Metal organic framework zif-8 immobilized enzyme, method for preparing the same and application thereof in preparing diglyceride

Abstract

The application discloses a kind of metal organic framework ZIF-8 fixed enzyme and its preparation method and application in preparation of diglyceride, to provide a kind of can effectively improve enzyme stability and the reuse of fixed enzyme, with the characteristics of simple operation, mild condition, can effectively promote edible oil preparation rich in diglyceride component edible oil while retaining enzyme activity, its prepared diglyceride edible oil byproduct is less, active ingredient is higher;Its technical scheme is that aspergillus niger lipase is added to zinc nitrate solution, is uniformly dispersed, then, slowly 2-methylimidazole solution is added, uniform speed stirring 60min, stationary 20min, the product is centrifuged, washed, freeze-dried to obtain fixed enzyme;Then raw material oil and glycerol are mixed, emulsified, then ZIF-8 fixed enzyme is added, glycerolysis reaction is carried out at 40~70℃ for 2~12h, after reaction, fixed enzyme is centrifuged, liquid is purified by molecular distillation, and DAG-rich edible oil is obtained;It is related to food technology field.

Description

Technical Field

[0001] This invention relates to the field of food technology, specifically to a metal-organic framework ZIF-8 immobilized enzyme, its preparation method, and its applications. Background Technology

[0002] Structural lipids, also known as reconstructed lipids, are oils with special nutritional functions synthesized through modification or structural reorganization of ordinary oils. Generally speaking, structural lipids are glycerides formed by the esterification of fatty acids of different chain lengths. These novel lipids can fulfill the physiological functions and nutritional value of fatty acids. Typically, structural lipids include triglycerides, diglycerides, monoglycerides, and phospholipids.

[0003] Diacylglycerol (DAG) is a natural component of various edible oils, existing as three different glycerol isomers: Sn-1,2-DAG, Sn-2,3-DAG, and Sn-1,3-DAG. Natural vegetable oils contain relatively low levels of DAG, generally less than 10%. Recent studies have found that the health benefits of DAG are related to its unique metabolic pathway. DAG can reduce the accumulation of fat in the abdomen and internal organs, lower serum TAG concentration, promote fatty acid β-oxidation, alleviate diabetes, and prevent arteriosclerosis. Furthermore, because DAG is metabolized differently in the human body than TAG, triglycerides (TAG) first form emulsion droplets in the intestines. Pancreatic lipase specifically recognizes and acts on the Sn-1 and Sn-3 ester bonds of the glycerol backbone, regioselectively hydrolyzing them to cleave two fatty acid molecules, generating one molecule of 2-monoglycerate (2-MAG) and two molecules of free fatty acid (FFA). After being absorbed by the intestinal epithelial cells, the 2-monoglycerate and fatty acid are efficiently re-esterified into triglycerides via a re-esterification pathway and released into the bloodstream, thus increasing blood lipid levels and becoming a key dietary factor contributing to obesity. In contrast, diglycerides exhibit a completely different metabolic pathway: their hydrolysis products are more likely to enter the liver for β-oxidation for energy rather than serving as precursors for fat storage.

[0004] Therefore, compared to triglycerides, consuming diglycerides can reduce the chance of triglycerides being resynthesized in the body, thus effectively preventing the accumulation of fat. Furthermore, DAG molecules have strong lipophilicity and some hydrophilicity; their unique molecular structure endows DAG with lubricating and emulsifying properties. In addition, it has advantages such as safety and nutrition, and is widely used in the food, pharmaceutical, and chemical industries, showing promising industrial application prospects.

[0005] Methods for synthesizing diacylglycerol (DAG) are divided into chemical and enzymatic methods. Based on different reaction mechanisms, they can also be classified as direct esterification, glycerolysis, and partial hydrolysis of oils and fats. Traditional chemical catalysis typically utilizes inorganic catalysts to drive glycerolysis or hydrolysis reactions under high temperature, vacuum, or inert gas protection. This process has stringent requirements for reaction conditions; high temperatures easily lead to the degradation and isomerization of heat-sensitive unsaturated fatty acids and other nutrients in oils and fats, triggering complex side reactions that result in a darker color, deteriorated flavor, and a significant decline in sensory quality of the final product. Furthermore, chemical catalysts lack site specificity, making it impossible to achieve the controlled synthesis of lipids with specific structures. Enzymatic synthesis processes are increasingly favored due to their mild reaction conditions, excellent product flavor, and environmental friendliness. Lipases, as highly efficient biocatalysts, possess high catalytic efficiency, strong regioselectivity, and reusability. Based on these significant advantages, the production of diacylglycerol (DAG) using lipase catalysis has become the mainstream research and application direction, gradually replacing traditional chemical synthesis methods.

[0006] The high sensitivity of free enzymes to environmental conditions (such as temperature, pH, and organic solvents) makes them highly susceptible to irreversible inactivation under suboptimal conditions. This inherent instability makes them unable to withstand the harsh operating conditions commonly found in industrial reactors, greatly limiting their potential for widespread application in numerous fields such as synthetic chemistry, biocatalysis, and food processing. Summary of the Invention

[0007] To address the aforementioned problems, the first objective of this invention is to provide a metal-organic framework ZIF-8 immobilized enzyme (ANL@ZIF-8), which exhibits higher activity, stability, and catalytic efficiency than the free enzyme; furthermore, it is easily separated and recovered, enabling multiple cyclic catalysis and significantly reducing costs.

