KR100850646B1 - Manufacturing method of phospholipid composition - Google Patents

Abstract

The present invention comprises the steps of administering a phospholipid, a solvent and a polyunsaturated fatty acid to a reactor, administering at least one enzyme selected from the phospholipase A1, phospholipase A2 and 1-3 lipase to the reactor, and It relates to a method for producing a phospholipid composition comprising the step of replacing or changing the fatty acid bound to phospholipid with polyunsaturated fatty acid through transesterification and acyl exchange reaction to obtain a recombinant phospholipid bound to polyunsaturated fatty acid. . According to the present invention, a phospholipid composition combined with polyunsaturated fatty acid having excellent human absorption can be obtained.

Phospholipids, DHA, EPA, ARA, 1,3-Lipase, Phospholipase A1, Phospholipase A2

Description

Manufacturing method of phospholipid composition

 1 is a graph showing the results of analyzing the content of DHA and EPA in the phospholipid composition.

Figure 2 is a graph showing the results of analyzing the content of DHA and EPA in the phospholipid composition.

Figure 3 is a graph showing the results of analyzing the content of DHA, EPA and ARA in the phospholipid composition.

Figure 4 is a graph showing the results of analyzing the content of phosphatidylcholine and phosphatidylethanolamine in the phospholipid composition.

The present invention relates to a method for preparing a phospholipid composition, and more particularly, a human absorption rate through a transesterfication and an acyl exchange reaction using phospholipase A1, phospholipase A2 or lipase. The present invention relates to a method for producing a recombinant phospholipid bound to this excellent polyunsaturated fatty acid.

Polyunsaturated fatty acids are largely divided into omega-6 and omega-3. Omega-6 fatty acids include linoleic acid (C18: 3, LA), linoleic acid (C18: 2, LA), γ-linoleic acid (C18: 3, GLA), dihomo-γ-linoleic acid ( C20: 3, DHLA) or arachidonic acid (C20: 4, ARA). Omega-3 fatty acids include ω-3 fatty acids such as α-linoleic acid (C18: 3, ALA), eicosapentaenoic acid (C20: 5, EPA), and docosahexaenoic acid. (C22: 6, DHA) and the like. They are 2-prostaglandins, 4-leukotrienes, 2-thromboxanes, 5-leukotriens, 3-prostaglandins, 3-throm, which play a physiologically important function during human metabolism. Synthesize thromboxanes and the like. Substances that occur during this metabolic process are involved in the body, ranging from diseases of the circulatory system, the respiratory system, the digestive system, the kidneys, the skin and the immune system, the development of the brain and the eye, and the development of cancer.

Eicosapentaenoic acid (cis-5,8,11,14,17-Eicosapenta-enoic acid; EPA, C20: 5), doco, a type of polyunsaturated fatty acid (PUFA) of the omega-3 family Tetrahexanoic acid (cis-4,7,10,13,16,19-Docosahexaenoic acid; DHA, C22: 6) and omega-6-based arachidonic acid (cis-5,8,11,14-Arachidonic acid; ARA, C20: 4) are generally well known.

In general, omega-3 long-chain polyunsaturated fatty acids, EPA and DHA, respectively, inhibit platelet aggregation, prevent blood clots, do not lower blood HDL cholesterol, and lower VLDL and LDL-cholesterol. In addition, it is known to have pharmacological effects such as lowering neutral lipids in the blood, lowering the viscosity of blood, and improving blood circulation.

DHA, which is contained locally in the gray matter, retina, nerve, heart, sperm and breast milk of the human brain, has the effect of improving memory learning function. In particular, the analysis of the fatty acids that make up the brain contains a large amount of DHA. This means that among the omega-3 fatty acids in the human body, fatty acids that can cross the brain barrier are the most DHA. However, in order for DHA to cross the brain barrier, it is easier for phospholipid-bound forms to cross the brain barrier than for triglyceride form of DHA. In addition, in the case of phospholipids containing DHA, the membrane fluidity of the cells is excellent, the activation of neurons and the activation of neurotransmitters is improved, the brain function, anticancer action, cholesterol and triglyceride lowering action, anti allergic action, Many reports have been made on the pharmacological effects of the retina's ability to improve reflexes (such as suppressing vision lowering), lowering blood pressure, and anti-inflammatory effects. It is widely used as a material.

