WO2019132549A1 - Method for preparing choline alfoscerate, usable as food raw material, from phosphatidylcholine or lecithin - Google Patents

Abstract

The present invention relates to a method for preparing choline alfoscerate, usable as a food raw material, from phosphatidylcholine or lecithin, and a choline alfoscerate preparation method according to the present invention uses a specific reaction medium and a specific catalytic enzyme, thereby enabling a high purity choline alfoscerate to be effectively prepared. Particularly, the preparation method allows a mixed solvent of water and hexane, which have high substrate solubility and an extraction solvent usable in the preparation of food raw materials, to be used as a reaction medium, and enables high purity purification of choline alfoscerate by only using a layer separation process of the reaction medium, thereby enhancing the productivity of choline alfoscerate to be greater than that of a conventional enzymatic synthesis method, having, unlike a conventional chemical synthesis method and a conventional purification method, very simple purification, and enabling the preparation of choline alfoscerate which is usable as a food raw material. Specifically, lecithin is a raw material of fractionated lecithin used as a substrate in the preparation method and is an inexpensive raw material obtained as a by-product in a soybean oil refining process and the like, and thus is expected to be greatly helpful in the commercialization of the preparation method in the future. In addition, choline alfoscerate prepared by the method according to the present invention can be effectively usable as a material for foods, pharmaceuticals and the like for improving cognitive function in patients having had a stroke and having dementia such as Alzheimer's disease.

Description

Process for the production of choline alfoscerate, which is available as a food source from phosphatidylcholine or lecithin

The present invention relates to a process for the preparation of choline alfoscerate which is available as a food source from phosphatidylcholine or 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, in previous reports, choline alfoscerate was prepared by hydrolyzing 0.5 g of egg yolk phosphatidylcholine in 100 mL of Tris buffer using sn -1,3 -position specific lipase and phospholipase A ₂ as reaction catalysts.

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 and removing substances having lower polarity than choline alfoscerate using ether, methanol or the like. 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.

The present invention relates to a method for producing choline alfoscerate usable as a food raw material from phosphatidylcholine and choline alfoscerate prepared thereby. More specifically, the present invention relates to a method of 1) hydrolyzing phosphatidylcholine under enzyme catalysis using a reaction medium in which water and hexane, which are extraction solvents usable for the production of food raw materials, are used to obtain choline alfoscerate, and 2) The present invention relates to a method for separating and purifying choline alfoscerate with high purity by extracting choline alfoscerate obtained only by the separation process or by extracting choline alfoscerate obtained by using water which is an additional extraction solvent which can be used for producing food raw materials .

The present invention also relates to a process for preparing choline alfoscerate which can be used as a food raw material from lecithin and 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 accomplish the above object, the present invention provides a method for producing a lipase, comprising: 1) adding a mixed solvent of water and hexane and a lipase enzyme to phosphatidylcholine, followed by hydrolysis; 2) removing the lipase enzyme from the reaction product; 3) separating the reactants in step 2); And 4) recovering the water layer from the layered reactant to obtain a water fraction.

The present invention also relates to a method for producing lecithin comprising the steps of 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 the production of choline alfoscerate which can be used as a food raw material from phosphatidylcholine or lecithin. The process for producing choline alfoscerate according to the present invention is characterized by high purity (98 to 99 Weight%) of choline alfoscerate can be effectively produced. In addition, the reaction medium is composed of solvents which can be used for food production. After completion of the reaction, it is possible to separate and purify choline alfoscerate with high purity only by simply separating the reaction medium from the reaction medium. Therefore, choline alpase Rate 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 representation of the lipase catalytic hydrolysis of phosphatidylcholine for the production of choline alfoscerate.

FIG. 2 is a flow chart generally showing a process for producing choline alfoscerate including an enzyme reaction step of phosphatidylcholine and an isolation purification step.

Figure 3 shows the choline alfoscerate content in the reaction product of the hydrolysis of soybean phosphatidylcholine according to the enzyme type (Novozyme 435, Lipozyme® RM IM, Lipozyme® TL IM, immobilized Candida rugosa lipase, Lecitase® Ultra) Fig.

4 is a graph showing the content of choline alfoscerate in the reaction product of the hydrolysis reaction of soybean phosphatidylcholine as a function of reaction time according to the ratio of water in the reaction medium (20-400% relative to the weight of soybean phosphatidylcholine).

Figure 5 shows the results of analysis of the soybean phosphatidylcholine substrate used in the production 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 This is the chromatogram obtained. Abbreviation: PC, phosphatidylcholine; FFA, free fatty acid.

