KR20180118996A - Manufacturing method of monogalactosyldiacylglycerol containing high phenolic acid content - Google Patents

Abstract

The present invention relates to a method for manufacturing monogalactosyldiacylglycerol having high phenolic acid content by an enzyme catalyzed reaction and a monogalactosyldiacylglycerol having high phenolic acid content manufactured thereby. The method for manufacturing monogalactosyldiacylglycerol containing phenolic acid according to the present invention can effectively manufacture monogalactosyldiacylglycerol containing a high concentration of phenolic acid by using a specific reaction medium and a specific catalytic enzyme. In addition, the monogalactosyldiacylglycerol manufactured by the method of the present invention contains a high content of phenolic acid having various functions, and can be usefully used as a material for health assisting.

Description

{Manufacturing method of monogalactosyldiacylglycerol high phenolic acid content containing monogalactosyldiacylglycerol having high phenolic acid content}

The present invention relates to a method for producing monogalactosyl diacyl glycerol having a high content of phenolic acid by using an enzyme catalyzed reaction and a monogalactosyl diacyl glycerol having a high content of the produced phenolic acid.

Pine nuts are seeds found in the cones of Pinus koraiensis belonging to the Pinus (Pinus), indicating that the name of the Pinus koraiensis is Korean endemic. There are about 115 kinds of plants in the pine tree, but only about 20 species including pine trees make edible seeds. In general, pine nut contains about 70% fat, and it is made up of 15% protein, 5% carbohydrate and 3% ash.

In pinolenic acid (c5, c9, c12-18: 3), the double bond closest to the carboxy group is on carbon number 5, and the double bond and the carboxyl group and the closest double bond are separated by two or more methylene groups (polymethylene-interrupted)? 5-unsaturated fatty acid. The phenolic acid present in nature is mainly contained in the pine nut obtained from the Korean endemic pine tree and the siberian pine obtained from the siberian pine (Pinus sibirica). Especially, the pine nut oil obtained from these pine nut contains 13 ~ 18% (Xie K et al., J. Funct. Foods 23, 464-473, 2016).

Phenolic acid has health effects and properties unlike other 18: 3 fatty acids (18 carbon atoms, three of which have double bonds). First, it is known that phenolic acid has a lipid-lowering effect in blood by changing the expression level of apolipoprotein genes (apo genes). Secondly, phenolic acid has been reported to lower the serum LDL cholesterol level by increasing the absorption of LDL cholesterol in the liver. Finally, phenolic acid has an appetite-suppressing effect and acts as an appetite suppressant by promoting the secretion of the appetite-regulating hormone cholecystokinin and glucagon-like peptide-1 secreted from the small intestine (Pasman et al., Lipids Health Dis. 7, 10, 2008).

Monogalactosyldiacylglycerols, on the other hand, are a type of glycoglycerolipids in which one galactose is linked to the sn-3 position of 1,2-diacyl-sn-3-glycerol. Monogalactosyl diacyl glycerol accounts for 50% of the lipids present in the chloroplasts, and is present in many photosynthetic tissues of tuberous plants, seaweeds and some bacteria.

The ingestion of the thylakoid membrane of chloroplast composed of glycolipids containing monogalactosyl diacyl glycerol promotes the secretion of cholecystokinin and glucagon like peptide-1 in clinical trials, (Ghrelin et al., 1989), and the regulation of the secretion of appetite-related hormones in glycolipids (Ghrelin et al. The mechanism is thought to be closely related to the increased digestion time of fat in the diet induced by glycolipid. Monogalactosyl diacyl glycerol has been reported to inhibit the activity of porcine pancreatic lipase in in vitro experiments (Banskota AH et al., J Appl. Phycol. 28, 169-175, 2016). Inhibition of pancreatic lipase activity inhibits the appetite by decreasing the rate of hydrolysis of dietary fat in the small intestine and by increasing the amount of undigested dietary fat at the end of the small intestine (Chu et al., Langmuir 25 , 9352-9360, 2009).

As a result, both the phenolic acid and monogalactosyl diacyl glycerol are lipids showing an appetite suppressing effect. Monogalactosyldiacylglycerol is an amphipathic substance having both a nonpolar region composed of fatty acid acyl groups and a polar region composed of galactose, and also has a surface activity and emulsifying ability similar to glycerol phospholipids such as phosphatidylcholine.

Therefore, the inventors of the present invention have found that monogalactosyl diacyl glycerol having a high content of phenolic acid, which can be used as an emulsifier having an appetite suppressing effect, can be used as an enzyme catalyst To obtain the desired product. When the reaction conditions for increasing the content of phenolic acid in the monogalactosyldiacyl glycerol were studied, only when the specific enzyme and the specific organic solvent were used as the catalyst and medium for the reaction, monogalactosyldiacylglycerol The present inventors have completed the present invention.

Korean Patent Publication No. 10-1993-0009532

Accordingly, an object of the present invention is to provide a method for effectively producing monogalactosyl diacyl glycerol with high content of phenolic acid.

Another object of the present invention is to provide monogalactosyl diacyl glycerol having high content of phenolic acid produced by the above method.

It is still another object of the present invention to provide a health supplement containing monogalactosyl diacyl glycerol having a high content of phenolic acid as an active ingredient.

It is still another object of the present invention to provide a method for producing monogalactosyl diacyl glycerol having an intermediate fatty acid content.

In order to achieve the above-mentioned object of the present invention,

The present invention relates to a process for preparing a phenolic acid concentrate comprising the steps of: i) preparing a phenolic acid concentrate; And ii) adding phenolic acid concentrate and monogalactosyl diacyl glycerol to acetone and an enzyme, and then reacting the resultant monogalactosyl diacyl glycerol with monogalactosyl diacyl glycerol.

In one embodiment of the present invention, the concentrate of phenolic acid may contain 30 to 90 mol% of phenolic acid.

In one embodiment of the present invention, the enzyme may be a sn-1, 3 position-specific lipase.

In one embodiment of the present invention, the sn -1 , 3- position specific lipase may be Lipozyme RM IM or Lipozyme TL IM.