EPA, DHA and ARA are being used as dietary supplements worldwide. Of these, high purity EPA (95%) has been approved by the US FDA for the treatment of hyperlipidemia. Generally supplied EPA or DHA is their ethyl ester or triacyl glycerol (TG) in general form. However, they lose their solubility in water, which is combined with phospholipids in the small intestine in the human body and absorbed into the body. In other words, polyunsaturated fatty acids in the form of phospholipids are highly absorbed by the body. In general, the DHA efficacy of phospholipids containing 25% DHA is reported to be similar to ethyl esters containing 95% DHA. However, since the extraction method using a large amount of organic solvent is mainly used in the manufacturing method, it is difficult to carry out on a large scale and has a disadvantage that the cost is high.

Therefore, there is a constant need to develop a technology for producing phospholipids containing a large amount of EPA, DHA or ARA, which is easily absorbed in the body, in a more economical manner. To this end, various attempts have been made at home and abroad to increase the content of PUFAs (EPA, DHA and ARA) in phospholipids.

In Japan, Suntory and Bizen Chemical (PCT / JP 96/01453), ARA, EPA and DHA contents were added to the poultry eggs by adding cultured cells with high content of omega-3 and omega-6 fatty acids. The technology to produce this increased yolk lipid is introduced. In the Republic of Korea Patent Publication (10-1998-013757), there is an attempt to adjust the ratio of omega-3 fatty acids by using sesame jelly, wild jelly, etc. as a feed additive, and in the Republic of Korea Patent Publication (10-1993-0005548) Katsuobushi B. The method for producing eggs containing 0.5g or more of DHA per 100g of egg yolk is introduced by feeding and breeding the waste obtained from the manufacture of squid by adding 0.5 to 5.0 wt% to the feed. However, the content of EPA and DHA is still low, and no method for preparing phospholipid compositions in the form in which these omega-3 fatty acids are bound to phospholipids has not been introduced. In addition, there have been attempts to increase the content of omega-3 fatty acids by combining fatty acids having a high content of omega-3 fatty acids with egg yolk lipids (see Korean Patent Application Laid-Open No. 10-2001-0035040). It is difficult to call it a phospholipid composition containing a fatty acid.

Recent studies have shown that EPA, DHA (Omega-3) and ARA (Omega-6) -bound phospholipids in combination with these phospholipids, omega-3 and omega-6 PUFAs, improve cell membrane fluidity. , And physiological effects by inhibiting the production of inflammatory substances (Eicosanoid, etc.), and in particular, the effects of DHA and EPA-bound phospholipids have been confirmed for brain reactivation function, increasing sleep time, improving learning ability, and improving memory. . As such, EPA and DHA bound to phospholipids are not only higher in physiological activity than EPA and DHA in natural fats and oils, but also have unique effects as EPA and DHA-bound phospholipids. However, the fact that the phospholipids present in nature is low in the content of phospholipids combined with EPA, DHA and ARA, it is difficult to expect a physiological activity effect based on the normal intake. In addition, most of the omega-6-based arachidonic acid (ARA) is present in the human body in the form of phospholipids, it is expected to have a superior physiological activity effect when ingested compared to triacylglycerol (TG) type oil.

The technical problem to be achieved by the present invention is phospholipase A1, phospholipase A2 or lipase (Lipase) using a transesterfication (transesterfication) and acyl exchange (Transacylation) through the combination of polyunsaturated fatty acids with excellent human absorption rate To provide a method for producing a recombinant phospholipid.

The present invention comprises administering a polyunsaturated fatty acid having a phospholipid, a solvent and an acyl group to a reactor, and administering at least one enzyme selected from phospholipase A1, phospholipase A2 and lipase to the reactor. And replacing or changing the fatty acid bound to the phospholipid into a polyunsaturated fatty acid through transesterification and acyl exchange by the enzyme to obtain a recombinant phospholipid bound to the polyunsaturated fatty acid. Provided is a method for preparing a phospholipid composition.

Natural or phosphatidylcholine, phosphatidylethanolamine, phosphatidyl acid, phosphatidylserine, phosphatidyl inositol, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylserine and lysophosphatidyl inositol as phospholipids administered to the reactor. Synthetic phospholipids can be used.

As the solvent, an organic solvent including at least one of n-hexane, ethanol, acetone, chloroform, and ethyl acetate may be used. Acetate buffer or phosphate buffer, which is a buffer, may be further included in the solvent.

As the polyunsaturated fatty acid administered to the reactor, polyunsaturated fatty acids having an acyl group containing at least one of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and arachidonic acid (ARA) may be used. .

The enzyme may substitute or change fatty acids at positions sn-1 and sn-2 of the phospholipid to polyunsaturated fatty acids through transesterification and acyl exchange.