FIG. 6 is a flow chart that entirely illustrates the preparation process of choline alfoscerate, including an enzymatic reaction step of lecithin and an isolated purification step.

7 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 8 shows the chromatograms obtained by analyzing the substrate used for the production 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 > Also, after completion of the reaction, it was confirmed that 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, thereby completing the present invention.

In order to investigate the commercialization possibility of the enzyme reaction system developed in the follow-up study, a method of using soybean lecithin as raw material substrate which is much cheaper instead of soybean phosphatidylcholine has been sought. However, when soybean lecithin is used as a substrate, . 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.

The present invention relates to a method for producing a polyhydroxyalkanoate comprising the steps of: 1) adding a mixed solvent of water and hexane and a lipase enzyme to phosphatidylcholine, followed by hydrolysis; 2) removing the lipase enzyme from the reaction product; 3) separating the reactants in step 2); And 4) recovering the water layer from the layered reactant to obtain a water fraction.

Preferably, the lipase can be a sn-position nonspecific lipase, more preferably Candida antarctica lipase B, and even more preferably a macroporous anionic 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 400% 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.

More preferably, in the hydrolysis reaction, the amount of water in the mixed solvent of water and hexane is 100 to 200% by weight based on the weight of phosphatidylcholine, the amount of the lipase enzyme is 5 to 10% by weight based on the weight of phosphatidylcholine, For 6 hours, but is not limited thereto.

Preferably, the step 3) 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 4) may be to remove the free fatty acid present in the hexane layer.

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

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, and even more preferably a macroporous anionic 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 phosphatidylcholine is a substrate, the mixed solvent of water and hexane is a reaction medium, and the lipase enzyme acts as a catalyst. Hexane in the reaction medium serves to 1) increase the efficiency and productivity of the phosphatidylcholine, which is a substrate, by increasing the efficiency of the reaction and 2) to dissolve the free fatty acid in the reaction product during the separation of the reaction medium for purification of the reaction product . The water in the reaction medium serves to 1) be used as a substrate for the hydrolysis reaction and 2) to dissolve the choline alfoscerate in the reaction product in the layer separation process of the reaction medium for the purification of the reaction product.

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 preferably be 20 to 400% by weight relative to the weight of phosphatidylcholine used as the substrate, but the lower the amount of water, the slower the reaction rate, while if the amount of water is too high, the leaching from the solid support promotes , And more preferably 100 to 200% by weight (that is, 1 to 2 mL / g of phosphatidylcholine) relative to the weight of phosphatidylcholine. Thus, the reaction medium may be composed of hexane and water in a volume ratio of 4: 1.

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 method for producing choline alfoscerate from the phosphatidylcholine of the present invention, the steps 3) and 4) are steps of separating the reaction medium which is a mixed solvent of water and hexane to obtain choline alfoscerate produced from phosphatidylcholine And recovering the choline alfoscerate dissolved in the water layer after layer separation. After completion of the reaction, the reaction product obtained from the phosphatidylcholine can be filtered to obtain a reaction product from which the enzyme has been removed. The reaction product is dissolved in a reaction medium composed of water and hexane, which are solvents that can be used in food production. Especially, the free fatty acid generated by the hydrolysis of phosphatidylcholine is present in the hexane layer and the choline alfoscerate is dissolved in the water layer. The reaction medium is allowed to stand 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 choline alfoscerate available as a food raw material. The choline alfoscerate content of the purified product (water fraction) obtained after the separation and purification step of the reaction product obtained from the phosphatidylcholine according to the present invention may be 98% by weight or more.

In the method for producing choline alfoscerate from the lecithin of the present invention, the steps 4) and 5) are a step 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 choline alfoscerate dissolved in the water layer after layer separation. 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 phosphatidylcholine may contain 98-99% by weight of choline alfoscerate.

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 phosphatidylcholine contains choline alfoscerate in a high purity (98 to 99% by weight), so that it can have a major physiological activity of choline alfoscerate.

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. In particular, the purified product of the reaction product obtained from phosphatidylcholine or lecithin in the present invention 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), Lipozyme RM IM ( Rhizomucor miehei lipase immobilized on an ion-exchange resin, Lipozyme TL IM ( Thermomyces lanuginosus lipase immobilized on silica gel) and Lecitase Ultra (phospholipase A1 from T. lanuginosus / Fusarium oxysporum ) were purchased from Novozymes A / S (Bagsvaerd, Denmark); Candida rugosa lipase immobilized on Immobead 150 was purchased from Sigma-Aldrich Chemical Co. (St. Louis, Mo., USA).