In one embodiment of the present invention, the concentrated phenolic acid comprises (a) dissolving nuts oil fatty acid in a solvent and then adding and dissolving the element; (b) crystallizing the solution while maintaining the solution at room temperature at 4 ° C to 5 ° C, removing the crystallized product, and separating the uncrystallized filtrate; (c) removing the solvent from the separated filtrate, adding hydrochloric acid to the reaction mixture, and then adding hexane to extract the fatty acid.

In one embodiment of the present invention, the step (ii) comprises the step of mixing the concentrate of the phenolic acid as the substrate and the monogalactosyl diacyl glycerol in a molar ratio of 1:30 to 1:40, Followed by addition of the enzyme and reaction at 25 < 0 > C.

Further, the present invention provides monogalactosyl diacyl glycerol containing phenolic acid prepared by the above method.

In one embodiment of the present invention, the monogalactosyl diacyl glycerol containing the phenolic acid may contain the phenolic acid in a concentration of 30 to 50 mol%.

Further, the present invention provides a health supplement containing monogalactosyl diacyl glycerol containing phenolic acid as an active ingredient.

Also, the present invention provides a method for producing monogalactosyldiacyl glycerol containing an intermediate fatty acid, which comprises adding acetone and an enzyme to a saturated fatty acid having 6 to 12 carbon atoms and monogalactosyl diacyl glycerol, followed by reaction .

In one embodiment of the present invention, the saturated fatty acid having 6 to 12 carbon atoms may be capric acid.

In one embodiment of the present invention, the enzyme may be a sn-1, 3 position-specific lipase.

In one embodiment of the present invention, the sn -1 , 3- position specific lipase may be Lipozyme RM IM or Lipozyme TL IM.

In one embodiment of the present invention, the saturated fatty acid having 6 to 12 carbon atoms and the monogalactosyl diacyl glycerol are constituted in a molar ratio of 1:30 to 1:40 as a substrate, and 20% by weight of enzyme And then reacted at 25 ° C.

In one embodiment of the present invention, the intermediate fatty acid-containing monogalactosyl diacyl glycerol may contain an intermediate fatty acid in a concentration of 30 to 50 mol%.

The method for producing monogalactosyl diacyl glycerol containing phenolic acid according to the present invention can effectively produce monogalactosyl diacyl glycerol containing a high concentration of phenolic acid by using a specific reaction medium and a specific catalytic enzyme. In addition, the monogalactosyl diacyl glycerol prepared by the method according to the present invention contains a high content of phenolic acid having various functions, and can be usefully used as a material for health assisting.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram showing a process for producing monogalactosyl diacyl glycerol having a high content of phenolic acid according to the present invention.
2 is a graph showing the content of phenolic acid in monogalactosyl diacyl glycerol in the reaction product as a function of reaction time according to the type of lipase (Lipozyme RM IM, Lipozyme TM TL IM, Novozyme 435, Candida rugosa lipase).
FIG. 3 is a graph showing the content of phenolic acid in monogalactosyl diacyl glycerol as a function of reaction time in the reaction product of liposome RM IM catalytic acid decomposition reaction according to the type of medium (Acetone, Toluene, Bemzene, Ethanol).
4 is a graph showing the content of phenolic acid of monogalactosyl diacyl glycerol in the reaction product as a function of reaction time according to the reaction temperature (25, 30, 35, 40 ° C).
Figure 5 shows the results of the reaction of the liposome RM IM catalytic acid with monogalactosyl diacyl glycerol and the concentrate of phenolic acid (1:10, 1:20, 1:30, 1:40) A graph showing the content of phenolic acid in lactosyl diacyl glycerol as a function of reaction time.
6 is a graph showing the content of phenolic acid in monogalactosyl diacyl glycerol as a function of reaction time in the reaction product of the liposome RM IM catalytic acid decomposition reaction according to the amount of enzyme (5, 10, 15, 20 wt%).
FIG. 7 is a graph showing the content of phenolic acid in monogalactosyl diacyl glycerol as a function of reaction time in the reaction product of the liposome RM IM catalytic acid decomposition reaction carried out under optimum reaction conditions. FIG.
8 is a graph showing the content of capric acid of monogalactosyl diacyl glycerol in the reaction product of the liposome RM IM catalytic acid decomposition reaction as a function of reaction time.

The present invention relates to a process for preparing a phenolic acid concentrate comprising the steps of: i) preparing a phenolic acid concentrate; And ii) adding phenolic acid concentrate and monogalactosyl diacyl glycerol to acetone and an enzyme, and then reacting the resultant monogalactosyl diacyl glycerol with monogalactosyl diacyl glycerol.

In the present invention, "pinolenic acid" is an unsaturated fatty acid frequently found in pine trees, and is a unique component having an active function such as polyunsaturated fatty acids such as EPA or DHA. Phenolic acid (5, 9, 12 C18: 3 fatty acids, i.e. fatty acids having 18 carbon atoms with three double bonds at positions 5, 9 and 12) is added to the pine nut oil or fraction thereof to about 13-18 weight % ≪ / RTI > amount and is represented by the following formula (1).

[Chemical Formula 1]

In the present invention, "monogalactosyldiacylglycerol" is a kind of glycoglycerolipids in which one galactose is bonded to the sn-3 position of 1,2-diacyl-sn-3-glycerol, And 50% of the lipids present in the body, and it is known to exist in many photosynthetic tissues of the vascular plants, seaweeds and some bacteria.

(2)

In the present invention, "monogalactosyl diacyl glycerol containing a phenolic acid" is a compound in which the fatty acid at the sn-1 position of the monogalactosyl diacyl glycerol represented by the formula (2) is replaced with the phenolic acid of the formula (1) Can be prepared through the process of Scheme 1.

[Reaction Scheme 1]

The method for producing the monogalactosyl diacyl glycerol containing the phenolic acid of the present invention comprises the steps of: i) preparing a concentrated phenolic acid; And ii) adding acetone and an enzyme to the concentrate of the phenolic acid and the monogalactosyl diacyl glycerol and then reacting them.