The recombinant phospholipid bound to the polyunsaturated fatty acid is preferably controlled so that the content of phosphatidylcholine is 30 to 95% by weight relative to the recombinant phospholipid bound to the polyunsaturated fatty acid.

The recombinant phospholipid can be obtained by making the gas atmosphere of the reactor into a nitrogen gas atmosphere and setting the temperature of the reactor in the range of 20 to 95 ° C. for 12 to 90 hours in consideration of activation of enzymes.

The recombinant phospholipid combined with polyunsaturated fatty acid has 15 to 75% by weight of polyunsaturated fatty acid with respect to the total fatty acids of recombinant phospholipid, and 15 to 75% by weight of DHA as polyunsaturated fatty acid with respect to all fatty acids of recombinant phospholipid. % Or polyunsaturated fatty acid is preferably controlled so that the content of ARA is 5 to 45% by weight based on the total fatty acids of the recombinant phospholipid.

The present invention also provides a phospholipid composition as a food, cosmetic or pharmaceutical material prepared by the method for producing a phospholipid composition according to the present invention.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, the following embodiments are provided to those skilled in the art to fully understand the present invention, and may be modified in various forms, and the scope of the present invention is limited to the embodiments described below. It doesn't happen. Like numbers refer to like elements in the figures.

The present invention converts phospholipid fatty acids into polyunsaturated fatty acids through transesterification and acyl exchange using enzymes, and then DHA (docosahexaenoic acid), EPA (Eicosapentaenoic acid) or ARA (Arachidonic acid). A method of preparing phospholipids rich in content is presented.

Phospholipid composition according to a preferred embodiment of the present invention, phospholipid (Phospholipase) A1, phospholipase A2 or lipase (Lipase) using the phospholipids sn-1 and sn-2 site of the phospholipids Saturated fatty acids or unsaturated fatty acids are converted to polyunsaturated fatty acids such as EPA, DHA or ARA by transesterification or acyl exchange to obtain phospholipids bound to DHA, EPA or ARA.

Phospholipids are known as major components of human brain, optic nerve and central nervous system tissues. Phospholipids exist in the form of a mixture of several phospholipid homologues such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Recombinant phospholipids with a very high content of PUFAs (DHA, EPA and ARA) in phospholipids can be prepared based on the conversion of fatty acids using enzymes to existing phospholipids.

Hereinafter, the method for preparing the phospholipid composition combined with EPA, DHA or ARA according to the present invention will be described in detail.

Administer phospholipids to the reactor. The phospholipid is a natural or synthetic phospholipid comprising at least one of phosphatidylcholine, phosphatidylethanolamine, phosphatidyl acid, phosphatidylserine, phosphatidyl inositol, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidyl acid, lysophosphatidylserine and lysophosphatidyl inositol ( phospholipid), for example soybean or egg yolk phospholipid.

The solvent is administered to the reactor. The solvent may be an organic solvent including at least one of n-hexane, ethanol, acetone, chloroform and ethyl acetate. The weight ratio of the phospholipid and the solvent is preferably 1: 1 to 1: 5. Meanwhile, a buffer may be further included in the solvent. For example, an ideal system in which n-hexane and a buffer are mixed may be used as the solvent. The ratio of n-hexane and the buffer is preferably 5: 1 to 50: 1 by weight. Acetate buffer or phosphate buffer may be used as the buffer, and the pH is preferably 3.5 to 8.5. The solvent can be used to dissolve the phospholipids, and as a result, the dissolution is smooth and easy to recover the reactants after the reaction.

Phospholipids are generally graded by the content of phosphatidyl choline (PC). Among them, high purity phospholipid and phosphatidylcholine having a content of 20-40% by weight of phosphatidyl choline and 20-30% by weight of phosphatidylethanolamine (PE) are 40-95% by weight. Phospholipids may not melt well depending on the nature of the organic solvent. In particular, in the case of ethanol, ethyl acetate, acetone, when a phospholipid is used at a certain concentration or more, agitation is not smooth and phospholipid aggregation occurs and the reactivity is very low.

In a preferred embodiment of the present invention, however, n-hexane, ethanol, acetone, chloroform or ethyl acetate is used as the solvent, and at the same time, temperature change and composition change of the solvent are considered to obtain proper solubility. The present invention is to increase the content of PUFA combined with high-purity phospholipids by changing the fatty acid composition of the high-purity phospholipids as well as medium-low purity phospholipids (phospholipid content of 50% by weight or less). It also facilitates the recovery of the phospholipid reactants produced as reaction residues after the reaction.

