Process for the production of glycerides using immobilized enzymes

Abstract

The application discloses a method for producing glycerol diester by using immobilized enzyme catalysis, and relates to the technical field of biological enzyme, which comprises the following steps: step I, obtaining mesoporous silica; step II, preparing an immobilized carrier; step III, preparing an immobilized enzyme; and step IV, preparing glycerol diester, and the following conditions are met: condition A, in the step I, the pore size of the mesoporous silica is 5nm to 30nm, the specific surface area is 500m 2 / g to 1200m 2 / g, and the pore volume is 0.8cm 3 / g to 2.0 cm 3 / g; and condition B, in the step II, the molecular weight of the water-soluble chitosan is 5kDa to 50kDa, and the degree of deacetylation is 70% to 95%. The immobilized carrier with a flexible composite network is prepared, the flexible cover of porcine pancreatic lipase is combined, the cover can be pulled away from the active center to expose the active site, and the cover is not easy to close again.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological enzymes, and particularly relates to a method for catalytically producing diglyceride by using immobilized enzyme. BACKGROUND

[0002] The enzyme method for producing diglyceride usually takes lipase as a catalyst to catalyze the esterification reaction of glycerol and fatty acid or the partial hydrolysis reaction of triglyceride. However, free lipase has the problems of poor stability, difficult recycling and reuse, and difficult separation from products after reaction, which limits its industrial application.

[0003] In order to solve the above problems, the immobilized enzyme technology is widely used. By fixing the lipase on the carrier, the stability of the enzyme can be significantly improved, the reuse of the enzyme can be realized, and the product separation process can be simplified. However, the catalytic activity of lipase (such as porcine pancreatic lipase) depends on the exposure of its active center, and the active center is usually covered by its own flexible cap structure. The cap needs to be opened through the interaction with the substrate, but the initial binding efficiency of the cap and the substrate is limited, which leads to insufficient exposure. Moreover, when the substrate molecule leaves the active center or the local concentration fluctuates, the cap is easy to close again, which restricts the catalytic efficiency of the lipase and leads to low yield. SUMMARY

[0004] Therefore, the present application provides a method for catalytically producing diglyceride by using immobilized enzyme. The immobilized carrier with flexible composite network is prepared, which can pull the flexible cap of porcine pancreatic lipase away from the active center to expose the active site, and the cap is not easy to close again.

[0005] A method for catalytically producing diglyceride by using immobilized enzyme, comprising the following steps: step I, obtaining mesoporous silica; step II, preparing an immobilized carrier: mixing the mesoporous silica in step I, glutaraldehyde solution and water-soluble chitosan, stirring at 100 rpm to 300 rpm at 20℃ to 40℃ for 2 hours to 12 hours, and obtaining the immobilized carrier; step III, preparing an immobilized enzyme: mixing the immobilized carrier in step II and a lipase solution, and performing a solidification reaction at 100 rpm to 200 rpm at 20℃ to 37℃ for 2 hours to 24 hours, and obtaining the immobilized enzyme; step IV, catalytically preparing diglyceride: mixing an unsaturated long-chain fatty acid, glycerol and the immobilized enzyme in step III, and stirring at 100 rpm to 500 rpm at 30℃ to 60℃ and pH 6.0-8.0 for 4 hours to 48 hours, and obtaining the diglyceride.

[0006] and at least one of the following conditions is met: condition A, in step I, the pore size of the mesoporous silica is 5nm to 30nm, the specific surface area is 500m 2 / g to 1200 m 2 / g, pore volume is 0.8 cm 3 / g to 2.0 cm 3 / g; in condition B, step II, the molecular weight of the water-soluble chitosan is 5 kDa to 50 kDa, and the degree of deacetylation is 70% to 95%. In this way, the water-soluble chitosan forms a stable flexible composite network on the surface and in the pores of the mesoporous silica, and an immobilized carrier with a flexible composite network is prepared.

[0007] Preferably, at least one of the following conditions is met: condition a, the pore size of the mesoporous silica is 15 nm to 30 nm, the specific surface area is 800 m 2 / g to 1200 m 2 / g, pore volume is 0.8 cm 3 / g to 1.5 cm 3 / g; condition b, the molecular weight of the water-soluble chitosan is 10 kDa to 30 kDa, and the degree of deacetylation is 80% to 90%; condition c, the particle size of the mesoporous silica is 100 nm to 300 nm. In this way, the stability of the flexible composite network of the prepared immobilized carrier is more optimal.

[0008] The preparation process of the mesoporous silica includes: dissolving a surfactant as a template agent in a solvent, promoting the template agent to self-assemble into micelles with a specific structure by adjusting the pH value of the solution, adding a silicon source under stirring to make the silicon source hydrolyze and polycondensate around the template agent micelles, and then performing crystallization treatment on the reaction system to promote the ordering and stabilization of the mesoporous structure. After crystallization, the initial product is obtained by separation, washing, and drying, and then the template agent is removed by calcination or solvent extraction, and the mesoporous silica is obtained. By adjusting the type and amount of the template agent, the concentration of the silicon source and the ratio of the template agent, the pH value of the reaction system, the crystallization temperature and time, and the calcination conditions, the particle size, pore size, specific surface area, and pore volume of the product mesoporous silica can be controlled within the above range.

