KR101995643B1 - Manufacturing method of food-grade choline alfocerate from lecithin - Google Patents

Abstract

The present invention relates to a method of preparing choline alfoscerate available as a food raw material from lecithin. According to the present invention, it is possible to effectively prepare choline alfoscerate by using a specific reaction medium and a specific catalytic enzyme. Particularly, since the method uses a mixed solvent of water and hexane as an extraction solvent which has high solubility to a substrate and can be used for manufacturing a food raw material is used as a reaction medium and choline alfoscerate can be purified only by a layer separation process of a reaction medium, the method has dynamically improved productivity of choline alfoscerate compared to a conventional catalytic synthesis method, enables very simple purification compared to a conventional chemical synthesis method and a conventional purification method, and enables preparation of choline alfoscerate available as a food raw material. In particular, lecithin, which is a raw material of a fractional lecithin used as a substrate in the preparation method, is an inexpensive raw material obtained as a byproduct in a soybean oil purification process or the like, thereby being expected to be a great help in commercialization of the preparation method in the future. In addition, the choline alfoscerate prepared by the method according to the present invention can be useful as a material for food, medicine, or the like for improving cognitive function of dementia patients such as stroke, Alzheimer′s disease and the like.

Description

[0001] The present invention relates to a method for preparing choline alfoscerate as a food raw material from lecithin,

The present invention relates to a process for the preparation of choline alfoscerate which is available as a food source from lecithin.

Choline alfoscerate is a substance called L-α-glycerylphosphorylcholine ( sn -glycero-3-phosphocholine), which is a phosphatidylcholine (1,2-diacyl- sn -glycero -3-phosphocholine) in which two acyl groups are removed. Choline alfoscerate is an in vivo precursor of the neurotransmitter acetylcholine and phosphatidylcholine, a major component of the cell membrane, and is used as a cognitive enhancer in patients with dementia such as stroke and Alzheimer's disease. Choline alfoscerate was approved by the US FDA as GRAS in November 2012 (GRN No. 419). On the other hand, the use of choline alfoscerate as a food additive or health functional food raw material has not yet been recognized in Korea.

Currently, there are chemical and enzymatic methods for the synthesis of choline alfoscerate. Chemical synthesis can be carried out by deacylation of phosphatidylcholine or condensation reaction of glycerol derivative (eg, glycidol, solketal) with phosphocholine donor using an organic solvent as a reaction medium in the presence of a base (eg, Tetrabutylammonium hydroxide (TBAI) . The chemical synthesis method has advantages of high reaction efficiency and high productivity but it is difficult to use it as a food material because it uses a toxic substrate and a catalyst and does not meet the recent trend of strengthening environmental regulations and development of environmentally friendly manufacturing technology. In contrast, enzymatic synthesis has attracted attention as an eco-friendly alternative to chemical synthesis. As a reaction catalyst, phospholipase A ₁ (phospholipase A ₁ ) alone or sn -1, 3-site specific lipase ( sn- 3 specific lipase) and phospholipase A ₂ (phospholipase A ₂ ) are commonly used to produce choline alfoscerate by hydrolyzing pure phosphatidylcholine or lecithin in an aqueous medium such as a pH buffer solution. However, in the conventional enzyme reaction system, there is a problem that the reaction efficiency and productivity are significantly lower than those of the chemical synthesis method because the substrate such as lecithin has a low solubility in the aqueous medium, and thus there are many limitations in industrial application. For example, Blasi et al. (2006) produced choline alfoscerate by hydrolyzing 0.5 g of egg yolk phosphatidylcholine in 100 mL of Tris buffer using sn -1,3 -specific lipase and phospholipase A ₂ as a reaction catalyst.

Organic solvent extraction and column chromatography are mainly used for the separation and purification of choline alfoscerate synthesized by chemical or enzymatic methods. The organic solvent extraction method is a method of separating and purifying choline alfoscerate by selectively extracting substances having lower polarity than choline alfoscerate using ether, methanol or the like (McArthur, US Patent, US 2864848, 1958). Column chromatography is a method of sequentially separating and purifying substances in a reaction product such as choline alfoscerate using a polar phase fixed bed (silica, ion exchange resin, etc.) using an organic solvent such as methanol or chloroform as a mobile phase . However, organic solvents such as ether, methanol and chloroform are not defined as extraction solvents that can be used in the production of functional food ingredients in food circulation or food additive circulation. Inevitably, The standard must be set to control the amount of residual solvent in the production of the product.

