Method for catalytically producing diglyceride by utilizing immobilized enzyme

Abstract

The invention discloses a method for producing diglyceride through catalysis of an immobilized enzyme, and relates to the technical field of bio-enzymes, the method comprises the following steps: step I, obtaining mesoporous silica, step II, preparing an immobilized carrier, step III, preparing the immobilized enzyme, and step IV, preparing the diglyceride, and the following conditions are met: condition A, in the step I, the aperture of the mesoporous silica is 5-30nm, and in the step II, the aperture of the immobilized carrier is 5-30nm; the specific surface area is 500 m < 2 > / g to 1200 m < 2 > / g, the pore volume is 0.8 cm < 3 > / g to 2.0 cm < 3 > / g, and in the condition B and the step II, the molecular weight of the water-soluble chitosan is 5 kDa to 50 kDa, and the deacetylation degree is 70% to 95%. The immobilized carrier with the flexible composite network is prepared, the immobilized carrier is combined with a flexible cover of porcine pancreatic lipase, the cover can be pulled to be away from an active center to expose active sites, and the cover is not prone to being closed 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 water-soluble chitosan has a molecular weight of 5 kDa to 50 kDa and a degree of deacetylation of 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 water-soluble chitosan has a molecular weight of 10 kDa to 30 kDa and a degree of deacetylation of 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 mesoporous silica is obtained by the following process: a surfactant is used as a template agent, which is dissolved in a solvent, and the template agent is self-assembled to form micelles with a specific structure by adjusting the pH value of the solution, and a silicon source is added under stirring to cause hydrolysis and polycondensation of the silicon source around the template agent micelles, and then the reaction system is crystallized to promote the ordering and stabilization of the mesoporous structure, and 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 template agent, the concentration of silicon source and the ratio of 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 porcine pancreatic lipase, which in turn 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 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, it is beneficial 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 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: 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

[0017] 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.

[0018] A method for catalytically producing diglyceride by using immobilized enzyme Step I, obtaining mesoporous silica; A 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.

[0019] 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. 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; 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.

[0020] Step III: Preparation of immobilized enzyme: Mix the immobilized carrier from Step II with porcine pancreatic lipase solution, and carry out the immobilization reaction at 20℃ to 37℃ at 100 rpm to 200 rpm to obtain the enzyme. For example, in step III, the reaction temperature is 20°C, 25°C, 30°C, 35°C, 37°C, or any value within the range 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 of any two of the above values.

[0021] Step IV, Preparation of diglycerides: The immobilized enzyme, unsaturated long-chain fatty acids and glycerol from Step III are 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.

[0022] For example, 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 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 of any two of the above values; and the pH is 6.0, 6.3, 6.5, 7.0, 7.5, 8.0, or any value within the range of any two of the above values.

[0023] And at least one of the following conditions must be met: In condition A and 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; In condition B and step II, the molecular weight of the water-soluble chitosan is 5kDa to 50kDa, and the degree of deacetylation is 70% to 95%.

[0024] For example, the pore size of 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 a range consisting of any two of the above values. The specific surface area of ​​mesoporous silica is 500 m². 2 / g、600m 2 / g、800m 2 / g、900m 2 / g, 1000m 2 / g, 1100 m 2 / g, 1200 m 2 / g or any value within a range derived from any two of the above values. The pore volume of the mesoporous silica is 0.8 cm 3 / g, 1.0 cm 3 / g, 1.2 cm 3 / g, 1.3 cm 3 / g, 1.5 cm 3 / g, 1.8 cm 3 / g, 2.0 cm 3 / g or any value within a range derived from any two of the above values.

[0025] Illustratively, the water-soluble chitosan has a molecular weight of 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa or any value within a range derived from any two of the above values. The water-soluble chitosan has a degree of deacetylation of 70%, 75%, 80%, 85%, 90%, 95% or any value within a range derived from any two of the above values.

[0026] Illustratively, 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 derived from any two of the above values.

