Diglyceride prepared by enzyme method as well as preparation method and application thereof

Abstract

The invention discloses diglyceride prepared by an enzymic method as well as a preparation method and application thereof, and belongs to the technical field of deep processing of grease. The method comprises the following steps: adding fatty acid into a choline chloride-glycerol-urea ternary eutectic solvent, uniformly mixing, and then adding candida antarctica lipase B for esterification reaction to obtain a reaction mixture; separating the reaction mixture, and recovering an upper-layer oil phase; and separating and purifying the upper-layer oil phase to obtain the diglyceride. According to the method, the reaction rate is high, the selectivity of the diglyceride in the esterification process is high, the yield of the diglyceride can reach 65% or above by reacting for 1 h, the selectivity of the diglyceride is 75% or above, and the purity of the separated and purified diglyceride is 90% or above; in addition, the method is simple to operate, and the reaction product and the eutectic solvent can be separated through simple centrifugation after the reaction is finished; in addition, the monoglyceride content in the product is low, and the subsequent separation and purification difficulty is greatly reduced.

Description

Technical Field

[0001] This invention belongs to the field of deep processing technology of oils and fats, specifically relating to an enzymatically prepared diglyceride, its preparation method, and its application. Background Technology

[0002] Diacylglycerols are lipid molecules formed by the esterification of one molecule of glycerol and two molecules of fatty acids, and their content in everyday animal and vegetable oils is usually less than 10%. Based on the different binding sites of fatty acids on the glycerol backbone, diglycerides can be divided into 1,3-diglycerides and 1,2(2,3)-diglycerides. After ingestion, diglycerides undergo a different metabolic pathway than triglycerides in the body, resulting in effects such as lowering postprandial blood lipids, inhibiting fat accumulation, reducing uric acid, and improving insulin resistance. As a new generation of healthy edible oils, diglycerides are currently widely used in food, medicine, cosmetics, and other fields. Therefore, research on diglyceride preparation technology has always been a hot topic and frontier in the field of oleochemistry.

[0003] Enzymatic and chemical methods are commonly used for the preparation of diglycerides. Compared to chemical methods, enzymatic methods have advantages such as mild reaction conditions, strong catalytic specificity, and environmental friendliness, making them the preferred industrial method for diglyceride preparation. Enzymatic hydrolysis, esterification, glycerolization, and alcoholysis are all common methods for preparing diglycerides. Among them, enzymatic esterification is a commonly used method for the industrial preparation of high-purity diglycerides. However, in the actual operation of enzymatic esterification for the preparation of diglycerides, many challenges are still faced. For example, poor mixing of glycerol and fatty acids, heterogeneous reaction systems, and low diglyceride selectivity during esterification result in slow reaction rates, low fatty acid conversion rates, low diglyceride content, high monoglyceride content in the product, and difficulty in final separation and purification, leading to low diglyceride yields and a diglyceride purity that is difficult to achieve above 90%.

[0004] CN116376990A discloses a method for the enzymatic preparation of diglycerides in a eutectic solvent system. The method uses eutectic solvents such as choline chloride-xylitol, betaine-xylitol, or choline chloride-glucose-water as the reaction medium, and glycerol and fatty acids as substrates. Diglycerides are synthesized under lipase-catalyzed esterification. After 6 hours of reaction, the product mixture is separated by centrifugation. The crude product has a diglyceride content >82%. After molecular distillation purification, the final product has a diglyceride purity of over 90%. CN117802169A discloses a two-step enzymatic method for the preparation of diglycerides, in which immobilized lipase catalyzes the alcoholysis of fats and oils in ethanol for 6 hours. h. A method for preparing diglycerides using a two-step enzymatic process is disclosed. First, moderate enzymatic hydrolysis is performed, controlling the fatty acid content in the hydrolysis product to be >45wt% and the triglyceride content to <22wt%. Then, the hydrolysis product is enzymatically esterified with glycerol, resulting in a diglyceride content of 50%–56% in the final esterified product. CN105400837A discloses an enzyme-catalyzed method for preparing diglycerides, which involves moderately hydrolyzing oils enzymatically, strictly controlling the fatty acid content in the hydrolysis product to be 26%–30wt%. Then, the hydrolysis product is enzymatically esterified with glycerol, resulting in a diglyceride content of 60%–65% in the final product.

[0005] In summary, existing enzymatic esterification techniques for preparing diglycerides mainly suffer from problems such as complex multi-step processes, the need for strict control of intermediate products, excessively long reaction times, and limited purity or yield of diglycerides. There is an urgent need to develop alternative enzymatic esterification techniques for preparing diglycerides. Summary of the Invention

[0006] To address the shortcomings of the existing technology, the present invention aims to provide and develop an enzymatic method for preparing diglycerides in a ternary eutectic solvent system, along with its preparation method and applications. This method for preparing diglycerides exhibits a fast reaction rate, high diglyceride selectivity, high diglyceride content in the product, and low content of monoglycerides and triglycerides. Furthermore, the reaction products are easily separated, and subsequent purification is relatively simple, demonstrating promising prospects for industrial application.

[0007] This invention is achieved through the following technical solution: This invention provides a method for preparing diglycerides using an enzymatic process, comprising the following steps: After adding fatty acids to a ternary eutectic solvent and mixing them evenly, Candida antarcticis lipase B was added to carry out an esterification reaction to obtain a reaction mixture. The ternary eutectic solvent was choline chloride-glycerol-urea. The reaction mixture was separated, and the upper oil phase was recovered; the upper oil phase was further separated and purified to obtain diglycerides.

[0008] The molar ratio of choline chloride-glycerol-urea is 1:(0.5~2):(0.5~2), wherein the molar ratio of glycerol to urea is 1:1.

[0009] Fatty acids include one or more of animal fats, vegetable oils, marine fish oils, algal oils, or deodorized distillates.