Korean Patent Laid-Open No. 10-2009-0084194 (published on Aug. 5, 2009)

The present invention relates to a process for preparing choline alfoscerate which is available as a food source from lecithin and to the choline alfoscerate prepared thereby. More specifically, the present invention relates to a method for preparing lecithin as a raw material substrate from lecithin, 2) hydrolyzing fractionated lecithin under enzyme catalysis using a reaction medium in which water and hexane, which are extractable solvents, And 3) extracting choline alfoscerate obtained by simply separating the reaction medium from the reaction medium, or adding choline alfoscerate obtained by using water as an extraction solvent which can additionally be used for the production of food ingredients To extract and purify choline alfoscerate.

In order to achieve the above object, the present invention provides a process for producing lecithin comprising: 1) adding ethanol to lecithin and obtaining an ethanol fraction to prepare fractionated lecithin; 2) adding a mixed solvent of water and hexane and a lipase enzyme to the prepared fractionated lecithin, and hydrolyzing the fraction; 3) removing the lipase enzyme from the reaction product; 4) separating the reaction product of step 3); And 5) recovering the water layer from the layered reactant to obtain a water fraction.

In addition, the present invention provides choline alfoscerate which can be used as a food raw material produced by the above method.

In addition, the present invention provides a health functional food for improving cognitive function containing choline alfoscerate as an active ingredient.

The present invention relates to a process for preparing choline alfoscerate which can be used as a food raw material from lecithin. The process for producing choline alfoscerate according to the present invention is a process for producing choline alfoscerate efficiently by using a specific reaction medium and a specific catalytic enzyme can do. In addition, the reaction medium is composed of solvents that can be used for food production. After completion of the reaction, it is possible to separate and purify choline alfoscerate only by simply separating the reaction medium from the reaction medium. Therefore, choline alfoscerate Can be effectively produced. Also, the choline alfoscerate prepared by the method of the present invention can be usefully used as a health-aid material such as a cognitive function improving agent for dementia patients.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram showing a lipase catalytic hydrolysis reaction of phosphatidylcholine contained in lecithin for producing choline alfoscerate; FIG.
Figure 2 is a flow chart generally showing the preparation of choline alfoscerate comprising an enzyme reaction step and an isolated purification step.
FIG. 3 is a graph showing the content of choline alfoscerate and the content of glycerophosphodiester in a reaction product obtained by hydrolysis of soybean phosphatidylcholine and fractionated soybean lecithin under the Novozym 435 catalyst, which is a reaction-compatible enzyme, as a function of reaction time.
Figure 4 shows the chromatograms obtained by analyzing the substrate used for the preparation of choline alfoscerate and the reaction products and the purified products obtained during the production of choline alfoselate using a liquid chromatography-vaporization light scattering detector (LC-ELSD) system Grams. 1, 2 and 3 in the chromatogram are unknown component peaks. Abbreviation: PC, phosphatidylcholine; FFA, free fatty acid; GPL, glycerophospholipids; GD, glycerophosphodiesters.

The inventors of the present invention found that when choline alfoscerate is prepared by hydrolyzing soybean phosphatidylcholine using a mixed solvent of water and n- hexane as a reaction medium and phospholipase A ₁ as a reaction catalyst, (Bang et al., Food Chem., 190, 201-206, 2016) that the solubility of soybean phosphatidylcholine in the reaction medium was greatly increased (by dissolving phosphatidylcholine to 21 g per 100 mL of the reaction medium). In the previous study, high-purity (> 99% by weight) choline alfoscerate was separated and purified by column chromatography using a mixed solvent of methanol and water as a mobile phase by extracting and removing the free fatty acids produced by using ether after the reaction Respectively. However, the method of producing choline alfoscerate used in this prior study is not suitable for food production because the reaction time is long (eg, 30 hours) and choline alfoscerate is separated and purified using an extraction solvent that is not usable for food production. It is difficult to use it as a food raw material. In a subsequent study to solve this problem, it was found that when the water content in the mixed reaction medium of water and hexane was more than a certain volume ratio, only soybean phosphatidylcholine contained choline alpase in a short reaction time (eg, 5-6 hours) Rate. ≪ / RTI > After completion of the reaction, it was confirmed that the choline alfoscerate was separated and purified to a high purity (98 to 99% by weight) only by simply separating the mixed reaction medium of water and hexane. However, in order to investigate the possibility of commercialization of the enzyme reaction system developed in the above-mentioned study, a method of using soybean lecithin as raw material substrate which is much cheaper instead of soybean phosphatidylcholine has been sought. However, there is a problem in that enzyme reaction does not occur at all when using soybean lecithin as a substrate there was. In a subsequent study to solve this problem, the present inventors completed the present invention by confirming that the fractionated lecithin prepared by removing the neutral lipid from lecithin is dispersed and dissolved in the mixed reaction medium of water and hexane and the enzyme reaction occurs well.

