A method for the segmented production of a diglyceride edible oil and products thereof

Abstract

This invention discloses a method for preparing diglyceride edible oil in stages and the resulting product. Addressing the poor compatibility between glycerol and oils, and the problem that excessive glycerol easily adsorbs onto enzyme surfaces and affects enzyme activity, this invention improves the solubility of glycerol in the reaction system by adding monoglycerides to the reaction system and by adding glycerol in stages combined with high-pressure homogenization. Utilizing the characteristic that the metaglycerol lipase Lipase G50 is active only for metaglycerols, including monoglycerides and diglycerides, Lipase G50 is used as a catalyst to induce esterification of monoglycerides with fatty acids in the glycerol ester reaction product under vacuum conditions, thereby increasing the yield of diglycerides. Simultaneously, only fatty acids need to be removed during the molecular distillation stage, thus reducing the content of harmful substances.

Description

Technical Field

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

[0002] Diacylglycerol (DAG) is an ester formed when a fatty acid in the triglyceride (TAG) backbone is replaced by a hydroxyl group. Diacylglycerols are found in nature primarily through several sources: First, they exist naturally in some common edible oils, but the mass fraction is generally no more than 10%, with cottonseed oil, palm oil, and olive oil being the most abundant, and their relative content depending on the properties of the oilseeds. Second, small amounts of diglycerol are also produced during the hydrolysis of oils and fats during processing, storage, and transportation. Additionally, they are also produced in very small amounts during the digestion of oils and fats in the human body and during the incomplete synthesis of triglycerides and phospholipids in plants. Therefore, currently, diglycerols are mainly obtained through modification techniques in the oil and fat industry and are a type of structural lipid.

[0003] Based on the different positions of the acyl groups in their molecular structure, diglycerides can be divided into 1,3-diglycerides and 1,2(2,3)-diglycerides. Under normal conditions, 1,3-diglycerides account for about 70% of the total mass. Structurally, diglycerides contain two long-chain fatty acids, exhibiting good lipophilicity, and also have a hydroxyl group, giving them some hydrophilicity. In terms of appearance and flavor, diglycerides are pale yellow, have a mild taste, and are no different from triglycerides, so they can be used in cooking, frying, and baking like ordinary cooking oils. In terms of efficacy, the unique structure of diglycerides causes their digestion and metabolism in the body to differ from that of triglycerides. Diglycerides have the effects of inhibiting the rise of serum triglycerides, preventing the accumulation of fat in the body, increasing β-oxidation, and reducing weight. These functions are all related to the unique digestive and metabolic pathways of diglycerides in the body, which differ from those of triglycerides.

[0004] After entering the human body, conventional triglycerides are first hydrolyzed by pancreatic lipases in the intestinal lumen into 2-monoglyceride and two free fatty acids. Subsequently, in the small intestinal epithelial cells, 2-monoglyceride and most of the free fatty acids are re-esterified into triglycerides under the action of relevant enzymes via the 2-monoglyceride pathway and the α-glycerophosphate pathway, respectively. Then, under the action of microsomal transport proteins, they reach the liver in the form of chylomicrons via the lymphatic and circulatory systems. Part of this triglyceride provides energy for the body, while another part is stored as body fat. A small portion of the free fatty acids also undergoes further degradation. The 1,3-diglyceride enters the liver via the portal vein and is used for energy through β-oxidation. The products of 1,3-diglyceride breakdown in the intestinal lumen are 1(3) monoglyceride and one free fatty acid. 1(3) monoglyceride cannot be resynthesized into triglycerides via the 2-monoglyceride pathway; instead, it travels to the liver via the portal vein along with some free fatty acids, where it is used for energy through β-oxidation in the mitochondria of hepatocytes. The remaining free fatty acids, although they can be synthesized into triglycerides via the α-glycerophosphate pathway, cannot form chylomicrons but are stored in the small intestinal epithelial cells to supply energy. Therefore, diglycerides have a higher utilization rate in the body and are less likely to be resynthesized into fat, thus helping to lower blood lipid levels. The caloric value and digestibility of diglycerides are roughly equivalent to those of triglycerides. Therefore, replacing some conventional fats with diglycerides in the daily diet can gradually reduce fat accumulation while maintaining energy supply, thus aiding in weight loss.

[0005] Currently, the main method for synthesizing diglycerides is enzymatic glycerolysis, which utilizes lipase to catalyze the glycerolysis reaction between edible oils and glycerol to obtain a mixture of glycerides rich in monoglycerides and diglycerides. Monoglycerides are then removed by molecular distillation to obtain a high-purity diglyceride product. However, problems exist: the solubility of glycerol and oils in ordinary glycerolysis reaction systems is low, resulting in low reaction efficiency and difficulty in reaching equilibrium. Furthermore, the molecular distillation conditions used to remove monoglycerides are too harsh, leading to the formation of a large amount of glycidyl esters and chloropropanol esters in the product. Simultaneously, because monoglycerides are a byproduct of the reaction and are expelled through molecular distillation, the yield of diglycerides is low.

[0006] Therefore, improving the compatibility of glycerol with oils, increasing reaction efficiency, and enhancing the purity of diglycerides during the reaction process are urgent problems that need to be solved. Summary of the Invention

[0007] 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.

[0008] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0009] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing diglyceride edible oil in stages.