[0010] The molar ratio of fatty acids to glycerol in the ternary eutectic solvent is 1:(3~5).

[0011] The amount of Candida antarcticis lipase B added is 0.3% to 0.6% of the total mass of glycerol and fatty acids.

[0012] The esterification reaction temperature is 40~60℃.

[0013] The upper oil phase contains >65% diglycerides, <15% monoglycerides, <7% triglycerides, and <12% fatty acids.

[0014] The separation and purification process employs molecular distillation with a feed flow rate of 80-90 L / h, a primary evaporation surface temperature of 145-150℃, a secondary evaporation surface temperature of 155-165℃, a tertiary evaporation surface temperature of 165-170℃, and a condensation surface temperature of 41-43℃.

[0015] The present invention also provides a method for preparing diglycerides using the above-described enzymatic method.

[0016] This invention also provides applications of the above-mentioned diglycerides in the fields of food, medicine, and cosmetics.

[0017] Compared with the prior art, the present invention has the following beneficial technical effects: This invention discloses an enzymatic method for preparing diglycerides, using a ternary eutectic solvent composed of choline chloride-glycerol-urea as the reaction substrate and reaction medium, to prepare diglycerides under the catalysis of free lipase. Enzyme activity analysis combined with molecular dynamics simulations revealed that in the ternary eutectic solvent of choline chloride-glycerol-urea, the number of hydrogen bonds between His224 and Ser105 of *Candida antarcticis* lipase B decreases, increasing the degree of freedom of Ser105 and making it easier to bind to the substrate oleic acid. This results in a 1-2 times increase in the esterification activity of *Candida antarcticis* lipase B compared to the solvent-free system. Figure 3Furthermore, in this system, the number of α-Helix residues in *Candida antarcticis* lipase B significantly increased, with the secondary structures of key regions such as Ala141-Val149 residues and Gln211-Cys216 residues transforming from irregular to regular. Regarding the stability of flexible regions, the RMSF values ​​of catalytic triplet-related residues (Trp104, Ser105, Asp187, His224) decreased, and the volatility of the α-5 Helix region also decreased, resulting in a 1-2 fold increase in the thermal stability of *Candida antarcticis* lipase B compared to the solvent-free system. Figure 5 Furthermore, in this system, the electrostatic interactions between His224 and Asp187, and His224 and Ser105 of *Candida antarcticis* lipase B are weakened, leading to an expansion of the catalytic active pocket and improved substrate binding channel spatial adaptability. This enhances its selectivity for diglycerides and promotes the conversion of monoglycerides to diglycerides. With the synergistic improvement in enzyme activity, thermal stability, and diglyceride selectivity, *Candida antarcticis* lipase B can catalyze rapid and highly selective esterification of diglycerides in this system. The product contains high levels of diglycerides and low levels of monoglycerides and triglycerides, which not only reduces the difficulty of subsequent separation and purification but also significantly improves the purity of the purified diglycerides.

[0018] Furthermore, the ternary eutectic solvent is choline chloride-glycerol-urea, and the molar ratio of the three is 1:(0.5~2):(0.5~2), wherein the molar ratio of glycerol and urea is 1:1; this ensures that Candida antarctica lipase B has high activity, thermal stability and diglyceride selectivity in this system, thereby ensuring that diglycerides can be synthesized rapidly and with high selectivity during enzymatic esterification.

[0019] Furthermore, stirring at 2000~3000 rpm for 2~4 min ensures that the reaction system is pseudo-homogeneous, promotes mass transfer, and increases the esterification rate.

[0020] Furthermore, a molar ratio of fatty acids to glycerol in a ternary eutectic solvent of 1:3~5 is beneficial not only to improving the activity, thermal stability, and diglyceride selectivity of Candida antarctica lipase B, but also to the separation and purification of subsequent products.

[0021] Furthermore, the conditions for enzymatic esterification reaction can ensure high enzyme activity, thermal stability, and diglyceride selectivity, and are also beneficial for the separation of enzymatic products. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below: Figure 1 This is a schematic diagram illustrating the principle of the enzymatic preparation of diglycerides according to the present invention. Figure 2 This is an HPLC chromatogram of the reaction mixture of Example 2 of the present invention; Figure 3 Bar charts showing the enzyme activity of Candida antarcticis lipase B in different solvents for embodiments and comparative examples of the present invention; Figure 4 The figure shows the results of a molecular dynamics simulation to explain the high activity of Candida antarctica lipase B, based on an embodiment of the present invention. Figure 5 The bar chart shows the relative enzyme activity (enzyme activity after incubation / initial enzyme activity) of Candida antarcticis lipase B after incubation in different solvents in the examples and comparative examples at 60°C for 12 h. Figure 6 This is a molecular dynamics simulation result diagram based on an embodiment of the present invention, used to explain the thermal stability of Candida antarctica lipase B. Detailed Implementation

[0023] To make the technical problem to be solved, the technical solution, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0024] In this invention, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0025] In this invention, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items. For example, "at least one of a, b, or c", or "at least one of a, b, and c", can both represent: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0026] It should be understood that in various embodiments of the present invention, the order of the above-mentioned processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present invention.

[0027] The terminology used in the embodiments of this invention is for the purpose of describing particular embodiments only and is not intended to limit the invention. The singular forms “a,” “the,” and “the” as used in the embodiments of this invention and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0028] The weights of the relevant components mentioned in the embodiments of this invention can refer not only to the specific content of each component, but also to the proportional relationship between the weights of the components. Therefore, any scaling up or down of the content of the relevant components according to the embodiments of this invention is within the scope disclosed in the embodiments of this invention. Specifically, the mass described in the embodiments of this invention can be a mass unit known in the chemical industry, such as μg, mg, g, or kg.

