Structural phospholipid rich in functional fatty acid and preparation method thereof
Hollow double-layer mesoporous silica microspheres were prepared by sol-gel method and phospholipase A1 was immobilized, and structural phospholipids rich in functional fatty acids were prepared by catalyzing the transesterification reaction, which solved the synthesis difficulties in the prior art and achieved efficient, stable and environmentally friendly preparation of functional fatty acids.
Patent Information
- Application Number
- CN202510500241.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-21
- Publication Date
- 2025-07-22
AI Technical Summary
It is difficult to efficiently synthesize structural phospholipids rich in functional fatty acids such as neuric acid and DHA, and traditional methods have problems such as high cost, cumbersome operation, harsh conditions or pollute the environment.
Hollow bilayer mesoporous silica microspheres were prepared by sol-gel method, and phospholipase A1 was immobilized by bifunctional group modification and physical adsorption method, and structural phospholipids rich in functional fatty acids were prepared by catalyzing transesterification reaction.
It improves the immobilization efficiency and stability of phospholipase, has a wide range of substrates, mild reaction conditions, and is suitable for large-scale production. The content of functional fatty acids reaches 39%-82%. The immobilized phospholipase can be reused for 9 times and the enzyme activity remains above 80%.
Smart Images
Figure CN120350073A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of oil modification, and more specifically, to a structured phospholipid rich in functional fatty acids and a preparation method thereof. Background Art
[0002] Functional fatty acids such as docosahexaenoic acid (DHA), arachidonic acid (ARA), eicosapentaenoic acid (EPA), nervonic acid, and medium-chain fatty acids (MCFA) are of great significance to human health. For example, DHA is a key component of brain cell membranes, participates in the formation and development of brain cells and the normal physiological activities of nerve cells, and is also closely related to the formation of brain thinking and memory. Nervonic acid is the core natural component of brain nerve fibers and nerve cells, which can repair and dredge damaged brain nerve fibers and promote the regeneration of nerve cells. Medium-chain fatty acids have the functions of reducing blood lipids and cholesterol and improving obesity-related metabolic disorders. However, the human body is difficult to synthesize these fatty acids with special structures and functions by itself and mainly relies on external intake to supplement. However, currently common functional fatty acid products are mostly ethyl ester type and glyceride type, which have the problem of low absorption and utilization rate.
[0003] Phospholipids are a class of important biomolecules, widely present in animal and plant tissues and microbial cell membranes, and possess a variety of key biological functions. Cephalin group can carry key brain nutrients to break through the blood-brain barrier and improve the absorption rate of nutrients. Research shows that phospholipid DHA has a higher absorption and utilization rate in the brain and nerve cells compared with other types of DHA. Therefore, combining functional fatty acids with phospholipids to prepare structured phospholipids rich in functional fatty acids such as nervonic acid and DHA to provide nutrients for the brain and nerves may be a more effective strategy for supplementing functional fatty acids.
[0004] Currently, the preparation methods of structured phospholipids mainly include natural extraction method, chemical synthesis method, microbial fermentation method, and enzymatic synthesis method. Among them, the natural extraction method has limitations such as high cost, cumbersome operation, and low extraction rate; the reaction conditions of the chemical synthesis method are relatively harsh, often requiring high temperature, strong acid or strong base environments, which are likely to damage the natural configuration of phospholipids, and the content of by-products is relatively high, limiting its application in industries such as food, medicine, and cosmetics; the microbial fermentation method is in the preliminary research stage; the enzymatic synthesis method has attracted wide attention due to its mild reaction conditions, environmental friendliness, and pollution-free characteristics.
[0005] Therefore, how to develop an efficient and green enzymatic synthesis method for preparing structured phospholipids rich in functional fatty acids such as nervonic acid and DHA to improve the absorption and utilization rate of functional fatty acids by the human body is an urgent problem to be solved by those skilled in the art. Summary of the Invention
[0006] In view of this, the purpose of the present invention is to provide a structured phospholipid rich in functional fatty acids and its preparation method to solve the deficiencies in the prior art.
[0007] To achieve the above purpose, the present invention adopts the following technical solutions:
[0008] A preparation method of a structured phospholipid rich in functional fatty acids specifically includes the following steps:
[0009] (1) Preparation of hollow double-layer mesoporous silica microspheres
[0010] ① Mix ethanol, water, and ammonia water evenly, add tetraethyl orthosilicate, and stir to react to obtain solution A;
[0011] ② Mix water and ethanol evenly, add cetyltrimethylammonium bromide (CTAB), and stir to dissolve to obtain solution B;
[0012] ③ Stir and mix solution A and solution B, add tetraethyl orthosilicate, react, filter, wash, and dry to obtain core / shell nanosilica microspheres;
[0013] ④ Disperse the core / shell nanosilica microspheres in a sodium carbonate solution, react, filter, wash, and dry to obtain hollow double-layer nanosilica microspheres with the silicon core removed;
[0014] ⑤ Disperse the hollow double-layer nanosilica microspheres with the silicon core removed in a hydrochloric acid absolute ethanol solution, stir, filter, wash, and dry to obtain hollow double-layer mesoporous silica microspheres (HdlMS);
[0015] (2) Preparation of bifunctional group-modified hollow double-layer mesoporous silica microspheres
[0016] Disperse the hollow double-layer mesoporous silica microspheres in anhydrous toluene, dropwise add 3-[2-(2-aminoethylamino)ethylamino]propyl-trimethoxysilane and n-octyltrichlorosilane successively, sonicate, perform surface modification, filter, wash the filter, and dry to obtain bifunctional group-modified hollow double-layer mesoporous silica microspheres (NH2 / C8-HdlMS);
[0017] (3) Preparation of immobilized phospholipase
[0018] First, mix phospholipase A1 and PBS buffer solution to obtain a phospholipase solution; then mix the bifunctional group-modified hollow double-layer mesoporous silica microspheres and the phospholipase solution, incubate, centrifuge, filter, wash, and freeze-dry to obtain immobilized phospholipase (PLA1@NH2 / C8-HdlMS);
[0019] (4) Preparation of structured phospholipids rich in functional fatty acids
[0020] Add the immobilized phospholipase into a n-hexane solution containing phospholipids and functional fatty acid ethyl esters, add water, carry out transesterification reaction, separate and recover the immobilized phospholipase, add acetone, and precipitate, which is the structured phospholipid rich in functional fatty acids.