[0008] A second objective of this invention is to provide a method for preparing a metal-organic framework ZIF-8 immobilized enzyme.

[0009] The method is simple to operate, operates under mild conditions, and can effectively achieve enzyme immobilization.

[0010] The third objective of this invention is to provide a method for preparing diglyceride edible oil, which produces diglyceride edible oil with lower byproducts, higher effective components, and physiological effects such as reducing body fat accumulation and improving blood lipid levels.

[0011] To achieve the above objectives, the first technical solution provided by this invention is as follows:

[0012] Aspergillus niger lipase was added to a 2-methylimidazole solution and dispersed evenly. Then, zinc nitrate solution was slowly added and stirred at a constant speed for 20-60 min. After standing for 20-60 min, the product was centrifuged, the solid was collected, washed, and freeze-dried to obtain the immobilized enzyme.

[0013] The molar ratio of zinc nitrate hexahydrate to 2-methylimidazole is 1:4 to 1:8;

[0014] The amount of Aspergillus niger lipase added is 10-40% of the weight of 2-methylimidazole.

[0015] Furthermore, in the above-mentioned method for preparing the metal-organic framework ZIF-8 immobilized enzyme, the concentration of the zinc nitrate solution is 20-40 mM; and the concentration of the 2-methylimidazole solution is 80-400 mM.

[0016] Furthermore, in the above-mentioned method for preparing the metal-organic framework ZIF-8 immobilized enzyme, the washing process involves washing with deionized water 3 to 6 times.

[0017] Furthermore, in the above-mentioned method for preparing the metal-organic framework ZIF-8 immobilized enzyme, the stirring speed during uniform stirring is 100-400 r / min; the centrifugation speed during centrifugation is 8000 r / min.

[0018] Furthermore, in the above-mentioned method for preparing the metal-organic framework ZIF-8 immobilized enzyme, the freeze-drying temperature is -40℃ and the freeze-drying time is 8-12h.

[0019] The second technical solution provided by the present invention is a metal-organic framework ZIF-8 immobilized enzyme, which is prepared by the method described in the first technical solution.

[0020] The third technical solution provided by this invention is a method for preparing diglyceride edible oil, which includes the following steps in sequence:

[0021] (1) Mix the raw oil and glycerol, stir and emulsify, then add metal-organic framework ZIF-8 immobilized enzyme, and carry out glycerolysis reaction at 40-70℃ for 2-12 hours. After the reaction is completed, centrifuge to separate the immobilized enzyme. The resulting liquid mixture is a crude product rich in diglycerides.

[0022] (2) The crude diglyceride product obtained in step (1) is purified by molecular distillation to obtain edible oil rich in DAG.

[0023] The molar ratio of raw oil to glycerin is 1:1 to 1:5;

[0024] The amount of the metal-organic framework ZIF-8 immobilized enzyme added is 1% to 5% of the weight of the raw oil.

[0025] Furthermore, in the above-mentioned method for preparing diglyceride edible oil, the raw material oil is rapeseed oil.

[0026] Furthermore, in the above-mentioned method for preparing diglyceride edible oil, the molecular distillation is a two-stage molecular distillation.

[0027] Furthermore, the preparation method of the above-mentioned diglyceride edible oil, the primary molecular distillation conditions are as follows: distillation temperature is 170-200℃, vacuum degree is 10-50Pa, and film scraping speed is 300-400r / min;

[0028] The conditions for secondary molecular distillation are as follows: distillation temperature 200-230℃, vacuum degree 1-10Pa, and film scraping speed 300-400r / min.

[0029] Compared with existing technologies, the present invention achieves the following beneficial effects:

[0030] 1. The immobilized enzyme catalyst prepared by this invention can provide a rigid framework shield for enzyme molecules, resisting environments such as high temperature, acid and alkali and organic solvents. It has the characteristics of high specific surface area, high porosity and excellent mass transfer efficiency. Its activity, stability and catalytic efficiency are higher than those of free enzymes. Moreover, it is easy to separate and recover, and can realize multiple catalytic cycles, significantly reducing costs.

[0031] 2. The process employed in this invention is based entirely on a solvent-free enzyme-catalyzed glycerol hydrolysis system. This method has significant advantages such as mild reaction conditions, short reaction time, and simple post-processing. It also minimizes side reactions and avoids the generation of large amounts of wastewater and other secondary waste, thereby significantly reducing production costs and environmental burden.

[0032] 3. The diglyceride edible oil prepared by this invention has fewer byproducts and more active ingredients, and has physiological effects such as reducing body fat accumulation and improving blood lipid levels. Attached Figure Description

[0033] Figure 1 This is a gas chromatogram of a diglyceride product;

[0034] Among them, FFA represents free fatty acids, MAG represents monoglycerides, DAG represents diglycerides, and TAG represents triglycerides.

