CN114934080A

CN114934080A - Preparation method of phospholipid type DHA

Info

Publication number: CN114934080A
Application number: CN202210413365.1A
Authority: CN
Inventors: 李冰麟; 张甜甜; 赵宇科; 刘媛媛; 蒋沛阳; 张小里; 李海宁; 郭美静; 戚歆宇
Original assignee: Northwest University
Current assignee: Northwest University
Priority date: 2022-04-06
Filing date: 2022-04-06
Publication date: 2022-08-23

Abstract

The embodiment of the application discloses a preparation method of phospholipid type DHA, which comprises the following steps: performing ester exchange reaction under the action of biological enzyme by taking acyl donor of phospholipid and DHA as reactant to obtain phospholipid type DHA; wherein the acyl donor of DHA is a fatty acid salt rich in DHA. According to the method, the DHA is synthesized by catalyzing fatty acid salt rich in DHA and phospholipid through a biological enzyme to perform ester exchange reaction, wherein the acyl donor DHA in the form of fatty acid salt has stronger nucleophilicity, so that the acyl donor DHA has stronger combination efficiency and combination rate with phospholipid, the synthesis efficiency of the phospholipid DHA can be improved, and the DHA content in the product can be improved.

Description

Preparation method of phospholipid type DHA

Technical Field

The application relates to the technical field of biology, in particular to a preparation method of phospholipid DHA.

Background

Docosahexaenoic acid (DHA) belongs to one of polyunsaturated fatty acids (PUFA), is a constituent of cell membranes in human brain and retina tissues, has a vital effect on the development of infant vision and nervous systems, and also has the effects of preventing and treating cardiovascular diseases, delaying aging, preventing senile dementia, resisting cancer, resisting inflammation and the like. DHA is morphologically classified into triglyceride type, methylethyl type, and phospholipid type, wherein triglyceride type and methylethyl type are absorbed in vivo in a passive diffusion manner, resulting in low absorption rate, for example, triglyceride type absorption rate is about 50%, and ethyl ester type absorption rate is only 20%. The phospholipid DHA is absorbed in vivo in an active absorption mode, is considered to be an effective form for human body absorption, has an absorption rate close to 100 percent, and has extremely high application value.

Typical methods for producing phospholipid-type DHA include a method of extracting a naturally occurring substance and a method of synthesizing a phospholipid as a raw material. As the former method, extraction from marine organisms such as krill, yellow croaker, shellfish, etc. is performed, however, this method cannot satisfy the increasing demand of people because the raw materials are expensive and not necessarily stably supplied, and the organic solvent used in the extraction process poses a risk of food safety. The latter method may be, for example, chemical or enzymatic, wherein a toxic and harmful catalyst, such as dimethylaminopyridine, is used in the chemical synthesis of phospholipid-type DHA, which has a great safety problem, and most of the chemical syntheses are carried out at high reaction temperatures and under severe conditions, which easily destroy the natural structure of phospholipid molecules. The enzymatic method has the advantages of mild reaction conditions, few byproducts and the like, so that the enzymatic preparation of phospholipid DHA has a good application prospect, the enzymatic preparation of phospholipid DHA generally adopts esterified DHA or fatty acid DHA as an acyl donor to react with phospholipid in a double-liquid-phase system consisting of an organic solvent and water or a solvent-free system to synthesize DHA, on one hand, the bonding rate of the esterified DHA or fatty acid DHA and the phospholipid is low (lower than 20%), so that the content of DHA in the product is low, on the other hand, the use of a large amount of toxic and harmful solvents in the double-liquid-phase system undoubtedly affects the safety of the product, and the solvent-free system often causes poor reaction efficiency due to large mass transfer resistance.

Therefore, there is a need in the art to develop a rapid, efficient and safe method for preparing phospholipid-type DHA, and in particular, a method for preparing phospholipid-type DHA that can increase the content of DHA in the product.

Disclosure of Invention

It is an object of a preferred embodiment of the present application to provide a method for the preparation of a phospholipid-type DHA.

The above object of the present application is at least achieved by a specific selection or treatment of DHA as acyl donor.

Objects of the present application are not limited to the above objects, and other objects and advantages of the present application, which are not mentioned above, can be understood from the following description and more clearly understood through embodiments of the present application. Further, it is easily understood that the objects and advantages of the present application can be achieved by the features disclosed in the claims and the combinations thereof.

As one aspect of the present application, there is provided a method for preparing phospholipid-type DHA, comprising:

performing ester exchange reaction under the action of biological enzyme by taking acyl donor of phospholipid and DHA as reactant to obtain phospholipid type DHA;

wherein the acyl donor of DHA is a fatty acid salt rich in DHA.

