JP5836025B2 - Method for producing highly unsaturated fatty acid concentrated oil - Google Patents

Description

  The present invention relates to a method for producing fats and oils containing polyunsaturated fatty acid (hereinafter abbreviated as PUFA) at a high concentration by a hydrolysis reaction using lipase.

Eicosapentaenoic acid (hereinafter abbreviated as EPA) and docosahexaenoic acid (hereinafter abbreviated as DHA), which are n-3 PUFAs, have various physiological functions and are used as pharmaceuticals, health foods, food materials, and the like. EPA ethyl ester is used as a therapeutic agent for arteriosclerosis and hyperlipidemia, and beverages containing fish oil containing EPA and DHA are also approved as food for specified health use. Furthermore, the demand for supplements in Japan and overseas is also very high.
PUFA is very unstable to oxidation due to its large number of double bonds. Therefore, it is highly desirable to use an enzyme reaction in which the reaction proceeds under mild conditions such as normal temperature and pressure in the production process of PUFA-containing fats and oils.
Some industrial lipase products obtained mainly from microorganisms are known to have such a property that they hardly act on PUFA. By using a lipase having such properties to release fatty acids with a small number of carbons preferentially and removing them, it is possible to produce oils and fats enriched in PUFA. Patent Document 1 discloses a method for producing fats and oils in which DHA is concentrated by hydrolyzing tuna oil using a lipase of Candida cylindoracea and then removing free fatty acids.

  A method of concentrating PUFA using an alcoholysis reaction using lipase has also been reported. Patent Document 2 discloses a method using magnesium oxide, magnesium hydroxide, calcium oxide, or calcium hydroxide as a reaction additive for the alcoholysis reaction using lipase.

JP 58-165796 WO2007 / 119811

  Concentrated oils that have concentrated PUFA such as fish oil by applying the properties of lipase as described above are already on the market, but the concentration is limited and it is difficult to obtain high concentration products or very many enzymes. Need. Specifically, most of the products sold as DHA concentrated oil are concentrated products of about 46%, and those exceeding 50% are very few. They cannot be produced by a single lipase reaction, and are extremely expensive because they require repeated reactions and removal of unnecessary components. An object of the present invention is to provide a method for highly concentrating PUFA, particularly DHA, EPA, and the like contained in a raw material oil without much effort.

  The inventors have studied the reaction using lipase for industrial use from various angles. As a result, in the alcoholysis reaction, lipase is added by adding a small amount of magnesium oxide, magnesium hydroxide, calcium oxide, calcium hydroxide, etc. It has been found that the efficiency of the alcoholysis reaction can be drastically improved even if the use amount of is small. In addition, in this method, the property of lipase, which is difficult to act on PUFAs such as EPA and DHA, which are target products, is strictly maintained during the reaction.

  The present invention relates to a hydrolysis reaction, not an alcoholysis reaction. The inventors have found that the hydrolysis reaction tends to be completely different from the alcoholysis reaction. That is, PUFA was concentrated as a reaction additive only in any one of calcium hydroxide, magnesium chloride, ammonium sulfate, calcium carbonate, potassium dihydrogen phosphate, and ammonium chloride. Compared with the overwhelming effect. The present invention is any one of calcium hydroxide, magnesium chloride, ammonium sulfate, calcium carbonate, potassium dihydrogen phosphate, and ammonium chloride, in particular, after subjecting the PUFA-containing fat to hydrolysis with lipase in the presence of calcium hydroxide, The gist is a method for producing a PUFA concentrated oil, which is obtained by separating a glyceride fraction.

  The present invention improves the selectivity of the enzyme by increasing the reactivity of the enzyme by adding an inexpensive additive and hardly acting on PUFA that is ester-bonded to glycerides. As a result, a concentrated oil containing a high content of PUFA can be produced at a low cost by a simple operation.

FIG. 1 is a graph showing the DHA content of a glyceride fraction in a calcium hydroxide-free reaction. FIG. 2 is a diagram showing the DHA recovery rate of the glyceride fraction in the reaction without addition of calcium hydroxide. FIG. 3 is a diagram showing a comparison of the DHA content in the glyceride fraction of the reaction with and without the addition of calcium hydroxide. FIG. 4 is a graph showing the DHA content and recovery rate in the glyceride fraction with and without calcium hydroxide added.