The polyunsaturated fatty acid having an acyl group is administered to the reactor to which the phospholipid is administered. The polyunsaturated fatty acid having an acyl group may be an omega-3 polyunsaturated fatty acid or an omega-6 polyunsaturated fatty acid. The polyunsaturated fatty acid of the omega-3 series may be eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA), and the polyunsaturated fatty acid of the omega-6 series may be arachidonic acid (ARA).

The reactor is administered with at least one enzyme selected from phospholipase A1, phospholipase A2 and 1-3 lipases. The enzyme replaces or changes fatty acids at positions sn-1 and sn-2 of fatty acids bound with phospholipids to polyunsaturated fatty acids with excellent human absorption through transesterification or acyl exchange.

The enzymes used in the preferred examples of the present invention are shown in Table 1.

Source Enzyme Source Supplier Specificity      Lipase Aspergillus niger A, B 1,3-specific Candida antarctica E Nonspecific Candida rugosa (Former Candida cylindracea) A, D, E, F Nonspecific Mucoja vanicusa A 1,3-specific Penicillium cyclopium B 1,3-specific Rhizopus sp. A, H 1,3-specific Rhizopus delemar A, F 1,3-specific Rizomucor miehei (Former Mucor miehei) E 1,3-specific Rhizopus oryzae (Former Rhizopus arrhizus) A, C 1,3-specific Thermomyces lanuginosa (Former Humicola lanuginosa) B, N 1,3-specific Phospholipase Bee venom G Sn-2 Guncine Pancreas A, N Sn-2

In the suppliers of Table 1, A is Amano Pharmaceutical Co. (Nagoya, Japan), B is Biocatalysts (Pontyprid, UK), and C is Gist-Brocade. Brocades (Delft, Netherlands), D for Meito Sangyo (Aichi, Japan), E for Novozymes A / S (Bagsvaerd, Denmark), F for Seikagaku Koyosa Seikagaku Kogyo Co. (Tokyo, Japan), G stands for Sigma Chemical (St. Louis, MO), and H stands for Tanabe Seiyaku (Osaka, Japan).

The atmosphere in the reactor is made into a nitrogen gas atmosphere and reacted. That is, the reactor is purged with nitrogen gas to remove oxygen in the reactor and reacted in a nitrogen gas atmosphere. By this reaction, the composition of saturated or unsaturated fatty acids bound to the sn-1 and sn-2 sites of the phospholipid is replaced or changed to EPA, DHA or ARA by transesterification or acyl exchange. In addition, a recombinant phospholipid with a high content of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), or arachidonic acid (ARA) can be obtained. The temperature of the reaction is preferably in the range of 20 ℃ to 95 ℃ considering the activation temperature of the enzyme, the reaction time is 12hr ~ 90hr in consideration of transesterification or acyl exchange (transacylation) by the enzyme It is preferable. In addition, RPM (revolution per minute) of the reactor is preferably about 200 to 1000 in consideration of transesterification or acyl exchange (transacylation) by the enzyme. In addition, the recombinant phospholipid obtained by the reaction is preferably controlled so that the content of phosphatidylcholine is 30 to 95% by weight relative to the recombinant phospholipid. In addition, the recombinant phospholipid obtained by the reaction is 15 to 75% by weight of the total polyunsaturated fatty acid of EPA of polyunsaturated fatty acid, and the content of DHA of polyunsaturated fatty acid to all the fatty acids of recombinant phospholipid. It is preferable to control so that content of ARA which is 15-75 weight% with respect to polyunsaturated fatty acid, or 5 to 45 weight% with respect to the whole fatty acid of recombinant phospholipid is with respect to.

Analytical conditions for measuring the amount of EPA, DHA and ARA of the final product of the present invention is as follows.

<Analysis Condition>

In order to measure the content of EPA, DHA and ARA bound to phospholipids, the reactants were extracted and purified by acetone insolubles in the phospholipid content measurement method in food industry, and then methyl-esterification gas-chromatography ( gas-chromatography).

To 0.2 g of the extracted sample, 6 ml of methanol containing 0.5 N NaOH was added, followed by agitation at 100 ° C., followed by 7 ml of methanol containing 10% BF ₃ (Fluka Co., Switzerland). . After 5 minutes, 6 ml of n-hexane was added and allowed to stand for 2 minutes, after which the solution was transferred to a separatory filter. 2 ml of saturated NaCl solution is added to the transferred solution, shaken for 30 seconds, and then centrifuged. Water was removed by adding anhydrous sodium sulfate to the centrifuged supernatant, followed by injection into 1 μl gas-chromatography (Hewlett Packard, USA, 5890 II).

