Process for the production of diglyceride oils in one step

Abstract

This application discloses a one-step process for preparing diglyceride oil, relating to the field of oil and fat production technology. The process includes the following steps: S1: Mixing vegetable oil and water, adding a hydrolytic enzyme for enzymatic hydrolysis, and separating to obtain a hydrolyzed supernatant; S2: Distilling the hydrolyzed supernatant to obtain a light phase and a heavy phase; S3: Performing an enzymatic hydrolysis on the heavy phase, and separating to obtain a hydrolyzed supernatant; S4: Repeating steps S2 and S3 to complete N distillations and N enzymatic hydrolysis reactions, collecting the light phase obtained from each distillation to obtain a light phase mixture; S5: Performing an enzymatic esterification reaction, and separating to obtain an esterification supernatant; the esterification supernatant is then distilled to obtain diglyceride oil. This application's process, by combining enzymatic cyclic hydrolysis with enzymatic esterification, improves product purity and reduces production costs.

Description

Technical Field

[0001] This application relates to the field of oil and fat production technology, specifically to a one-step process for preparing diglyceride oil. Background Technology

[0002] Diacylglycerol (DAG), as a functional oil, has significant application value in the food, pharmaceutical, and health product fields due to its unique physiological functions such as regulating lipid metabolism, inhibiting weight gain, reducing serum lipids, and improving insulin sensitivity. In recent years, the market demand for high-purity diacylglycerol has continued to grow, especially driven by the trend of health consumption, making high-purity diacylglycerol a key direction for the research and development of high-end oil products.

[0003] The preparation methods of diglycerides mainly include chemical synthesis and enzymatic catalysis. Chemical synthesis is usually carried out under high temperature and high pressure conditions, which easily leads to side reactions and causes the product to darken in color. The purity of the product is poorly controllable, and subsequent separation and purification are costly. Enzymatic synthesis of diglycerides has attracted much attention due to its advantages such as mild reaction conditions, high selectivity, and environmental friendliness. However, current enzymatic processes are still insufficient to prepare diglyceride oils with a diglyceride content ≥95%.

[0004] For example, patent CN105400837A uses a two-step method of "hydrolysis-esterification of vegetable oil" to prepare diglyceride oil. Although it alleviates the problem of excessive hydrolysis to some extent, it still has obvious limitations: the diglyceride content in its final product is only 60%-65%, which cannot meet the current market demand for high-purity products.

[0005] Although patent CN202210168223.3 can obtain high-concentration diglyceride oil, the process uses concentrated sulfuric acid and ethanol for acidification, which not only increases the cost of subsequent waste liquid treatment, but also seriously restricts the application of this process in the preparation of high-quality diglycerides due to the potential risk of solvent residue. Summary of the Invention

[0006] In view of the shortcomings of the above-mentioned related technologies, this application aims to provide a one-step production process for preparing diglyceride oil, which combines enzymatic cyclic hydrolysis with enzymatic esterification to improve product purity and reduce production costs. This process is suitable for large-scale industrial production and solves the current problems of low purity and high cost of diglyceride oil.

[0007] To achieve the above and other related objectives, this application provides a one-step process for preparing diglyceride oil, comprising the following steps: S1: Mix vegetable oil and water, add hydrolytic enzyme to carry out enzymatic hydrolysis, and then separate to obtain the hydrolysis supernatant; S2: The hydrolysate supernatant is distilled to obtain a light phase and a heavy phase; S3: Using the aforementioned heavy phase as a substrate, add hydrolytic enzyme and water to continue the enzymatic hydrolysis reaction, and obtain the hydrolysis supernatant after separation; S4: Repeat steps S2 and S3 to complete N distillation processes and N enzymatic hydrolysis reactions, collect the light phase obtained from each distillation process, and obtain a light phase mixture. S5: The light phase mixture from step S4 is mixed with a protic solvent and lipase to carry out an enzymatic esterification reaction. After separation, the esterification supernatant is obtained. The esterification supernatant is then distilled to obtain diglyceride oil.

[0008] In some specific implementations, step S1 satisfies at least one of the following conditions: (1) The hydrolytic enzyme is at least one of Lipozyme RMIM, Lipozyme TL100 L, and Palatase 20000 L; (2) Based on the mass of the vegetable oil, the mass of the hydrolytic enzyme added is 1% to 5%; (3) Based on the mass of the vegetable oil, the mass of water added is 1% to 3.5%; (4) The temperature of the enzymatic hydrolysis reaction is 55℃~65℃; (5) The time for the enzymatic hydrolysis reaction is 1h to 4h; (6) The vegetable oil includes at least one of corn oil, soybean oil, rapeseed oil, peanut oil, sunflower oil, olive oil and flaxseed oil.

[0009] In some specific embodiments, in step S2, the temperature of the distillation process is 180℃~200℃.

[0010] In some specific implementations, step S3 satisfies at least one of the following conditions: (1) The hydrolytic enzyme is at least one of Lipozyme RMIM, Lipozyme TL100 L, and Palatase 20000 L; (2) Based on the mass of the heavy phase, the mass of the added hydrolytic enzyme is 1% to 5%; (3) Based on the mass of the heavy phase, the mass of water added is 1% to 3.5%; (4) The temperature of the enzymatic hydrolysis reaction is 55℃~65℃; (5) The time for the enzymatic hydrolysis reaction is 1h to 4h.

[0011] In some specific implementations, in step S4, N≥2; Preferably, 3 ≤ N ≤ 6.

[0012] In some specific implementations, step S5 satisfies at least one of the following conditions: (1) The lipase is at least one of Lipase G50, Candida antarcticis lipase B, and lipase SMG1; (2) The protic solvent includes at least one of glycerol, ethylene glycol, and methanol; (3) Based on the mass of the light phase mixture, the mass of the protic solvent added is 15%~40%; (4) Based on the mass of the light phase mixture, the mass of the added lipase is 1% to 10%; (5) The temperature of the enzymatic esterification reaction is 55℃~65℃; (6) The time for the enzymatic esterification reaction is 1h to 4h; (7) The distillation process includes primary distillation and secondary distillation; The temperature of the first-stage distillation is 150℃~170℃, and the vacuum degree during the first-stage distillation is less than 10Pa. The temperature of the secondary distillation is 200℃~280℃, and the vacuum degree during the secondary distillation is less than 2Pa.

[0013] In some specific embodiments, in step S5, the temperature of the first-stage distillation is 155°C to 170°C, and the vacuum degree during the first-stage distillation is 1 Pa to 9 Pa. The temperature of the secondary distillation is 220℃~280℃, and the vacuum degree during the secondary distillation is 0.1Pa to 1.5Pa.

[0014] In some specific embodiments, the separation described in steps S1, S3, and S5 is centrifugal separation, and the centrifugal rotation speed is 1000 rpm to 4000 rpm.

[0015] In some specific embodiments, the diglyceride oil satisfies at least one of the following conditions: (1) The mass content of diglycerides in the diglyceride oil is ≥90%; (2) The oleic acid value of the diglyceride is 0.12 mg KOH / g to 0.17 mg KOH / g; (3) The triglyceride content in the diglyceride oil is ≤5.17% by mass; (4) The total residual solvent in the diglyceride oil is ≤10ppm.

