Process for the preparation of a glyceride edible oil and products thereof

Abstract

The application discloses a preparation method of a glyceride edible oil and the product thereof, and belongs to the technical field of enzyme catalysis. The preparation method comprises the following steps: catalyzing a glycerolysis reaction of glycerol and oil by using a bio-imprinted lipase as a catalyst, and adding glycerol in stages; and removing monoglyceride and fatty acid by using two-stage molecular distillation to obtain the glyceride edible oil. In the glycerolysis stage, in order to increase the solubility of glycerol and oil and avoid glycerol covering the surface of the enzyme, the glycerol is added in stages, the adding rate of the glycerol in different stages is controlled, the monoglyceride and the glyceride produced in the reaction are effectively utilized, and the solubility of the glycerol in the whole reaction process is improved, so that the reaction efficiency is remarkably improved.

Description

Technical Field

[0001] This invention belongs to the field of oil preparation technology, specifically relating to a method for preparing diglyceride edible oil and its product. Background Technology

[0002] Diacylglycerol (DAG) is the most abundant natural component of edible oils besides TAG. It exists in two isomers: 1,3-DAG and 1,2-DAG, with a ratio of approximately 7:3 in natural oils. The safety of DAG oils has been extensively assessed. DAG's metabolic pathway differs from that of TAG, making it less likely to be converted into fat and stored in the body. This avoids various metabolic syndromes caused by long-term TAG intake; therefore, DAG is a natural and healthy alternative to TAG.

[0003] Currently, the main methods for synthesizing diglycerides include chemical synthesis and enzymatic synthesis. Chemical catalysts in chemical synthesis mainly include sodium methoxide, potassium hydroxide, magnesium oxide, and lithium oxide. Using alkaline catalysts allows for saponification reactions with oils, increasing the solubility of glycerol in the system and accelerating the reaction process. The reaction mechanism mainly involves glycerolysis and esterification. The advantages of chemical methods are simple reaction routes, short reaction times, relatively low costs, and ease of scalability. However, their disadvantages are also significant. For example, the reaction products are usually a mixture of monoglycerides, diglycerides, and glycerol, lacking specificity and resulting in a low yield of the target diglyceride. Strict requirements are placed on temperature and pressure during the reaction, making side reactions prone to occur. The chemical catalysts used may pollute the environment and raise consumer concerns about food safety. Furthermore, the target products obtained by chemical methods often have poor flavor and color, limiting their application in various fields. Enzymatic synthesis methods mainly include enzymatic esterification, enzymatic glycerolysis, and enzymatic selective hydrolysis. The lipases used in enzymatic methods are mainly derived from eukaryotes such as plants, animals, and fungi, as well as prokaryotes such as bacteria, which contain fats. These enzymes possess multiple catalytic capabilities and can catalyze the hydrolysis, alcoholysis, esterification, transesterification, and reverse synthesis of esters under certain conditions. They are characterized by their specificity, high efficiency, and environmental friendliness. Enzymatic glycerol hydrolysis is currently the most widely used and economical method. Glycerol hydrolysis mainly utilizes the rearrangement of fatty acids in glycerol and triglycerides under certain conditions to form new glycerides. From the perspective of raw material types and reaction mechanisms, it belongs to a type of transesterification reaction. Glycerol hydrolysis is characterized by short reaction time and easy product separation, and its application technology in the oil and fat industry is becoming increasingly mature.

[0004] Currently, the main problems in the process of producing diglycerides through enzymatic glycerolysis are low enzyme activity, poor compatibility between glycerol and oils, and the tendency for excessive glycerol to adsorb onto the enzyme surface, which further limits enzyme activity. Summary of the Invention

[0005] 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.

[0006] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0007] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing diglyceride edible oil.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a method for preparing diglyceride edible oil, comprising,

[0009] A nonionic surfactant-imprinted lipase dissolved in isopropanol was used as a catalyst to catalyze the glycerol-oil glycerolysis reaction, and glycerol was injected in stages.

[0010] Diglyceride edible oil is obtained by removing monoglycerides and fatty acids through two-stage molecular distillation.

[0011] As a preferred embodiment of the preparation method described in this invention, the staged injection of glycerol includes pumping it into the reaction system in three stages.

[0012] The process involves pumping in 10-30% glycerol at a constant rate over 1 hour, followed by pumping in 25-50% glycerol over 0.5 hours, and then pumping in 20-60% glycerol over 1 hour. After the glycerol is fully pumped in, the reaction continues for 3-8 hours. The lipase is then filtered to obtain a mixture of glycerides.

[0013] As a preferred embodiment of the preparation method described in this invention, the glycerol hydrolysis reaction includes an amount of imprinted lipase added at 5-20 wt% of the weight of the oil, a reaction temperature of 40-70°C, a stirring speed of 500-800 rpm, and a molar ratio of glycerol to oil of 1:0.4-4.