[0029] The enzymatic method for preparing diglycerides provided by this invention includes the following steps: Step 1: Add a ternary eutectic solvent to the reaction vessel, add fatty acids and mix thoroughly by high-speed stirring; then add Candida antarcticis lipase B, and carry out an esterification reaction under the catalysis of Candida antarcticis lipase B to obtain the reaction mixture; wherein, the ternary eutectic solvent is choline chloride-glycerol-urea; the ternary eutectic solvent is used as the reaction substrate and reaction medium; Step 2: After the reaction is complete, the reaction mixture is separated and the upper oil phase is recovered. The upper oil phase is rich in diglycerides and the lower layer is lipase and eutectic solvent phase. Step 3: Separate and purify the upper oil phase to obtain diglycerides.

[0030] In some embodiments, the molar ratio of choline chloride-glycerol-urea is 1:(0.5~2):(0.5~2), wherein the molar ratio of glycerol to urea is 1:1.

[0031] In some embodiments, the fatty acids include one or more of animal oils, vegetable oils, marine fish oils, algal oils, or deodorized distillates, and the vegetable oils include soybean oil, pomegranate seed oil, flaxseed oil, rice bran oil, walnut oil, and olive oil.

[0032] In some embodiments, the molar ratio of fatty acid to glycerol in a ternary eutectic solvent is 1:(3~5).

[0033] In some embodiments, the esterification reaction is carried out at a temperature of 40-60°C, for a reaction time of 0.5-1 h, and at a stirring speed of 400-600 rpm.

[0034] In some embodiments, the amount of Candida antarcticis lipase B added is 0.3% to 0.6% of the total mass of glycerol and fatty acids.

[0035] In some embodiments, centrifugation is used to separate the reaction mixture, with a centrifugation speed of 8000~10000 rpm.

[0036] In some embodiments, the content of diglycerides in the upper oil phase is >65%, the content of monoglycerides is <15%, the content of triglycerides is <7%, and the content of fatty acids is <12%.

[0037] In some embodiments, separation and purification are performed using molecular distillation, with a feed flow rate of 80-90 L / h, a primary evaporation surface temperature of 145-150°C, a secondary evaporation surface temperature of 155-165°C, a tertiary evaporation surface temperature of 165-170°C, and a condensation surface temperature of 41-43°C.

[0038] In the following embodiments, unless otherwise specified, all materials used can be obtained through ordinary channels; the testing methods used are conventional methods in the art.

[0039] Example 1 151.96 g of eutectic solvent (choline chloride-glycerol-urea, molar ratio of 1:1:1) was added to a 500 mL Erlenmeyer flask, along with 48.09 g of soybean oil fatty acids (molar ratio of fatty acids to glycerol of 1:3), resulting in a total mass of 95.84 g of fatty acids and glycerol. The mixture was homogenized at 2000 rpm for 2 min. The reaction mixture was then heated to 50 °C, followed by the addition of 575 µL of CALB (0.6% of the total mass of glycerol and fatty acids). Esterification was then carried out at 500 rpm. After reacting for 30 min, the reaction mixture was centrifuged at 10,000 rpm for 1 min, and the upper oil phase was collected. The upper oil phase was analyzed by liquid chromatography, and it was found that the product contained 67.56% diglycerides, of which 1,3-diglycerides contained 53.95%, monoglycerides contained 14.31%, fatty acids contained 11.43%, triglycerides contained 6.70%, and the selectivity of diglycerides was 76.28%. The above product was separated and purified by molecular distillation, [1] with a feed flow rate of 80 L / h, a first-stage evaporation surface temperature of 145℃, a second-stage evaporation surface temperature of 155℃, a third-stage evaporation surface temperature of 165℃, and a condensation surface temperature of 41℃. The final product contained 90.97% diglycerides. Figure 3 It can be seen that the enzyme activity of CALB in this system is 8397 U / mL, which is 2 times higher than that of CALB in the solvent-free system (2793 U / mL); Figure 5It can be seen that after CALB was incubated at 60℃ for 12 h, its enzyme activity was 102% of the initial enzyme activity, while after CALB was incubated in a solvent-free system for 12 h, the enzyme activity was 34% of the initial enzyme activity, indicating that the thermal stability of CALB in this system was 2 times higher than that in the solvent-free system.

[0040] Example 2 167.77 g of eutectic solvent (choline chloride-glycerol-urea, molar ratio of 1:0.5:0.5) was added to a 500 mL Erlenmeyer flask, followed by 32.23 g of marine fish oil fatty acids (molar ratio of fatty acids to glycerol of 1:3.5), with a total mass of 68.02 g of fatty acids and glycerol. The mixture was homogenized at 3000 rpm for 1 min, and the reaction mixture was heated to 40 °C. Then, 204 µL of CALB (0.3% of the total mass of glycerol and fatty acids) was added, and the esterification reaction was carried out at 400 rpm. After reacting for 60 min, the reaction mixture was centrifuged at 9500 rpm for 1 min, and the upper oil phase was collected. Liquid chromatography analysis of the upper oil phase revealed a diglyceride content of 67.42%, including 48.39% 1,3-diglycerides, 14.82% monoglycerides, 11.46% fatty acids, and 6.30% triglycerides, with a diglyceride selectivity of 76.15%. The product was then purified by molecular distillation at a feed flow rate of 80 L / h, a first-stage evaporation surface temperature of 145℃, a second-stage evaporation surface temperature of 155℃, a third-stage evaporation surface temperature of 165℃, and a condensation surface temperature of 41℃. The final product contained 91.45% diglycerides. Figure 3 It can be seen that the enzyme activity of CALB in this system is 8232 U / mL, which is 1.9 times higher than that of CALB in the solvent-free system (2793 U / mL); Figure 5 It can be seen that after CALB was incubated at 60℃ for 12 h, its enzyme activity was 100% of the initial enzyme activity, while after CALB was incubated in a solvent-free system for 12 h, its enzyme activity was 34% of the initial enzyme activity, indicating that the thermal stability of CALB in this system was 1.9 times higher than that in the solvent-free system.

