A method for preparing high-purity diglyceride edible oil by continuous enzyme reaction

Abstract

This invention discloses a method for preparing high-purity diglyceride edible oil via a continuous enzymatic reaction. The method employs lipase-catalyzed glycerol hydrolysis to prepare a mixture of glycerides. To avoid excessive glycerol being incompatible with oils and to deactivate the enzyme, glycerol is added in stages at a controlled rate during the glycerol hydrolysis reaction. This improves the compatibility of glycerol with oils and increases reaction efficiency. Furthermore, slow stirring at high temperature allows the glycerol and glycerides to separate into layers. The activity of a metaglycerol lipase (Lipase G50) is enhanced using bioimprinting. Under optimal reaction pH conditions, Lipase G50 is imprinted with a nonionic surfactant, and the imprinted lipase acts as a catalyst to catalyze the esterification reaction of monoglycerides and fatty acids in the glycerol hydrolysis products. Finally, molecular distillation is used to remove the fatty acids, yielding a high-purity diglyceride product.

Description

Technical Field

[0001] This invention belongs to the field of oil and fat technology, specifically relating to a method for preparing high-purity diglyceride edible oil via a continuous enzymatic reaction. Background Technology

[0002] Diacylglycerol (DAG) is a natural oil, present in relatively low amounts in natural oils, reaching a maximum of nearly 10%. DAG has two isomers: 1,3-DAG and 1,2(2,3)-DAG. Generally, with the same fatty acid composition, 1,3-DAG has a melting point about 10°C higher than 1,2(2,3)-DAG, and is also more stable. Under normal conditions, the ratio of 1,3-DAG to 1,2(2,3)-DAG in natural oils is approximately 7:3. Due to structural differences from TAG, DAG also exhibits significant differences in its metabolic pathway. When TAG is ingested, due to the selectivity of lipases, it is hydrolyzed primarily to produce 2-monoacylglycerol esters (2-MAG) and free fatty acids. After the product is absorbed into the small intestinal epithelial cells, a portion (approximately 80% of the product) is resynthesized into TAG under the action of monoacylglycerol acyltransferase and diacylglycerol acyltransferase, and the reaction is rapid; the other portion (approximately 20%) enters the glycerol-3-phosphate pathway to synthesize TAG, and the process is slow. The resynthesized TAG is then assembled into chylomicrons via microsomal triglyceride transport proteins, and these chylomicrons are transported to the bloodstream via the intestinal lymphatic system. Excessive TAG intake leads to a rapid increase in blood lipids in a short period, while long-term hyperlipidemia can lead to a series of chronic diseases. When DAG is ingested, 1,2-DAG is metabolized in the same way as TAG, mainly producing 2-MAG and free fatty acids under the action of lipases; while 1,3-DAG is metabolized to produce 1(3)-MAG and free fatty acids. 1(3)-MAG can hardly synthesize TAG; most of the free fatty acids enter the liver for metabolism to produce energy, and a very small portion of the product enters the glycerol-3-phosphate pathway to synthesize TAG.

[0003] Therefore, compared to TAG, DAG intake significantly reduces postprandial blood lipid levels and the rate of increase. Thus, compared to regular cooking oils, DAG's unique metabolic pathway enables it to inhibit postprandial blood lipid elevation, reduce body fat accumulation, lower weight, and regulate blood sugar.

[0004] The safety of DAG has been extensively assessed. The U.S. Food and Drug Administration (FDA) and the Japanese Ministry of Health and Welfare have listed DAG as a generally safe (GRAS) substance. In 2009, my country's Ministry of Health listed DAG as a new resource food. In 2021, the National Health Commission of China revised the quality requirements for DAG oil, clarifying that its production process uses soybean oil, rapeseed oil, etc. as raw materials, and lipase preparations, water, glycerin, etc. as main auxiliary materials. It is produced through lipase catalysis, distillation separation, decolorization, deodorization and other processes. The DAG content in DAG oil should be ≥40%.

[0005] Currently, the main methods for synthesizing diglycerides are enzymatic and chemical methods. Enzymatic synthesis, due to its relatively few byproducts and high safety, has been applied in actual production. According to the difference in reaction form, enzymatic synthesis of diglycerides mainly includes glycerol hydrolysis, esterification, hydrolysis, and combinations of various methods. Enzyme-catalyzed glycerol hydrolysis is the mainstream method due to its high product yield and easy separation. However, there are still some problems in the process of producing diglycerides by glycerol hydrolysis: (1) the poor compatibility between glycerol and oils leads to a decrease in reaction efficiency; (2) since the enzyme-catalyzed glycerol hydrolysis reaction is a reversible reaction, in addition to producing diglycerides, a large amount of monoglycerides will also be produced in the reaction system. The traditional method uses molecular distillation to remove monoglycerides, producing a considerable amount of glycidyl esters and chloropropanol esters, thus causing safety issues. Summary of the Invention

[0006] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0007] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0008] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing high-purity diglyceride edible oil by continuous enzymatic reaction.