[0021] Further, in the step (1) ① above, the dosage ratio of ethanol, water, ammonia water and tetraethyl orthosilicate is 400 mL: 32 mL: 24 mL: 12 mL; the stirring reaction time is 6 h.
[0022] Further, in the step (1) ② above, the dosage ratio of water, ethanol and cetyltrimethylammonium bromide is 880 mL: 40 mL: 4.8 g; the stirring and dissolving time is 6 h;
[0023] Further, in the step (1) ③ above, the stirring and mixing time is 30 min; the addition amount of tetraethyl orthosilicate is 4 mL; the reaction time is 12 h;
[0024] Further, in the step (1) ④ above, the concentration of the sodium carbonate solution is 0.2 - 0.4 M; the dosage ratio of the core / shell nano silica microspheres and the sodium carbonate solution is 3 g: 500 mL; the reaction temperature is 55 °C and the time is 4 - 7 h.
[0025] Further, in the step (1) ⑤ above, the concentration of the hydrochloric acid anhydrous ethanol solution is 2.4 M; the dosage ratio of the hollow double-layer nano silica microspheres removing the silicon core and the hydrochloric acid anhydrous ethanol solution is 2 g: 100 mL; the stirring temperature is 60 °C and the time is 12 h.
[0026] Further, in the step (2) above, the dosage ratio of the hollow double-layer mesoporous silica microspheres, anhydrous toluene, 3-[2-(2-aminoethylamino)ethylamino]propyl-trimethoxysilane and n-octyltrichlorosilane is 0.5 g: 15 mL: 10 - 60 μL: 120 μL; the ultrasonic time is 10 min; the surface modification equipment is a shaker, the temperature is 30 °C, the rotation speed is 220 rpm, and the time is 2 h; the drying temperature is 60 °C.
[0027] Further, in the step (3) above, phospholipase A1 is first obtained from Chaetomium thermophilum / Fusarium oxysporum through protein engineering to get carboxylic ester hydrolase, and then obtained by deep fermentation of genetically modified Aspergillus oryzae micro; the pH of the PBS buffer solution is 6.0; the volume ratio of phospholipase A1 and the PBS buffer solution is 1:1 - 5; the dosage ratio of the bifunctional group modified hollow double-layer mesoporous silica microspheres and the phospholipase solution is 1 g: 100 mL; the incubation equipment is a shaker, the temperature is 30 °C, and the time is 1 h.
[0028] Further, in the above step (4), the phospholipid is at least one of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidic acid monophospholipid; the functional fatty acid ethyl ester is at least one of nervonic acid ethyl ester (Cis-15-Tatracosanic Ethyl Ester), arachidonic acid ethyl ester (ARA Ethyl Ester), docosahexaenoic acid ethyl ester (DHA Ethyl Ester), eicosapentaenoic acid ethyl ester (EPA Ethyl Ester), ethyl caprylate, and ethyl caprate; the mass ratio of the phospholipid to the functional fatty acid ethyl ester is 1:3 - 7; the addition amount of the immobilized phospholipase is 2.5% - 12.5% of the substrate mass; the addition amount of water is 0.25% - 1.5% of the substrate mass; the temperature of the transesterification reaction is 30 - 60 °C, and the time is 0.3 - 12 h. After the reaction is completed, 6 mL of acetone is added, and after centrifugal separation, the precipitate is washed 3 times with acetone. Then, 6 mL of chloroform-methanol (2:1, v:v) is added to the precipitate to dissolve the phospholipid, and the immobilized phospholipase is separated by centrifugation. Then, the immobilized phospholipase is washed with n-hexane. Finally, it is freeze-dried by a freeze dryer and then recycled. After 9 cycles of use, the relative enzyme activity is still 81% of the initial enzyme activity.
[0029] The present invention also claims to protect a structured phospholipid rich in functional fatty acids prepared by the above preparation method.