Example 1 Analysis of Fatty Acid Composition of Soybean Phosphatidylcholine

The fatty acid composition of commercial soybean phosphatidylcholine used as a substrate of the enzyme reaction for producing choline alfoscerate in the present invention is shown in Table 1 below.

Fatty acid composition of soybean phosphatidylcholine

Fatty acid	Content (mol%)
14: 0	0.1 ± 0.0
16: 0	15.1 ± 0.1
16: 1n-7	0.1 ± 0.0
18: 0	3.5 ± 0.0
18: 1n-9	11.0 0.0
18: 1n-7	1.7 ± 0.0
18: 2n-6	62.6 ± 0.1
18: 3n-3	5.8 ± 0.0
20: 1	0.1 ± 0.0
Total SFA	18.7 ± 0.1
Total USFA	81.3 ± 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 1, the main fatty acids of soybean phosphatidylcholine were linoleic acid (62.6 mol%), palmitic acid (15.1 mol%) and oleic acid (11.0 mol%) and the total unsaturated fatty acid content was 81.3 mol%.

Example 2 Preparation of choline alfoscerate using enzyme catalysis

The choline alfoscerate of the present invention was prepared by hydrolysis using commercially available soybean phosphatidylcholine as a substrate and a mixed solvent of water and hexane as a reaction medium and using lipase as a catalyst.

<2-1> Selection of enzymes suitable for the production of choline alfoscerate

In this experiment, in order to select a commercial immobilized lipase suitable as a catalyst for the reaction for producing choline alfoscerate by hydrolyzing phosphatidylcholine, the reaction temperature was maintained at 50 ° C, the enzyme amount was adjusted to 10% of the weight of soybean phosphatidylcholine as a substrate, The choline alfoscerate content in the reaction product was measured. Commercial immobilized enzymes that evaluated the suitability of the reaction were Novozym 435, Lipozyme RM IM, Lipozyme TL IM, Candida lyase, rugosa lipase immobilized on Immobead 150) and Lecitase Ultra with phospholipase A1 activity. Among these lipases, lipozyme RM IM and lipozyme TL IM have sn -1,3 position specificity of neutral fat or phospholipid. Novogym 435 and Candida lucosa lipase are lipases having no specificity in the sn positions of neutral fat or phospholipids.

Specifically, 4 g of soybean phosphatidylcholine 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 each lipase (10% of substrate weight) Followed by stirring at a reaction temperature of 50 DEG C at 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 determine the content of choline alfosceride in the reaction product according to the kind of lipase, 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). The solvent was removed under a nitrogen stream and the solution was dissolved in 0.2 mL of 93 v / v% methanol used as a mobile phase of liquid chromatography. The resulting solution was filtered with a 0.45 μm syringe filter (13 mm), and then filtered through a liquid chromatograph (JASCO Corp., 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.

As a result, as shown in FIG. 3, the hydrolysis reaction of phosphatidylcholine occurred only when noborg 435 and Lecitase Ultra were used as catalysts. Especially, in the case of noborg 435 catalytic hydrolysis, phosphatidylcholine completely hydrolyzed And converted to choline alfoscerate. On the other hand, in the case of lipozyme RM IM, lipozyme TL IM and Candida lucosa lipase, the hydrolysis reaction of phosphatidylcholine did not occur within the reaction time range.

From the above results, it was confirmed that noborg 435, an sn -position specific lyticase, was an enzyme suitable for producing choline alfoscerate.

Therefore, the amount of water in the medium suitable for the Novogym 435 catalytic hydrolysis reaction and the optimal reaction conditions were further investigated in the following experiment, and the suitability of the phosphatidylcholine as a substrate was compared and evaluated.

&Lt; 2-2 > Selection of the amount of water in the medium suitable for producing choline alfoscerate

For the production of choline alfoscerate, hydrolysis reaction was carried out using noborg 435, which was shown to have the best suitability for reaction with soybean phosphatidylcholine as a substrate.

In order to determine the optimum amount of water required for the reaction in a mixed solvent of water and hexane, which is the medium of the hydrolysis of phosphatidylcholine, the reaction temperature was maintained at 50 ° C and the amount of water in the medium was maintained while maintaining the amount of enzyme at 10% of the weight of the substrate phosphatidylcholine (20-400% relative to the weight of phosphatidylcholine) in the reaction product.