1) adding ethanol to lecithin and obtaining an ethanol fraction to prepare fractionated lecithin; 2) adding a mixed solvent of water and hexane and a lipase enzyme to the prepared fractionated lecithin, and hydrolyzing the fraction; 3) removing the lipase enzyme from the reaction product; 4) separating the reaction product of step 3); And 5) recovering the water layer from the layered reactant to obtain a water fraction.

Preferably, the fractionated lecithin is prepared by adding ethanol to lecithin, stirring the mixture at 60 to 65 ° C for 10 minutes to 1 hour, removing ethanol from the supernatant obtained by centrifuging to obtain an ethanol fraction , But is not limited thereto.

Preferably, the fractionated lecithin may be removed to neutral lipids.

Preferably, the lipase can be a sn-position nonspecific lipase, more preferably Candida antarctica lipase B, even more preferably a macroporous acrylic resin, but not limited to, Candida antarctic lipase B, which uses a resin as an immobilization carrier.

Preferably, the amount of water in the mixed solvent of water and hexane may be 20 to 200% by weight based on the weight of phosphatidylcholine, but is not limited thereto.

Preferably, the amount of the lipase enzyme may be 5-15 wt% based on the weight of phosphatidylcholine, but is not limited thereto.

Preferably, the hydrolysis reaction is carried out at 50 to 55 ° C for 2 to 12 hours, but is not limited thereto.

Preferably, the step 4) may further include a step of further separating by adding water and hexane in a volume ratio of 1: 1, but the present invention is not limited thereto.

Preferably, the step of obtaining the water fraction of step 5) may be to remove the free fatty acid present in the hexane layer.

Preferably, the water fraction in step 5) may contain glycerophosphodiesters containing choline alfoscerate.

On the other hand, choline alfoscerate prepared by the above method can be used as a food raw material.

In the present invention, "phosphatidylcholine (1,2-diacyl- sn -glycero-3-phosphocholine)" is a kind of glycerophospholipids, and glycerol, fatty acid, phosphoric acid and choline [CH ₂ CH ₂ N ⁺ ₃ ) ₃ ], and is a major component of the cell membrane of all animals and plants.

[Chemical Formula 1]

The phosphatidylcholine may be purchased and used in the market, or may be directly separated and purified from soybean oil or yolk by a known method in the art.

In the present invention, " lecithin " is a by-product obtained when purifying egg yolk or soybean oil, and contains glycine phospholipids such as phosphatidylcholine in a large amount and is mainly used as an emulsifier in processed foods such as margarine, chocolate and ice cream, and pharmaceuticals, cosmetics and feed.

The fractionated lecithin of the present invention can be prepared either directly or by purchasing fractionated lecithin, which is commercially available. When fractionated lecithin is directly prepared and used, fractionated lecithin containing a large amount of phosphatidylcholine can be prepared by completely removing neutral lipids from lecithin. It is preferable that the content of phosphatidylcholine is as high as possible. Thus, soybean lecithin having a phosphatidylcholine content of 10 to 20% by weight or egg yolk lecithin containing 60 to 80% by weight of a higher content of phosphatidylcholine can be used.