In one embodiment, the DHA-rich fatty acid salt is prepared by saponification of a DHA-containing substance with an alkali, wherein the DHA-containing substance is an ester derivative of DHA and/or a fat containing the ester derivative;

optionally, the ester derivative of DHA is selected from any one of a methyl ester type derivative, an ethyl ester type derivative, a glyceride type derivative of DHA, or any combination of the above ester derivatives;

optionally, the oil is selected from any one of fish oil, algae oil and microbial oil, or any combination of the above oils.

In one embodiment, the phospholipid is at least one of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and diphosphatidylglycerol.

In one embodiment thereof, the biological enzyme comprises a lipase and/or a phospholipase;

optionally, the lipase comprises at least one of novozyme 435, Lipozyme TL IM, Lipozyme RM IM, Lipozyme TL 100L, Novocor ADL, novozyme 51032, Palatase 20000L, and Lipozyme CALB;

optionally, the phospholipase comprises at least one of phospholipase a1 and phospholipase a 2.

In one embodiment, the phospholipid is present in the reaction system in a carrier-immobilized form.

In one embodiment thereof, the phospholipid is immobilized to a carrier by adsorption or covalent bonding to form the phospholipid in the carrier-immobilized form;

optionally, the carrier is at least one of silica gel, silicon dioxide, calcium sulfate, cellulose microcrystals and activated carbon particles.

In one embodiment, the phospholipid is present in the reaction system in a form dissolved in a low-polarity organic solvent;

optionally, the low polarity organic solvent is selected from any one or any combination of the group consisting of ethyl acetate, n-hexane, dichloromethane, chloroform, hexafluoroisopropanol and acetone.

In one embodiment, the molar ratio of the acyl donor to the phospholipid is 1-3: 1.

In one embodiment, the transesterification reaction is carried out at a temperature of 25 to 55 ℃ and a pH of 7.5 to 9.0.

The embodiment of the application also provides the phospholipid DHA prepared by the method, and the bonding rate of DHA in the phospholipid DHA is not lower than 9%.

The technical scheme provided by the embodiment of the application can have the following beneficial effects:

according to the method, the DHA is synthesized by catalyzing fatty acid salt rich in DHA and phospholipid through transesterification reaction, wherein the acyl donor DHA in the form of fatty acid salt has stronger nucleophilicity, so that the acyl donor DHA has stronger combination efficiency and combination rate with phospholipid, the synthesis efficiency of the phospholipid DHA can be improved, and the content of DHA in the product can be improved.

Drawings

Other features, objects and advantages of the present application will become more apparent upon reading of the following detailed description of non-limiting embodiments thereof, made with reference to the accompanying drawings in which:

FIG. 1 is a graph showing the effect of different pH values on the DHA binding rate of the product.

FIG. 2 is a graph showing the effect of different lipases on the DHA binding rate of the product.

FIG. 3 is a graph showing the effect of different immobilized lipases on the DHA binding rate of the product.

FIG. 4 is a graph showing the effect of molar ratio of acyl donor to phospholipid on the DHA binding rate of the product.

FIG. 5 is a graph showing the effect of reaction temperature on the DHA binding rate of the product.

FIG. 6 is a graph showing the effect of the catalysis of immobilized enzyme and free enzyme on the DHA binding rate of the product.

FIG. 7 is a graph showing the effect of different acyl donors on the DHA binding rate of the product.

Detailed Description

The present application will be described in further detail with reference to examples. It is to be understood that the specific embodiments described herein are merely illustrative of the relevant invention and not restrictive of the invention.

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict.

It is noted that the endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and that such ranges or values are understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.

The examples do not specify particular techniques or conditions, and are performed according to techniques or conditions described in literature in the art or according to the product specification. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

In the present application, the term "phospholipid-type DHA" refers to the presence of DHA in the fatty acids that make up the phospholipids, also known as DHA-type phospholipids, which have a dual role of DHA and phospholipids.

In the present application, the term "acyl donor" refers to a chemical entity or molecule capable of transferring an acyl group to an acyl acceptor by an enzymatic transfer reaction, such as a molecule having R-m (o) -wherein the acyl acceptor is one or more selected from the group consisting of esters, carbohydrates, proteins, protein subunits, or hydroxy acids.

