WO2012071768A1

WO2012071768A1 - Method for preparing high quality euphausia superba oil with phosphatidylserine enriched of polyunsaturated double bond fatty acyl group

Info

Publication number: WO2012071768A1
Application number: PCT/CN2011/000678
Authority: WO
Inventors: 张永志; 王秀兰; 沈连红; 梁水英; 吴萍; 谢春定
Original assignee: Zhang Yongzhi
Priority date: 2010-12-03
Filing date: 2011-04-18
Publication date: 2012-06-07
Also published as: CN102485899A

Abstract

Method for preparing high quality Euphausia superba oil with phosphatidylserine enriched of polyunsaturated double bond fatty acyl group, which includes the following steps: 1) solvent extracting Euphausia superba oil to obtain phosphatidylcholine and oils containing vitamin A, vitamin E and astaxanthin; 2) preparing aqueous phase enzyme catalyzing reaction system; 3) adding said reaction system into reactor, stirring after adding phosphatidase D continuously; 4) extracting resulting reaction liquid repeatedly, filtering and freeze drying resulting solid to obtain phosphatidylserine enriched of polyunsaturated double bond fatty acyl group; 5) mixing said phosphatidylserine enriched of polyunsaturated double bond fatty acyl group with oils of step 1) to obtain high quality Euphausia superba oil.

Description

Preparation of phosphatidylserine rich in polyunsaturated double bond fatty acyl group

High quality Antarctic krill oil method

Technical field:

The invention relates to a method for preparing high quality Antarctic krill oil rich in polyunsaturated double bond fatty acyl phosphatidylserine, in particular to enzymatic catalysis of phosphatidylcholine in Antarctic krill A method of preparing a phosphatidylserine rich in a polyunsaturated double bond fatty acyl group and compounding it to obtain a high quality Antarctic krill oil is prepared.

Background technique:

In the prior art, there are various methods for preparing phosphatidylserine, and the extraction method and the enzyme conversion method are mainly used. The extraction method is to extract phosphatidylserine from animal phospholipids, mainly from the brain of animals. Patent CN1583766A extracts phosphatidylserine from animal brain. However, due to reasons such as mad cow disease, the safety of its products has been suspected and has been eliminated. In recent years, the preparation of phosphatidylserine by soy lecithin as a raw material has been widely used, for example, the patent CN101230365A, but the product formed by this patent does not contain the phosphatidylserine of the polyunsaturated double bond long chain fatty acyl group. Patent CN101818179A synthesizes phosphatidylserine containing polyunsaturated double bond fatty acyl group from common lecithin by two-step enzymatic method. The phospholipid in common lecithin does not contain Omega-3 polyunsaturated fatty acyl group. The reaction is to use phospholipase Al, A2 or lipase to catalyze the phosphatidylcholine containing a polyunsaturated double bond fatty acyl group in a fatty acid containing a polyunsaturated double bond and a phosphatidylcholine in lecithin, followed by phospholipase D catalysis. The reaction of serine with phosphatidylcholine containing a polyunsaturated double bond fatty acyl group to finally form a phosphatidylserine containing a polyunsaturated double bond fatty acyl group, the invention has the following disadvantages: Phospholipids and polyunsaturated fatty acids are complex mixtures, and the effective content of phosphatidylcholine and polyunsaturated fatty acids is not high. In the case of not highly concentrated, the effective content of these two raw materials generally does not exceed 50%, and the probability of producing a phosphatidylcholine containing a polyunsaturated double bond fatty acyl group at the SN2 position of the target product catalyzed by phospholipase A2 or a lipase specific to the SN2 position is low, resulting in a final product. The concentration of the phosphatidylserine containing the polyunsaturated double bond fatty acyl group is lower, which is not conducive to the quality of the final product. If the active ingredient concentration of the phosphatidylserine of the polyunsaturated double bond fatty acyl group in the product is to be increased, it will be severe. Increase production costs. The patent CN100402656C is based on the phospholipid extracted from the liver of the fish. The patent CN101157946A extracts the phospholipid from the carp and uses the phospholipase D enzyme method to catalyze the phosphatidylcholine in the liver of the fish liver or the phosphatidylcholine in the carp. Preparation of phosphatidylserine rich in polyunsaturated double bond fatty acyl group by L-serine reaction, wherein phosphatidylcholine rich in polyunsaturated double bond fatty acyl group in fish liver phospholipid and salmon phospholipid is prepared with polyunsaturated double It is an excellent raw material for the phosphatidylserine of the fatty acyl group, but these two raw materials have limited sources, which is not conducive to large-scale production. After years of research and exploration, the present inventors have found that phospholipid compounds in Antarctic krill or Antarctic krill oil contain extremely abundant eicosapentaenoic acid EPA and docosahexaenoic acid DHA, which is Omega-3. A rich source of saturated fatty acids, these Omega-3 polyunsaturated fatty acids are mainly bound to the Sn2 position of the phospholipid compound molecule, forming a polyphosphoric acid-rich phospholipid at the Sn2 position, which is rich in polyunsaturated fatty acyl groups. Acylcholine accounts for about 30-40% of the total phospholipid content of Antarctic krill oil, while the content of phosphatidylserine in the total phospholipid of Antarctic krill is very low, about 1% to 3% of the phospholipid content. Therefore, the conversion of phosphatidylcholine rich in polyunsaturated fatty acyl groups in Antarctic krill into phosphatidylserine rich in polyunsaturated fatty acyl groups is a very significant subject.

Summary of the invention:

The object of the present invention is to provide an existing Antarctic krill in view of the deficiencies of the prior art. High-quality Antarctic krill oil process for the preparation of phosphatidylserine rich in polyunsaturated double bond fatty acyl phosphatidylserine.

It is also an object of the present invention to provide a process for preparing a phosphatidylcholine rich in polyunsaturated double bond fatty acyl groups from existing Antarctic krill oil.

It is also an object of the present invention to provide a process for preparing a phosphatidylserine rich in a polyunsaturated double bond fatty acyl group.