[0014] In a preferred embodiment of the preparation method described in this invention, the glycerol hydrolysis reaction involves oils including soybean oil, rapeseed oil, sunflower seed oil, flaxseed oil, peanut oil, olive oil, and camellia seed oil.

[0015] As a preferred embodiment of the preparation method described in this invention, the method further includes heating the glycerol ester mixture obtained from the reaction to 80-90°C, stirring slowly at a speed of 10-40 rpm for 0.5-2 hours to accelerate the separation of glycerol and glycerol esters, and recovering the glycerol as a raw material for a new glycerolysis reaction.

[0016] In a preferred embodiment of the preparation method described in this invention, fatty acids and monoglycerides are removed by two-stage molecular distillation to obtain diglyceride edible oil, wherein the diglyceride content is greater than 40%.

[0017] As a preferred embodiment of the preparation method described in this invention, the conditions for removing fatty acids by primary molecular distillation are: distillation temperature of 150°C, pressure of 3 Pa, and condenser temperature of 50°C.

[0018] The conditions for removing monoglycerides by secondary molecular distillation are: distillation temperature of 210℃ and pressure of 1 Pa.

[0019] As a preferred embodiment of the preparation method described in this invention, the preparation method of the bio-imprinted lipase includes:

[0020] The surfactant was dissolved in isopropanol at a concentration of 10–100 mg / L and dispersed thoroughly to obtain mixed solution 1;

[0021] Add 10-50% by mass of commercial immobilized lipase to mixed solution 1, stir the mixture at 100-400 rpm for 10-60 min at 25°C, and filter to obtain lipase;

[0022] The surfactant-imprinted template on the immobilized lipase was eluted using a nonpolar solvent, n-hexane or octane, with the solvent amount being 30-80% of the mixed solution 1. The lipase was then filtered and dried in a vacuum desiccator at room temperature for 8-24 hours to remove the organic solvent, thus obtaining the imprinted lipase.

[0023] In a preferred embodiment of the preparation method described in this invention, the surfactant is a nonionic surfactant, including Tween 20, 40, 60, and 80; the commercially available immobilized lipase includes Novozym 435, LipozymeRM IM, LipozymeTL IM, and NS 40086.

[0024] Another objective of this invention is to overcome the shortcomings of the prior art and provide a method for preparing diglyceride edible oil, which yields a diglyceride edible oil.

[0025] Beneficial effects of this invention:

[0026] (1) In the glycerol hydrolysis stage, in order to increase the solubility of glycerol and fat and avoid glycerol covering the enzyme surface, the present invention adds glycerol in stages and controls the rate of glycerol addition at different stages, effectively utilizing the monoglyceride and diglyceride produced by the reaction itself, thereby improving the solubility of glycerol in the entire reaction process and significantly improving the reaction efficiency.

[0027] (2) The present invention uses nonionic surfactants dissolved in isopropanol as imprint templates to enhance the activity and catalytic stability of lipases through bioimprinting.

[0028] (3) This invention establishes a predictive model for the composition of products in the equilibrium stage of enzyme-catalyzed glycerolysis reaction, predicting the composition of glycerolysis products at equilibrium under different substrate ratios, so that a suitable substrate ratio can be selected according to product requirements. Detailed Implementation

[0029] 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.

[0030] 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.

[0031] 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.

[0032] The glycerololysis reaction between glycerol and lipids catalyzed by enzymes is actually a stochastic and reversible reaction. The composition of the final product of a reversible reaction is related to the substrate ratio; other reaction parameters, such as temperature and catalyst dosage, only affect the time to reach equilibrium and have little impact on the composition of the final product. Based on this, a model of the product composition under different substrate ratios can be established using the theory of random distribution of fatty acids. According to the position selectivity of lipases, it can be divided into two cases: a stochastic transesterification reaction and a directional transesterification reaction at the sn-1,3 position.

[0033] Assuming a molar ratio of glycerol to fat of 1:r, the H atoms on glycerol exchange with the fatty acid R atoms in triglycerides. Among all exchangeable functional groups in the reaction system, the proportion of H atoms is 1 / (1+r), and the proportion of fatty acids is r / (1+r). According to the theory of random distribution of H and R under enzyme catalysis, under a molar ratio of glycerol to fat of 1:r, the proportion of substitutable H atoms is 3 / (3+3r), and the proportion of substitutable R atoms is 3r / (3+3r). Based on this random arrangement, the proportion of components containing different functional groups in the product can be calculated.