In the present application, the term "lipase" refers to any enzyme capable of catalyzing transesterification or esterification, and in the present application, the lipase may be a non-immobilized product or an immobilized product, and exemplary non-immobilized products may be liquid lipases, including, for example, but not limited to, a solution comprising lipase (e.g., commercially available lipase enzyme activity or a dilution thereof), fermentation broth of a strain expressing lipase or a recombinant strain, fermentation supernatant, and the like. In the present application, the lipase used may be a lipase expressed by fermentation using a strain, for example, including but not limited to: candida antarctica lipase (Candida antarctica lipase), thermomyces lanuginosa lipase, Pseudomonas fluorescens lipase (Pseudomonas fluorescens lipase), Pseudomonas Pseudomonas lipase (Pseudomonas cepacia lipase), Chromobacterium viscosum lipase, P.aeruginosa lipase, P.fluoroscens lipase, Pseudomonas lipase, B.cepacia lipase, C.viscosum lipase, Bacillus subtilis lipase, Achromobacter sp.lipase, Alcaligenes lipase and Serratia marcescens lipase, as well as C.antarctica B lipase and C.rugosa lipase from fungi, and the like, commercially available lipases such as Novozym L, Novozyme B lipase, Lipomyces lipolase, and Lipomyces lipolase, can also be used, and commercially available lipases such as Novozym L including but not limited to Novozym, Novozyme B, Lipomyces 5102, Lipomyces lipolase, Lipomyces L435, Lipomyces L32, and Lipomyces L.

In the present application, the transesterification reaction is an enzymatic transesterification, and the specific process is well known to those skilled in the art or can be determined experimentally. For example, in one embodiment herein, the reaction temperature for the transesterification reaction is 20 to 100 ℃, preferably 25 to 55 ℃, more preferably 30 to 50 ℃.

In a first aspect, the present application provides a method for producing phospholipid-type DHA, comprising:

wherein the acyl donor of DHA is a fatty acid salt rich in DHA.

In the present example, DHA as the acyl donor introduced into the 1-or 2-position of the phospholipid in the transesterification reaction with the biological enzyme is preferably used in the form of a fatty acid salt. The applicant has found that the efficiency with which the enzymes catalyze the transesterification of DHA with phospholipids is affected by the steric hindrance caused by the presence of multiple double bonds in DHA itself. In particular, compared with DHA in the form of ethyl ester, methyl ester and triglyceride, DHA in the form of fatty acid salt has smaller steric hindrance, and DHA in the form of ethyl ester, methyl ester, triglyceride and free fatty acid has stronger nucleophilicity, so that DHA can be bonded to phospholipid with higher efficiency and binding rate, and at least the reaction efficiency of transesterification reaction is improved, the content of DHA in the product is improved, and the industrial feasibility is higher.

The phospholipid as the acyl acceptor in the transesterification reaction with the biological enzyme is not particularly limited, and glycerophospholipids are preferably used.

Further, in an embodiment of the present application, the DHA-rich fatty acid salt is prepared by saponification of a DHA-containing material with an alkali, wherein the DHA-containing material is an ester derivative of DHA and/or a fat containing the ester derivative;

optionally, the ester derivative of DHA is selected from any one of a methyl ester derivative, an ethyl ester derivative, a glyceride derivative of DHA, or any combination of the above ester derivatives, wherein the content of DHA in the ester derivative of DHA is not less than 40 wt%, preferably not less than 50 wt%, and more preferably not less than 75%, and the ester derivative of DHA may be derived from fish oil, algae oil, or microbial oil.

Optionally, the oil is selected from any one of fish oil, algae oil, microbial oil, or any combination thereof, wherein the microbial oil may be derived from bacteria, fungi, yeast, schizochytrium, and the like. In the grease, the content of DHA is not less than 10 wt%, preferably not less than 20 wt%.

That is, in the present application, the DHA-containing material may be esterified DHA, fatty acid DHA, fish oil, algae oil, or the like, or a mixture thereof, and the esterified DHA and fatty acid DHA may be a commercially available standard or a purified product extracted from fish oil or the like.

Illustratively, the DHA-rich fatty acid salt is prepared by the following method:

dissolving a substance containing DHA in a solvent, sequentially adding an ascorbic acid solution and an alkali solution, heating and refluxing until complete hydrolysis, removing the solvent by vacuum rotary evaporation, and adjusting the pH value to 7.5-9.0 to obtain the fatty acid salt rich in DHA.

The solvent is not particularly limited, and may be any solvent capable of dissolving the DHA-containing substance, and is preferably absolute ethanol. Wherein the alkali solution is potassium hydroxide solution or sodium hydroxide solution, preferably potassium hydroxide solution.

Further, in one embodiment of the present application, the phospholipid is at least one of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and diphosphatidylglycerol.