The invention of the invention is achieved by the following measures:

A method for preparing a high quality Antarctic krill oil rich in polyunsaturated double bond fatty acyl phosphatidylserine, which is characterized in that the preparation is carried out as follows:

Step one, solvent extraction of existing Antarctic krill oil to obtain phosphatidylcholine and containing vitamins

A, vitamin E and astaxanthin oil;

Step 2, preparing an enzyme-catalyzed reaction system, the reaction system is an aqueous phase reaction system; Step 3, adding the reaction system of the second step to the reactor, in a nitrogen atmosphere, at a temperature of 35-55 degrees Celsius, and inputting the phospholipid plum D After continuous mechanical stirring, stirring time is 1~15 hours;

Step 4, the reaction liquid obtained in the third step is repeatedly extracted 2~3 times, and after filtration, the solid is freeze-dried to obtain a phosphatidylserine rich in a polyunsaturated double bond fatty acyl group;

In the fifth step, the phosphatidylserine rich in polyunsaturated double bond fatty acyl group obtained in the fourth step is mixed with the oil containing vitamin A, vitamin E and astaxanthin obtained in the first step to obtain high quality Antarctic krill oil, and the preparation is completed.

In the first step, the existing Antarctic krill oil is mixed with the solvent in the reactor, and then subjected to mechanical stirring in a nitrogen atmosphere at 0 to 30 degrees Celsius for 0.5 to 15 hours. After filtration, the solid is freeze-dried to be rich. A phosphatidylcholine of a polyunsaturated double bond fatty acyl group. The phosphatidylcholine obtained in the first step is a phosphatidylcholine family rich in polyunsaturated double bond fatty acyl groups, and the phosphatidylcholine family contains a dozen series of phosphatidylcholine molecules, each of which is a series of molecules. The chemical structures are the same and different: (1) a choline is attached to the 3-position of the glyceryl group, that is, Sn-3; (2) each is attached to the 1-position Sn-1 and the 2-position Sn-2 of the glyceryl group. a fatty acyl group, and a long-chain fatty acyl group containing a polyunsaturated double bond, which is rich in a polyunsaturated double bond fatty acyl group, which is a fat linked at the Sn-2 position, is usually attached to the 2-position Sn-2. More than 5% of the fatty acyl groups in the acyl group contain two or more unsaturated double bonds, or a fatty acyl group having a higher than 3% or more of the fatty acyl group attached at the Sn-2 position, containing an Omega-3 unsaturated double (3) The unsaturated double bond fatty acyl group contains 18 to 22 carbon atoms, and the number of double bonds is usually three to six. The unsaturated double bond fatty acyl group is an Omega-3 unsaturated fatty acid family. Refers to a docosahexaenoic acid fatty acyl group and a twenty carbon five acid fatty acyl group.

In the second step, the aqueous phase reaction system is an aqueous solution reaction system prepared by phosphatidylcholine rich in polyunsaturated double bond fatty acyl group, L-serine, calcium salt and a buffer solution, and the mixture of the substances in the aqueous solution reaction system The ratio is as follows: The weight percentage of phosphatidylcholine added per liter of the reaction solution is 100, and the weight percentage of L-serine added per liter of the reaction solution is 60 to 300, and the weight of the calcium salt added per liter of the reaction solution The percentage is 0.05 to 10, the concentration of the buffer salt in the buffer solution is 0 to 2 mol/liter, and the pH of the aqueous solution is in the range of 5.0 to 8.5.

Another object of the present invention is a method for preparing a phosphatidylcholine rich in a polyunsaturated double bond fatty acyl group from an existing Antarctic krill oil, the preparation step being: solvent extraction of an existing Antarctic krill oil; The solvent may be optionally selected from the group consisting of food safety grade n-heptane or n-hexane, edible ethanol or acetone, or a mixture thereof; the extraction is after the existing Antarctic krill oil is mixed with the solvent in the reactor. In a nitrogen atmosphere, at 0~30 degrees Celsius, mechanically stirred, reacted for 0.5 to 15 hours, filtered to solid Lyophilized to obtain a phosphatidylcholine rich in a polyunsaturated double bond fatty acyl group.

A third object of the present invention is a process for preparing a phosphatidylserine rich in a polyunsaturated double bond fatty acyl group, which is prepared by the following steps:

Step one, solvent extraction of existing Antarctic krill oil or direct purchase of phosphatidylcholine from the market; the extraction is carried out according to the following steps: The extraction solvent can be selected among the following items: food safety grade n-heptane or positive Alkane, edible ethanol or acetone, or a mixture thereof; the extraction is that the existing Antarctic krill oil is mixed with the solvent in a reactor, and then subjected to mechanical stirring in a nitrogen atmosphere at 0 to 30 degrees Celsius, and the reaction is 0.5- After 15 hours, after filtration, the solid was freeze-dried to obtain a phosphatidylcholine rich in polyunsaturated double bond fatty acyl;

Step two, preparing an enzyme catalytic reaction system, comprising an aqueous phase reaction system or a two-phase reaction system composed of an aqueous phase and an organic solvent;

Step 3, the second step of the aqueous solution reaction system is added to the reactor, in a nitrogen atmosphere, the temperature is 35-55 degrees Celsius, after the phospholipid plum D is continuously mechanically stirred, after the holding time of 1 to 15 hours, the resulting reaction liquid The extraction was repeated 2 to 3 times, and after filtration, the solid was freeze-dried to obtain a phosphatidylserine rich in a polyunsaturated double bond fatty acyl group, and the preparation was completed.