[0034]

[0035] Therefore, under this reaction system, the content of diglycerides (sn-1,2-diglycerides + sn-1,3-diglycerides + sn-2,3-diglycerides) can be calculated as follows:

[0036]

[0037] The content of monoglycerides (sn-1 monoglyceride + sn-2 monoglyceride + sn-3 monoglyceride) can be calculated as follows:

[0038]

[0039] The triglyceride content can be calculated as follows:

[0040]

[0041] The remaining glycerol content can be calculated as follows:

[0042]

[0043] In the sn-1,3 directional transesterification reaction, without considering acyl transfer, since lipase does not act on the sn-2 position, the functional groups at the sn-2 positions of glycerol and fat do not interact in the system. The proportion of H at the sn-2 position is 1 / (1+r), the proportion of R at the sn-2 position is r / (1+r), the proportion of H at the sn-1,3 position is 2 / (2+2r), and the proportion of R at the sn-1,3 position is 2r / (2+2r). Based on the random arrangement, the proportion of the composition containing different functional groups in the product can be calculated.

[0044]

[0045] Therefore, under this reaction system, the content of diglycerides (sn-1,2-diglycerides + sn-1,3-diglycerides + sn-2,3-diglycerides) can be calculated as follows:

[0046]

[0047] The content of monoglycerides (sn-1 monoglyceride + sn-2 monoglyceride + sn-3 monoglyceride) can be calculated as follows:

[0048]

[0049] The triglyceride content can be calculated as follows:

[0050]

[0051] The remaining glycerol content can be calculated as follows:

[0052]

[0053] Therefore, although the functional group at the sn-2 position in sn-1,3 transesterification does not participate in the reaction, its proportion is consistent with that of random transesterification. Thus, when the products of random transesterification and sn-1,3 directional transesterification reach equilibrium during the reaction, the final product composition is consistent, as shown in the table below.

[0054] Table 1. Composition of products at equilibrium of enzyme-catalyzed glycerol hydrolysis

[0055] sn-1 monoglyceride <![CDATA[r / (1+r) 3 ]]> SN-2 monoglyceride <![CDATA[r / (1+r) 3 ]]> SN-3 monoglyceride <![CDATA[r / (1+r) 3 ]]> sn-1,2-diglyceride <![CDATA[r 2 / (1+r) 3 <!-- 4 -->]]> sn-1,3-glycerol diglyceride <![CDATA[r 2 / (1+r) 3 ]]> sn-2,3-glycerol diglyceride <![CDATA[r 2 / (1+r) 3 ]]> Triglycerides <![CDATA[r 3 / (1+r) 3 ]]> glycerin <![CDATA[1 / (1+r) 3 ]]> monoglycerides <![CDATA[3r / (1+r) 3 ]]> diglycerides <![CDATA[3r 2 / (1+r) 3 ]]>

[0056] By inputting different r values, the composition of the products at equilibrium under different substrate ratios can be calculated, as shown in the table below. These results can guide and predict the reaction process and outcomes in enzyme-catalyzed glycerol hydrolysis, facilitating the optimization of reaction conditions.

[0057] Table 2. Product composition (%) at different substrate ratios

[0058] 0.1 2.25 22.54 24.79 0.08 75.13 0.2 6.94 34.72 41.67 0.46 57.87 0.3 12.29 40.96 53.25 1.23 45.52 0.4 17.49 43.73 61.22 2.33 36.44 0.5 22.22 44.44 66.67 3.70 29.63 0.6 26.37 43.95 70.31 5.27 24.41 0.7 29.92 42.74 72.66 6.98 20.35 0.8 32.92 41.15 74.07 8.78 17.15 1 37.50 37.50 75.00 12.50 12.50 2 44.44 22.22 66.67 29.63 3.70 3 42.19 14.06 56.25 42.19 1.56 4 38.40 9.60 48.00 51.20 0.80 5 34.72 6.94 41.67 57.87 0.46 6 31.49 5.25 36.73 62.97 0.29 7 28.71 4.10 32.81 66.99 0.20 8 26.34 3.29 29.63 70.23 0.14

[0059] Since glycerol cannot be detected during the testing process, and it is necessary to remove glycerol first during the preparation process, and to obtain the final product, monoglyceride must also be removed, the composition of the product after glycerol removal and after removal of glycerol and monoglyceride is listed in the table below based on calculations.

[0060] Table 3. Composition of products after removal of glycerol and monoglycerides

[0061]

[0062] The above data represents the equilibrium values ​​of different glycerides in the system when glycerol hydrolysis reaches equilibrium, calculated theoretically. Comparing actual and theoretical data allows for guidance on whether the reaction has reached equilibrium in actual reactions, and on determining the appropriate reactant ratio to obtain a certain proportion of product. This avoids extensive optimization of the reaction process in actual reactions and significantly improves efficiency.