Further, in one embodiment herein, the biological enzyme comprises a lipase and/or a phospholipase;

optionally, the lipase comprises at least one of Novozym 435, Lipozyme TL IM, Lipozyme RM IM, Lipozyme TL 100L, Novocor ADL, Novozym 51032, Palatase 20000L, and Lipozyme CALB, preferably the lipase is from Candida Antarctica lipase B (Candida Antarctica lipase B), Candida Antarctica lipase A (Candida Antarctica A), Thermomyces lanuginosus (Thermomyces lanuginosus), Mucor miehei (Rhizomucor miehei) or Aspergillus oryzae (Aspergillus oryzae);

Further, in one embodiment of the present application, the phospholipids are present in the reaction system in a carrier-immobilized form, i.e. the phospholipids are first immobilized on a carrier and then mixed with the DHA-rich fatty acid salt solution to form a solid-liquid reaction system.

In this example, the biological enzyme and acyl donor are in the aqueous phase, and the contact is easier, the mass transfer resistance is smaller, and the yield is higher.

Wherein the phospholipid is immobilized to a carrier by an adsorption method or a covalent bonding method to form the phospholipid in a form immobilized on the carrier; optionally, the carrier is at least one of silica gel, silicon dioxide, calcium sulfate, cellulose microcrystals and activated carbon particles.

As an example of the adsorption method, a method of immobilizing phospholipid on a carrier is as follows:

dissolving phospholipid in organic solvent, mixing with carrier, stirring, adding precipitant, precipitating, and washing with water to obtain phospholipid adsorbed on carrier, wherein the precipitant can be acetone.

As an example of the covalent bonding method, a method of immobilizing phospholipid on a carrier is as follows:

adding phospholipid into phosphate buffer solution, performing ultrasonic oscillation, stirring at normal temperature for several hours, adding a carrier modified by a surfactant, stirring at normal temperature until a liquid phase is transparent, precipitating, and washing with water to obtain the phospholipid covalently bonded and adsorbed on the carrier, wherein the surfactant can be at least one of triton 100 or epoxide thereof, sodium deoxycholate and sodium cholate, and is preferably triton X-100 epoxide.

Further, in another embodiment of the present application, the phospholipids are present in the reaction system in a form dissolved in a low polar organic solvent, i.e. the phospholipids are first dissolved in a low polar organic solvent and then mixed with a DHA-rich fatty acid salt solution to form a liquid-liquid reaction system (two liquid phase reaction system).

Wherein the low polarity organic solvent is selected from any one or any combination of the group consisting of ethyl acetate, n-hexane, dichloromethane, chloroform, hexafluoroisopropanol and acetone, preferably ethyl acetate and/or n-hexane.

Further, in one embodiment herein, the molar ratio of the acyl donor to the phospholipid is 1-3: 1.

Further, in one embodiment of the present application, the reaction temperature of the transesterification reaction is 25 to 55 ℃, preferably 30 to 50 ℃, and the pH of the reaction is 7.5 to 9.0, preferably 7.5 to 8.0.

Illustratively, the preparation method of phospholipid-type DHA of the present application specifically comprises the following steps:

1) dissolving a DHA-containing substance in a solvent, sequentially adding an ascorbic acid solution and an alkali solution, heating and refluxing until complete hydrolysis, removing the solvent by vacuum rotary evaporation, and adjusting the pH value to 7.5-9.0 by using 4N hydrochloric acid to obtain a DHA-rich fatty acid salt solution;

2) dissolving phospholipid in an organic solvent, mixing and stirring the solution and a carrier, adding a precipitator for precipitation, and washing the precipitate with water to obtain the phospholipid adsorbed on the carrier;

3) mixing the phospholipid obtained in the step 2) with the fatty acid salt solution rich in DHA obtained in the step 1), adding a certain amount of phosphate buffer solution to form a mixed solution, then adding lipase, heating to a certain temperature, and stirring for reaction; wherein the reaction temperature is 25-55 ℃, the reaction time is 0.5-24h, and the addition amount of the lipase is 0.1-20% of the mixed solution;

4) after the reaction is finished, immediately inactivating at high temperature, then centrifuging at high speed, washing the obtained solid with deionized water, and drying in vacuum;

5) dissolving the solid obtained in the step 4) in an eluent (such as methanol), extracting the product adsorbed on the carrier, and evaporating the solvent to obtain the phospholipid type DHA.

In a second aspect, the present invention relates to a phospholipid-type DHA obtained according to the process of the first aspect. Preferably, the DHA content in the product is not lower than 9%. The binding rate is the peak area percentage content of DHA in the phospholipid DHA of the final product in the total fatty acids, namely the DHA content in the product, and can be measured by gas chromatography analysis.