[0010] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a method for preparing diglyceride edible oil in stages, comprising,

[0011] Using lipase imprinted with a nonionic surfactant dissolved in isopropanol as a catalyst, and adding monoglycerides as a solubilizer, the compatibility of glycerol and oil is improved by adding glycerol in stages and homogenizing under high pressure. The glycerolysis reaction yields the glycerolysis product.

[0012] Glycerol and oil are separated in the glycerol hydrolysis product. Free fatty acids are added to the glycerol hydrolysis product after glycerol separation, and the reaction is carried out under vacuum with Lipase G50 as a catalyst to esterify monoglycerides and fatty acids in the system.

[0013] Molecular distillation is used to remove fatty acids, thereby obtaining high-purity diglyceride products.

[0014] As a preferred embodiment of the method described in this invention, the process of segmented addition of glycerol and high-pressure homogenization to improve compatibility is as follows:

[0015] In the first stage, add 10-30% glycerol, homogenize at 20-40 MPa for 2-5 minutes, add imprinted lipase, stir at 500-800 rpm, react for 0.5-1.5 hours, and filter out the lipase.

[0016] In the second stage, add 30-50% glycerol, homogenize at 20-40 MPa for 2-5 minutes with a stirring speed of 500-800 rpm, add lipase again and react for 0.5-1.5 hours, then filter out the lipase.

[0017] In the third stage, add 30-60% glycerol, stir at 600-800 rpm, and add lipase to react for 3-5 hours.

[0018] In a preferred embodiment of the method described in this invention, the amount of monoglyceride added in the glycerolysis reaction is 0.5% to 1.5% of the substrate by mass.

[0019] In a preferred embodiment of the method described in this invention, the amount of imprinted lipase added in the glycerol hydrolysis reaction is 8-20 wt%, the reaction temperature is 50-70°C, and the molar ratio of glycerol to oil is 1:0.4-3.

[0020] As a preferred embodiment of the method described in this invention, the oil includes, but is not limited to, any one of sunflower seed oil, walnut oil, and olive oil.

[0021] In a preferred embodiment of the method described in this invention, after the glycerol hydrolysis reaction is completed, the method further includes:

[0022] Heat the temperature to 85-95℃, stir slowly at 20-50 rpm for 0.5-1.5 hours to accelerate the separation of glycerol and glycerides.

[0023] In a preferred embodiment of the method described in this invention, the esterification reaction is carried out in a ratio of fatty acid addition to glycerol backbone in the glycerol ester product of 1 to 3:1, the amount of Lipase G50 added is 3 to 8 wt% of the substrate weight, the reaction temperature is 30 to 50°C, the vacuum degree is 10 to 30 mbar, and the reaction time is 12 to 24 h.

[0024] In a preferred embodiment of the method described in this invention, the imprinted lipase is prepared by a method comprising:

[0025] A nonionic surfactant is dissolved in isopropanol at a concentration of 10–100 mg / L to obtain a mixed solution;

[0026] Add 20-50% (w / w) of immobilized lipase to the mixed solution, stir the mixture at 100-300 rpm for 20-50 min at 25°C, and filter to obtain the lipase.

[0027] The immobilized lipase was eluted with a nonpolar solvent, the solvent volume being 20-50% of the volume of the mixed solution. The lipase was then filtered and dried under vacuum to obtain the imprinted lipase.

[0028] In a preferred embodiment of the method described in this invention, the nonionic surfactant includes Tween 20, Tween 40, and Tween 60; the lipase includes, but is not limited to, Lipozyme RM IM, Novozyme 435, and NS40086.

[0029] Another objective of this invention is to overcome the shortcomings of the prior art and provide a method for preparing diglyceride edible oil in stages, wherein the diglyceride edible oil obtained has a glycidyl ester content of less than 1.6 mg / kg and a chloropropanol ester content of less than 0.6 mg / kg.

[0030] Beneficial effects of this invention:

[0031] (1) In view of the poor compatibility between glycerol and oils and the problem that excessive glycerol is easily adsorbed on the enzyme surface and affects enzyme activity, this invention improves the solubility of glycerol in the reaction system by adding monoglycerides to the reaction system and by adding glycerol in stages and mixing under high pressure homogenization, thereby further improving the reaction efficiency; taking advantage of the characteristic that the lipase Lipase G50 is only active for glycerol esters, including monoglycerides and diglycerides, Lipase G50 is used as a catalyst to make the monoglycerides in the glycerol ester reaction product undergo esterification reaction with fatty acids under vacuum conditions, thereby improving the yield of diglycerides. At the same time, only fatty acids need to be removed in the molecular distillation stage, thereby reducing the content of harmful substances;

[0032] (2) In this invention, imprinted lipase, a nonionic surfactant dissolved in isopropanol, is used as a catalyst. Isopropanol has appropriate polarity and has a good dissolving effect on nonionic surfactants, with minimal impact on enzyme activity. This is more conducive to the interaction between nonionic surfactants and lipases. The surfactant opens the cap of the active center of lipase, thereby improving lipase activity. At the same time, the nonionic surfactant interacts with the surface of lipase hydrophobically, changing the surface properties of lipase and making the surface of lipase change from hydrophilic to hydrophobic. On the one hand, this avoids excessive adsorption of glycerol, which leads to a decrease in lipase activity. On the other hand, it avoids lipase dehydration caused by excessive contact between lipase and polar substances in the system, thereby protecting the catalytic structure of lipase and improving the catalytic stability of lipase. Detailed Implementation

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038]

[0039] 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:

[0040]

[0041] The content of monoglycerides (sn-1 monoglyceride + sn-2 monoglyceride + sn-3 monoglyceride) can be calculated as follows:

[0042]

[0043] The triglyceride content can be calculated as follows:

[0044]

[0045] The remaining glycerol content can be calculated as follows:

[0046]

[0047] 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.