Specifically, 4 g of soybean phosphatidylcholine was placed in a batch reactor (15 cm × 5 cm id), and 16.8 to 32 mL of a mixed solvent of water and hexane (16 mL of hexane + 0.8, 1.6, 2.4, 3.2, ), Novogym 435 0.4 g (10% of substrate weight) was added and stirred at a reaction temperature of 50 캜 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 1 hour to 6 hours. The reaction product samples were taken at 0, 1, 2, 3, 4, 5 and 6 hours.

The content of choline alfoscerate in the reaction product was measured in the same manner as in Example <2-1>, and the content of choline alfoscerate in the reaction product according to the amount of water in the medium was measured by using phosphatidylcholine, lysophosphatidylcholine, The percentage of total content of serrate was calculated as a function of reaction time.

As a result, as shown in FIG. 4, when the amount of water was 20% or more based on the weight of the substrate, phosphatidylcholine was hydrolyzed to produce choline alfoscerate within the reaction time period, and choline alpocellate was formed as the amount of water increased. The choline alfoscerate contents of the reaction products obtained at reaction time of 6 hours when the amount of water was 20%, 40%, and 60% based on the weight of the substrate were 2.4 wt%, 21.4 wt%, and 46.9 wt%, respectively. The choline alfoscerate content of the reaction product obtained at the reaction time of 5 hours when the amount of water was 80% based on the weight of the substrate was 97.2 wt%, and the choline alfoscerate content of the reaction product obtained at the reaction time of 6 hours was 100.0 wt% . When the amount of water was 100%, 200%, 300%, and 400% of the substrate weight, the choline alfoscerate contents of the reaction products obtained at the reaction time of 4 hours were 79.3 wt%, 99.7 wt%, 99.7 wt%, 99.9 wt% %, And the choline alfoscerate content of the reaction product obtained at the reaction time of 5 hours was 100.0% by weight. As a result, it was confirmed that the phosphatidylcholine completely converted to choline alfoscerate within 5 hours of reaction time when the amount of water was 100% or more of the substrate weight. Based on the above results, the amount of water in the medium suitable for producing choline alfoscerate was selected as 100% or more of the substrate weight.

&Lt; Example 3 > Optimum reaction conditions for the production of choline alfoscerate

In order to prepare choline alfoscerate using enzyme catalysis in Example 2, Novozym 435 was used as the enzyme, and a mixed solvent of water and hexane was used as a medium. The amount of water in the medium was 100% or more In addition, the optimal reaction conditions for the production of choline alfoscerate were further analyzed in this experiment.

<3-1> Establishment of optimal reaction conditions using reaction surface analysis method

In this experiment, the optimal reaction conditions (reaction temperature, reaction time, water content in the medium) of novolac 435 catalytic hydrolysis of phosphatidylcholine to maximize the production of choline alfoscerate by using the principle of response surface methodology (RSM) Amount, and enzyme amount).

In detail, the experiment was designed using a central composite circumscribed (CCC) design with a star point at a distance of 1.414 from the center point of the central composite design. The independent variables for the optimal reaction conditions for the production of choline alfoscerate are the reaction temperature (50-55 ° C), the reaction time (3-5 hours), the amount of water in the medium (100-200% of the weight of phosphatidylcholine) 5 to 15% by weight), and the dependent variable was the choline alfoscerate content (wt%) of the reaction product. The experimental range of each independent variable was coded in five stages of +1.414, +1, 0, -1, -1.414, and 27 items including 16 factorial points, 8 axis points, and 3 center points After setting the conditions, enzyme reaction was performed under each condition. The second-order regression model, which predicts the effect of the above four reaction conditions on choline alfoscerate content, is as follows.

Y was the dependent variable, ie, choline alfoscerate content, X i was the independent variable and was the temperature (Te), the reaction time (RT), the amount of water in the medium (WC) and the enzyme amount (En).

The results of the reaction surface analysis were analyzed using the Design Expert 8.0 program of Stat-Ease (Minneapolis, MN, USA).

The content of choline alfoscerate in the reaction product was measured in the same manner as in Example <2-1>, and the content of choline alfoscerate in the reaction product according to the amount of water in the medium was measured by using phosphatidylcholine, lysophosphatidylcholine, Calculated as a percentage of the total content of serrate.

As a result, as shown in Table 2, the range of the choline alfoscerate content of the reaction product obtained at 27 reaction conditions was 1.0 to 100.0% by weight.

