KR101115283B1 - Process for preparing a complex of omega-3 unsaturated fatty acid and ascorbic acid - Google Patents

Abstract

The present invention provides a method for producing a complex of omega-3 unsaturated fatty acid and ascorbic acid by condensing omega-3 unsaturated fatty acid and ascorbic acid in the presence of a lipase, wherein the condensation is an ionic liquid; Or it provides a method for producing a complex of omega-3 unsaturated fatty acid and ascorbic acid, characterized in that carried out in a mixed solvent of an ionic solvent and an organic solvent. In the production method according to the present invention, by using a solvent system capable of dissolving high concentrations of ascorbic acid, the yield and productivity per volume can be significantly increased, thereby significantly improving the productivity.

Omega-3 Unsaturated Fatty Acids, Ascorbic Acid, Complex

Description

Process for preparing a complex of omega-3 unsaturated fatty acid and ascorbic acid}

The present invention relates to a method for preparing a complex of an omega-3 unsaturated fatty acid and ascorbic acid, and more particularly, an omega that can significantly increase the yield by using a specific solvent system in the enzymatic condensation reaction using lipase. An improved process for the preparation of complexes of -3 unsaturated fatty acids with ascorbic acid.

Omega-3 unsaturated fatty acids are also called omega-3 highly unsaturated fatty acids or polyunsaturated fatty acids (PUFAs) and are typically docosahexaene. Acids (Docosahexaenoic acid (DHA), Eicosapentaenoic aicd (EPA), Arachidonic acid (ARA), Docosapentaenoic acid (DocApentaenoic aicd, DPA), and α-linolenic acid are known. Docosahexaenoic acid and eicosapentaenoic acid are essential fatty acids that are not synthesized in the body and should be consumed mainly through food.

Docosahexaenoic acid is abundant in the retina, semen and brain tissue of humans and animals, and is an essential fatty acid that makes up 60% of brain fat. Docosahexaenoic acid, along with arachidonic acid (ARA), is known to be important for the healthy development of the brain, eyes and nervous system of infants. In recent years, it has been reported to be effective in the prevention and treatment of a number of diseases ranging from cancer to arthritis, cardiovascular diseases and mental disorders (Pak Deok-cheon, Food World, 5 , 87-93 (2004)).

Omega-3 unsaturated fatty acids are easily acidified by oxygen, and in the case of fish oil processed foods, fishy fish used as a raw material recurs, resulting in a remarkable deterioration in flavor (Terao et al. 1992, Adachi et al. 1995).

In order to overcome this disadvantage, it has been suggested to use a combination of fat-soluble natural antioxidant alpha-tocopherol in the form of a composition, but alpha-tocopherol also has a problem that is unstable to external factors such as heat and oxygen. In other words, alpha-tocopherol is oxidized to produce an unstable epoxide that can be irreversibly converted to alpha-topoferol quinone, thereby oxidizing it before it can function in food (Food Science and Industry June, 2002).

In addition, it has been proposed to complex ascorbic acid called vitamin C by ester-bonding with an omega-3 unsaturated fatty acid as an antioxidant. That is, ascorbic acid has a water-soluble property due to its chemical structure in spite of its powerful antioxidant effect, causing limitations in the application of food and cosmetics (Youchun Yan et al. 1999). It has been proposed to prepare in the form of fatty acid esters of corvinic acid. The obtained complex, that is, fatty acid ester, is a form in which omega-3 unsaturated fatty acid is bound to 6-position of ascorbic acid by ester bond, and is known to enhance solubility and antioxidant effect in fat-soluble environment due to its amphoteric nature. (XY Liu et al. 1996).

The complex of ascorbic acid and omega-3 unsaturated fatty acid may be prepared by chemical synthesis or enzymatic synthesis. For example, chemical synthesis methods are prepared by condensation of omega-3 unsaturated fatty acids and ascorbic acid using a coupling agent such as dicyclohexyl carbodiimide, or acid chlorides of omega-3 unsaturated fatty acids. It can be prepared by converting to a form and reacting with ascorbic acid (see US Pat. No. 6,069,168). However, the chemical synthesis method should use an organic solvent harmful to health, such as benzene, there is a disadvantage that the reaction by-products are generated due to the instability of ascorbic acid.