[0048]

[0049] 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:

[0050]

[0051] The content of monoglycerides (sn-1 monoglyceride + sn-2 monoglyceride + sn-3 monoglyceride) can be calculated as follows:

[0052]

[0053] The triglyceride content can be calculated as follows:

[0054]

[0055] The remaining glycerol content can be calculated as follows:

[0056]

[0057] 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.

[0058] Table 1. Composition of products at equilibrium of enzyme-catalyzed glycerol hydrolysis

[0059] Product composition Proportion 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 ]]> 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 ]]>

[0060] 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.

[0061] Table 2. Product composition (%) at different substrate ratios

[0062] r value DAG MAG DAG+MAG TAG G 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

[0063] 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.

[0064] Table 3. Composition of products after removal of glycerol and monoglycerides

[0065]

[0066] DAG: diglyceride; MAG: monoglyceride; TAG: triglyceride

[0067] Determination of lipase glycerol hydrolysis activity: The catalytic activity of lipase was evaluated by performing a glycerol hydrolysis reaction between soybean oil and glycerol.

[0068] The reaction substrate was a mixture of soybean oil and glycerol in 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 a temperature of 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.

[0069] Example 1

[0070] The nonionic surfactant Tween 40 was dissolved in isopropanol at a concentration of 10 mg / L. Isopropanol has better solubility for surfactants and can better disperse them. After thorough dispersion, a mixed solution 1 was obtained. 20% (w / w) of immobilized lipase Lipozyme RM IM was added to mixed solution 1. The mixture was stirred at 100 rpm for 30 min at 25 °C and filtered to obtain the lipase. Excess surfactant imprinted template on the immobilized lipase Lipozyme RMIM was eluted with the nonpolar solvent octane. The amount of n-hexane used was 20% of mixed solution 1. The lipase was then filtered and dried in a vacuum desiccator at room temperature for 8 h to remove organic solvents, yielding the imprinted lipase.

[0071] The activities of imprinted and non-imprinted lipases are shown below.

[0072] Table 4. Comparison of activities of imprinted and non-imprinted lipases

[0073] Lipase Imprint LipozymeRMIM Non-imprinted LipozymeRMIM Increase in vitality (%) Enzyme activity 34.3％ 21.6％ 58.8％

[0074] Sunflower seed oil was added to a batch reactor, nitrogen gas was introduced for protection, the temperature was raised to 60°C, 0.5% monoglyceride was added, and the mixture was stirred to fully dissolve the monoglyceride in the sunflower seed oil. Glycerin was added in three stages according to a molar ratio of glycerol to sunflower seed oil of 1:3.

[0075] In the first stage, add 10% glycerol and homogenize at 20 MPa for 5 minutes to fully mix the glycerol with the sunflower seed oil. Add imprinted lipase at a weight of 8 wt% of the sunflower seed oil and start stirring at 600 rpm. React for 1 hour and then filter out the lipase.

[0076] In the second stage, 30% glycerol was added, homogenized at 20 MPa for 5 minutes, and then lipase was added again for 1 hour of reaction with stirring at 600 rpm. The lipase was then filtered out.

[0077] In the third stage, 60% glycerol was added, and lipase was added to react for 3 hours with a stirring speed of 600 rpm.

[0078] After the reaction was complete, the system temperature was heated to 90℃, and the mixture was slowly stirred at 20 rpm for 1 hour to accelerate the separation of glycerol and glycerides. The glycerol and glycerides were then separated to obtain the glyceride product, and the recovered glycerol was used as a raw material for a new glycerolysis reaction. The obtained glycerides contained 11.4% monoglycerides, 41.2% diglycerides, and 47.4% triglycerides.

[0079] Compared with the theoretical value, it can be seen that the reaction system has basically reached equilibrium after 5 hours of reaction.

[0080] Free fatty acids from sunflower seed oil were added to the glycerol ester product at a ratio of 1:1 to the glycerol backbone in the glycerol ester product. Lipase G50 was added to catalyze the reaction of monoglycerides and fatty acids in the glycerol ester product to form diglycerides. The amount of Lipase G50 added was 3 wt% of the substrate weight. The reaction was carried out at 50°C under a vacuum of 10 mbar for 12 h. Fatty acids were removed by molecular distillation at 160°C, 3 Pa, and 30°C. The resulting product contained 0.9% monoglyceride, 52.5% diglyceride, 46.6% triglyceride, and a diglyceride yield of 52.5%. The content of glycidyl esters was 1.55 mg / kg, and the content of chloropropanol esters was 0.42 mg / kg.

[0081] Example 2

[0082] The nonionic surfactant Tween 20 was dissolved in isopropanol at a concentration of 50 mg / L. Isopropanol has better solubility for surfactants and can better disperse them. After thorough dispersion, a mixed solution 1 was obtained. Immobilized lipase Novozym 435 at a concentration of 30% was added to mixed solution 1. The mixture was stirred at 300 rpm for 20 min at 25 °C and filtered to obtain the lipase. Excess surfactant imprinted template on the immobilized lipase Novozym 435 was eluted with the nonpolar solvent n-hexane. The amount of n-hexane used was 50% of the mixed solution 1. The lipase was then filtered and dried in a vacuum desiccator at room temperature for 24 h to remove organic solvents, thus obtaining the imprinted lipase.