[0009] In some embodiments, the water-soluble chitosan is hydroxyethyl chitosan and / or hydroxypropyl chitosan, and the mass concentration of the glutaraldehyde solution is 0.5% to 5%. In this way, the hydroxyl groups, amino groups, and unreacted aldehyde groups on the molecular chain of the water-soluble chitosan form specific and directional hydrogen bonds with the polar residues on the cover structure surface of the porcine pancreatic lipase, which further pulls the cover structure away from the active center.

[0010] In some embodiments, in step III, the porcine pancreatic lipase solution is a solution with a concentration of 0.1 mg / mL to 5 mg / mL obtained by dissolving porcine pancreatic lipase in a phosphate buffer with a pH of 6.0-8.0. In this way, it is beneficial to ensure that the porcine pancreatic lipase maintains its catalytic activity before being combined with the immobilized carrier, and a suitable concentration is beneficial for its specific binding with the active sites on the surface of the immobilized carrier (such as the amino and hydroxyl groups of water-soluble chitosan).

[0011] In some embodiments, the porcine pancreatic lipase has an enzyme activity of 1000 U / g to 10000 U / g.

[0012] In some embodiments, the mass ratio of the mesoporous silica, the glutaraldehyde solution, and the water-soluble chitosan is 1:(0.5-5):(10-50). The above three have a synergistic effect, and controlling the mass ratio of the above three within the above range is beneficial on the one hand for the water-soluble chitosan to form a stable flexible composite network on the surface and in the pores of the mesoporous silica, and on the other hand for the hydroxyl, amino, and unreacted aldehyde groups on the molecular chain of the water-soluble chitosan to specifically and directionally bind with the polar residues on the surface of the lid structure of the porcine pancreatic lipase to form an open conformation of the lid structure away from the active center, thereby improving the yield of diglyceride. Preferably, the mass ratio of the mesoporous silica, the glutaraldehyde solution, and the water-soluble chitosan is 1:(1-3):(15-25).

[0013] In some embodiments, in step III, the immobilized carrier and the lipase solution are mixed at a mass-volume ratio of 1 g:(5-50 mL). In this way, it is beneficial to achieve both high immobilization efficiency and high activity of the immobilized enzyme.

[0014] In some embodiments, the unsaturated long-chain fatty acid is oleic acid, linoleic acid, linolenic acid, or arachidonic acid, and the carbon chain length is C16 to C22. Preferably, the carbon chain length is C18 to C20.

[0015] In some embodiments, the mass ratio of the unsaturated long-chain fatty acid, the glycerol, and the immobilized enzyme is 1:(0.2-2):(0.01-0.2). By adjusting the ratio of glycerol to unsaturated long-chain fatty acid, the selectivity of the product is high, and the generation of monoglyceride and triglyceride is reduced. By adjusting the ratio of the three, it is beneficial to achieve efficient catalysis and further improve the yield of diglyceride. Preferably, the mass ratio of the unsaturated long-chain fatty acid, the glycerol, and the immobilized enzyme is 1:1:(0.05-0.1).

[0016] Compared with the prior art, the present application has the following advantages and beneficial effects:

[0017] The application first prepares an immobilized carrier with a flexible composite network by mixing mesoporous silica, glutaraldehyde solution and water-soluble chitosan under the condition of 20-40°C and 100-300 rpm. Specifically, the silicon hydroxyl groups on the surface of mesoporous silica can form hydrogen bonds with the hydroxyl groups and amino groups of water-soluble chitosan, thereby achieving preliminary adsorption. The aldehyde groups of glutaraldehyde solution as a bifunctional crosslinking agent can react with the amino groups of chitosan to form Schiff base (-C=N-), so that the water-soluble chitosan forms a stable flexible composite network on the surface and in the pores of mesoporous silica. The application further adjusts the structure parameters of mesoporous silica and the performance parameters of water-soluble chitosan to be within a suitable range. In this way, the flexible composite network is conducive to forming specific and directional hydrogen bonds with the polar residues on the surface of the lid structure of porcine pancreatic lipase (PPL), thereby pulling the lid structure away from the active center to expose the active site, and the flexible composite network can further support the open conformation of the lid structure through spatial restriction. DETAILED DESCRIPTION

[0018] In order to make the purpose, technical scheme and advantages of the application more clear, the application will be further described in detail below with examples. It should be understood that the specific examples described herein are only used to explain the application and not to limit the application.

[0019] A method for catalytically producing diglyceride by using immobilized enzyme

[0020] Step I, obtaining mesoporous silica;

[0021] The surfactant is used as a template agent, which is dissolved in a solvent. The pH value of the solution is adjusted to promote the self-assembly of the template agent into micelles with a specific structure. Under stirring conditions, a silicon source is added to make the silicon source hydrolyze and polymerize around the template agent micelles. Then the reaction system is subjected to crystallization treatment to promote the ordering and stabilization of the mesoporous structure. After crystallization, the initial product is obtained by separation, washing and drying. Then the template agent is removed by calcination or solvent extraction, and mesoporous silica is obtained.