The aqueous phase reaction system is a phosphatidylcholine-enriched enzyme-catalyzed reaction system prepared from an existing Antarctic krill oil, which is rich in polyunsaturated double bond fatty acyl groups, and is composed of a polyphosphoryl amide group rich in polyunsaturated double bond fatty acyl groups. An aqueous solution reaction system prepared by using a base, an L-serine, a calcium salt and a buffer solution, wherein the ratio of each substance in the aqueous reaction system is as follows: the weight percentage of phosphatidylcholine added per liter of the reaction solution is 100, L - the weight percentage of serine added per liter of the reaction solution is 60 to 300, the weight percentage of the calcium salt added per liter of the reaction solution is 0.05 to 10, and the concentration of the buffer salt in the buffer solution is 0 to 2 moles per liter. The pH of the aqueous solution is in the range of 5.0 to 8.5. The two-phase reaction system composed of the aqueous phase and the organic solvent phase is a phosphatidylcholine-formulated enzyme catalytic reaction system commercially available in the first step, which is a solution of a phosphatidylcholine rich in a polyunsaturated double bond fatty acyl group. The C5-C9 terpene hydrocarbon organic solvent is added to an aqueous solution containing L-serine, a calcium salt, a short-chain hydrocarbon or a short-chain hydrocarbon ketone and a buffer solution to form a mixed phase of the aqueous phase and the organic solvent. In the reaction system, the ratio of each substance in the mixed reaction system of the aqueous solution and the organic solvent is as follows: The phosphatidylcholine is added in a weight percentage of 100 per liter of the mixed reaction solution, and the L-serine is in the reaction liquid per liter. The weight percentage added is 60~300, the weight percentage of the calcium salt added in each liter of the reaction liquid is 0.05~10, and the concentration of the buffer salt in the buffer solution is 0~2 mol/liter, and the C5-C9 hydrazine hydrocarbon organic solvent is The volume fraction of the mixed reaction solution is 5% to 95%, and the short-chain hydrocarbon or short-chain hydrocarbon ketone accounts for 1% to 80% by volume of the total mixed reaction solution, and the mixed reaction system of the aqueous solution and the organic solvent is mixed. pH value is within 5.0~8.5 .

The C5-C8 alkane organic solvent is any one of the following: pentane, hexane, cyclohexanthene, heptane, octone, decane, hydrazine or any of their isomers. The solvent or a mixture of these solvents is formed in any ratio to form a mixed solvent.

The short-chain hydrocarbon or short-chain hydrocarbon ketone is any one of the following: ethanol, propanol, butanol, pentanol, acetone, methyl ethyl ketone or any of their isomers or these The solvent is mixed in any ratio to form a mixed solvent.

Compared with the prior art, the present invention has the following advantages: 1) The first advantage of the present invention is to utilize phosphatidylcholine or Antarctic krill oil rich in polyunsaturated double bond fatty acyl derived from Antarctic krill oil. The phosphatidylserine rich in polyunsaturated double bond fatty acyl is prepared by itself using a phospholipase D biotransformation method as a reactant. The raw materials of the product are easy to obtain and the reaction is simple and practical. 2) The second advantage of the present invention is the phosphatidylserine of the polyunsaturated double bond fatty acyl group and the Antarctic krill oil or the extraction of phosphatidylcholine The latter Antarctic krill oil is mixed to increase the content of the phosphatidylserine of the polyunsaturated double bond fatty acyl group in the Antarctic krill oil product. The resulting product can be used in premium nutrition and health supplements, as well as in food, pharmaceuticals or cosmetics.

BRIEF DESCRIPTION OF THE DRAWINGS:

Figure 1 shows the structural differences between the phosphatidylcholine of Antarctic krill and the phosphatidylserine formed by transformation.

Fig. 2 is a schematic diagram showing the reaction of producing phosphatidylserine from phosphatidylcholine under the catalysis of phospholipase D.

For a better understanding of the contents of the present invention, first, FIG. 1 is illustrated. In the figure, Sn-1, Sn-2 and Sn-3 respectively guide the 1st and 2nd positions of the glyceride group in the phospholipid in the krill. And 3 positions; R1 and R2 are long carbon chain fatty acyl groups, and a R1 at the Sn-1 position is common: a C ₁₅ H ₃₁ , a C ₁₇ H ₃₅ , a C ₁₇ H ₃₃ , if it is a C ₁₅ H ₃₁ , indicating that R1 is derived from hexadecane carbonate or palmitic acid, and a carbonyl group to which C ₁₅ H ₃₁ is attached constitutes a hexadecane fatty acyl group, indicating that at the Sn-1 position is a 16-1 carbonic acid and glycerol at the Sn-1 position. An ester formed by an alcohol; if it is a C ₁₇ H ₃₅ , it means that R1 is derived from octadecyl carbonate or stearic acid, and a carbonyl group to which C ₁₇ H ₃₅ is attached constitutes an octadecyl fatty acyl group, which is represented by Sn-1. The position is an ester of octadecyl carbonate and glycerol at the Sn-1 position; if it is a C ₁₇ H ₃₃ , it means that R1 is derived from octadecanoic acid or oleic acid, and a C ₁₇ H _{33 is} bonded to the carbonyl group. An octadecyl fatty acyl group, which indicates an ester of an 18-carbonic acid at the Sn-1 position with an alcohol at the Sn-1 position of glycerol. A common R2 at the Sn-2 position is: -C ₁₇ H ₃₁ , a C ₁₉ H ₂₉ , _C ₁₉ H ₃₁ , - C ₂₁ H ₃₁ , - C ₁₇ H ₃₃ , if it is a C ₁₇ H ₃₁ , It is indicated that R2 is derived from octadecane dibasic acid or linoleic acid, and one ( ₁₇ 13⁄4 ₁ attached to the carbonyl group constitutes an octadecyl di-fatty fatty acyl group, indicating that it is an 18-carbon diacid at the Sn-2 position. An ester formed from the alcohol at the Sn-2 position of glycerol; if it is a C ₁₉ H ₂₉ , it means that R 2 is derived from eicosapentaic acid or EPA, and a C ₁₉ H ₂₉ is attached thereto. a carbonyl group constituting a twenty-carbon five-fat fatty acyl group, which represents an ester of a sesquicarbon acid at the Sn-2 position with an alcohol of the Sn-2 position of glycerol; if it is a C ₁₉ H ₃₁ , DESCRIPTION R2 from arachidonic dilute or arachidonic acid, -C ₁₉ H ₃₁ carbonyl group connected thereto constitute a dilute fatty acyl eicosatetraenoic, Sn-2 represents the bit is a dilute acid eicosatetraenoic An ester formed with the alcohol at the Sn-2 position of glycerol; if it is a C ₂₁ H ₃₁ , it means that R 2 is derived from docosahexaenoic acid, and a carbonyl group to which C ₂₁ H ₃₁ is attached constitutes a twenty-two carbon Hexa-fatty fatty acyl group, which means an ester of a sesquicarbon acid at the Sn-2 position with a Sn-2 position alcohol of glycerol; if it is a C ₁₇ H ₃₃ , it means that R 2 is derived from octadecanoic acid. Or oleic acid, a carbonyl group to which C ₁₇ H ₃₃ is attached constitutes an octadecyl fatty acyl group, which means an ester of an 18-carbon acid at the Sn-2 position with an alcohol of the Sn-2 position of glycerol. -X is a different linking group at the Sn-3 position, and if it is a CH ₂ CH ₂ N(CH ₃ ) ₃ , it means that the phospholipid compound is phosphatidylcholine; if it is a CH ₂ CH (NH ₂ ) COOH, the phospholipid compound is phosphatidylserine.