[0035] Figure 2 Comparison of diglycerides obtained from immobilized enzyme (ANL@ZIF-8), free Aspergillus niger lipase (ANL), and commercial enzyme (Novozym 435) at different reaction temperatures;

[0036] Figure 3Comparison of diglycerides obtained from immobilized enzyme (ANL@ZIF-8), free Aspergillus niger lipase (ANL), and commercial enzyme (Novozym 435) at different reaction times;

[0037] Figure 4 A comparison of diglycerides obtained from immobilized enzyme (ANL@ZIF-8), free Aspergillus niger lipase (ANL), and commercial enzyme (Novozym 435) at different enzyme addition amounts;

[0038] Figure 5 A comparison of diglycerides obtained from immobilized enzyme (ANL@ZIF-8), free Aspergillus niger lipase (ANL), and commercial enzyme (Novozym 435) at different substrate molar ratios; Detailed Implementation

[0039] The embodiments of the present invention will be clearly and completely described below. It should be understood that the embodiments described herein are merely some examples of the present invention, and are intended to illustrate the technical solutions in conjunction with specific embodiments to ensure full coverage of the protection scope of the present invention, and to cover all changes and modifications that may arise based on the claims of the present invention.

[0040] Example 1

[0041] This embodiment provides a metal-organic framework ZIF-8 immobilized enzyme, which is prepared by the following method:

[0042] 1) Add 0.297g of zinc nitrate hexahydrate crystals to a 100ml beaker, add deionized water to 50ml, stir to dissolve, and prepare 50ml of 20mM zinc nitrate solution; dissolve 0.328g of 2-methylimidazole in 50ml of deionized water to prepare 80mM 2-methylimidazole solution.

[0043] 2) Add 5 ml of Aspergillus niger lipase to the zinc nitrate solution prepared in step 1), stirring constantly during the addition process to ensure uniform dispersion. Then, add all of the 2-methylimidazole solution prepared in step 1), stir at 300 r / min for 1 h, and then stop stirring. Let the mixture stand for 20 min, pour it into a centrifuge tube, and centrifuge at 8000 r / min for 10 min. After centrifugation, remove the centrifuge tube, pour off the supernatant, and wash with deionized water. Repeat the operation 3 times.

[0044] 3) Finally, scrape off the precipitate from the bottom of the centrifuge tube, place it in a petri dish, and freeze-dry it at -40℃ for 8 hours to obtain ZIF-8 immobilized enzyme.

[0045] Example 2

[0046] This embodiment provides a metal-organic framework ZIF-8 immobilized enzyme, which is prepared by the following method:

[0047] 1) Add 0.297g of zinc nitrate hexahydrate crystals to a 100ml beaker, add deionized water to 50ml, stir to dissolve, and prepare 50ml of zinc nitrate solution with a concentration of 20mM; dissolve 0.657g of 2-methylimidazole in 50ml of deionized water to prepare 2-methylimidazole solution with a concentration of 160mM.

[0048] 2) Add 5 ml of Aspergillus niger lipase to the zinc nitrate solution prepared in step 1), stirring constantly during the addition process to ensure uniform dispersion. Then, add all of the 2-methylimidazole solution prepared in step 1), stir at 300 r / min for 1 h, and then stop stirring. Let the mixture stand for 20 min, pour it into a centrifuge tube, and centrifuge at 8000 r / min for 10 min. After centrifugation, remove the centrifuge tube, pour off the supernatant, and wash with deionized water. Repeat the operation 3 times.

[0049] 3) Finally, scrape off the precipitate from the bottom of the centrifuge tube, place it in a petri dish, and freeze-dry it at -40℃ for 8 hours to obtain ZIF-8 immobilized enzyme.

[0050] Example 3

[0051] This embodiment provides a metal-organic framework ZIF-8 immobilized enzyme, which is prepared by the following method:

[0052] 1) Add 0.297g of zinc nitrate hexahydrate crystals to a 100ml beaker, add deionized water to 50ml, stir to dissolve, and prepare 50ml of zinc nitrate solution with a concentration of 20mM. Dissolve 0.985g of 2-methylimidazole in 50ml of deionized water to prepare 2-methylimidazole solution with a concentration of 240mM.

[0053] 2) Add 5 ml of Aspergillus niger lipase to the zinc nitrate solution prepared in step 1), stirring constantly during the addition process to ensure uniform dispersion. Then, add all of the 2-methylimidazole solution prepared in step 1), stir at 300 r / min for 1 h, and then stop stirring. Let the mixture stand for 20 min, pour it into a centrifuge tube, and centrifuge at 8000 r / min for 10 min. After centrifugation, remove the centrifuge tube, pour off the supernatant, and wash with deionized water. Repeat the operation 3 times.

[0054] 3) Finally, scrape off the precipitate from the bottom of the centrifuge tube, place it in a petri dish, and freeze-dry it at -40℃ for 10 hours to obtain ZIF-8 immobilized enzyme.