Table 2 is a table showing the analysis conditions for gas chromatography.

Gas chromatography parameter Column HP-Innowax (30m * 0.32mm id; 0.15μm) Detector temperature (℃) 300 ℃ / FID Oven Temperature (℃) Hold at 150 ° C for 10 minutes, rise to 3 ° C / min, hold at 210 ° C for 12 minutes, rise to 3 ° C / min, hold at 250 ° C for 15 minutes Injector temperature (℃) 250 ℃

The invention is described in more detail with reference to the following experimental examples, which do not limit the invention.

Experimental Example 1

10 g of phospholipid was administered to the reactor, and 10 g of a solvent, n-hexane, was administered to the reactor. In addition, 20 g of polyunsaturated fatty acid DHA (70%) was administered to the reactor, and 10 g of enzyme 1-3 lipase (Lipase) was administered. In addition, 2% (weight / volume) of the total substrate reaction solution was administered to the reactor using a phosphate buffer (Phosphate Buffer) as a buffer. After filling the reactor with nitrogen gas to remove oxygen in the reactor, the reaction was conducted at 400 RPM for 30 hours at a temperature of 45 ° C. in a sealed state. After the reaction, the enzyme having completed the reaction was removed by using a filter having a pore size of 100 μm, and concentrated in vacuo under nitrogen to remove n-hexane, which was immersed in acetone at −20 ° C. for 2 hours. After washing with 500 ml of acetone at room temperature, phospholipids were recovered as an acetone insoluble matter. Thus obtained recombinant phospholipid was 25% by weight of the total fatty acid of the recombinant phospholipid, yield 78% by gas-chromatographic analysis.

Experimental Example 2

10 g of phospholipid was administered to the reactor, and 10 g of a solvent, n-hexane, was administered to the reactor. In addition, 20 g of polyunsaturated fatty acid DHA (70%) was administered to the reactor, and 10 g of enzyme 1-3 lipase (Lipase) was administered. In addition, 4% (weight / volume) of the total substrate reaction solution was administered to the reactor using a phosphate buffer (Phosphate Buffer) as a buffer. After filling the reactor with nitrogen gas to remove oxygen in the reactor, the reaction was carried out for 45 hours at 400 RPM at a temperature of 35 ° C. in a sealed state. After the reaction, the enzyme having completed the reaction was removed by using a filter having a pore size of 100 μm, and concentrated in vacuo under nitrogen to remove n-hexane, which was immersed in acetone at −20 ° C. for 2 hours. After washing with 500 ml of acetone at room temperature, phospholipids were recovered as an acetone insoluble matter. The recombinant phospholipids thus obtained were obtained by gas-chromatographic analysis. The DHA content was 22% by weight based on the total fatty acids of the recombinant phospholipids, and the yield was 71%.

Experimental Example 3

10 g of phospholipid was administered to the reactor, and 30 g of a solvent, n-hexane, was administered to the reactor. In addition, 20 g of polyunsaturated fatty acid DHA (70%) was administered to the reactor, and 2 g of phospholipase A2, an enzyme, was administered. In addition, 4% (weight / volume) of the total substrate reaction solution was administered to the reactor using a phosphate buffer (Phosphate Buffer) as a buffer. After filling the reactor with nitrogen gas to remove oxygen in the reactor, the reaction was carried out for 40 hours at 400 RPM at a temperature of 40 ℃ in a closed state. After the reaction, the enzyme having completed the reaction was removed by using a filter having a pore size of 100 μm, and concentrated in vacuo under nitrogen to remove n-hexane, which was immersed in acetone at −20 ° C. for 2 hours. After washing with 500 ml of acetone at room temperature, phospholipids were recovered as an acetone insoluble matter. The recombinant phospholipids thus obtained were found to have a DHA content of 36% by weight based on the total fatty acid of the recombinant phospholipids, and a yield of 64%.