[0022] Step II, preparing an immobilized carrier: mixing mesoporous silica, glutaraldehyde solution and water-soluble chitosan in step I, and stirring at 100-300 rpm at 20-40°C to obtain the immobilized carrier.

[0023] For example, in step II, the stirring temperature is 20°C, 25°C, 30°C, 35°C, 37°C, 39°C, 40°C or any value within the range composed of any two of the above values; and the stirring speed is 100 rpm, 120 rpm, 150 rpm, 180 rpm, 200 rpm, 250 rpm, 300 rpm or any value within the range composed of any two of the above values.

[0024] Step III, preparing immobilized enzyme: mixing the immobilized carrier in step II with porcine pancreatic lipase solution, and carrying out solidification reaction at 20-37°C at 100-200 rpm, to obtain;

[0025] Exemplarily, in step III, the reaction temperature is 20°C, 25°C, 30°C, 35°C, 37°C, or any value within the range consisting of any two of the above values; the stirring speed is 100 rpm, 120 rpm, 150 rpm, 180 rpm, 200 rpm, or any value within the range consisting of any two of the above values.

[0026] Step IV, preparing diglyceride: mixing the immobilized enzyme in step III, unsaturated long-chain fatty acid, and glycerol, and stirring at 100-500 rpm at 30-60°C and pH 6.0-8.0, to obtain diglyceride.

[0027] Exemplarily, in step IV, the stirring temperature is 30°C, 35°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, or any value within the range consisting of any two of the above values; the stirring speed is 100 rpm, 120 rpm, 150 rpm, 180 rpm, 200 rpm, 300 rpm, 350 rpm, 400 rpm, 500 rpm, or any value within the range consisting of any two of the above values; the pH is 6.0, 6.3, 6.5, 7.0, 7.5, 8.0, or any value within the range consisting of any two of the above values.

[0028] and at least one of the following conditions is met:

[0029] Condition A: in step I, the pore size of the mesoporous silica is 5-30 nm, the specific surface area is 500-1200 m 2 / g, and the pore volume is 0.8-2.0 cm 2 / g. 3 / g. 3 / g; Condition B: in step II, the molecular weight of the water-soluble chitosan is 5-50 kDa, and the degree of deacetylation is 70-95%.

[0030] Exemplarily, the pore size of the mesoporous silica is 5 nm, 8 nm, 10 nm, 15 nm, 16 nm, 18 nm, 19 nm, 20 nm, 25 nm, 30 nm, or any value within the range consisting of any two of the above values. The specific surface area of the mesoporous silica is 500 m 2 / g, 600 m 2 / g, 800 m 2 / g、900m 2 / g, 1000m 2 / g、1100m 2 / g、1200m 2 / g or any value within the range of any two of the above values. The pore volume of mesoporous silica is 0.8 cm³. 3 / g, 1.0cm 3 / g, 1.2cm 3 / g, 1.3cm 3 / g, 1.5cm 3 / g, 1.8cm 3 / g, 2.0cm 3 / g or any value within the range consisting of any two of the above values.

[0031] For example, the molecular weight of water-soluble chitosan is 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, or any value within the range of any two of the above values. The degree of deacetylation of water-soluble chitosan is 70%, 75%, 80%, 85%, 90%, 95%, or any value within the range of any two of the above values.

[0032] Preferably, the mesoporous silica has a particle size of 100 nm to 300 nm. Exemplarily, the mesoporous silica has a particle size of 100 nm, 150 nm, 180 nm, 200 nm, 250 nm, 300 nm, or any value within a range consisting of any two of the above values.

[0033] Preferably, the water-soluble chitosan is hydroxyethyl chitosan and / or hydroxypropyl chitosan, and the mass concentration of the glutaraldehyde solution is 0.5% to 5%. Exemplarily, the mass concentration of the glutaraldehyde solution is 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, or any value within the range of any two of the above values.

[0034] Preferably, in step III, the porcine pancreatic lipase solution is a solution with a concentration of 0.1 mg / mL to 5 mg / mL obtained by dissolving porcine pancreatic lipase in a phosphate buffer with a pH of 6.0-8.0. Exemplarily, the pH of the phosphate buffer is 6.0, 6.5, 6.8, 7.0, 7.5, 7.8, 8.0, or any value within the range of any two of the above values. The concentration of the porcine pancreatic lipase solution is 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 5 mg / mL, or any value within the range of any two of the above values.

[0035] Preferably, the porcine pancreatic lipase has an enzyme activity of 1000 U / g to 10000 U / g. Exemplarily, the porcine pancreatic lipase has enzyme activities of 1000 U / g, 2000 U / g, 3000 U / g, 5000 U / g, 8000 U / g, 9000 U / g, 10000 U / g, or any value within a range consisting of any two of the above values.