The step i) of the present invention is a step of preparing a concentrated phenolic acid, which can be prepared by directly producing a concentrated phenolic acid or by purchasing a commercially available condensed phenolic acid.

In the case of using the concentrate of pinoleic acid directly, it is possible to produce a concentrate containing a large amount of pinoleic acid from the nourishing oil, and it is preferable that the content of the pinolein acid is 13 mol% or more. Thus, Pinus koraiensjs, a Korean native species containing more than 13 mol% phenolic acid, or Pinus sibirica seeds containing 18 mol% higher levels of pinoleic acid, may be used .

(A) dissolving nuts oil fatty acid in a solvent and then adding and dissolving the component; (b) crystallizing the solution while maintaining the solution at room temperature at 4 ° C to 5 ° C, removing the crystallized product, and separating the uncrystallized filtrate; (c) removing the solvent from the separated filtrate, adding hydrochloric acid to the reaction mixture, and then adding hexane to extract the fatty acid.

In the step (a), the nuts oil fatty acid can be prepared by saponifying the nuts oil with alkali. The alkali may be sodium hydroxide or potassium hydroxide. After the saponification, hydrochloric acid is added to convert the salt of natto oil fatty acid into free fatty acid form and extracted with hexane. The hexane may be removed by a method such as concentration under reduced pressure to prepare a liquid nut oil fatty acid. The content of phenolic acid in the nutshell fatty acid obtained from the nourishing oil according to the present invention may be 13 mol% or more.

The concentrate of pinoleic acid of the present invention is obtained by reacting the above-mentioned nut oil fatty acid with a urea in a solvent to crystallize an unsaturated fatty acid having a low saturated or unsaturated fatty acid as a urea adduct and then filtration, And concentrating the relatively unevenly high level of the pinoleic acid into a liquid phase. Wherein the solvent is methanol or ethanol. The mixing ratio of the nuts fatty acids and urea is 1: 1 to 1: 5. The lower the proportion of urea in the mixing ratio, the lower the concentration of the phenolic acid in the concentrate and the higher the proportion of the urea, the more the content of the phenolic acid in the concentrate increases but the recovery of the phenolic acid decreases. Therefore, when both the content of the phenolic acid and the content of the phenolic acid in the concentrate of the obtained phenolic acid are taken into account, the mixing ratio of the nut oil fatty acid and the urea is most preferably about 1: 4.

The concentrate of the present invention prepared by the above method may contain the phenolic acid at a concentration of 30 to 90 mol%.

Step ii) of the present invention is a step of adding acetone and an enzyme to the concentrate of phenolic acid and monogalactosyl diacyl glycerol and then reacting them. The concentrate of phenolic acid and the monogalactosyl diacyl glycerol are substrates, Is the reaction medium, and the enzyme acts as a catalyst.

The monogalactosyldiacylglycerol may be purchased and used from the market, or may be directly isolated and purified from the green leaf, seaweed, and bacterial culture by known methods in the art.

In one embodiment of the present invention, the enzyme may be a sn-1, 3- position specific lipase, preferably a Rhizomucor miehei microorganism-derived lipase or Thermomyces lanuginosus, May be a microbial-derived lipase. The lipase may be used in the form of a liquid preparation, a solid preparation and an immobilization preparation. In the following examples of the present invention, commercially available Lipozyme RM IM (Lipozyme RM IM) and Thermomyces lanuginosus-derived lipase commercially available as lipase derived from Rhizomucor miehei Lipozyme® TL IM, a commercially available liposome, was used.

In the present invention, the term "sn-1, 3 position specificity" means that the enzyme reaction selectively occurs only in sn-1, sn-3 among sn-1, sn-2 and sn-3 of the neutral substance constituting the nourishing oil.

In another embodiment of the present invention, the step (ii) comprises a step in which the concentration of the phenolic acid as the substrate and the monogalactosyl diacyl glycerol are in a molar ratio of 1:30 to 1:40, acetone is used as the reaction medium, Adding 20% by weight of enzyme to the substrate, and reacting at 25 < 0 > C.

Further, the present invention provides monogalactosyl diacyl glycerol containing phenolic acid prepared by the above method.

Since the monogalactosyl diacyl glycerol containing the phenolic acid of the present invention contains the phenolic acid at a high concentration (42 mol% or more), it can have various physiological activities of the phenolic acid. For example, it is known that phenolic acid lowers low-density lipoprotein (LDL cholestrol) and not only has a thrombolytic effect, but also promotes secretion of cholestyocinin, which is known as a hormone having an action of suppressing hunger and inducing satiety, The monogalactosyl diacyl glycerol having a high content of the pinoleic acid of the present invention is excellent in its physiological activity and can be usefully used in the production field of foods, medicines, cosmetics and the like containing the above-mentioned effects.

The present invention also provides a composition comprising monogalactosyl diacyl glycerol containing phenolic acid as an active ingredient.

The composition may be a composition for controlling body fat and body weight, a pharmaceutical composition for preventing, treating or improving obesity, a food composition, a food additive composition or a health supplement food composition; A skin external composition (including a cosmetic composition) for skin moisturizing effect or skin tone improvement.

Further, the present invention provides a health supplement containing monogalactosyl diacyl glycerol containing phenolic acid as an active ingredient.

The health supplements are intended for body fat and weight control.

The form of the health supplement food in the present invention is not particularly limited. It can be manufactured in various forms including beverage, granule, tablet, powder, ring, capsule, wire, noodle, confectionery, meat, fish, herb, pot or rice by mixing known ingredients, food additives and the like.

The health supplements of the present invention may contain conventional food additives and, unless otherwise specified, whether or not they are suitable for use as food additives are classified according to the General Rules and General Test Methods approved by the Food and Drug Administration 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, and nutrients, vitamins, edible electrolytes, flavors, , Cheese, 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 food supplement in tablet form can be prepared by granulating a mixture obtained by mixing the active ingredient (monogalactosyl diacyl glycerol containing phenolic acid) of the present invention with excipients, binders, disintegrants and other additives in a usual manner Next, a lubricant or the like may be put in a compression mold, or the mixture may be directly compression molded. In addition, the above-mentioned tablets may contain a mating agent or the like if necessary.