[0083] The activities of imprinted and non-imprinted lipases are shown below.

[0084] Table 5. Comparison of activities of imprinted and non-imprinted lipases

[0085] Lipase Imprint Novozym435 Novozym435 (non-imprint) Increase in vitality (%) Enzyme activity 39.4％ 23.5％ 67.7％

[0086] Walnut oil was added to a batch reactor, which was then purged with nitrogen for protection. The temperature was raised to 70°C, and 0.8% monoglyceride was added. The mixture was stirred until the monoglyceride was fully dissolved in the walnut oil. Glycerin was added in three stages at a molar ratio of 1:0.8 for glycerol to walnut oil. In the first stage, 20% glycerol was added, and the mixture was homogenized at 30 MPa for 3 minutes to ensure thorough mixing with the walnut oil. Imprinted lipase was then added at a rate of 10 wt% of the walnut oil weight, and stirring was initiated at 800 rpm. The reaction was carried out for 0.5 hours, after which the lipase was filtered out. In the second stage, 50% glycerol was added, and the mixture was homogenized at 30 MPa for 3 minutes. The lipase was reintroduced, and the reaction was carried out for 0.5 hours at 800 rpm. The lipase was then filtered out. In the third stage, 30% glycerol (based on the molar ratio of walnut oil) was added, and the lipase was introduced. The reaction was carried out for 5 hours at 800 rpm. After the reaction was complete, the system temperature was heated to 95℃, and the mixture was slowly stirred at 30 rpm for 0.5 h to accelerate the separation of glycerol and glycerides. The glycerol and glycerides were then separated to obtain the glyceride product, and the recovered glycerol was used as a raw material for a new glycerolysis reaction. The obtained glycerides contained 45.9% monoglycerides, 41.5% diglycerides, and 12.6% triglycerides.

[0087] Compared with the theoretical value, it can be seen that the reaction system has basically reached equilibrium after 6 hours of reaction.

[0088] Walnut oil free fatty acids were added to the glycerol ester product at a ratio of 2:1 to the glycerol backbone in the glycerol ester product. Lipase G50 was added to catalyze the reaction of monoglycerides and fatty acids in the glycerol ester product to form diglycerides. The amount of Lipase G50 added was 5 wt% of the substrate weight. The reaction was carried out at 40°C under a vacuum of 20 mbar for 16 h. Fatty acids were removed by molecular distillation at 160°C, 3 Pa, and 30°C. The resulting product contained 0.7% monoglyceride, 86.5% diglyceride, 12.8% triglyceride, and a diglyceride yield of 86.5%. The glycidyl ester content was 1.46 mg / kg, and the chloropropanol ester content was 0.47 mg / kg.

[0089] Example 3

[0090] The nonionic surfactant Tween 60 was dissolved in isopropanol at a concentration of 100 mg / L. Isopropanol has better solubility for surfactants and can better disperse surfactants. After sufficient dispersion, mixed solution 1 was obtained.

[0091] Add 50% by mass of immobilized lipase NS40086 to mixed solution 1, stir the mixture at 200 rpm for 50 min at 25 °C, and filter to obtain lipase.

[0092] Excess surfactant-imprinted template on immobilized lipase NS40086 was eluted with the nonpolar solvent n-hexane. The amount of n-hexane used was 40% of mixed solution 1. The lipase was then filtered and dried in a vacuum desiccator at room temperature for 12 h to remove organic solvent, thus obtaining imprinted lipase.

[0093] The activities of imprinted and non-imprinted lipases are shown below.

[0094] Table 6. Comparison of activities of imprinted and non-imprinted lipases

[0095] Lipase Imprint NS40086 Non-imprinted NS40086 Increase in vitality (%) Enzyme activity 32.9％ 20.8％ 58.2％

[0096] Olive oil was added to the batch reactor, nitrogen gas was introduced for protection, the temperature was raised to 50°C, 1.5% monoglyceride was added, and the mixture was stirred to fully dissolve the monoglyceride in the olive oil. Glycerin was added in three stages according to a molar ratio of glycerin to olive oil of 1:0.4.

[0097] In the first stage, add 30% glycerol and homogenize at 40 MPa for 2 minutes to fully mix the glycerol with the olive oil. Add imprinted lipase at a rate of 20 wt% of the weight of the olive oil and start stirring at 500 rpm. React for 1.5 hours and then filter out the lipase.

[0098] In the second stage, 40% glycerol was added, and the mixture was homogenized at 40 MPa for 2 minutes. The lipase was then added back in and reacted for 1.5 hours with a stirring speed of 500 rpm. The lipase was then filtered out.

[0099] In the third stage, 30% glycerol was added, and lipase was introduced for a reaction at 500 rpm for 4 hours. After the reaction, the system temperature was heated to 85°C, and the mixture was slowly stirred at 50 rpm for 1.5 hours to accelerate the separation of glycerol and glycerides. The glycerol and glycerides were then separated to obtain the glyceride product, and the recovered glycerol was used as a raw material for a new glycerolysis reaction. The resulting glycerides contained 63.8% monoglycerides, 30.7% diglycerides, and 5.5% triglycerides.