[0036] Preferably, the mass ratio of the mesoporous silica, the glutaraldehyde solution, and the water-soluble chitosan is 1:(0.5~5):(10~50). More preferably, the mass ratio of the mesoporous silica, the glutaraldehyde solution, and the water-soluble chitosan is 1:(1~3):(15~25).

[0037] Preferably, in step III, the immobilized carrier and the lipase solution are mixed at a mass-to-volume ratio of 1 g:(5~50 mL). More preferably, the immobilized carrier and the lipase solution are mixed at a mass-to-volume ratio of 1 g:(25~35 mL).

[0038] Preferably, the unsaturated long-chain fatty acid is oleic acid, linoleic acid, linolenic acid, or arachidonic acid, and the carbon chain length is C16 to C22. More preferably, the unsaturated long-chain fatty acid is oleic acid, linoleic acid, linolenic acid, or arachidonic acid, and the carbon chain length is C18 to C20.

[0039] Preferably, the mass ratio of the unsaturated long-chain fatty acid, the glycerol, and the immobilized enzyme is 1:(0.2~2):(0.01~0.2). More preferably, the mass ratio of the unsaturated long-chain fatty acid, the glycerol, and the immobilized enzyme is 1:1:(0.05~0.1).

[0040] The following specific embodiments illustrate a method for producing diglycerides using immobilized enzyme catalysis. Those skilled in the art will understand that the preparation methods described in this application are merely examples, and any other suitable preparation methods are within the scope of this application.

[0041] Preparation of mesoporous silica

[0042] Step 1: Dissolve 1g of P123 (polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer, molecular weight 5800) in 30mL of deionized water, stir at 30℃ until completely dissolved, add 0.5mL of concentrated hydrochloric acid (mass fraction 37%) to adjust the pH to 1.5-2.0, and continue stirring for 30 minutes;

[0043] Step 2: While stirring, slowly add 4 mL of tetraethyl orthosilicate (TEOS) dropwise to the above solution, controlling the dropping rate at 0.5 mL / min. After the addition is complete, stir at a constant temperature of 30°C for 24 hours to form a white emulsion (at this time, the molar ratio of TEOS to P123 is 5:1).

[0044] Step 3: Transfer the emulsion to a polytetrafluoroethylene reactor and statically crystallize at 80°C for 48 hours to order the mesoporous structure;

[0045] Step 4: After crystallization, cool to room temperature and collect the solid product by centrifugation (4000 rpm, 10 minutes). Wash the product three times alternately with deionized water and anhydrous ethanol (50 mL each time) to remove unreacted silicon source and free template agent.

[0046] Step 5: The washed product was vacuum dried at 60°C for 12 hours, then placed in a muffle furnace and heated to 550°C at a rate of 2°C / min. It was then calcined at a constant temperature for 6 hours and naturally cooled to obtain mesoporous silica.

[0047] Product performance can be precisely controlled by adjusting the following parameters:

[0048] (1) Pore size adjustment: If a pore size of 5~15nm is required, maintain the above amount of P123; if a pore size of 15~30nm is required, the amount of P123 can be increased to 1.5g;

[0049] (2) Adjustment of specific surface area and pore volume: When the amount of TEOS is increased to 5 mL, the specific surface area can be reduced to 500~800 m². 2 / g, pore volume increased to 1.5~2.0cm 3 / g; if the TEOS dosage is reduced to 3mL, the specific surface area can increase to 800~1200m². 2 / g, pore volume adjusted to 0.8~1.5cm 3 / g.

[0050] Among them, the pore size is 5~15nm and the specific surface area is 500~800m². 2 / g, pore volume 1.5~2.0cm 3 The product of / g is denoted as first mesoporous silica;

[0051] Pore ​​size of 15~30nm and specific surface area of ​​800~1200m 2 / g, pore volume 0.8~1.5cm 3 The product per g is denoted as second mesoporous silica (preferred);

[0052] Aperture range exceeding 30 nm and specific surface area greater than 1200 m² 2 / g, pore volume greater than 2.0cm 3 The product of / g is denoted as third mesoporous silica;

[0053] Hydroxyethyl chitosan with a viscosity-average molecular weight of 5kDa to 8kDa and a degree of deacetylation of 70% is designated as first-hydroxyethyl chitosan (commonly available).

[0054] Hydroxyethyl chitosan with a viscosity-average molecular weight of 10kDa to 30kDa and a degree of deacetylation of 70% is designated as second hydroxyethyl chitosan (commonly available in the market).

[0055] Hydroxyethyl chitosan with a viscosity-average molecular weight of 35kDa to 50kDa and a degree of deacetylation of 70% is designated as third hydroxyethyl chitosan (commonly available).

[0056] Hydroxyethyl chitosan with a viscosity-average molecular weight of 10kDa to 30kDa and a degree of deacetylation of 80% is designated as hydroxyethyl chitosan (commonly available).