Central composite circumscribed design applied to novolac 435 catalytic hydrolysis of phosphatidylcholine and production of choline alfoscerate by reaction conditions

Exp. no.	Factor				Response
	Te (占 폚)	RT (h)	WC (wt%)	En (wt%)	Choline alfocerate content (wt%)
One	50.0	3.0	100.0	5.0	1.0 ± 0.3
2	55.0	3.0	100.0	5.0	1.5 ± 0.1
3	50.0	5.0	100.0	5.0	18.6 ± 3.7
4	55.0	5.0	100.0	5.0	44.1 ± 3.8
5	50.0	3.0	100.0	10.0	10.4 ± 2.5
6	55.0	3.0	100.0	10.0	23.7 ± 3.6
7	50.0	5.0	100.0	10.0	99.7 ± 0.5
8	55.0	5.0	100.0	10.0	100.0 0.0
9	50.0	3.0	200.0	5.0	2.2 ± 1.5
10	55.0	3.0	200.0	5.0	10.9 ± 4.2
11	50.0	5.0	200.0	5.0	53.9 ± 3 5
12	55.0	5.0	200.0	5.0	100.0 0.0
13	50.0	3.0	200.0	10.0	27.0 ± 2.1
14	55.0	3.0	200.0	10.0	99.7 ± 0.3
15	50.0	5.0	200.0	10.0	100.0 0.0
16	55.0	5.0	200.0	10.0	100.0 0.0
17	49.0	4.0	150.0	7.5	43.8 ± 4.0
18	56.0	4.0	150.0	7.5	100.0 0.0
19	52.5	2.6	150.0	7.5	4.8 ± 1.4
20	52.5	5.4	150.0	7.5	100.0 0.0
21	52.5	4.0	150.0	4.0	7.0 ± 0.4
22	52.5	4.0	150.0	11.0	100.0 0.0
23	52.5	4.0	79.3	7.5	19.1 ± 2.3
24	52.5	4.0	220.7	7.5	99.9 ± 0.1
25	52.5	4.0	150.0	7.5	91.3 ± 1.0
26	52.5	4.0	150.0	7.5	99.5 ± 0.1
27	52.5	4.0	150.0	7.5	99.8 ± 0.1

All values were expressed as means ± SD (n = 3) Abbreviations: Te, temperature; RT, reaction time; WC, water content in the medium; And En, and enzyme loading.

Second order polynomial regression model of choline alfoscerate content of reaction products according to reaction temperature (Te), reaction time (RT), amount of water (WC) and enzyme amount (En) Analysis of Variance (ANOVA) was performed to evaluate the goodness-of-fit of the test.

As a result, as shown in Table 3, the quadratic polynomial regression model showing the relationship between the four reaction factors and the choline alfoscerate content was statistically significant at the 0.01% significance level and evaluated as appropriate at the 5% significance level. The coefficient of determination of the model was calculated as another index to evaluate the fit of the second polynomial regression model. Regression coefficient indicator decision indicating how well the fit to the data used in the derivation of the regression model (R ²⁾ value and the adjusted coefficient of determination (adjusted R ²⁾ value respectively 0.8298 and 0.7787 were regression model the response to the new observation (Q ² ), which is an indicator of how well we predicted, is 0.6965. Therefore, the explanatory power and the predictive power of the regression model were evaluated to be excellent.

Variance analysis of regression model

Variable	df	SS	MS	F -value	p-value
Corrected total	26	47242.36
Model	6	39200.97	6533.49	16.25	<0.0001
Residual	20	8041.39	402.07
Lack of fit	18	7994.86	444.16	19.09	0.0509
Pure error	2	46.53	23.26

Abbreviation: df, degree of freedom; SS, sum of squares; And MS, mean square.

The independent variables used in the derivation of the regression model for the novolac 435 catalytic hydrolysis of phosphatidylcholine for the production of choline alfoscerate were selected using the backward elimination method. In the present study, the reaction temperature (Te), the reaction time (RT), the amount of water in the medium (WC) and the amount of enzyme (En) considered as reaction factors affecting the choline alfoscerate content of the hydrolyzed product of phosphatidylcholine The linear term, the quadratic term that is the square of these factors, and the interaction term between these factors are all inserted into the model as explanatory variables. Regression models for choline alfoscerate content were derived by leaving only significant explanatory variables in the model. The absolute value of the coefficients of the finally selected independent variables in each regression model means the relative influence on the dependent variable, choline alfoscerate content.

