Method for efficiently producing diglyceride through nano-enzyme catalytic glycerolysis

Abstract

The invention belongs to the technical field of biological catalysis and food grease, discloses a method for efficiently producing diglyceride through nano-enzyme catalysis glycerolysis, and solves the technical problems of low reaction efficiency, high enzyme dosage and poor enzyme reutilization capability in preparation of diglyceride through enzyme catalysis. The preparation method comprises the following steps: carrying out covalent cross-linking on lauryl gallate (LG) and chitosan (CS) by a free radical induction method to form an LGCS cross-linked substance; forming nano particles (LGCS NPs) through a self-assembly method; lGCS NPs nano-particles are used as a carrier, genipin is used as a biological cross-linking agent, and free lipase is fixed on the nano-particle carrier to form the nano-enzyme. The prepared nano-enzyme has the characteristics of good hydrophobicity, good dispersibility, high stability and high catalytic activity, and can effectively improve the problem of insufficient contact between the traditional immobilized enzyme and a fat-soluble substrate, so that the glycerolysis reaction efficiency and the reutilization rate of a biocatalyst are remarkably improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological catalysis and food oil, and particularly relates to a method for efficiently producing diglyceride. BACKGROUND

[0002] Diglyceride (DAG) is a kind of fatty molecule combined by glycerol and two fatty acid chains, mainly including 1,3-diglyceride and 1,2-diglyceride two isomers, wherein 1,3-diglyceride has higher nutritional value. Studies have shown that the metabolic mode of diglyceride is different from that of traditional triglyceride (TAG), which can effectively reduce the accumulation of fat in the body, reduce the levels of triglyceride, cholesterol and uric acid in the blood, and thus has wide application prospect and high market value in the food and health industry.

[0003] Current preparation methods of diglyceride mainly include chemical catalysis method and enzyme catalysis method. The chemical catalysis method usually promotes glycerol and oil to occur glycerolysis reaction by an alkaline catalyst to generate diglyceride, and has the advantages of simple reaction path, short reaction time and relatively low cost. However, this method has defects such as harsh reaction conditions, easy production of by-products, possible environmental pollution and food safety hazards caused by catalysts, and the quality of the prepared diglyceride product in flavor and color is also difficult to meet the needs of the food industry.

[0004] In comparison, the enzyme catalysis method gradually becomes an important method for preparing diglyceride in the fields of food and medicine due to its mild reaction conditions, high selectivity and excellent product quality. The enzyme catalysis method for preparing diglyceride mainly includes glycerolysis method, esterification method and hydrolysis method, etc. Among them, the glycerolysis method is to catalyze glycerol and triglyceride to occur glycerolysis reaction by lipase to prepare diglyceride, which has shown good prospects in industrial application. However, some immobilized lipases have poor dispersibility in the glycerolysis reaction system due to the properties of the immobilized carriers, so the catalytic efficiency is low, the price of the commercial immobilized enzyme is expensive, the stability and reusability of the free enzyme are not high, etc. The cost of industrial production is relatively high, which limits the further popularization and application of the enzyme catalysis method. SUMMARY

[0005] In view of the above technical problems, the present application provides a method for efficiently producing diglyceride by nano-enzyme catalysis glycerolysis.

[0006] To achieve the above purpose, the technical scheme of the present application is as follows:

[0007] A method for efficiently producing diglyceride by nano-enzyme catalysis glycerolysis, the steps are as follows:

[0008] (1) In the redox system of ascorbic acid and hydrogen peroxide, gallic acid lauryl ester (LG) and chitosan (CS) are covalently cross-linked by free radical induction method to form LGCS cross-linking material; LGCS cross-linking material is formed by self-assembly method to form LGCS NPs. The free radical induction method is to covalently graft gallic acid lauryl ester molecules to the amino and hydroxyl groups on the chitosan molecules in a free radical manner in the redox system of ascorbic acid and hydrogen peroxide.

[0009] (2) The LGCS NPs prepared in step (1) are used as carriers, and genipin (Genipin) is used as a biological cross-linking agent for cross-linking reaction; then, the free lipase is immobilized on the surface of the cross-linked LGCS NPs by immobilization reaction to form a nano-enzyme.

[0010] The preparation principle is that the amino group on the LGCS NPs first performs nucleophilic attack on the C3 site on the Genipin to cause ring opening of the Genipin cyclic structure to form an amine intermediate, the intermediate is further rearranged to generate an aldehyde group carrier, the primary amino group in the lipase reacts with the aldehyde group of the activated carrier to form a Schiff base (imine bond), and finally the lipase is covalently fixed to the carrier through the -CH=N- bond (a blue complex is generated, which is a characteristic of Genipin cross-linking).

[0011] (3) The nano-enzyme obtained in step (2) is dispersed in a mixture of oil and glycerol to perform glycerolysis reaction, and then glycerol diester is obtained after centrifugal separation and molecular distillation.