In the present invention, "choline alfoscerate" is a substance called L-α-glycerylphosphorylcholine ( sn -glycero-3-phosphocholine), which is obtained from phosphatidylcholine by two acyl groups group is removed and is represented by the following formula (2). Choline alfoscerate is an in vivo precursor of the neurotransmitter acetylcholine and phosphatidylcholine, a major component of the cell membrane, and is used as a cognitive enhancer in patients with dementia such as stroke and Alzheimer's disease.

(2)

In the present invention, the fractionated lecithin is a substrate, the mixed solvent of water and hexane is a reaction medium, and the lipase enzyme acts as a catalyst. In the reaction medium, hexane increases the solubility of the fractionated lecithin as a substrate to increase the efficiency and productivity of the reaction, and 2) dissolves the free fatty acid in the reaction product in the separation of the reaction medium for purification of the reaction product. do. The water in the reaction medium serves to 1) be used as a substrate for the hydrolysis reaction and 2) to obtain glycerophosphodiesters containing choline alfoscerate in the reaction product by layer separation of the reaction medium for purification of the reaction product .

In the examples of the present invention, a mixed solvent of water and hexane was used as the reaction medium, and the amount of hexane in the mixed solvent was preferably about 260% (i.e., 4 mL per gram of the substrate) of the fractionated lecithin used as the substrate, The amount may be 20 to 200% by weight based on the weight of the fractionated lecithin. However, the lower the amount of water, the slower the reaction rate. On the other hand, if the amount of water is too high, the stability of the enzyme is accelerated by leaching from the solid support More preferably from 100 to 200 parts by weight (i.e., from 1 to 2 mL per gram of phosphatidylcholine) relative to the weight of fractionated lecithin. Thus, the reaction medium may be composed of hexane and water in a volume ratio of 4: 1.

In the present invention, step 4) and step 5) are steps of separating the reaction medium, which is a mixed solvent of water and hexane, to obtain choline alfoscerate produced from the fractionated lecithin, And recovering the serrate. After completion of the reaction, the reaction product is allowed to stand overnight at 4 to 23 ° C, and then filtered to obtain a reaction product from which the enzyme has been removed. The reason for this is that the final purified product of the reaction product obtained through the settling process exhibits a higher glycerophosphoester content than the final purified product obtained by filtration immediately after the completion of the reaction. The reaction product is dissolved in a reaction medium composed of water and hexane, which are solvents that can be used for food production. Particularly, the free fatty acid generated by the hydrolysis of phosphatidylcholine is present in the hexane layer and the glycerophosphodiester Is dissolved in the water layer. Therefore, the reaction medium is left at room temperature for 30 minutes to separate the water layer, and then dried using a rotary vacuum concentrator or the like to separate and purify only the glycerophosphoester containing choline alfoscerate can do. The glycerophosphodiester content of the purified product (water fraction) obtained after the separation and purification step of the reaction product obtained from the fractionated lecithin according to the present invention, including choline alfoscerate, may be 50% by weight or more.

In an embodiment of the present invention, the final purified product of the reaction product obtained from the fractionated lecithin may contain, in addition to choline alfoscerate, L-? -Glycerylphosphorylinositol, L-? -Glycerylphosphoryl And 50-70% by weight of glycerophosphodiesters such as L-alpha-glycerylphosphorylethanolamine.

In addition, the present invention provides choline alfoscerate which can be used as a food raw material produced by the above method.

In the present invention, the purified product of the reaction product obtained from the fractionated lecithin contains 50 to 70% by weight of glycerophosphoester including choline alfoscerate, and thus may have a major physiological activity of choline alfoscerate. Since choline alfoscerate is known to improve the cognitive function of dementia patients such as stroke and Alzheimer's disease as a neurotransmitter acetylcholine and phosphatidylcholine in vivo as a main component of the cell membrane, Rate may be usefully used in the field of production of foods, medicines and the like containing the above-mentioned effects. Particularly, in the present invention, the purified product of the reaction product obtained from the fractionated lecithin is water-soluble as a water fraction, and thus may be usefully used in the production of liquid foods (drinks, tea, etc.) and pharmaceuticals.

In addition, the present invention provides a health functional food for improving cognitive function containing choline alfoscerate as an active ingredient. In addition, the composition can be used as a pharmaceutical composition for improving cognitive function, a food composition, a food additive composition or a health supplement food composition containing the choline alfoscerate as an active ingredient.