[0041] like Figure 2 As shown in the HPLC chromatogram, the components elute in order of increasing polarity (retention time from shortest to longest): triglyceride (TAG), free fatty acid (FFA), 1,3-diglyceride (1,3-DAG), 1,2-diglyceride (1,2-DAG), 1-monoglyceride (1-MAG), and 2-monoglyceride (2-MAG).

[0042] Example 3 166.56 g of eutectic solvent (choline chloride-glycerol-urea, molar ratio of 1:0.8:0.8) was added to a 500 mL Erlenmeyer flask, followed by 33.44 g of algal oil fatty acids (molar ratio of fatty acids to glycerol of 1:4.5), with a total mass of 80.37 g of fatty acids and glycerol. The mixture was homogenized at 2700 rpm for 2 min. The reaction mixture was then heated to 60 °C, and 321 µL of CALB (0.4% of the total mass of glycerol and fatty acids) was added. The esterification reaction was carried out at 600 rpm. After reacting for 55 min, the reaction mixture was centrifuged at 9000 rpm for 1 min, and the upper oil phase was collected. Liquid chromatography analysis of the upper oil phase revealed a diglyceride content of 66.47%, including 43.12% 1,3-diglycerides, 14.97% monoglycerides, 11.76% fatty acids, and 6.80% triglycerides, with a diglyceride selectivity of 75.32%. Molecular distillation was used to separate and purify the product. The feed flow rate was 80 L / h, the first-stage evaporation surface temperature was 145℃, the second-stage evaporation surface temperature was 155℃, the third-stage evaporation surface temperature was 165℃, and the condensation surface temperature was 41℃. The final product obtained contained 90.72% diglycerides. Figure 3 It can be seen that the enzyme activity of CALB in this system is 8370 U / mL, which is 2 times higher than that of CALB in the solvent-free system (2793 U / mL); Figure 5 It can be seen that after CALB was incubated at 60℃ for 12 h, its enzyme activity was 102% of the initial enzyme activity, while after CALB was incubated in a solvent-free system for 12 h, the enzyme activity was 34% of the initial enzyme activity, indicating that the thermal stability of CALB in this system was 2 times higher than that in the solvent-free system.

[0043] Example 4 160.43 g of eutectic solvent (choline chloride-glycerol-urea, molar ratio of 1:1.2:1.2) was added to a 500 mL Erlenmeyer flask, along with 39.57 g of pomegranate seed oil fatty acids (molar ratio of fatty acids to glycerol of 1:4.2), with a total mass of 94.55 g of fatty acids and glycerol. The mixture was homogenized at 2500 rpm for 2 min. The reaction mixture was then heated to 45 °C, followed by the addition of 472 µL of CALB (0.5% of the total mass of glycerol and fatty acids). Esterification was carried out at 400 rpm. After reacting for 45 min, the reaction mixture was centrifuged at 8500 rpm for 2 min, and the upper oil phase was collected. Liquid chromatography analysis of the upper oil phase revealed a diglyceride content of 65.96%, including 53.20% 1,3-diglycerides, 14.68% monoglycerides, 13.12% fatty acids, and 6.24% triglycerides, with a diglyceride selectivity of 75.92%. The product was then purified by molecular distillation at a feed flow rate of 90 L / h, a first-stage evaporation surface temperature of 150℃, a second-stage evaporation surface temperature of 165℃, a third-stage evaporation surface temperature of 170℃, and a condensation surface temperature of 42℃. The final product contained 91.36% diglycerides. Figure 3 It can be seen that the enzyme activity of CALB in this system is 8265 U / mL, which is 1.9 times higher than that of CALB in the solvent-free system (2793 U / mL); Figure 5 It can be seen that after CALB was incubated at 60℃ for 12 h, its enzyme activity was 98% of the initial enzyme activity, while after CALB was incubated in a solvent-free system for 12 h, its enzyme activity was 34% of the initial enzyme activity, indicating that the thermal stability of CALB in this system was 1.9 times higher than that in the solvent-free system.

[0044] Example 5 147.64 g of eutectic solvent (choline chloride-glycerol-urea, molar ratio of 1:1.5:1.5) was added to a 500 mL Erlenmeyer flask, along with 52.36 g of flaxseed oil fatty acids (molar ratio of fatty acids to glycerol of 1:3.2), with a total mass of fatty acids and glycerol of 107.81 g. The mixture was homogenized at 2800 rpm for 1 min. The reaction mixture was then heated to 55 °C, followed by the addition of 646 µL of CALB (0.6% of the total mass of glycerol and fatty acids). Esterification was carried out at 500 rpm. After reacting for 35 min, the reaction mixture was centrifuged at 9000 rpm for 2.5 min, and the upper oil phase was collected. Liquid chromatography analysis of the upper oil phase revealed a diglyceride content of 66.86%, including 41.65% 1,3-diglycerides, 14.93% monoglycerides, 11.92% fatty acids, and 6.29% triglycerides, with a diglyceride selectivity of 75.88%. The product was then purified by molecular distillation at a feed flow rate of 90 L / h, a first-stage evaporation surface temperature of 145℃, a second-stage evaporation surface temperature of 155℃, a third-stage evaporation surface temperature of 165℃, and a condensation surface temperature of 41℃. The final product contained 91.40% diglycerides. Figure 3 It can be seen that the enzyme activity of CALB in this system is 8284 U / mL, which is 1.9 times higher than that of CALB in the solvent-free system (2793 U / mL); Figure 5 It can be seen that after CALB was incubated at 60℃ for 12 h, its enzyme activity was 101% of the initial enzyme activity, while after CALB was incubated in a solvent-free system for 12 h, the enzyme activity was 34% of the initial enzyme activity, indicating that the thermal stability of CALB in this system was 2 times higher than that in the solvent-free system.

