolysis process> The fatty acid obtained by the hydrolysis step has a high purity, but water and enzymes remain. If the aqueous phase separation at the end of the hydrolysis step is insufficient, glycerin and the like remain, so it is preferable to wash the oil phase with water in order to remove water-soluble components such as glycerin and enzymes. The washing is performed by adding water to the oil phase, stirring, leaving the mixture to stand, and removing the aqueous phase.

[0030] In addition, it is preferable to carry out a drying process in order to remove moisture. Examples of the drying process include methods of removing moisture using an adsorbent and drying under reduced pressure. In addition, it is preferable to carry out a heating process in order to inactivate the enzyme remaining in the fatty acid. Drying under reduced pressure accompanied by heating is preferable because it can remove moisture and inactivate the enzyme. For example, drying under reduced pressure at 80°C or higher can simultaneously inactivate the enzyme and remove moisture. The degree of vacuum for drying under reduced pressure may be 47.5 kPa or less, which is the vapor pressure of water at 80°C, preferably 30 kPa or less, and more preferably 15 kPa or less. In addition, the upper limit temperature for drying under reduced pressure may be a temperature at which fatty acids are not distilled off by drying under reduced pressure and are not trans-converted, and is preferably 180°C or less, and in the case of fatty acids with a large amount of unsaturated fatty acids such as linseed fatty acid, it is more preferable to carry out a heating process in order to inactivate the enzyme and remove moisture at the same time. The preferred temperature range for drying under reduced pressure is 80 to 110°C, and more preferably 90 to 110°C.

[0031] In addition, in order to increase the specific fatty acid purity of the obtained fatty acid, the purity of the fatty acid can be increased by distillation, if necessary.

[0032] Furthermore, if necessary, the fatty acids can be subjected to an adsorption treatment or filtration to remove trace components. EXAMPLES

[0033] The present invention will be specifically described below based on examples, but the present invention is not limited to these examples.

[0034] Comparative Example 1 15 kL of NB linseed oil, 4.5 kL of water, and 1.5 kg of lipase OF (3.6 × 105 U / g, manufactured by Meito Sangyo Co., Ltd.) were stirred in a tank at 30 ° C. for 7.5 hours, and 2 kL of water was added and stirred for 4 hours. After standing for 3 hours, the aqueous phase was removed to obtain an oil phase 1-1 that had undergone the first hydrolysis step. 5 kL of water and 1.5 kg of lipase OF (3.6 × 105 U / g, manufactured by Meito Sangyo Co., Ltd.) were added to the oil phase 1-1 and stirred at 30 ° C. for 15 hours. After standing for 3 hours, the aqueous phase was removed to obtain an oil phase 1-2 that had undergone the second hydrolysis step.

[0035] 5 kL of water was added to the oil phase 1-2, stirred for 1 hour, allowed to stand for 7 hours, the aqueous phase was removed, and another 5 kL of water was added, stirred for 1 hour, allowed to stand for 48 hours, the aqueous phase was removed, and the oil phase 1-3 was obtained after washing with water. The oil phase 2-3 was dried under reduced pressure at 100 to 110° C. for 15 hours and filtered through a 0.5 μm cartridge filter to obtain linseed oil fatty acid 1.

[0036] Example 1 NB linseed oil, 15 kL, water, 4.5 kL, Lipase OF (Meito Sangyo Co., Ltd., 3.6 × 10 5 The mixture was stirred in a tank at 30° C. for 7.5 hours, and then 2 kL of water was added and stirred for 4 hours. After standing for 3 hours, the aqueous phase was removed to obtain an oil phase 2-1 that had been subjected to the first hydrolysis step. The oil phase 2-1 was mixed with 5 kL of water, Lipase OF (Meito Sangyo Co., Ltd., 3.6×10 5 0.5 kg of ethyl acetate (U / g) was added and stirred at 30° C. for 15 hours. After standing for 3 hours, the aqueous phase was removed to obtain an oil phase 2-2 that had been subjected to the second hydrolysis step. 5 kL of water was added to the oil phase 2-2 and stirred at 30° C. for 15 hours. After standing for 3 hours, the aqueous phase was removed to obtain an oil phase 2-3 that had been subjected to the third hydrolysis step.