In the present invention, polyunsaturated fatty acid (PUFA) refers to a fatty acid having 20 or more carbon atoms and 3 or more double bonds. Specific examples include eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), arachidonic acid, dihomo-γ-linolenic acid, linolenic acid and the like. The highly unsaturated fatty acid-containing oil / fat of the present invention is not particularly limited as long as it contains the highly unsaturated fatty acid as a fatty acid constituting the oil / fat, and includes fish oils such as fish oil, microbial oil, and algae including PUFA. Examples include oil and vegetable oil. When used as a raw material of the present invention, those crude oils (extracted or squeezed themselves) may be used, or those having undergone some refining process. The method of the present invention is suitable for the concentration of fatty acids having 20 or more carbon atoms and 4 to 6 double bonds, particularly 20 to 22 carbon atoms and 4 to 6 double bonds, among highly unsaturated fatty acids. ing. Examples of fatty acids suitable for the method of the present invention include EPA, DHA, arachidonic acid, and docosapentaenoic acid.
Fats and oils usually mean triglycerides of fatty acids, but in the present invention means glycerides including diglycerides and monoglycerides. In the present invention, glyceride is a general term for triglycerides, diglycerides and monoglycerides of fatty acids.
In the present invention, the concentration of highly unsaturated fatty acid means to increase “the amount of highly unsaturated fatty acid / the total amount of fatty acid” after the reaction from “amount of highly unsaturated fatty acid / the total amount of fatty acid” of the raw oil and fat, Fats and oils whose “amount of highly unsaturated fatty acids / total amount of fatty acids” is larger than the raw oils and fats are highly unsaturated fatty acid concentrated oils.
By the method of the present invention, it is possible to obtain fats and oils whose highly unsaturated fatty acids in the fatty acids contained in the fats and oils are 55 area% or more. In particular, it is possible to obtain fats and oils in which docosahexaenoic acid in the fatty acid contained in the fats and oils is 46 area% or more, preferably 50 area% or more. Furthermore, by repeating the method of the present invention two or more times, it is possible to produce an oil having 70% by area or more of docosahexaenoic acid in the fatty acid more easily than the conventional method.

  The lipase used in the present invention is not particularly limited as long as it has a property of catalyzing a hydrolysis reaction and hardly acting on PUFA. Lipases that react more easily with saturated fatty acids than PUFA and lipases with 1,3-position selectivity can be used. For example, a lipase obtained from a microorganism belonging to Candida cylindoracea (lipase OF, manufactured by Meika Sangyo Co., Ltd.), a lipase obtained from a microorganism belonging to Alcaligenes sp. (Lipase QLM, lipase QLC, Lipase PL (both manufactured by Meika Sangyo Co., Ltd.), lipase obtained from microorganisms belonging to Burkholderia cepacia (Lipase PS, manufactured by Amano Enzyme Co., Ltd.), Pseudomonas fluorescens Examples include lipases obtained from microorganisms belonging to them (Lipase AK, manufactured by Amano Enzyme Co., Ltd.), lipases obtained from microorganisms belonging to Thermomyces lanuginosa (Lipozyme TLIM, manufactured by Novozyme). The amount of lipase used is not particularly limited, but for powdered lipase, it is preferably 10 units / g or more for fats and oils, and 30 units / g or more is preferred in view of practicality considering the reaction rate. 0.01% (w / w) or more is preferable with respect to fats and oils. In the case of lipase OF, 100-2000 unit / g is preferable with respect to fats and oils.

As a reaction additive, any of calcium hydroxide, magnesium chloride, ammonium sulfate, calcium carbonate, potassium dihydrogen phosphate, and ammonium chloride is used. In particular, calcium hydroxide is preferable. Powders, fine granules, granules and the like are easy to handle, and those commercially available for industrial use can be used. The addition amount of the reaction additive is not particularly limited, but is preferably 0.01% (w / w) to 15% (w / w), more preferably 0.05% (w / w) to 10% (w / w).
Since it is hydrolysis, water is also added. The amount of water is not particularly limited as long as it is equal to or greater than the reaction equivalent, but it is preferable to use a volume of 0.1 to 2 times the amount of oil or fat, preferably about 0.3 to 1 times the amount of oil or fat.