[0045] Example 6 149.39 g of eutectic solvent (choline chloride-glycerol-urea, molar ratio of 1:2:2) was added to a 500 mL Erlenmeyer flask, followed by 50.61 g of rice bran oil fatty acids (molar ratio of fatty acids to glycerol of 1:3.7), with a total mass of 104.19 g of fatty acids and glycerol. The mixture was homogenized at 1500 rpm for 1.5 min, and the reaction mixture was heated to 40 °C. Then, 416 µL of CALB (0.4% of the total mass of glycerol and fatty acids) was added, and the esterification reaction was carried out at 400 rpm. After reacting for 40 min, the reaction mixture was centrifuged at 8000 rpm for 2 min, and the upper oil phase was collected. Liquid chromatography analysis of the upper oil phase revealed a diglyceride content of 66.78%, including 54.23% 1,3-diglycerides, 14.68% monoglycerides, 11.72% fatty acids, and 6.82% triglycerides, with a diglyceride selectivity of 75.64%. The product was then purified by molecular distillation at a feed flow rate of 85 L / h, a first-stage evaporation surface temperature of 150℃, a second-stage evaporation surface temperature of 160℃, a third-stage evaporation surface temperature of 170℃, and a condensation surface temperature of 41℃. The final product contained 90.73% diglycerides. Figure 3 It can be seen that the enzyme activity of CALB in this system is 8100 U / mL, which is 1.9 times higher than that of CALB in the solvent-free system (2793 U / mL); Figure 5 It can be seen that after CALB was incubated at 60℃ for 12 h, its enzyme activity was 98% of the initial enzyme activity, while after CALB was incubated in a solvent-free system for 12 h, its enzyme activity was 34% of the initial enzyme activity, indicating that the thermal stability of CALB in this system was 1.9 times higher than that in the solvent-free system.

[0046] Example 7 163.81 g of eutectic solvent (choline chloride-glycerol-urea, molar ratio of 1:1.4:1.4) was added to a 500 mL Erlenmeyer flask, followed by 36.19 g of walnut oil fatty acids (molar ratio of fatty acids to glycerol of 1:5), with a total mass of fatty acids and glycerol of 95.98 g. The mixture was homogenized at 2000 rpm for 2 min, and the reaction mixture was heated to 60 °C. Then, 479 µL of CALB (0.5% of the total mass of glycerol and fatty acids) was added, and the esterification reaction was carried out at 600 rpm. After reacting for 50 min, the reaction mixture was centrifuged at 10,000 rpm for 1 min, and the upper oil phase was collected. Liquid chromatography analysis of the upper oil phase revealed a diglyceride content of 67.78%, including 54.20% 1,3-diglycerides, 14.26% monoglycerides, 11.28% fatty acids, and 6.68% triglycerides, with a diglyceride selectivity of 76.39%. Molecular distillation was used to separate and purify the product. The feed flow rate was 85 L / h, the first-stage evaporation surface temperature was 148℃, the second-stage evaporation surface temperature was 155–165℃, the third-stage evaporation surface temperature was 168℃, and the condensation surface temperature was 43℃. The final product contained 90.66% diglycerides. Figure 3 It can be seen that the enzyme activity of CALB in this system is 7920 U / mL, which is 1.8 times higher than that of CALB in the solvent-free system (2793 U / mL); Figure 5 It can be seen that after CALB was incubated at 60℃ for 12 h, its enzyme activity was 99% of the initial enzyme activity, while after CALB was incubated in a solvent-free system for 12 h, the enzyme activity was 34% of the initial enzyme activity, indicating that the thermal stability of CALB in this system was 1.9 times higher than that in the solvent-free system.

[0047] Example 8 169.42 g of eutectic solvent (choline chloride-glycerol-urea, molar ratio of 1:0.6:0.6) was added to a 500 mL Erlenmeyer flask, followed by 30.58 g of olive oil fatty acids (molar ratio of fatty acids to glycerol of 1:4), with a total mass of fatty acids and glycerol of 71.06 g. The mixture was homogenized at 2000 rpm for 2 min, and the reaction mixture was heated to 45 °C. Then, 213 µL of CALB (0.3% of the total mass of glycerol and fatty acids) was added, and the esterification reaction was carried out at 600 rpm. After reacting for 60 min, the reaction mixture was centrifuged at 10,000 rpm for 1 min, and the upper oil phase was collected. Liquid chromatography analysis of the upper oil phase revealed a diglyceride content of 67.68%, including 53.12% 1,3-diglycerides, 14.39% monoglycerides, 11.79% fatty acids, and 6.14% triglycerides, with a diglyceride selectivity of 76.73%. The product was then purified by molecular distillation at a feed flow rate of 85 L / h, with primary evaporation surface temperatures of 145℃, secondary evaporation surface temperatures of 165℃, tertiary evaporation surface temperatures of 165℃, and a condensation surface temperature of 43℃. The final product obtained contained 91.68% diglycerides. Figure 3 It can be seen that the enzyme activity of CALB in this system is 7865 U / mL, which is 1.8 times higher than that of CALB in the solvent-free system (2793 U / mL); Figure 5 It can be seen that after CALB was incubated at 60℃ for 12 h, its enzyme activity was 100% of the initial enzyme activity, while after CALB was incubated in a solvent-free system for 12 h, its enzyme activity was 34% of the initial enzyme activity, indicating that the thermal stability of CALB in this system was 1.9 times higher than that in the solvent-free system.