[0012] Further specifically, a method for efficiently producing glycerol diester by nano-enzyme catalytic glycerolysis, the specific steps are as follows:

[0013] (1) a. Chitosan (chitosan molecular weight is 100 KDa, and degree of deacetylation is 90%) is dissolved in acetic acid solution (volume fraction is 1-2%) to obtain chitosan acetic acid solution, and nitrogen protection is performed. Ascorbic acid is dissolved in hydrogen peroxide (H2O2), and then added to the chitosan acetic acid solution prepared in step a, and reacted for 30 min to obtain solution A. b. The ethanol solution containing gallic acid lauryl ester is added to solution A, and the reaction is carried out under light protection and nitrogen protection for 16-24 h to obtain solution B. c. The pH of solution B is adjusted to 7, and after filtration, washing and drying, modified chitosan powder (LGCS) is obtained, that is, LGCS cross-linking material. d. The LGCS cross-linking material powder is dissolved in acetic acid solution, and self-assembly is formed in the reaction under the condition of 400-800 rpm / min stirring speed. Then, large aggregates are removed by filtering through a 220 nm microporous membrane, and LGCS NPs are obtained after freeze-drying treatment.

[0014] (2) Under stirring, the LGCS NPs prepared in step (1) are dispersed into acetic acid, and a Genipin solution (solvent: anhydrous ethanol) is slowly added dropwise thereto, and a cross-linking reaction is carried out under certain conditions to obtain a reaction solution containing the cross-linked LGCS NPs. Subsequently, free lipase is dissolved in a PBS buffer solution with a pH of 7.0-8.5 to obtain a free lipase solution, and the free lipase solution is added to the above reaction solution to carry out an immobilization reaction. After the reaction is completed, the nanoscale enzyme is separated by centrifugation, washed with PBS, and freeze-dried to obtain the nanoscale enzyme.

[0015] (3) The oil and fat and glycerol are stirred under a magnetic stirrer, and the nanoscale enzyme obtained in step (2) is added to carry out glycerolysis. After the reaction is completed, the enzyme is separated from the oil phase by centrifugation, and the upper oil phase is removed to carry out molecular distillation to obtain a heavy phase, which is the target product, diglyceride.

[0016] In the above step (1), the mass fraction of chitosan in the chitosan acetic acid solution is 0.5-2.5%; and the molar ratio of ascorbic acid to hydrogen peroxide is 1:(5-10).

[0017] In the above step (1), the mass ratio of chitosan to lauryl gallate is 1:(0.4-2); and the mass ratio of ascorbic acid to chitosan is 1:(1.5-7).

[0018] In the above step (2), the mass ratio of LGCS NPs to Genipin is 3:(0.5-5), the cross-linking reaction temperature is 25-40℃, and the reaction time is 30-90 min.

[0019] Further, the magnetic stirring speed of the cross-linking reaction is 600-800 rpm.

[0020] In the above step (2), the mass ratio of LGCS NPs to free lipase is 100:(1-10).

[0021] In the above step (2), the immobilization reaction pH is 7.0-8.5, the temperature is 20-35℃, and the time is 4-10 h. In addition, the magnetic stirring speed of the immobilization reaction is 150-300 rpm.

[0022] In the above step (2), the centrifugal speed for separating the nanoscale enzyme is 4000-8000 rpm, and the centrifugal temperature is 4-25℃.

[0023] In the above step (2), the free lipase is any one of Lipozyme RM, Lipozyme TL 100L, and Candida antarctica lipase B (CALB).

[0024] In step (3) above, the molar ratio of glycerol to oil is (1.5-3):1, and the amount of nanozyme used is 1-5% of the total mass of oil and glycerol.

[0025] In step (3) above, the temperature of the glycerol hydrolysis reaction is 40-60℃ and the time is 6-24h; the stirring speed is 500-1000rpm.

[0026] In step (3) above, the oil is vegetable oil or animal oil; the centrifugation speed is 8000-12000 rpm and the time is 10-20 min; the conditions for molecular distillation are: temperature 160-180℃, vacuum degree 0.1-1 Pa, condenser temperature 20-30℃.

[0027] Furthermore, the aforementioned vegetable oils are one or more of flaxseed oil, soybean oil, rapeseed oil, corn oil, and peanut oil, and the animal oils are lard or horse fat.

[0028] The beneficial effects of this invention are:

[0029] (1) This invention crosslinks food-grade antioxidant lauryl gallate (LG) onto natural chitosan (CS) using a green free radical method to form self-assembled nanoparticles (LGCS NPs), and further immobilizes lipase with genipin to prepare novel nanozymes. These nanoparticles have 12-carbon hydrophobic alkyl chains on their surface, allowing them to disperse well in lipophilic media. Therefore, the prepared nanozymes exhibit good hydrophobicity and dispersibility, effectively improving the problem of insufficient contact between traditional immobilized enzymes and lipophilic media, thereby increasing the glycerol hydrolysis reaction rate.

[0030] (2) The immobilized nanozymes prepared by the present invention can effectively reduce the mass transfer resistance at the phase interface during enzyme catalysis, and exhibit superior catalytic efficiency, catalytic stability and reusability compared to free enzymes.

[0031] (3) This invention provides a new, efficient, green and economical technical solution for the industrial production of diglycerides.