[0027] Illustratively, the glutaraldehyde solution has a mass concentration of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5% or any value within a range derived from any two of the above values.

[0028] Illustratively, the phosphate buffer has a pH of 6.0, 6.5, 6.8, 7.0, 7.5, 7.8, 8.0 or any value within a range derived from any two of the above values. The porcine pancreatic lipase solution has a concentration of 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 a range derived from any two of the above values.

[0029] Preferably, the porcine pancreatic lipase has an enzyme activity of 1000 U / g to 10000 U / g. Illustratively, the porcine pancreatic lipase has an enzyme activity 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 the range consisting of any two of the aforementioned values.

[0030] 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).

[0031] Preferably, in step III, the immobilization carrier and the lipase solution are mixed at a mass volume ratio of 1 g: (5-50 mL). More preferably, the immobilization carrier and the lipase solution are mixed at a mass volume ratio of 1 g: (25-35 mL).

[0032] Preferably, the unsaturated long-chain fatty acid is oleic acid, linoleic acid, linolenic acid, or arachidonic acid, and has a carbon chain length of C16 to C22. More preferably, the unsaturated long-chain fatty acid is oleic acid, linoleic acid, linolenic acid, or arachidonic acid, and has a carbon chain length of C18 to C20.

[0033] 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).

[0034] The method for producing diacylglycerol using immobilized enzyme is described below in conjunction with specific examples. Those skilled in the art will understand that the preparation method described in the present application is only an example, and any other suitable preparation method is within the scope of the present application.

[0035] Preparation of mesoporous silica Step 1: Dissolve 1 g of P123 (polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer, molecular weight 5800) in 30 mL of deionized water, stir at 30°C until completely dissolved, add 0.5 mL of concentrated hydrochloric acid (mass fraction 37%) to adjust the pH to 1.5-2.0, continue stirring for 30 minutes; Step 2: Under stirring, 4 mL of tetraethyl orthosilicate (TEOS) was added slowly into the above solution, the dropping rate was controlled at 0.5 mL / min, and after the completion of dropping, the solution was stirred at 30°C for 24 hours to form a white emulsion (at this time, the molar ratio of TEOS to P123 was 5:1); Step 3: The emulsion was transferred into a polytetrafluoroethylene reactor, and static crystallization was carried out at 80°C for 48 hours to make the mesoporous structure ordered; Step 4: After crystallization, the temperature was cooled to room temperature, and the solid product was collected by centrifugation (4000 rpm, 10 minutes), and then washed with deionized water and anhydrous ethanol alternately for 3 times (50 mL each time) to remove unreacted silicon source and free template agent; Step 5: The washed product was vacuum dried at 60°C for 12 hours, and then heated to 550°C at a rate of 2°C / min in a muffle furnace, and calcined at constant temperature for 6 hours, and then naturally cooled to obtain mesoporous silica.

[0036] The product performance can be precisely controlled by adjusting the following parameters: (1) Pore size adjustment: if the pore size is 5-15 nm, the above P123 dosage is maintained; if the pore size is 15-30 nm, the P123 dosage can be increased to 1.5 g; (2) Specific surface area and pore volume adjustment: when the TEOS dosage is increased to 5 mL, the specific surface area can be reduced to 500-800 m 2 / g, and the pore volume can be increased to 1.5-2.0 cm 3 / g; if the TEOS dosage is reduced to 3 mL, the specific surface area can be increased to 800-1200 m 2 / g, and the pore volume can be adjusted to 0.8-1.5 cm 3 / g.