In the application, the content of fatty acid is detected by gas chromatography, and before detection, the phospholipid DHA product is prepared according to ISO 5509: 2000(E) methyl esterification and subsequent gas phase detection. The detection conditions of the gas chromatography were as follows:

the chromatographic column is SH-RTX-WAX (30m × 0.25mm × 0.25 μm); keeping the column temperature at 165 ℃ for 1min, heating to 210 ℃ at the speed of 6.5 ℃/min, heating to 240 ℃ at the speed of 1.5 ℃/min, and keeping for 2 min; the sample injection temperature is 250 ℃; the detector temperature is 280 ℃, the sample injection volume is 1 mu L, the nitrogen flow rate is 24mL/min, the hydrogen flow rate is 32mL/min, and the air flow rate is 200 mL/min.

Example 1

Weighing 100mg of algae oil, adding 2mL of absolute ethyl alcohol, 1mL of 10% ascorbic acid solution and 1mL of 40% potassium hydroxide solution, and carrying out reflux reaction at 90 ℃ until no oil drops appear; after cooling to room temperature, adding 4mL of deionized water; after ethanol was completely removed by vacuum rotary evaporation, 6mL of deionized water was added, and 3mL of the mixture was sequentially adjusted to pH 9.0, 8.5, 8.0, and 7.5 with 4N HCl.

Dissolving 5mg of Phosphatidylcholine (PC) in 1mL of ethyl acetate, stirring until the phosphatidylcholine is fully dissolved, adding 10mg of silica gel G, precipitating with 1mL of acetone, stirring at 200rpm for 3 hours, and washing with deionized water for 3 times to obtain PC adsorbed and precipitated on the silica gel G; parallel adsorption of 4 groups of PC, 1mL of pH 9.0, 8.5, 8.0, 7.5 phosphate buffer suspension.

Taking 1mL of the suspension, adding 0.8mL of DHA fatty acid salt solution with corresponding pH value, adding 0.2mL

CALB (enzyme activity 5000LU/mL), reacting at 40 deg.C and 400rpm for 18h, inactivating at high temperatureAnd then centrifuging at a high speed, washing the precipitate with deionized water for 3 times, drying in vacuum overnight, eluting with 1.0mL of methanol, and measuring the DHA binding rate of the products of different pH systems as shown in figure 1 after removing the methanol.

It can be seen that the DHA and the phospholipid have higher binding rate when the pH value of the reaction system is 7.5-9.0, and especially the binding rate reaches more than 15% when the pH value is 7.5 and 8.0.

Example 2

Weighing 100mg of algae oil, adding 2mL of absolute ethyl alcohol, 1mL of 10% ascorbic acid solution and 1mL of 40% potassium hydroxide solution, and carrying out reflux reaction at 90 ℃ until no oil drops appear; after cooling to room temperature, adding 4mL of deionized water; after complete removal of ethanol by rotary evaporation under vacuum, 6mL of deionized water was added and the pH was adjusted to 7.5 with 4N HCl.

50mg of PC is dissolved in 5mL of ethyl acetate, stirred until the solution is fully dissolved, then 100mg of silica gel G is added, and the solution is precipitated by 5mL of acetone, stirred at 200rpm for 3 hours and then washed by deionized water for 3 times, so that the PC adsorbed and precipitated on the silica gel G is obtained and suspended by 10mL of phosphate buffer with the pH value of 7.5.

1mL of each suspension was added with 2mL of DHA fatty acid salt solution with pH 7.5, and 1LU of lipase (1 LU)

TL 100L, enzyme activity 100 KLU/g;

ADL, enzyme activity 6000 LU/mL;

51032, enzyme activity is 15 KLU/mL;

20000L, and the enzyme activity is 20000 LU/mL;

HT, enzyme activity of 50KLU/mL), reacting at 30 ℃ and 400rpm for 12h, inactivating at high temperature, centrifuging at high speed, washing precipitate with deionized water for 3 times, vacuum drying overnight, eluting with 1.0mL of methanol, and removing methanolThe DHA binding rate of the products of different lipase catalysis is shown in FIG. 2.

It can be seen that when using

ADL、

51032 and

20000L, DHA has high binding rate with phospholipid, and the binding rate reaches more than 19%.

Example 3

Weighing 100mg of algae oil, adding 2mL of absolute ethyl alcohol, 1mL of 10% ascorbic acid solution and 1mL of 40% potassium hydroxide solution, and carrying out reflux reaction at 90 ℃ until no oil drops appear; after cooling to room temperature, adding 4mL of deionized water; after complete removal of ethanol by rotary evaporation in vacuo, 6mL of deionized water was added, and 3mL was adjusted to pH 8.0 with 4N HCl.