[0032] (4) This invention uses genipin, a naturally derived crosslinking agent, which has been widely used in edible membrane materials, enzyme immobilization, biosensors, drug carriers, etc., and is more in line with the food industry's requirements for catalyst safety. Furthermore, the nanozyme of this invention retains 92% of its enzyme activity after 40 reuses, with a lower enzyme loss rate. In the glycerol hydrolysis system, the carrier of the nanozyme of this invention is lauryl gallate, which has a hydrophobic chain and is more suitable for the glycerol hydrolysis reaction system. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 TEM particle size images of LGCS NPs prepared in Example 1 of this invention and images of the dispersion of LGCS NPs in the aqueous phase.

[0035] Figure 2 TEM particle size distribution of the Enzyme-LGCS NPs prepared in Example 1 of this invention and a photograph showing the dispersion of the Enzyme-LGCS NPs in the oil phase.

[0036] Figure 3 The graph shows the relationship between reaction time and DAG content for the nanozyme Enzyme-LGCS NPs (Example 3), free enzyme CALB (Comparative Example 2), and commercially available immobilized enzyme Novozym 435 (Comparative Example 3) prepared in this invention.

[0037] Figure 4 The graph shows the relationship between the number of cycles of glycerol hydrolysis of nanozymes prepared with Novozym 435 and in Example 1 and the yield of DAG.

[0038] Figure 5 DAG yield diagrams of the nanozymes (Enzyme-LGCS NPs) and free enzymes (CALB) prepared in Example 1 at different temperatures.

[0039] Figure 6 The DAG yield graphs show the reaction of the nanozymes (Enzyme-LGCS NPs) and free enzymes (CALB) prepared in Example 1 after incubation in buffer systems at different pH for 30 min. Detailed Implementation

[0040] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] Example 1

[0042] The method for efficient production of diglycerides via nanozyme-catalyzed glycerol hydrolysis in this embodiment comprises the following steps:

[0043] I. The preparation method of the nanozyme in this embodiment is as follows:

[0044] (1) Dissolve 1 g of chitosan (molecular weight of chitosan is 100 kDa, degree of deacetylation is 90%) in 50 mL of acetic acid solution (volume fraction is 1%), stir for 3 h, and replace with N2 for 30 min to obtain a chitosan acetic acid solution with a chitosan mass fraction of 2%. Dissolve 0.176 g of ascorbic acid powder in 1 mL of 5 M H2O2, and slowly add it to the above chitosan acetic acid solution. Continue stirring under N2 protection in the dark for 30 min to obtain solution A. Slowly add 30 mL of LG ethanol solution with a concentration of 0.0133 g / mL to solution A, and react under nitrogen protection at room temperature for 24 h in the dark. After the reaction is completed, obtain solution B. Adjust the pH of solution B to 7 with NaOH, and add excess ethanol to precipitate unmodified chitosan. Wash with ethanol solution and filter to remove unreacted LG. Freeze dry for 24 h to obtain LGCS crosslinked product.

[0045] (2) Weigh 0.5 g of LGCS crosslinking and dissolve it in 50 mL of 1% (volume fraction) acetic acid solution. Stir at 600 rpm / min for 2.5 h until clear and transparent. During this period, LGCS NPs are self-assembled. Then, filter through a 0.22 μm microporous membrane to remove large aggregates and obtain a dispersion containing LGCS NPs. Figure 1 ).Depend on Figure 1 It can be seen that the prepared LGCS NPs have a particle size of 60-80 nm and good dispersibility in water. The dispersion containing LGCS NPs was freeze-dried to obtain LGCS NPs.

[0046] (3) Under stirring at 800 rpm, 0.1 g of LGCS NPs were dispersed in 10 mL of 1% acetic acid, and 2 mL of Genipin solution (8.33 mg / mL, anhydrous ethanol) was slowly added dropwise to control the mass ratio of LGCS NPs to Genipin at 3:0.5. The reaction was carried out at 25 °C for 30 min to obtain a reaction solution containing cross-linked LGCS NPs. Subsequently, Candida antarcticis lipase B (CALB) was dissolved in PBS buffer at pH=7 to obtain a free lipase solution, which was added to the above reaction solution (mass ratio of lipase to LGCS NPs was 1:100). The mixture was gently stirred at 25 °C for 4 hours at 150 rpm. After the reaction was completed, the nanozyme was separated by centrifugation at 6000 rpm at 4 °C, washed with PBS, and lyophilized for 24 h to obtain the nanozyme (Enzyme-LGCS NPs). Figure 2The images show TEM particle size distribution of Enzyme-LGCS NPs and their dispersion in the oil phase. The TEM images show that the free enzyme and LGCS NPs were successfully cross-linked to form nanozymes. Furthermore, Enzyme-LGCS NPs contain hydrophobic chains, which is beneficial for the catalytic production of diglycerides from glycerol by the good dispersion of the nanozymes in the oil phase.