[0037] The product with a pore size of 5-15 nm, a specific surface area of 500-800 m 2 / g, and a pore volume of 1.5-2.0 cm 3 / g is referred to as the first mesoporous silica; The product with a pore size of 15-30 nm, a specific surface area of 800-1200 m 2 / g, and a pore volume of 0.8-1.5 cm 3 / g is referred to as the second mesoporous silica (preferred); The product with a pore size greater than 30 nm, a specific surface area greater than 1200 m 2 / g, and a pore volume greater than 2.0 cm 3 / g is referred to as the third mesoporous silica; The hydroxyethyl chitosan with a viscosity-average molecular weight of 5 kDa to 8 kDa and a degree of deacetylation of 70% is referred to as the first hydroxyethyl chitosan (commonly sold on the market); Hydroxyethyl chitosan with a viscosity average molecular weight of 10 kDa to 30 kDa and a degree of deacetylation of 70% is referred to as the second hydroxyethyl chitosan (commonly commercially available); Hydroxyethyl chitosan with a viscosity average molecular weight of 35 kDa to 50 kDa and a degree of deacetylation of 70% is referred to as the third hydroxyethyl chitosan (commonly commercially available); Hydroxyethyl chitosan with a viscosity average molecular weight of 10 kDa to 30 kDa and a degree of deacetylation of 80% is referred to as the fourth hydroxyethyl chitosan (commonly commercially available); Hydroxyethyl chitosan with a viscosity average molecular weight of 10 kDa to 30 kDa and a degree of deacetylation of 90% is referred to as the fifth hydroxyethyl chitosan (commonly commercially available); Hydroxyethyl chitosan with a viscosity average molecular weight of 10 kDa to 30 kDa and a degree of deacetylation of 95% is referred to as the sixth hydroxyethyl chitosan (commonly commercially available); Hydroxypropyl chitosan with a viscosity average molecular weight of 10 kDa to 30 kDa and a degree of deacetylation of 80% is referred to as the seventh hydroxypropyl chitosan (commonly commercially available); Hydroxypropyl chitosan with a viscosity average molecular weight of 10 kDa to 30 kDa and a degree of deacetylation of 65% is referred to as the eighth hydroxypropyl chitosan (commonly commercially available) The porcine lipase solution is a solution with a concentration of 1.5 mg / mL obtained by dissolving porcine pancreatic lipase (commercially available) in a phosphate buffer with a pH of 6.0-8.0. Example 1

[0038] A method for catalytically producing diglyceride by using immobilized enzyme, comprising the following steps: Step I, obtaining first mesoporous silica; Step II, preparing an immobilized carrier; The first mesoporous silica in step I, a glutaraldehyde solution with a mass concentration of 2.5%, and the fourth hydroxyethyl chitosan are mixed (mass ratio 1:2:20), and stirred at 35°C and 150 rpm for 2 hours; Step III, preparing an immobilized enzyme; The immobilized carrier in step II is mixed with the porcine pancreatic lipase solution (1 g:30 mL), and a solidification reaction is carried out at 25°C and 150 rpm for 4 hours; Step IV, preparing diglyceride; The immobilized enzyme in step III, oleic acid (carbon chain length C18), and glycerol are mixed (mass ratio: 1:1:0.1), and stirred at 40°C and pH 6.0-8.0 and 300 rpm for 12 hours, and then filtered to obtain the product.

[0039] The product is analyzed by HPLC-ELSD, and it is found that the content of diglyceride in the product is 83.11%, and the content of 1,3-diglyceride is 77.56%. Example 2

[0040] A method for producing diglyceride by using immobilized enzyme, comprising the following steps: Step I, obtaining second mesoporous silica; Step II, preparing immobilized carrier; The second mesoporous silica in step I, a 2.5% glutaraldehyde solution and the fourth hydroxyethyl chitosan were mixed (mass ratio 1:2:20), and stirred at 150 rpm at 35°C for 2 hours; Step III, preparing immobilized enzyme; The immobilized carrier in step II was mixed with a porcine pancreatic lipase solution (1 g:30 mL), and a curing reaction was carried out at 150 rpm at 25°C for 4 hours; Step IV, preparing diglyceride; The immobilized enzyme in step III, oleic acid (carbon chain length C18) and glycerol were mixed (mass ratio: 1:1:0.1), and stirred at 300 rpm at 40°C and pH 6.0-8.0 for 12 hours, and then filtered to obtain the product.