The form of the health functional food in the present invention is not particularly limited. It can be manufactured in various forms including beverage, granule, tablet, powder, ring, capsule, wire, noodles, confectionery, meat, fish, herb, stew, rice and the like by mixing known ingredient, food additive and the like.

The health functional foods of the present invention may contain conventional food additives and, unless otherwise specified, whether or not they are suitable as food additives are subject to the provisions of the General Food and Drug Administration approved by the Food and Drug Administration, Shall be determined according to the relevant standards and standards.

Examples of the food additives include sugars such as monosaccharides, disaccharides, polysaccharides and sugar alcohols and flavorings such as tau martin, stevia extract, saccharin and aspartame, nutrients, vitamins, edible electrolytes, flavors, coloring agents, , Chocolate, etc.), pectic acid, alginic acid, organic acid, protective colloid thickening agent, pH adjusting agent, stabilizer, preservative, glycerin, alcohol and carbonating agent.

For example, the health functional food in tablet form may be prepared by granulating a mixture obtained by mixing the active ingredient (choline alfoscerate) of the present invention with an excipient, a binder, a disintegrant, and other additives in a usual manner, Or the mixture can be directly compression-molded. The health functional food of the tablet form may contain a sweetener or the like as needed.

The hard capsule of the capsule type health functional food can be prepared by filling the normal hard capsule with the active ingredient of the present invention (choline alfoscerate), and the soft capsule is prepared by mixing the active ingredient with an additive such as an excipient Such as gelatin, into a capsule base. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative and the like, if necessary.

The ring-shaped health functional food can be prepared by molding a mixture of the active ingredient (choline alfoscerate) of the present invention, an excipient, a binder, a disintegrant, and the like by a conventionally known method and, if necessary, It may be applied to the skin, or the surface may be coated with a substance such as starch or talc.

The granular health functional food may be prepared by granulating a mixture of the active ingredient (skin care agent) of the present invention with an excipient, a binder, a disintegrant, etc. by a conventionally known method and, if necessary, adding a flavoring agent, ≪ / RTI >

The health functional food of the present invention is manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and rings, and is easy to carry, and is easy to take at anytime and anywhere.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

< Experimental Example >

The following experimental examples are intended to provide experimental examples that are commonly applied to the respective embodiments according to the present invention.

1. Reagents

Soybean phosphatidylcholine (purity> 97%) used in the following experiments was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA); Soybean lecithin is available from Ilshinwells Co. (Cheongwon, Korea); Novozym 435 [ Candida antarctica lipase B immobilized on a macroporous anionic resin (Lewatit VP OC 1600) was purchased from Novozymes A / S (Bagsvaerd, Denmark).

< Example  1> Preparation of fractionated soybean lecithin from soybean lecithin

<1-1> Chemical composition of soybean lecithin

The chemical composition of soybean lecithin used as a raw material in the present invention is shown in Table 1 below. The total lipid content including the glycerophospholipids of the soybean lecithin was ~90 wt%. The content of phosphatidylcholine (PC) was the highest at ~ 15 wt%, and the content of phosphatidylinositol (PI; ~ 11 wt%), phosphatidylethanolamine (PE; weight%). And phosphatidic acid (PA: ~ 4% by weight). The content of lipids other than phospholipids such as neutral lipids was ~ 45 wt%.

Chemical composition of soybean lecithin Component Content (wt%) Fats 90.0  Phospholipids 44.5   Phosphatidylcholine (PC) 14.8   Lysophosphatidylcholine (LPC) 1.0   Phosphatidylinositol (PI) 10.8   Lysophosphatidylinositol (LPI) 0.3   Phosphatidylserine (sodium salt) (PS) 0.3   Phosphatidylethanolamine (PE) 8.8   Lysophosphatidylethanolamine (LPE) 0.3   N-acyl phosphatidylethanolamine 1.9   Phosphatidylglycerol (PG) 0.5   Diphosphatidylglycerol (DPG) 0.6   Phosphatidic acid (PA) 4.3   Lysophosphatidic acid (LPA) 0.2   Other phospholipids 0.8  Other fats including triacylglycerols 45.5 Carbohydrates 5.0 Ash 4.5

<1-2> Production of fractionated soybean lecithin

In our previous study, we confirmed that neutral lipids in lecithin should be removed in order to hydrolyze lecithin to produce choline alfoscerate in a mixed reaction medium of water and hexane. Accordingly, in order to improve the suitability of soybean lecithin, which is a raw material substrate, in the present invention, fractionation conditions capable of effectively removing neutral lipids were established.