Dissolving 5mg of PC in 1mL of ethyl acetate, stirring until the solution is sufficiently dissolved, adding 10mg of silica gel G, precipitating with 1mL of acetone, stirring at 200rpm for 3 hours, washing with deionized water for 3 times to obtain PC adsorbed and precipitated on silica gel G, and suspending with 1mL of phosphate buffer solution with the pH of 8.0.

And (2) adding 2mL of DHA fatty acid salt solution with the pH value of 8.0 into 1mL of the suspension, adding 0.1mL of PLA1 (with the enzyme activity of 10KLU/mL), reacting at 30 ℃ and 400rpm for 10min, inactivating at high temperature, centrifuging at high speed, washing precipitates with deionized water for 3 times, drying in vacuum overnight, eluting with 1.0mL of methanol, and measuring the DHA binding rate in the product to be 14.7% after removing the methanol.

Example 4

Taking 1mL of the suspension, adding 2mL of DHA fatty acid salt solution with the pH value of 8.0, adding 0.1mL of PLA2 (the specific activity is 600U/mg protein), reacting at 35 ℃ and 400rpm for 10min, inactivating at high temperature, centrifuging at high speed, washing precipitates with deionized water for 3 times, drying in vacuum overnight, eluting with 1.0mL of methanol, and measuring the DHA binding rate in the product to be 10.3% after removing the methanol.

Example 5

Dissolving 5mg PC in 1mL n-hexane, adding 2mL DHA fatty acid salt solution with pH of 7.5, and sequentially adding 1mg

435 (enzyme activity 10000PLU/g), 40mg

TL IM (enzyme activity 250IUN/g), 36mg

RM IM (enzyme activity 275IUN/g), reacting for 18h at 30 ℃ and 400rpm, separating out an n-hexane phase after the reaction is finished, washing a lower-layer water phase for 3 times by using 1mL of n-hexane, collecting and combining the n-hexane phase, and measuring the DHA binding rate in the product obtained by the 3 immobilized enzyme catalysis two-liquid-phase system after the solvent is removed by rotary evaporation, wherein the DHA binding rate is shown in figure 3.

It can be seen that when using

435 and

in RM IM, DHA and phospholipid have high binding rate, and the binding rate reaches more than 20%.

Example 6

Weighing 100mg of algae oil, adding 2mL of absolute ethyl alcohol, 1mL of 10% ascorbic acid solution and 1mL of 40% potassium hydroxide solution, and carrying out reflux reaction at 90 ℃ until no oil drops appear; after cooling to room temperature, adding 4mL of deionized water; after ethanol was removed by rotary evaporation in vacuo, 6mL of deionized water was added and 3mL was adjusted to pH 7.5 with 4N HCl.

5mg of PC was added to 2mL of phosphate buffer pH 5.5, and the mixture was stirred at 500rpm for 4 hours under ultrasonic agitation for 5min at room temperature. Adding 10mg of Triton X-100 epoxide-modified silica gel G into the mixed solution, stirring at 30 ℃ and 500rpm for 70min, washing the precipitate with water for 3 times to obtain the phospholipid covalently bonded on the silica G, and drying in vacuum for later use.

Suspending the covalently bound PC by using 1mL of phosphate buffer solution with the pH value of 7.5, adding 0.8mL of DHA fatty acid salt solution with the pH value of 7.5, adding 0.2mL of CalB, reacting at 40 ℃ and 400rpm for 12h, inactivating at high temperature, centrifuging at high speed, washing the precipitate with deionized water for 3 times, drying in vacuum overnight, eluting with 1.0mL of methanol, and measuring the DHA binding rate in the product to be 9.2% after removing the methanol.

Example 7

Weighing 100mg of algae oil, adding 2mL of absolute ethyl alcohol, 1mL of 10% ascorbic acid solution and 1mL of 40% potassium hydroxide solution, and carrying out reflux reaction at 90 ℃ until no oil drops appear; after cooling to room temperature, adding 4mL of deionized water; after complete removal of ethanol by rotary evaporation in vacuo, 6mL of deionized water was added, and 3mL was adjusted to pH 7.5 with 4N HCl.

50mg of PC is dissolved in 5mL of ethyl acetate, stirred until the PC is fully dissolved, then 100mg of nano SiO2 is added, and the mixture is precipitated by 5mL of acetone, stirred at 200rpm for 3 hours, and washed by deionized water for 3 times to obtain the PC adsorbed and precipitated in the nano SiO2, and the PC is suspended by 10mL of phosphoric acid buffer solution with the pH value of 7.5.