[0100] Compared with the theoretical value, it can be seen that the reaction system has basically reached equilibrium after 7 hours of reaction.

[0101] Free fatty acids from olive oil were added to the glycerol ester product at a ratio of 3:1 to the glycerol backbone in the glycerol ester product. Lipase G50 was added to catalyze the reaction of monoglycerides and fatty acids in the glycerol ester product to form diglycerides. The amount of Lipase G50 added was 8 wt% of the substrate weight. The reaction was carried out at 30°C under a vacuum of 30 mbar for 24 hours. Fatty acids were removed by molecular distillation at 160°C, 3 Pa, and 30°C. The resulting product contained 1.1% monoglyceride, 92.8% diglyceride, 6.1% triglyceride, and a diglyceride yield of 92.8%. The glycidyl ester content was 1.51 mg / kg, and the chloropropanol ester content was 0.52 mg / kg.

[0102] Comparative Example 1

[0103] A control experiment was conducted following the reaction in Example 1, with the addition of monoglycerides and the addition of glycerol in stages, followed by homogenization.

[0104] Sunflower seed oil was added to the batch reactor, nitrogen gas was introduced for protection, the temperature was raised to 60°C, glycerol was added at a molar ratio of 1:3 to sunflower seed oil, imprinted lipase was added at a weight of 8 wt% of sunflower seed oil, and stirring was started at a speed of 600 rpm for 5 hours.

[0105] After the reaction was completed, the system temperature was heated to 90℃ and stirred slowly at 20 rpm for 1 hour to accelerate the separation of glycerol and glycerides. The glycerol was then recovered and used as a raw material for a new glycerolysis reaction.

[0106] The composition of the obtained glycerides is shown in Table 7.

[0107] Comparative Example 2

[0108] Following the reaction in Example 2, a control experiment was conducted with the addition of monoglycerides and the addition of glycerol in stages, followed by homogenization.

[0109] Walnut oil was added to a batch reactor, nitrogen gas was introduced for protection, the temperature was raised to 70°C, glycerol was added at a molar ratio of glycerol to walnut oil of 1:0.8, imprinted lipase was added at a weight of 10 wt% of walnut oil, and stirring was started at 800 rpm for 6 hours.

[0110] After the reaction was complete, the system temperature was heated to 95℃, and the mixture was slowly stirred at 30 rpm for 0.5 h to accelerate the separation of glycerol and glycerides. The glycerol was then recovered and used as a raw material for a new glycerolysis reaction. The composition of the obtained glycerides is shown in Table 7.

[0111] Comparative Example 3

[0112] A control experiment was conducted following the reaction in Example 3, with the addition of monoglycerides and the addition of glycerol in stages, followed by homogenization.

[0113] Olive oil was added to the batch reactor, nitrogen gas was introduced for protection, the temperature was raised to 50°C, glycerol was added at a molar ratio of glycerol to walnut oil of 1:0.4, imprinted lipase was added at a weight of 20 wt% of sunflower seed oil, and stirring was started at a speed of 500 rpm for 7 hours.

[0114] After the reaction was complete, the system temperature was heated to 85℃, and the mixture was slowly stirred at 50 rpm for 1.5 h to accelerate the separation of glycerol and glycerides. The glycerol was then recovered and used as a raw material for a new glycerolysis reaction. The composition of the obtained glycerides is shown in Table 7.

[0115] Table 7. Glyceryl ester content in Comparative Examples 1-3

[0116] content(%) Comparative Example 1 Comparative Example 2 Comparative Example 3 monoglycerides 9.5 34.3 44.4 diglycerides 31.1 38.9 36.8 Triglycerides 59.4 26.7 18.8

[0117] Judging from the triglyceride content in Comparative Examples 1-3, after the reaction, the content was far from reaching equilibrium compared to the theoretical value.

[0118] Comparative Example 4

[0119] Referring to the reaction conditions in Example 3, instead of using lipase G50 for esterification, the fatty acids were directly removed by molecular distillation. After removing the fatty acids by molecular distillation, monoglycerides were removed by secondary molecular distillation.

[0120] The nonionic surfactant Tween 60 was dissolved in isopropanol at a concentration of 100 mg / L. Isopropanol has better solubility for surfactants and can better disperse surfactants. After sufficient dispersion, mixed solution 1 was obtained.

[0121] Add 50% by mass of immobilized lipase NS40086 to mixed solution 1, stir the mixture at 200 rpm for 50 min at 25 °C, and filter to obtain lipase.

[0122] The surfactant-imprinted template on the immobilized lipase NS40086 was eluted with the nonpolar solvent n-hexane. The amount of n-hexane used was 40% of the mixed solution 1. The lipase was then filtered and dried in a vacuum desiccator at room temperature for 12 h to remove the organic solvent, thus obtaining the imprinted lipase.

[0123] Olive oil was added to the batch reactor, nitrogen gas was introduced for protection, the temperature was raised to 50°C, 1.5% monoglyceride was added, and the mixture was stirred to fully dissolve the monoglyceride in the olive oil. Glycerin was added in three stages according to a molar ratio of glycerin to olive oil of 1:0.4.

[0124] In the first stage, add 30% glycerol and homogenize at 40 MPa for 2 minutes to fully mix the glycerol with the olive oil. Add imprinted lipase at a rate of 20 wt% of the weight of the olive oil and start stirring at 500 rpm. React for 1.5 hours and then filter out the lipase.