[0041] The product was analyzed by HPLC-ELSD, and it was found that the content of diglyceride in the product was 88.35%, and the content of 1,3-diglyceride was 82.16%. Example 3

[0042] A method for producing diglyceride by using immobilized enzyme, comprising the following steps: Step I, obtaining second mesoporous silica; Step II, preparing immobilized carrier; The second mesoporous silica in step I, a 2.5% glutaraldehyde solution and the first hydroxyethyl chitosan were mixed (mass ratio 1:2:20), and stirred at 150 rpm at 35°C for 2 hours; Step III, preparing immobilized enzyme; The immobilized carrier in step II was mixed with a porcine pancreatic lipase solution (1 g:30 mL), and a curing reaction was carried out at 150 rpm at 25°C for 4 hours; Step IV, preparing diglyceride; The immobilized enzyme in step III, oleic acid (carbon chain length C18) and glycerol were mixed (mass ratio: 1:1:0.1), and stirred at 300 rpm at 40°C and pH 6.0-8.0 for 12 hours, and then filtered to obtain the product.

[0043] The product was analyzed by HPLC-ELSD, and it was found that the content of diglyceride in the product was 80.11%, and the content of 1,3-diglyceride was 74.36%. Example 4

[0044] A method for producing diglyceride by using immobilized enzyme, comprising the following steps: Step I, obtaining second mesoporous silica; Step II, preparing immobilized carrier; The second mesoporous silica in step I, a glutaraldehyde solution with a mass concentration of 2.5%, and the third hydroxyethyl chitosan were mixed (mass ratio 1:2:20), and stirred at 35°C and 150 rpm for 2 hours; Step III, preparing immobilized enzyme; The immobilized carrier in step II was mixed with a porcine pancreatic lipase solution (1 g:30 mL), and a curing reaction was carried out at 25°C and 150 rpm for 4 hours; Step IV, preparing diglyceride; The immobilized enzyme in step III, oleic acid (carbon chain length C18), and glycerol were mixed (mass ratio: 1:1:0.1), and stirred at 40°C and pH 6.0-8.0 and 300 rpm for 12 hours, and then filtered to obtain the product.

[0045] The product was analyzed by HPLC-ELSD, and it was found that the content of diglyceride in the product was 80.32%, and the content of 1,3-diglyceride was 73.66%. Example 5

[0046] A method for producing diglyceride by using immobilized enzyme, comprising the following steps: Step I, obtaining second mesoporous silica; Step II, preparing immobilized carrier; The second mesoporous silica in step I, a glutaraldehyde solution with a mass concentration of 2.5%, and the third hydroxyethyl chitosan were mixed (mass ratio 1:2:20), and stirred at 35°C and 150 rpm for 2 hours; Step III, preparing immobilized enzyme; The immobilized carrier in step II was mixed with a porcine pancreatic lipase solution (1 g:30 mL), and a curing reaction was carried out at 25°C and 150 rpm for 4 hours; Step IV, preparing diglyceride; The immobilized enzyme in step III, oleic acid (carbon chain length C18), and glycerol were mixed (mass ratio: 1:1:0.1), and stirred at 40°C and pH 6.0-8.0 and 300 rpm for 12 hours, and then filtered to obtain the product.

[0047] The product was analyzed by HPLC-ELSD, and it was found that the content of diglyceride in the product was 78.32%, and the content of 1,3-diglyceride was 71.11%. Example 6

[0048] A method for producing diglyceride by using immobilized enzyme catalysis, comprising the following steps: Step I, obtaining second mesoporous silica; Step II, preparing an immobilized carrier; The second mesoporous silica in step I, a glutaraldehyde solution with a mass concentration of 2.5%, and the fifth hydroxyethyl chitosan were mixed (mass ratio 1:2:20), and stirred at 35°C and 150 rpm for 2 hours; Step III, preparing an immobilized enzyme; The immobilized carrier in step II was mixed with a porcine pancreatic lipase solution (1g:30mL), and a curing reaction was carried out at 25°C and 150 rpm for 4 hours; Step IV, preparing diglyceride; The immobilized enzyme in step III, oleic acid (carbon chain length C18), and glycerol were mixed (mass ratio: 1:1:0.1), and stirred at 40°C and pH 6.0-8.0 and 300 rpm for 12 hours, and then filtered to obtain the product.