And (3) adding 2mL of DHA fatty acid salt solution with the pH value of 7.5 into 1mL of the suspension, adding 0.2mL of CalB, reacting for 12 hours at 30 ℃ and 400rpm, inactivating at high temperature, centrifuging at high speed, washing precipitates with deionized water for 3 times, drying in vacuum for overnight, eluting with 1.0mL of methanol, and measuring the DHA binding rate in the product to be 22.8% after removing the methanol.

Example 8

50mg of PC was dissolved in 5mL of ethyl acetate and stirred until sufficiently dissolved.

And (3) adding 2mL of DHA fatty acid salt solution with the pH value of 7.5 into 1mL of the PC solution, adding 0.2mL of CalB, reacting at 30 ℃ and 400rpm for 12h, carrying out high-temperature inactivation after the reaction is finished, carrying out vacuum rotary evaporation at 40 ℃, dissolving again by using methanol, separating out a methanol phase, and measuring the DHA binding rate in the product to be 28.3% after the solvent is evaporated.

Example 9

Dissolving 50mg of PC in 5mL of ethyl acetate, stirring until the PC is fully dissolved, adding 100mg of silica gel G, precipitating with 5mL of acetone, stirring at 200rpm for 3h, washing with deionized water for 3 times to obtain PC adsorbed and precipitated on the silica gel G, and drying in vacuum for later use.

Taking 5mg of phospholipid/10 mg of silica gel after adsorption precipitation, respectively adding a DHA-rich fatty acid potassium salt solution with pH of 7.5 and the molar ratio of the DHA to the phospholipid of 1.0: 1, 1.5: 1, 2.0: 1, 2.5: 1 and 3.0: 1, adding a certain amount of phosphate buffer solution, adding 0.2mL of CalB, reacting for 8 hours at 40 ℃ and 400rpm, performing high-speed centrifugation after high-temperature inactivation, washing the precipitate with deionized water for 3 times, performing vacuum drying overnight, eluting with 1.0mL of methanol, and measuring the change of the DHA binding rate in the product after removing the methanol, wherein the change is shown in figure 4.

It can be seen that when the molar ratio of the acyl donor to the phospholipid is 1-3: 1, the binding rate of DHA to the phospholipid is higher, reaching more than 10%, and especially when the molar ratio of the acyl donor to the phospholipid is 2.5: 1 and 3.0: 1, the binding rate can reach more than 20%.

Example 10

50mg of PC was dissolved in 5mL of ethyl acetate, and stirred to be sufficiently dissolved, 100mg of silica gel G was added, and precipitated with 5mL of acetone, and after stirring at 200rpm for 3 hours, washed with deionized water 3 times to obtain PC adsorbed and precipitated on silica gel G, which was suspended in 10mL of phosphate buffer solution having a pH of 7.5.

Taking 1mL of the suspension, adding 2mL of DHA fatty acid salt solution with pH of 7.5, adding 0.2mL of CalB, reacting at 25-60 ℃ for 8h at 400rpm, inactivating at high temperature, centrifuging at high speed, washing the precipitate with deionized water for 3 times, vacuum drying overnight, eluting with 1.0mL of methanol, and measuring the DHA binding rate of the product at different temperatures as shown in FIG. 5.

It can be seen that when the reaction temperature is 25-55 ℃, the DHA and the phospholipid have higher binding rate which is more than 10%, and particularly when the reaction temperature is 30-50 ℃, the binding rate is higher.

Example 11

Dissolving 5mg PC in 1mL n-hexane, adding 2mL DHA fatty acid salt solution with pH of 7.5, and adding 1mg

435, 0.2mL of CalB, 30 ℃, reaction for 8h at 400rpm, separating out an n-hexane phase after the reaction is finished, washing a lower-layer water phase for 3 times by using 1mL of n-hexane, collecting and combining the n-hexane phases,the immobilized candida antarctica lipase (a) is respectively measured after the solvent is removed by rotary evaporation

435) And the binding rate of the product DHA in the free Candida antarctica lipase (CalB) catalyzed two-liquid phase system is shown in FIG. 6.

As can be seen, the free Candida antarctica lipase (CalB) has a higher binding rate when used for catalyzing a two-liquid phase system, so that the non-immobilized biological enzyme is preferably used for catalyzing the reaction of DHA and phospholipid.

Comparative example 1

Weighing 100mg of algae oil, adding 2mL of absolute ethyl alcohol, 1mL of 10% ascorbic acid solution and 1mL of 40% potassium hydroxide solution, and carrying out reflux reaction at 90 ℃ until no oil drops appear; after cooling to room temperature, adding 4mL of deionized water; after complete removal of ethanol by rotary evaporation in vacuo, 6mL of deionized water was added, and 2mL was adjusted to pH 7.5 with 4N HCl. And adjusting the pH value of 2mL of the obtained product to 1.0 by using 4N HCl, extracting the separated fatty acid by using 1mL of N-hexane for 2 times, collecting an N-hexane phase, and performing vacuum rotary evaporation to remove the solvent to obtain the DHA-rich free fatty acid.