Figure 2 shows a reaction scheme for the production of phosphatidylserine from phosphatidylcholine catalyzed by phospholipase D. Under the reaction conditions disclosed in the present invention, phospholipase D can catalyze the production of phosphatidylcholine by phosphatidylcholine. The molecular formula means that a molecule of phosphatidylcholine can react with a molecule of L-serine to form a molecule. The phosphatidylserine simultaneously produces a molecule of choline.

detailed description:

A method for preparing a high quality Antarctic krill oil rich in polyunsaturated double bond fatty acyl phosphatidylserine, which is prepared by the following steps:

In the first step, the existing Antarctic krill oil is solvent-extracted to obtain phosphatidylcholine and the filtrate is degreased. The solvent may be selected from the group consisting of: food safety grade n-glycol or n-hexane, edible ethanol or acetone, or a mixture thereof. The extraction is an Antarctic krill oil mixed with a solvent in a reactor, in a nitrogen atmosphere at 0 to 30 degrees Celsius, mechanically stirred, reacted for 0.5 to 15 hours, and filtered. The solid was freeze dried to give phosphatidylcholine. The obtained phosphatidylcholine is a phospholipid group rich in a polyunsaturated double bond fatty acyl group, and the phosphatidylcholine group contains a dozen series of phosphatidylcholine molecules, wherein the chemical structure of each molecular series has the same And different: (1) a choline is attached to the 3 position of the glyceryl group, that is, Sn-3; (2) a fatty acyl group is attached to each of the sylylene group at the 1-position Sn-Ι and the 2-position Sn-2, and The 2-position Sn-2 is usually bonded to a long-chain fatty acyl group containing a polyunsaturated double bond, which is rich in a polyunsaturated double bond fatty acyl group, which means that the fatty acyl group attached at the Sn-2 position is higher than 5 More than or equal to the fatty acyl group contains two or more unsaturated double bonds, or a fatty acyl group having a higher than 3% or more of the fatty acyl group linked to the Sn-2 position, which contains an Omega-3 unsaturated double bond; (3) The unsaturated double bond fatty acyl group has 18 to 22 carbon atoms, and the number of double bonds is usually three to six. The unsaturated double bond fatty acyl group belongs to the Omega-3 unsaturated fatty acid family. Dilute fatty acyl and eicosocene fatty acid acyl.

Step two, preparing an enzyme-catalyzed reaction system. The reaction system is an aqueous phase reaction system prepared from an existing Antarctic krill oil, which is rich in polyunsaturated double bond fatty acyl phosphatidylcholine, which is rich in polyunsaturated double bond fatty acyl group. An aqueous solution reaction system prepared by phosphatidylcholine, L-serine, calcium salt and a buffer solution, wherein the ratio of each substance in the aqueous reaction system is as follows: Weight percentage of phosphatidylcholine added per liter of reaction solution 100, L-serine is added in a weight percentage of 60 to 300 per liter of the reaction solution, the weight percentage of the calcium salt added per liter of the reaction solution is 0.05 to 10, and the concentration of the buffer salt in the buffer solution is 0 to 2 The molar value of the aqueous solution is in the range of 5.0 to 8.5.

In this step, L-serine: commercialized biological product; calcium salt: is a soluble calcium salt, and calcium chloride and calcium sulfate are used in this embodiment, and the concentration in water may be 0.1 gram per liter to 20 gram per liter. Buffer: It is a conventional water buffer such as phosphate buffer or acetate buffer, usually 0 to 0.2 mole per liter of salt concentration system. Step three, adding the reaction system of step two to the reactor, in a nitrogen atmosphere, the temperature is

35~55 degrees Celsius, after the input of phospholipid plum D, mechanical stirring is continued, the stirring speed is lOO rpm, and the stirring time is 2~15 hours.

In this step, phospholipase D: is an enzyme derived from a commercially available enzyme preparation of Streptomyces and/or plants, such as phospholipase D sold by Sigma-Aldrich (St. Louis, MO, USA). ), the enzyme can also be purified from the fermentation broth of Streptomyces.

Step 4, the reaction solution obtained in the third step is repeatedly extracted 2 to 3 times, and after filtration, the solid is freeze-dried to obtain a phosphatidylserine rich in a polyunsaturated double bond fatty acyl group. The extraction solvent and extraction method in this step are the same as in the first step.

In the fifth step, the phosphatidylserine rich in polyunsaturated double bond fatty acyl group obtained in the fourth step is mixed with the oil after removing the solvent in the step-filter to obtain high-quality Antarctic krill oil, and the preparation is completed.

The phosphatidylserine content of the high quality Antarctic krill oil prepared according to this method can be higher than 40%.