[0063] Determination of lipase glycerol hydrolysis activity: The catalytic activity of lipase was evaluated by performing a glycerol hydrolysis reaction between soybean oil and glycerol. The substrate was a mixture of soybean oil and glycerol at a molar ratio of 1:1 (soybean oil / glycerol), and the amount of lipase added was 4% relative to the total mass of the substrate. The catalytic reaction was carried out in a round-bottom flask at 50°C and a stirring rate of 600 rpm. After 1 hour of reaction, the product was collected to assess its initial activity, and the enzyme activity was evaluated based on the percentage of soybean oil reacted after 1 hour of reaction.

[0064] Example 1

[0065] The nonionic surfactant Tween 20 was dissolved in isopropanol at a concentration of 50 mg / L and thoroughly dispersed to obtain mixed solution 1. Immobilized lipase Novozym 435 (30% by mass) was added to mixed solution 1. The mixture was stirred at 200 rpm for 60 min at 25 °C and filtered to obtain the lipase. The surfactant-imprinted template on the immobilized lipase Novozym 435 was eluted with the nonpolar solvent n-hexane (50% of mixed solution 1). The lipase was then filtered and dried in a vacuum desiccator at room temperature for 24 h to obtain imprinted lipase. The activities of imprinted and unimprinted lipases are shown below.

[0066] Table 1. Comparison of activities of imprinted and non-imprinted lipases

[0067] Enzyme activity 37.3％ 22.6％ 65％

[0068] Soybean oil was added to a batch reactor and purged with nitrogen for protection. Simultaneously, imprinted lipase was added at a rate of 10 wt% of the soybean oil weight. The temperature was raised to 60°C and stirring was started at a speed of 600 rpm. At the same time, food-grade glycerol was pumped into the reactor at a molar ratio of 1:1 to soybean oil.

[0069] The glycerol was pumped in at three stages: the first stage was to pump in 20% glycerol at a constant rate for 1 hour; the second stage was to pump in 30% glycerol for 0.5 hours; and the third stage was to pump in 50% glycerol for 1 hour. After the glycerol was pumped in, the reaction continued for 4 hours. The lipase was then filtered to obtain a mixture of glycerides.

[0070] The resulting glycerol ester mixture was heated to 90°C and stirred slowly at 30 rpm for 0.5 h to accelerate the separation of glycerol and glycerol esters. The glycerol was then separated from the glycerol esters, and the recovered glycerol was used as a raw material for a new glycerolysis reaction. The composition of the resulting glycerol esters is shown below.

[0071] Table 2. Composition of reaction products

[0072] diglycerides 41.86 monoglycerides 43.55 Triglycerides 14.59

[0073] Compared with the theoretical value, it can be seen that the reaction system has basically reached equilibrium after 6.5 hours of reaction.

[0074] The glycerides contain triglycerides, diglycerides, monoglycerides, and some fatty acids. Fatty acids and monoglycerides are removed by two-stage molecular distillation. The conditions for removing fatty acids by the first-stage molecular distillation are: distillation temperature 150℃, pressure 3 Pa, and condenser temperature 50℃. The conditions for removing monoglycerides by the second-stage molecular distillation are: distillation temperature 210℃ and pressure 1 Pa. The resulting product contains 73.65% diglycerides, 1.32% monoglycerides, and 25.51% triglycerides.

[0075] Example 2

[0076] The nonionic surfactant Tween 40 was dissolved in isopropanol at a concentration of 10 mg / L and thoroughly dispersed to obtain mixed solution 1. 10% (w / w) of immobilized lipase Lipozyme RM IM was added to mixed solution 1. The mixture was stirred at 300 rpm for 30 min at 25 °C and filtered to obtain the lipase. The surfactant-imprinted template on the immobilized lipase Lipozyme RM IM was eluted with the nonpolar solvent octane (30% of the volume of mixed solution 1). The lipase was then filtered and dried in a vacuum desiccator at room temperature for 12 h to obtain imprinted lipase. The activities of imprinted and unimprinted lipases are shown below.

[0077] Table 3. Comparison of activities of imprinted and non-imprinted lipases

[0078] Enzyme activity 33.7％ 20.5％ 64.4％

[0079] Rapeseed oil was added to a batch reactor and purged with nitrogen for protection. Simultaneously, imprinted lipase was added at a rate of 5 wt% of the rapeseed oil weight. The temperature was raised to 50°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 1:0.4 between the amount of glycerol pumped and the amount of rapeseed oil.