[0057] Hydroxyethyl chitosan with a viscosity-average molecular weight of 10kDa to 30kDa and a degree of deacetylation of 90% is designated as fifth hydroxyethyl chitosan (commonly available in the market).

[0058] Hydroxyethyl chitosan with a viscosity-average molecular weight of 10kDa to 30kDa and a degree of deacetylation of 95% is designated as sixth hydroxyethyl chitosan (commonly available).

[0059] Hydroxypropyl chitosan with a viscosity-average molecular weight of 10kDa to 30kDa and a degree of deacetylation of 80% is designated as VII hydroxypropyl chitosan (commonly available in the market).

[0060] Hydroxypropyl chitosan with a viscosity-average molecular weight of 10kDa to 30kDa and a degree of deacetylation of 65% is designated as hydroxypropyl chitosan (commercially available).

[0061] The porcine lipase solution is a 1.5 mg / mL solution obtained by dissolving porcine pancreatic lipase (commercially available) in a phosphate buffer solution with a pH of 6.0-8.0. Example 1

[0062] A method for producing diglycerides using immobilized enzyme catalysis includes the following steps:

[0063] Step 1: Obtain the first mesoporous silica;

[0064] Step II: Preparation of immobilized carrier;

[0065] The first mesoporous silica, the 2.5% glutaraldehyde solution and the fourth hydroxyethyl chitosan from step I were mixed (mass ratio 1:2:20) and stirred at 150 rpm for 2 hours at 35°C.

[0066] Step III: Preparation of immobilized enzyme;

[0067] The immobilization carrier from step II was mixed with porcine pancreatic lipase solution (1g:30mL), and the solidification reaction was carried out at 25°C and 150rpm for 4 hours.

[0068] Step IV: Preparation of diglycerides;

[0069] The immobilized enzyme, oleic acid (carbon chain length C18), and glycerol from step III were mixed (mass ratio: 1:1:0.1) and stirred at 300 rpm for 12 hours at 40℃ and pH 6.0~8.0. The mixture was then filtered to obtain the product.

[0070] HPLC-ELSD analysis of the product revealed that the content of diglycerides in the product was 83.11%, of which the content of 1,3-diglycerides was 77.56%. Example 2

[0071] A method for producing diglycerides using immobilized enzyme catalysis includes the following steps:

[0072] Step 1: Obtain the second mesoporous silica;

[0073] Step II: Preparation of immobilized carrier;

[0074] The second mesoporous silica, the 2.5% glutaraldehyde solution and the fourth hydroxyethyl chitosan from step I were mixed (mass ratio 1:2:20) and stirred at 150 rpm for 2 hours at 35°C.

[0075] Step III: Preparation of immobilized enzyme;

[0076] The immobilization carrier from step II was mixed with porcine pancreatic lipase solution (1g:30mL), and the solidification reaction was carried out at 25°C and 150rpm for 4 hours.

[0077] Step IV: Preparation of diglycerides;

[0078] The immobilized enzyme, oleic acid (carbon chain length C18), and glycerol from step III were mixed (mass ratio: 1:1:0.1) and stirred at 300 rpm for 12 hours at 40℃ and pH 6.0~8.0. The mixture was then filtered to obtain the product.

[0079] HPLC-ELSD analysis of the product revealed that the content of diglycerides in the product was 88.35%, of which the content of 1,3-diglycerides was 82.16%. Example 3

[0080] A method for producing diglycerides using immobilized enzyme catalysis includes the following steps:

[0081] Step 1: Obtain the second mesoporous silica;

[0082] Step II: Preparation of immobilized carrier;

[0083] The second mesoporous silica, the 2.5% glutaraldehyde solution and the first hydroxyethyl chitosan from step I were mixed (mass ratio 1:2:20) and stirred at 150 rpm for 2 hours at 35°C.

[0084] Step III: Preparation of immobilized enzyme;

[0085] The immobilization carrier from step II was mixed with porcine pancreatic lipase solution (1g:30mL), and the solidification reaction was carried out at 25°C and 150rpm for 4 hours.

[0086] Step IV: Preparation of diglycerides;

[0087] The immobilized enzyme, oleic acid (carbon chain length C18), and glycerol from step III were mixed (mass ratio: 1:1:0.1) and stirred at 300 rpm for 12 hours at 40℃ and pH 6.0~8.0. The mixture was then filtered to obtain the product.

[0088] HPLC-ELSD analysis of the product revealed that the content of diglycerides in the product was 80.11%, of which the content of 1,3-diglycerides was 74.36%. Example 4

[0089] A method for producing diglycerides using immobilized enzyme catalysis includes the following steps:

[0090] Step 1: Obtain the second mesoporous silica;

[0091] Step II: Preparation of immobilized carrier;

[0092] The second mesoporous silica, the 2.5% glutaraldehyde solution and the second hydroxyethyl chitosan from step I were mixed (mass ratio 1:2:20) and stirred at 150 rpm for 2 hours at 35°C.

[0093] Step III: Preparation of immobilized enzyme;

[0094] The immobilization carrier from step II was mixed with porcine pancreatic lipase solution (1g:30mL), and the solidification reaction was carried out at 25°C and 150rpm for 4 hours.