[0047] II. A method for the efficient production of diglycerides from vegetable oils via nanozyme-catalyzed glycerol hydrolysis, the steps of which are as follows:

[0048] Rapeseed oil and glycerol were mixed at a molar ratio of 1:3 to obtain a mixed system. Enzyme-LGCS NPs prepared in this example (3.0% of the mass of the mixed system) were added to the mixed system, and the mixture was placed in a magnetic stirrer (1000 rpm) at 50°C for 10 hours. After the reaction was completed, the mixture was centrifuged at 10000 rpm for 15 min to separate the oil from the nanozyme. A trace amount of the upper oil phase was taken for GC analysis, and the yield was calculated to be 58.4%. The remaining upper oil phase was subjected to molecular distillation at 170°C, a vacuum of 0.5 Pa, and a condenser temperature of 25°C to obtain the final product. GC analysis showed that the diglyceride content was 70.3%.

[0049] Example 2

[0050] The method for efficient production of diglycerides via nanozyme-catalyzed glycerol hydrolysis in this embodiment comprises the following steps:

[0051] I. The preparation method of the nanozyme in this embodiment is as follows:

[0052] (1) Dissolve 1 g of chitosan (molecular weight of chitosan is 100 kDa, degree of deacetylation is 90%) in 50 mL of acetic acid solution (volume fraction is 1%), stir for 3 h, and replace with N2 for 30 min to obtain a chitosan acetic acid solution with a chitosan mass fraction of 2%. Dissolve 0.176 g of ascorbic acid powder in 1 mL of 7.5 M H2O2, and slowly add it to the above chitosan acetic acid solution. Continue stirring under N2 protection in the dark for 30 min to obtain solution A. Slowly add 30 mL of LG ethanol solution with a concentration of 0.0267 g / mL to solution A, and react under nitrogen protection at room temperature for 24 h in the dark. After the reaction is completed, obtain solution B. Adjust the pH of solution B to 7 with NaOH, and add excess ethanol to precipitate unmodified chitosan. Wash with ethanol solution and filter to remove unreacted LG. Freeze dry for 24 h to obtain LGCS crosslinked product.

[0053] (2) Weigh 0.5g of LGCS crosslinking and dissolve it in 50mL of 1% (volume fraction) acetic acid solution. Stir at 600rpm / min for 2.5h until clear and transparent. During this period, LGCS NPs are self-assembled. Then, the large aggregates are removed by filtration through a 0.22μm microporous membrane. After freeze-drying, LGCS NPs are obtained.

[0054] (3) Under stirring at 800 rpm, 0.1 g LGCS NPs were dispersed in 10 mL of 1% acetic acid, and 2 mL of Genipin solution (solvent: anhydrous ethanol) with a concentration of 33.32 mg / mL was slowly added dropwise, controlling the mass ratio of LGCS NPs to Genipin to be 3:2. The reaction was carried out at 30 °C for 60 min to obtain a reaction solution containing cross-linked LGCS NPs. Subsequently, 100 L of Lipozyme TL was dissolved in PBS buffer at pH = 8 to obtain a free lipase solution, which was added to the above reaction solution (mass ratio of lipase to LGCS NPs was 5:100). The mixture was gently stirred at 25 °C for 7 hours at 150 rpm. After the reaction was completed, the nanozyme was separated by centrifugation at 4 °C and 6000 rpm, washed with PBS, and lyophilized for 24 h to obtain the nanozyme (Enzyme-LGCSNPs).

[0055] II. The method for efficiently producing diglycerides from animal oils using nanoenzyme catalysis in this embodiment comprises the following steps:

[0056] Lard and glycerol were mixed at a molar ratio of 1:1.5 to obtain a mixed system. The nanozyme prepared in this example (3.0% of the mass of the mixed system) was added to the mixed system, and the mixture was placed in a magnetic stirrer (500 rpm) at a constant temperature of 60°C for 10 hours. After the reaction was completed, the mixture was centrifuged at 10,000 rpm for 10 minutes to separate the oil from the nanozyme. A trace amount of the upper oil phase was taken for GC analysis, and the calculated yield was 59.3%. The remaining upper oil phase was subjected to molecular distillation at 170°C, a vacuum of 0.5 Pa, and a condenser temperature of 25°C to obtain the final product. GC analysis showed that the diglyceride content was 72.9%.

[0057] Example 3

[0058] The method for efficient production of diglycerides via nanozyme-catalyzed glycerol hydrolysis in this embodiment comprises the following steps:

[0059] I. The preparation method of the nanozyme in this embodiment is as follows:

[0060] (1) Dissolve 1 g of chitosan (molecular weight of chitosan is 100 kDa, degree of deacetylation is 90%) in 50 mL of acetic acid solution (volume fraction is 1%), stir for 3 h, and replace with N2 for 30 min to obtain a chitosan acetic acid solution with a chitosan mass fraction of 2%. Dissolve 0.176 g of ascorbic acid powder in 1 mL of 10 M H2O2, and slowly add it to the above chitosan acetic acid solution. Continue stirring under N2 protection in the dark for 30 min to obtain solution A. Slowly add 30 mL of LG ethanol solution with a concentration of 0.067 g / mL to solution A, and react under nitrogen protection at room temperature for 24 h in the dark. After the reaction is completed, obtain solution B. Adjust the pH of solution B to 7 with NaOH, and add excess ethanol to precipitate unmodified chitosan. Wash with ethanol solution and filter to remove unreacted LG. Freeze dry for 24 h to obtain LGCS crosslinked product.