[0016] In some specific embodiments, the diglyceride oil satisfies at least one of the following conditions: (1) Preferably, the mass content of diglycerides in the diglyceride oil is 94.78%~98.83%; (2) The triglyceride content in the diglyceride oil is 1.14%~5.17% by mass; (3) The total solvent residue in the diglyceride oil was not detected.

[0017] The beneficial effects of this application are as follows: 1. The production process provided in this application uses hydrolytic enzymes to catalyze the hydrolysis of vegetable oil in a circulating manner. The heavy phase obtained from the hydrolysis is recycled back to the hydrolysis kettle, which gradually concentrates the hydrolysis product. This eliminates the need for multiple intermediate material changes and avoids the problems of oil oxidation and enzyme inactivation caused by single long-term high-temperature deep hydrolysis. It can achieve the one-step preparation of high-concentration diglyceride oil and effectively improve production efficiency.

[0018] 2. The production process provided in this application, after multiple enzymatic hydrolysis and enzymatic esterification reactions, separation and purification, can produce a diglyceride content of over 90%, with a maximum of 98.83%.

[0019] 3. The production process provided in this application avoids side reactions caused by single deep hydrolysis by batching and gradually converting raw materials, thereby improving the utilization rate of raw materials, reducing by-product loss, and lowering production costs.

[0020] Other features and advantages of this application will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the application. It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only, and are not intended to limit this disclosure. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The process flow diagram (N=2) shows the one-step preparation process of diglyceride oil described in this application. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions of this application will be clearly and completely described below in conjunction with embodiments. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. The embodiments described herein are illustrative in nature and are used to provide a basic understanding of this application. The embodiments of this application should not be construed as limiting this application.

[0024] Unless otherwise expressly stated, the terms used herein have the meanings indicated below.

[0025] In this document, a list of items connected by the terms "at least one of," "at least one of," "at least one of," or other similar terms may mean any combination of the listed items. For example, if items A and B are listed, then the phrase "at least one of A and B" means only A; only B; or A and B. In another instance, if items A, B, and C are listed, then the phrase "at least one of A, B, and C" means only A; or only B; only C; A and B; A and C; B and C; or all of A, B, and C. Item A may contain a single element or multiple elements. Item B may contain a single element or multiple elements. Item C may contain a single element or multiple elements.

[0026] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0027] To address the current problems of low purity, high cost, and high solvent residue in diglyceride oil, this application provides a one-step production process for preparing diglyceride oil. By combining enzymatic cyclic hydrolysis with enzymatic esterification, the purity of the product is improved, resulting in virtually no solvent residue and reduced production costs, making it suitable for large-scale industrial production.

[0028] A one-step process for preparing diglyceride oil includes the following steps: S1: Mix vegetable oil and water, add hydrolytic enzyme to carry out enzymatic hydrolysis, and then separate to obtain the hydrolysis supernatant; S2: The hydrolysate supernatant is distilled to obtain a light phase and a heavy phase; S3: Using the aforementioned heavy phase as a substrate, add hydrolytic enzyme and water to continue the enzymatic hydrolysis reaction, and obtain the hydrolysis supernatant after separation; S4: Repeat steps S2 and S3 to complete N distillation processes and N enzymatic hydrolysis reactions, collect the light phase obtained from each distillation process, and obtain a light phase mixture. S5: The light phase mixture from step S4 is mixed with a protic solvent and lipase to carry out an enzymatic esterification reaction. After separation, the esterification supernatant is obtained. The esterification supernatant is then distilled to obtain diglyceride oil.

[0029] In the above technical solution, the production process first replaces the traditional chemical hydrolysis process with an enzymatic hydrolysis reaction, avoiding side reactions and oil oxidation problems caused by high temperature and high pressure conditions. Simultaneously, distillation is used to promptly separate the generated diglycerides and free fatty acids, reducing the risk of degradation of the target product in subsequent reactions. The remaining heavy-phase substrate after distillation is repeatedly fed into the hydrolysis reaction, gradually consuming unreacted triglycerides, improving raw material utilization, avoiding enzyme inactivation and byproduct accumulation problems caused by single-stage deep hydrolysis, and eliminating multiple intermediate material changeover steps, simplifying the production process. Then, a specific lipase is used to catalyze a directed esterification reaction between free fatty acids and glycerol, further increasing the diglyceride production. Two-stage distillation removes residual impurities, yielding a product with a purity of over 90%, while reducing the risk of solvent residue. Compared to traditional processes, this production process can improve product quality while reducing overall production costs, making it more suitable for large-scale industrial production applications.

[0030] In some embodiments, in step S1, the hydrolase is at least one selected from Lipozyme RMIM, Lipozyme TL100 L, and Palatase 20000 L. Using this specific hydrolase effectively ensures the catalytic specificity of the hydrolysis reaction for the triglyceride substrate, enabling the directional cleavage of the ester bonds of triglycerides under mild reaction conditions to generate the target product, free fatty acids, while significantly reducing the probability of side reactions such as over-hydrolysis. Furthermore, this type of hydrolase exhibits excellent catalytic stability, adapting to the process requirements of multi-cycle hydrolysis reactions, reducing the amount of enzyme preparation used per unit yield, further compressing the material costs of the entire production process, and meeting the application requirements of industrial continuous production.

[0031] In some embodiments, in step S1, the added mass of the hydrolytic enzyme is 1% to 5% based on the mass of the vegetable oil. When the added mass of the hydrolytic enzyme in step S1 meets the above conditions, it can ensure that the hydrolysis reaction has a sufficient catalytic rate, avoiding a slow hydrolysis reaction rate and a long reaction cycle due to too low an amount of hydrolytic enzyme, thereby reducing the overall production efficiency. At the same time, it will not waste enzyme preparation due to too high an amount of hydrolytic enzyme, avoiding unnecessary increase in production costs. It can also reduce the adverse effects of excessive enzyme protein aggregation on catalytic activity and reduce the risk of increasing the processing load of subsequent separation processes after introducing excess impurities.

[0032] In some embodiments, in step S1, the mass of water added is 1% to 3.5% based on the mass of the vegetable oil; for example, the mass of water added can be 1.0%, 1.2%, 1.5%, 1.8%, 2.0%, 2.3%, 2.5%, 2.8%, 3.0%, 3.2%, 3.5%, or a range consisting of any two of these values. When the mass of water added in step S1 is adjusted to meet the above conditions, on the one hand, the reaction medium environment required for the hydrolytic enzyme to catalyze the hydrolysis of triglycerides can be guaranteed, which is beneficial to maintaining the catalytic activity of the hydrolytic enzyme and helping the hydrolysis reaction to proceed at a relatively stable rate; on the other hand, this range of added mass makes it easier for the hydrolysis reaction to remain in the controllable hydrolysis stage, tending to generate free fatty acids and diglycerides, reducing the possibility of excessive hydrolysis generating large amounts of monoglycerides and glycerol, reducing the processing burden of subsequent separation and purification, and also helping to improve the overall yield of the target product. When the amount of water added is too low, it may be difficult to meet the water molecule requirements of the substrate for the hydrolysis reaction, which may lead to insufficient hydrolysis. When the amount of water added is too high, it may dilute the concentration of enzyme and substrate in the system and affect the reaction rate.