[0030] As can be seen from the above technical solutions, compared with the prior art, the beneficial effects of the present invention are as follows:
[0031] The present invention first prepares core / shell nanosilica microspheres by the sol-gel method, and prepares hollow double-layer mesoporous silica microspheres (HdlMS) by selectively etching the core; then, through the modification of the surface with hydrophobic alkyl silane (n-octyltrichlorosilane) and nitrogen-containing alkyl chain (3-[2-(2-aminoethylamino)ethylamino]propyl-trimethoxysilane), the hollow double-layer mesoporous silica microspheres modified with bifunctional groups (NH2 / C8-HdlMS) are obtained; then, using it as a carrier, phospholipase A1 is immobilized by the physical adsorption method to obtain an immobilized phospholipase (PLA1@NH2 / C8-HdlMS); finally, it is added to the reaction system of "phospholipid-functional fatty acid ethyl ester-n-hexane" to catalyze the transesterification reaction to obtain a structured phospholipid product rich in functional fatty acids such as nervonic acid and DHA. The preparation method of the present invention has the advantages of high efficiency of immobilized phospholipase, good stability, wide range of applicable substrates, mild conditions, and suitability for large-scale production. At the same time, the content of functional fatty acids in the structured phospholipid product prepared by the present invention reaches 39% - 82%. In addition, the immobilized phospholipase (PLA1@NH2 / C8-HdlMS) prepared by the present invention can be reused, and the relative enzyme activity remains above 80% after 9 times of use.
[0032] The carrier of the bifunctional group - modified hollow double - layer mesoporous silica microspheres (NH2 / C8 - HdlMS) used in the present invention provides abundant immobilization sites for phospholipase. Its unique mesoporous channel structure optimizes the mass transfer efficiency of the substrate and significantly improves the catalytic performance.
[0033] The bifunctional group modification strategy adopted in the present invention significantly improves the activity and stability of phospholipase and creates an ideal micro - environment for its catalytic reaction. Under this strategy, the reaction time is greatly shortened and the product yield is significantly increased. Description of the Drawings
[0034] Figure 1 SEM image (left) and TEM image (right) of the bifunctional group - modified hollow double - layer mesoporous silica microspheres prepared in Example 1.
[0035] Figure 2 Relative activity of the immobilized phospholipase after recovery and reuse prepared in Example 1. Detailed Description of the Invention
[0036] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.
[0037] Example 1
[0038] A preparation method of structured phospholipids rich in functional fatty acids specifically includes the following steps:
[0039] (1) Preparation of hollow double - layer mesoporous silica microspheres
[0040] ① Mix 400 mL of ethanol, 32 mL of water and 24 mL of ammonia water evenly, add 12 mL of tetraethyl orthosilicate, and stir - react at room temperature for 6 h to obtain solution A;
[0041] ② Mix 880 mL of water and 40 mL of ethanol evenly, add 4.8 g of cetyltrimethylammonium bromide, and stir for 6 h until completely dissolved to obtain solution B;
[0042] ③ Stir - mix solution A and solution B for 30 min, add 4 mL of tetraethyl orthosilicate, react for 12 h, filter and collect the white particles, wash with anhydrous ethanol, and dry to obtain core / shell nano - silica microspheres;
[0043] ④Disperse 3 g of core / shell nano-silica microspheres in 500 mL of sodium carbonate solution with a concentration of 0.2 M, react at 55 °C for 6 h to remove the silicon core of the solid silica, collect the white particles by suction filtration, wash with absolute ethanol, and dry to obtain hollow double-layer nano-silica microspheres with the silicon core removed;
[0044] ⑤Disperse 2 g of hollow double-layer nano-silica microspheres with the silicon core removed in 100 mL of hydrochloric acid absolute ethanol solution with a concentration of 2.4 M, stir at 60 °C for 12 h, collect the white particles by suction filtration, wash twice with absolute ethanol to remove tetraethyl orthosilicate, and dry to obtain hollow double-layer mesoporous silica microspheres;
[0045] (2) Preparation of bifunctional group-modified hollow double-layer mesoporous silica microspheres
[0046] Disperse 0.5 g of hollow double-layer mesoporous silica microspheres in 15 mL of anhydrous toluene, dropwise add 10 μL of 3-[2-(2-aminoethylamino)ethylamino]propyl-trimethoxysilane and 120 μL of n-octyltrichlorosilane successively, ultrasonicate for 10 min, shake on a shaker at 220 rpm at 30 °C for 2 h, filter, wash three times with absolute ethanol by filtration, and dry at 60 °C to obtain bifunctional group-modified hollow double-layer mesoporous silica microspheres;
[0047] (3) Preparation of immobilized phospholipase
[0048] First, mix 50 mL of phospholipase A1 and 50 mL of PBS buffer solution with a pH of 6.0 to obtain a phospholipase solution; then mix 1 g of bifunctional group-modified hollow double-layer mesoporous silica microspheres and 100 mL of the phospholipase solution, incubate on a shaker at 30 °C for 1 h, centrifuge, filter, wash three times with PBS, and freeze-dry to obtain immobilized phospholipase;
[0049] (4) Preparation of structured phospholipids rich in functional fatty acids
[0050] Add 150 mg of immobilized phospholipase to a 3 mL n-hexane solution containing 1 g of phosphatidylcholine and 5 g of ethyl docosahexaenoate, add 45 μL of water, carry out transesterification reaction at 50 °C for 8 h, after the reaction, add 6 mL of acetone, precipitate, centrifuge and separate, wash the precipitate three times with acetone, add 6 mL of chloroform / methanol solution with a volume ratio of 2:1 to the precipitate, dissolve the precipitate, recover the immobilized phospholipase, and carry out vacuum distillation on the organic phase to obtain structured phospholipids rich in functional fatty acids.