[0009] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a method for preparing high-purity diglyceride edible oil by continuous enzymatic reaction, comprising,

[0010] The oil is added to a batch reactor, nitrogen gas is introduced for protection, lipase is added, and glycerol is added in stages at a controlled rate to catalyze the glycerolysis reaction to prepare a glycerol ester mixture. The glycerol and glycerol ester are separated by slow stirring at high temperature to obtain the glycerol ester product.

[0011] Using imprinted lipase as a catalyst, the monoglyceride in the glycerol ester product is catalyzed to undergo an esterification reaction with fatty acids.

[0012] Molecular distillation is used to remove fatty acids, resulting in a high-purity diglyceride product.

[0013] As a preferred embodiment of the method described in this invention, the segmented, rate-controlled addition of glycerin includes,

[0014] After the addition of lipase, the pumping rate of glycerol is divided into three stages;

[0015] The process involves pumping in 10-20% glycerol at a constant rate over 0.5-1.5 hours, followed by pumping in 20-30% glycerol over 0.5-1.5 hours, and then pumping in 50-70% glycerol over 0.5-1.5 hours. After the glycerol is fully pumped in, the reaction continues for 3-5 hours.

[0016] In a preferred embodiment of the method described in this invention, the enzyme-catalyzed glycerol hydrolysis reaction is carried out in the following manner: the amount of lipase added is 10-20 wt% of the weight of the oil, the reaction temperature is 50-70°C, the stirring speed during the glycerol hydrolysis reaction is 600-800 rpm, and the molar ratio of glycerol pumped in to oil is 2-5:1.

[0017] As a preferred embodiment of the method described in this invention, the enzyme catalyzes the glycerol hydrolysis reaction, wherein the lipase is a commercially available lipase, including Lipozyme RM IM, Novozyme 435, and NS40086;

[0018] The oils are edible animal and vegetable oils, including rapeseed oil, soybean oil and sunflower seed oil;

[0019] The glycerin is food-grade glycerin.

[0020] In a preferred embodiment of the method described in this invention, the step of separating glycerol and glycerides by slow stirring at high temperature is wherein the temperature is 85-95°C, the stirring speed is 20-40 rpm, and the stirring time is 0.5-1.5 h.

[0021] In a preferred embodiment of the method described in this invention, the imprinted lipase is prepared by a method comprising:

[0022] Prepare a buffer solution with a pH of 5.5, add 10–30% Lipase G50, stir at 25°C for 20–40 min, filter and vacuum dry to obtain pH-controlled lipase; dissolve a nonionic surfactant at a concentration of 30–100 mg / mL in isopropanol, add 10–30% pH-controlled Lipase G50 lipase, stir at 25°C for 20–40 min, filter, freeze dry for 12–36 h, elute excess imprinted template with a nonpolar solvent, and vacuum dry to obtain double-imprinted lipase.

[0023] In a preferred embodiment of the method described in this invention, the nonionic surfactant includes Tween 20, 40, and 80, and the nonpolar solvent includes n-hexane and octane.

[0024] As a preferred embodiment of the method described in this invention, the esterification reaction conditions are as follows: the molar ratio of free fatty acid to glycerol ester backbone is 2-5:1, the temperature is 30-50°C, the stirring speed is 500-800 rpm, the amount of imprinted Lipase G50 added is 4-8 wt%, the vacuum degree is 10-30 mbar, and the reaction time is 6-12 h.

[0025] As a preferred embodiment of the method described in this invention, the conditions for removing fatty acids by molecular distillation are: distillation temperature of 150-170°C, pressure of 2-5 Pa, and condenser temperature of 25-35°C.

[0026] Another objective of this invention is to overcome the shortcomings of the prior art and provide a method for preparing high-purity diglyceride edible oil by continuous enzymatic reaction. The resulting diglyceride edible oil product has a diglyceride content of greater than 90%, a glycidyl ester content of less than 2 mg / Kg, and a chloropropanol ester content of less than 0.5 mg / Kg.

[0027] Beneficial effects of this invention:

[0028] (1) This invention utilizes lipase to catalyze the hydrolysis of glycerol to prepare glycerol ester products. During the reaction, the rate of glycerol addition is controlled by adding glycerol in stages. Due to the continuous generation of monoglycerides and diglycerides during the reaction, the rate of glycerol addition is controlled to effectively improve the compatibility between glycerol and the substrate, while avoiding the inhibition of lipase activity by excessive glycerol, thus greatly improving the reaction efficiency.

[0029] (2) This invention utilizes the principle that the macromolecular structure of biological enzymes exhibits flexible characteristics in an aqueous environment and rigid characteristics in a non-aqueous system. It enhances the activity and stability of lipase through double imprinting. First, the pH value of the glycerol lipase Lipase G50 is adjusted to the optimal reaction value. Then, a nonionic surfactant dissolved in isopropanol is used to cover the surface of the lipase and open the cap of the active center of the lipase to imprint the lipase, thereby enhancing the activity and stability of Lipase G50. Utilizing the substrate selectivity of Lipase G50, the monoglyceride in the glycerolysis product is catalyzed to undergo an esterification reaction with free fatty acids under a certain vacuum, thereby increasing the yield of diglycerides and obtaining a high-purity diglyceride product. At the same time, after the esterification reaction, since the content of monoglycerides in the system is extremely low, only relatively mild molecular distillation conditions are used for deacidification. The harmful components such as glycidyl esters and chloropropanol esters in the obtained product are also much lower than those of traditional methods. Detailed Implementation

[0030] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0031] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0032] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0033] The determination of the esterification activity of Lipase G50 in this invention: Glycerol and oleic acid were added to a batch reactor at a molar ratio of 1:1, and 4% Lipase G50 was added. The reaction was carried out at a temperature of 30°C and a stirring rate of 600 rpm for 1 hour. The initial activity of the enzyme was evaluated based on the esterification rate (%) of fatty acids in the system after 1 hour of reaction.