  The reaction method is not particularly limited as long as a predetermined amount of raw oil and fat, water, enzyme, and calcium hydroxide can be mixed, but the reaction time is about 1 to 24 hours at the optimal reaction temperature of the enzyme (for example, 20 ° C to 60 ° C). In general, the mixture is stirred so as to be mixed well. An immobilized enzyme packed in a column or the like may be used for the reaction. After the reaction, calcium hydroxide and enzyme are removed by filtration and washing with an aqueous solution, and glyceride is separated and purified to obtain a PUFA concentrated oil as a glyceride fraction. The separation method of the glyceride fraction is not particularly limited. For example, distillation methods such as molecular distillation and short-path distillation, separation methods using various chromatographies, and solvent deoxidation methods that remove free fatty acids using a solvent are used. it can. As a purification method, a method usually used for purification of fats and oils may be used, and various chromatography, steam distillation and the like are exemplified.

  Hereinafter, the present invention will be specifically described with reference to examples. The present invention is not limited to these examples. In the examples, the fatty acid composition, acid value, and lipid composition were measured by the following methods.

Fatty acid composition measurement The fatty acid composition of the fish oil used as a raw material was measured by gas chromatography after converting the fish oil to ethyl ester. That is, 1 mL of 1N sodium ethylate / ethanol solution was added to 40 μL of fish oil and stirred for about 30 seconds. Thereafter, 1 mL of 1N hydrochloric acid was added for neutralization, 2 mL of hexane and 3 mL of saturated aqueous ammonium sulfate solution were added, and after stirring and standing, the upper layer was measured by gas chromatography. The percentage of each peak area relative to the total peak area was taken as the fatty acid composition.

  The fatty acid composition of the glyceride fraction of the oil subjected to the enzyme reaction was measured by gas chromatography after removing the free fatty acid, which is a byproduct of the enzyme reaction, after ethyl esterifying the glyceride fraction. That is, 1 mL of a 1N sodium ethylate / ethanol solution was added to 70 μL of the reaction oil and stirred for about 30 seconds. Thereafter, 1 mL of 1N hydrochloric acid was added to neutralize, 700 μL of hexane and 3 mL of a saturated aqueous ammonia sulfate solution were added, stirred and allowed to stand, and then the upper layer containing ethyl ester and free fatty acid was recovered. In order to remove free fatty acid from the obtained upper layer, 1-2 drops of triethylamine was added to 250 μL of the upper layer, and then loaded onto a silica gel column (Varian, BOND ELUT SI, 100 mg, 1 mL) and mixed with hexane and ethyl acetate. The ethyl ester was eluted with 1 mL of the solution (hexane: ethyl acetate = 50: 1 volume ratio) and measured by gas chromatography.

Gas chromatographic analysis conditions for measuring fatty acid composition; Agilent 6850 GC system (Agilent)
Column; DB-WAX J & W 122-7032
Column temperature: 200 ° C
Injection temperature
; 300 ° C
Injection method
Split split ratio; 50: 1
Detector temperature: 300 ° C
Detector: FID
Carrier gas: Helium (2.9 mL / min, constant flow)

About 0.5 g of acid value (AV) measurement oil was dissolved in ethanol, 1 drop of phenolphthalein was added, neutralization titration was performed with a 1N sodium hydroxide aqueous solution, and the following formula was calculated.
AV = titer (mL) × 56.11 / sample weight (g)
For the reaction oil obtained by the reaction with the addition of calcium hydroxide, the value corresponding to the amount of added calcium hydroxide is corrected when it is clear that the added calcium hydroxide has been transferred to the oil. It was.
AV = titer (mL) × 56.11 / sample weight (g) + calcium hydroxide added weight per 1 g of oil (mg) × 1.515

Measurement of lipid composition The lipid composition was measured by thin layer chromatography / flame ionization detector (TLC / FID, Iatroscan, Mitsubishi Chemical Yatron Corporation). 20 μL of oil was dissolved in 1 mL of hexane, and a chroma rod was loaded with 0.4 to 0.8 μL. The mixed solution (hexane: diethyl ether: acetic acid = 65: 35: 1, volume ratio) was used as a developing solvent and developed for 35 minutes. This was analyzed by Iatroscan.