On the other hand, the enzymatic synthesis method dissolves omega-3 unsaturated fatty acid and ascorbic acid in an organic solvent such as acetone, and then condenses with lipase to condense ascorbic acid and omega-3 unsaturated fatty acid, that is, 6 of ascorbic acid. A complex is prepared in which the omega-3 unsaturated fatty acid is bonded to the position by an ester bond (Yoshiyuki Watanabe et al., JAOCS, 2001, 78, 823). The complex can be optionally purified by silica gel chromatography. However, the conventional enzymatic synthesis method has a problem in that the yield is very low and the productivity per volume (Volumetric Productivity, VP) is low. For example, the productivity per volume (g / L) in the known production method is only about 28 g / L.

The present inventors have conducted various studies in order to develop an improved manufacturing method that can solve problems in the enzymatic synthesis method, that is, low yield and low volumetric productivity (VP). While examining various conditions, the inventors found that the decrease in productivity per volume was caused by the low solubility of ascorbic acid in the organic solvent, and the dissolution of high concentration of ascorbic acid using a specific ionic liquid. It has been found that this problem can be solved if the solvent system is configured to allow.

Accordingly, an object of the present invention is to provide an enzymatic synthesis method capable of producing a complex of omega-3 unsaturated fatty acid and ascorbic acid with high yield and productivity per volume.

According to one aspect of the invention, in the method for producing a complex of omega-3 unsaturated fatty acid and ascorbic acid by condensing omega-3 unsaturated fatty acid and ascorbic acid in the presence of lipase, the condensation is an ionic solvent (ionic liquid); Or a method for producing a complex of omega-3 unsaturated fatty acid and ascorbic acid, which is carried out in a mixed solvent of an ionic solvent and an organic solvent.

In the preparation method of the present invention, the ionic solvent is trioctylmethylammonium trifluoroacetate, 1-butyl-3-methyl imidazolium CF ₃ SO ₃ (1-butyl-3- methylimidazolium? CF ₃ SO ₃ ), or mixtures thereof. In addition, the organic solvent may be at least one selected from the group consisting of methanol, ethanol, t-butanol, 2-methyl-2-butanol, and acetone. The mixed solvent of the ionic solvent and the organic solvent may be a mixed solvent having a volume ratio of 1: 1 to 3 of the ionic solvent and the organic solvent. In one embodiment, the mixed solvent may be a mixed solvent of 1: 1 (volume ratio) of trioctylmethylammonium trifluoroacetate and t-butanol. In addition, the concentration of ascorbic acid in the solvent may be 20 to 250 g / L, the molar ratio of the omega-3 unsaturated fatty acid and ascorbic acid may be in the range of 1: 1 to 6: 1.

In addition, in the preparation method of the present invention, the product obtained through the condensation is separated, and then the hexane extraction process is performed to remove the unreacted material, and then water is added to separate the complex of precipitated omega-3 unsaturated fatty acid and ascorbic acid. It may further comprise the step.

The production method according to the invention is an ionic solvent; Or a complex of an omega-3 unsaturated fatty acid and ascorbic acid by an enzymatic condensation reaction using a mixed solvent system of an ionic solvent and an organic solvent. The solvent system is capable of dissolving high concentrations of ascorbic acid, thereby significantly increasing the yield and productivity per volume, thereby significantly improving the productivity.

As used herein, "omega-3 unsaturated fatty acid" refers to omega-3 highly unsaturated fatty acid or polyunsaturated fatty acid (PUFA). which includes both the free acid form and also, acyl ester form acid-terminal carboxyl of the fatty acid to form an ester bond as a C ₁ ~ C ₄ alcohol. Specifically, docosahexaenoic acid (DHA), eicosapentaenoic aicd (EPA), arachidonic acid (ARA), docosapentaenoic acid (Docosapentaenoic aicd, DPA), and α Linolenic acid and the like, and C ₁ to C ₄ alcohols thereof and ester linkages, preferably DHA or EPA. When referred to herein as "PUFA", it means the aforementioned omega-3 unsaturated fatty acid.

In addition, "complex of omega-3 unsaturated fatty acid and ascorbic acid" means that the 6-position hydroxy of ascorbic acid is ester-bonded with omega-3 unsaturated fatty acid. It means a compound bound through, it is also referred to herein as "6-O-PUFA ascorbic acid".

The present invention provides a method for producing a complex of omega-3 unsaturated fatty acid and ascorbic acid by condensing omega-3 unsaturated fatty acid and ascorbic acid in the presence of a lipase, wherein the condensation is an ionic liquid; Or it provides a method for producing a complex of omega-3 unsaturated fatty acid and ascorbic acid, characterized in that carried out in a mixed solvent of an ionic solvent and an organic solvent.