To 10 g of the soybean lecithin, 25 mL of ethanol was added, and the mixture was stirred at 65 ° C at 600 rpm for 1 hour, followed by centrifugation (2,200 × g, 15 minutes) to obtain a supernatant. Ethanol was removed from the supernatant in a rotary vacuum concentrator to prepare fractionated soybean lecithin (ethanol fraction of soy lecithin) suitable for enzyme reaction for the production of choline alfoscerate. The fractionated soybean lecithin prepared under the above fractionation conditions was confirmed to have completely removed the neutral lipids. The yield of the obtained fractionated soybean lecithin was ~ 48 wt% based on the weight of soybean lecithin. The obtained fractionated soybean lecithin was refrigerated at 4 ° C until use.

< Example  2> Fatty acid composition analysis of soybean lecithin and fractionated soybean lecithin

The fatty acid compositions of the fractionated soybean lecithin and the raw soybean lecithin obtained in Example 1 are shown in Table 2 below.

Fatty acid composition of soybean lecithin and fractionated soybean lecithin Fatty acid Soy lecithin (mol%) Ethanol-soluble fraction of
strain lecithin (mol%) 16: 0 18.9 ± 0.1 16.5 ± 0.1 16: 1n-7 0.2 ± 0.0 0.1 ± 0.0 18: 0 3.3 ± 0.0 3.1 ± 0.0 18: 1n-9 21.6 ± 0.0 23.3 ± 0.1 18: 2n-6 50.8 ± 0.0 51.4 ± 0.0 18: 3n-3 5.0 ± 0.0 5.2 ± 0.0 20: 0 0.2 ± 0.0 0.2 ± 0.0 20: 1 0.2 ± 0.0 0.2 ± 0.0 Total SFA 22.4 ± 0.1 19.8 ± 0.1 Total USFA 77.6 ± 0.1 80.2 ± 0.1

All values were expressed as mean ± SD (n = 2).

Abbreviations: SFA, saturated fatty acids; And USFA, unsaturated fatty acids.

As shown in Table 2, the major fatty acids of the fractionated soybean lecithin were 51.4 mol% linoleic acid (18: 2n-6), 23.3 mol% oleic acid (18: 1 n-9) and 16.5 mol% palmitic acid : 0) and the total unsaturated fatty acid content was 80.2 mol%.

< Example  3> Evaluation of reaction suitability of substrate

The amount of choline alfoscerate produced in the Novogam 435 catalytic hydrolysis reaction was determined by using the fractionated soybean lecithin and the commercial soybean phosphatidylcholine prepared in Example <1-2> as the substrate and the mixed solvent of water and hexane as the reaction medium. Respectively.

Specifically, 4 g of soybean phosphatidylcholine or fractionated soybean lecithin was placed in a batch reactor (15 cm × 5 cm id), and 20 mL of a mixed solvent of water and hexane (16 mL of hexane + 4 mL of water) and 0.4 g of Novogym 435 ) Was added thereto, followed by stirring at a reaction temperature of 55 ° C at a speed of 600 rpm. The reaction temperature was kept constant by a constant temperature circulating water bath connected to the water jacket of the reactor. The reaction time was set at least 2 hours to 12 hours. The reaction product samples were taken at 0.2, 0.2, and 0.2 hours at reaction times 0, 2, 4, 6, 8, 10, and 12 hours, respectively.

On the other hand, in order to measure the content of choline alfosceride in the reaction product depending on the type of substrate, the content of phosphatidylcholine, lysophosphatidylcholine and choline alfoscerate in the reaction product produced by the enzyme reaction was analyzed by liquid chromatography.