[0080] The glycerol was pumped in at three stages: the first stage involved a uniform pumping of 30% glycerol over 1 hour; the second stage involved pumping in 50% glycerol over 0.5 hours; and the third stage involved pumping in 20% glycerol over 1 hour. After the glycerol pumping was complete, the reaction continued for 6 hours. Lipase was then filtered to obtain a mixture of glycerides. The resulting glyceride mixture was heated to 80°C and slowly stirred at 20 rpm for 2 hours to accelerate the separation of glycerol and glycerides. The glycerol was then separated from the glycerides, and the recovered glycerol was used as a raw material for further glycerolysis reactions. The composition of the obtained glycerides is shown below.

[0081] Table 4. Composition of reaction products

[0082] diglycerides 30.52 monoglycerides 65.31 Triglycerides 4.17

[0083] Compared with the theoretical value, it can be seen that the reaction system has basically reached equilibrium after 8.5 hours of reaction.

[0084] Glycerides contain triglycerides, diglycerides, monoglycerides, and some fatty acids. Fatty acids and monoglycerides are removed by two-stage molecular distillation.

[0085] The conditions for primary molecular distillation to remove fatty acids were: distillation temperature 160℃, pressure 4 Pa, and condenser temperature 40℃; the conditions for secondary molecular distillation to remove monoglycerides were: distillation temperature 190℃ and pressure 2 Pa. The resulting product contained 86.98% diglycerides, 1.14% monoglycerides, and 11.88% triglycerides.

[0086] Example 3

[0087] The nonionic surfactant Tween 80 was dissolved in isopropanol at a concentration of 100 mg / L and thoroughly dispersed to obtain mixed solution 1. Immobilized lipase NS40086 (50% by mass) was added to mixed solution 1. The mixture was stirred at 100 rpm for 20 min at 25 °C and filtered to obtain the lipase. The surfactant-imprinted template on the immobilized lipase NS40086 was eluted with the nonpolar solvent n-hexane (60% of the volume of mixed solution 1). The lipase was then filtered and dried in a vacuum desiccator at room temperature for 16 h to obtain imprinted lipase. The activities of imprinted and unimprinted lipases are shown below.

[0088] Table 5. Comparison of activities of imprinted and non-imprinted lipases

[0089] Enzyme activity 31.4％ 18.3％ 71.6％

[0090] Sunflower seed oil was added to a batch reactor under nitrogen protection. Simultaneously, imprinted lipase was added at a rate of 15 wt% of the sunflower seed oil weight. The temperature was raised to 70°C, and stirring was initiated at 500 rpm. Food-grade glycerol was pumped into the reactor at a molar ratio of glycerol to sunflower seed oil of 1:4. The glycerol was pumped in three stages: 15% glycerol was pumped in at a uniform rate over 1 hour; 25% glycerol was pumped in over 0.5 hours; and 60% glycerol was pumped in over 1 hour. After the glycerol injection was complete, the reaction continued for 8 hours. The lipase was filtered to obtain a mixture of glycerides. The resulting glyceride mixture was heated to 85°C and slowly stirred at 10 rpm for 1.5 hours to accelerate the separation of glycerol and glycerides. The glycerol was then separated from the glycerides, and the recovered glycerol was used as a raw material for further glycerolysis reactions. The composition of the obtained glycerides is shown below.

[0091] Table 6. Composition of reaction products

[0092] diglycerides 39.21 monoglycerides 10.85 Triglycerides 49.94

[0093] Compared with the theoretical value, it can be seen that the reaction system has basically reached equilibrium after 10.5 hours of reaction.

[0094] The glycerides contain triglycerides, diglycerides, monoglycerides, and some fatty acids. Fatty acids and monoglycerides are removed by two-stage molecular distillation. The conditions for removing fatty acids by the first-stage molecular distillation are: distillation temperature 140℃, pressure 2 Pa, and condenser temperature 30℃. The conditions for removing monoglycerides by the second-stage molecular distillation are: distillation temperature 200℃ and pressure 3 Pa. The resulting product contains 43.56% diglycerides, 0.97% monoglycerides, and 55.48% triglycerides.

[0095] Example 4

[0096] The nonionic surfactant Tween 60 was dissolved in isopropanol at a concentration of 80 mg / L and thoroughly dispersed to obtain mixed solution 1. Immobilized lipase Lipozyme TL IM (30% by mass) was added to mixed solution 1. The mixture was stirred at 400 rpm for 10 min at 25 °C and filtered to obtain the lipase. The surfactant-imprinted template on the immobilized lipase Lipozyme TL IM was eluted with the nonpolar solvent n-hexane (80% of the volume of mixed solution 1). The lipase was then filtered and dried in a vacuum desiccator at room temperature for 8 h to remove the organic solvent, yielding imprinted lipase. The activities of imprinted and unimprinted lipases are shown below.