[0051] Example 2
[0052] A preparation method of structured phospholipids rich in functional fatty acids, specifically including the following steps:
[0053] (1) Preparation of Hollow Double-Layer Mesoporous Silica Microspheres
[0054] ① Mix 400 mL of ethanol, 32 mL of water, and 24 mL of ammonia water evenly, add 12 mL of tetraethyl orthosilicate, and stir and react at room temperature for 6 h to obtain solution A;
[0055] ② Mix 880 mL of water and 40 mL of ethanol evenly, add 4.8 g of cetyltrimethylammonium bromide, and stir for 6 h to completely dissolve to obtain solution B;
[0056] ③ Stir and mix solution A and solution B for 30 min, add 4 mL of tetraethyl orthosilicate, react for 12 h, filter and collect the white particles, wash with absolute ethanol, and dry to obtain core / shell nano-silica microspheres;
[0057] ④ Disperse 3 g of core / shell nano-silica microspheres in 500 mL of a sodium carbonate solution with a concentration of 0.2 M, react at 55 °C for 6 h to remove the silica core of the solid silica, filter and collect the white particles, wash with absolute ethanol, and dry to obtain hollow double-layer nano-silica microspheres with the silica core removed;
[0058] ⑤ Disperse 2 g of hollow double-layer nano-silica microspheres with the silica core removed in 100 mL of a hydrochloric acid absolute ethanol solution with a concentration of 2.4 M, stir at 60 °C for 12 h, filter and collect the white particles, wash twice repeatedly with absolute ethanol to remove tetraethyl orthosilicate, and dry to obtain hollow double-layer mesoporous silica microspheres;
[0059] (2) Preparation of Double-Functional Group Modified Hollow Double-Layer Mesoporous Silica Microspheres
[0060] Disperse 0.5 g of hollow double-layer mesoporous silica microspheres in 15 mL of anhydrous toluene, dropwise add 20 μL of 3-[2-(2-aminoethylamino)ethylamino]propyl-trimethoxysilane and 120 μL of n-octyltrichlorosilane successively, ultrasonicate for 10 min, shake on a shaker at 220 rpm at 30 °C for 2 h, filter, wash repeatedly with absolute ethanol three times, and dry at 60 °C to obtain double-functional group modified hollow double-layer mesoporous silica microspheres;
[0061] (3) Preparation of Immobilized Phospholipase
[0062] First, mix 50 mL of phospholipase A1 and 100 mL of a PBS buffer solution with a pH of 6.0 to obtain a phospholipase solution; then mix 1 g of double-functional group modified hollow double-layer mesoporous silica microspheres and 100 mL of the phospholipase solution, incubate on a shaker at 30 °C for 1 h, centrifuge, filter, wash three times repeatedly with PBS, and freeze-dry to obtain immobilized phospholipase;
[0063] (4) Preparation of Structured Phospholipids Rich in Functional Fatty Acids
[0064] 200 mg of immobilized phospholipase was added to a 3 mL n - hexane solution containing a mixture of 1 g of phosphatidylcholine and phosphatidylethanolamine and 3 g of ethyl eicosapentaenoate. 20 μL of water was added, and the transesterification reaction was carried out at 30 °C for 6 h. After the reaction, 6 mL of acetone was added to precipitate, and then centrifuged. The precipitate was washed three times with acetone. 6 mL of a chloroform / methanol solution with a volume ratio of 2:1 was added to the precipitate, and the precipitate dissolved. The immobilized phospholipase was recovered, and the organic phase was distilled under reduced pressure to obtain structured phospholipids rich in functional fatty acids.