[0125] In the second stage, 40% glycerol was added, and the mixture was homogenized at 40 MPa for 2 minutes. The lipase was then added back in and reacted for 1.5 hours with a stirring speed of 500 rpm. The lipase was then filtered out.

[0126] In the third stage, 30% glycerol was added, and lipase was introduced to react for 4 hours at a stirring rate of 500 rpm. After the reaction was completed, the system temperature was heated to 85°C, and the mixture was slowly stirred at 50 rpm for 1.5 hours to accelerate the separation of glycerol and glycerides. The glycerol and glycerides were then separated to obtain the glyceride product, and the glycerol was recovered and used as a raw material for a new glycerolysis reaction.

[0127] The obtained glycerides contained 63.8% monoglycerides, 30.7% diglycerides, and 5.6% triglycerides. Fatty acids were removed by molecular distillation at 160°C, 3 Pa, and 30°C. The resulting glycerides contained 62.6% monoglycerides, 31.5% diglycerides, and 5.9% triglycerides, with a diglyceride yield of 31.5%.

[0128] Two-stage molecular distillation was used to remove monoglycerides. The distillation conditions were: distillation temperature 200℃, pressure 2Pa, and condenser temperature 25℃. The glyceride composition of the product was: monoglyceride 0.8%, diglyceride 86.3%, triglyceride 15.6%, and the diglyceride yield, calculated based on the addition of triglyceride reactants, was consistent with the glycerolysis reaction and was 31.5%. The content of glycidyl esters was 5.88 mg / kg, and the content of chloropropanol esters was 1.37 mg / kg.

[0129] Therefore, secondary molecular distillation can also obtain high-purity diglycerides. However, since diglycerides are obtained by removing monoglycerides through evaporation, the yield of diglycerides is significantly lower than that of enzymatic esterification, and the purity is also lower. In addition, the conditions for secondary molecular distillation are more stringent, with higher temperatures, and the resulting product contains higher levels of harmful substances.

[0130] Glycerol's poor miscibility with oils and fats leads to a slow reaction rate in the enzymatic glycerol hydrolysis process. In traditional reactions, a large amount of glycerol is added to the reaction system initially, resulting in most of it existing in a free state and not mixing well with the oils and fats. This results in a high system viscosity, and the presence of a large amount of free glycerol also coats the enzyme surface, limiting enzyme activity and further reducing the reaction rate. This patent improves reaction efficiency by adding monoglyceride surfactants to the oils and fats to increase the system's polarity, adding glycerol in stages, and homogenizing to improve the mixing of glycerol with the oils and fats. In the staged addition of glycerol, a small amount of glycerol is added and homogenized in the first stage. Under the condition of monoglycerides in the oils and fats, the system is completely mixed, resulting in a rapid reaction that produces monoglycerides and diglycerides. In the second stage, a sufficient amount of glycerol is added and homogenized. The monoglycerides and diglycerides produced in the system are used to completely mix the added glycerol with the oils and fats. In the third stage, since the system has already produced sufficient monoglycerides and diglycerides, the remaining glycerol is added and homogenized to achieve a completely homogeneous mixture. By adding glycerol in stages and homogenizing it, very little free glycerol exists during the entire reaction process, and glycerol mixes well with the oil, which is beneficial to improving the efficiency of enzyme-catalyzed glycerolysis.

[0131] Comparative Example 5

[0132] Under the conditions of Example 3, monoglycerides were not added, and other conditions were the same as in Example 3.

[0133] The imprinted lipase from Example 3 was used as a catalyst.

[0134] Olive oil was added to the batch reactor, nitrogen gas was introduced for protection, the temperature was raised to 50°C, and glycerol was added in three stages according to the molar ratio of glycerol to olive oil of 1:0.4.

[0135] In the first stage, add 30% glycerol and homogenize at 40 MPa for 2 minutes to fully mix the glycerol with the olive oil. Add imprinted lipase at a rate of 20 wt% of the weight of the olive oil and start stirring at 500 rpm. React for 1.5 hours and then filter out the lipase.

[0136] In the second stage, 40% glycerol was added, and the mixture was homogenized at 40 MPa for 2 minutes. The lipase was then added back in and reacted for 1.5 hours with a stirring speed of 500 rpm. The lipase was then filtered out.

[0137] In the third stage, 30% glycerol was added, and lipase was introduced for a reaction at 500 rpm for 4 hours. After the reaction, the system temperature was heated to 85°C, and the mixture was slowly stirred at 50 rpm for 1.5 hours to accelerate the separation of glycerol and glycerides. The glycerol and glycerides were then separated to obtain the glyceride product, and the glycerol was recovered and used as a raw material for a new glycerolysis reaction. The resulting glycerides contained 60.5% monoglycerides, 28.8% diglycerides, and 10.7% triglycerides.

[0138] Therefore, without the addition of monoglycerides, the triglyceride content in the reaction system is higher than that in Example 3, and the reaction has not reached final equilibrium, which will ultimately affect the purity of the final product. The main purpose of adding monoglycerides is to, in the first stage, allow the oil and glycerol to form a better emulsion under homogeneous conditions through the action of monoglycerides. This is beneficial for lipase reaction, while preventing glycerol from covering the lipase surface and affecting enzyme activity.

[0139] Comparative Example 6

[0140] Under the conditions of Example 3, the amount of glycerol added was changed, while other conditions remained the same as in Example 3.