2 mL of chloroform / methanol (1: 2, v / v) was added to 0.2 mL of the reaction product produced by the enzyme reaction, and the enzyme was removed by filtration with a 0.45 μm syringe filter (13 mm). After removing the solvent under a nitrogen stream, the solution was dissolved in 0.2 mL of 93 v / v% methanol used as a mobile phase of liquid chromatography and dissolved at a concentration of 1 mg / mL. The solution was filtered through a 0.45 μm syringe filter (13 mm) Tokyo, Japan). A LiChrosorb Si 60 column (250 mm × 4 mm i.d., 5 μm particle size, Merck, Darmstadt, Germany) was used as a column and detected using an evaporative light scattering detector (ELSD). The sample injection amount was 20 μL and the flow rate of the mobile phase was 1 mL / min. The drift tube temperature of ELSD was 60 ° C and nitrogen was supplied at a flow rate of 1.5 L / min.

The content of choline alfoscerate in the reaction product measured by the above method was calculated as a function of the reaction time as a percentage of the total content of phosphatidylcholine, lysophosphatidylcholine and choline alfoscerate in the reaction product. However, glycerophospholipids (GPL) such as phosphatidylinositol (PI) and phosphatidylethanolamine (PE) are present in the fractionated soybean lecithin in addition to phosphatidylcholine (PC), and the glycerophospholipid species are all produced by phosphatidylcholine It was eluted at the same time without separation. Lysoglycerophospholipids (LPL) such as lysophosphatidylinositol (LPI), lysophosphatidyl ethanolamine (LPE), and the like, which are produced from the glycerophospholipid species by the hydrolysis reaction of fractionated soybean lecithin and L- Glycerophosphodiesters (GD) such as L-α-glycerylphosphorylinositol and L-α-glycerylphosphorylethanolamine are known as lysophosphatidylcholine and choline alfoscerate The total amount of phospholipids (including phosphatidylcholine), the total phospholipid content (including phospholipid content), and the total glycerol content (including phospholipid content) in the reaction products obtained using the fractionated soybean lecithin as a substrate. The phosphodiester content (including choline alfoscerate content) was measured and the content of each component The content was expressed as a function of the reaction time as a percentage of the total content of glycerophospholipid, lithoglycerol phospholipid and glycerophosphoester in the reaction product.

As a result, as shown in Fig. 4, before the reaction time of 2 hours, the amount of glycerophosphoester (20.0 wt%) including choline alfoscerate produced by the hydrolysis reaction of fractionated soybean lecithin was lowered to the hydrolysis reaction of soybean phosphatidylcholine Was higher than that of choline alfoscerate (3.7% by weight). These results indicate that fractionated soybean lecithin is more emulsifying than soybean phosphatidylcholine, and that the fractionated soybean lecithin is more soluble or dispersed in the reaction mixture of water and hexane, resulting in faster hydrolysis by Novogym 435. However, the rate of hydrolysis of soybean phosphatidylcholine was higher than that of fractionated soybean lecithin after 4 hours of reaction. Especially, soybean phosphatidylcholine is completely converted to choline alfoscerate after 6 hours of reaction, whereas when fractionated soybean lecithin is used as a substrate, glycerophospholipid (including phosphatidylcholine) is completely dissolved in glycerophosphate Ester (including choline alfoscerate).

On the other hand, as shown in Fig. 4, the reaction products obtained from the fractionated soybean lecithin contained three kinds of unknown components (1, 2, and 3 in addition to glycerophosphoester (including choline alfoscerate) Peak) were present together. Two of the three unknown components (peaks 1 and 2) were also contained in the fractionated soybean lecithin as a raw material substrate and one (peak 3) was determined to be produced during the reaction.

< Example  4> From the reaction product Choline alfoscerate  And Glycerophosphodiester  Separation purification

The glycerophosphoester (including choline alfoscerate) was separated from the reaction medium after the reaction by reacting the Novogem 435 catalytic hydrolysis product of the fractionated soybean lecithin prepared under the optimum conditions set up in the previous study Lt; / RTI &gt;

<4-1> Of the reaction product  water Fraction  Produce

In this experiment, a water fraction was prepared from the reaction product obtained by hydrolyzing fractionated soybean lecithin under Novogem 435 catalyst, and the content of glycerophosphoester (including choline alfoscerate) of the fraction was measured.