[0034] Example 1.

[0035] Lipase G50, a metaglycerol lipase, exhibits strong substrate specificity, showing activity only towards monoglycerides and diglycerides, but no activity towards triglycerides. Therefore, leveraging the characteristics of Lipase G50 to generate diglycerides from monoglycerides in an esterification reaction system under specific vacuum conditions can significantly improve the content and yield of diglycerides. Buffer solutions with pH values ​​of 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, and 9 were prepared. 10% (w / w) of Lipase G50 was added to each buffer solution, and the mixture was stirred at 25°C for 20 min to obtain a mixed solution. The resulting lipase was filtered and then vacuum-dried to remove moisture, yielding pH-controlled Lipase G50 lipase. The optimal catalytic pH value for Lipase G50 was then explored.

[0036] Table 1 Enzymatic activity of Lipase G50 under different pH values

[0037] pH value Enzyme catalytic activity 4 11.1％ 4.5 17.3％ 5 22.5％ 5.5 25.7％ 6 23.4％ 6.5 17.4％ 7 14.2％ 7.5 13.1％ 8 11.6％ 8.5 8.7％ Unadjusted pH 15.4％

[0038] pH alters the conformation of lipases by changing their surface charge, thus affecting their catalytic activity. Data analysis shows that Lipase G50 exhibits significantly higher activity after treatment with a buffer solution at pH 5-6 compared to untreated lipases. Furthermore, its catalytic activity is highest at pH 5.5, indicating that at this pH, the lipase exhibits its optimal catalytic conformation through electrostatic interactions.

[0039] Example 2

[0040] Rapeseed oil was added to a batch reactor and purged with nitrogen for protection. Lipozyme RM IM was added at a rate of 10 wt% of the rapeseed oil weight. The temperature was raised to 70°C and stirring was started at a speed of 800 rpm. At the same time, food-grade glycerol was pumped into the reactor at a molar ratio of 2:1 to rapeseed oil.

[0041] The glycerol was pumped in at three stages: the first stage was 20% glycerol pumped in at a constant rate over 0.5 hours; the second stage was 30% glycerol pumped in over 0.5 hours; and the third stage was 50% glycerol pumped in over 0.5 hours. After the glycerol was pumped in, the reaction continued for 4 hours. The lipase was then filtered to obtain a mixture of glycerides.

[0042] The obtained glycerol ester mixture was heated to 95°C and slowly stirred at 20 rpm for 0.5 h to accelerate the separation of glycerol and glycerol esters, thus obtaining glycerol ester product 1. The glycerol was recovered and used as a raw material for a new glycerolysis reaction. The product contained 58.9% monoglyceride, 35.0% diglyceride, and 6.1% triglyceride.

[0043] Lipase G50, a metaglycerol lipase, exhibits strong substrate specificity, showing activity only towards monoglycerides and diglycerides, but not towards triglycerides. Therefore, by utilizing the characteristics of Lipase G50, diglycerides can be generated from monoglycerides in an esterification reaction system under specific vacuum conditions, which can significantly improve the content and yield of diglycerides.

[0044] The activity and stability of lipase G50 were enhanced through double imprinting. Lipase G50 exhibited optimal activity at pH 5.5. A pH 5.5 buffer solution was prepared, and 10% (w / w) of Lipase G50 was added. The mixture was stirred at 25°C for 20 min, filtered, and vacuum-dried to remove moisture, yielding pH-controlled Lipase G50 lipase. The nonionic surfactant Tween 20 was dissolved in isopropanol at a concentration of 30 mg / mL, and 10% of the pH-controlled Lipase G50 lipase was added. The mixture was stirred at 25°C for 20 min, filtered, and freeze-dried for 24 h to obtain nonionic surfactant-imprinted lipase. Excess template was eluted with the nonpolar solvent n-hexane, and n-hexane was removed by vacuum drying, yielding pH- and nonionic surfactant-imprinted lipase. The activity of imprinted lipase G50 was measured to be 34.2%, while that of non-imprinted lipase G50 was only 15.4%. Imprinting increased the activity of lipase G50 by 122.1%.

[0045] Glyceryl ester product 1 was added to a batch reactor, and free rapeseed oil fatty acids were added based on the glycerol backbone content of the glyceryl ester. The molar ratio of free fatty acids to the glycerol backbone of the glyceryl ester was 2:1. The temperature was raised to 30°C, and stirring was started at a speed of 600 rpm. Imprinted Lipase G50 lipase was added at a rate of 4 wt%. Vacuum was applied at a vacuum degree of 30 mbar, and the reaction was carried out for 8 hours to obtain glyceryl ester product 2. The product contained 0.7% monoglyceride, 92.7% diglyceride, and 6.6% triglyceride, with a diglyceride yield of 92.7%.