As a result, as shown in Table 4, all of the four reaction factors showed a significant effect on the choline alfoscerate content and the reaction time (RT; 28.73), the enzyme amount (En; 22.99) WC; 15.45) and the reaction temperature (Te; 12.33), the reaction time was the most significant variable for the content of choline alfoscerate. The second term of reaction time (RT * RT; -14.69) and enzyme amount (WC * WC; -14.14) showed significant explanatory power at 5% level and negative coefficient value. On the other hand, the quadratic term of the reaction temperature (Te * Te) and the amount of water in the medium (WC * WC) did not affect the choline alfoscerate content at the 5% significance level. On the other hand, all the cross terms between the four response factors did not significantly affect the choline alfoscerate content at the 5% level. These results indicate that when the reaction temperature (50 to 55 ° C), the reaction time (3 to 5 hours), the amount of water in the medium (100 to 200% by weight) and the enzyme amount (5 to 15% And the amount of choline alfoscerate in the reaction product increases with increasing amount of enzyme, while the content tends to decrease again after reaching the maximum value under certain conditions. On the other hand, in case of reaction temperature and water amount in medium, , The choline alfoscerate content is continuously increased.

Second-order regression model for Novogem 435 catalytic hydrolysis of phosphatidylcholine

Effect	Coefficient	p-value
Intercept	79.06	<0.0001
Linear term
Te	12.33	0.0124
RT	28.73	<0.0001
WC	15.45	0.0026
En	22.99	<0.0001
Quadratic term
RT * RT	-14.69	0.0396
En * En	-14.14	0.0468

Abbreviation: degree of freedom; SS, sum of squares; And MS, mean square.

In order to verify the validity of the regression model derived from the present invention, ten reaction conditions other than the 27 reaction conditions in Table 2 were additionally set in a given range of the independent variables, and the content of choline alfoscerate in the reaction products obtained under these conditions was regressed Were compared with the choline alfoscerate content predicted using the parental format.

As a result, as shown in Table 5, the prediction error of the regression model for the choline alfoscerate content ranged from 0.6 to 61.4%. Especially, the prediction error of choline alfoscerate content of reaction products obtained at 5 ~ 10 conditions with relatively large predicted values was relatively low as 0.6 ~ 24.6%. The use of the second regression model derived from the present invention establishes an optimal reaction condition that maximizes the amount of choline alfoscerate produced, so the above regression model is evaluated to be valid.

Production of choline alfoscerate by reaction conditions predicted by the second-order regression model for the catalytic hydrolysis of norbom 435 of phosphatidylcholine and production of choline alfoscerate

Exp. no.	Factor				Response
	Te (占 폚)	RT (h)	WC (wt%)	En (wt%)	Choline alfocerate content (wt%)
					Observed	Predicted	% PE
One	50.3	3.1	169.3	5.0	2.2 ± 0.7	2.1	4.6
2	51.0	3.0	121.9	7.0	9.4 ± 3.2	14.8	58.2
3	50.1	4.8	103.2	5.0	20.4 ± 5.3	29.6	45.4
4	51.5	4.9	102.9	5.1	23.1 ± 7.5	37.2	61.4
5	54.2	4.9	100.2	5.0	45.3 ± 13.6	48.8	7.6
6	54.3	4.8	110.5	5.2	50.5 ± 2.5	56.8	12.5
7	50.3	4.9	197.2	5.3	69.3 ± 4.4	66.0	4.8
8	50.0	3.5	198.3	9.9	95.0 ± 4.2	71.6	24.6
9	50.4	4.0	194.4	8.5	98.5 ± 2.2	89.6	9.0
10	54.2	4.2	186.6	7.0	100.0 0.0	99.4	0.6

All values were expressed as means ± SD (n = 3) Abbreviations: Te, temperature; RT, reaction time; WC, water content in the medium; En, enzyme loading; And% PE, and a percentage prediction error.

The optimal reaction conditions for the Novogem 435 catalytic hydrolysis of phosphatidylcholine, which maximizes the production of choline alfoscerate, were established using the regression model derived from the present invention. The optimum conditions for the reaction temperature, reaction time, amount of water in the medium and amount of enzyme in the reaction products were determined. The optimum conditions were determined by using Optimization function of Design Expert 8.0 program. The content of choline alfoscerate in the reaction product obtained in the above experiment was examined.