[0055] Example 4

[0056] This embodiment provides a metal-organic framework ZIF-8 immobilized enzyme, which is prepared by the following method:

[0057] 1) Add 0.297g of zinc nitrate hexahydrate crystals to a 100ml beaker, add deionized water to 50ml, stir to dissolve, and prepare 50ml of 20mM zinc nitrate solution; dissolve 1.314g of 2-methylimidazole in 50ml of deionized water to prepare 320mM 2-methylimidazole solution.

[0058] 2) Add 5 ml of Aspergillus niger lipase to the zinc nitrate solution prepared in step 1), stirring constantly during the addition process to ensure uniform dispersion. Then, add all of the 2-methylimidazole solution prepared in step 1), stir at 400 r / min for 1 h, and then stop stirring. Let the mixture stand for 20 min, pour it into a centrifuge tube, and centrifuge at 8000 r / min for 10 min. After centrifugation, remove the centrifuge tube, pour off the supernatant, and wash with deionized water. Repeat the operation 6 times.

[0059] 3) Finally, scrape off the precipitate from the bottom of the centrifuge tube, place it in a petri dish, and freeze-dry it at -40℃ for 8 hours to obtain ZIF-8 immobilized enzyme.

[0060] Example 5

[0061] This embodiment provides a metal-organic framework ZIF-8 immobilized enzyme, which is prepared by the following method:

[0062] 1) Add 0.595g of zinc nitrate hexahydrate crystals to a 100ml beaker, add deionized water to 50ml, stir to dissolve, and prepare a zinc nitrate solution with a concentration of 40mM. Dissolve 1.642g of 2-methylimidazole in 50ml of deionized water to prepare a 2-methylimidazole solution with a concentration of 400mM.

[0063] 2) Add 5 ml of Aspergillus niger lipase to the 2-methylimidazole solution prepared in step 1), stirring continuously during the addition process to ensure uniform dispersion. Then, add all of the zinc nitrate solution prepared in step 1), stir at 400 r / min for 1 h, and then stop stirring. Let the mixture stand for 20 min, pour it into a centrifuge tube, and centrifuge at 8000 r / min for 10 min. After centrifugation, remove the centrifuge tube, pour off the supernatant, and wash with deionized water. Repeat the operation 6 times.

[0064] 3) Finally, scrape off the precipitate from the bottom of the centrifuge tube, place it in a petri dish, and freeze-dry it at -40℃ for 8 hours to obtain ZIF-8 immobilized enzyme.

[0065] Example 6

[0066] This embodiment provides a metal-organic framework ZIF-8 immobilized enzyme, which is prepared in a manner similar to that of Example 1, except that the stirring speed is 350 r / min and the enzyme is freeze-dried for 9 h.

[0067] Example 7

[0068] This embodiment provides a metal-organic framework ZIF-8 immobilized enzyme, which is prepared in a manner similar to that of Example 1, except that the stirring speed is 200 r / min and the enzyme is freeze-dried for 12 h.

[0069] Example 8

[0070] This embodiment provides a metal-organic framework ZIF-8 immobilized enzyme, which is prepared in a manner similar to that of Example 1, except that the zinc nitrate solution concentration is 35 mM.

[0071] Application Example 1

[0072] This application example provides a method for preparing diglyceride edible oil. The method is as follows: 50g of rapeseed oil and 7.87g of glycerol (molar ratio = 1:1.5) are placed in a 250ml flask and stirred for emulsification. The amount of metal-organic framework ZIF-8 prepared in Example 1 is added at 1% of the weight of the raw material oil (rapeseed oil and glycerol). The reaction is carried out for 10.5h at a temperature of 53℃. After the reaction is completed, the mixture is collected and centrifuged, and the supernatant is collected. The gas phase detection method is as follows: 50mg of glycerol ester sample is taken and dissolved in n-hexane solution. Then, the solution is drawn up with a 1ml syringe, filtered through a 0.22μm organic needle filter membrane, and injected into a gas phase detection bottle for gas phase analysis. The gas chromatography detection conditions were as follows: injection volume 1 μL, split ratio 40:1, column oven initial temperature 50℃, injection port temperature 360℃, flame ionization detector (FID) temperature 360℃, carrier gas nitrogen at a flow rate of 4.21 mL / min, and a programmed temperature ramp: initial column oven temperature 50℃ held for 1 min, then ramped to 200℃ at a rate of 20℃ / min and held for 1 min, then ramped to 240℃ at a rate of 10℃ / min and held for 1 min, then ramped to 360℃ at a rate of 40℃ / min and held at 360℃ for 15 min. The gas chromatogram of the obtained product is shown below. Figure 1 As shown, through Figure 1 The peak elution order of the substances can be seen as FFA (9.2–11.3 min), MAG (12.2–15.8 min), DAG (17.8–20.8 min), and TAG (27.4–32.2 min).

[0073] The content of glycerides was analyzed and calculated using the peak area normalization method. The calculation results are shown in Table 1.

[0074] Separation and purification: The crude product was purified by molecular distillation. The first-stage molecular distillation conditions were as follows: distillation temperature 170℃, vacuum degree 10Pa, and scraping speed 300 r / min. The second-stage molecular distillation conditions were as follows: distillation temperature 200℃, vacuum degree 1Pa, and scraping speed 300 r / min.