Another object of the present invention is a method for preparing a phosphatidylcholine rich in a polyunsaturated double bond fatty acyl group from an existing Antarctic krill oil, the preparation step being: solvent extraction of an existing Antarctic krill oil; The solvent may be optionally selected from the group consisting of food safety grade n-glycan or ruthenium, edible ethanol or acetone, or a mixture thereof; the extraction is after the existing Antarctic krill oil is mixed with the solvent in the reactor. The reaction was carried out for 0.5 to 15 hours under mechanical stirring at 0 to 30 ° C in a nitrogen atmosphere. After filtration, the solid was freeze-dried to obtain a phosphatidylcholine rich in a polyunsaturated double bond fatty acyl group.

A third object of the present invention is a process for preparing a phosphatidylserine rich in a polyunsaturated double bond fatty acyl group, which is prepared by the following steps

Step one, and preparing a phosphatidylcholine rich in polyunsaturated double bond fatty acyl groups from existing Antarctic krill oil The method of alkali is the same;

Step two, preparing an enzyme catalytic reaction system, comprising an aqueous phase reaction system or a two-phase reaction system composed of an aqueous phase and an organic solvent phase; the aqueous phase reaction system is directly rich by the existing Antarctic krill oil Formulation of the polyunsaturated double bond fatty acyl phosphatidylcholine, which is the same as step 2 of the method for preparing a high quality Antarctic krill oil rich in polyunsaturated double bond fatty acyl phosphatidylserine, except that The following measures can be taken: the two-phase reaction system in which the aqueous phase is mixed with an organic solvent is prepared from a commercially available phosphatidylcholine, which is first dissolved in a C5-C9 alkane organic solvent by phosphatidylcholine, and then added to a two-phase mixed reaction system containing an aqueous solution of an L-serine, a calcium salt, a short-chain hydrocarbon or a short-chain hydrocarbon ketone and a buffer solution, and an organic solvent, and a ratio of each substance in the two-phase mixed reaction system of the aqueous solution and the organic solvent The relationship is as follows: The weight percentage of phosphatidylcholine added per liter of the mixed reaction solution is 100, and the weight percentage of L-serine added per liter of the reaction liquid is 60 to 300, and the calcium salt is per The weight percentage added to the reaction solution is 0.05 to 10, the concentration of the buffer salt in the buffer solution is 0 to 2 mol/liter, and the volume percentage of the C5-C9 hydrazine hydrocarbon organic solvent in the mixed reaction solution is 5% to 95%. The short-chain hydrocarbon or short-chain hydrocarbon ketone accounts for 1%-80% by volume in the total mixed reaction solution, and the pH of the mixed reaction system of the aqueous solution and the organic solvent is within 5.0-8.5.

The C5-C8 alkane organic solvent is any one of the following: pentane, hexane, cyclohexane, heptane, octane, hydrazine, hydrazine or any of their isomers. The solvent or a mixture of these solvents is formed in any ratio to form a mixed solvent. The short-chain hydrocarbon or short-chain hydrocarbon ketone is any solvent selected from the group consisting of: ethanol, propanol, butanol, pentanol, acetone, butanone or an isomer thereof or these The solvent is mixed in any ratio to form a mixed solvent.

Step 3, the reaction system of the second step is added to the reactor, and the temperature is 35-55 degrees Celsius in a nitrogen atmosphere, and the mechanical stirring is continued after the phospholipid D is put into the reactor, and the holding time is 1 to 15 hours. The obtained reaction solution was repeatedly extracted 2 to 3 times, and after filtration, the solid was freeze-dried to obtain a phosphatidylserine rich in a polyunsaturated double bond fatty acyl group, and the preparation was completed.

The present invention employs the following analysis conditions:

The method for analyzing the relative content of polyunsaturated fatty acids is described in the method described in CN101818178A, and the specifics are as follows:

The sample was dissolved in an appropriate amount of chloroform, and then centrifuged at 6000 rpm for 20 min, and the organic phase layer was prepared into a 20-100 mg/mL sample chloroform solution, and the organic phase was spotted on a thin-wave layer silica gel plate, and then developed through a developing agent, and the developing agent was: Chloroform: Water: Methanol = 63.5: 3.5: 25 (V: V: V), developed with 2, 7,-dichlorofluorescein, and then banded with silica gel to remove the band of phosphatidylserine, methyl esterification After gas chromatography (GC) analysis, the relative content of various fatty acids was calculated by the area normalization method.

Chromatographic conditions are - Instruments: Agilent 6890 Gas Chromatograph

Detector: Hydrogen Flame Ion Detector (FID)

Capillary column: FFAP30.0mx2.5mmx0.10

Inlet temperature: 220 °C; column temperature: 200 ° C _; detector temperature: 250 ° C _;

Injection volume: l L; injection split ratio: 20: 1; nitrogen flow rate: 0.6 mL/min

The analysis method of phosphatidylcholine and phosphatidylserine in the sample is as follows:

Phosphatidylcholine and phosphatidylserine standards were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.) and the remaining reagents were of liquid chromatography purity grade.

Phosphatidylcholine standard curve mapping:

300mg phosphatidylcholine standard was dissolved in 3mL chloroform solvent to prepare 5mg/mL, lOmg/mL, 20mg/mL, 40mg/mL, 80mg/mL, 100mg/mL phosphatidylcholine chloroform to be tested For the sample, the above series of samples were analyzed by high-phase liquid chromatography (HPLC) to draw a phosphatidylcholine mass-integrated area standard curve.

Determination of phosphatidylcholine content in the sample to be tested:

The sample to be tested is dissolved in an appropriate amount of chloroform, and then centrifuged at 6000 rpm for 20 min, and the organic phase layer is prepared into a sample chloroform solution of 20-40 mg/mL, and the sample is analyzed by high-phase liquid chromatography (HPLC), according to the test The integrated area of the phosphatidylcholine peak in the sample, and the absolute content of phosphatidylcholine in the sample to be tested is calculated using the above-described phosphatidylcholine mass-integrated area standard curve. The specific calculation method is -

PC%=PC quality / sample quality χΐοο%

Phosphatidylserine standard curve mapping:

The 300 mg phosphatidylserine standard was dissolved in 3 mL of chloroform solvent to prepare 5 mg/mL, 10 mg/mL, 20 mg/mL, 40 mg/mL, 80 mg/mL, 100 mg/mL phosphatidylserine chloroform sample to be tested. The above series of samples were analyzed by high-phase liquid chromatography (HPLC) to draw a phosphatidylserine mass-integral area standard curve.