[0095] Step IV: Preparation of diglycerides;

[0096] The immobilized enzyme, oleic acid (carbon chain length C18), and glycerol from step III were mixed (mass ratio: 1:1:0.1) and stirred at 300 rpm for 12 hours at 40℃ and pH 6.0~8.0. The mixture was then filtered to obtain the product.

[0097] HPLC-ELSD analysis of the product revealed that the content of diglycerides in the product was 80.32%, of which the content of 1,3-diglycerides was 73.66%. Example 5

[0098] A method for producing diglycerides using immobilized enzyme catalysis includes the following steps:

[0099] Step 1: Obtain the second mesoporous silica;

[0100] Step II: Preparation of immobilized carrier;

[0101] The second mesoporous silica, the 2.5% glutaraldehyde solution and the third hydroxyethyl chitosan from step I were mixed (mass ratio 1:2:20) and stirred at 150 rpm for 2 hours at 35°C.

[0102] Step III: Preparation of immobilized enzyme;

[0103] The immobilization carrier from step II was mixed with porcine pancreatic lipase solution (1g:30mL), and the solidification reaction was carried out at 25°C and 150rpm for 4 hours.

[0104] Step IV: Preparation of diglycerides;

[0105] The immobilized enzyme, oleic acid (carbon chain length C18), and glycerol from step III were mixed (mass ratio: 1:1:0.1) and stirred at 300 rpm for 12 hours at 40℃ and pH 6.0~8.0. The mixture was then filtered to obtain the product.

[0106] HPLC-ELSD analysis of the product revealed that the content of diglycerides in the product was 78.32%, of which the content of 1,3-diglycerides was 71.11%. Example 6

[0107] A method for producing diglycerides using immobilized enzyme catalysis includes the following steps:

[0108] Step 1: Obtain the second mesoporous silica;

[0109] Step II: Preparation of immobilized carrier;

[0110] The second mesoporous silica, the 2.5% glutaraldehyde solution and the fifth hydroxyethyl chitosan from step I were mixed (mass ratio 1:2:20) and stirred at 150 rpm for 2 hours at 35°C.

[0111] Step III: Preparation of immobilized enzyme;

[0112] The immobilization carrier from step II was mixed with porcine pancreatic lipase solution (1g:30mL), and the solidification reaction was carried out at 25°C and 150rpm for 4 hours.

[0113] Step IV: Preparation of diglycerides;

[0114] The immobilized enzyme, oleic acid (carbon chain length C18), and glycerol from step III were mixed (mass ratio: 1:1:0.1) and stirred at 300 rpm for 12 hours at 40℃ and pH 6.0~8.0. The mixture was then filtered to obtain the product.

[0115] HPLC-ELSD analysis of the product revealed that the content of diglycerides in the product was 86.88%, of which the content of 1,3-diglycerides was 80.86%. Example 7

[0116] A method for producing diglycerides using immobilized enzyme catalysis includes the following steps:

[0117] Step 1: Obtain the second mesoporous silica;

[0118] Step II: Preparation of immobilized carrier;

[0119] The second mesoporous silica from step I, the 2.5% glutaraldehyde solution, and the sixth hydroxyethyl chitosan were mixed (mass ratio 1:2:20) and stirred at 150 rpm for 2 hours at 35°C.

[0120] Step III: Preparation of immobilized enzyme;

[0121] The immobilization carrier from step II was mixed with porcine pancreatic lipase solution (1g:30mL), and the solidification reaction was carried out at 25°C and 150rpm for 4 hours.

[0122] Step IV: Preparation of diglycerides;

[0123] The immobilized enzyme, oleic acid (carbon chain length C18), and glycerol from step III were mixed (mass ratio: 1:1:0.1) and stirred at 300 rpm for 12 hours at 40℃ and pH 6.0~8.0. The mixture was then filtered to obtain the product.

[0124] HPLC-ELSD analysis of the product revealed that the content of diglycerides in the product was 80.23%, of which the content of 1,3-diglycerides was 74.61%. Example 8

[0125] A method for producing diglycerides using immobilized enzyme catalysis includes the following steps:

[0126] Step 1: Obtain the second mesoporous silica;

[0127] Step II: Preparation of immobilized carrier;

[0128] The second mesoporous silica from step I, the 2.5% glutaraldehyde solution, and the seventh hydroxypropyl chitosan were mixed (mass ratio 1:2:20) and stirred at 150 rpm for 2 hours at 35°C.

[0129] Step III: Preparation of immobilized enzyme;

[0130] The immobilization carrier from step II was mixed with porcine pancreatic lipase solution (1g:30mL), and the solidification reaction was carried out at 25°C and 150rpm for 4 hours.

[0131] Step IV: Preparation of diglycerides;

[0132] The immobilized enzyme, oleic acid (carbon chain length C18), and glycerol from step III were mixed (mass ratio: 1:1:0.1) and stirred at 300 rpm for 12 hours at 40℃ and pH 6.0~8.0. The mixture was then filtered to obtain the product.