Experimental Example 4

10 g of phospholipid was administered to the reactor, and 30 g of a solvent, n-hexane, was administered to the reactor. In addition, 20 g of polyunsaturated fatty acid DHA (70%) was administered to the reactor, and 10 g of phospholipase A2, an enzyme, was administered. In addition, 4% (weight / volume) of the total substrate reaction solution was administered to the reactor using a phosphate buffer (Phosphate Buffer) as a buffer. After filling the reactor with nitrogen gas to remove oxygen in the reactor, the reaction was conducted at 400 RPM for 30 hours at a temperature of 45 ° C. in a sealed state. After the reaction, the enzyme having completed the reaction was removed by using a filter having a pore size of 100 μm, and concentrated in vacuo under nitrogen to remove n-hexane, which was immersed in acetone at −20 ° C. for 2 hours. After washing with 500 ml of acetone at room temperature, phospholipids were recovered as an acetone insoluble matter. The recombinant phospholipids thus obtained had a DHA content of 52% by weight based on the total fatty acids of the recombinant phospholipids, and a yield of 70%.

Experimental Example 5

10 g of phospholipid was administered to the reactor, and 10 g of a solvent, n-hexane, was administered to the reactor. In addition, 20 g of polyunsaturated fatty acid DHA (70%) was administered to the reactor, and 5 g of enzyme 1-3 Lipase and 0.1 g of phospholipase A2 were administered. In addition, 4% (weight / volume) of the total substrate reaction solution was administered to the reactor using a phosphate buffer (Phosphate Buffer) as a buffer. After filling the reactor with nitrogen gas to remove oxygen in the reactor, the reaction was performed at 400 RPM at a temperature of 60 ° C. for 90 hours in a sealed state. After the reaction, the enzyme having completed the reaction was removed by using a filter having a pore size of 100 μm, and concentrated in vacuo under nitrogen to remove n-hexane, which was immersed in acetone at −20 ° C. for 2 hours. After washing with 500 ml of acetone at room temperature, phospholipids were recovered as an acetone insoluble matter. The recombinant phospholipids thus obtained were found to have a DHA content of 36 wt% based on the total fatty acids of the recombinant phospholipids, and a yield of 63%.

Experimental Example 6

10 g of phospholipid was administered to the reactor, and 10 g of a solvent, n-hexane, was administered to the reactor. In addition, 20 g of polyunsaturated fatty acid EPA (70%) was administered to the reactor, and 10 g of enzyme 1-3 lipase (Lipase) was administered. In addition, 2% (weight / volume) of the total substrate reaction solution was administered to the reactor using a phosphate buffer (Phosphate Buffer) as a buffer. After filling the reactor with nitrogen gas to remove oxygen in the reactor, the reaction was carried out for 72 hours at 400 RPM at a temperature of 60 ℃ in a closed state. After the reaction, the enzyme having completed the reaction was removed by using a filter having a pore size of 100 μm, and concentrated in vacuo under nitrogen to remove n-hexane, which was immersed in acetone at −20 ° C. for 2 hours. After washing with 500 ml of acetone at room temperature, phospholipids were recovered as an acetone insoluble matter. The recombinant phospholipid thus obtained was found to be 28.3% by weight, and the yield was 76.4% based on gas-chromatographic analysis of the total fatty acid of the recombinant phospholipid.

Experimental Example 7

10 g of phospholipid was administered to the reactor, and 10 g of a solvent, n-hexane, was administered to the reactor. In addition, 10 g of polyunsaturated fatty acid EPA (70%) was administered to the reactor, and 10 g of enzyme 1-3 lipase (Lipase) was administered. In addition, 4% (weight / volume) of the total substrate reaction solution was administered to the reactor using a phosphate buffer (Phosphate Buffer) as a buffer. After filling the reactor with nitrogen gas to remove oxygen in the reactor, the reaction was carried out at 400 RPM at a temperature of 60 ° C. for 24 hours in a closed state. After the reaction, the enzyme having completed the reaction was removed by using a filter having a pore size of 100 μm, and concentrated in vacuo under nitrogen to remove n-hexane, which was immersed in acetone at −20 ° C. for 2 hours. After washing with 500 ml of acetone at room temperature, phospholipids were recovered as an acetone insoluble matter. The recombinant phospholipid thus obtained was found to be 24.2% by weight and the yield was 71% based on the gas-chromatographic analysis of the total fatty acid of the recombinant phospholipid.

Experimental Example 8

10 g of phospholipid was administered to the reactor, and 30 g of a solvent, n-hexane, was administered to the reactor. In addition, 20 g of polyunsaturated fatty acid EPA (70%) was administered to the reactor, and 10 g of phospholipase A2, an enzyme, was administered. In addition, 4% (weight / volume) of the total substrate reaction solution was administered to the reactor using a phosphate buffer (Phosphate Buffer) as a buffer. The reactor was filled with nitrogen gas to remove oxygen in the reactor, and the reaction was carried out at 1000 RPM for 60 hours at a temperature of 60 ° C. in a sealed state. After the reaction, the enzyme having completed the reaction was removed by using a filter having a pore size of 100 μm, and concentrated in vacuo under nitrogen to remove n-hexane, which was immersed in acetone at −20 ° C. for 2 hours. After washing with 500 ml of acetone at room temperature, phospholipids were recovered as an acetone insoluble matter. Thus obtained recombinant phospholipid was found to be 44.9% by weight and 64% of the total fatty acid of the recombinant phospholipid by gas-chromatographic analysis.