The hard capsule of the capsule type health supplement food can be prepared by filling the normal hard capsule with the active ingredient of the present invention (monogalactosyl diacyl glycerol containing the phenolic acid), and the soft capsule may contain the active ingredient as an excipient, And mixing the mixture with an additive into a capsule base such as gelatin. 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 supplement food can be prepared by molding a mixture of the active ingredient (monogalactosyl diacyl glycerol containing the phenolic acid) of the present invention with excipients, binders, disintegrants, etc. by a conventionally known method, If necessary, it may be coated with white sugar or other skin care agent, or the surface may be coated with a substance such as starch or talc.

The granular form of the health supplements can be prepared by granulating a mixture of the active ingredient (monogalactosyl diacyl glycerol containing phenolic acid) of the present invention with an excipient, a binder, a disintegrant and the like by a conventionally known method, If necessary, a flavoring agent, a mating agent, and the like may be contained.

The health supplement 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.

According to still another aspect of the present invention, there is provided a health supplement containing monogalactosyl diacyl glycerol containing phenolic acid as an active ingredient.

Also, the present invention provides a method for producing monogalactosyldiacyl glycerol containing an intermediate fatty acid, which comprises adding acetone and an enzyme to a saturated fatty acid having 6 to 12 carbon atoms and monogalactosyl diacyl glycerol, followed by reaction .

In the present invention, "intermediate fatty acid" means a saturated fatty acid having 6 to 12 carbon atoms.

In one embodiment of the present invention, the saturated fatty acid having 6 to 12 carbon atoms may be capric acid, but the type thereof is not particularly limited.

In another embodiment of the present invention, the enzyme may be a sn-1, 3 position-specific lipase, preferably Lipozyme RM IM or Lipozyme TL IM.

In another embodiment of the present invention, the method for preparing monogalactosyldiacylglycerol containing an intermediate fatty acid is characterized in that a saturated fatty acid having 6 to 12 carbon atoms and monogalactosyldiacyl glycerol as a substrate is contained at a molar ratio of 1:30 to 1:40 , And adding 20 wt% of enzyme to the substrate, followed by reaction at 25 < 0 > C.

The monogalactosyl diacyl glycerol prepared by the process of the present invention may contain an intermediate fatty acid in a concentration of 30 to 50 mol%.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.

< Example >

reagent

The monogalactosyl diacyl glycerol (purity> 99%) used in the following experiments was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA); Lipozyme RM IM, Lipozyme TL IM and Novozym 435 were purchased from Novozymes A / S (Bagsvaerd, Denmark); Candida rugoselipase immobilized on Immobead 150 and capric acid (purity > 98%) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, Mo., USA).

< Example  1>

From nuts oil Phenolic acid Concentrate  Produce

<1-1> Pine nut extract

The nourishing oil used as a raw material in the present invention was extracted from seeds of Pinus koraiensjs, a Korean native species purchased or obtained in Korea.

In detail, the pine nuts were pulverized with a blender, and the crude oil and the solvent ratio were adjusted to about 1:10 (w / v) using n-hexane to sufficiently agitate for about 6 hours to extract the nuts. .

<1-2> Nut oil fatty acid production

400 g of 75% ethanol containing 10 wt% of sodium hydroxide was added to 100 g of the pine nut extracted from Example <1-1>, and the mixture was heated for 30 minutes at 250 ° C. using a reflux condenser. After the saponification, the reaction product was transferred to a separatory funnel, and 200 mL of distilled water and 250 mL of 6N hydrochloric acid were added to convert the fatty acid salt into the free fatty acid form. After layer separation, 500 mL of hexane and 300 mL of distilled water were added, and the layer was separated to obtain a hexane layer. Since hydrochloric acid may remain in the hexane layer, it was washed five times with 100 mL of distilled water, and then the pH of the washing water was determined to be neutral by a pH test paper. After filtering once the anhydrous sodium sulfate to remove water remaining in the hexane layer, hexane was removed at 40 ° C using a rotary vacuum concentrator. The obtained nut oil fatty acid was stored frozen at -80 ° C until use.

<1-3> Phenolic acid Concentrate  Produce

100 g of the nutshell fatty acid obtained in the above Example <1-2> was dissolved in 1 L of methanol, and 400 g of the urea was added thereto. The resulting solution was heated to 250 캜 in a reflux apparatus and stirred for 30 minutes to 1 hour to completely dissolve the urea . The solution was allowed to cool slowly at room temperature for 30 minutes, and then allowed to stand at 4 ° C for 16 hours to crystallize it as an urea adduct, which was then removed using filter paper (Whatman no. 1 and Buchner funnel) to separate the uncrystallized fatty acid filtrate. The filtrate of methanol was removed at 40 ° C using a rotary evaporator and 800 mL of 0.1 N hydrochloric acid was added to dissolve the remaining components and transferred to a separatory funnel. The uncrystallized fatty acids were extracted twice with 1 L of hexane. Since hydrochloric acid may remain in the hexane layer, it was washed twice with 200 mL of distilled water, and then the pH of the washing water was determined to be neutral by using a pH test paper. After filtering once the anhydrous sodium sulfate to remove water remaining in the hexane layer, hexane was removed at 40 ° C using a rotary vacuum concentrator. The resulting phenolic acid concentrate was stored frozen at -80 DEG C until use.

< Example  2>

Pine nut oil, Phenolic acid Concentrate  And Of monogalactosyl diacyl glycerol  Fatty acid composition analysis

In addition to the fatty acid composition of each of the pine nut, nut oil fatty acid and pinoleic acid concentrate obtained in Example 1 above, the monogalactosyl diacyl glycerol used in the present invention (commercial monogalactosyl diacyl glycerol purity separated and purified from spinach 99 wt% or more) are shown in detail in Tables 1 and 2 below.