[0075] Application Example 2

[0076] This application example provides a method for preparing diglyceride edible oil. The only difference between this method and application example 1 is that an equal amount of metal-organic framework ZIF-8 immobilized enzyme prepared in example 2 is used instead of metal-organic framework ZIF-8 immobilized enzyme prepared in example 1.

[0077] Application Example 3

[0078] This application example provides a method for preparing diglyceride edible oil. The only difference between this method and application example 1 is that an equal amount of metal-organic framework ZIF-8 immobilized enzyme prepared in example 3 is used instead of metal-organic framework ZIF-8 immobilized enzyme prepared in example 1.

[0079] Application Example 4

[0080] This application example provides a method for preparing diglyceride edible oil. The only difference between this method and application example 1 is that an equal amount of metal-organic framework ZIF-8 immobilized enzyme prepared in example 4 is used instead of metal-organic framework ZIF-8 immobilized enzyme prepared in example 1.

[0081] Application Example 5

[0082] This application example provides a method for preparing diglyceride edible oil. The only difference between this method and application example 1 is that an equal amount of metal-organic framework ZIF-8 immobilized enzyme prepared in example 5 is used instead of the metal-organic framework ZIF-8 immobilized enzyme prepared in example 1.

[0083] Application Example 6

[0084] This application example provides a method for preparing diglyceride edible oil. The only difference between this method and application example 1 is that an equal amount of metal-organic framework ZIF-8 immobilized enzyme prepared in example 6 is used instead of the metal-organic framework ZIF-8 immobilized enzyme prepared in example 1.

[0085] Application Example 7

[0086] This application example provides a method for preparing diglyceride edible oil. The only difference between this method and application example 1 is that an equal amount of metal-organic framework ZIF-8 immobilized enzyme prepared in example 7 is used instead of metal-organic framework ZIF-8 immobilized enzyme prepared in example 1.

[0087] Application Example 8

[0088] This application example provides a method for preparing diglyceride edible oil. The only difference between this method and application example 1 is that an equal amount of metal-organic framework ZIF-8 immobilized enzyme prepared in example 8 is used instead of the metal-organic framework ZIF-8 immobilized enzyme prepared in example 1.

[0089] The specific experimental results of Application Examples 1 to 8 are shown in Table 1.

[0090] Table 1

[0091] Group Free fatty acids % monoglycerides % diglycerides % Triglycerides % Application Example 1 9.63 4.2 40.48 45.69 Application Example 2 13.52 8.21 39.7 38.57 Application Example 3 9.66 5.01 44.5 40.83 Application Example 4 7.53 6.8 35.81 49.86 Application Example 5 8.52 7.16 46.18 38.14 Application Example 6 5.74 5.89 42.66 45.71 Application Example 7 7.21 5.1 39.73 47.96 Application Example 8 6.39 7.89 41.85 43.87

[0092] As can be seen from Table 1, when the metal-organic framework ZIF-8 immobilized enzyme provided in this application is applied to the preparation of diglyceride edible oil, the content of diglycerides is relatively high, with the highest content of diglycerides reaching 46.18%.

[0093] Application Examples 9-15

[0094] This application example provides a method for preparing diglyceride edible oil. The only difference between this method and Application Example 5 is the reaction temperature. Specific reaction temperatures and experimental results are shown in Table 2 and... Figure 2 .

[0095] Table 2

[0096] Group Reaction temperature ℃ diglycerides % Application Example 9 40 39.51 Application Example 10 45 44.83 Application Example 11 50 49.55 Application Example 12 55 53.6 Application Example 13 60 51.05 Application Example 14 65 46.23 Application Example 15 70 43.44

[0097] Through Table 2 and Figure 2 Between 40 and 70°C, it can be seen that the content of diglycerides gradually increases with increasing temperature. At 55°C, the content of diglycerides is 53.6%. If the temperature is further increased, the content of diglycerides decreases instead. This is because as the reaction temperature increases, the collision between enzyme and substrate molecules intensifies, the reaction rate increases, and above a certain temperature, the enzyme gradually becomes inactive.

[0098] Application Examples 16-21

[0099] Application Examples 16-21 provide a method for preparing diglyceride edible oil. The only difference between this method and Application Example 1 is the reaction time. Specific reaction times and experimental results are shown in Table 3. Figure 3 .

[0100] Table 3

[0101] Group reaction time h diglycerides % Application Example 16 2 36.7 Application Example 17 4 40.42 Application Example 18 6 46.37 Application Example 19 8 47.58 Application Example 20 10 51.63 Application Example 21 12 43.55

[0102] Through Table 3 and Figure 3 It can be seen that the content of diglycerides gradually increases with the increase of reaction time. When the reaction time is 10h, the content of diglycerides is 57.32%. If the reaction time is extended further, the content of diglycerides does not change significantly. This is because there is an equilibrium between reactants and products. Diglycerides and monoglycerides will also be converted into triglycerides again under the catalysis of lipase.

[0103] Application Examples 22-26

[0104] Application Examples 22-26 provide a method for preparing diglyceride edible oil. The only difference between this method and Application Example 1 is the amount of metal-organic framework ZIF-8 immobilized enzyme used. Specific enzyme dosages and experimental results are shown in Table 4. Figure 4 .