Dissolving 5mg of PC in 1mL of n-hexane, adding 2mL of DHA fatty acid salt solution with the pH value of 7.5, sequentially adding 0.2mL of CalB, reacting at 30 ℃ and 400rpm for 8h, separating an n-hexane phase after the reaction is finished, washing a lower-layer water phase for 3 times by using 1mL of n-hexane, collecting and combining the n-hexane phase, and measuring the DHA binding rate after removing the solvent by rotary evaporation.

Dissolving 5mg of PC and the DHA-rich free fatty acid solid obtained in the previous step in 1mL of n-hexane, adding 2mL of phosphate buffer with pH of 7.5, sequentially adding 0.2mL of CalB, reacting at 30 ℃ and 400rpm for 8h, separating an n-hexane phase after the reaction is finished, washing a lower-layer water phase for 3 times by using 1mL of n-hexane, collecting and combining the n-hexane phase, and measuring the DHA binding rate after the solvent is removed by rotary evaporation. The binding rates of the acyl donors, i.e., DHA-rich fatty acid solution and DHA-product of free fatty acid rich in DHA, are shown in fig. 7.

It can be seen that the binding rate is much higher with DHA-rich fatty acid salts than with DHA-rich free fatty acids, indicating that acyl donors in the form of fatty acid salts are the preferred form for increasing the DHA content in phospholipid-type DHA.

Comparative example 2

Dissolving 100mg of algae oil and 5mg of PC in 1mL of n-hexane, adding 2mL of phosphate buffer with the pH value of 7.5, sequentially adding 0.2mL of CalB, reacting at 30 ℃ and 400rpm for 8h, separating an n-hexane phase after the reaction is finished, washing a lower-layer water phase for 3 times by using 1mL of n-hexane, collecting and combining the n-hexane phase, concentrating the n-hexane phase, analyzing the n-hexane phase by using a thin-layer chromatography (a silica gel G plate, a developing agent system is chloroform, methanol and water, 13: 5: 0.8(v/v)), scraping a strip of DHA-PC, eluting the DHA-PC by using methanol, and removing the solvent by rotary evaporation to obtain the DHA binding rate of 2.1% in the product.

It can be seen that the incorporation efficiency is much higher when using fatty acid salts rich in DHA than when using esterified DHA and/or triglyceride DHA, indicating that acyl donors in the form of fatty acid salts are the preferred form for increasing the DHA content in phospholipid DHA.

In the description herein, reference to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the application. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present application have been shown and described above, it should be understood that the above embodiments are exemplary and should not be construed as limiting the present application, and that modifications, substitutions, and alterations may be made to the above embodiments by those of ordinary skill in the art within the scope of the present application.

Claims

1. A method for producing phospholipid-type DHA, comprising:

wherein the acyl donor of DHA is a fatty acid salt rich in DHA.

2. The preparation method according to claim 1, wherein the DHA-rich fatty acid salt is prepared by saponification of a DHA-containing substance with an alkali, wherein the DHA-containing substance is an ester derivative of DHA and/or an oil or fat containing the ester derivative;

3. The method according to claim 1, wherein the phospholipid is at least one of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and diphosphatidylglycerol.

4. The method according to claim 1, wherein the biological enzyme comprises lipase and/or phospholipase;

optionally, the lipase comprises at least one of Novozym 435, Lipozyme TL IM, Lipozyme RM IM, Lipozyme TL 100L, Novocor ADL, Novozym 51032, Palatase 20000L, and Lipozyme CALB;

5. The method according to claim 1, wherein the phospholipid is present in the reaction system in a carrier-immobilized form.

6. The production method according to claim 5, wherein the phospholipid is immobilized to a carrier by an adsorption method or a covalent bonding method to form the phospholipid in a form immobilized on the carrier;

7. The production method according to claim 1, wherein the phospholipid is present in the reaction system in a form dissolved in a low-polarity organic solvent;

optionally, the low-polarity organic solvent is selected from any one or any combination of the group consisting of ethyl acetate, n-hexane, dichloromethane, chloroform, hexafluoroisopropanol and acetone.

8. The method according to claim 1, wherein the molar ratio of the acyl donor to the phospholipid is 1-3: 1.

9. The method according to claim 1, wherein the reaction temperature of the transesterification is 25 to 55 ℃ and the pH value of the reaction is 7.5 to 9.0.

10. The phospholipid-type DHA according to any one of claims 1 to 9.