[0097] Table 7. Comparison of activities of imprinted and non-imprinted lipases

[0098] Enzyme activity (U) 25.3％ 14.6％ 73.3％

[0099] Flaxseed oil was added to a batch reactor under nitrogen protection. Simultaneously, imprinted lipase was added at a rate of 20 wt% of the flaxseed oil weight. The temperature was raised to 40°C, and stirring was initiated at 800 rpm. Food-grade glycerol was pumped into the reactor at a molar ratio of glycerol to flaxseed oil of 1:0.6. The glycerol was pumped in three stages: 10% glycerol was pumped in at a uniform rate over 1 hour; 40% glycerol was pumped in over 0.5 hours; and 50% glycerol was pumped in over 1 hour. After the glycerol injection was complete, the reaction continued for 3 hours. The lipase was filtered to obtain a mixture of glycerides. The resulting glyceride mixture was heated to 80°C and slowly stirred at 40 rpm for 1 hour to accelerate the separation of glycerol and glycerides. The glycerol was then separated from the glycerides, and the recovered glycerol was used as a raw material for further glycerolysis reactions. The composition of the obtained glycerides is shown below.

[0100] Table 8. Composition of reaction products

[0101] diglycerides 35.24 monoglycerides 56.78 Triglycerides 7.98

[0102] Compared with the theoretical value, it can be seen that the reaction system has basically reached equilibrium after 5.5 hours of reaction.

[0103] The glycerides contain triglycerides, diglycerides, monoglycerides, and some fatty acids. Fatty acids and monoglycerides are removed by two-stage molecular distillation. The conditions for removing fatty acids by the first-stage molecular distillation are: distillation temperature 160℃, pressure 5 Pa, and condenser temperature 40℃. The conditions for removing monoglycerides by the second-stage molecular distillation are: distillation temperature 190℃ and pressure 2 Pa. The resulting product contains 80.51% diglycerides, 1.26% monoglycerides, and 18.23% triglycerides.

[0104] Comparative Example 1

[0105] The imprinted lipase Novozym 435 obtained in Example 1 was used as the catalyst. Glycerol was added directly in one step instead of in stages. Other conditions were the same as in Example 1.

[0106] Soybean oil was added to a batch reactor, and food-grade glycerol was pumped into the reactor at a molar ratio of 1:1 to soybean oil. Nitrogen gas was introduced for protection. Imprinted lipase was added at a weight of 10 wt% of soybean oil. The temperature was raised to 60°C and stirring was started at 600 rpm. The reaction time was 6.5 h.

[0107] The resulting glycerol ester mixture was heated to 90°C and stirred slowly at 30 rpm for 0.5 h to accelerate the separation of glycerol and glycerol esters. The glycerol was then separated from the glycerol esters, and the recovered glycerol was used as a raw material for a new glycerolysis reaction. The composition of the resulting glycerol esters is shown below.

[0108] Table 9. Composition of reaction products

[0109] diglycerides 33.75 monoglycerides 29.48 Triglycerides 33.77

[0110] The comparison shows that after 6.5 hours of reaction, the triglyceride content in Comparative Example 1 is much higher than that in Example 1, indicating that the glycerolysis reaction has not reached equilibrium.

[0111] The main reason may be that excessive glycerol in the early stage led to excessive viscosity of the system, and the system was not fully mixed with oils, causing glycerol to adsorb onto the surface of lipase, thereby inhibiting the activity of the enzyme.

[0112] Comparative Example 2

[0113] The imprinted lipase Lipozyme RM IM obtained in Example 2 was used as the catalyst. Glycerol was added directly in one step instead of in stages. Other conditions were the same as in Example 2.

[0114] Rapeseed oil was added to a batch reactor and purged with nitrogen for protection. At the same time, food-grade glycerol was pumped into the reactor at a molar ratio of glycerol to rapeseed oil of 1:0.4. Imprinted lipase was added at a weight of 5 wt% of the rapeseed oil. The temperature was raised to 50°C and stirring was started at 800 rpm for 8.5 h.

[0115] The resulting glycerol ester mixture was heated to 80°C and stirred slowly at 20 rpm for 2 hours to accelerate the separation of glycerol and glycerol esters. The glycerol was then separated from the glycerol esters, and the recovered glycerol was used as a raw material for a new glycerolysis reaction. The composition of the obtained glycerol esters is shown below.

[0116] Table 10. Composition of reaction products

[0117] diglycerides 22.75 monoglycerides 46.03 Triglycerides 31.22

[0118] The comparative results above show that staged addition of glycerol can significantly improve the reaction efficiency of the reaction system. When glycerol is added all at once, most of it remains in a free state due to the poor miscibility between glycerol and oils. Higher concentrations of added glycerol will increase the viscosity of the system, reduce mass transfer, and cause glycerol to coat the enzyme surface, thus reducing enzyme activity.