Analysis of Fatty Acid Composition of Pine Nut and Nut Oil fatty acid Pine nut (mol%) Nut oil fatty acid (mol%) 16: 0 5.3 ± 0.0 4.5 ± 0.0 16: 1n-7 0.1 ± 0.0 - 18: 0 2.1 ± 0.0 2.3 ± 0.0 18: 1n-9 25.7 ± 0.1 27.1 + - 0.1 18: 1n-7 0.5 ± 0.0 0.5 ± 0.0 c5, c9-18: 2 2.2 ± 0.0 2.3 ± 0.0 18: 2n-6 46.0 ± 0.1 45.3 ± 0.2 c5, c9, c12-18: 3 (phenolic acid) 13.7 ± 0.0 13.0 ± 0.1 18: 3n-3 0.6 ± 0.0 0.2 ± 0.0 20: 0 0.3 ± 0.0 0.5 ± 0.0 20: 1 1.1 ± 0.0 1.5 ± 0.1 20: 2 0.6 ± 0.0 0.8 ± 0.0 c5, c11, c14-20: 3 1.1 ± 0.0 1.3 ± 0.0 Unidentified 0.7 ± 0.1 0.7 ± 0.0

Analysis of Fatty Acid Composition of Concentrated Pinoleic Acid and Monogalactosyldiacyl Glycerol fatty acid Concentrated phenolic acid (mol%) Monogalactosyl
Diacylglycerol (mol%) 16: 0 - 1.4 ± 0.0 16: 3n-3 - 37.5 ± 0.1 18: 0 - 0.1 ± 0.0 18: 1n-9 0.2 ± 0.0 0.4 ± 0.0 18: 1t - 0.1 ± 0.0 18: 2n-6 4.6 ± 0.0 2.6 ± 0.0 c5, c9, c12-18: 3 (phenolic acid) 85.5 ± 0.2 - 18: 3n-3 - 57.7 ± 0.3 18: 3t - 0.2 ± 0.0 20: 0 0.9 ± 1.2 - c5, c11, c14-20: 3 1.7 ± 0.0 - Unidentified 7.0 ± 1.4 -

As shown in Table 1, the content of all fatty acids including phenolic acid was not significantly different (p <0.05) between the raw nuts oil and the nuts oil fatty acid. The major fatty acids in the nutshell fatty acids were 45.3 mol% linoleic acid (18: 2n-6) and 27.1 mol% oleic acid (18: 1 n-9). The content of phenolic acid in the nut oil fatty acid was 13.0 mol%.

On the other hand, as shown in Table 2, the fatty acid composition of the concentrate of phenolic acid decreased to 4.6 mol% and 0.2 mol% in the concentrate of linoleic acid and oleic acid, which are major unsaturated fatty acids of the nutshell fatty acid, Saturated fatty acids such as stearic acid (18: 0) and arachidic acid (20: 0) were all removed and the content of pinoleic acid increased to 85.5 mol%. On the other hand, the contents of alpha-linolenic acid (18: 3n-3) and hexadecatrienoic acid (16: 3n-3) as major fatty acids were higher in commercial monogalactosyl diacyl glycerol , And phenolic acid were not contained.

< Example  3>

Phenolic acid High content Monogalactosyl diacyl glycerol  Produce

Using a concentrate of phenolic acid and monogalactosyl diacyl glycerol prepared in Example <1-3> as a substrate and using an organic solvent (acetone, toluene, benzene, ethanol) as a reaction medium, Lipozyme RM IM, Lipozyme TL IM, Novozym 435, Candida rugoselipase immobilized on Immobead 150) were used as catalysts to prepare monogalactosyl diacyl glycerol of the present invention. In the present invention, commercial monogalactosyl diacyl glycerol (purity 99 wt% or more) separated and purified from spinach was used.

<3-1> Phenolic acid High content Monogalactosyl diacyl glycerol  Selection of suitable enzymes for production

In this experiment, the molar ratio of monogalactosyl diacyl glycerol to the concentrate of phenolic acid was maintained at 1:40, and the molar ratio of monogalactosyl The content of phenolic acid in diacylglycerol was measured. Commercial immobilized lipases that evaluated the suitability of the reaction were Lipozyme RM IM, Lipozyme TL IM, Novozym 435 and Candida rugoselipase immobilized on Immobead 150 Four species were used. Among these lipases, lipozyme RM IM and lipozyme TL IM have sn-1, 3 position specificity of neutral fat. Novogym 435 and Candida lucosa lipase are lipases having no specificity in the sn position of neutral fat.

Specifically, 5 mg of monogalactosyldiacylglycerol and 74.7 mg of the condensation product of phenolic acid obtained in Example <1-3> were placed in a batch reactor (15 cm × 5 cm id), 4 mL of acetone and 4 mL of each lipase 15.9 mg of the enzyme (20% of the substrate weight) was added and stirred at a reaction temperature of 25 캜 at a speed of 500 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 to a minimum of 3 hours to 60 hours. The reaction product samples were taken at 0.2, 0.2, and 0.2 hours at reaction times 0, 3, 6, 12, 24, 36, 48, and 60 hours, respectively.

On the other hand, in order to measure the content of phenolic acid in the monogalactosyl diacyl glycerol in the reaction product according to the type of lipase, the reaction product produced by the enzyme reaction was separated using TLC (thin layer chromatography) And analyzed using gas chromatography.

The reaction products produced by the enzymatic reaction were separated by TLC. 2 mL of chloroform / methanol (1: 2, v / v) was added to 0.2 mL of the reaction product and the enzyme was removed by filtration through a 0.45 μm syringe filter (13 mm). The solvent was removed under a nitrogen stream and the residue was dissolved in 0.2 mL of chloroform / methanol (1: 2, v / v) and spotted on a TLC plate (silica gel 60 F254, Merck) to remove chloroform / methanol / water / ethyl acetate / 50: 20: 10: 50: 50, v / v / v / v / v) as a solvent. The monogalactosyl diacyl glycerol separated from the TLC plate was recovered by scrapping.