[Example 1], [Comparative Example 1]
Add 4mL of purified fish oil 1 (distilled deacidified tuna oil, Nihon Suisan Co., Ltd.) with 2mL of water and 6.7mg of lipase OF (Meito Sangyo Co., Ltd., 660unit / g oil) and 40mg of calcium hydroxide (1.1wt) % To oil). This was stirred for 14 hours with a magnetic stirrer in a thermostat at 50 ° C. After stirring for 14 hours, the reaction oil was heated at 80 ° C. for 10 minutes to deactivate the lipase, and then the oil layer and the aqueous layer were separated by a centrifuge (40 ° C., 1800 g, 10 minutes) to obtain a reaction oil. .
As a comparative example, the reaction was performed without adding calcium hydroxide under the conditions of Example 1 above.
Fatty acid composition of purified fish oil 1 (area%), acid value of the reaction oil obtained, glyceride content calculated from acid value (%, oleic acid equivalent), fatty acid composition of glyceride fraction (area%), calculated from glyceride content Table 1 shows the recovery rate (%) of EPA and DHA and the lipid composition of the glyceride fraction. Here, the glyceride content was calculated by subtracting the free fatty acid content from the acid value as equivalent to oleic acid and subtracting from the whole reaction oil.

  The DHA content in the glyceride fraction of the reaction oil obtained by the reaction of Example 1 to which calcium hydroxide was added was 51.7%. Compared with the result of Comparative Example 1 to which no calcium hydroxide was added, the content of DHA Increased by 3.2%. The effect of DHA concentration by adding calcium hydroxide was confirmed.

[Comparative Example 2]
To 6 mL of purified fish oil 1, 3 mL of water and 10 mg, 20 mg, or 30 mg of lipase OF (660, 1300, 2000 unit / g oil) were added. This was stirred with a magnetic stirrer in a thermostat at 50 ° C. After stirring for 2 hours, 5 hours, 8 hours, 24 hours, 48 hours, 96 hours, 2 mL was sampled, and the reaction oil was heated at 80 ° C. for 10 minutes to inactivate the lipase, and then centrifuged (40 ° C. 1800 g, 10 minutes), the oil layer and the aqueous layer were separated to obtain a reaction oil.
Fatty acid composition of purified fish oil 1 (area%), acid value of the reaction oil obtained, glyceride content calculated from acid value (%, oleic acid equivalent), fatty acid composition of glyceride fraction (area%), calculated from glyceride content Table 2 shows the EPA and DHA recovery (%). Changes in DHA content and DHA recovery are shown in FIGS.
In the lipase reaction without addition of calcium hydroxide, the DHA content in the glyceride fraction did not reach 50% even when the amount of lipase was increased or the reaction time was extended.

[Example 2] (Influence of calcium hydroxide amount)
Add 2mL of water and 6.7mg of lipase OF (660unit / g oil) to 4mL of purified fish oil 1 and add 4, 8, 20, 40, 60, 80, 120, 200, 320mg (0.11, 0.22, 0.55, 1.1, 1.7, 2.2, 3.3, 5.5, 8.8 wt% oil). This was stirred with a magnetic stirrer in a thermostat at 50 ° C. After stirring for 24 hours, the reaction oil was heated at 80 ° C. for 10 minutes to deactivate the lipase, and then the oil layer and the aqueous layer were separated by a centrifuge (40 ° C., 1800 g, 10 minutes) to obtain a reaction oil. .
Acid value of the obtained reaction oil, glyceride content calculated from the acid value (%, oleic acid equivalent), fatty acid composition of glyceride fraction (area%), recovery rate of EPA and DHA calculated from glyceride content (%), Table 3 shows the lipid composition of glycerides.
As a result of changing the addition amount of calcium hydroxide in the range of 0.11 to 8.8% by weight with respect to oil, it was shown that the higher the addition amount, the more concentrated the DHA.