[0048] Comparative Example 1 47.75 g of glycerol was added to a 500 mL Erlenmeyer flask, followed by 48.09 g of soybean oil fatty acids (molar ratio of fatty acids to glycerol was 1:3), resulting in a total mass of 95.84 g of fatty acids and glycerol. The mixture was homogenized at 2000 rpm for 2 min. The reaction mixture was then heated to 50 °C, and 575 µL of CALB (0.6% of the total mass of glycerol and fatty acids) was added. Esterification was carried out at 500 rpm. After reacting for 30 min, the reaction mixture was centrifuged at 10000 rpm for 1 min, and the upper oil phase was collected. Liquid chromatography analysis of the upper oil phase revealed that the product contained 40.66% diglycerides, including 36.43% 1,3-diglycerides, 24.49% monoglycerides, 28.65% fatty acids, and 6.20% triglycerides, with a diglyceride selectivity of 59.98%. The above products were separated and purified by molecular distillation at a feed flow rate of 80 L / h, a first-stage evaporation surface temperature of 145℃, a second-stage evaporation surface temperature of 155℃, a third-stage evaporation surface temperature of 165℃, and a condensation surface temperature of 41℃. The final product contained 84.76% diglycerides.

[0049] As shown in Table 1, Comparative Example 1 and Example 1 were prepared under identical reaction conditions except for the reaction system. The difference between Comparative Example 1 and Example 1 is that pure glycerol was used as the reaction medium and substrate, resulting in a significantly lower diglyceride content (40.66% vs 67.56%) compared to the diglyceride content in the ternary eutectic solvent system of choline chloride-glycerol-urea (molar ratio 1:1:1). The diglyceride selectivity was also significantly lower than that in Example 1 (59.98% vs 76.28%). Furthermore, the content of monoglycerides (24.49% vs 14.31%) and free fatty acids (28.65% vs 11.43%) in the esterification product of Comparative Example 1 was higher than that in Example 1. Moreover, the content of purified diglycerides in Comparative Example 1 (84.76% vs 90.97%) was also significantly lower than that in Example 1. The above results indicate that the ternary low co-solvent component has a regulatory effect on the activity, thermal stability and diglyceride selectivity of Candida antarctica lipase, which is beneficial to the rapid and highly selective synthesis of diglycerides. At the same time, it can effectively avoid the accumulation of monoglycerides and triglycerides, thereby reducing the difficulty of diglyceride separation and purification.

[0050] Comparative Example 2 In a 500 mL Erlenmeyer flask, 83.98 g of a eutectic solvent (choline chloride-glycerol, molar ratio 1:2) and 48.09 g of soybean oil fatty acids (oleic acid to glycerol, molar ratio 1:3) were added, resulting in a total fatty acid and glycerol mass of 95.84 g. The mixture was homogenized at 2000 rpm for 2 min. The reaction mixture was then heated to 50 °C, and 575 µL CALB (0.6% of the total mass of glycerol and fatty acids) was added. Esterification was then carried out at 200 rpm. After 30 min of reaction, the reaction mixture was centrifuged at 10000 rpm for 2 min, and the upper oil phase was collected. Liquid chromatography analysis of the upper oil phase revealed a diglyceride content of 45.78%, including 41.72% 1,3-diglycerides, 15.49% monoglycerides, 31.63% fatty acids, and 7.10% triglycerides. The selectivity for diglycerides was 66.96%. The above products were separated and purified by molecular distillation at a feed flow rate of 80 L / h, a first-stage evaporation surface temperature of 145℃, a second-stage evaporation surface temperature of 155℃, a third-stage evaporation surface temperature of 165℃, and a condensation surface temperature of 41℃. The final product contained 83.42% diglycerides.

[0051] As shown in Table 1, Comparative Example 2 and Example 1 prepared diglycerides under identical reaction conditions except for the reaction system. The difference from Example 1 is that Comparative Example 2 used choline chloride-glycerol (molar ratio 1:2) as the reaction medium and substrate. The resulting diglyceride content was significantly lower than that in the ternary eutectic solvent system choline chloride-glycerol-urea (molar ratio 1:1:1) (45.78% vs 67.56%), and the diglyceride selectivity was also significantly lower than that in Example 1 (59.98% vs 76.28%). Furthermore, the content of monoglycerides (15.49% vs 14.31%) and free fatty acids (31.63% vs 11.43%) in the esterification product of Comparative Example 2 was higher than those in Example 1. Moreover, the content of purified diglycerides in Comparative Example 2 (83.42% vs 67.56%) was significantly lower than that in Example 1. The content of diglycerides (90.97%) was also significantly lower than that in Example 1. The above results indicate that when 1 mol of glycerol is used to replace 1 mol of urea in the ternary eutectic solvent choline chloride-glycerol-urea (molar ratio 1:1:1) in Example 1 to construct a binary eutectic solvent system of choline chloride-glycerol (molar ratio 1:2), although the synthesis of diglycerides can be achieved, the following problems exist: (1) the content and selectivity of diglycerides in the product are low; (2) the content of monoglycerides and free fatty acids is significantly increased; (3) the yield of diglycerides after final separation and purification is significantly lower than that of the ternary eutectic solvent system. Comprehensive analysis shows that the ternary eutectic solvent system (choline chloride-glycerol-urea) has significant advantages: (1) the synergistic effect among the three components creates a more optimized solvent environment; (2) the specific interaction between the solvent and Candida antarcticis lipase B can effectively regulate the enzyme's activity and thermal stability; (3) this system can significantly improve the selectivity of diglycerides and promote their rapid synthesis; (4) it can simultaneously inhibit the accumulation of monoglycerides and triglycerides, thereby greatly reducing the difficulty of subsequent separation and purification. These characteristics make the ternary eutectic solvent system exhibit significantly better performance than the binary system in diglyceride synthesis. In conclusion, the above results indicate that the differences in solvent components of the eutectic solvent can significantly affect the catalytic effect of the enzyme.