[0049] The product was analyzed by HPLC-ELSD, and it was found that the content of diglyceride in the product was 86.88%, and the content of 1,3-diglyceride was 80.86%. Example 7

[0050] A method for producing diglyceride by using immobilized enzyme catalysis, comprising the following steps: Step I, obtaining second mesoporous silica; Step II, preparing an immobilized carrier; The second mesoporous silica in step I, a glutaraldehyde solution with a mass concentration of 2.5%, and the sixth hydroxyethyl chitosan were mixed (mass ratio 1:2:20), and stirred at 35°C and 150 rpm for 2 hours; Step III, preparing an immobilized enzyme; The immobilized carrier in step II was mixed with a porcine pancreatic lipase solution (1g:30mL), and a curing reaction was carried out at 25°C and 150 rpm for 4 hours; Step IV, preparing diglyceride; The immobilized enzyme in step III, oleic acid (carbon chain length C18), and glycerol were mixed (mass ratio: 1:1:0.1), and stirred at 40°C and pH 6.0-8.0 and 300 rpm for 12 hours, and then filtered to obtain the product.

[0051] The product was analyzed by HPLC-ELSD, and it was found that the content of diglyceride in the product was 80.23%, and the content of 1,3-diglyceride was 74.61%. Example 8

[0052] A method for producing diglyceride by using immobilized enzyme catalysis, comprising the following steps: Step I, obtaining second mesoporous silica; Step II, preparing an immobilized carrier; The second mesoporous silica in step I, a 2.5% glutaraldehyde solution, and the seventh hydroxypropyl chitosan were mixed (mass ratio 1:2:20), and stirred at 35°C and 150 rpm for 2 hours; Step III, preparing an immobilized enzyme; The immobilized carrier in step II was mixed with a porcine pancreatic lipase solution (1g:30mL), and a solidification reaction was carried out at 25°C and 150 rpm for 4 hours; Step IV, preparing diglyceride; The immobilized enzyme in step III, oleic acid (carbon chain length C18), and glycerol were mixed (mass ratio: 1:1:0.1), and stirred at 40°C and pH 6.0-8.0 and 300 rpm for 12 hours, and then filtered to obtain the product.

[0053] The product was analyzed by HPLC-ELSD, and it was found that the content of diglyceride in the product was 87.87%, and the content of 1,3-diglyceride was 81.96%.

[0054] As can be seen from Examples 1 to 8, the structural parameters of mesoporous silica and the parameters of water-soluble chitosan are in the appropriate range, which is beneficial to promote the synergy between mesoporous silica, glutaraldehyde solution, and water-soluble chitosan, so that the hydroxyl, amino, and unreacted aldehyde groups on the molecular chain of water-soluble chitosan specifically and directionally bind to the polar residues on the cover structure surface of porcine pancreatic lipase to form an open conformation of the cover structure away from the active center. When the structural parameters of mesoporous silica and the parameters of water-soluble chitosan are in the preferred range, the synergy of the three is better, and the yield of diglyceride can be as high as 88.35%. Example 9

[0055] A method for producing diglyceride by using immobilized enzyme catalysis, comprising the following steps: Step I, obtaining second mesoporous silica; Step II, preparing an immobilized carrier; The second mesoporous silica in step I, a 2.5% glutaraldehyde solution, and the fourth hydroxyethyl chitosan are mixed (mass ratio 1:4:40), and stirred at 150 rpm at 35°C for 2 hours; Step III, preparation of immobilized enzyme; The immobilized carrier in step II is mixed with a porcine pancreatic lipase solution (1 g:30 mL), and a curing reaction is carried out at 150 rpm at 25°C for 4 hours; Step IV, preparation of diglyceride; The immobilized enzyme in step III, oleic acid (carbon chain length C18), and glycerol are mixed (mass ratio: 1:1:0.1), and stirred at 300 rpm at 40°C and pH 6.0-8.0 for 12 hours, and filtered to obtain the product.