Experimental Example 9

10 g of phospholipid was administered to the reactor, and 10 g of a solvent, n-hexane, was administered to the reactor. In addition, 20 g of polyunsaturated fatty acid EPA (70%) was administered to the reactor, and 5 g of enzymes 1-3 lipase and 10 g of phospholipase A2 were administered. In addition, 4% (weight / volume) of the total substrate reaction solution was administered to the reactor using a phosphate buffer (Phosphate Buffer) as a buffer. After filling the reactor with nitrogen gas to remove oxygen in the reactor, the reaction was carried out for 48 hours at 1000 RPM at a temperature of 40 ℃ in a closed state. After the reaction, the enzyme having completed the reaction was removed by using a filter having a pore size of 100 μm, and concentrated in vacuo under nitrogen to remove n-hexane, which was immersed in acetone at −20 ° C. for 2 hours. After washing with 500 ml of acetone at room temperature, phospholipids were recovered as an acetone insoluble matter. The recombinant phospholipid thus obtained was found to be 53.8% by weight and 63% of the total fatty acid of the recombinant phospholipid as a result of gas chromatography analysis.

Experimental Example 10

10 g of phospholipid was administered to the reactor, and 10 g of a solvent, n-hexane, was administered to the reactor. In addition, 20 g of polyunsaturated fatty acid EPA (70%) was administered to the reactor, and 5 g of enzymes 1-3 lipase and 0.1 g of phospholipase A2 were administered. In addition, 4% (weight / volume) of the total substrate reaction solution was administered to the reactor using a phosphate buffer (Phosphate Buffer) as a buffer. After filling the reactor with nitrogen gas to remove oxygen in the reactor, the reaction was carried out for 72 hours at 1000 RPM at a temperature of 50 ℃ in a sealed state. After the reaction, the enzyme having completed the reaction was removed by using a filter having a pore size of 100 μm, and concentrated in vacuo under nitrogen to remove n-hexane, which was immersed in acetone at −20 ° C. for 2 hours. After washing with 500 ml of acetone at room temperature, phospholipids were recovered as an acetone insoluble matter. The recombinant phospholipids thus obtained were gas-chromatographically analyzed. The EPA content was 42.2% by weight based on the total fatty acids of the recombinant phospholipids, and the yield was 63%.

Experimental Example 11

10 g of phospholipid was administered to the reactor, and 10 g of a solvent, n-hexane, was administered to the reactor. In addition, 20 g of polyunsaturated fatty acid ARA (70%) was administered to the reactor, and 10 g of enzyme 1-3 lipase was administered. In addition, 2% (weight / volume) of the total substrate reaction solution was administered to the reactor using a phosphate buffer (Phosphate Buffer) as a buffer. After filling the reactor with nitrogen gas to remove oxygen in the reactor, the reaction was carried out for 72 hours at 1000 RPM at a temperature of 60 ℃ in a sealed state. After the reaction, the enzyme having completed the reaction was removed by using a filter having a pore size of 100 μm, and concentrated in vacuo under nitrogen to remove n-hexane, which was immersed in acetone at −20 ° C. for 2 hours. After washing with 500 ml of acetone at room temperature, phospholipids were recovered as an acetone insoluble matter. The recombinant phospholipids thus obtained were found to have a ARA content of 28.3% by weight and a yield of 76.4% with respect to the total fatty acids of the recombinant phospholipids by gas-chromatographic analysis.

1 is a graph showing the results of analyzing the content of DHA and EPA in the phospholipid composition, which is the result when the experiment according to the Experimental Example 4.

Figure 2 is a graph showing the results of analyzing the content of DHA and EPA in the phospholipid composition, which is the result when the experiment according to the experimental example 9.

Figure 3 is a graph showing the results of analyzing the content of DHA, EPA and ARA in the phospholipid composition, which is the result when the experiment according to Experimental Example 10.

Figure 4 is a graph showing the results of analyzing the content of phosphatidylcholine and phosphatidylethanolamine in the phospholipid composition.