Determination of phosphatidylserine content in the sample to be tested:

The sample to be tested is dissolved in an appropriate amount of chloroform, and then centrifuged at 6000 rpm for 20 min, and the organic phase layer is prepared into a sample chloroform solution of 20-40 mg/mL, and the sample is analyzed by high-phase liquid chromatography (HPLC), according to The integrated area of the phosphatidylserine peak in the sample to be tested, and the absolute content of phosphatidylserine in the sample to be tested is calculated using the above-described phosphatidylserine mass-integration area standard curve. The specific calculation method is:

Phosphatidylserine % = phosphatidylserine mass / sample quality χΐοο% Chromatographic conditions are:

Instrument: Agilent 1200 High Performance Liquid Chromatograph; Detector: G1314B ultraviolet detector; detection wavelength: 205nm;

Analytical column: Kromasil, KR100-5SIL, (250mm x 4.6mm); column temperature: 30 ° C;

Mobile phase: acetonitrile: methanol: 85% phosphoric acid = 900: 95: 5 (V: V: V); flow rate: l.OmL / min The following is a detailed description of the preferred embodiment of the invention in a specific application, these examples It is not intended to limit the scope of the invention.

Example 1 : Extraction of phosphatidylcholine rich in polyunsaturated double bond fatty acyl groups.

Weigh 20 kg of Antarctic krill oil, which contains: EPA of 19.3%, DHA of 11.5%, PC content of 36.3%, phosphatidylserine content of 1.5%, 40 liters of acetone, adding the above two raw materials to 100 In the liter reactor, the reactor is filled with nitrogen gas, at zero degrees Celsius, mechanical stirring is continued, 80 rpm, the reaction is carried out for 2 to 15 hours, the reaction is terminated, filtered, and the solid is freeze-dried to obtain a polyunsaturated double bond fatty acyl group. 13 kg of phosphatidylcholine, of which: EPA was 18.3%, DHA was 8.1%, PC content was 47.8%, phosphatidylserine content was 2.4%, and the filtrate was concentrated under reduced pressure at 20 ° C to remove the solvent to obtain 7 kg. Reddish brown grease.

Example 2: Refined Antarctic Krill Oil Rich in Phosphatidylserine

Weigh 10 kg of Antarctic krill oil, which contains: EPA of 19.3%, DHA of 11.5%, PC content of 36.3%, and phosphatidylserine content of 1.5 °/. 4 to 6 kg of L-serine, 0.1 to 0.8 kg of calcium chloride, 20 to 100 liters of water buffer in the range of pH 5.5 to pH 8.5, added to a 150 liter reactor, and placed under nitrogen protection 0.1 to 1 liter of phospholipase D (about 20,000 to 500,000 units), in the range of 45-53 degrees Celsius, continuous mechanical stirring, 100 rpm, reaction 2 to 15 hours to stop stirring, end the reaction, add acetone 10 to 50 kg , add 10 to 50 kg of n-gum, continue mechanical stirring, 30 rpm, stop stirring after 0.5 hours, let stand for stratification, take the organic phase, repeat according to the above method The extract was extracted 2 to 3 times, and the organic extract was collected and concentrated under reduced pressure at 20 ° C to obtain 9.5 kg of phosphatidylserine-rich Antarctic krill oil. The content of unsaturated fatty acids in the phospholipid was 26.7% by GC, of which EPA was 17.2%, DHA was 9.5%, and the PC content of phospholipids was 2.7% by HPLC and 31.8% by phosphatidylserine.

Example 3: Phosphatidylserine rich in polyunsaturated double bond fatty acyl

10 kg of Antarctic krill phosphatidylcholine prepared in Example 1 was weighed, wherein the phospholipid had an EPA of 18.3%, a DHA of 8.1%, a PC content of 47.8%, a phosphatidylserine content of 2.4%, and 12 to 20 kg. L-serine, 0.4-1.5 kg of calcium chloride, 50 liters to 120 liters of water buffer in the range of pH 5.5 to pH 8.5, added to a 150 liter reactor, and 0.1 to 2 liters of phospholipid under nitrogen protection Enzyme D 1 million units, in the range of 45-53 degrees Celsius, continuous mechanical stirring, lOO rpm, reaction 2 to 15 hours, end the reaction, add 10 to 60 kg of ethanol, 10 to 60 kg of hexane, continuous mechanical stirring, 30 rpm After 0.5 hours, the stirring was stopped, the layer was allowed to stand, and the organic phase was taken. The extraction was repeated 2 to 4 times according to the method described above, and the organic extracts were collected and concentrated under reduced pressure at 20 ° C to obtain a fatty acid rich in polyunsaturated double bonds. Crude phosphatidylserine crude, add acetone 10 to 50 kg, continue mechanical stirring, 80 rpm, stop stirring after 0.5 hours, filter, freeze solid to obtain 8.6 kg of polyunsaturated double bond fat Phosphatidylserine quality group which PC content 5.5% by HPLC, phosphatidylserine content of 43.9% by HPLC, phospholipid EPA detected by GC was 17.5%, DHA was 7.9%.

Example 4: Phosphatidylserine rich in polyunsaturated double bond fatty acyl

The phosphatidylcholine prepared in Example 1 was 3 kg, which contained: phospholipid with EPA of 18.3%, DHA of 8.1%, PC content of 47.8%, phosphatidylserine content of 2.4%, and 4 to 10 kg of L-serine. , 0.2 to 0.8 kg of calcium chloride, 20 to 40 liters of water buffer in the range of pH 5.5 to pH 8.5 Add the liquid to the 100 liter reactor, fill the reactor with nitrogen, and add 0.1 to 1 liter of phospholipase D from about 20,000 to 500,000 units in the range of 45-53 degrees Celsius, mechanical stirring, lOO rpm, reaction. 2 to 15 hours, the reaction is completed, add 5 to 25 kg of acetone, stir, add 5 to 25 kg of n-heptane, continue mechanical stirring, 30 rpm, stop stirring after 0.5 hours, let stand for stratification, take the organic phase, according to The above method was repeatedly extracted 2 to 3 times, and the organic extract was collected and concentrated under reduced pressure at 20 ° C to obtain 2.8 kg of oily yellow phosphatidylserine rich in polyunsaturated double bond fatty acyl group, and the PC content was determined by HPLC to be 8.5%. The content of phosphatidylserine was 36.4% by HPLC, and the EPA of phospholipid was 17.1% by GC and 7.3% by DHA.