[0133] HPLC-ELSD analysis of the product revealed that the content of diglycerides in the product was 87.87%, of which the content of 1,3-diglycerides was 81.96%.

[0134] As can be seen from Examples 1 to 8, the structural parameters of mesoporous silica and the parameters of water-soluble chitosan are all within a suitable range, which is conducive to promoting the synergy among mesoporous silica, glutaraldehyde solution and water-soluble chitosan. This allows the hydroxyl, amino and unreacted aldehyde groups on the water-soluble chitosan molecular chain to specifically and directionally bind with the polar residues on the surface of the cap structure of porcine pancreatic lipase to form an open conformation of the cap structure away from the active center. When the structural parameters of mesoporous silica and the parameters of water-soluble chitosan are both within the preferred range, the synergistic effect of the three is better, and the yield of diglyceride can reach up to 88.35%. Example 9

[0135] A method for producing diglycerides using immobilized enzyme catalysis includes the following steps:

[0136] Step 1: Obtain the second mesoporous silica;

[0137] Step II: Preparation of immobilized carrier;

[0138] The second mesoporous silica, the 2.5% glutaraldehyde solution and the fourth hydroxyethyl chitosan from step I were mixed (mass ratio 1:4:40) and stirred at 150 rpm for 2 hours at 35°C.

[0139] Step III: Preparation of immobilized enzyme;

[0140] The immobilization carrier from step II was mixed with porcine pancreatic lipase solution (1g:30mL), and the solidification reaction was carried out at 25°C and 150rpm for 4 hours.

[0141] Step IV: Preparation of diglycerides;

[0142] The immobilized enzyme, oleic acid (carbon chain length C18), and glycerol from step III were mixed (mass ratio: 1:1:0.1) and stirred at 300 rpm for 12 hours at 40℃ and pH 6.0~8.0. The mixture was then filtered to obtain the product.

[0143] HPLC-ELSD analysis of the product revealed that the content of diglycerides in the product was 75.55%, of which the content of 1,3-diglycerides was 69.82%.

[0144] Combining Examples 2 and 9, it can be seen that a suitable mass ratio of mesoporous silica, glutaraldehyde solution, and water-soluble chitosan can improve the yield of diglycerides. The inventors speculate that the possible reason is that mesoporous silica, glutaraldehyde solution, and water-soluble chitosan are more conducive to synergy within a suitable range. The resulting immobilized carrier is conducive to specific and directional binding with the polar residues on the surface of the cap structure of porcine pancreatic lipase, causing the cap structure to move away from the active center. Example 10

[0145] A method for producing diglycerides using immobilized enzyme catalysis includes the following steps:

[0146] Step 1: Obtain the second mesoporous silica;

[0147] Step II: Preparation of immobilized carrier;

[0148] The second mesoporous silica, the 2.5% glutaraldehyde solution and the fourth hydroxyethyl chitosan from step I were mixed (mass ratio 1:2:20) and stirred at 150 rpm for 2 hours at 35°C.

[0149] Step III: Preparation of immobilized enzyme;

[0150] The immobilization carrier from step II was mixed with porcine pancreatic lipase solution (1g:30mL), and the solidification reaction was carried out at 25°C and 150rpm for 4 hours.

[0151] Step IV: Preparation of diglycerides;

[0152] The immobilized enzyme, oleic acid (carbon chain length C18), and glycerol from step III were mixed (mass ratio: 1:1.5:0.2), stirred at 300 rpm for 12 hours at 40℃ and pH 6.0~8.0, and then filtered to obtain the product.

[0153] HPLC-ELSD analysis of the product revealed that the content of diglycerides in the product was 82.22%, of which the content of 1,3-diglycerides was 76.89%.

[0154] As can be seen from Examples 2 and 10, regulating the mass ratio of immobilized enzyme, unsaturated long-chain fatty acid, and glycerol within a suitable range is beneficial to further improve the yield of diglycerides. Comparative Example 1

[0155] A method for producing diglycerides using immobilized enzyme catalysis includes the following steps:

[0156] Step 1: Obtain the third mesoporous silica;

[0157] Step II: Preparation of immobilized carrier;

[0158] Mix the third mesoporous silica, the 2.5% glutaraldehyde solution and the fourth hydroxyethyl chitosan from step I (mass ratio 1:2:20), and stir at 150 rpm for 2 hours at 35°C.

[0159] Step III: Preparation of immobilized enzyme;

[0160] The immobilization carrier from step II was mixed with porcine pancreatic lipase solution (1g:30mL), and the solidification reaction was carried out at 25°C and 150rpm for 4 hours.

[0161] Step IV: Preparation of diglycerides;

[0162] The immobilized enzyme, oleic acid (carbon chain length C18), and glycerol from step III were mixed (mass ratio: 1:1:0.1) and stirred at 300 rpm for 12 hours at 40℃ and pH 6.0~8.0. The mixture was then filtered to obtain the product.