[0033] In some embodiments, in step S1, the temperature of the enzymatic hydrolysis reaction is 55℃~65℃; for example, the temperature of the enzymatic hydrolysis reaction can be 55℃, 56℃, 57℃, 58℃, 59℃, 60℃, 61℃, 62℃, 63℃, 64℃, 65℃, or a range consisting of any two of these values. In step S1, when the temperature of the hydrolysis reaction is controlled within the above range, this temperature range has a high degree of matching with the catalytic temperature of the selected hydrolase, which is beneficial to maintaining the spatial conformational stability of the hydrolase, ensuring its catalytic activity is fully utilized, and keeping the hydrolysis reaction rate within a reasonable range; at the same time, the substrate molecule motion rate is suitable under this temperature condition, which can increase the probability of substrate binding to the enzyme active site, promote the directional breakage of triglyceride ester bonds, and reduce the risk of slow reaction rate at low temperature and enzyme protein denaturation and inactivation at high temperature; in addition, this temperature range also helps to reduce the probability of side reactions of lipid oxidation, ensuring the overall quality of the hydrolysis product.

[0034] In some embodiments, in step S1, the enzymatic hydrolysis reaction time is 1 h to 4 h; for example, the enzymatic hydrolysis reaction time can be 1.0 h, 1.5 h, 2.0 h, 2.5 h, 3.0 h, 3.5 h, 4.0 h, or a range of any two of these values. When the hydrolysis reaction time is controlled within the above range in step S1, sufficient contact reaction time can be ensured for the hydrolytic enzyme, which is beneficial for achieving appropriate hydrolysis and conversion, and reducing the problem of low raw material conversion rate caused by insufficient reaction. At the same time, this time range can reduce the risk of over-hydrolysis caused by excessively long reaction time, reduce the probability of the formation of byproducts such as monoglycerides, and retain a suitable reaction basis for subsequent cyclic hydrolysis.

[0035] In some embodiments, in step S1, the vegetable oil includes at least one selected from corn oil, soybean oil, rapeseed oil, peanut oil, sunflower seed oil, olive oil, and flaxseed oil. Using the above-mentioned vegetable oil raw materials in step S1 results in better compatibility of their fatty acid composition with the substrate of the selected hydrolytic enzyme, which is beneficial for improving the directionality of the hydrolysis reaction and reducing the probability of non-target byproduct formation. Furthermore, these vegetable oil raw materials are widely available and cost-controllable, effectively reducing raw material procurement costs in the industrial production stage.

[0036] In the embodiments of this application, step S1 employs the principle of enzyme-catalyzed hydrolysis. A specific hydrolytic enzyme is added to a mixture of vegetable oil and water, initiating a controllable enzymatic hydrolysis reaction under mild temperature conditions. The resulting supernatant is then obtained by centrifugation. Relying on the selective hydrolysis of triglycerides by the hydrolytic enzyme, which breaks some ester bonds, this step is easier to control the reaction direction and reduces the generation of side reactions compared to traditional chemical hydrolysis processes. Step S1 solves the problems of oil oxidation, enzyme activity loss, and excessive byproducts that easily occur in single-stage deep hydrolysis in existing processes, while also providing an initial reaction system for subsequent cyclic hydrolysis. Furthermore, by controlling the enzyme dosage, reaction temperature, and reaction time, step S1 can ensure adequate hydrolysis efficiency while avoiding problems such as product color deepening and increased impurities caused by over-hydrolysis. This is beneficial for improving the enrichment efficiency of diglycerides in subsequent steps, increasing raw material utilization, and laying the foundation for obtaining high-purity products.

[0037] Specifically, step S1 is implemented as follows: after mixing vegetable oil with water, a hydrolytic enzyme is added to carry out a hydrolysis reaction, and a hydrolyzed supernatant is obtained after separation treatment; the hydrolytic enzyme is at least one of Lipozyme RMIM, Lipozyme TL100 L, and Palatase 20000 L; based on the mass of the vegetable oil, the mass percentage of the added hydrolytic enzyme is 1%~5%, and the mass percentage of the added water is 1%~3.5%; the temperature of the hydrolysis reaction is controlled at 55℃~65℃, and the hydrolysis reaction time is 1h~4h; the vegetable oil includes at least one of corn oil, soybean oil, rapeseed oil, peanut oil, sunflower seed oil, olive oil, and flaxseed oil.

[0038] In some embodiments, the distillation temperature in step S2 is 180°C to 200°C; for example, the distillation temperature can be 180°C, 182°C, 185°C, 188°C, 190°C, 192°C, 195°C, 198°C, 200°C, or a range of any two of these values. Step S2, based on the principle of differences in boiling points between different components, uses distillation to separate the initial enzymatic hydrolysis products. The diglycerides and free fatty acids generated from the decomposition are separated by distillation and collected as the light phase, while the incompletely hydrolyzed triglycerides and remaining substrate are retained in the heavy phase. In step S2, adjusting the distillation temperature to meet the above conditions can adapt to the boiling point difference between diglycerides and triglycerides, achieving efficient separation of the target product and unreacted substrate, while avoiding excessively high temperatures that could lead to diglyceride degradation or lipid oxidation, ensuring the activity of the heavy phase substrate can support subsequent cyclic hydrolysis reactions.

[0039] In some embodiments, in step S3, the hydrolase is at least one of Lipozyme RMIM, Lipozyme TL100 L, and Palatase 20000 L. Using the above-mentioned hydrolase in step S3 effectively maintains the catalytic specificity required for triglyceride hydrolysis in the heavy-phase system, ensuring the hydrolase's ability to directionally break the ester bonds of residual triglycerides. Since the triglyceride concentration in the heavy-phase substrate is relatively high after the previous distillation process, adding this type of specific hydrolase can maintain stable catalytic activity, allowing insufficiently converted triglycerides to continue undergoing controlled hydrolysis, avoiding low conversion rates due to insufficient enzyme activity. Simultaneously, it maintains directional catalytic characteristics under mild conditions, reducing side reactions such as over-hydrolysis, which is beneficial for improving overall raw material utilization and providing a better substrate basis for subsequent esterification reactions.

[0040] In some embodiments, in step S3, the added mass of the hydrolytic enzyme is 1% to 5% based on the mass of the heavy phase; for example, the added mass of the hydrolytic enzyme can be 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, or a range of any two of these values. When the added mass of the hydrolytic enzyme in step S3 meets the above conditions, it can ensure that the hydrolysis reaction in the heavy phase system obtains sufficient catalytic activity, avoiding the problem that the hydrolysis rate of triglycerides in the heavy phase substrate is too slow and the conversion efficiency is insufficient due to too low an amount of hydrolytic enzyme, thereby reducing the overall enrichment effect of the cyclic hydrolysis; at the same time, it will not cause waste of enzyme preparation due to too high an amount of hydrolytic enzyme, avoiding unnecessary increase in production costs, and can also reduce the risk of increasing the processing load of subsequent centrifugation separation processes after excessive enzyme protein is introduced into the system.