[0105] Table 4

[0106] Group Metal-organic framework ZIF-8 immobilized enzymes as a percentage of crude oil content (wt%) diglycerides % Application Example 22 1 15.5 Application Example 23 2 32.33 Application Example 24 3 43.85 Application Example 25 4 49.41 Application Example 26 5 42.58

[0107] Through Table 4 and Figure 4 It can be seen that as the amount of catalyst added increases, the content of diglycerides gradually increases. When the amount of enzyme added is 4%, the content of diglycerides is 49.51%. If the amount of enzyme added is further increased, the content of diglycerides will decrease instead. This is because excessive lipase content causes aggregation. This aggregation will mask the active site of the enzyme, thereby reducing the proportion of enzyme molecules that can effectively participate in the catalytic reaction.

[0108] Application Examples 27-31

[0109] Application Examples 27-31 provide a method for preparing diglyceride edible oil. The only difference between this method and Application Example 1 is the substrate molar ratio. The specific substrate molar ratio and experimental results are shown in Table 5. Figure 5 .

[0110] Table 5

[0111] Group substrate molar ratio diglycerides % Application Example 27 1:1 15.47 Application Example 28 1:2 32.7 Application Example 29 1:3 42.31 Application Example 30 1:4 50.49 Application Example 31 1:5 49.53

[0112] Through Table 5 and Figure 5 It can be seen that as the content of glycerol in the system increases, the content of diglycerides increases significantly. When the substrate molar ratio is 1:4, the content of diglycerides reaches 50.49%. This is because the increase in the relative content of glycerol can drive the reaction forward and increase the conversion rate of TAG.

[0113] Examples of proportional application 1-7

[0114] A method for preparing diglyceride edible oil is provided. The only difference between this method and Application Examples 9-15 is that, at the same temperature, free Aspergillus niger lipase ANL is used instead of the metal-organic framework ZIF-8 immobilized enzyme. Specific reaction temperatures, catalysts, and experimental results are shown in Table 5 and [Table data would be inserted here]. Figure 2 .

[0115] The free Aspergillus niger lipase ANL was purchased from Aladdin Chemical Reagent Co., Ltd.

[0116] Table 5

[0117] Group catalyst Reaction temperature ℃ diglycerides % Comparative Application Example 1 ANL 40 21.87 Comparative Application Example 2 ANL 45 35.79 Comparative Application Example 3 ANL 50 42.03 Comparative Application Example 4 ANL 55 45.37 Comparative Application Example 5 ANL 60 43.31 Comparative Application Example 6 ANL 65 39.33 Comparative Application Example 7 ANL 70 37.36

[0118] Through Table 2, Table 5 and Figure 2 It can be seen that, under the same reaction temperature conditions, the diglyceride content prepared using the metal-organic framework ZI-8 immobilized enzyme is significantly higher than that prepared using free Aspergillus niger lipase ANL. For example, at 40℃, the diglyceride content prepared using the metal-organic framework ZI-8 immobilized enzyme is 39.51%, while that prepared using free Aspergillus niger lipase ANL is only 21.87%; at 55℃, the former reaches 53.6% diglyceride content, while the latter is only 45.37%.

[0119] This indicates that ZIF-8 metal-organic framework immobilized enzymes can catalyze the reaction more effectively, increasing the production of diglycerides. This may be because the immobilized enzyme structure is more stable, maintaining high activity during the reaction and reducing enzyme inactivation and aggregation.

[0120] Examples of proportional application 8-14

[0121] Examples 8-14 provide a method for preparing diglyceride edible oil. The only difference between this method and Examples 9-15 is that the metal-organic framework ZIF-8 immobilized enzyme is replaced with Novozym 435 catalyst at the same temperature. Specific reaction temperatures, catalysts, and experimental results are shown in Table 6. Figure 2 .

[0122] The Novozym 435 lipase mentioned was purchased from Novozymes Biotechnology Co., Ltd.

[0123] Table 6

[0124] Group catalyst Reaction temperature ℃ diglycerides % Comparative Application Example 8 Novozym 435 40 35.07 Comparative Application Example 9 Novozym 435 45 39.86 Comparative Application Example 10 Novozym 435 50 46.71 Comparative Application Example 11 Novozym 435 55 49.68 Comparative Application Example 12 Novozym 435 60 50.18 Comparative Application Example 13 Novozym 435 65 42.64 Comparative Application Example 14 Novozym 435 70 39.94

[0125] Through Table 2, Table 6 and Figure 2It can be seen that, under the same reaction temperature conditions, the content of diglycerides prepared using the metal-organic framework ZIF-8 immobilized enzyme provided in this application is basically the same as that prepared using the Novozym 435 catalyst. However, the price of the metal-organic framework ZIF-8 immobilized enzyme provided in this application is much lower than that of the Novozym 435 catalyst (the market price of 10g of Novozym435 is about RMB 1275, while the price of 10g of raw material free Aspergillus niger lipase is RMB 280).