The ionic solvent is trioctylmethylammonium trifluoroacetate (tOMATFA), 1-butyl-3-methylimidazolium CF ₃ SO ₃ (1-butyl-3-methylimidazolium CF ₃ SO ₃ , BMIM.CF ₃ SO ₃ ), or a mixture thereof. Preferably it is tOMA * TFA.

In addition, the organic solvent may be at least one selected from the group consisting of methanol, ethanol, t-butanol, 2-methyl-2-butanol, and acetone, and more preferably t-butanol.

The mixed solvent of the ionic solvent and the organic solvent may be a mixed solvent having a volume ratio of 1: 1 to 3 of the ionic solvent and the organic solvent. In one embodiment, the mixed solvent may be a mixed solvent of 1: 1 (volume ratio) of trioctylmethylammonium trifluoroacetate and t-butanol.

The production method according to the present invention enables the reaction of a high concentration of omega-3 unsaturated fatty acids and ascorbic acid compared to the reaction system using only the conventional organic solvent by employing the solvent system as described above, it is possible to significantly increase the productivity per volume. Therefore, in the condensation reaction, the concentration of ascorbic acid in the solvent may be in the range of 20 to 250 g / L, more preferably 100 to 250 g / L, particularly preferably about 200 g / L. In addition, the molar ratio of the PUFA and ascorbic acid may be in the range of 1: 1 to 6: 1, more preferably in the range of about 5: 1.

In the preparation method of the present invention, the lipase may be a known lipase derived from a microorganism, more specifically 1,3-specific or nonspecific lipase. The lipase can be recovered and reused after being used in the reaction, and thus can be referred to as 'immobilized lipase'. Lipase of the microorganism derived from Aspergillus (Aspergillus) genus Candida (Candida) genus Pseudomonas (Pseudomonas) in, chromotherapy tumefaciens (Chromobacterium) genus, Rhizopus crispus (Rhizopus) of the origin known from microorganisms such as genus lipase Can be. In addition, the lipases are commercially available enzymes such as Chirazyme L-2, c.-f. Or C2 (Roche Molecular Biochemicals) or Novozyme 435 (Novozymes Korea Co., Ltd.).

The condensation reaction can be carried out at 40 ~ 80 ℃, preferably 50 ~ 70 ℃, more preferably about 70 ℃, the content of the pure portion of the reaction system is maintained at 0.1% by weight or less, more preferably 0.01% by weight or less It is desirable to. If necessary, the reaction may be carried out by adding a molecular sieve, for example 4 kPa, to remove moisture. 6-O-PUFA ascorbic acid obtained through the condensation reaction can be purified by conventional chromatography using silica gel. More preferably, the product obtained through the condensation is separated, followed by hexane extraction to remove unreacted material (eg, unreacted PUFA), and then water is added to precipitate the omega-3 unsaturated fatty acid and The complex with corbic acid can be separated. Separation of the product from the condensation reaction mixture can be carried out by removing solids such as molecular sieves by a conventional method such as centrifugation and the like to obtain a supernatant. In addition, when water is added to the aqueous phase obtained by performing the hexane extraction process, the complex is precipitated, and the precipitate can be separated by conventional methods, for example, by filtration, centrifugation, and the like. The precipitate may be dried by a conventional method, for example, by drying under reduced pressure, to finally obtain a composite.

The productivity of 6-O-PUFA ascorbic acid according to the manufacturing method of the present invention as described above is in the range of 80g / L to 150g / L, preferably 120g / L to 150g / L, very good productivity can be achieved have.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are intended to illustrate the invention, but not to limit the scope of the invention.

Example 1 Evaluation of Influence of PUFA and Ascorbic Acid Molar Ratios

Productivity was evaluated by changing the molar ratio of EPA and L-ascorbic acid from 1: 1 to 9: 1. In other words, acetone dehydrated to 0.01% by weight or less of water content was used as a solvent, and Novozyme 435 (Novozymes Korea Co., Ltd.) was used as a lipase to 1 mass (ie, the total weight of EPA and L-ascorbic acid). It was added in a volume ratio of 1: 1. The reaction temperature was 50 ° C., the stirring speed was 500 rpm, and the reaction time was 36 hours. Samples were taken over time for yield analysis and analyzed based on the amount of ascorbic acid initially added. The result is shown in FIG. From FIG. 1, the reaction rate and yield change according to the molar ratio tended to increase as the molar ratio increased, that is, as the EPA content increased, but decreased after showing the maximum value (48%) at the molar ratio 5: 1. Indicated.