The fatty acid composition of the monogalactosyl diacyl glycerol was analyzed by gas chromatography after methyl esterification. The monogalactosyl diacyl glycerol recovered from the TLC plate and 1.5 mL of 0.5% NaOH in 95 vol% methanol were placed in a 50 mL tube, vortexed, and then boiled for 10 minutes in a 85 ° C water bath. After boiling, 2 mL of BF3 solution was added to the tube which had been sufficiently cooled at 20 DEG C, vortexed, and then the mixture was incubated at 85 DEG C for 10 minutes. After boiling, the tube was sufficiently cooled at 20 ° C, and 3 ml of isooctane and 5 ml of saturated NaCl solution were added to extract the methyl esterified fatty acid, followed by sufficient vortexing. The supernatant was collected and passed through anhydrous sodium sulfate to remove moisture and foreign substances. The isooctane layer thus obtained was analyzed by injecting it into a gas chromatograph (Agilent 7890A, Agilent Technologies Inc., Palo Alto, CA, USA). SP-2560 (100 m × 0.25 mm i.d. × 0.2 μm film thickness, Supelco, Bellefonte, PA, USA) was used as a column and detected using a flame ionization detector. The temperature of the column was maintained at 100 ° C for 4 minutes, then increased to 240 ° C at 3 ° C per minute and maintained for 17 minutes. The carrier gas was flowed at a flow rate of 1 mL per minute using helium. The temperatures of the injector and the detector were set at 225 ° C and 285 ° C, respectively. Each sample was analyzed by injecting 1 μL, and the split ratio was 5: 1.

The content of phenolic acid in the monogalactosyl diacyl glycerol in the reaction product was measured as a function of reaction time.

As a result, as shown in FIG. 2, acid decomposition reaction of monogalactosyl diacyl glycerol by phenolic acid occurred only when liposomes RM IM and liposomes TL IM were used as catalysts. In particular, Showed a significantly higher content of phenolic acid in the total reaction time versus the propylene TL IM. On the other hand, in the case of Novogym 435 and Candida lucosa lipase, acid decomposition reaction of monogalactosyl diacyl glycerol by phenolic acid did not occur.

As a result, the sn -1,3 -specific lipase was determined to be an enzyme suitable for producing monogalactosyldiacylglycerol having a high content of phenolic acid. In particular, lipozyme RM IM, which exhibited the highest activity, It was confirmed to be the most suitable enzyme for the production of monogalactosyldiacylglycerol.

In the following experiment, the medium suitable for the decomposition reaction of the liposome RM IM catalyst and the optimum reaction conditions were further investigated.

<3-2> Phenolic acid High content Monogalactosyl diacyl glycerol  Selection of media suitable for manufacturing

For the preparation of monogalactosyl diacyl glycerol with high content of phenolic acid, the liposomes RM IM, which has the highest reactivity suitability with commercial monogalactosyl diacyl glycerol and phenolic acid concentrates as substrates, Respectively.

In order to select a suitable solvent as the medium of the reaction, the molar ratio of the monogalactosyl diacyl glycerol to the phenolic acid concentrate was maintained at 1:40, and the amount of the monogalactosyl diacyl glycerol, And the content thereof was measured. The solvents used for evaluating the reaction suitability were acetone, toluene, benzene, and ethanol.

Specifically, 5 mg of monogalactosyl diacyl glycerol and 74.7 mg of the condensed phenolic acid obtained in Example <1-3> were placed in a batch reactor (15 cm × 5 cm id), and acetone, toluene, benzene, ethanol 4 mL of each and liposome RM IM 15.9 mg (20% of substrate weight) were added and stirred at a reaction temperature of 25 ° C at a speed of 500 rpm. The reaction time was set from a minimum of 12 hours to a maximum of 48 hours. Samples of the reaction products were taken at 0, 12, 24, 36 and 48 hours, respectively.

The content of phenolic acid in monogalactosyldiacyl glycerol in the reaction product was measured in the same manner as in Example <3-1>, and the content of monogalactosyl diacyl glycerol in the reaction product Is a function of reaction time.

As a result, as shown in Fig. 3, acid decomposition reaction of monogalactosyl diacyl glycerol with phenolic acid occurred only when acetone, toluene and benzene were used as a medium. Especially, when acetone was reacted with toluene or benzene And showed a remarkably superior content of phenolic acid. On the other hand, the acid decomposition reaction of monogalactosyl diacyl glycerol by phenolic acid did not occur in ethanol.

From the above results, it was confirmed that acetone was a suitable reaction medium for preparing monogalactosyl diacyl glycerol having a high content of phenolic acid.

< Example  4>

Phenolic acid High content Monogalactosyl diacyl glycerol  Optimum reaction conditions for production

It was confirmed through the above Example 3 that acetone was used as a reaction medium for preparing monogalactosyl diacyl glycerol having a high content of phenolic acid and that liposome RM IM as a lipase enzyme was effective as a catalyst. In addition, optimal reaction conditions for the preparation of monogalactosyl diacyl glycerol with high content of phenolic acid were analyzed.

<4-1> Phenolic acid High content Monogalactosyl diacyl glycerol  Optimal for manufacturing reaction Temperature

In this experiment, the molar ratio of monogalactosyl diacyl glycerol to the concentrate of phenolic acid was maintained at 1:40, and the amount of monogalactur The content of phenolic acid in the lactosyl diacyl glycerol was measured.

In detail, 5 mg of monogalactosyl diacyl glycerol and 74.7 mg of the condensed phenolic acid obtained in Example <1-3> were placed in a batch reactor (15 cm × 5 cm id), 4 mL of acetone and 15.9 mg of Lipozyme RM IM (20% of substrate weight) was added thereto, and the mixture was stirred at a reaction temperature of 25, 30, 35 and 40 ° C at 500 rpm, respectively. The reaction time was set to a minimum of 3 hours to 60 hours. The reaction product samples were taken at 0.2, 0.2, and 0.2 hours at reaction times 0, 3, 6, 12, 24, 36, 48, and 60 hours, respectively.

The content of phenolic acid in monogalactosyldiacylglycerol in the reaction product was measured in the same manner as in Example <3-1>, and the content of monogalactosyldiacylglycerol in the reaction product according to the reaction temperature was measured As a function of reaction time.