[0065] Example 3
[0066] A method for preparing structured phospholipids rich in functional fatty acids specifically includes the following steps:
[0067] (1) Preparation of hollow double - layer mesoporous silica microspheres
[0068] ① 400 mL of ethanol, 32 mL of water and 24 mL of ammonia water were mixed evenly, and 12 mL of tetraethyl orthosilicate was added. The mixture was stirred at room temperature for 6 h to obtain solution A;
[0069] ② 880 mL of water and 40 mL of ethanol were mixed evenly, and 4.8 g of cetyltrimethylammonium bromide was added. The mixture was stirred for 6 h until completely dissolved to obtain solution B;
[0070] ③ Solution A and solution B were stirred and mixed for 30 min, 4 mL of tetraethyl orthosilicate was added, and the reaction was carried out for 12 h. The white particles were collected by suction filtration, washed with absolute ethanol, and dried to obtain core / shell nano - silica microspheres;
[0071] ④ 3 g of core / shell nano - silica microspheres were dispersed in 500 mL of a 0.2 M sodium carbonate solution, and the reaction was carried out at 55 °C for 6 h to remove the silicon core of the solid silica. The white particles were collected by suction filtration, washed with absolute ethanol, and dried to obtain hollow double - layer nano - silica microspheres with the silicon core removed;
[0072] ⑤ 2 g of hollow double - layer nano - silica microspheres with the silicon core removed were dispersed in 100 mL of a 2.4 M hydrochloric acid - absolute ethanol solution, and the mixture was stirred at 60 °C for 12 h. The white particles were collected by suction filtration, and washed twice with absolute ethanol to remove tetraethyl orthosilicate, and then dried to obtain hollow double - layer mesoporous silica microspheres;
[0073] (2) Preparation of hollow double - layer mesoporous silica microspheres modified with bifunctional groups
[0074] Disperse 0.5 g of hollow double-layer mesoporous silica microspheres in 15 mL of anhydrous toluene, dropwise add 40 μL of 3-[2-(2-aminoethylamino)ethylamino]propyl-trimethoxysilane and 120 μL of n-octyltrichlorosilane successively, ultrasonicate for 10 min, shake on a shaker at 220 rpm at 30 °C for 2 h, filter, repeat the filtration and washing three times with anhydrous ethanol, and dry at 60 °C to obtain bifunctional group-modified hollow double-layer mesoporous silica microspheres;
[0075] (3) Preparation of immobilized phospholipase
[0076] First, mix 50 mL of phospholipase A1 with 150 mL of PBS buffer solution with a pH of 6.0 to obtain a phospholipase solution; then mix 1 g of bifunctional group-modified hollow double-layer mesoporous silica microspheres with 100 mL of the phospholipase solution, incubate on a shaker at 30 °C for 1 h, centrifuge, filter, repeat the washing three times with PBS, and freeze-dry to obtain immobilized phospholipase;
[0077] (4) Preparation of structured phospholipids rich in functional fatty acids
[0078] Add 1.0 g of immobilized phospholipase to a 3 mL n-hexane solution containing 1 g of a mixture of phosphatidylcholine and phosphatidylinositol and 7 g of ethyl octanoate, add 60 μL of water, carry out a transesterification reaction at 60 °C for 12 h. After the reaction is completed, add 6 mL of acetone, precipitate, centrifuge and separate, wash the precipitate three times with acetone, add 6 mL of a chloroform / methanol solution with a volume ratio of 2:1 to the precipitate, dissolve the precipitate, recover the immobilized phospholipase, and carry out vacuum distillation on the organic phase to obtain structured phospholipids rich in functional fatty acids.
[0079] Example 4
[0080] A preparation method of structured phospholipids rich in functional fatty acids, specifically comprising the following steps:
[0081] (1) Preparation of hollow double-layer mesoporous silica microspheres
[0082] ① Mix 400 mL of ethanol, 32 mL of water and 24 mL of ammonia water evenly, add 12 mL of tetraethyl orthosilicate, and stir and react at room temperature for 6 h to obtain solution A;
[0083] ② Mix 880 mL of water and 40 mL of ethanol evenly, add 4.8 g of cetyltrimethylammonium bromide, and stir for 6 h until completely dissolved to obtain solution B;
[0084] ③ Stir and mix solution A and solution B for 30 min, add 4 mL of tetraethyl orthosilicate, react for 12 h, filter and collect the white particles, wash with anhydrous ethanol, and dry to obtain core / shell nanosilica microspheres;
[0085] ④Disperse 3 g of core / shell nano-silica microspheres in 500 mL of sodium carbonate solution with a concentration of 0.2 M, react at 55 °C for 6 h to remove the silica core of the solid silica, filter with suction to collect the white particles, wash with absolute ethanol, and dry to obtain hollow double-layer nano-silica microspheres with the silica core removed;
[0086] ⑤Disperse 2 g of hollow double-layer nano-silica microspheres with the silica core removed in 100 mL of hydrochloric acid absolute ethanol solution with a concentration of 2.4 M, stir at 60 °C for 12 h, filter with suction to collect the white particles, wash twice repeatedly with absolute ethanol to remove tetraethyl orthosilicate, and dry to obtain hollow double-layer mesoporous silica microspheres;
[0087] (2) Preparation of bifunctional group-modified hollow double-layer mesoporous silica microspheres
[0088] Disperse 0.5 g of hollow double-layer mesoporous silica microspheres in 15 mL of anhydrous toluene, dropwise add 60 μL of 3-[2-(2-aminoethylamino)ethylamino]propyl-trimethoxysilane and 120 μL of n-octyltrichlorosilane successively, ultrasonicate for 10 min, shake on a shaker at 220 rpm at 30 °C for 2 h, filter, wash repeatedly with absolute ethanol three times, and dry at 60 °C to obtain bifunctional group-modified hollow double-layer mesoporous silica microspheres;
[0089] (3) Preparation of immobilized phospholipase
[0090] First, mix 50 mL of phospholipase A1 and 200 mL of PBS buffer solution with a pH of 6.0 to obtain a phospholipase solution; then mix 1 g of bifunctional group-modified hollow double-layer mesoporous silica microspheres and 100 mL of the phospholipase solution, incubate on a shaker at 30 °C for 1 h, centrifuge, filter, wash repeatedly with PBS three times, and freeze-dry to obtain immobilized phospholipase;
[0091] (4) Preparation of structured phospholipids rich in functional fatty acids
[0092] Add 800 mg of immobilized phospholipase to 3 mL of n-hexane solution containing 1 g of phospholipids and 5 g of ethyl caprate, add 60 μL of water, carry out transesterification reaction at 55 °C for 12 h, after the reaction, add 6 mL of acetone, precipitate, centrifuge and separate, wash the precipitate three times with acetone, add 6 mL of chloroform / methanol solution with a volume ratio of 2:1 to the precipitate, the precipitate dissolves, recover the immobilized phospholipase, and distill the organic phase under reduced pressure to obtain structured phospholipids rich in functional fatty acids.