[0061] (2) Weigh 0.5g of LGCS crosslinking and dissolve it in 50mL of 1% (volume fraction) acetic acid solution. Stir at 600rpm / min for 2.5h until clear and transparent. During this period, LGCS NPs are self-assembled. Then, the large aggregates are removed by filtration through a 0.22μm microporous membrane. After freeze-drying, LGCS NPs are obtained.

[0062] (3) Under stirring at 800 rpm, 0.1 g of LGCS NPs were dispersed in 10 mL of 1% acetic acid, and 2 mL of Genipin solution (83.3 mg / mL, anhydrous ethanol) was slowly added dropwise, controlling the mass ratio of LGCS NPs to Genipin to be 3:5. The reaction was carried out at 40 °C for 90 min to obtain a reaction solution containing cross-linked LGCS NPs. Subsequently, Candida antarcticis lipase B (CALB) was dissolved in PBS buffer at pH 8.5 to obtain a free lipase solution, which was added to the above reaction solution (mass ratio of lipase to LGCS NPs was 10:100). The mixture was gently stirred at 25 °C for 4 hours at 150 rpm. After the reaction was completed, the nanozyme was separated by centrifugation at 6000 rpm at 4 °C, washed with PBS, and lyophilized for 24 h to obtain the nanozyme (Enzyme-LGCS NPs).

[0063] II. The method for efficient production of diglycerides by nanozyme-catalyzed glycerol hydrolysis in this embodiment comprises the following steps:

[0064] Rapeseed oil and glycerol were mixed at a molar ratio of 1:3 to obtain a series of mixed systems. The nanozyme prepared in this example (3.0% of the mass of the mixed system) was added to the mixed system, and the mixture was placed in a magnetic stirrer (750 rpm) at a constant temperature of 60°C. Samples (20 μL each time) were collected periodically at different time points (e.g., 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, ..., 24h). The diglyceride content was analyzed by GC to track the reaction progress. The diglyceride content at each time point is shown below. Figure 3 As shown.

[0065] Example 4

[0066] The method for efficient production of diglycerides via nanozyme-catalyzed glycerol hydrolysis in this embodiment comprises the following steps:

[0067] I. The preparation method of the nanozyme in this embodiment is as follows:

[0068] (1) Dissolve 0.25 g of chitosan (molecular weight of chitosan is 100 kDa, degree of deacetylation is 90%) in 50 mL of acetic acid solution (volume fraction is 2%), stir for 3 h, and replace with N2 for 30 min to obtain a chitosan acetic acid solution with a chitosan mass fraction of 0.5%. Dissolve 0.176 g of ascorbic acid powder in 1 mL of 5 M H2O2, and slowly add it to the above chitosan acetic acid solution. Continue stirring under N2 protection in the dark for 30 min to obtain solution A. Slowly add 30 mL of LG ethanol solution with a concentration of 0.0133 g / mL to solution A, and react under nitrogen protection at room temperature in the dark for 16 h. After the reaction is completed, obtain solution B. Adjust the pH of solution B to 7 with NaOH, and add excess ethanol to precipitate unmodified chitosan. Wash with ethanol solution and filter to remove unreacted LG. Freeze dry for 24 h to obtain LGCS crosslinked product.

[0069] (2) Weigh 0.5g of LGCS crosslinking and dissolve it in 50mL of 1% (volume fraction) acetic acid solution. Stir at 800rpm / min for 2.5h until clear and transparent. During this period, LGCS NPs are self-assembled. Then, the large aggregates are removed by filtration through a 0.22μm microporous membrane. After freeze-drying, LGCS NPs are obtained.

[0070] (3) Under stirring at 700 rpm, 0.1 g of LGCS NPs were dispersed in 10 mL of 1% acetic acid, and 2 mL of Genipin solution (solvent: anhydrous ethanol) with a concentration of 33.32 mg / mL was slowly added dropwise, controlling the mass ratio of LGCS NPs to Genipin to be 3:2. The reaction was carried out at 40 °C for 30 min to obtain a reaction solution containing cross-linked LGCS NPs. Subsequently, 100 L of Lipozyme TL was dissolved in PBS buffer at pH = 8 to obtain a free lipase solution, which was added to the above reaction solution (mass ratio of lipase to LGCS NPs was 5:100). The mixture was gently stirred at 20 °C for 10 h at 300 rpm. After the reaction was completed, the nanozyme was separated by centrifugation at 8000 rpm at 4 °C, washed with PBS, and lyophilized for 24 h to obtain the nanozyme (Enzyme-LGCSNPs).