4 is 0.5g of samples (phospholipids) obtained through the experimental examples, dissolved in methanol and extracted for 5 minutes with ultrasonic wave, and then justified to 100ml. The solution thus obtained is taken up to 10ml with methanol and filtered to 100ml. As a test solution, the result was analyzed by High Pressure Liquid Chromatography (HPLC). Equipment analysis conditions using high pressure liquid chromatography are shown in Table 3 below.

High Pressure Liquid Chromatography Parameter High Pressure Liquid Chromatography (HPLC) Waters 600 Detector Waters 2420 Evaporative Light-Scattering Detector (ELSD) Column Novapak Silica C18 (150mm * 2.1mm) Mobile Phase n-hexane: isopropanol: Deionized Water (30: 60:10) Nebulization Temperature 60 ℃ Pressure (N ₂ gas) 30 psi Drift Tube Temperature 75 ℃ Injection Volume 20 μl Flow Rate 0.5 ml / min

According to the present invention, phospholipids combined with polyunsaturated fatty acids (PUFA) that are safe even for application to food and other industries can be obtained.

The present invention uses saturated or unsaturated fatty acids at the sn-1 and sn-2 site sites of phospholipids using enzymes such as 1,3-lipase, phospholipase A1 or phospholipase A2. EPA, DHA or omega-6 long chain highly unsaturated fatty acids ARA (arachidonic acid), which is a high concentration of phospholipids, are contained in a phospholipid by esterification or acyl exchange reaction. You can get it.

In addition, the phospholipid composition according to the present invention can be used as a food, cosmetic or pharmaceutical material.

As mentioned above, although preferred embodiment of this invention was described in detail, this invention is not limited to the said embodiment, A various deformation | transformation by a person of ordinary skill in the art within the scope of the technical idea of this invention is carried out. This is possible.

Claims (11)

Administering an unsaturated fatty acid having a phospholipid, a solvent, and an acyl group to the reactor; Administering at least one enzyme selected from phospholipase A1, phospholipase A2, and lipase to the reactor; Preparing a phospholipid composition comprising the step of replacing or changing the fatty acid bound to the phospholipid with an unsaturated fatty acid through transesterification and acyl exchange (transacylation) by the enzyme to obtain a recombinant phospholipid bound to the unsaturated fatty acid Way. The phospholipid administered to the reactor is phosphatidylcholine, phosphatidylethanolamine, phosphatidyl acid, phosphatidylserine, phosphatidyl inositol, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidyl acid, lysophosphatidyl inositol Method for producing a phospholipid composition, characterized in that using at least one natural or synthetic phospholipid (phospholipid). The method of claim 1, wherein an organic solvent including at least one of n-hexane, ethanol, acetone, chloroform and ethyl acetate is used as the solvent. The method of claim 3, further comprising the step of introducing an acetate buffer (acetate buffer) or phosphate buffer (acid buffer) having a pH of the acid to the reactor. According to claim 1, Unsaturated fatty acid administered to the reactor, unsaturated having an acyl group containing at least one of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and arachidonic acid (ARA) Method for producing a phospholipid composition, characterized in that using fatty acids. According to claim 1, wherein the enzyme is characterized in that to replace or change the fatty acid at the position of sn-1 and sn-2 of the phospholipid with unsaturated fatty acid through transesterification and acyl exchange (transacylation) Phospholipid composition manufacturing method. The phospholipid composition according to claim 1, wherein the recombinant phospholipid bound to unsaturated fatty acid contains phosphatidylcholine and the content of the phosphatidylcholine is controlled to be 30 to 95% by weight relative to the recombinant phospholipid bound to the unsaturated fatty acid. Manufacturing method. According to claim 1, wherein the recombinant phospholipid bound to unsaturated fatty acids, the content of eicosapentaenoic acid (EPA) is unsaturated fatty acid 15 to 75% by weight relative to the total fatty acids of the recombinant phospholipid, docosahexaene is unsaturated fatty acid The content of the acid (DHA) is 15 to 75% by weight relative to the total fatty acids of the recombinant phospholipid, or the content of arachidonic acid (ARA), which is an unsaturated fatty acid, is controlled to be 5 to 45% by weight relative to the total fatty acids of the recombinant phospholipid. Phospholipid composition production method. The method of claim 1, wherein the gas atmosphere of the reactor is a nitrogen gas atmosphere, the temperature of the reactor is set in the range of 20 to 95 ℃ considering the activation of the enzyme to react for 12 to 90 hours to obtain the recombinant phospholipids Phospholipid composition production method. delete delete

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