[0052] Comparative Example 3 104.21 g of eutectic solvent (choline chloride-urea, molar ratio 1:2) and 48.09 g of soybean oil fatty acids (molar ratio of oleic acid to glycerol 1:3) were added to a 500 mL Erlenmeyer flask, with a total mass of fatty acids and glycerol of 95.84 g. The mixture was homogenized at 2000 rpm for 2 min. The reaction mixture was then heated to 50 °C, and 575 µL CALB (0.6% of the total mass of glycerol and fatty acids) was added. The esterification reaction was carried out at 500 rpm. After reacting for 30 min, the reaction mixture was centrifuged at 10,000 rpm for 1 min, and the upper oil phase was collected. Liquid chromatography analysis of the upper oil phase revealed a diglyceride content of 42.71%, including 32.19% 1,3-diglycerides, 18.81% monoglycerides, 31.92% fatty acids, and 6.56% triglycerides, with a diglyceride selectivity of 62.73%. The product was then purified by molecular distillation at a feed flow rate of 80 L / h, with primary evaporation surface temperatures of 145℃, secondary evaporation surface temperatures of 155℃, tertiary evaporation surface temperatures of 165℃, and a condensation surface temperature of 41℃. The final product contained 86.68% diglycerides.

[0053] As shown in Table 1, Comparative Example 3 and Example 1 were prepared under identical reaction conditions except for the reaction system. The difference between Comparative Example 3 and Example 1 was that Comparative Example 3 used choline chloride-urea (molar ratio 1:2) as the reaction medium, resulting in a significantly lower diglyceride content (42.70% vs 67.56%) compared to the ternary eutectic solvent system of choline chloride-glycerol-urea (molar ratio 1:1:1). The diglyceride selectivity was also significantly lower than that in Example 1 (66.96% vs 76.28%). Furthermore, the content of monoglycerides (18.81% vs 14.31%) and free fatty acids (31.92% vs 11.43%) in the esterification product of Comparative Example 3 was higher than those in Example 1. Moreover, the content of purified diglycerides in Comparative Example 3 was 86.68%. The diglyceride content (vs90.97%) was also significantly lower than that in Example 1. The above results indicate that when 1 mol of urea is used to replace 1 mol of glycerol in the ternary eutectic solvent choline chloride-glycerol-urea (molar ratio 1:1:1) in Example 1 to construct a choline chloride-glycerol (molar ratio 1:2) binary eutectic solvent system, although the synthesis of diglycerides can be achieved, the following problems exist: (1) the content and selectivity of diglycerides in the product are low; (2) the content of monoglycerides and free fatty acids is significantly increased; (3) the yield of diglycerides after final separation and purification is significantly lower than that of the ternary eutectic solvent system. Comprehensive analysis shows that the ternary eutectic solvent system (choline chloride-glycerol-urea) has significant advantages: (1) the synergistic effect among the three components creates a more optimized solvent environment; (2) the specific interaction between the solvent and Candida antarcticis lipase B can effectively regulate the enzyme's activity and thermal stability; (3) this system can significantly improve the selectivity of diglycerides and promote their rapid synthesis; (4) it can simultaneously inhibit the accumulation of monoglycerides and triglycerides, thereby greatly reducing the difficulty of subsequent separation and purification. These characteristics make the ternary eutectic solvent system exhibit significantly better performance than the binary system in diglyceride synthesis. In conclusion, the above results indicate that the differences in solvent components of the eutectic solvent can significantly affect the catalytic effect of the enzyme.

[0054] Comparative Example 4 286.10 g of eutectic solvent (choline chloride-glycerol-urea, molar ratio of 1:0.5:1.5) was added to a 500 mL Erlenmeyer flask, along with 48.09 g of soybean oil fatty acids (molar ratio of fatty acids to glycerol of 1:3), with a total mass of fatty acids and glycerol of 95.84 g. The mixture was homogenized at 2000 rpm for 2 min. The reaction mixture was then heated to 50 °C, followed by the addition of 575 µL of CALB (0.6% of the total mass of glycerol and fatty acids), and the esterification reaction was carried out at 500 rpm. After reacting for 30 min, the reaction mixture was centrifuged at 10,000 rpm for 1 min, and the upper oil phase was collected. Liquid chromatography analysis of the upper oil phase revealed a diglyceride content of 46.64%, including 39.48% 1,3-diglycerides, 15.19% monoglycerides, 31.73% fatty acids, and 6.44% triglycerides, with a diglyceride selectivity of 68.36%. The product was then purified by molecular distillation at a feed flow rate of 80 L / h, with primary evaporation surface temperatures of 145℃, secondary evaporation surface temperatures of 155℃, tertiary evaporation surface temperatures of 165℃, and a condensation surface temperature of 41℃. The final product contained 84.09% diglycerides.

[0055] As shown in Table 1, both Comparative Example 4 and Example 1 prepared diglycerides in a ternary eutectic solvent composed of choline chloride, glycerol, and urea. The difference was that the molar ratio of choline chloride, glycerol, and urea in Comparative Example 4 was 1:0.5:1.5, while the molar ratio in Example 1 was 1:1:1. Comparing the results of Comparative Example 4 and Example 1, it can be seen that the content of diglycerides obtained in Comparative Example 4 is significantly lower than that in Example 1 (46.64% vs 67.56%), and the selectivity of diglycerides is also significantly lower than that in Example 1 (68.36% vs 76.28%). Furthermore, the contents of monoglycerides (15.19% vs 14.31%) and free fatty acids (31.73% vs 11.43%) in the esterification product of Comparative Example 4 are also higher than those in Example 1. Moreover, the content of diglycerides after purification in Comparative Example 4 (84.09% vs 90.97%) is also significantly lower than that in Example 1. These results indicate that in the ternary eutectic solvent choline chloride-glycerol-urea system, the molar ratio of each component has a significant impact on the synthesis of diglycerides. When the composition of the ternary eutectic solvent was adjusted from the initial 1:1:1 (choline chloride: glycerol: urea) to 1:0.5:1.5, although diglyceride synthesis could still be achieved, three significant problems arose: (1) the content and selectivity of diglycerides in the product decreased significantly; (2) the content of monoglycerides and free fatty acids increased significantly; and (3) the purity of the final purified diglyceride was significantly lower than that of the 1:1:1 system. These results indicate that changes in the ratio of glycerol to urea in the ternary eutectic solvent system significantly affect the catalytic performance of Candida antarctica lipase B, and also confirm the important structure-activity relationship between solvent composition and enzyme catalytic performance.