[0126] Examples of proportional application 15-20

[0127] Examples 15-20 provide a method for preparing diglyceride edible oil. The only difference between this method and Examples 16-23 is that, under the same time conditions, free Aspergillus niger lipase ANL is used instead of the metal-organic framework ZIF-8 immobilized enzyme. Specific reaction temperatures, catalysts, and experimental results are shown in Table 7. Figure 3 .

[0128] Table 7

[0129] Group catalyst reaction time h diglycerides % Comparative Application Example 15 ANL 2 26.26 Comparative Application Example 16 ANL 4 33.22 Comparative Application Example 17 ANL 6 36.56 Comparative Application Example 18 ANL 8 41.62 Comparative Application Example 19 ANL 10 43.56 Comparative Application Example 20 ANL 12 39.51

[0130] Through Table 2, Table 7 and Figure 3 It can be seen that, under the same reaction time conditions, the diglyceride content prepared using the metal-organic framework ZIF-8 immobilized enzyme is significantly higher than that prepared using free Aspergillus niger lipase ANL. For example, after 2 hours of reaction, the diglyceride content prepared using the metal-organic framework ZIF-8 immobilized enzyme is 36.7%, while that prepared using free Aspergillus niger lipase ANL is only 26.26%; after 10 hours of reaction, the former reaches 57.32% diglyceride content, while the latter is only 43.56%.

[0131] Examples of proportional application 21-26

[0132] A method for preparing diglyceride edible oil is provided. The only difference between this method and Application Examples 16-23 is that the metal-organic framework ZIF-8 immobilized enzyme is replaced with Novozym 435 catalyst at the same reaction time. Specific reaction times, catalysts, and experimental results are shown in Table 8. Figure 3 .

[0133] Table 8

[0134] Group catalyst reaction time h diglycerides % Comparative Application Example 21 Novozym 435 2 35.66 Comparative Application Example 22 Novozym 435 4 38.62 Comparative Application Example 23 Novozym 435 6 40.77 Comparative Application Example 24 Novozym 435 8 43.64 Comparative Application Example 25 Novozym 435 10 48.19 Comparative Application Example 26 Novozym 435 12 43.9

[0135] Through Table 3, Table 8 and Figure 3It can be seen that, under the same reaction time conditions, the content of diglycerides prepared using the metal-organic framework ZIF-8 immobilized enzyme provided in this application is close to that prepared using the Novozym 435 catalyst. However, the price of the metal-organic framework ZIF-8 immobilized enzyme provided in this application is much lower than that of the Novozym 435 catalyst (the market price of 10g of Novozym 435 is about RMB 1275, while the price of 10g of raw material free Aspergillus niger lipase is RMB 280).

[0136] Comparative examples 27-31

[0137] A method for preparing diglyceride edible oil is provided. The only difference between this method and application examples 22-26 is that, at the same enzyme dosage, free Aspergillus niger lipase ANL is used instead of the metal-organic framework ZIF-8 immobilized enzyme. Specific enzyme dosage, catalyst, and experimental results are shown in Table 9. Figure 4 .

[0138] Table 9

[0139] Group catalyst ANL accounts for wt% of crude oil volume diglycerides % Comparative Application Example 27 ANL 1 13.98 Comparative Application Example 28 ANL 2 28.5 Comparative Application Example 29 ANL 3 32.32 Comparative Application Example 30 ANL 4 35.36 Comparative Application Example 31 ANL 5 31.9

[0140] By analyzing Tables 4, 9, and... Figure 4 It can be seen that, under the same enzyme addition conditions, the content of diglycerides prepared using the metal-organic framework ZIF-8 immobilized enzyme is significantly higher than that prepared using free Aspergillus niger lipase ANL.

[0141] Comparative examples 32-36

[0142] A method for preparing diglyceride edible oil is provided. The only difference between this method and application examples 22-26 is that, at the same enzyme dosage, Novozym 435 lipase is used instead of the metal-organic framework ZIF-8 immobilized enzyme. Specific enzyme dosage, catalyst, and experimental results are shown in Table 10. Figure 4 .

[0143] Table 10

[0144] Group catalyst Novozym 435 as a percentage of crude oil volume (wt%) diglycerides % Comparative Application Example 32 Novozym 435 1 13.26 Comparative Application Example 33 Novozym 435 2 30.99 Comparative Application Example 34 Novozym 435 3 37.3 Comparative Application Example 35 Novozym 435 4 44.33 Comparative Application Example 36 Novozym 435 5 36.28

[0145] Through Table 5, Table 10 and Figure 4 It can be seen that, under the same enzyme addition conditions, the content of diglycerides prepared using the metal-organic framework ZIF-8 immobilized enzyme provided in this application is similar to that prepared using Novozym 435 lipase. However, the price of the metal-organic framework ZIF-8 immobilized enzyme provided in this application is much lower than that of the Novozym 435 catalyst (the market price of 10g of Novozym435 is about RMB 1275, while the price of 10g of raw material free Aspergillus niger lipase is RMB 280).

[0146] Comparative examples 37-41

[0147] A method for preparing diglyceride edible oil is provided. The only difference between this method and Application Examples 27-31 is that, at the same substrate molar ratio, free Aspergillus niger lipase ANL is used instead of the metal-organic framework ZIF-8 immobilized enzyme. Specific substrate molar ratios, catalysts, and experimental results are shown in Table 11 and... Figure 5 .