Example 5: phosphatidylserine Antarctic krill oil rich in polyunsaturated double bond fatty acyl 2.8 kg of phosphatidylserine prepared in Example 4 and 7 kg of reddish brown fat prepared in Example 1 were added to 20 liters of reaction. In the reactor, the reactor is filled with nitrogen gas, at 0 to 10 degrees Celsius, mechanical stirring is continued, 100 rpm, and the reaction is carried out for 2 to 15 hours, and the reaction is terminated to obtain a phosphatidylserine Antarctic phosphorus rich in polyunsaturated double bond fatty acyl group. The prawn oil was 9.8 kg, the PC content was 10.5% by HPLC, the phosphatidylserine content was 10.8% by HPLC, the EPA in phosphatidylserine was 17.1% by GC, and DHA was 7.3%.

Example 6: Phospholipid serine-rich Antarctic krill oil rich in polyunsaturated double bond fatty acyl is weighed 10 kg of Antarctic krill oil, which contains: EPA content of phospholipid is 19.3%, DHA content is 11.5%, PC content 36.3%, phosphatidylserine content was 1.5%; 5 kg of phosphatidylserine was prepared from Example 3, wherein the phosphatidylserine content was 43.9%, the EPA content was 17.5%, the DHA content was 7.9%; and the 20 liter reactor was added. The reactor is filled with nitrogen gas, at zero degrees Celsius, mechanical stirring is continued, 100 rpm, and the reaction is carried out for 2 to 15 hours, and the reaction is terminated to obtain a phosphatidylserine Antarctic krill oil rich in polyunsaturated double bond fatty acyl group 15 Kilogram, phosphatidylserine content by HPLC The test was 18.6%, and the EPA in phospholipids was 17.6% by GC and 7.8% by DHA.

Example 7: Phosphatidylserine rich in polyunsaturated double bond fatty acyl

10 kg of Antarctic krill phosphatidylcholine prepared in Example 1 was weighed, wherein EPA was 18.3%, DHA was 8.1%, PC content was 47.8%, phosphatidylserine content was 2.4%, and dissolved in 15 to 40 liters. Just add 12 to 20 kg of L-serine, 0.4-1.5 kg of calcium chloride, 5 to 20 liters of acetone or propanol, 50 liters to 100 liters of water buffer pH 5.5 to pH 8.5, add In a 150 liter reactor, under a nitrogen atmosphere, put 0.1 to 2 liters of phospholipase D into 1 million units in the range of 45-53 degrees Celsius, continue mechanically stirring, lOO rpm, react for 2 to 15 hours, and end the reaction. Add 0 to 50 liters of n-hexane, continue mechanical stirring, 30 rpm, stop stirring after 0.5 hours, let stand for stratification, take the organic phase, repeat the extraction 2 to 4 times according to the above method, and collect the organic extract at 20 ° C. Concentrate under reduced pressure to obtain crude phosphatidylserine rich in polyunsaturated double bond fatty acyl group, add 10 to 30 liters of ethanol, continue mechanical stirring, 80 rpm, stop stirring after 0.5 hours, filter, freeze solid to obtain 8.6 kg Double bonds containing polyunsaturated fatty acyl phosphatidylserine quality, its PC content by HPLC of 5.5 ° /. The phosphatidylserine content was determined by HPLC to be 43.5%, and the EPA in the phospholipid was 17.3% by GC and 7.6% by DHA.

In this specification, the invention has been described with reference to specific embodiments thereof. However, it will be apparent that various modifications and changes can be made without departing from the spirit and scope of the invention, and one of ordinary skill in the art can still be practiced within the scope of the present invention. Other embodiments, but any simple modifications, equivalent changes and modifications to the above embodiments in accordance with the technical spirit of the present invention are still within the scope of the technical solutions of the present invention.

Claims

A method for producing a high quality Antarctic phosphorus shrimp oil rich in polyunsaturated double bond fatty acyl phosphatidylserine, characterized in that:

Step one, solvent extraction of the existing Antarctic krill oil to obtain phosphatidylcholine and a fat containing vitamin A, vitamin E and astaxanthin;

Step two, preparing an enzyme catalytic reaction system, the reaction system is an aqueous phase reaction system;

Step 3, the reaction system of the second step is added to the reactor, and the temperature is 35-55 degrees Celsius in a nitrogen atmosphere, and the mechanical stirring is continued after the phospholipid D is added, and the stirring time is 1 to 15 hours;

The method for preparing high quality Antarctic krill oil rich in polyunsaturated double bond fatty acyl phosphatidylserine according to claim 1, wherein the extraction in step 1 is an existing Antarctic krill oil and The solvent is mixed in the reactor, and then subjected to mechanical stirring in a nitrogen atmosphere at 0 to 30 ° C for 0.5 to 15 hours. After filtration, the solid is freeze-dried to obtain a phosphatidylcholine rich in a polyunsaturated double bond fatty acyl group.