[0056] The product is analyzed by HPLC-ELSD, and it is found that the content of diglyceride in the product is 75.55%, and the content of 1,3-diglyceride is 69.82%.

[0057] In combination with Example 2 and Example 9, it can be seen that the mass ratio of mesoporous silica, glutaraldehyde solution, and water-soluble chitosan is appropriate, which can improve the yield of diglyceride. The inventors speculate that the reason may be that the three of mesoporous silica, glutaraldehyde solution, and water-soluble chitosan are more conducive to synergy in a suitable range, and the obtained immobilized carrier is conducive to specific and directional binding with the polar residue on the surface of the lid structure of porcine pancreatic lipase, which promotes the lid structure to be away from the active center. Example 10

[0058] A method for producing diglyceride by using immobilized enzyme catalysis, comprising the following steps: Step I, obtaining second mesoporous silica; Step II, preparation of immobilized carrier; The second mesoporous silica in step I, a 2.5% glutaraldehyde solution, and the fourth hydroxyethyl chitosan are mixed (mass ratio 1:2:20), and stirred at 150 rpm at 35°C for 2 hours; Step III, preparation of immobilized enzyme; The immobilized carrier in step II is mixed with a porcine pancreatic lipase solution (1 g:30 mL), and a curing reaction is carried out at 150 rpm at 25°C for 4 hours; Step IV, preparation of diglyceride; The immobilized enzyme in step III, oleic acid (carbon chain length C18), and glycerol are mixed (mass ratio: 1:1.5:0.2), and stirred at 300 rpm at 40°C and pH 6.0-8.0 for 12 hours, and filtered to obtain the product.

[0059] The product was analyzed by HPLC-ELSD, and it was found that the content of diglyceride in the product was 82.22%, and the content of 1,3-diglyceride was 76.89%.

[0060] It can be seen from the combination of Example 2 and Example 10 that the mass ratio of the immobilized enzyme, the unsaturated long-chain fatty acid, and the glycerol is in a suitable range, which is conducive to further improving the yield of diglyceride. Comparative Example 1

[0061] A method for producing diglyceride by using immobilized enzyme catalysis, comprising the following steps: Step I, obtaining third mesoporous silica; Step II, preparing an immobilized carrier; The third mesoporous silica in step I, a 2.5% glutaraldehyde solution, and a fourth hydroxyethyl chitosan were mixed (mass ratio 1:2:20), and stirred at 35°C and 150 rpm for 2 hours; Step III, preparing an immobilized enzyme; The immobilized carrier in step II was mixed with a porcine pancreatic lipase solution (1g:30mL), and a solidification reaction was carried out at 25°C and 150 rpm for 4 hours; Step IV, preparing diglyceride; The immobilized enzyme in step III, oleic acid (carbon chain length C18), and glycerol were mixed (mass ratio: 1:1:0.1), and stirred at 40°C and pH 6.0-8.0 and 300 rpm for 12 hours, and then filtered to obtain the product.

[0062] The product was analyzed by HPLC-ELSD, and it was found that the content of diglyceride in the product was 38.63%, and the content of 1,3-diglyceride was 33.62%.