Example 2 Evaluation of Effect According to Water Content

Acetone was used as the reaction solvent, and the effect on the yield was evaluated while changing the water content of acetone from 0.01% by weight to 2% by weight. The molar ratio of EPA and ascorbic acid was 5: 1, and other conditions were the same as Example 1. The result is shown in FIG. 2, the yield was 0% when the water content is 2% by weight, which indicates that the direction of the esterification reaction is biased toward the reactant due to the presence of water. If the water content is 0.01% by weight, the yield is 25% or more, it can be seen that the removal of water has a significant effect on the yield or reactivity of the reaction.

As can be seen from Examples 1 and 2, when acetone was used as the solvent, there was a limit to the increase in productivity. This was confirmed to be due to the low solubility of ascorbic acid in acetone (data not shown). As a solvent, a condensation reaction was performed by configuring an ionic solvent or a mixed solvent system of an ionic solvent and an organic solvent.

Example 3 Preparation of 6-O-PUFA Ascorbic Acid Using tOMA-TFA

After dissolving 0.2148 g of ascorbic acid and 2.08 g of DHA in 1 ml of tOMA.TFA, 200 mg of molecular sieve (4 kPa) and 200 mg of lipase (Novozyme 435) were added, followed by stirring at 70 ° C. at 300 rpm for 24 hours. During the condensation reaction. The reaction mixture was centrifuged at 3000 rpm for 10 minutes to give a supernatant. 1 ml of hexane was added to the obtained supernatant, followed by extraction twice to remove unreacted PUFA. 1 ml of water was added to the remaining aqueous layer to form a precipitate, and then the centrifuged precipitate was recovered at 3000 rpm. The obtained precipitate was dried under reduced pressure to obtain 0.1 g of 6-O-PUFA ascorbic acid (productivity per volume: 100 g / L).

Example 4 Preparation of 6-O-PUFA Ascorbic Acid Using BMIM.CF ₃ SO ₃

The reaction was carried out in the same manner as in Example 3, except that BMIM.CF ₃ SO ₃ was used instead of 1 ml of tOMA.TFA to obtain 0.112 g of 6-O-PUFA ascorbic acid (productivity per volume: 112 g / L) .

Example 5 Preparation of 6-O-PUFA Ascorbic Acid Using a Mixed Solvent of tOMA.TFA and t-BuOH

6-O-PUFA ascorbic acid was carried out in the same manner as in Example 3, except that 1 ml (1: 1 v / v) of a mixed solvent of tOMA, TFA and t-BuOH was used instead of 1 ml of tOMA and TFA. 0.178 g was obtained (productivity per volume: 178 g / L).

Comparative example. Preparation of 6-O-PUFA Ascorbic Acid Using t-BuOH

The reaction was carried out in the same manner as in Example 3, except that 1 ml of t-BuOH was used instead of 1 ml of tOMA.TFA to obtain 0.0062 g of 6-O-PUFA ascorbic acid (productivity per volume: 62 g / L).

Figure 1 shows the results of evaluating the reactivity according to the molar ratio of PUFA and ascorbic acid.

Figure 2 shows the results of evaluating the change in yield according to the amount of water in the reaction solvent.

Claims (8)

In the method for producing a complex of omega-3 unsaturated fatty acid and ascorbic acid by condensing omega-3 unsaturated fatty acid and ascorbic acid in the presence of lipase, the condensation is trioctylmethylammonium trifluoroacetate and t-butanol 1: 1 to 3 (volume ratio) A method for producing a complex of omega-3 unsaturated fatty acid and ascorbic acid, characterized in that carried out in a mixed solvent. delete delete delete delete The method according to claim 1, wherein the concentration of ascorbic acid in the mixed solvent is 20 to 250 g / L. The method of claim 1, wherein the molar ratio of the omega-3 unsaturated fatty acid and ascorbic acid is 1: 1 to 6: 1. The method of claim 1, 6 or 7, wherein the product obtained through the condensation is separated, and then the hexane extraction process is performed to remove the unreacted material, and then water is added to precipitate the omega-3 unsaturated fatty acid and The method of claim 1, further comprising the step of separating the complex with cholic acid.

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