[0093] Example 5
[0094] A preparation method of structured phospholipids rich in functional fatty acids specifically includes the following steps:
[0095] (1) Preparation of hollow double-layer mesoporous silica microspheres
[0096] ① Mix 400 mL of ethanol, 32 mL of water, and 24 mL of ammonia water evenly, add 12 mL of tetraethyl orthosilicate, and stir and react at room temperature for 6 h to obtain solution A;
[0097] ② Mix 880 mL of water and 40 mL of ethanol evenly, add 4.8 g of cetyltrimethylammonium bromide, and stir for 6 h until completely dissolved to obtain solution B;
[0098] ③ Stir and mix solution A and solution B for 30 min, add 4 mL of tetraethyl orthosilicate, react for 12 h, filter with suction to collect white particles, wash with absolute ethanol, and dry to obtain core / shell nano-silica microspheres;
[0099] ④ Disperse 3 g of core / shell nano-silica microspheres in 500 mL of sodium carbonate solution with a concentration of 0.2 M, react at 55 °C for 6 h to remove the silicon core of solid silica, filter with suction to collect white particles, wash with absolute ethanol, and dry to obtain hollow double-layer nano-silica microspheres with the silicon core removed;
[0100] ⑤ Disperse 2 g of hollow double-layer nano-silica microspheres with the silicon core removed in 100 mL of hydrochloric acid absolute ethanol solution with a concentration of 2.4 M, stir at 60 °C for 12 h, filter with suction to collect white particles, wash twice repeatedly with absolute ethanol to remove tetraethyl orthosilicate, and dry to obtain hollow double-layer mesoporous silica microspheres;
[0101] (2) Preparation of bifunctional group-modified hollow double-layer mesoporous silica microspheres
[0102] Disperse 0.5 g of hollow double-layer mesoporous silica microspheres in 15 mL of anhydrous toluene, dropwise add 60 μL of 3-[2-(2-aminoethylamino)ethylamino]propyl-trimethoxysilane and 120 μL of n-octyltrichlorosilane successively, ultrasonicate for 10 min, shake on a shaker at 220 rpm at 30 °C for 2 h, filter, wash repeatedly with absolute ethanol three times, and dry at 60 °C to obtain bifunctional group-modified hollow double-layer mesoporous silica microspheres;
[0103] (3) Preparation of immobilized phospholipase
[0104] First, mix 50 mL of phospholipase A1 and 250 mL of PBS buffer solution with a pH of 6.0 to obtain a phospholipase solution; then mix 1 g of bifunctional group-modified hollow double-layer mesoporous silica microspheres and 100 mL of the phospholipase solution, incubate on a shaker at 30 °C for 1 h, centrifuge, filter, wash three times repeatedly with PBS, and freeze-dry to obtain immobilized phospholipase;
[0105] (4) Preparation of structured phospholipids rich in functional fatty acids
[0106] 100 mg of immobilized phospholipase was added to a 3 mL n - hexane solution containing 1 g of a mixture of phosphatidylcholine, phosphatidylethanolamine, and phosphatidic acid monophosphate and 3 g of ethyl linolenate. 30 μL of water was added, and the transesterification reaction was carried out at 40 °C for 5 h. After the reaction, 6 mL of acetone was added to precipitate the product, followed by centrifugation. The precipitate was washed three times with acetone. 6 mL of a chloroform / methanol solution with a volume ratio of 2:1 was added to the precipitate, and the precipitate dissolved. The immobilized phospholipase was recovered, and the organic phase was distilled under reduced pressure to obtain structured phospholipids rich in functional fatty acids.