[0119] By adding glycerol in stages and controlling the glycerol infusion rate at each stage, the glycerol is injected more slowly in the early stages and more quickly in the later stages. In the early stages, due to the small amount of glycerol injected, the glycerol and oil are fully dissolved under stirring conditions. During the reaction, diglycerides and monoglycerides are continuously generated, which further increases the polarity of the system and increases the solubility of glycerol in the system. In the later stages, the injection of glycerol is accelerated. Due to the presence of diglycerides and monoglycerides in the system, the injected glycerol can quickly dissolve with the oil, thereby improving the reaction efficiency and avoiding a large amount of glycerol covering the enzyme surface, which would cause enzyme activity loss.

[0120] Therefore, by injecting glycerol in stages, the reaction efficiency can be significantly improved, allowing the glycerol hydrolysis reaction to reach equilibrium more quickly.

[0121] Comparative Example 3

[0122] Referring to Example 1, in the segmented addition of glycerol, the amount of glycerol added at different stages was changed, while other conditions remained unchanged.

[0123] The glycerol was pumped in at three stages: the first stage involved a uniform pumping of 5% glycerol over 1 hour; the second stage involved pumping in 20% glycerol over 0.5 hours; and the third stage involved pumping in 75% glycerol over 0.5 hours. After the glycerol pumping was complete, the reaction continued for 5 hours. The lipase was then filtered to obtain a mixture of glycerides. The final composition of the reactants is shown in the table below.

[0124] Table 11. Composition of reaction products

[0125] diglycerides 40.03 monoglycerides 39.73 Triglycerides 20.24

[0126] The triglyceride content in Comparative Example 3 was higher than that in Example 1, indicating that the triglycerides did not react sufficiently, which means that the reaction in Comparative Example 3 did not reach equilibrium after the same reaction time.

[0127] Because the amount of glycerol added in the early stage was small and the amount of glycerol added in the later stage was excessive, the glycerol did not react completely in the system. As a result, some glycerol covered the surface of the lipase, causing the lipase to lose some activity and ultimately reducing the reaction efficiency.

[0128] Comparative Example 4

[0129] Referring to Example 1, in the segmented addition of glycerol, the amount of glycerol added at different stages was changed, while other conditions remained unchanged.

[0130] The glycerol was pumped in at three stages: the first stage involved a uniform pumping of 40% glycerol over 1 hour; the second stage involved pumping in 55% glycerol over 0.5 hours; and the third stage involved pumping in 5% glycerol over 0.5 hours. After the glycerol pumping was complete, the reaction continued for 5 hours. The lipase was then filtered to obtain a mixture of glycerol esters. The final composition of the reactants is shown in the table below.

[0131] Table 12. Composition of reaction products

[0132] diglycerides 39.03 monoglycerides 37.73 Triglycerides 23.24

[0133] The triglyceride content in Comparative Example 4 was higher than that in Example 1, indicating that the triglycerides did not react sufficiently, which means that the reaction in Comparative Example 4 did not reach equilibrium after the same reaction time.

[0134] Because too much glycerol was added in the early stage, the glycerol and oil could not be completely mixed. At the same time, the excess glycerol did not react fully with the oil and covered the surface of the lipase, which eventually led to the partial loss of lipase activity and reduced reaction efficiency.

[0135] Comparative Example 5

[0136] Referring to Example 2, instead of isopropanol, other solvents such as ethanol, methanol, n-hexane, and octane were used to dissolve the lipase. All other conditions remained unchanged. The activity of the lipase Lipozyme RM IM was then tested, and all other conditions were the same as in Example 2.

[0137] Table 13. Comparison of activities of lipases imprinted with different solvents

[0138] methanol 16.1％ 20.50％ -21.5％ ethanol 23.4％ 20.50％ 14.1％ Butanol 30.6％ 20.50％ 49.3％ n-Hexane 25.3％ 20.50％ 23.4％ Octane 24.7％ 20.50％ 20.5％ Example 2 33.70％ 20.50％ 64.4％

[0139] It can be seen that using nonionic surfactants dissolved in isopropanol as imprint templates to enhance lipase activity through bioimprinting is more effective.

[0140] Comparative Example 6

[0141] Referring to Example 3, multiple batches of the lipase NS40086, both imprinted and non-imprinted, were used to compare the activities of different lipases in different batches, thereby comparing their catalytic stability. Other conditions were the same as in Example 3.

[0142] 1 31.4％ 18.3％ 2 30.6％ 17.7％ 3 30.1％ 16.9％ 4 29.5％ 16.1％ 5 28.7％ 15.4％ 6 27.4％ 14.2％ 7 26.8％ 13.4％ 8 26.2％ 11.7％ 9 25.5％ 10.2％ 10 24.1％ 9.3％ Enzyme inactivation rate 23.2％ 49.2％

[0143] It can be seen that using nonionic surfactants dissolved in isopropanol as imprint templates to improve the catalytic stability of lipases through bioimprinting is more effective.