[0041] In some embodiments, in step S3, the mass of water added is 1% to 3.5% based on the mass of the heavy phase; for example, the mass of water added can be 1.0%, 1.2%, 1.5%, 1.8%, 2.0%, 2.3%, 2.5%, 2.8%, 3.0%, 3.2%, 3.5%, or a range of any two of these values. When the mass of water added in step S3 is adjusted to meet the above conditions, on the one hand, it ensures that the heavy phase system has suitable water activity, providing a sufficient reaction medium environment for the hydrolytic enzyme, which is beneficial for maintaining the spatial conformational stability of the hydrolytic enzyme and ensuring that its catalytic activity is fully utilized. At the same time, it avoids the problems of phase separation and uneven distribution of enzyme proteins caused by excessive water addition, reducing the risk of fluctuations in catalytic efficiency. On the other hand, this range of water addition ensures the directionality of the hydrolysis reaction, avoiding the defects of insufficient water leading to insufficient triglyceride bond breaking reaction and low substrate conversion, and reducing problems such as excessive hydrolysis and byproduct accumulation caused by excessive water. Since the components of the heavy phase substrate are relatively concentrated after the preceding distillation treatment, the amount of water added can form a good synergistic effect with the supplemented hydrolytic enzyme, ensuring the stable conversion efficiency of each cycle of hydrolysis, providing uniform hydrolysis products for subsequent distillation separation, and helping to improve the overall operational stability of the process.

[0042] In some embodiments, in step S3, the temperature of the enzymatic hydrolysis reaction is 55℃~65℃; for example, the temperature of the enzymatic hydrolysis reaction can be 55℃, 56℃, 57℃, 58℃, 59℃, 60℃, 61℃, 62℃, 63℃, 64℃, 65℃, or a range consisting of any two of these values. When the temperature of the hydrolysis reaction is controlled to meet the above conditions, this temperature range has a high degree of matching with the catalytic temperature of the selected hydrolytic enzyme, which is beneficial to maintaining the spatial conformational stability of the added hydrolytic enzyme, ensuring its catalytic activity is fully utilized, and keeping the hydrolysis reaction rate in the heavy phase system within a reasonable range. Since the original hydrolytic enzyme has been basically inactivated after the heavy phase substrate has undergone high-temperature distillation treatment at 180℃~200℃, this temperature condition can quickly activate the newly added hydrolytic enzyme, avoiding problems such as enzyme protein denaturation and inactivation, and oil oxidation and browning caused by excessively high temperatures. At the same time, it can prevent situations such as slow hydrolysis reaction rate and incomplete substrate conversion caused by excessively low temperatures. In addition, this temperature range can keep the conversion rate of each cycle of hydrolysis stable, reduce the probability of excessive hydrolysis to generate side reactions such as monoglycerides, ensure the uniformity of the quality of the light phase components separated by subsequent distillation, and provide a stable substrate basis for subsequent enzymatic esterification reactions.

[0043] In some embodiments, in step S3, the enzymatic hydrolysis reaction time is 1 h to 4 h. For example, the enzymatic hydrolysis reaction time can be 1.0 h, 1.5 h, 2.0 h, 2.5 h, 3.0 h, 3.5 h, 4.0 h, or a range of any two of these values. When the hydrolysis reaction time is controlled to meet the above conditions, on the one hand, it can ensure that the added hydrolytic enzyme has sufficient reaction time, so that the residual triglycerides in the heavy phase substrate can fully contact the enzyme and undergo directional hydrolysis, reducing the residual triglycerides caused by insufficient reaction time, which is beneficial to improving the overall conversion rate of raw materials and ensuring the stability of the conversion efficiency of each cycle of hydrolysis; on the other hand, this time range can effectively avoid excessive hydrolysis caused by excessively long hydrolysis reaction time, reduce the generation of unnecessary by-products, reduce the accumulation of non-target components such as monoglycerides in the system, and ensure that the composition of hydrolysis products meets the requirements of subsequent processes. Since the original hydrolytic enzymes are basically inactivated after the heavy-phase substrate is distilled at high temperature, the hydrolysis reaction is controlled within the above time range after the enzyme is added. This can not only give full play to the catalytic effect of the newly added hydrolytic enzyme, but also avoid enzyme conformational changes and activity loss caused by long-term reaction, maintain the stability of the entire cycle hydrolysis process, and provide a substrate with uniform composition for the subsequent esterification reaction.

[0044] In step S3 of this application, the principle of enzyme-catalyzed hydrolysis is continued. The heavy phase remaining after distillation is used as the substrate. By adding a certain proportion of hydrolytic enzyme and water, a controlled enzymatic hydrolysis reaction continues under suitable temperature conditions. After the reaction is complete, a new hydrolysis supernatant is obtained by centrifugation. Step S3 addresses the characteristic that a large amount of unhydrolyzed triglycerides remain in the heavy phase substrate. By supplementing enzyme and water, the required catalytic activity and water content of the reaction system are maintained, ensuring that triglycerides can continue to undergo selective cleavage. Step S3 solves the problems of insufficient conversion of raw materials in a single hydrolysis, low raw material utilization, and waste caused by the direct disposal of unhydrolyzed substrate. It also avoids the risks of side reactions and lipid oxidation caused by prolonged single-stage hydrolysis. By performing secondary catalytic conversion on the incompletely hydrolyzed substrate, step S3 can further improve the conversion rate of triglycerides in the raw material to the target product, gradually enrich the diglyceride component, provide more reaction precursors for subsequent esterification reactions, and improve the total yield of diglycerides in the final product, reducing production costs and enhancing the overall economic efficiency of the process.

[0045] Specifically, step S3 is implemented as follows: using the heavy phase as a substrate, adding hydrolytic enzyme and water to continue the enzymatic hydrolysis reaction, and obtaining the hydrolysis supernatant after separation; the hydrolytic enzyme is at least one of Lipozyme RMIM, Lipozyme TL100L, and Palatase 20000L; based on the heavy phase of the vegetable oil, the added mass percentage of hydrolytic enzyme is 1%~5%, and the added mass percentage of water is 1%~3.5%; the temperature of the hydrolysis reaction is controlled at 55℃~65℃, and the hydrolysis reaction time is 1h~4h.