[0107] Example 6
[0108] A method for preparing structured phospholipids rich in functional fatty acids specifically includes the following steps:
[0109] (1) Preparation of hollow double - layer mesoporous silica microspheres
[0110] ① 400 mL of ethanol, 32 mL of water, and 24 mL of ammonia water were mixed evenly, and 12 mL of tetraethyl orthosilicate was added. The mixture was stirred at room temperature for 6 h to obtain solution A;
[0111] ② 880 mL of water and 40 mL of ethanol were mixed evenly, and 4.8 g of cetyltrimethylammonium bromide was added. The mixture was stirred for 6 h until completely dissolved to obtain solution B;
[0112] ③ Solution A and solution B were stirred and mixed for 30 min, 4 mL of tetraethyl orthosilicate was added, and the reaction was carried out for 12 h. The white particles were collected by suction filtration, washed with absolute ethanol, and dried to obtain core / shell nanosilica microspheres;
[0113] ④ 3 g of core / shell nanosilica microspheres were dispersed in 500 mL of a 0.2 M sodium carbonate solution, and the reaction was carried out at 55 °C for 6 h to remove the silica core of the solid silica. The white particles were collected by suction filtration, washed with absolute ethanol, and dried to obtain hollow double - layer nanosilica microspheres with the silica core removed;
[0114] ⑤ 2 g of hollow double - layer nanosilica microspheres with the silica core removed were dispersed in 100 mL of a 2.4 M hydrochloric acid - absolute ethanol solution, stirred at 60 °C for 12 h, the white particles were collected by suction filtration, and washed twice with absolute ethanol to remove tetraethyl orthosilicate, and then dried to obtain hollow double - layer mesoporous silica microspheres;
[0115] (2) Preparation of hollow double - layer mesoporous silica microspheres modified with bifunctional groups
[0116] Disperse 0.5 g of hollow double-layer mesoporous silica microspheres in 15 mL of anhydrous toluene, dropwise add 60 μL of 3-[2-(2-aminoethylamino)ethylamino]propyl-trimethoxysilane and 120 μL of n-octyltrichlorosilane successively, ultrasonicate for 10 min, shake on a shaker at 220 rpm at 30 °C for 2 h, filter, wash repeatedly with anhydrous ethanol three times, and dry at 60 °C to obtain bifunctional group-modified hollow double-layer mesoporous silica microspheres;
[0117] (3) Preparation of immobilized phospholipase
[0118] First, mix 50 mL of phospholipase A1 and 50 mL of PBS buffer solution with a pH of 6.0 to obtain a phospholipase solution; then mix 1 g of bifunctional group-modified hollow double-layer mesoporous silica microspheres and 100 mL of the phospholipase solution, incubate on a shaker at 30 °C for 1 h, centrifuge, filter, wash repeatedly with PBS three times, and freeze-dry to obtain immobilized phospholipase;
[0119] (4) Preparation of structured phospholipids rich in functional fatty acids
[0120] Add 180 mg of immobilized phospholipase to a 3 mL n-hexane solution containing 1 g of phosphatidylcholine and phosphatidic acid monophospholipid and 5 g of ethyl nervonate, add 30 μL of water, carry out transesterification reaction at 50 °C for 0.3 h. After the reaction, add 6 mL of acetone to precipitate, centrifuge and separate, wash the precipitate three times with acetone, add 6 mL of chloroform / methanol solution with a volume ratio of 2:1 to the precipitate to dissolve the precipitate, recover the immobilized phospholipase, and distill the organic phase under reduced pressure to obtain structured phospholipids rich in functional fatty acids.
[0121] Material characterization and catalytic performance
[0122] 1. SEM and TEM characterization
[0123] Carry out SEM and TEM characterization on the bifunctional group-modified hollow double-layer mesoporous silica microspheres prepared in Example 1 respectively.
[0124] It can be seen from Figure 1 that the hollow double-layer mesoporous silica microspheres exhibit a uniform double-layer hollow spherical morphology, with an average diameter of about 469 nm. The shell thickness of the hollow double-layer mesoporous silica microspheres is about 56 nm, while the inner shell thickness is about 26 nm, showing good structural regularity.
[0125] 2. Incorporation rate of functional fatty acids
[0126] Take the structured phospholipids rich in functional fatty acids prepared in Examples 1-6 respectively, carry out methylation respectively, detect by GC, and calculate the incorporation rate of functional fatty acids.
[0127] The results are shown in Table 1.
[0128] Table 1 Incorporation Rates of Functional Fatty Acids in Structured Phospholipids of Examples 1 - 6
[0129] Structural phospholipid Functional fatty acid Incorporation rate / % Example 1 Docosahexaenoic acid 67 Example 2 Eicosapentaenoic acid 49 Example 3 Caprylic acid 39 Example 4 Capric acid 49 Example 5 Linolenic acid 60 Example 6 Nervonic acid 82
[0130] As can be seen from Table 1, phospholipase A1 has a greater preference for long - chain and unsaturated fatty acids, and the incorporation rate of medium - chain fatty acids is less than 50%; while the incorporation rate of long - chain unsaturated fatty acids all exceeds 60%.
[0131] 3. Composition Content of Phospholipid Fatty Acids
[0132] After different functional phospholipids are incorporated, the composition content of phospholipid fatty acids is shown in Table 2.
[0133] Table 2 Composition Content of Phospholipid Fatty Acids
[0134]
[0135]
[0136] 4. Relative Activity of Immobilized Phospholipase after Recycling and Reuse
[0137] The immobilized phospholipase recovered from Example 1 was washed 3 times with n - hexane solution, dried, and put into a new reaction for repetition. The conditions were the same as those in Example 1, and the results are as Figure 2 shown.
[0138] As Figure 2 can be seen, taking the first functional fatty acid incorporation rate as 100% activity, after repeating 9 times, the relative enzyme activity of the immobilized phospholipase remains above 80%.
[0139] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather to the broadest scope consistent with the principles and novel features disclosed herein.