[0148] Table 11

[0149] Group catalyst substrate molar ratio diglycerides % Comparative Application Example 37 ANL 1:1 13.26 Comparative Application Example 38 ANL 1:2 27.65 Comparative Application Example 39 ANL 1:3 32.59 Comparative Application Example 40 ANL 1:4 39.43 Comparative Application Example 41 ANL 1:5 41.99

[0150] Through Table 6, Table 11 and Figure 5 It can be seen that, under the same substrate molar ratio, the content of diglycerides prepared using the metal-organic framework ZIF-8 immobilized enzyme is significantly higher than that prepared using free Aspergillus niger lipase ANL.

[0151] Comparative examples 42-46

[0152] A method for preparing diglyceride edible oil is provided. The only difference between this method and Application Examples 27-31 is that, at the same substrate molar ratio, Novozym 435 lipase is used instead of the metal-organic framework ZIF-8 immobilized enzyme. Specific substrate molar ratios, catalysts, and experimental results are shown in Table 12 and... Figure 5 .

[0153] Table 12

[0154] Group catalyst substrate molar ratio diglycerides % Comparative Application Example 42 Novozym 435 1:1 12.98 Comparative Application Example 43 Novozym 435 1:2 29.51 Comparative Application Example 45 Novozym 435 1:3 37.89 Comparative Application Example 46 Novozym 435 1:4 44.78 Comparative Application Example 47 Novozym 435 1:5 46.86

[0155] Through Table 7, Table 12 and Figure 5 It can be seen that, under the same substrate molar ratio conditions, the content of diglycerides prepared using the metal-organic framework ZIF-8 immobilized enzyme provided in this application is close to that prepared using Novozym 435 lipase. However, the price of the metal-organic framework ZIF-8 immobilized enzyme provided in this application is much lower than that of the Novozym 435 catalyst (the market price of 10g of Novozym435 is about RMB 1275, while the price of 10g of raw material free Aspergillus niger lipase is RMB 280).

[0156] The embodiments disclosed herein, as described above, are sufficient to enable those skilled in the art to make or apply the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention should not be limited to the embodiments shown herein, but should be given the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a metal-organic framework ZIF-8 immobilized enzyme, characterized in that, Aspergillus niger lipase was added to a 2-methylimidazole solution and dispersed evenly. Then, zinc nitrate solution was slowly added and stirred at a constant speed for 20-60 min. After standing for 20-60 min, the product was centrifuged, the solid was collected, washed, and freeze-dried to obtain the immobilized enzyme. The molar ratio of zinc nitrate hexahydrate to 2-methylimidazole is 1:4 to 1:8; The amount of Aspergillus niger lipase added is 10-40% of the weight of 2-methylimidazole.

2. The method for preparing the metal-organic framework ZIF-8 immobilized enzyme according to claim 1, characterized in that, The zinc nitrate solution has a concentration of 20–40 mM; the 2-methylimidazole solution has a concentration of 80–400 mM.

3. The method for preparing the metal-organic framework ZIF-8 immobilized enzyme according to claim 1, characterized in that, The cleaning process involves rinsing with deionized water 3 to 6 times.

4. The method for preparing the metal-organic framework ZIF-8 immobilized enzyme according to claim 1, characterized in that, The stirring speed for uniform stirring is 100–400 r / min; the centrifugation speed for centrifugation is 8000 r / min.

5. The method for preparing the metal-organic framework ZIF-8 immobilized enzyme according to claim 1, characterized in that, The freeze-drying temperature is -40℃, and the freeze-drying time is 8 to 12 hours.

6. A metal-organic framework ZIF-8 immobilized enzyme, characterized in that, Prepared using the method described in any one of claims 1 to 5.

7. A method for preparing a diglyceride edible oil, characterized in that, The steps are as follows: (1) Mix the raw oil and glycerol, stir and emulsify, then add the metal-organic framework ZIF-8 immobilized enzyme described in claim 6, and carry out glycerolysis reaction at 40-70℃ for 2-12 h. After the reaction is completed, centrifuge to separate the immobilized enzyme. The resulting liquid mixture is a crude product rich in diglycerides. (2) The crude diglyceride product obtained in step (1) is purified by molecular distillation to obtain edible oil rich in DAG. The molar ratio of raw oil to glycerin is 1:1 to 1:5; The amount of the metal-organic framework ZIF-8 immobilized enzyme added is 1% to 5% of the weight of the raw oil.

8. The method for preparing diglyceride edible oil according to claim 7, characterized in that, The raw material oil is rapeseed oil.

9. The method for preparing diglyceride edible oil according to claim 7, characterized in that, The molecular distillation is a two-stage molecular distillation.

10. The method for preparing diglyceride edible oil according to claim 9, characterized in that, The conditions for primary molecular distillation are as follows: distillation temperature 170-200℃, vacuum degree 10-50Pa, and film scraping speed 300-400r / min. The conditions for secondary molecular distillation are as follows: distillation temperature 200-230℃, vacuum degree 1-10Pa, and film scraping speed 300-400r / min.

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