[0141] Olive oil was added to the batch reactor, nitrogen gas was introduced for protection, the temperature was raised to 50°C, 1.5% monoglyceride was added, and the mixture was stirred to fully dissolve the monoglyceride in the olive oil. Glycerin was added in three stages according to a molar ratio of glycerin to olive oil of 1:0.4.

[0142] In the first stage, add 5% glycerol and homogenize at 40 MPa for 2 minutes to fully mix the glycerol with the olive oil. Add imprinted lipase at a rate of 20 wt% of the weight of the olive oil and start stirring at 500 rpm. React for 1.5 hours and then filter out the lipase.

[0143] In the second stage, 45% glycerol was added, and the mixture was homogenized at 40 MPa for 2 minutes. The lipase was then added back in and reacted for 1.5 hours with a stirring speed of 500 rpm. The lipase was then filtered out.

[0144] In the third stage, 50% glycerol was added, and lipase was introduced for a reaction at 500 rpm for 4 hours. After the reaction, the system temperature was heated to 85°C, and the mixture was slowly stirred at 50 rpm for 1.5 hours to accelerate the separation of glycerol and glycerides. The glycerol and glycerides were then separated to obtain the glyceride product, and the recovered glycerol was used as a raw material for a new glycerolysis reaction. The resulting glycerides contained 56.6% monoglycerides, 31.9% diglycerides, and 11.5% triglycerides.

[0145] The triglyceride content in Comparative Example 6 was higher than that in Example 3, indicating that the reaction was not complete, which will lead to a decrease in the purity of the final product. Because the amount of glycerol added in the first stage was too low, insufficient emulsifier was not generated during the reaction. As a result, after adding glycerol in the second stage, a large amount of glycerol remained in a free state and continued to coat the enzyme surface, ultimately affecting the activity of the lipase.

[0146] Comparative Example 7

[0147] Under the conditions of Example 3, the amount of glycerol added was changed, while other conditions remained the same as in Example 3.

[0148] Olive oil was added to the batch reactor, nitrogen gas was introduced for protection, the temperature was raised to 50°C, 1.5% monoglyceride was added, and the mixture was stirred to fully dissolve the monoglyceride in the olive oil. Glycerin was added in three stages according to a molar ratio of glycerin to olive oil of 1:0.4.

[0149] In the first stage, add 40% glycerol and homogenize at 40 MPa for 2 minutes to fully mix the glycerol with the olive oil. Add imprinted lipase at a rate of 20 wt% of the weight of the olive oil and start stirring at 500 rpm. React for 1.5 hours and then filter out the lipase.

[0150] In the second stage, 30% glycerol was added, and the mixture was homogenized at 40 MPa for 2 minutes. The lipase was then added back in and reacted for 1.5 hours with a stirring speed of 500 rpm. The lipase was then filtered out.

[0151] In the third stage, 30% glycerol was added, and lipase was introduced for a reaction at 500 rpm for 4 hours. After the reaction, the system temperature was heated to 85°C, and the mixture was slowly stirred at 50 rpm for 1.5 hours to accelerate the separation of glycerol and glycerides. The glycerol and glycerides were then separated to obtain the glyceride product, and the recovered glycerol was used as a raw material for a new glycerolysis reaction. The resulting glycerides contained 53.5% monoglycerides, 33.2% diglycerides, and 13.3% triglycerides.

[0152] The triglyceride content in Comparative Example 7 was higher than that in Example 3, indicating that the reaction was not complete, which would lead to a decrease in the purity of the final product.

[0153] Because the amount of glycerol added in the first stage was too high, it could not be completely dissolved with the fats, resulting in a large amount of glycerol being in a free state. This caused a large amount of glycerol to cover the surface of the lipase after its addition, ultimately affecting the activity of the lipase.

[0154] Comparative Example 8

[0155] Under the conditions of Example 3, isopropanol was not used; instead, methanol, ethanol, butanol, n-hexane, and octane were used as solvents to compare the effects of different solvents on the activity of imprinted lipase NS40086. Other conditions were the same as in Example 3.

[0156] Table 8. Lipase activity enhancement values ​​when dissolved in different solvents

[0157] Dissolve Enzyme activity Increased vitality (%) methanol 18.1％ -13.0％ ethanol 21.1％ 1.4％ Butanol 28.2％ 35.6％ n-Hexane 24.6％ 18.3％ Octane 22.5％ 8.2％ Isopropanol 32.9％ 58.2％ No trace 20.8％

[0158] Because methanol and ethanol are too polar, they will deplete the surface water of lipases, and at the same time, they have relatively weak ability to dissolve surfactants, thus making their enzyme activity lower than that of isopropanol. Meanwhile, hexane and octane have a weak effect on lipases, making it difficult for surfactants to interact with lipases.

[0159] Butanol is less polar than isopropanol, and its interaction with lipases and its ability to dissolve surfactants are weaker than those of isopropanol. Therefore, lipases imprinted with isopropanol as a solvent exhibit the highest activity.

[0160] Comparative Example 9

[0161] Under the conditions of Example 1, the enzyme was blotted and reacted in different batches, with repeated use of the blotted enzyme compared to the repeated use of the unblotted enzyme.