As a result, as shown in Table 6, it was confirmed that the phosphatidylcholine completely converted to choline alfoscerate in all of the above conditions. The most difficult thing in industrial use of enzyme reaction is that most enzyme catalysts are expensive and economical. Therefore, in order to industrially produce choline alfoscerate, which is the ultimate goal of the present invention, it is necessary to recover the novoid 435 enzyme after the hydrolysis reaction and reuse it. The immobilized carrier of the Novojim 435 enzyme is easily ruptured by shear force such as stirring and the possibility of leaching of the enzyme from the immobilized carrier by the surfactant or polar solvent is high. Therefore, as the amount of water in the reaction medium according to the present invention increases, the stability of the noborg 435 decreases, which makes it difficult to recover the noborg 435 after the reaction. Therefore, in the present invention, the amount of water in the medium is relatively low (Reaction temperature 55 ° C, reaction time 4.9 hours, amount of water 105.9% by weight in the medium, amount of enzyme 9.4% by weight) or similar conditions (reaction temperature 55 ° C, reaction time 6 hours, amount of water in the medium 100% Was set as the optimal reaction condition for the Novogem 435 catalytic hydrolysis of phosphatidylcholine to maximize the production of choline alfoscerate.

Optimal reaction conditions derived from the second regression model for novolac 435 catalytic hydrolysis of phosphatidylcholine and production of choline alfoscerate

Exp. no.	Factor				Response
	Te (占 폚)	RT (h)	WC (wt%)	En (wt%)	Choline alfocerate content (wt%)
					Observed	Predicted	% PE
One	54.1	3.9	185.2	8.8	100.0 0.0	100.0	0.0
2	55.0	5.0	199.2	6.2	100.0 0.0	100.0	0.0
3	50.0	4.4	198.7	8.9	100.0 0.0	100.0	0.0
4	55.0	4.9	105.9	9.4	100.0 0.0	100.0	0.0

All values were expressed as means ± SD (n = 3) Abbreviations: Te, temperature; RT, reaction time; WC, water content in the medium; En, enzyme loading; And% PE, and a percentage prediction error.

Example 4 Separation of choline alfoscerate from the reaction product Purification

Choline alfoscerate was isolated and purified by subjecting the crude product of soybean phosphatidylcholine produced by Novojim 435 catalytic hydrolysis reaction product prepared under the optimal reaction conditions set forth in the above Example <3-1> to reaction and then separating the reaction medium.

<4-1> Preparation of water fraction of reaction product

In this experiment, a water fraction was prepared from the reaction product obtained by hydrolyzing soybean phosphatidylcholine under novolac 435 catalyst, and the choline alfoscerate content of the fraction was measured.

Specifically, 4 g of soybean phosphatidylcholine 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) The reaction was carried out at a reaction temperature of 55 ° C at 600 rpm for 6 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 reaction product was placed in a Buchner funnel with a filter paper, and filtration under reduced pressure was conducted to obtain a reaction product from 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. At this time, since the free fatty acid generated by the hydrolysis of the substrate is present in the hexane layer and the choline alfoscerate is dissolved in the water layer, the water layer is recovered and dried at 40 ° C using a rotary vacuum concentrator to purify the choline alpocellate Water (water fraction) was prepared.

The content of phosphatidylcholine, lysophosphatidylcholine and choline alfoscerate in the water fraction obtained from the reaction product of soybean phosphatidylcholine was measured in the same manner as in Example <2-1>, and the content of each component was measured by phosphatidylcholine, Phosphatidylcholine and choline alfoscerate as a percentage of the total content.

As a result, as shown in Fig. 5, it was confirmed that the free fatty acid present in the reaction product was almost completely removed from the water fraction obtained from the reaction product of soybean phosphatidylcholine.

As a result, as shown in Table 7, the yield of the water fraction obtained from the reaction product of soybean phosphatidylcholine was 31.8% by weight with respect to the weight of soybean phosphatidylcholine choline as a raw material substrate and the choline alfoscerate content of the water fraction was 98.6% by weight.

The yield of the water fraction obtained from the Novogam 435 hydrolysis product of soybean phosphatidylcholine and the choline alfoscerate content

Products	Product yield (wt% of substrate)	Choline alfocerate content (wt%)
Water-soluble fraction	31.8 ± 5.6	98.6 ± 0.3

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

< Example  5> Preparation of fractionated soybean lecithin from soybean lecithin

<5-1> Chemical Composition of Soybean Lecithin

The chemical composition of soybean lecithin used as a raw material in the present invention is shown in detail in Table 8 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

<5-2> Preparation 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  6> 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 5 are shown in Table 9 below.