[0056] Table 1 compares the data on the reaction system, raw materials, and resulting products of each embodiment and comparative example.

[0057] In a eutectic solvent composed of choline chloride, glycerol, and urea, *Candida antarcticus* lipase B catalyzes the esterification of glycerol and oleic acid to prepare diglycerides. Choline chloride, glycerol, and urea serve as both the solvent and substrate. After 1 hour of reaction, the oleic acid conversion rate reaches over 88%, and the diglyceride content in the product reaches over 65%, with a selectivity of over 75%. Furthermore, the content of monoglycerides is less than 15%, and the content of triglycerides is less than 7%. Analysis of the catalytic activity of *Candida antarcticus* lipase B in the eutectic solvent showed that the esterification activity of *Candida antarcticus* lipase B was 1-2 times higher in this solvent compared to the solvent-free system. Figure 4As shown, molecular dynamics studies indicate that in the ternary eutectic solvent composed of choline chloride-glycerol-urea, the number of hydrogen bonds between His224 and Ser105 of *Candida antarcticis* lipase B decreases, increasing the degree of freedom of Ser105 and making it easier to bind to the substrate oleic acid, thereby enhancing the esterification activity of *Candida antarcticis* lipase B. Analysis of the thermal stability of *Candida antarcticis* lipase B in the eutectic solvent of choline chloride-glycerol-urea revealed that the thermal stability of *Candida antarcticis* lipase B after incubation in this solvent was 1-2 times higher than that in the solvent-free system. Figure 6 As shown, molecular dynamics studies indicate that the number of α-Helix residues in *Candida antarcticis* lipase B is significantly increased in this system, with the secondary structure of key regions such as Ala141-Val149 residues and Gln211-Cys216 residues transitioning from irregular to regular structures. Regarding the stability of flexible regions, the RMSF values ​​of catalytic triplet-related residues (Trp104, Ser105, Asp187, His224) decrease, and α-5... The reduced volatility of the Helix region enhances the thermal stability of *Candida antarcticus* lipase B. Analysis of the catalytic glycerol ester selectivity of *Candida antarcticus* lipase B in a eutectic solvent composed of choline chloride-glycerol-urea revealed that incubation in this solvent increased the diglyceride selectivity [diglyceride / (triglyceride + diglyceride + monoglyceride)] by over 20%. Molecular dynamics studies showed that the electrostatic interactions between His224 and Asp187, and His224 and Ser105 of *Candida antarcticus* lipase B, were weakened in this system, leading to an expansion of the catalytic active pocket and improved substrate binding channel spatial adaptability, thus enhancing its diglyceride selectivity. With the synergistic improvement in enzyme activity, thermal stability, and diglyceride selectivity, the enzymatic esterification synthesis of diglycerides in this system significantly increased the synthesis rate and selectivity, resulting in a high diglyceride content and low monoglyceride and triglyceride content in the product.

[0058] The endpoints and any values ​​of the ranges disclosed in this invention are not limited to the precise ranges or values; these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein. In the following, various technical solutions can, in principle, be combined with each other to obtain new technical solutions, which should also be considered as specifically disclosed herein.

[0059] The above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still make modifications or equivalent substitutions to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention are within the protection scope of the claims of the present invention pending approval.

Claims

1. A method for preparing diglycerides using an enzymatic process, characterized in that, Includes the following steps: After adding fatty acids to a ternary eutectic solvent and mixing them evenly, Candida antarcticis lipase B was added to carry out an esterification reaction to obtain a reaction mixture. The ternary eutectic solvent was choline chloride-glycerol-urea. The reaction mixture was separated, and the upper oil phase was recovered; the upper oil phase was further separated and purified to obtain diglycerides.

2. The method for preparing diglycerides by enzymatic method according to claim 1, characterized in that, The molar ratio of choline chloride-glycerol-urea is 1:(0.5~2):(0.5~2), wherein the molar ratio of glycerol to urea is 1:

1.

3. The method for preparing diglycerides by enzymatic method according to claim 1, characterized in that, The fatty acids include one or more of animal oils, vegetable oils, marine fish oils, algal oils, or deodorized distillates.

4. The method for preparing diglycerides by enzymatic method according to claim 1, characterized in that, The molar ratio of the fatty acid to glycerol in the ternary eutectic solvent is 1:(3~5).

5. The method for preparing diglycerides by enzymatic method according to claim 1, characterized in that, The amount of Antarctic Candida lipase B added is 0.3% to 0.6% of the total mass of glycerol and fatty acids.

6. The method for preparing diglycerides by enzymatic method according to claim 1, characterized in that, The esterification reaction is carried out at a temperature of 40~60℃.

7. The method for preparing diglycerides by enzymatic method according to claim 1, characterized in that, The upper oil phase contains >65% diglycerides, <15% monoglycerides, <7% triglycerides, and <12% fatty acids.

8. A method for preparing diglycerides using an enzymatic process according to claim 1, characterized in that, The separation and purification process employs molecular distillation, with a feed flow rate of 80-90 L / h, a primary evaporation surface temperature of 145-150℃, a secondary evaporation surface temperature of 155-165℃, a tertiary evaporation surface temperature of 165-170℃, and a condensation surface temperature of 41-43℃.

9. A diglyceride, characterized in that, The glycerol is prepared by the enzymatic method for preparing diglycerides according to any one of claims 1-8.

10. The application of the diglyceride according to claim 9 in the fields of food, medicine, and cosmetics.

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