Specifically, 4 g of fractionated soybean lecithin was placed in a batch reactor (15 cm × 5 cm id), 20 mL of a mixed solvent of water and hexane (16 mL of hexane + 4 mL of water) and 0.4 g of Novogym 435 (10% of substrate weight) , And the mixture was reacted at a reaction temperature of 55 ° C at 600 rpm for 8 hours with stirring. The reaction temperature was kept constant by a constant temperature circulating water bath connected to the water jacket of the reactor. After completion of the reaction, the obtained reaction product was allowed to stand overnight at 4 to 23 ° C. The following reaction product was placed in a Buchner funnel with a filter paper and filtered under reduced pressure to obtain a reaction product in which novoid 435 enzyme was removed. The remaining reaction product in the reactor was further washed with 20 mL of water into the Buchner funnel and filtered. After filtration, the reaction product was put into a separatory funnel, and further, 20 mL of water and 20 mL of hexane were added, and the mixture was allowed to stand at room temperature for 30 minutes, and the reaction medium was separated into a hexane layer and a water layer. Since the free fatty acid generated by hydrolysis of the substrate is present in the hexane layer and the glycerophosphoester including choline alfoscerate is dissolved in the water layer, the water layer is recovered and dried using a rotary vacuum concentrator at 40 ° C To give a purified product (water fraction) of glycerophosphoester (including choline alfoscerate).

The total glycerol content (including phosphatidylcholine content), the total lithoglycerol phospholipid content (including lysophosphatidylcholine content) and the total glycerophosphoester content (including choline alfoscerate content) in the water fraction obtained from the reaction product of fractionated soybean lecithin ) Was measured in the same manner as in Example 3, and the content of each component was expressed as a percentage of the total content of glycerophospholipid, lithoglycerophospholipid and glycerophosphoester in the water fraction.

As a result, as shown in Fig. 4, in the water fraction obtained from the reaction product of the fractionated soybean lecithin, most of the free fatty acid present in the reaction product was removed, but the three unknown components (1, 2 And No. 3 peaks) remained unremoved and the relative content of component 2 was increased.

As a result, as shown in Table 3, the yield of the water fraction obtained in the reaction product of the fractionated soybean lecithin was 12.0% by weight based on the weight of the fractionated soybean lecithin as the raw material substrate, and the glycerophosphate The ester content was 52.4 wt%.

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The yields of the ethanol fractions of the water fraction and the water fraction obtained from the Novogem 435 hydrolysis reaction product of fractionated soybean lecithin and the content of glycerophosphoester (including choline alpocerate) Products Product yield
(wt% of substrate) Content of glycerophosphodiesters including choline alfocerate (wt%) Water-soluble fraction 12.0 + - 0.4 52.4 ± 1.3

All values were expressed as mean ± SD (n = 3).

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (15)

1) adding ethanol to lecithin and obtaining an ethanol fraction to prepare a neutral lecithin-free fractionated lecithin;
2) To the fractionated lecithin from which the neutral lipid was removed, a mixed solvent in which hexane, 100 to 200% by weight of water based on the weight of the fractionated lecithin was mixed, and Candida antarc, which used macroporous acrylic resin, Adding a ticar lipase B enzyme, and hydrolyzing the mixture at 50 to 55 ° C for 2 to 12 hours;
3) removing the lipase B enzyme from the reaction product of the hydrolysis reaction;
4) further adding water and hexane in a volume ratio of 1: 1 to the reaction product in which the lipase B enzyme in step 3) has been removed, thereby separating the reaction product; And
5) recovering the water layer from the layered reactant to obtain a water fraction. The method of claim 1, wherein the neutral lipid-free fractionated lecithin is prepared by adding ethanol to lecithin, stirring the mixture at 60 to 65 ° C for 10 minutes to 1 hour, removing ethanol from the supernatant obtained by centrifugation, Ethanol fraction of the choline alfoscerate is obtained. delete delete delete delete delete [3] The method according to claim 1, wherein the amount of Candida antarctica lipase B enzyme using the macroporous acrylic resin as an immobilization carrier is 5 to 15% by weight relative to the weight of phosphatidylcholine. Gt; delete delete The method of claim 1, wherein the step of obtaining the water fraction of step 5) is for removing free fatty acid present in the hexane layer. The method for producing choline alfoscerate according to claim 1, wherein the water fraction in step 5) contains glycerophosphodiester containing choline alfoscerate.
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