[0163] HPLC-ELSD analysis of the product revealed that the content of diglycerides in the product was 38.63%, of which the content of 1,3-diglycerides was 33.62%.

[0164] Comparing Comparative Example 1 and Example 1, it can be seen that the structural parameters of the mesoporous silica are unsuitable. Even with the selection of water-soluble chitosan with appropriate parameters, the synergy among the mesoporous silica, glutaraldehyde solution, and water-soluble chitosan is not conducive. The inventors speculate that the unsuitable structural parameters of the mesoporous silica result in insufficient spatial constraint of the flexible composite network of the immobilized carrier on the cap structure, which is not conducive to pulling the cap structure away from the active center to expose the active sites. Comparative Example 2

[0165] A method for producing diglycerides using immobilized enzyme catalysis includes the following steps:

[0166] Step 1: Obtain the second mesoporous silica;

[0167] Step II: Preparation of immobilized carrier;

[0168] The second mesoporous silica from step I, the 2.5% glutaraldehyde solution, and the eighth hydroxypropyl chitosan were mixed (mass ratio 1:2:20) and stirred at 150 rpm for 2 hours at 35°C.

[0169] Step III: Preparation of immobilized enzyme;

[0170] The immobilization carrier from step II was mixed with porcine pancreatic lipase solution (1g:30mL), and the solidification reaction was carried out at 25°C and 150rpm for 4 hours.

[0171] Step IV: Preparation of diglycerides;

[0172] The immobilized enzyme, oleic acid (carbon chain length C18), and glycerol from step III were mixed (mass ratio: 1:1:0.1) and stirred at 300 rpm for 12 hours at 40℃ and pH 6.0~8.0. The mixture was then filtered to obtain the product.

[0173] HPLC-ELSD analysis of the product revealed that the content of diglycerides in the product was 57.65%, of which the content of 1,3-diglycerides was 48.89%.

[0174] Comparing Comparative Example 2 and Example 1, it can be seen that the parameters of water-soluble chitosan are not suitable, and it is not conducive to the synergistic effect among mesoporous silica, glutaraldehyde solution and water-soluble chitosan, resulting in a low yield of diglycerides.

[0175] The above are merely preferred embodiments of this application and are not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for producing diglycerides using immobilized enzyme catalysis, characterized in that, Includes the following steps: Step 1: Obtain mesoporous silica; Step II: Preparation of immobilized carrier; Mix the mesoporous silica, glutaraldehyde solution and water-soluble chitosan from step I, and stir at 100 rpm to 300 rpm at 20°C to 40°C to obtain the final product. The mass ratio of the mesoporous silica, the glutaraldehyde solution, and the water-soluble chitosan is 1:(0.5~5):(10~50). Step III: Preparation of immobilized enzyme; The immobilized carrier from step II is mixed with porcine pancreatic lipase solution and solidified at 20°C to 37°C at 100 rpm to 200 rpm to obtain the product. Step IV: Preparation of diglycerides; The immobilized enzyme, oleic acid, and glycerol from step III were mixed and stirred at 100 rpm to 500 rpm under conditions of 30°C to 60°C and pH 6.0 to 8.0 to obtain diglycerides. And it meets the following conditions: Condition A: In step I, the mesoporous silica has a pore size of 5 nm to 30 nm and a specific surface area of ​​500 m². 2 / g to 1200m 2 / g, pore volume 0.8cm 3 / g to 2.0 cm 3 / g; Condition B: In step II, the water-soluble chitosan has a molecular weight of 5kDa to 50kDa and a degree of deacetylation of 70% to 95%, and the water-soluble chitosan is hydroxyethyl chitosan or hydroxypropyl chitosan.

2. The method for producing diglycerides using immobilized enzyme catalysis according to claim 1, characterized in that, At least one of the following conditions must be met: Condition a: The mesoporous silica has a pore size of 15 nm to 30 nm and a specific surface area of ​​800 m². 2 / g to 1200m 2 / g, pore volume 0.8cm 3 / g to 1.5cm 3 / g; Condition b: The water-soluble chitosan has a molecular weight of 10 kDa to 30 kDa and a degree of deacetylation of 80% to 90%.

3. The method for producing diglycerides using immobilized enzyme catalysis according to claim 1, characterized in that, The mass concentration of the glutaraldehyde solution is 0.5% to 5%.

4. The method for producing diglycerides using immobilized enzyme catalysis according to claim 1, characterized in that, In step III, the porcine pancreatic lipase solution is a solution with a concentration of 0.1 mg / mL to 5 mg / mL obtained by dissolving porcine pancreatic lipase in a phosphate buffer solution with a pH of 6.0 to 8.

0.

5. The method for producing diglycerides using immobilized enzyme catalysis according to claim 4, characterized in that, The activity of the porcine pancreatic lipase is 1000 U / g to 10000 U / g.

6. The method for producing diglycerides using immobilized enzyme catalysis according to claim 1, characterized in that, In step III, the immobilized carrier and the lipase solution are mixed at a mass-to-volume ratio of 1g:(5~50mL).