[0046] Fatty acids were removed by molecular distillation. The conditions for removing fatty acids by molecular distillation were: distillation temperature of 150℃, pressure of 2Pa, and condenser temperature of 30℃. The resulting product contained 0.5% monoglycerides, 92.8% diglycerides, 6.7% triglycerides, 1.22 mg / kg glycidyl esters, and 0.21 mg / kg chloropropanol esters.

[0047] Example 3

[0048] Soybean oil was added to a batch reactor and purged with nitrogen for protection. At the same time, lipase Novozym435 was added at a rate of 15 wt% of the soybean oil weight. The temperature was raised to 60°C and stirring was started at a speed of 600 rpm. Simultaneously, food-grade glycerol was pumped into the reactor at a molar ratio of 3:1 between the glycerol and soybean oil.

[0049] The glycerol was pumped in at three stages: the first stage was to pump in 15% glycerol at a constant rate for 1 hour; the second stage was to pump in 25% glycerol for 1 hour; and the third stage was to pump in 60% glycerol for 1 hour. After the glycerol was pumped in, the reaction continued for 5 hours. The lipase was then filtered to obtain a mixture of glycerides.

[0050] The obtained glycerol ester mixture was heated to 90°C and slowly stirred at 30 rpm for 1 hour to accelerate the separation of glycerol and glycerol esters. The glycerol and glycerol esters were then separated to obtain glycerol ester product 1. The glycerol was recovered and used as a raw material for a new glycerolysis reaction. The product contained 64.8% monoglyceride, 29.7% diglyceride, and 5.5% triglyceride.

[0051] Lipase G50, a metaglycerol lipase, exhibits strong substrate specificity, showing activity only towards monoglycerides and diglycerides, but not towards triglycerides. Therefore, by utilizing the characteristics of Lipase G50, diglycerides can be generated from monoglycerides in an esterification reaction system under specific vacuum conditions, which can significantly improve the content and yield of diglycerides.

[0052] The activity and stability of lipase G50 were enhanced through double imprinting. Lipase G50 exhibited optimal activity at pH 5.5. A pH 5.5 buffer solution was prepared, and 20% (w / w) of Lipase G50 was added. The mixture was stirred at 25°C for 30 min, filtered, and vacuum-dried to remove moisture, yielding pH-controlled Lipase G50 lipase. The nonionic surfactant Tween 40 was dissolved in isopropanol at a concentration of 60 mg / mL, and 20% of the pH-controlled Lipase G50 lipase was added. The mixture was stirred at 25°C for 30 min, filtered, and freeze-dried for 12 h to obtain nonionic surfactant-imprinted lipase. Excess template was eluted with the nonpolar solvent n-hexane, and the hexane was removed by vacuum drying, yielding pH- and nonionic surfactant-imprinted lipase. The activity of imprinted lipase G50 was measured to be 35.5%, while that of non-imprinted lipase G50 was only 15.4%. Imprinting increased the activity of lipase G50 by 130.5%.

[0053] Glyceryl ester product 1 was added to a batch reactor, and free soybean oil fatty acids were added based on the glycerol backbone content of the glyceryl ester. The molar ratio of free fatty acids to the glycerol backbone of the glyceryl ester was 3:1. Stirring was started at 800 rpm, and the temperature was raised to 40°C. Imprinted Lipase G50 lipase was added at a rate of 6 wt%, and vacuum was applied at a vacuum degree of 20 mbar. The reaction was carried out for 6 h to obtain glyceryl ester product 2. The product contained 0.9% monoglyceride, 93.2% diglyceride, and 5.9% triglyceride, with a diglyceride yield of 93.2%.

[0054] Fatty acids were removed by molecular distillation. The conditions for removing fatty acids by molecular distillation were: distillation temperature of 160℃, pressure of 3Pa, and condenser temperature of 25℃. The resulting product contained 0.6% monoglyceride, 93.1% diglyceride, 6.3% triglyceride, 1.44 mg / kg glycidyl ester, and 0.35 mg / kg chloropropanol ester.

[0055] Example 4

[0056] Sunflower seed oil was added to a batch reactor and protected with nitrogen. At the same time, lipase NS40086 was added at a rate of 20 wt% of the sunflower seed weight. The temperature was raised to 50°C and stirring was started at a speed of 700 rpm. Simultaneously, food-grade glycerol was pumped into the reactor at a molar ratio of 5:1 between the glycerol and soybean oil.

[0057] The glycerol was pumped in at three stages: the first stage was to pump in 10% glycerol at a constant rate over 1.5 hours; the second stage was to pump in 20% glycerol over 1.5 hours; and the third stage was to pump in 70% glycerol over 1.5 hours. After the glycerol was pumped in, the reaction continued for 3 hours. The lipase was then filtered to obtain a mixture of glycerides.