The method for preparing high quality Antarctic krill oil rich in polyunsaturated double bond fatty acyl phosphatidylserine according to claim 1, wherein the phosphatidylcholine obtained in the first step is rich A phosphatidylcholine family containing a polyunsaturated double bond fatty acyl group containing more than a dozen phosphatidylcholine molecular series, wherein the chemical structure of each molecular series has the same and different: (1) The glyceryl group is attached to a choline at the 3-position of Sn-3; (2) at the 1-position of the glyceryl group, Sn-1 and 2 Each of Sn-2 is bonded to a fatty acyl group, and at the 2-position Sn-2, a long-chain fatty acyl group containing a polyunsaturated double bond is usually attached, which is rich in a polyunsaturated double bond fatty acyl group, which means Sn More than 5% of the fatty acyl groups in the 2-position-attached fatty acyl group contain two or more unsaturated double bonds, or a fatty acyl group having a higher than 3% or more of the fatty acyl group linked at the Sn-2 position Omega-3 unsaturated double bond; (3) Containing an unsaturated double bond fatty acyl group having 18 to 22 carbon atoms, and the number of double bonds is usually three to six, and the unsaturated double bond fatty acyl group is Omega- The 3 unsaturated fatty acid family refers to a docosahexaenoic acid fatty acyl group and a pentane carbonic acid fatty acyl group.

The method for preparing high quality Antarctic krill oil rich in polyunsaturated double bond fatty acyl phosphatidylserine according to claim 1, wherein the aqueous phase reaction system is rich in a plurality of steps. An aqueous solution reaction system prepared by mixing a phosphatidylcholine, a L-serine, a calcium salt and a buffer solution of a saturated double bond fatty acyl group, wherein the ratio of each substance in the aqueous solution reaction system is as follows: phosphatidylcholine per liter of the reaction solution The weight percentage added is 100, the weight percentage of L-serine added per liter of the reaction liquid is 60 to 300, and the weight percentage of the calcium salt added per liter of the reaction liquid is 0.05 to 10, and the concentration of the buffer salt in the buffer solution is It is 0~2 mol/L, and the pH of the aqueous solution is within 5.0~8.5.

A method for preparing a phosphatidylcholine rich in a polyunsaturated double bond fatty acyl group from an existing Antarctic krill oil, characterized in that a solvent extraction is performed on an existing Antarctic krill oil; the solvent may be in the following Optional: food safety grade n-heptane or n-hexane, edible ethanol or acetone, or a mixture thereof; the extraction is the existing Antarctic krill oil mixed with the solvent in a reactor in a nitrogen atmosphere At 0-30 degrees Celsius, the mixture is mechanically stirred for 0.5 to 15 hours. After filtration, the solid is freeze-dried to obtain a phosphatidylcholine rich in polyunsaturated double bond fatty acyl groups.

6. A method of preparing a phosphatidylserine rich in a polyunsaturated double bond fatty acyl group, characterized in that the following steps are taken: Step one, solvent extraction of existing Antarctic krill oil or direct purchase of phosphatidylcholine from the market; the extraction is carried out according to the following steps: The extraction solvent can be selected among the following items: food safety grade Already, edible ethanol or acetone, or a mixture thereof; the extraction is the existing Antarctic krill oil mixed with the solvent in the reactor, in a nitrogen atmosphere, 0 ~ 30 degrees Celsius, mechanically stirred, the reaction 0.5- After 15 hours, after filtration, the solid was freeze-dried to obtain a phosphatidylcholine rich in polyunsaturated double bond fatty acyl;

The method for preparing a phosphatidylserine rich in a polyunsaturated double bond fatty acyl group according to claim 6, wherein the second step of the aqueous phase reaction system is rich in the preparation of the existing krill oil. An aqueous solution reaction system prepared by mixing a phosphatidylcholine, a L-serine, a calcium salt and a buffer solution of a saturated double bond fatty acyl group, wherein the ratio of each substance in the aqueous solution reaction system is as follows: phosphatidylcholine in each liter of the reaction solution The weight percentage added is 100, the weight percentage of L-serine added per liter of the reaction liquid is 60 to 300, and the weight percentage of the calcium salt added per liter of the reaction liquid is 0.05 to 10, and the concentration of the buffer salt in the buffer solution is It is 0~2 mol/L, and the pH of the aqueous solution is within 5.0~8.5.

The method for preparing a phosphatidylserine rich in a polyunsaturated double bond fatty acyl group according to claim 6, wherein in the second step, the two-phase reaction system consisting of the aqueous phase and the organic solvent phase is in the first step. Commercially available phosphatidylcholine formulated enzyme catalyzed reaction system, which is first rich in polyunsaturated double bonds The fatty acyl phosphatidylcholine is dissolved in a C5-C9 hydrazine organic solvent, and then added to an aqueous solution containing L-serine, a calcium salt, a short-chain hydrocarbon or a short-chain hydrocarbon ketone and a buffer solution to form water. a reaction system in which two phases are mixed with an organic solvent, and the ratio of each substance in the mixed reaction system of the aqueous solution and the organic solvent is as follows: the weight percentage of phosphatidylcholine added per liter of the mixed reaction solution is 100, L - the weight percentage of serine added per liter of the reaction liquid is 60 to 300, the weight percentage of the calcium salt added per liter of the reaction liquid is 0.05 to 10, and the concentration of the buffer salt in the buffer solution is 0 to 2 mol/liter. The C5-C9 alkane organic solvent occupies 5% to 95% by volume of the mixed reaction solution, and the short-chain hydrocarbon or short-chain hydrocarbon ketone accounts for 1% to 80% by volume of the total mixed reaction solution, aqueous solution and organic solvent. The pH of the solvent two-phase mixed reaction system is in the range of 5.0 to 8.5.

The method for preparing a polyphosphonyl-rich fatty acid-rich phosphatidylserine according to claim 8, wherein the C5-C8 terpene hydrocarbon organic solvent is selected from the group consisting of pentane, Any solvent selected from the group consisting of hexane, cyclohexamidine, hydrazine, octane, decane, hydrazine or an isomer thereof or a mixture of these solvents in any ratio.

The method for producing a polyunsaturated double bond fatty acyl group-containing phosphatidylserine according to claim 8, wherein the short-chain hydrocarbon or short-chain hydrocarbon ketone is optional from the following: ethanol Any one of propanol, butanol, pentanol, acetone, methyl ethyl ketone or an isomer thereof or a mixed solvent of these solvents in any ratio.