[0071] II. The method for efficiently producing diglycerides from animal oils using nanoenzyme catalysis in this embodiment comprises the following steps:

[0072] Lard and glycerol were mixed at a molar ratio of 1:2 to obtain a mixed system. The nanozyme prepared in this example (5.0% of the mass of the mixed system) was added to the mixed system, and the mixture was placed in a magnetic stirrer (500 rpm) at a constant temperature of 40°C for 10 hours. After the reaction was completed, the mixture was centrifuged at 8000 rpm for 20 min to separate the oil from the nanozyme. A trace amount of the upper oil phase was taken for GC analysis, and the calculated yield was 57.8%. The remaining upper oil phase was subjected to molecular distillation at 160°C, a vacuum of 1 Pa, and a condenser temperature of 20°C to obtain the final product. GC analysis showed that the diglyceride content was 71.3%.

[0073] Example 5

[0074] The method for efficient production of diglycerides via nanozyme-catalyzed glycerol hydrolysis in this embodiment comprises the following steps:

[0075] I. The preparation method of the nanozyme in this embodiment is as follows:

[0076] (1) 1.25 g of chitosan (molecular weight of chitosan is 100 kDa, degree of deacetylation is 90%) was dissolved in 50 mL of acetic acid solution (volume fraction of 1.5%), stirred for 3 h, and then replaced with N2 for 30 min to obtain a chitosan acetic acid solution with a chitosan mass fraction of 2.5%. 0.176 g of ascorbic acid powder was dissolved in 1 mL of 7.5 M H2O2 and slowly added to the above chitosan acetic acid solution. The mixture was stirred under N2 protection in the dark for 30 min to obtain solution A. 30 mL of 0.0267 g / mL LG ethanol solution was slowly added to solution A, and the reaction was carried out at room temperature under nitrogen protection for 20 h. After the reaction was completed, solution B was obtained. The pH of solution B was adjusted to 7 with NaOH, and excess ethanol was added to precipitate unmodified chitosan. The solution was washed with ethanol solution and filtered to remove unreacted LG. The product was lyophilized for 24 h to obtain LGCS crosslinked material.

[0077] (2) Weigh 0.5g of LGCS crosslinking and dissolve it in 50mL of 1% (volume fraction) acetic acid solution. Stir at 400rpm / min for 2.5h until clear and transparent. During this period, LGCS NPs are self-assembled. Then, the large aggregates are removed by filtration through a 0.22μm microporous membrane. After freeze-drying, LGCS NPs are obtained.

[0078] (3) Under stirring at 600 rpm, 0.1 g of LGCS NPs were dispersed in 10 mL of 1% acetic acid, and 2 mL of Genipin solution (solvent: anhydrous ethanol) with a concentration of 16.66 mg / mL was slowly added dropwise, controlling the mass ratio of LGCS NPs to Genipin to be 3:1. The reaction was carried out at 25 °C for 90 min to obtain a reaction solution containing cross-linked LGCS NPs. Subsequently, Candida antarcticis lipase B was dissolved in PBS buffer at pH=8 to obtain a free lipase solution, which was added to the above reaction solution (mass ratio of lipase to LGCS NPs was 5:100). The mixture was gently stirred at 35 °C for 4 hours at 200 rpm. After the reaction was completed, the nanozyme was separated by centrifugation at 4 °C and 4000 rpm, washed with PBS, and lyophilized for 24 h to obtain the nanozyme (Enzyme-LGCSNPs).

[0079] II. The method for efficiently producing diglycerides from animal oils using nanoenzyme catalysis in this embodiment comprises the following steps:

[0080] Lard and glycerol were mixed at a molar ratio of 1:1.5 to obtain a mixed system. The nanozyme prepared in this example (3.0% of the mass of the mixed system) was added to the mixed system, and the mixture was placed in a magnetic stirrer (500 rpm) at a constant temperature of 60°C for 10 hours. After the reaction was completed, the mixture was centrifuged at 10,000 rpm for 10 minutes to separate the oil from the nanozyme. A trace amount of the upper oil phase was taken for GC analysis, and the calculated yield was 57.3%. The remaining upper oil phase was subjected to molecular distillation at 180°C, a vacuum of 0.1 Pa, and a condenser temperature of 30°C to obtain the final product. GC analysis showed that the diglyceride content was 70.2%.

[0081] Comparative Example 1

[0082] The comparative method for producing diglycerides by nanozyme-catalyzed glycerol hydrolysis includes the following steps:

[0083] Rapeseed oil and glycerol were mixed at a molar ratio of 1:3 to obtain a mixture. Antarctic Candida lipase B (CALB) (3.0% of the mixture mass) was added to the mixture, and the mixture was reacted at 50°C for 10 hours under a magnetic stirrer (500 rpm). After the reaction was complete, the mixture was centrifuged at 10,000 rpm for 10 minutes to separate the oil from the Antarctic Candida lipase B. A trace amount of the upper oil phase was taken for GC analysis, and the calculated yield was 44.3%. The remaining upper oil phase was subjected to molecular distillation at 170°C, a vacuum of 0.5 Pa, and a condenser temperature of 25°C to obtain the final product. GC analysis showed that the diglyceride content was 56.3%.