[0058] The obtained glycerol ester mixture was heated to 85°C and slowly stirred at 40 rpm for 1.5 h to accelerate the separation of glycerol and glycerol esters, thus obtaining glycerol ester product 1. The glycerol was recovered and used as a raw material for a new glycerolysis reaction. The product contained 71.8% monoglyceride, 25.1% diglyceride, and 3.1% triglyceride.

[0059] Lipase G50, a metaglycerol lipase, exhibits strong substrate specificity, showing activity only towards monoglycerides and diglycerides, but not towards triglycerides. Therefore, by utilizing the characteristics of Lipase G50, diglycerides can be generated from monoglycerides in an esterification reaction system under specific vacuum conditions, which can significantly improve the content and yield of diglycerides.

[0060] The activity and stability of lipase G50 were enhanced through double imprinting. Lipase G50 exhibited optimal activity at pH 5.5. A pH 5.5 buffer solution was prepared, and 30% (w / w) of Lipase G50 was added. The mixture was stirred at 25°C for 40 min, filtered, and vacuum-dried to remove moisture, yielding pH-controlled Lipase G50 lipase. The nonionic surfactant Tween 80 was dissolved in isopropanol at a concentration of 100 mg / mL, and 30% of the pH-controlled Lipase G50 lipase was added. The mixture was stirred at 25°C for 40 min, filtered, and freeze-dried for 36 h to obtain nonionic surfactant-imprinted lipase. Excess template was eluted with the nonpolar solvent n-hexane, and n-hexane was removed by vacuum drying, yielding pH- and nonionic surfactant-imprinted lipase. The activity of imprinted lipase G50 was measured to be 33.8%, while that of non-imprinted lipase G50 was only 15.4%. Imprinting increased the activity of lipase G50 by 119.5%.

[0061] Glyceryl ester mixture 1 was added to a batch reactor, and free sunflower seed oil fatty acids were added based on the glyceryl skeleton content of the glyceryl esters. The molar ratio of free fatty acids to the glyceryl skeleton of the glyceryl esters was 5:1. Stirring was started at a speed of 500 rpm, the temperature was raised to 50°C, and 8 wt% imprinted Lipase G50 lipase was added. Vacuum was applied at a vacuum degree of 30 mbar, and the reaction was carried out for 12 h to obtain glyceryl ester product 2. The product contained 0.6% monoglyceride, 95.8% diglyceride, and 3.6% triglyceride, with a diglyceride yield of 95.8%.

[0062] Fatty acids were removed by molecular distillation. The conditions for removing fatty acids by molecular distillation were: distillation temperature of 170℃, pressure of 5Pa, and condenser temperature of 35℃. The resulting product contained 0.4% monoglyceride, 95.6% diglyceride, 4.0% triglyceride, 1.63 mg / kg glycidyl ester, and 0.44 mg / kg chloropropanol ester.

[0063] Comparative Example 1

[0064] The reaction conditions of Example 2 were used, but instead of adding glycerol in stages, glycerol was added all at once. Other conditions were the same as in Example 2.

[0065] Rapeseed oil was added to a batch reactor, and food-grade glycerol was pumped into the reactor at a molar ratio of glycerol to rapeseed oil of 2:1. Nitrogen gas was introduced for protection. Lipozyme RM IM was added at a rate of 10 wt% of the weight of rapeseed oil. The temperature was raised to 70°C and stirring was started at a speed of 800 rpm. The reaction time was 5.5 h.

[0066] The resulting glycerol ester mixture was heated to 95°C and stirred slowly at 20 rpm for 0.5 h to accelerate the separation of glycerol and glycerol esters. The glycerol was then separated from the glycerol esters and recovered as a raw material for a new glycerolysis reaction.

[0067] The obtained glyceride products contained 49.4% monoglycerides, 30.9% diglycerides, and 19.6% triglycerides.

[0068] The triglyceride content in the product of Comparative Example 1 was much higher than that in Example 1, indicating that Comparative Example 1 was far from reaching reaction equilibrium.

[0069] Comparative Example 2

[0070] The reaction conditions of Example 3 were used, but Lipase G50 was not used for blotting; the reaction was carried out directly with Lipase G50. Other reaction conditions were the same as in Example 3.

[0071] Soybean oil was added to a batch reactor and purged with nitrogen for protection. At the same time, lipase Novozym435 was added at a rate of 15 wt% of the soybean oil weight. The temperature was raised to 60°C and stirring was started at a speed of 600 rpm. Simultaneously, food-grade glycerol was pumped into the reactor at a molar ratio of 3:1 between the glycerol and soybean oil.

[0072] The glycerol was pumped in at three stages: the first stage was to pump in 15% glycerol at a constant rate for 1 hour; the second stage was to pump in 25% glycerol for 1 hour; and the third stage was to pump in 60% glycerol for 1 hour. After the glycerol was pumped in, the reaction continued for 5 hours. The lipase was then filtered to obtain a mixture of glycerides.

[0073] The obtained glycerol ester mixture was heated to 90°C and slowly stirred at 30 rpm for 1 hour to accelerate the separation of glycerol and glycerol esters. The glycerol and glycerol esters were then separated to obtain glycerol ester product 1. The glycerol was recovered and used as a raw material for a new glycerolysis reaction. The product contained 64.8% monoglyceride, 29.7% diglyceride, and 5.5% triglyceride.