[0046] In some embodiments, in step S4, N ≥ 2; preferably, 3 ≤ N ≤ 6; for example, N can be 2, 3, 4, 5, 6, 7, 8, or a range consisting of any two of these values. Specifically, step S4, based on the principle of cyclic transformation and stepwise enrichment, repeatedly feeds the heavy phase obtained from a single distillation into the next round of hydrolysis reaction, completing at least two "hydrolysis-distillation" cycle operations (e.g., Figure 1 As shown (N=2), the newly generated light phase component is collected in each cycle, and the light phases collected in all cycles are combined to obtain a light phase mixture. Step S4, by carrying out multiple cyclic catalytic conversions of the insufficiently hydrolyzed substrate, solves the defects of incomplete raw material conversion and low utilization rate in traditional single-cycle hydrolysis processes. It also overcomes the problems of low raw material utilization rate, limited diglyceride content in a single hydrolysis, and low production efficiency caused by multiple material changes in existing processes. Simultaneously, it reduces the risk of side reactions and oil oxidation caused by long-term single-batch reactions. Step S4 gradually enriches the diglyceride component through cyclic conversion, significantly improving the overall raw material conversion rate and achieving effective accumulation of total diglyceride content in the light phase mixture. It provides sufficient high-content precursor raw materials for subsequent esterification reactions without the need for additional complex intermediate purification steps, simplifying the process and reducing production costs.

[0047] In some embodiments, in step S5, the lipase is at least one of Lipase G50, Candida antarcticis lipase B, and lipase SMG1. By selecting the specific lipase mentioned above, the esterification reaction between free fatty acids in the light phase and the hydroxyl groups of glycerol can be selectively catalyzed, directionally generating the target product, significantly reducing the generation of non-target byproducts such as monoglycerides, and increasing the proportion of the target product in the reaction system. Simultaneously, this type of lipase exhibits excellent catalytic stability under mild reaction conditions of 55℃~65℃, can tolerate the influence of trace impurities in the light phase mixture, has a low enzyme activity loss rate during the reaction, does not require frequent replenishment of enzyme preparations, and can meet the needs of large-scale continuous industrial production.

[0048] In some embodiments, in step S5, the protic solvent includes at least one of glycerol, ethylene glycol, and methanol. Using these protic solvents allows for full utilization of their polar properties and hydroxyl donor function, shifting the equilibrium of the enzymatic esterification reaction towards the formation of diglycerides. Glycerol, as a natural substrate for esterification, plays a dual role as both solvent and reactant. Using it as a protic solvent not only provides excess hydroxyl groups, promoting the full esterification conversion of free fatty acids, but also avoids the introduction of exogenous toxic and harmful solvents, ensuring the safety of the final product for consumption. Compared to non-protic solvents, protic solvents can stabilize the catalytic active center of lipases through hydrogen bonding, maintaining the spatial conformational stability of the enzyme molecule, reducing enzyme inactivation and denaturation during the reaction, extending the effective period of enzyme catalysis, and reducing the amount of enzyme preparation used and production costs. Ethylene glycol and methanol have strong polarity, which can improve the fluidity of the reaction system, reduce system viscosity, promote sufficient contact between the substrate and enzyme molecules, improve mass transfer efficiency, and facilitate the full progress of the esterification reaction. If a protic solvent is not used, the viscosity of the reaction system will be too high, resulting in greater mass transfer resistance. This can lead to insufficient contact between the lipase and the substrate, resulting in a low esterification conversion rate. At the same time, it will be unable to effectively shift the reaction equilibrium towards the product, thus affecting the yield of diglycerides.

[0049] In some embodiments, in step S5, the mass of the protic solvent added is 15% to 40% based on the mass of the light phase mixture; for example, the mass of the protic solvent added can be 15%, 18%, 20%, 22%, 25%, 28%, 30%, 32%, 35%, 38%, 40%, or a range of any two of these values. Controlling the mass of the protic solvent added to meet the above conditions provides sufficient hydroxyl donors for the esterification reaction, shifting the reaction equilibrium towards the formation of diglycerides and ensuring the esterification conversion rate of free fatty acids. It also avoids excessive solvent addition leading to excessive dilution of the substrate concentration in the reaction system, reducing the probability of contact between lipase and free fatty acids, thereby affecting the reaction rate and production efficiency. Simultaneously, this addition range allows the system to maintain suitable polarity and viscosity, stabilizing the lipase conformation through hydrogen bonding to ensure stable catalytic activity, while avoiding increased load on subsequent distillation and removal processes due to excessive solvent, thus reducing energy consumption and solvent residue risks.

[0050] In some embodiments, in step S5, the mass of lipase added is 1% to 10% based on the mass of the light phase mixture; for example, the mass of lipase added can be 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10.0%, or a range of any two values ​​therein. When the mass of lipase added is controlled to meet the above conditions, it can provide sufficient catalytic active sites for the esterification reaction, ensuring sufficient contact between the lipase and the free fatty acids and glycerol substrate, maintaining a suitable conversion rate for the esterification reaction, and avoiding problems such as slow reaction kinetics and incomplete esterification of free fatty acids due to insufficient enzyme addition; it can also prevent excessive lipase addition leading to enzyme waste, reducing production costs, while reducing the problem of increased mass transfer resistance caused by the aggregation of excess enzyme protein in the reaction system, reducing the processing load of subsequent separation and purification processes, and avoiding the introduction of impurities by excess enzyme protein residue, ensuring the purity and safety of the final product.

[0051] In some embodiments, in step S5, the temperature of the enzymatic esterification reaction is 55℃~65℃; the temperature of the enzymatic esterification reaction can be 55℃, 56℃, 57℃, 58℃, 59℃, 60℃, 61℃, 62℃, 63℃, 64℃, 65℃, or a range of any two of these values. When the temperature of the enzymatic esterification reaction is controlled to meet the above conditions, this temperature range is highly matched with the catalytic temperature range of the specific lipase selected in this application, which is beneficial to maintaining the spatial conformational stability of the lipase's catalytic active center. This effectively avoids the problems of insufficient enzyme catalytic activity, slow esterification reaction rate, and incomplete conversion of free fatty acids caused by excessively low temperatures, while also preventing lipase thermal denaturation and inactivation caused by excessively high temperatures, reducing the decrease in catalytic selectivity caused by irreversible destruction of the enzyme molecule conformation, thereby reducing the probability of the formation of byproducts such as monoglycerides and triglycerides. At the same time, this temperature condition can maintain the esterification reaction at a suitable rate, shorten the reaction cycle, and increase the production capacity per unit time.

[0052] In some embodiments, in step S5, the enzymatic esterification reaction time is 1 to 4 hours; for example, the enzymatic esterification reaction time can be 1.0 h, 1.5 h, 2.0 h, 2.5 h, 3.0 h, 3.5 h, 4.0 h, or a range of any two of these values. Regulating the enzymatic esterification reaction time to meet the above conditions ensures sufficient contact time between the lipase and the free fatty acids and glycerol substrate, allowing the esterification reaction to proceed fully and avoiding incomplete conversion of free fatty acids and low diglyceride production due to insufficient reaction time, thus guaranteeing the esterification conversion rate. It also prevents excessive esterification caused by excessively long reaction times, reducing the further combination of free diglycerides with fatty acids to form byproducts such as triglycerides. Simultaneously, it avoids conformational changes and decreased activity of the lipase due to prolonged reaction, reducing the efficiency of enzyme reuse. This time range also allows for reasonable control of the single-batch reaction cycle, adapting to the rhythm of continuous industrial production, increasing production capacity per unit time, and reducing production and operating costs.