[0084] Comparative Example 2

[0085] The comparative method for producing diglycerides by nanozyme-catalyzed glycerol hydrolysis includes the following steps:

[0086] Rapeseed oil and glycerol were mixed at a molar ratio of 1:3 to obtain a series of mixtures. Antarctic Candida lipase B (CALB) (3.0% of the mixture mass) was added to the mixtures, and the mixtures were reacted at a constant temperature of 50°C using a magnetic stirrer (500 rpm). Samples (20 μL each time) were collected periodically at different time points (e.g., 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, ..., 24 h). The diglyceride content was analyzed by GC to track the reaction progress. The diglyceride content at each time point is shown in the figure. Figure 3 As shown.

[0087] Comparative Example 3

[0088] The comparative method for producing diglycerides by immobilized enzyme Novozym 435 catalyzing glycerol hydrolysis is as follows:

[0089] Rapeseed oil and glycerol were mixed at a molar ratio of 1:3 to obtain a series of mixtures. Commercially available immobilized enzyme Novozym 435 (3.0% of the mixture mass) was added to each mixture, and the mixture was reacted at 50°C under a magnetic stirrer (500 rpm). Samples (20 μL each time) were collected periodically at different time points (e.g., 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, ..., 24 h) to analyze the diglyceride content and track the reaction progress. The diglyceride content at each time point is shown below. Figure 3 As shown.

[0090] Depend on Figure 3 It is known that because nanozymes have hydrophobic chains on their surface, they can be better dispersed in the glycerol hydrolysis system. The reaction time is between 0 and 12 hours, which makes the DAG content of the nanozyme reaction system significantly higher than that of the reaction systems using CALB and Novozym 435. Furthermore, nanozymes reach the plateau phase earlier (6 hours), while Novozym 435 reaches the plateau phase at about 8 hours. CALB shows a decrease in DAG content due to excessively long reaction time (12 hours). Therefore, nanozymes exhibit higher reaction efficiency than Novozym 435 and CALB.

[0091] Implementation Results Example

[0092] 1. Stability upon repeated use

[0093] Rapeseed oil and glycerol were mixed at a molar ratio of 1:3 to obtain a mixture. Novozym 435 (used in Comparative Example 3) or the nanozyme prepared in Example 1 (3.0% of the total mass) was added to the mixture, and the mixture was placed in a magnetic stirrer (500 rpm) at 50°C for 10 hours. After the reaction was complete, the mixture was centrifuged at 10,000 rpm for 10 minutes to separate the oil from the nanozyme. The Novozym 435 and nanozyme were then subjected to the above-mentioned catalytic glycerol hydrolysis forty times. The centrifuged products were then analyzed by GC. Figure 4 ),from Figure 4 The results show that the DAG yield of nanozymes decreased only from 56.1% to 52.1% after forty glycerol hydrolysis reactions, while the DAG yield of Novozym 435 decreased from 51.4% to 47.3% after forty glycerol hydrolysis reactions. Nanozymes exhibited the same good reusability as immobilized enzymes.

[0094] After washing and drying the nanozyme, its enzyme activity was measured by adding 5 mL of 0.025 mol·L⁻¹ enzyme to an Erlenmeyer flask. -1A phosphate buffer solution at pH 7.5 and 4 mL of polyvinyl alcohol-ol-ol emulsion were incubated in a 40°C water bath for 5 min. 1 g of nanozyme was added, and the reaction was allowed to proceed for 15 min. 15 mL of 95% ethanol was added to terminate the enzyme reaction. Three drops of phenolphthalein indicator were then added, and the reaction was stopped with 0.05 mol·L⁻¹ water. -1 Titrate with sodium hydroxide until the solution turns pink, then subtract the sodium hydroxide consumed in the blank reaction. One unit of lipase activity is defined as the amount of enzyme required per minute to release 1 μmol of free fatty acid from the substrate (olive oil). The enzyme activity of the solution = volume of sodium hydroxide consumed (mL) × 0.05 × 1000 / 15. The enzyme activity remains at 92% after forty reactions.

[0095] 2. Temperature stability

[0096] Rapeseed oil and glycerol were mixed at a molar ratio of 1:3. Antarctic Candida lipase B (CALB) used in Comparative Example 1 or the nanozyme prepared in Example 1 (3.0% of the total mass) were added to the mixture. The mixtures were placed on magnetic stirrers (500 rpm) and reacted at constant temperatures of 30, 40, 50, 60, and 70°C for 10 hours. After the reaction was complete, the mixture was centrifuged at 10,000 rpm for 10 minutes (the upper oil phase was collected) to separate the oil from the nanozyme. A trace amount of the upper oil phase was taken for GC analysis. Figure 5 The remaining upper oil phases, obtained under different temperature conditions, were subjected to molecular distillation at 170℃, a vacuum of 0.5 Pa, and a condenser temperature of 25℃ to yield the final product. Figure 5 It can be seen that the immobilized nanozymes have better catalytic effects than free enzymes at every temperature. This is because after immobilizing free enzymes with nanoparticles, the nanozymes are in more complete contact with the lipid-soluble medium, thereby improving the catalytic efficiency. Furthermore, excessively high temperatures cause the spatial structure of free enzymes to lose their catalytic activity, while nanozymes, due to their immobilization, are more resistant to high temperatures and have good temperature stability.