As a result, as shown in FIG. 4, the peak of reaction reached at a reaction time of 12 hours at all reaction temperature conditions examined, and there was no significant difference (p <0.05) in the content of phenolic acid according to the reaction temperature at all reaction times . The maximum content of phenolic acid by reaction temperature was 42.3 mol% at 25 ℃, 42.1 mol% at 30 ℃, 42.5 mol% at 35 ℃ and 42.8 mol% at 40 ℃.

Therefore, the optimal reaction temperature was chosen as 25 ℃ which is room temperature in terms of energy saving.

<4-2> Phenolic acid High content Monogalactosyl diacyl glycerol  Optimal interstratification for manufacturing Molar ratio

In this experiment, in order to determine the optimal molar ratio between the substrates of the liposome RM IM catalytic acid decomposition reaction, the molar ratio of the monogalactosyl diacyl glycerol to the phenolic acid concentrate The content of phenolic acid in the monogalactosyldiacyl glycerol was measured.

Specifically, the molar ratio of the monogalactosyl diacyl glycerol to the concentrate of the phenolic acid obtained in Example <1-3> was 1:10, 1:20, 1:30 or 1:40, 5 mg of silydiacyl glycerol and 18.7 mg, 37.3 mg, 56.0 mg or 74.7 mg of concentrated phenolic acid were placed in a batch reactor (15 cm x 5 cm id), and 4 mL of acetone and 4.7 mg, 8.5 mg, 12.2 mg or 15.9 mg of Lipozyme RM IM (20% of substrate weight), respectively, and stirred at a reaction temperature of 25 DEG C at 500 rpm. The reaction time was set to a minimum of 3 hours to 60 hours. The reaction product samples were taken at 0.2, 0.2, and 0.2 hours at reaction times 0, 3, 6, 12, 24, 36, 48, and 60 hours, respectively.

The content of phenolic acid in monogalactosyl diacyl glycerol in the reaction product was measured in the same manner as in Example <3-1>, and the content of monogalactosyl diacyl glycerol in the reaction product The content of phenolic acid in monogalactosyl diacyl glycerol was expressed as a function of reaction time.

As a result, as shown in FIG. 5, the content of pinoleic acid tended to increase with increasing molar ratio between the substrates at all reaction times. Especially after 36 hours of reaction time, molar ratios of 1:30 and 1:40 between substrates were significantly higher (p <0.05) than 1:10 or 1:20. After 6 hours of reaction time, there was no significant difference between the substrate molar ratios of 1:30 and 1:40 of phenolic acid content.

From the above results, '1:30 or more' was selected as the optimum molar ratio of the monogalactosyl diacyl glycerol and the concentrate of the phenolic acid.

<4-3> Phenolic acid High content Monogalactosyl diacyl glycerol  Optimal for Manufacturing Hyo handful

In this experiment, in order to set the optimal enzyme amount of the liposome RM IM catalytic acid decomposition reaction, the molar ratio of monogalactosyl diacyl glycerol to the concentrate of phenolic acid was maintained at 1:40 at a reaction temperature of 25 ° C, The content of phenolic acid in monogalactosyl diacyl glycerol in the reaction product was measured.

5 mg of monogalactosyl diacyl glycerol and 74.7 mg of the condensed phenolic acid obtained in Example <1-3> were placed in a batch reactor (15 cm × 5 cm id), 4 mL of acetone, 4.0 mg of liposome RM IM, 8.0 mg , 12.0 mg or 15.9 mg (5, 10, 15 or 20% of the substrate weight, respectively) were added thereto, followed by stirring at a reaction temperature of 25 ° C at 500 rpm. The reaction time was set to a minimum of 3 hours to 60 hours. The reaction product samples were taken at 0.2, 0.2, and 0.2 hours at reaction times 0, 3, 6, 12, 24, 36, 48, and 60 hours, respectively.

The content of phenolic acid in monogalactosyldiacyl glycerol in the reaction product was measured in the same manner as in Example <3-1>, and the content of phenolic acid in monogalactosyldiacyl glycerol in the reaction product according to the amount of enzyme was measured As a function of time.

As a result, as shown in FIG. 6, the content of the phenolic acid increased and the reaction peak reached a peak at the early stage of the reaction (3 to 24 hours) as the enzyme amount increased. Especially, the enzyme content of 15% and 20% was significantly higher (p <0.05) than that of 5% or 10% at 24 hours of reaction time. There was no significant difference between the contents of 15% and 20% of phenolic acid after 12 hours of reaction. Therefore, '15% or more' was selected as the optimum amount of enzyme.

< Example  5>

optimal Under the reaction conditions Phenolic acid High content Of monogalactosyl diacyl glycerol Produce

Using the optimal reaction temperature, molar ratio between the substrates and the amount of enzyme derived from Example 4, four reaction conditions were set as shown in Table 3 below, followed by acid decomposition reaction according to the method of Example 4, The content of phenolic acid in the monogalactosyldiacyl glycerol in the product was measured. The reaction time was set to a minimum of 3 hours to 60 hours. The reaction product samples were taken at 0.2, 0.2, and 0.2 hours at reaction times 0, 3, 6, 12, 24, 36, 48, and 60 hours, respectively.

The content of phenolic acid in monogalactosyldiacyl glycerol in the reaction product was measured in the same manner as in Example <3-1>.

Setting reaction conditions Condition Reaction temperature Molar ratio between substrates Amount of enzyme One 25 ℃ 1:30 15% 2 25 ℃ 1:40 15% 3 25 ℃ 1:30 20% 4 25 ℃ 1:40 20%

The content of phenolic acid in the monogalactosyl diacyl glycerol in the reaction product obtained under the above four reaction conditions was expressed as a function of reaction time.

As a result, as shown in Fig. 7, the reaction time and the maximum content of phenolic acid in the maximum phenolic acid content in each condition were 39.3 mol% in condition 1 for 60 hours; 48 hours at condition 2, 41.9 mol%; 36 hours in Condition 3, 42.1 mol%; The condition 4 was 36 hours, 42.3 mol%.