[0053] In some embodiments, in step S5, the distillation process includes primary distillation and secondary distillation; The temperature of the first-stage distillation is 150℃~170℃; for example, the temperature of the first-stage distillation can be 150℃, 152℃, 155℃, 158℃, 160℃, 162℃, 165℃, 168℃, 170℃, or any range of two of these values. The vacuum degree during the first-stage distillation is less than 10 Pa; for example, the vacuum degree during the first-stage distillation can be 1 Pa, 2 Pa, 3 Pa, 4 Pa, 5 Pa, 6 Pa, 7 Pa, 8 Pa, 9 Pa, or any other value less than 10 Pa.

[0054] The temperature of the secondary distillation is 200℃~280℃; for example, the temperature of the secondary distillation can be 200℃, 210℃, 220℃, 230℃, 240℃, 250℃, 260℃, 270℃, 280℃ or a range of any two of these values.

[0055] The vacuum degree during the second-stage distillation is less than 2 Pa. For example, the vacuum degree during the second-stage distillation can be 0.1 Pa, 0.2 Pa, 0.5 Pa, 0.8 Pa, 1.0 Pa, 1.2 Pa, 1.5 Pa, 1.8 Pa, or any other value less than 2 Pa, and the vacuum degree can be within the range of any two of the above values ​​(e.g., 0.1 Pa to 1.0 Pa, 0.5 Pa to 1.5 Pa, etc.).

[0056] Through the above embodiments, in step S5, primary and secondary distillations are performed. When the primary and secondary distillations meet the above conditions, the separation of impurities from the target product can be achieved based on the principle of fractional distillation. In the primary distillation stage, a temperature of 150℃~170℃ combined with a vacuum of less than 10Pa allows for the preferential distillation and removal of residual low-boiling-point free fatty acids in the esterification supernatant under relatively mild conditions, avoiding the impact of unreacted fatty acid residues on the final product's acid value. Subsequently, the secondary distillation uses a higher temperature (200℃~280℃) and a vacuum (less than 2Pa), which allows for the complete distillation of high-boiling-point diglycerides, while simultaneously achieving efficient separation from heavy impurities such as triglycerides. This fractional distillation process ensures both the recovery efficiency and product purity of diglycerides, and avoids oxidative denaturation caused by prolonged high-temperature treatment of the target product through gradient temperature control, which is beneficial for maintaining the good color and physicochemical stability of the diglyceride oil.

[0057] In some embodiments, in step S5, the temperature of the first-stage distillation is 155℃~170℃, and the vacuum degree during the first-stage distillation is 1Pa~9Pa. When the first-stage distillation meets the above temperature and vacuum conditions, this temperature range ensures that the residual unreacted free fatty acids have sufficient volatility, allowing them to be smoothly distilled out of the system under vacuum, effectively reducing the acid value of the final product. This vacuum condition can further lower the boiling point of the system, promoting the efficient separation of low-boiling-point free fatty acids at relatively low temperatures, avoiding thermal degradation of target components such as diglycerides, ensuring product quality stability, and simultaneously achieving preliminary efficient separation of free fatty acids and diglycerides, creating favorable conditions for deep purification by second-stage distillation.

[0058] In some embodiments, in step S5, the temperature of the secondary distillation is 220℃~280℃, and the vacuum degree during the secondary distillation is 0.1Pa to 1.5Pa. In step S5, the temperature range of 220℃~280℃ ensures that the high-boiling-point diglycerides have sufficient volatility, allowing them to be fully distilled from the system under high vacuum, avoiding incomplete distillation of diglycerides due to insufficient temperature, and reducing losses caused by the target product remaining in the heavy components. The vacuum condition of 0.1Pa~1.5Pa further lowers the boiling point of the diglycerides, enabling the target product to be separated at a relatively low temperature, effectively avoiding oxidative degradation of diglycerides caused by prolonged high temperatures, protecting the physiological activity of the diglycerides, and ensuring a clear product color, thus improving the quality of the final product.

[0059] In some embodiments, the separation described in steps S1, S3, and S5 is centrifugal separation, and the centrifugal speed is 1000 rpm to 4000 rpm. For example, the centrifugal speed can be 1000 rpm, 1500 rpm, 2000 rpm, 2500 rpm, 3000 rpm, 3500 rpm, 4000 rpm, or a range of any two of these values. When the centrifugal separation meets the above conditions, this speed range can effectively achieve gradient stratification separation of components with different densities, such as the oil phase, aqueous phase, and enzyme protein residue. Setting the lower limit of the speed to 1000 rpm provides sufficient centrifugal field strength, allowing denser enzyme protein particles and insoluble impurities to settle sufficiently to the bottom, preventing enzyme protein impurities from remaining in the oil supernatant, preventing impurities from being carried into subsequent esterification reactions and affecting the lipase catalytic efficiency, while ensuring that the purity of the final product is not affected by residual impurities. The maximum rotational speed is controlled at 4000 rpm to avoid excessive centrifugal force disrupting the emulsification balance of the system, reducing over-emulsification and mixing of the oil and water phases, minimizing entrainment losses of the target oil, and avoiding unnecessary energy consumption, thus helping to control the overall cost of industrial production. This rotational speed range balances separation efficiency and production cost, ensuring rapid and stable execution of each separation step.

[0060] In this application, step S5 is based on the separation principle of selective enzymatic esterification and fractional distillation. The light phase mixture obtained from previous cyclic hydrolysis enrichment is mixed with a protic solvent (such as glycerol) and lipase, and an enzymatic esterification reaction is carried out under mild temperature conditions. This allows the free fatty acids in the light phase to combine with glycerol to form diglycerides. After centrifugation to obtain the esterification supernatant, residual impurities are removed through stepwise distillation (first and second stage distillation) to obtain high-purity diglyceride oil. This step utilizes the specific catalytic ability of lipase to promote the esterification reaction under mild conditions, effectively reducing side reactions and product color changes compared to traditional chemical esterification processes. Simultaneously, the two-step distillation achieves the separation of components with different boiling points. Step S5 solves the problems of high acid value and insufficient purity in the product due to the high free fatty acid content in the light phase mixture, and the difficulty of efficiently separating high-purity diglycerides using existing processes. It also avoids the risk of solvent residue caused by the large amount of chemical reagents used in traditional processes. This process not only effectively reduces the acid value of the final product and improves the purity of diglycerides, ensuring that the diglyceride content in the product remains stable at over 90% (up to 98.83%), but also keeps the residual triglycerides at a low level and reduces the total solvent residue to less than 10 ppm. At the same time, the process has mild reaction conditions, causes less damage to the active components of the oil, results in better product quality, and can further reduce subsequent purification costs.

[0061] Through the production process of any of the above embodiments, diglyceride oil with a diglyceride content of up to 90% can be obtained.