[0097] 3. Stability at different pH levels

[0098] Several equal amounts of *Candida antarcticis* lipase B (CALB) and several equal amounts of the nanozyme prepared in Example 1 were incubated for 30 min in buffer systems of different pH values ​​(pH 5-11). After incubation, the *Candida antarcticis* lipase B (CALB) solution was added to a mixture of rapeseed oil and glycerol in a molar ratio of 1:3, and the mixture was kept at 50°C for 10 hours under a magnetic stirrer (500 rpm). After the reaction was completed, the mixture was centrifuged at 10000 rpm for 10 min to separate the oil from the nanozyme, and a trace amount of the upper oil phase was taken for GC analysis. Figure 6The remaining upper oil phases of different buffer solutions were subjected to molecular distillation at 170°C, a vacuum of 0.5 Pa, and a condenser temperature of 25°C to obtain the final product. Figure 6 As can be seen, the optimal pH for CALB is 7, while the optimal pH for nanozymes is 8. This is because after the enzyme is cross-linked with LGCS NPs, chitosan is present on the surface. Under acidic conditions, a large number of amino groups on the chitosan surface are protonated, causing the pH of the microenvironment on the carrier surface to be lower than the system pH. The acidic environment inhibits enzyme activity. Therefore, when the system pH is adjusted towards alkalinity, the degree of protonation of amino groups on the chitosan surface decreases, the microenvironment pH rises, and the enzyme catalytic activity reaches its peak. Thus, the optimal pH for nanozymes shifts towards alkalinity. Nanozymes exhibit higher catalytic activity at different pH levels, and the immobilized nanozymes have a wider pH adaptability range and better pH stability.

[0099] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for the efficient production of diglycerides from glycerol via nanozyme catalysis, characterized in that, The steps are as follows: (1) Gallic acid lauryl ester and chitosan were covalently cross-linked by free radical induction to form LGCS cross-linked material; LGCS cross-linked material was self-assembled to form nanoparticles, namely LGCS NPs; (2) Add genipin to the LGCS NPs obtained in step (1) to carry out cross-linking reaction; after the reaction is completed, add PBS buffer containing free lipase, carry out immobilization reaction under stirring, and obtain nanozyme after separation, washing and drying. (3) The nanozyme obtained in step (2) is dispersed in a mixture of oil and glycerol to carry out glycerolysis reaction, and then diglycerides are obtained after centrifugation and molecular distillation.

2. The method for efficient production of diglycerides via nanozyme-catalyzed glycerol hydrolysis according to claim 1, characterized in that, In step (1), the mass ratio of chitosan to gallic acid lauryl ester is 1:(0.4-2); the free radical induction method is carried out in the redox system of ascorbic acid and hydrogen peroxide, wherein the molar ratio of ascorbic acid to hydrogen peroxide is 1:(5-10); and the mass ratio of ascorbic acid to chitosan is 1:(1.5-7).

3. The method for high-efficiency production of diglycerides by nanozyme-catalyzed glycerol hydrolysis according to claim 2, characterized in that, The self-assembly method in step (1) refers to: dissolving LGCS crosslinked powder in acetic acid solution and stirring at 400-800 rpm / min to form nanoparticles.

4. The method for efficient production of diglycerides by nanozyme-catalyzed glycerol hydrolysis according to claim 3, characterized in that, In step (2), the mass ratio of LGCS NPs to genipin is 3:(0.5-5), the crosslinking reaction temperature is 25-40℃, and the time is 30-90 min.

5. The method for efficient production of diglycerides by nanozyme-catalyzed glycerol hydrolysis according to claim 4, characterized in that, In step (2), the mass ratio of LGCS NPs to free lipase is 100:(1-10); the pH of the immobilization reaction is 7.0-8.5, the temperature is 20-35℃, and the time is 4-10h.

6. The method for efficient production of diglycerides by nanozyme-catalyzed glycerol hydrolysis according to any one of claims 1-5, characterized in that, In step (2), the free lipase is any one of Lipozyme RM, Lipozyme TL 100L, and Candida antarcticis lipase B.

7. The method for efficient production of diglycerides via nanozyme-catalyzed glycerol hydrolysis according to claim 6, characterized in that, In step (3), the molar ratio of glycerol to oil is (1.5-3):1, and the amount of nanozyme used is 1-5% of the total mass of oil and glycerol.

8. The method for efficient production of diglycerides by nanozyme-catalyzed glycerol hydrolysis according to claim 7, characterized in that, The temperature of the glycerol hydrolysis reaction in step (3) is 40-60℃ and the time is 6-24h.

9. The method for efficient production of diglycerides by nanozyme-catalyzed glycerol hydrolysis according to claim 8, characterized in that, In step (3), the oil is vegetable oil or animal oil; the centrifugal separation speed is 8000-12000 rpm and the time is 10-20 min; the conditions for molecular distillation are: temperature 160-180℃, vacuum degree 0.1-1 Pa, condenser temperature 20-30℃.

10. The method for efficient production of diglycerides by nanozyme-catalyzed glycerol hydrolysis according to claim 9, characterized in that, The vegetable oil is one or more of flaxseed oil, soybean oil, rapeseed oil, corn oil, and peanut oil, and the animal oil is lard or horse fat.

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