[0074] Glyceryl ester product 1 was added to a batch reactor, and free soybean oil fatty acids were added based on the amount of glyceryl ester backbone. The molar ratio of free fatty acids to glyceryl ester backbone was 3:1. Stirring was started at 800 rpm, the temperature was raised to 40°C, and Lipase G50 lipase was added at a rate of 6 wt%. Vacuum was then applied at a vacuum level of 20 mbar, and the reaction was carried out for 6 hours to obtain glyceryl ester product 2, which contained 12.5% ​​monoglyceride.

[0075] Comparative Example 3

[0076] The reaction conditions of Example 4 were used, but the esterification reaction was not carried out using imprinted lipase G50.

[0077] Sunflower seed oil was added to a batch reactor and protected with nitrogen. At the same time, lipase NS40086 was added at a rate of 20 wt% of the sunflower seed weight. The temperature was raised to 50°C and stirring was started at a speed of 700 rpm. Simultaneously, food-grade glycerol was pumped into the reactor at a molar ratio of 5:1 between the glycerol and soybean oil.

[0078] The glycerol was pumped in at three stages: the first stage was to pump in 10% glycerol at a constant rate over 1.5 hours; the second stage was to pump in 20% glycerol over 1.5 hours; and the third stage was to pump in 70% glycerol over 1.5 hours. After the glycerol was pumped in, the reaction continued for 3 hours. The lipase was then filtered to obtain a mixture of glycerides.

[0079] The obtained glycerol ester mixture was heated to 85°C and stirred slowly at 40 rpm for 1.5 h to accelerate the separation of glycerol and glycerol esters. The glycerol and glycerol esters were separated to obtain glycerol ester product 1. The glycerol was recovered and used as a raw material for a new glycerolysis reaction.

[0080] The product contained 71.8% monoglycerides, 25.1% diglycerides, and 3.1% triglycerides.

[0081] Fatty acids were removed by molecular distillation. The conditions for fatty acid removal by molecular distillation were: distillation temperature of 170℃, pressure of 5 Pa, and condenser temperature of 35℃; the resulting product contained 69.9% monoglycerides, 26.8% diglycerides, and 3.3% triglycerides.

[0082] Traditional methods were employed, including two-stage molecular distillation, to further remove monoglycerides. The distillation temperature was 200℃, the pressure was 3 Pa, and the condenser temperature was 25℃. The resulting product contained 1.3% monoglycerides, 87.9% diglycerides, and 10.8% triglycerides. Due to the removal of monoglycerides, the yield of diglycerides obtained in this stage was 26.8% of the yield obtained in the first stage reaction. The glycidyl ester content was 6.95 mg / kg, and the chloropropanol ester content was 1.24 mg / kg.

[0083] Comparative Example 4

[0084] Under the conditions of Example 2, multiple batches of reactions were carried out using pH-controlled Lipase G50, double-blotted Lipase G50, and unblotted Lipase G50, respectively. The catalytic activity of the two lipases in different batches was analyzed to compare the catalytic stability of the lipases.

[0085] Table 2 Enzyme activity and enzyme inactivation rate of different lipases in different batches

[0086] batch pH adjustment Double Imprint No trace 1 25.7％ 34.2％ 15.4％ 2 22.3％ 32.3％ 13.1％ 3 17.1％ 31.4％ 12.7％ 4 13.2％ 30.2％ 11.2％ 5 10.3％ 28.5％ 9.7％ 6 8.1％ 26.3％ 6.3％ Enzyme inactivation rate 68.5％ 23.1％ 59.1％

[0087] Activity analysis of different batches of different lipases showed that the double-imprinted lipase Lipase G50 had a lower inactivation rate and better stability compared to pH-controlled and site-imprinted lipases. In the double-imprinted lipase process, pH control first brought the lipase to its optimal catalytic pH. In the second step, nonionic surfactant imprinting was used. Isopropanol has excellent dissolving effects on both the surfactant and the lipase, ensuring sufficient interaction between the surfactant and the lipase. Since the interaction between the nonionic surfactant and the lipase is mainly through hydrophobic interactions—the polar head of the surfactant binds to the hydrophilic group of the lipase, and the nonpolar head binds to the hydrophobic group—and since the lipase itself is water-soluble, the hydrophilic groups on the lipase surface are much larger than the hydrophobic groups. Therefore, the nonionic surfactant alters the hydrophilicity and hydrophobicity of the lipase surface, changing it from hydrophilic to hydrophobic. This modification avoids excessive contact between the lipase and polar substances in the reaction system, preventing these substances from drawing water from the lipase surface and causing structural changes that could lead to activity loss. Simultaneously, the surfactant interacts with the capping mechanism of the lipase, opening the cap and allowing water to escape, thus maintaining the lipase in its catalytic conformation. This not only improves the stability of the lipase but also enhances its activity. Furthermore, the nonionic surfactant coating on the lipase surface also improves its solubility in the reaction system, increasing reaction efficiency.