[0062] In some embodiments, the diglyceride oil contains ≥90% by mass; preferably, the diglyceride oil contains 94.78%~98.83% by mass. For example, the diglyceride content can be 94.78%, 95.5%, 96.0%, 96.5%, 97.0%, 97.5%, 98.0%, 98.5%, 98.83%, or a range of any two of these values ​​(e.g., 95.0% to 97.0%, 96.0% to 98.0%, etc.). When the diglyceride content in the diglyceride oil is controlled to meet the above conditions, it can ensure that the product fully exerts its physiological effects such as regulating lipid metabolism and inhibiting weight gain, meeting the application requirements of functional oils; it can also be adapted to the usage standards of multiple fields such as food, medicine, and health products, taking into account both the feasibility of the production process and market competitiveness.

[0063] In some embodiments, the oleic acid value of the diglyceride is 0.12 mgKOH / g to 0.17 mgKOH / g. For example, the acid value can be 0.12 mgKOH / g, 0.13 mgKOH / g, 0.14 mgKOH / g, 0.15 mgKOH / g, 0.16 mgKOH / g, 0.17 mgKOH / g, or a range of any two of these values ​​(e.g., 0.12 mgKOH / g to 0.15 mgKOH / g, 0.14 mgKOH / g to 0.17 mgKOH / g, etc.). When the oleic acid value of the diglyceride is controlled to meet the above conditions, the quality stability of the diglyceride oil is ensured, the product shelf life is extended, and flavor abnormalities caused by free fatty acids are avoided, making the product more suitable for applications in high-end food and pharmaceutical fields.

[0064] In some embodiments, the triglyceride content in the diglyceride oil is ≤5.17% by mass. For example, the triglyceride content can be 0.5%, 1.0%, 1.14%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.17%, or any other value less than or equal to 5.17%, and can be within a range of any two of the above values ​​(e.g., 0.5% to 1.0%, 1.14% to 5.17%, etc.). Controlling the triglyceride content within this range can fully guarantee the purity of the diglycerides in the product, indicating that after the cyclic hydrolysis and esterification process, the vast majority of the raw material triglycerides have been converted. A low triglyceride content not only allows diglycerides to fully exert their physiological activity and improve the product's functional properties, but also meets the quality standards for high-end functional oil products, enhancing the product's market competitiveness.

[0065] In some embodiments, the total solvent residue in the diglyceride oil is ≤10 ppm. For example, the total solvent residue can be 0 ppm, 1 ppm, 2 ppm, 3 ppm, 4 ppm, 5 ppm, 6 ppm, 7 ppm, 8 ppm, 9 ppm, 10 ppm, or a range of any two of these values ​​(e.g., 0 ppm to 5 ppm, 3 ppm to 10 ppm, etc., or the total solvent residue is undetectable). By controlling the total solvent residue within this limit, the food safety of the product can be fully guaranteed, fully complying with the strict requirements of national food and pharmaceutical standards for solvent residue, and avoiding health risks caused by exogenous solvent residue.

[0066] In some embodiments, the diglyceride oil contains ≥90% diglycerides by mass, has an oleic acid value of 0.12 mgKOH / g to 0.17 mgKOH / g, a triglyceride content of ≤5.17% by mass, and total solvent residue of ≤10 ppm. For example, the total solvent residue can be 0 ppm, 1 ppm, 2 ppm, 3 ppm, 4 ppm, 5 ppm, 6 ppm, 7 ppm, 8 ppm, 9 ppm, 10 ppm, or a range of any two of these values ​​(e.g., 0 ppm to 5 ppm, 3 ppm to 10 ppm, etc.). The high diglyceride content and low triglyceride content ensure the full functional activity of the product, the low acid value guarantees the product's flavor and storage stability, and the extremely low solvent residue meets stringent food safety standards, making the product suitable for various high-end application scenarios.

[0067] Example The following examples and comparative examples illustrate the implementation of the production process of this application in more detail. Those skilled in the art will understand that the preparation methods described in this application are merely examples, and any other suitable preparation methods are within the scope of this application. Unless otherwise specified, the reagents, materials, etc., used in the following examples are commercially available, and the apparatus or equipment used are purchased from conventional commercial sales channels. Various tests and evaluations were performed according to the methods described below. Furthermore, unless otherwise specified, "parts" and "%" refer to mass measurements.

[0068] Test methods and equipment Acid value test: The test was conducted in accordance with GB 5009.229-2025, "National Food Safety Standard - Determination of Acid Value in Food". Tests for triglycerides, 1,3-diglycerides, 1,2-diglycerides, and monoglycerides.

[0069] The determination of monoglycerides, diglycerides, triglycerides and glycerol in animal and vegetable oils was carried out in accordance with GB / T 46928-2025 "Determination of monoglycerides, diglycerides and triglycerides and glycerol by high performance size exclusion chromatography (HPSEC)".

[0070] Test for residual solvent: Refer to GB 5009.262-2016 "National Food Safety Standard: Determination of Solvent Residues in Food".

[0071] Example 1 The one-step process for preparing diglyceride oil includes the following steps: S1: Take 10 kg of corn oil, add 1% of Lipozyme TL100 L enzyme (by weight of corn oil) and 2% of water (by weight of corn oil) to a reaction vessel, set the stirring speed to 250 r / min, and carry out the enzymatic hydrolysis reaction at 55℃ for 3 h to obtain the hydrolysis product; centrifuge the hydrolysis product at 4000 r / min for 10 min to obtain the hydrolysis supernatant.

[0072] S2: The hydrolysate supernatant from step (1) or other steps is distilled to obtain a light phase and a heavy phase; the distillation temperature is 190℃.

[0073] S3: Using the heavy phase obtained from distillation as a substrate, add 2% water by mass of the heavy phase and 1% Lipozyme TL100 L enzyme by mass of the heavy phase for hydrolysis; During hydrolysis: the stirring rate was set to 250 r / min, the reaction temperature to 55℃, and the reaction time to 3 h to obtain the hydrolysis product; the aqueous phase of the hydrolysis product was separated by centrifugation (centrifuged at 4000 r / min for 10 min) to obtain the hydrolysis supernatant.

[0074] S4: Repeat steps S2 and S3 twice (N=2). This involves three rounds of enzymatic hydrolysis and distillation to separate the light phase. All collected light phases are then mixed to obtain a light phase mixture.

[0075] S5: Add 20% of the mass of glycerol and 2% of the mass of lipase SMG1 to the light phase mixture, place it in a reaction vessel, set the stirring speed to 250 r / min, and carry out the enzymatic esterification reaction at 60℃ for 3 h; after the reaction is completed, centrifuge (centrifuge at 4000 r / min for 10 min) to obtain the esterification supernatant. The esterified supernatant is then distilled; the distillation process includes primary distillation and secondary distillation, and the light phase obtained from the secondary distillation is the diglyceride oil product. The first-stage distillation was set at a distillation temperature of 160℃ and a vacuum degree of 8Pa. Two-stage distillation, with the distillation temperature set at 260℃ and the vacuum degree at 1Pa.