[Example 3] (Effect of enzyme amount)
Add 4 mL of purified fish oil 1 with 2 mL of water and lipase OF 3.3, 6.7, 10, 13.3 mg (330, 660, 990, 1300 unit / g oil), and then add 40 mg of calcium hydroxide (1.1% oil by weight) did. This was stirred with a magnetic stirrer in a thermostat at 50 ° C. After stirring for 24 hours, the sampled reaction oil is heated at 80 ° C for 10 minutes to deactivate the lipase, and then the oil layer and water layer are separated by a centrifuge (40 ° C, 1800 g, 10 minutes) to remove the reaction oil. Obtained.
Acid value of the reaction oil obtained, glyceride content calculated from the acid value (%, oleic acid equivalent), fatty acid composition of glyceride fraction (area%), recovery rate of EPA and DHA calculated from glyceride content (%) It is shown in Table 4.
When the amount of calcium hydroxide was constant and the amount of enzyme was changed, DHA was concentrated as the amount of enzyme increased.

When the results of Examples 1 and 3 and Comparative Examples 1 and 2 are compared, it is not only possible to concentrate DHA to 50 area% or more by one lipase reaction by adding calcium hydroxide. Even when producing DHA45-50% concentrated oil, the amount of enzyme is less, the reaction time is shorter, or the amount of DHA recovered is higher than when there is no calcium hydroxide. There is.
In addition, since lipase OF is an enzyme with an excellent ability to concentrate DHA, the concentration of EPA decreases as the reaction proceeds. However, when calcium hydroxide is added, the EPA concentration can be kept high as compared with the comparative example. That is, while the EPA concentration in the lipase OF330u / g oil condition of Example 3 is 8.1%, the EPA concentration at 45-49% in which the DHA concentrations in Comparative Examples 1 and 2 are similar is 7% or less. is there. When calcium hydroxide was added, the total amount of EPA and DHA tended to increase.

[Example 4] (Influence of reaction time)
To 6 mL of purified fish oil 1, 3 mL of water and 15 mg of lipase OF (990 unit / g oil) were added, and 60 mg of calcium hydroxide (1.1% by weight to oil) was further added. This was stirred with a magnetic stirrer in a thermostat at 50 ° C. After stirring for 2, 4, 8, 24, and 48 hours, the sampled reaction oil was heated at 80 ° C for 10 minutes to inactivate the lipase, and then the oil layer and water were centrifuged (40 ° C, 1800g, 10 minutes). The layers were separated to obtain a reaction oil.
Acid value of the reaction oil obtained, glyceride content calculated from the acid value (%, oleic acid equivalent), fatty acid composition of glyceride fraction (area%), recovery rate of EPA and DHA calculated from glyceride content (%) Table 5 shows. In order to compare with the result of Comparative Example 2 reacted without adding calcium hydroxide, the result of Comparative Example 2 and the result of Example 4 were combined, and FIG. 3 shows the DHA content of the glyceride fraction. Shows the relationship between the DHA content in the glyceride fraction and the DHA recovery rate.
As shown in Table 5, the DHA content of the glyceride fraction reached its maximum at a reaction time of 24 hours, and at 48 hours, the concentration of DHA did not proceed and only the hydrolysis reaction proceeded. As shown in FIG. 3, when calcium hydroxide was added as compared with Comparative Example 2 in which no calcium hydroxide was added, a result of easily exceeding the DHA content of 50% was obtained. Also, FIG. 4 plotting the relationship with the DHA recovery rate shows that the concentration of DHA can be increased with the same amount of lipase by adding calcium hydroxide.

Example 5 (Influence of temperature)
Add 4 mL of purified fish oil 1 with 2 mL of water and lipase OF 3.3, 6.7, 10, 13.3 mg (330, 660, 990, 1300 unit / g oil), and then add 40 mg of calcium hydroxide (1.1% oil by weight) did. This was stirred with a magnetic stirrer in a constant temperature bath at 40 ° C or 50 ° C. After stirring for 24 hours, the sampled reaction oil is heated at 80 ° C for 10 minutes to deactivate the lipase, and then the oil layer and water layer are separated by a centrifuge (40 ° C, 1800 g, 10 minutes) to remove the reaction oil. Obtained.
Acid value of the reaction oil obtained, glyceride content calculated from the acid value (%, oleic acid equivalent), fatty acid composition of glyceride fraction (area%), recovery rate of EPA and DHA calculated from glyceride content (%) Table 6 shows.
It was confirmed that almost the same results were obtained at a reaction temperature of 40 to 50 ° C.