[0037] 5 kL of water was added to the oil phase 2-3, stirred for 1 hour, allowed to stand for 7 hours, the aqueous phase was removed, and another 5 kL of water was added, stirred for 1 hour, allowed to stand for 48 hours, the aqueous phase was removed, and oil phase 2-4 was obtained after washing with water. The oil phase 2-4 was dried under reduced pressure at 100 to 110° C. for 15 hours and filtered through a 0.5 μm cartridge filter to obtain linseed oil fatty acid 2.

[0038] Example 2 NB linseed oil 15 kL, water 6 kL, Lipase OF (Meito Sangyo Co., Ltd. 3.6 × 10 5 The oil phase 1-1 was mixed with 5 kL of water, Lipase OF (Meito Sangyo Co., Ltd., 3.6 × 10 5 0.5 kg of ethanol (U / g) was added and stirred at 30° C. for 15 hours. After standing for 3 hours, the aqueous phase was removed to obtain an oil phase 3-2 that had been subjected to the second hydrolysis step. 5 kL of water was added to the oil phase 3-2 and stirred at 30° C. for 15 hours. After standing for 3 hours, the aqueous phase was removed to obtain an oil phase 3-3 that had been subjected to the third hydrolysis step.

[0039] 5 kL of water was added to the oil phase 3-3, stirred for 1 hour, allowed to stand for 7 hours, the aqueous phase was removed, and another 5 kL of water was added, stirred for 1 hour, allowed to stand for 48 hours, the aqueous phase was removed, and oil phase 3-4 was obtained after washing with water. The oil phase 3-4 was dried under reduced pressure at 100 to 110° C. for 15 hours and filtered through a 0.5 μm cartridge filter to obtain linseed oil fatty acid 3.

[0040] (Neutralization value) The neutralization values ​​of the oil phases 1-1 to 1-2, 2-1 to 2-3, and 3-1 to 3-3 that had been subjected to the first hydrolysis step (neutralization values ​​after the first to third hydrolysis steps) were measured and shown in Table 1. The increase rates of the neutralization values ​​in the second and third hydrolysis steps relative to the first hydrolysis step are also shown in Table 1. The neutralization value of linseed fatty acid is approximately 200, which indicates that the hydrolysis rate in the first hydrolysis step is 90% or more.

[0041] [Table 1]

[0042] From the results in Table 1, it can be confirmed that the reaction can be carried out even if the amount of enzyme is reduced in the second and subsequent hydrolysis reactions. It was also confirmed that Examples 1 and 2 had the same neutralization value as Comparative Example 1, despite the use of a smaller amount of lipase than Comparative Example 1.

Claims

1. In a method for producing fatty acids by hydrolysis of fats and oils, The hydrolysis comprises a hydrolysis step with a non-immobilized enzyme repeated three or more times, the fat or oil being linseed oil, the fatty acid being linseed fatty acid, and the enzyme being a lipase; With regard to the amount of the enzyme added in each hydrolysis step, the amount of the enzyme added in the first hydrolysis step is the largest, and the amount of the enzyme added in the second or subsequent hydrolysis steps is 0.5 times or less by mass of the amount added in the first hydrolysis step, The first hydrolysis step is carried out by applying the enzyme to the vegetable oil (oil phase) at a concentration of 4×10 ３ ~2×10 ６ The amount is added so that the ratio becomes U / L. In the first and second hydrolysis steps, hydrolysis is carried out by adding a non-immobilized enzyme, and in the third and subsequent hydrolysis steps, hydrolysis is carried out without adding the enzyme. A method for producing fatty acids.

2. The enzyme is a powdered lipase originating from Candida cylindracea, The second hydrolysis step is carried out by adding the enzyme in an amount of 0.2 to 0.5 times the amount of the enzyme added in the first hydrolysis step. A method for producing the fatty acid according to claim 1.

3. 3. The method for producing fatty acids according to claim 1 or 2, wherein the concentration of free fatty acids in the first hydrolysis step is 85% by mass or more.

4. The method for producing a fatty acid according to any one of claims 1 to 3, wherein the linseed oil does not undergo break in a break test (Standard Fats and Oils Analysis Test Method Reference 1.11-2013).

5. The method for producing a fatty acid according to any one of claims 1 to 4, wherein after the hydrolysis step, the oil phase is dried under reduced pressure at 80 to 110°C or less.

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