[0088] Comparative Example 5

[0089] Referring to Example 4, glycerol was added in three stages with controlled speed, but the glycerol addition rate was different from that in Example 3, while other conditions were the same as in Example 4.

[0090] Sunflower seed oil was added to a batch reactor and protected with nitrogen. At the same time, lipase NS40086 was added at a rate of 20 wt% of the sunflower seed weight. The temperature was raised to 50°C and stirring was started at a speed of 700 rpm. Simultaneously, food-grade glycerol was pumped into the reactor at a molar ratio of 5:1 between the glycerol and soybean oil.

[0091] The glycerol was pumped in at three stages: the first stage was 5% glycerol pumped in at a constant rate over 1.5 hours; the second stage was 15% glycerol pumped in over 1.5 hours; and the third stage was 80% glycerol pumped in over 1.5 hours. After the glycerol was pumped in, the reaction continued for 3 hours. The lipase was then filtered to obtain a mixture of glycerides.

[0092] The obtained glycerol ester mixture was heated to 85°C and slowly stirred at 40 rpm for 1.5 h to accelerate the separation of glycerol and glycerol esters, thus obtaining glycerol ester product 1. The glycerol was recovered and used as a raw material for a new glycerolysis reaction. The product contained 62.3% monoglyceride, 28.2% diglyceride, and 9.5% triglyceride.

[0093] The triglyceride content of the product in Comparative Example 5 was higher than that in Example 4. This indicates that the reaction in Comparative Example 5 was not as complete as in Example 4, and the high triglyceride content reduced the purity of the final diglyceride product.

[0094] Because the amount of glycerol pumped in the first stage was too low, too little emulsifying monoglyceride was formed. As a result, the injected glycerol could not mix well with the oil in the subsequent reaction. The free glycerol covered the surface of the lipase, which reduced the activity of the lipase.

[0095] Comparative Example 6

[0096] Referring to Example 2, glycerol was added in three stages with controlled speed, but the glycerol addition rate was different from that in Example 2, while other conditions were the same as in Example 2.

[0097] Rapeseed oil was added to a batch reactor and purged with nitrogen for protection. Lipozyme RM IM was added at a rate of 10 wt% of the rapeseed oil weight. The temperature was raised to 70°C and stirring was started at a speed of 800 rpm. At the same time, food-grade glycerol was pumped into the reactor at a molar ratio of 2:1 to rapeseed oil.

[0098] The glycerol was pumped in at three stages: the first stage was to pump in 30% glycerol at a constant rate for 0.5 h; the second stage was to pump in 40% glycerol for 0.5 h; and the third stage was to pump in 30% glycerol for 0.5 h. After the glycerol was pumped in, the reaction continued for 4 h. The lipase was then filtered to obtain a mixture of glycerides.

[0099] The obtained glycerol ester mixture was heated to 95°C and slowly stirred at 20 rpm for 0.5 h to accelerate the separation of glycerol and glycerol esters, thus obtaining glycerol ester product 1. The glycerol was recovered and used as a raw material for a new glycerolysis reaction. The product contained 52.7% monoglyceride, 33.5% diglyceride, and 13.8% triglyceride.

[0100] The triglyceride content of the product in Comparative Example 6 was higher than that in Example 2. This indicates that the reaction in Comparative Example 6 was not as complete as in Example 2, and the high triglyceride content reduced the purity of the final diglyceride product.

[0101] Because the glycerol infusion rate in the first stage is too high, the glycerol cannot be fully mixed with the fat, resulting in free glycerol covering the surface of the lipase and reducing its activity.

[0102] Comparative Example 7

[0103] Referring to Example 2, glycerol was added in two stages with controlled speed, but the glycerol addition rate was different from that in Example 1, while other conditions were the same as in Example 2.

[0104] Rapeseed oil was added to a batch reactor and purged with nitrogen for protection. Lipozyme RM IM was added at a rate of 10 wt% of the rapeseed oil weight. The temperature was raised to 70°C and stirring was started at a speed of 800 rpm. At the same time, food-grade glycerol was pumped into the reactor at a molar ratio of 2:1 to rapeseed oil.

[0105] The glycerol was pumped in two stages. In the first stage, 20% of the glycerol was pumped in at a constant rate over 0.5 hours. In the second stage, 80% of the glycerol was pumped in over 0.5 hours. After the glycerol was pumped in, the reaction continued for 4 hours. The lipase was then filtered to obtain a mixture of glycerides.

[0106] The obtained glycerol ester mixture was heated to 95°C and slowly stirred at 20 rpm for 0.5 h to accelerate the separation of glycerol and glycerol esters, thus obtaining glycerol ester product 1. The glycerol was recovered and used as a raw material for a new glycerolysis reaction. The product contained 50.8% monoglyceride, 32.7% diglyceride, and 16.5% triglyceride.

[0107] The triglyceride content of the product in Comparative Example 7 was higher than that in Example 2. This indicates that the reaction in Comparative Example 7 was not as complete as in Example 2, and the high triglyceride content reduced the purity of the final diglyceride product.

[0108] Because the glycerol infusion rate in the second stage is too high, the glycerol cannot mix sufficiently with the fat, resulting in free glycerol covering the surface of the lipase and reducing its activity.