[Example 6] (Influence of raw materials)
4mL of refined fish oil 2 (solvent deacidified tuna oil, Nippon Suisan Co., Ltd.) or refined fish oil 3 (refined (deacidified, degummed, decolorized and deodorized) bonito oil, Nippon Suisan Co., Ltd.) with 2mL of water and lipase OF 10mg (990unit) / g oil) was added and 40 mg (1.1 wt% oil to oil) of calcium hydroxide was added. This was stirred with a magnetic stirrer in a thermostat at 50 ° C. After stirring for 24 hours, the sampled reaction oil is heated at 80 ° C for 10 minutes to deactivate the lipase, and then the oil layer and water layer are separated by a centrifuge (40 ° C, 1800 g, 10 minutes) to remove the reaction oil. Obtained.
As a comparative example, a reaction oil was obtained by reacting in the same manner except that calcium hydroxide was not added.
Fatty acid composition (area%) of purified fish oil 2 and 3, acid value of the reaction oil obtained, glyceride content calculated from acid value (%, oleic acid equivalent), fatty acid composition (area%) of glyceride fraction, glyceride content Table 7 shows the recovery rate (%) of EPA and DHA calculated from the above.
It was confirmed that the DHA content in the glyceride fraction was improved by adding calcium hydroxide and reacting regardless of the fish oil purification method and fish species.

Example 7 (Other reaction additives)
To 4 mL of purified fish oil 1, 2 mL of water and 6.7 mg of lipase OF (660 unit / g oil) were added, and 40 mg (1.1% to oil) of one additive was further added. This was stirred with a magnetic stirrer in a thermostat at 50 ° C. After stirring for 14 hours, the reaction oil was heated at 80 ° C. for 10 minutes to deactivate the lipase, and then the oil layer and the aqueous layer were separated by a centrifuge (40 ° C., 1800 g, 10 minutes) to obtain a reaction oil. .
Table 8 shows the DHA content (area%) of the glyceride fraction of the obtained reaction oil. Among the various additives, calcium hydroxide, magnesium chloride, ammonium sulfate, calcium carbonate, potassium dihydrogen phosphate, and ammonium chloride have been shown to concentrate the DHA content. In particular, it has been shown that calcium hydroxide has a remarkable effect.

  According to the present invention, fats and oils containing high concentrations of PUFAs such as EPA and DHA can be provided. That is, when a certain amount of PUFA such as EPA or DHA is added to health food or the like, a smaller amount of oil or fat may be added than before.

Claims (8)

A highly unsaturated fatty acid-enriched oil comprising: a step of subjecting a fat containing a highly unsaturated fatty acid as a fatty acid constituting a fat to a hydrolysis reaction with lipase; and a step of separating a glyceride fraction enriched in the highly unsaturated fatty acid. A highly unsaturated fatty acid, characterized in that any one of calcium hydroxide, magnesium chloride, ammonium sulfate, calcium carbonate, potassium dihydrogen phosphate, and ammonium chloride is added as a reaction additive in the hydrolysis reaction with lipase A method for producing concentrated oil. However, lipase is a lipase obtained from a microorganism belonging to Candida cylindoracea.   The process of claim 1 wherein the reaction additive is calcium hydroxide.   The method according to claim 1 or 2, wherein the highly unsaturated fatty acid is a fatty acid having 20 to 22 carbon atoms and 3 to 6 double bonds.   The process according to claim 1 or 2, wherein the highly unsaturated fatty acid is docosahexaenoic acid and / or eicosapentaenoic acid.   The method according to claim 1 or 2, wherein the highly unsaturated fatty acid-containing fat is fish oil.   The method according to any one of claims 1 to 5, wherein the reaction additive is added in an amount of 0.01 to 15% by weight based on the highly unsaturated fatty acid-containing fat.   The method of Claim 6 that the addition amount of a reaction additive is 0.05 to 10 weight% with respect to highly unsaturated fatty acid containing fats and oils. The method according to any one of claims 1 to 7 , wherein the lipase is lipase OF (trade name; Meisho Sangyo Co., Ltd.).

Images