Claims
1. A preparation method of a structured phospholipid rich in functional fatty acids, characterized in that, Specifically, it includes the following steps: (1) Preparation of hollow double-layer mesoporous silica microspheres ① Mix ethanol, water and ammonia water evenly, add tetraethyl orthosilicate, and stir to react to obtain solution A; ② Mix water and ethanol evenly, add cetyltrimethylammonium bromide, and stir to dissolve to obtain solution B; ③ Stir and mix solution A and solution B, add tetraethyl orthosilicate, react, filter by suction, wash, and dry to obtain core / shell nanosilica microspheres; ④ Disperse the core / shell nanosilica microspheres in sodium carbonate solution, react, filter by suction, wash, and dry to obtain hollow double-layer nanosilica microspheres with the silicon core removed; ⑤ Disperse the hollow double-layer nanosilica microspheres with the silicon core removed in hydrochloric acid anhydrous ethanol solution, stir, filter by suction, wash, and dry to obtain hollow double-layer mesoporous silica microspheres; (2) Preparation of bifunctional group-modified hollow double-layer mesoporous silica microspheres Disperse the hollow double-layer mesoporous silica microspheres in anhydrous toluene, dropwise add 3-[2-(2-aminoethylamino)ethylamino]propyl-trimethoxysilane and n-octyltrichlorosilane successively, ultrasonically treat for surface modification, filter, wash the filter cake, and dry to obtain bifunctional group-modified hollow double-layer mesoporous silica microspheres; (3) Preparation of immobilized phospholipase First, mix phospholipase A1 and PBS buffer solution to obtain a phospholipase solution; then mix the bifunctional group-modified hollow double-layer mesoporous silica microspheres and the phospholipase solution, incubate, centrifuge, filter, wash, and freeze-dry to obtain immobilized phospholipase; (4) Preparation of structured phospholipids rich in functional fatty acids Add the immobilized phospholipase to a n-hexane solution containing phospholipids and functional fatty acid ethyl esters, add water, carry out transesterification reaction, separate and recover the immobilized phospholipase, add acetone, and precipitate, which is the structured phospholipids rich in functional fatty acids.
2. The preparation method of a structured phospholipid rich in functional fatty acids according to claim 1, wherein, In step (1)①, the dosage ratio of ethanol, water, ammonia water and tetraethyl orthosilicate is 400 mL: 32 mL: 24 mL: 12 mL; the stirring reaction time is 6 h.
3. The preparation method of a structured phospholipid rich in functional fatty acids according to claim 1, characterized in that, In step (1)②, the dosage ratio of water, ethanol and cetyltrimethylammonium bromide is 880 mL: 40 mL: 4.8 g; the stirring dissolution time is 6 h.
4. The preparation method of a structured phospholipid rich in functional fatty acids according to claim 1, characterized in that, In step (1)③, the stirring mixing time is 30 min; the addition amount of tetraethyl orthosilicate is 4 mL; the reaction time is 12 h.
5. The preparation method of a structured phospholipid rich in functional fatty acids according to claim 1, characterized in that, In step (1)④, the concentration of the sodium carbonate solution is 0.2 - 0.4 M; the dosage ratio of the core / shell nanosilica microspheres and the sodium carbonate solution is 3 g: 500 mL; the reaction temperature is 55 °C and the time is 4 - 7 h.
6. The preparation method of a structured phospholipid rich in functional fatty acids according to claim 1, characterized in that, In step (1)⑤, the concentration of the hydrochloric acid anhydrous ethanol solution is 2.4 M; the dosage ratio of the hollow double-layer nanosilica microspheres with the silicon core removed and the hydrochloric acid anhydrous ethanol solution is 2 g: 100 mL; the stirring temperature is 60 °C and the time is 12 h.
7. The preparation method of a structured phospholipid rich in functional fatty acids according to claim 1, characterized in that, In step (2), the dosage ratio of the hollow double-layer mesoporous silica microspheres, anhydrous toluene, 3-[2-(2-aminoethylamino)ethylamino]propyl-trimethoxysilane, and n-octyltrichlorosilane is 0.5 g: 15 mL: 10 - 60 μL: 120 μL; the time of the ultrasonic treatment is 10 min; the device for surface modification is a shaker, the temperature is 30 °C, the rotation speed is 220 rpm, and the time is 2 h; the drying temperature is 60 °C.
8. The preparation method of a structured phospholipid rich in functional fatty acids according to claim 1, characterized in that, In step (3), the pH of the PBS buffer solution is 6.0; the volume ratio of the phospholipase A1 to the PBS buffer solution is 1:1 - 5; the dosage ratio of the bifunctional group-modified hollow double-layer mesoporous silica microspheres to the phospholipase solution is 1 g: 100 mL; the device for incubation is a shaker, the temperature is 30 °C, and the time is 1 h.
9. The preparation method of a structured phospholipid rich in functional fatty acids according to claim 1, characterized in that, In step (4), the phospholipid is at least one of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidic acid monophosphate; the functional fatty acid ethyl ester is at least one of nervonic acid ethyl ester, eicosatetraenoic acid ethyl ester, docosahexaenoic acid ethyl ester, eicosapentaenoic acid ethyl ester, ethyl octanoate, and ethyl decanoate; the mass ratio of the phospholipid to the functional fatty acid ethyl ester is 1:3 - 7; the addition amount of the immobilized phospholipase is 2.5% - 12.5% of the substrate mass; the addition amount of water is 0.25% - 1.5% of the substrate mass; the temperature of the transesterification reaction is 30 - 60 °C, and the time is 0.3 - 12 h.
10. A structured phospholipid rich in functional fatty acids prepared by the preparation method according to any one of claims 1 - 9.