[0144] Isopropanol possesses suitable polarity, allowing it to effectively dissolve nonionic surfactants without stripping the lipase surface of essential water, thus maintaining lipase activity. Furthermore, the trace amounts of water in isopropanol help maintain the lipase's flexible conformation. In this system, the nonionic surfactant initially coats the lipase surface, interacting with it through hydrophobic interactions: the polar head interacts with the lipase's hydrophilic groups, and the hydrophobic head interacts with the lipase's hydrophobic groups. Since lipase itself is water-soluble, indicating a high proportion of hydrophilic groups on its surface, this hydrophobic interaction transforms the lipase surface from hydrophilic to hydrophobic, making it more readily interactable with hydrophobic substances in the system. This, to some extent, prevents glycerol from coating the lipase surface and further avoids excessive contact between polar substances and the lipase, which could lead to lipase degradation. The loss of active water and the resulting alteration of the catalytic structure can effectively enhance the catalytic stability of lipases. This hydrophobic interaction is relatively weak; unlike the electrostatic interaction between ionic surfactants and lipases, this hydrophobic interaction does not alter the conformation of the lipase, thus not affecting its activity. Simultaneously, due to the interfacial activation effect of lipases, the interaction between nonionic surfactants and the cap of the lipase's active center causes the cap to open, removing water and maintaining the lipase's active catalytic conformation, thereby further enhancing its catalytic activity and improving reaction efficiency.

[0145] 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 preparing a diglyceride edible oil, characterized in that: include, A nonionic surfactant-imprinted lipase dissolved in isopropanol was used as a catalyst to catalyze the glycerol hydrolysis reaction of glycerol and edible oils, and glycerol was injected in stages. Two-stage molecular distillation is used to remove monoglycerides and fatty acids to obtain diglyceride edible oil; The staged injection of glycerol includes pumping the reaction system in three stages; wherein, the first stage is to pump 10-30% of glycerol at a constant rate over 1 hour, the second stage is to pump 25-50% of glycerol over 0.5 hours, and the third stage is to pump 20-60% of glycerol over 1 hour. After the glycerol is pumped in, the reaction continues for 3-8 hours, and the lipase is filtered to obtain a mixture of glycerides. The preparation method of the nonionic surfactant-imprinted lipase includes: dissolving a surfactant in isopropanol at a concentration of 10-100 mg / L and dispersing it thoroughly to obtain a mixed solution 1; adding 10-50% by mass of commercially immobilized lipase to the mixed solution 1; stirring the mixture at 100-400 rpm for 10-60 min at 25°C; filtering to obtain the lipase; eluting the surfactant-imprinted template on the immobilized lipase with a nonpolar solvent such as n-hexane or octane, with the solvent amount being 30-80% of the mixed solution 1; then filtering the lipase and drying it in a vacuum desiccator at room temperature for 8-24 h to remove the organic solvent, thereby obtaining the imprinted lipase; The surfactant is Tween 20, Tween 40, Tween 60 or Tween 80; the commercially available immobilized lipase is Novozym 435, Lipozyme RM IM, Lipozyme TL IM or NS 40086.

2. The preparation method according to claim 1, characterized in that: The glycerol hydrolysis reaction is carried out in the following manner: the amount of imprinted lipase added is 5-20 wt% of the weight of the oil, the reaction temperature is 40-70℃, the stirring speed is 500-800 rpm, and the molar ratio of glycerol to oil is 1:0.4-4.

3. The preparation method according to claim 1, characterized in that: The glycerol hydrolysis reaction involves oils including soybean oil, rapeseed oil, sunflower seed oil, flaxseed oil, peanut oil, olive oil, and tea seed oil.

4. The preparation method according to claim 1, characterized in that: It also includes heating the glycerol ester mixture obtained from the reaction to 80~90℃, stirring slowly at a speed of 10~40 rpm for 0.5~2h to accelerate the separation of glycerol and glycerol esters, and recovering the glycerol as a raw material for new glycerolysis reactions.

5. The preparation method according to claim 1, characterized in that: The process involves removing fatty acids and monoglycerides through two-stage molecular distillation to obtain diglyceride edible oil, wherein the diglyceride content is greater than 40%.

6. The preparation method according to claim 1 or 5, characterized in that: The conditions for removing fatty acids by primary molecular distillation are: distillation temperature of 150℃, pressure of 3Pa, and condenser temperature of 50℃. The conditions for removing monoglycerides by secondary molecular distillation are: distillation temperature of 210℃ and pressure of 1 Pa.

7. Diglyceride edible oil prepared by any of the preparation methods described in claims 1 to 6.

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