[0063] Comparing Comparative Example 1 and Example 1, it can be seen that the structural parameters of mesoporous silica are not suitable, and even if a suitable parameter of water-soluble chitosan is selected, it is not conducive to the synergy between mesoporous silica, glutaraldehyde solution, and water-soluble chitosan. The inventors speculate that the possible reason is that the flexible composite network of the immobilized carrier formed by the unsuitable structural parameters of mesoporous silica does not limit the space of the cap structure enough, which is not conducive to pulling the cap structure away from the active center to expose the active site. Comparative Example 2

[0064] A method for producing diglyceride by using immobilized enzyme catalysis, comprising the following steps: Step I, obtaining second mesoporous silica; Step II, preparing an immobilized carrier; The second mesoporous silica in step I, a 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; Step III, preparation of immobilized enzyme; The immobilized carrier in step II was mixed with a porcine pancreatic lipase solution (1 g:30 mL), and a solidification reaction was carried out at 150 rpm for 4 hours at 25°C; Step IV, preparation of diglyceride; The immobilized enzyme in step III, oleic acid (carbon chain length C18), and glycerol were mixed (mass ratio: 1:1:0.1), and stirred at 300 rpm for 12 hours at 40°C and pH 6.0-8.0, filtered, and the product was obtained.

[0065] The product was analyzed by HPLC-ELSD, and it was found that the content of diglyceride in the product was 57.65%, and the content of 1,3-diglyceride was 48.89%.

[0066] Comparing Comparative Example 2 and Example 1, it can be seen that the parameters of water-soluble chitosan are not suitable and are not conducive to the synergy between mesoporous silica, glutaraldehyde solution, and water-soluble chitosan, resulting in a lower yield of diglyceride.

[0067] The above is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent replacements, and improvements made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for catalytic production of diglycerides using an immobilized enzyme, characterized in that, The method comprises the following steps: Step I, obtaining mesoporous silica; Step II, preparing an immobilized carrier; The mesoporous silica in step I, a glutaraldehyde solution, and water-soluble chitosan are mixed, and stirring is performed at 100 rpm to 300 rpm at 20 DEG C to 40 DEG C, and the mixture is obtained; Step III, preparing an immobilized enzyme; The immobilized carrier in step II and a porcine pancreatic lipase solution are mixed, and a solidification reaction is performed at 100 rpm to 200 rpm at 20 DEG C to 37 DEG C, and the immobilized enzyme is obtained; Step IV, preparing a diglyceride; The immobilized enzyme in step III, an unsaturated long-chain fatty acid, and glycerol are mixed, and stirring is performed at 100 rpm to 500 rpm at 30 DEG C to 60 DEG C and pH 6.0~8.0, and the diglyceride is obtained; And the following conditions are met: In Condition A, Step I, the mesoporous silica has a pore size of 5 nm to 30 nm, a specific surface area of 500 m 2 / g to 1200 m 2 / g, a pore volume of 0.8 cm 3 / g to 2.0 cm 3 / g; Condition B, in step II, the water-soluble chitosan has a molecular weight of 5 kDa to 50 kDa and a degree of deacetylation of 70% to 95%.

2. The method for catalytic production of diglycerides using immobilized enzymes according to claim 1, characterized in that, At least one of the following conditions is met: condition a, the pore diameter of the mesoporous silica is 15 nm to 30 nm, the specific surface area is 800 m 2 / g to 1200 m 2 / g, the pore volume is 0.8 cm 3 / g to 1.5 cm 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 catalytic production of diglycerides using immobilized enzymes according to claim 1, characterized in that, The water-soluble chitosan is hydroxyethyl chitosan and / or hydroxypropyl chitosan; The mass concentration of the glutaraldehyde solution is 0.5% to 5%.

4. The method for catalytic production of diglycerides using immobilized enzymes 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 with pH 6.0~8.

0.

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

6. The method for catalytic production of diglycerides using immobilized enzymes according to claim 1, characterized in that, The mass ratio of the mesoporous silica, the glutaraldehyde solution, and the water-soluble chitosan is 1:(0.5~5):(10~50).

7. The method for catalytic production of diglycerides using immobilized enzymes 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 1 g:(5~50 mL).

8. The method for catalytic production of diglycerides using immobilized enzymes according to claim 1, characterized in that, The unsaturated long-chain fatty acid is oleic acid, linoleic acid, linolenic acid, or arachidonic acid, and has a carbon chain length of C16 to C22.

9. The method for catalytic production of diglycerides using immobilized enzymes according to claim 8, characterized in that, 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).