Fatty acid composition of soybean lecithin and fractionated soybean lecithin

Fatty acid	Soy lecithin (mol%)	Ethanol-soluble fraction of soy 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 9, 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  7> 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 <5-2> 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 determine the content of choline alfoscerate 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). The solvent was removed under a nitrogen stream and the solution was dissolved in 0.2 mL of 93 v / v% methanol used as a mobile phase of liquid chromatography. The resulting solution was filtered with a 0.45 μm syringe filter (13 mm), and then filtered through a liquid chromatograph (JASCO Corp., 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. 8, the production amount of glycerophosphoester (20.0 wt%) including choline alfoscerate by the hydrolysis reaction of the fractionated soybean lecithin before the reaction time of 2 hours before 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. 8, 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 alfoselate) 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  8> 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;

<8-1> 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 7, 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. 8, in the water fraction obtained from the reaction product of the fractionated soybean lecithin, most of the free fatty acids present in the reaction product were 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 10, 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%.

The yield of the water fraction obtained from the Novogem 435 hydrolysis reaction product of the 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 (25)

1) adding a mixed solvent of water and hexane and a lipase enzyme to phosphatidylcholine, and hydrolyzing the phosphatidylcholine;

2) removing the lipase enzyme from the reaction product;

3) separating the reactants in step 2); And

4) recovering the water layer from the layered reactant to obtain a water fraction.

2. The method of claim 1, wherein the lipase is a sn-position nonspecific lipase.

3. The method of claim 2, wherein the lipase is Candida antarctica lipase B. 3. The method according to claim 2, wherein the lipase is Candida antarctica lipase B.

[4] The method of claim 3, wherein the lipase is Candida antarctica lipase B using a macroporous anionic resin as an immobilization carrier.

The method for producing choline alfoscerate according to claim 1, wherein the amount of water in the mixed solvent of water and hexane is 20 to 400% by weight based on the weight of phosphatidylcholine.

The method according to claim 1, wherein the amount of the lipase enzyme is 5 to 15% by weight based on the weight of phosphatidylcholine.

The method for producing choline alfoscerate according to claim 1, wherein the hydrolysis reaction is carried out at 50 to 55 ° C for 2 to 12 hours.

The method according to claim 1, wherein the amount of water in the mixed solvent of water and hexane is 100 to 200% by weight based on the weight of phosphatidylcholine, the amount of the lipase enzyme is 5 to 10% by weight based on the weight of phosphatidylcholine, Wherein the reaction is carried out for 3 to 6 hours.

[3] The method of claim 1, wherein the step 3) further comprises the step of further separating by adding water and hexane in a volume ratio of 1: 1.

The method of claim 1, wherein the step of obtaining the water fraction of step 4) is for removing free fatty acid present in the hexane layer.

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.

12. The method of claim 11, wherein the fractionated lecithin is prepared by adding ethanol to lecithin, stirring at 60 to 65 DEG C for 10 minutes to 1 hour, and then removing ethanol from the supernatant obtained by centrifugation to obtain an ethanol fraction &Lt; / RTI &gt;

12. The method of claim 11, wherein the fractionated lecithin is removed in neutral lipid.

12. The method of claim 11, wherein the lipase is a sn-position nonspecific lipase.

15. The method of claim 14, wherein the lipase is Candida antarctica lipase B. 15. The method of claim 14, wherein the lipase is Candida antarctica lipase B.

16. The method according to claim 15, wherein the lipase is Candida antarctica lipase B using a macroporous anionic resin as an immobilization carrier.

12. The method of claim 11, wherein the amount of water in the mixed solvent of water and hexane is 20 to 200% by weight based on the weight of phosphatidylcholine.

12. The method of claim 11, wherein the amount of the lipase enzyme is 5 to 15% by weight based on the weight of phosphatidylcholine.

12. The method according to claim 11, wherein the hydrolysis reaction is carried out at 50 to 55 DEG C for 2 to 12 hours.

[12] The method of claim 11, wherein the step 4) further comprises the step of adding water and hexane in a volume ratio of 1: 1 to separate the choline alfoscerate.

12. The method of claim 11, wherein the step of obtaining the water fraction of step 5) is for removing free fatty acid present in the hexane layer.

12. The method according to claim 11, wherein the water fraction in step 5) contains glycerophosphodiester containing choline alfoscerate.

23. The process according to any one of claims 1 to 22, wherein the choline alfoscerate is available as a food raw material.

22. A choline alfoscerate which can be used as a food raw material produced by the method of any one of claims 1 to 22.

A health functional food for improving cognitive function comprising choline alfoscerate according to claim 24 as an active ingredient.

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