[0162] Table 9. Reuse of Imprinted and Non-Imprinted Lipases

[0163] Number of repetitions Imprint LipozymeRMIM Non-imprinted LipozymeRMIM 1 34.3％ 21.6％ 2 33.1％ 20.2％ 3 31.3％ 19.2％ 4 31.1％ 18.4％ 5 30.2％ 17.2％ 6 28.7％ 15.6％ 7 28.2％ 14.1％ 8 26.1％ 13.5％ 9 25.4％ 13.2％ 10 23.2％ 11.5％ Enzyme inactivation rate 29.9％ 43.1％

[0164] Due to the action of nonionic surfactants, the surface of imprinted lipase changes from hydrophilic to hydrophobic, avoiding the interaction between lipase and polar substances, which would reduce the flow rate of water and cause denaturation, thus greatly improving its stability.

[0165] Isopropanol exhibits good solubility for both nonionic surfactants and lipases. Furthermore, the trace amounts of water in isopropanol help maintain the lipase in a relatively soft state. Under the influence of isopropanol, the nonionic surfactant first coats the lipase surface through hydrophobic interactions. Specifically, the polar head of the surfactant interacts with the hydrophilic groups of the lipase, while the nonpolar head interacts with the hydrophobic groups. Since lipase itself is water-soluble, meaning the hydrophilic groups on its surface are significantly more numerous than its hydrophobic groups, the nonionic surfactant alters the hydrophilic-hydrophobic nature of the lipase surface, making it more hydrophobic and facilitating its interaction with nonpolar substances. This prevents lipase deactivation caused by polar substances competing with the lipase for water, thus extending its lifespan (i.e., the number of times it can be reused) and reducing production costs.

[0166] Meanwhile, nonionic surfactants interact with the active site of lipase, opening the cap of the active site and allowing dehydration to bring the lipase into its active catalytic configuration, thus enhancing its activity. Since the interaction between nonionic surfactants and lipase is a hydrophobic interaction, their influence on the lipase's conformation is minimal, and therefore they do not affect its activity. Ionic surfactants, on the other hand, primarily interact with lipase through electrostatic interactions, altering the surface charge of the lipase and causing significant structural changes, ultimately leading to some degree of lipase inactivation.

[0167] 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 diglyceride edible oil in stages, characterized in that: include, Using lipase imprinted with a nonionic surfactant dissolved in isopropanol as a catalyst, and adding monoglycerides as a solubilizer, the compatibility of glycerol and oil is improved by adding glycerol in stages and homogenizing under high pressure. The glycerolysis reaction yields the glycerolysis product. Glycerol and oil are separated in the glycerol hydrolysis product. Free fatty acids are added to the glycerol hydrolysis product after glycerol separation, and the reaction is carried out under vacuum with Lipase G50 as a catalyst to esterify monoglycerides and fatty acids in the system. Molecular distillation is used to remove fatty acids, thereby obtaining high-purity diglyceride products; The process of segmented addition of glycerol and high-pressure homogenization to improve compatibility is as follows: in the first stage, 10-30% glycerol is added, homogenized at 20-40 MPa for 2-5 min, imprinted lipase is added, the stirring speed is 500-800 rpm, the reaction is carried out for 0.5-1.5 h, and the lipase is filtered out. In the second stage, add 30-50% glycerol, homogenize at 20-40 MPa for 2-5 minutes with a stirring speed of 500-800 rpm, add lipase again and react for 0.5-1.5 hours, then filter out the lipase. In the third stage, add 30-60% glycerol, stir at 600-800 rpm, and add lipase to react for 3-5 hours; The imprinted lipase is prepared by dissolving a nonionic surfactant in isopropanol at a mass fraction of 10-100 mg / L to obtain a mixed solution. Add 20-50% (w / w) of immobilized lipase to the mixed solution, stir the mixture at 100-300 rpm for 20-50 min at 25°C, and filter to obtain the lipase. The immobilized lipase was eluted with a nonpolar solvent, the solvent volume being 20-50% of the volume of the mixed solution. The lipase was then filtered and dried under vacuum to obtain the imprinted lipase. The esterification reaction is wherein the ratio of fatty acid added to the glycerol backbone in the glycerol ester product is 1~3:1, the amount of Lipase G50 added is 3~8wt% of the substrate weight, the reaction temperature is 30~50℃, the vacuum degree is 10~30mbar, and the reaction time is 12~24h. The nonionic surfactants include Tween 20, Tween 40, and Tween 60; the lipases are commercially available lipases, including Lipozyme RM IM, Novozyme 435, and NS40086. The amount of imprinted lipase added in the glycerol hydrolysis reaction is 8~20wt%, the reaction temperature is 50~70℃, and the molar ratio of glycerol to oil is 1:0.4~3.

2. The method as described in claim 1, characterized in that: The amount of monoglyceride added in the glycerolysis reaction is 0.5-1.5% of the substrate by mass.

3. The method as described in claim 1, characterized in that: The oils mentioned are edible oils, including sunflower seed oil, walnut oil, and olive oil.

4. The method as described in claim 1, characterized in that: After the glycerol hydrolysis reaction is complete, it also includes, Heat the temperature to 85~95℃, stir slowly at a speed of 20~50 rpm for 0.5~1.5h to accelerate the separation of glycerol and glycerides.

5. The diglyceride edible oil obtained by the method according to any one of claims 1 to 4, characterized in that: The diglyceride edible oil contains less than 1.6 mg / kg of glycidyl esters and less than 0.6 mg / kg of chloropropanol esters.

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