[0109] Enzymes are protein macromolecules that are flexible in aqueous solutions and rigid in organic solvents, and possess pH memory properties. Different enzymes have an optimal pH value, meaning they exhibit best catalytic activity within a specific pH range. Lipases differ from other enzymes in that they act at the oil-water interface and exhibit unique interfacial activity. Their active site has a unique structure: the active site is completely embedded under a "cap"-like structure, composed of one or two α-helices. The main principle of using surfactant bioimprinting to modify the catalytic activity of lipases is that in the presence of a surfactant, the "cap" is opened, exposing the hydrophobic groups containing the active site, thus facilitating substrate binding. Simultaneously, freeze-drying removes excess water, thereby solidifying the enzyme conformation. For Lipase G50, its optimal pH is 5.5. Therefore, Lipase G50 is first dissolved in a buffer solution with a pH of 5.5 to maintain its optimal catalytic conformation at the optimal pH. Then, a surfactant is added to the buffer solution to open the cap of the active site of Lipase G50. The water in the system is removed by freeze-drying to preserve the conformation of Lipase G50. Finally, the surfactant on the lipase is removed with an organic solvent. This puts Lipase G50 in the optimal catalytic state, thus significantly improving its catalytic efficiency.

[0110] Therefore, this invention employs segmented, rate-controlled addition of glycerol to fully utilize the monoglycerides and diglycerides produced in the reaction, thereby improving the solubility of glycerol and increasing reaction efficiency. Simultaneously, it utilizes lipase G50 to esterify the monoglycerides in the product into diglycerides, thereby increasing the product yield and purity, while also significantly reducing the production of harmful substances during the molecular distillation stage.

[0111] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. A method for the production of high purity diglyceride edible oil by continuous enzymatic reaction, characterized by: include, The oil is added to a batch reactor, nitrogen gas is introduced for protection, lipase is added, and glycerol is added in stages at a controlled rate to catalyze the glycerolysis reaction to prepare a glycerol ester mixture. The glycerol and glycerol ester are separated by slow stirring at high temperature to obtain the glycerol ester product. The activity and stability of the lipase were enhanced by double imprinting with pH and nonionic surfactants, and the esterification reaction of monoglycerides and fatty acids in the glycerol ester product was catalyzed. Molecular distillation is used to remove fatty acids to obtain high-purity diglyceride products; The segmented, rate-controlled addition of glycerin includes, After the addition of lipase, the pumping rate of glycerol is divided into three stages; The process involves pumping in 10-20% glycerol at a constant rate over 0.5-1.5 hours, followed by pumping in 20-30% glycerol over 0.5-1.5 hours, and then pumping in 50-70% glycerol over 0.5-1.5 hours. After the glycerol is pumped in, the reaction continues for 3-5 hours. The catalytic glycerol hydrolysis reaction is described in which the amount of lipase added is 10-20 wt% of the weight of the oil, the reaction temperature is 50-70℃, the stirring speed during the glycerol hydrolysis reaction is 600-800 rpm, and the molar ratio of glycerol pumped in to oil is 2-5:

1. The method for preparing the imprinted lipase includes: preparing a buffer solution with a pH of 5.5, adding 10-30% Lipase G50, stirring at 25°C for 20-40 min, filtering and vacuum drying to obtain a pH-controlled lipase; dissolving a nonionic surfactant at a concentration of 30-100 mg / mL in isopropanol, adding 10-30% pH-controlled Lipase G50 lipase, stirring at 25°C for 20-40 min, filtering, freeze-drying for 12-36 h, eluting excess imprint template with a nonpolar solvent, and vacuum drying to obtain a double-imprinted lipase; wherein the nonionic surfactant includes Tween 20, Tween 40, and Tween 80, and the nonpolar solvent includes n-hexane and octane; The esterification reaction conditions are as follows: the molar ratio of free fatty acid to glycerol ester backbone is 2~5:1, the temperature is 30~50℃, the stirring speed is 500~800rpm, the amount of imprinted Lipase G50 added is 4~8wt%, the vacuum degree is 10-30mbar, and the reaction time is 6-12h.

2. The method of claim 1, wherein: The enzyme catalyzes glycerol hydrolysis, wherein the lipase is a commercially available lipase, including Lipozyme RM IM, Novozyme 435 and NS40086; The oils are edible animal and vegetable oils, including rapeseed oil, soybean oil and sunflower seed oil; The glycerin is food-grade glycerin.

3. The method of claim 1, wherein: The process involves accelerating the separation of glycerol and glycerides through slow, high-temperature stirring. The temperature is 85-95°C, the stirring speed is 20-40 rpm, and the stirring time is 0.5-1.5 h.

4. The method as described in claim 1, characterized in that: The conditions for removing fatty acids by molecular distillation are: distillation temperature of 150~170℃, pressure of 2~5Pa, and condenser temperature of 25~35℃.

5. The diglyceride edible oil produced by the method of any one of claims 1 to 4, characterized by: The resulting diglyceride edible oil contains more than 90% diglycerides, less than 2 mg / kg of glycidyl esters, and less than 0.5 mg / kg of chloropropanol esters.