The reaction time for maximum content of phenolic acid by reaction conditions and the maximum content of phenolic acid Condition Reaction time Maximum phenolic acid content One 60 hours 39.3 mol% 2 48 hours 41.9 mol% 3 36 hours 42.1 mol% 4 36 hours 42.3 mol%

There was no significant difference between the maximum content of phenolic acid in Conditions 1 to 4. However, considering the production cost, it is desirable that the reaction time is short and the mole ratio between the substrates is low.

Thus, the optimum conditions for the decomposition reaction of the liposome RM IM catalyst for the production of the monogalactosyl diacyl glycerol having a high content of phenolic acid were determined at a reaction temperature of 25 ° C, an intermolecular molar ratio of 1:30 and an enzyme amount of 20% (condition 3) Respectively.

Analysis of fatty acid composition of monogalactosyl diacyl glycerol having high content of phenolic acid of the present invention produced through the above-mentioned condition 3 fatty acid (Mol%) of monogalactosyl diacyl glycerol with high content of phenolic acid 16: 0 8.0 ± 1.0 16: 3n-3 31.1 ± 0.8 18: 0 2.6 ± 0.1 18: 1n-9 1.4 ± 0.1 18: 1t - 18: 2n-6 7.3 ± 0.6 c5, c9, c12-18: 3 (phenolic acid) 42.1 ± 0.5 18: 3n-3 7.5 ± 0.3 18: 3t - 20: 0 - c5, c11, c14-20: 3 - Unidentified -

< Example  6>

Intermediate fatty acid High content Monogalactosyl diacyl glycerol  Produce

In this experiment, monogalactosyl diacyl glycerol and capric acid were used as substrates and lipozyme RM IM and acetone were used as catalysts and media for the production of monogalactosyl diacyl glycerol, Respectively. The conditions of the acid decomposition reaction were the same as the condition 4 of Example 5 (reaction temperature 25 ° C, intermolecular molar ratio 1:40, enzyme amount 20%).

Specifically, 5 mg of monogalactosyldiacyl glycerol and 45.0 mg of capric acid were placed in a batch reactor (15 cm × 5 cm id), and 4 mL of acetone and 10.0 mg of liposome RM IM (20% of the weight of the substrate) And stirred at a rate of 500 rpm at 25 캜. The reaction time was set from a minimum of 12 hours to 60 hours. Samples of the reaction products were taken at 0.2, 0.2, and 0.2 hours at reaction times 0, 12, 24, 36, 48, and 60 hours, respectively.

The capric acid content of the monogalactosyl diacyl glycerol in the reaction product was measured in the same manner as in the above Example <3-1>, and the content of the monogalactosyl diacyl glycerol in the reaction product of the liposome RM IM catalytic acid- The capric acid content of glycerol was expressed as a function of reaction time.

As a result, as shown in FIG. 8, the reaction peak reached 24 hours after the reaction time and the maximum content of capric acid was 41.2 mol%.

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)

i) preparing a concentrate of phenolic acid; And
And ii) adding phenolic acid concentrate and monogalactosyl diacyl glycerol to acetone and an enzyme, followed by a reaction, and then reacting the monogalactosyl diacyl glycerol with the phenolic acid. The method according to claim 1,
Wherein the concentrate of phenolic acid contains 30 to 90 mol% of phenolic acid. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt; The method according to claim 1,
Wherein the enzyme is a sn -1,3 -position-specific lipase. The method of claim 3,
Wherein the sn -1,3 -position specific lipase is Lipozyme RM IM or Lipozyme TL IM. [Claim 10] A method for producing monogalactosyl diacyl glycerol, The method according to claim 1,
(A) dissolving nuts oil fatty acid in a solvent and then adding and dissolving the component; (b) crystallizing the solution while maintaining the solution at room temperature at 4 ° C to 5 ° C, removing the crystallized product, and separating the uncrystallized filtrate; (c) removing the solvent from the separated filtrate, adding hydrochloric acid to the mixture, and then adding hexane to the mixture to extract the fatty acid. Glycerol. The method according to claim 1,
The step (ii) is a step in which a concentrate of a phenolic acid and a monogalactosyldiacyl glycerol as a substrate are constituted at a molar ratio of 1:30 to 1:40, 20% by weight of an enzyme is added to the substrate, Containing monogalactosyl diacyl glycerol is produced. 6. A monogalactosyl diacyl glycerol containing phenolic acid produced by the method according to any one of claims 1 to 6. 8. The method of claim 7,
A monogalactosyl diacyl glycerol containing phenolic acid characterized by containing pinoleic acid in a concentration of 30 to 50 mol%. A health supplement comprising the monogalactosyl diacyl glycerol containing the phenolic acid as claimed in claim 7 as an active ingredient. A method for producing monogalactosyldiacylglycerol containing an intermediate fatty acid, which comprises the step of adding acetone and an enzyme to a saturated fatty acid having 6 to 12 carbon atoms and monogalactosyldiacyl glycerol, followed by a reaction. 11. The method of claim 10,
Wherein the saturated fatty acid having 6 to 12 carbon atoms is capric acid. &Lt; RTI ID = 0.0 &gt; 8. &lt; / RTI &gt; 11. The method of claim 10,
Wherein the enzyme is a sn-1, 3- position specific lipase. 11. The method of claim 10,
Wherein said sn-1, 3- position specific lipase is Lipozyme RM IM or Lipozyme TL IM. 11. The method of claim 10,
Said saturated fatty acid having 6 to 12 carbon atoms and monogalactosyl diacyl glycerol are used as a substrate in a molar ratio of 1:30 to 1:40, and 20% by weight of enzyme is added to the substrate, followed by reaction at 25 ° C Wherein the fatty acid-containing monogalactosyl diacyl glycerol is at least one selected from the group consisting of glycerol, 11. The method of claim 10,
Wherein the intermediate fatty acid-containing monogalactosyl diacyl glycerol contains an intermediate fatty acid in a concentration of 30 to 50 mol%.

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