[0076] Example 2 The one-step process for preparing diglyceride oil includes the following steps: S1: Take 10 kg of soybean oil, add 1% Palatase 20000L enzyme (by weight of soybean oil) and 1.5% water (by weight of soybean oil), place in a reaction vessel, set the stirring speed to 300 r / min, and carry out the enzymatic hydrolysis reaction at 60℃ for 3 h to obtain the hydrolysis product. Centrifuge the hydrolysis product at 4000 r / min for 10 min to separate the aqueous phase and obtain the hydrolysis supernatant.

[0077] S2: The hydrolysate supernatant from step (1) or other steps is distilled to obtain a light phase and a heavy phase; the distillation temperature is 185℃.

[0078] S3: Using the heavy phase obtained from distillation as a substrate, add 1.5% water by weight of the heavy phase and 1% Palatase 20000L enzyme by weight of the heavy phase for hydrolysis; During hydrolysis: set the stirring speed to 300 r / min, the reaction temperature to 60℃, and the reaction time to 3 h to obtain the hydrolysis product; centrifuge the hydrolysis product to separate the aqueous phase (centrifuge at 4000 r / min for 10 min) to obtain the hydrolysis supernatant.

[0079] S4: Repeat steps S2 and S3 three times (N=3). This involves a total of four rounds of enzymatic hydrolysis and distillation to separate the light phase. All collected light phases are then mixed to obtain a light phase mixture. S5: Add 30% glycerol and 4% Candida antarcticis lipase B by weight of the light phase mixture to the light phase mixture, place it in a reaction vessel, set the stirring speed to 300 r / min, and carry out the enzymatic esterification reaction at 55℃ for 4 h; after the reaction is completed, centrifuge (centrifuge at 4000 r / min for 10 min) to obtain the esterification supernatant. The esterified supernatant is then distilled; the distillation process includes primary distillation and secondary distillation, and the light phase obtained from the secondary distillation is the diglyceride oil product. The first-stage distillation was set at a distillation temperature of 155℃ and a vacuum of 8Pa. Two-stage distillation, with a distillation temperature of 270℃ and a vacuum of less than 1 Pa (controlled between 0.5 and 0.8 Pa).

[0080] Example 3 The one-step process for preparing diglyceride oil includes the following steps: S1: Take 10 kg of rapeseed oil, add 1.2% (by weight of rapeseed oil) of Lipozyme RMIM enzyme and 3% (by weight of rapeseed oil) of water, place in a reaction vessel, set the stirring speed to 200 r / min, and carry out the enzymatic hydrolysis reaction at 65℃ for 1 h to obtain the hydrolysis product. Centrifuge the hydrolysis product at 4000 r / min for 10 min to separate the aqueous phase and obtain the hydrolysis supernatant.

[0081] S2: The hydrolysate supernatant from step (1) or other steps is distilled to obtain a light phase and a heavy phase; the distillation temperature is 200℃.

[0082] S3: Using the heavy phase obtained from distillation as a substrate, add 3% water by weight of the heavy phase and 1.2% Lipozyme RMIM enzyme by weight of the heavy phase for hydrolysis; During hydrolysis: set the stirring speed to 200 r / min and react at 65℃ for 1 h to obtain the hydrolysis product; centrifuge the hydrolysis product to separate the aqueous phase (centrifuge at 4000 r / min for 10 min) to obtain the hydrolysis supernatant.

[0083] S4: Repeat steps S2 and S3 twice (N=2). This involves three rounds of enzymatic hydrolysis and distillation to separate the light phase. All collected light phases are then mixed to obtain a light phase mixture. S5: Add 40% by weight of glycerol and 10% by weight of Lipase G50 to the light phase mixture, place it in a reaction vessel, set the stirring speed to 200 r / min, and carry out the enzymatic esterification reaction at 55℃ for 3 h; after the reaction is completed, centrifuge (centrifuge at 4000 r / min for 10 min) to obtain the esterification supernatant. The esterified supernatant is then distilled; the distillation process includes primary distillation and secondary distillation, and the light phase obtained from the secondary distillation is the diglyceride oil product. In the first stage of distillation, the distillation temperature is set at 170℃ and the vacuum degree is less than 5Pa (controlled between 2 and 4Pa). Two-stage distillation, with a distillation temperature of 280℃ and a vacuum of less than 0.5 Pa (controlled between 0.1 and 0.4 Pa).

[0084] Example 4 The one-step process for preparing diglyceride oil includes the following steps: S1: Take 10 kg of corn oil, add 5% (by weight of corn oil) of hydrolytic enzyme (including Lipozyme TL100 L enzyme and Palatase 20000 L enzyme, with a mass ratio of Lipozyme TL100 L enzyme and Palatase 20000 L enzyme of 1:1) and 3.5% (by weight of corn oil) of water to a reaction vessel, set the stirring speed to 250 r / min, and carry out the enzymatic hydrolysis reaction at 65℃ for 4 h to obtain the hydrolysis product; centrifuge the hydrolysis product at 1000 r / min for 30 min to obtain the hydrolysis supernatant.

[0085] S2: The hydrolysate supernatant from step (1) or other steps is distilled to obtain a light phase and a heavy phase; the distillation temperature is 200℃.

[0086] S3: Using the heavy phase obtained from distillation as a substrate, add 3.5% water by mass of the heavy phase, and add 5% hydrolytic enzyme by mass of the heavy phase (the hydrolytic enzymes include Lipozyme TL100 L enzyme and Palatase 20000 L enzyme, and the mass ratio of Lipozyme TL100 L enzyme to Palatase 20000 L enzyme is 1:1) to carry out hydrolysis; During hydrolysis: the stirring rate was set to 250 r / min, the reaction temperature to 65℃, and the reaction time to 4 h to obtain the hydrolysis product; the aqueous phase of the hydrolysis product was separated by centrifugation (centrifuged at 1000 r / min for 30 min) to obtain the hydrolysis supernatant.

[0087] S4: Repeat steps S2 and S3 twice (N=5). This involves a total of six rounds of enzymatic hydrolysis and distillation to separate the light phase. All collected light phases are then mixed to obtain a light phase mixture.

[0088] S5: Add 40% by weight of glycerol and 10% by weight of lipase (lipases include Lipase G50 and Candida antarcticis lipase B, with a mass ratio of Lipase G50 to Candida antarcticis lipase B of 1:1) to the light phase mixture, place it in a reaction vessel, set the stirring speed to 250 r / min, and carry out the enzymatic esterification reaction at 65℃ for 4 h; after the reaction is completed, centrifuge (centrifuge at 1000 r / min for 30 min) to obtain the esterification supernatant. The esterified supernatant is then distilled; the distillation process includes primary distillation and secondary distillation, and the light phase obtained from the secondary distillation is the diglyceride oil product. The first-stage distillation was set at a distillation temperature of 170℃ and a vacuum degree of 9Pa. Two-stage distillation, with a distillation temperature of 280℃ and a vacuum of 1.8Pa.

[0089] Example 5 The one-step process for preparing diglyceride oil includes the following steps: S1: Take 10 kg of corn oil, add 3% of Lipozyme TL100 L enzyme (by weight of corn oil) and 2% of water (by weight of corn oil) to a reaction vessel, set the stirring speed to 250 r / min, and carry out the enzymatic hydrolysis reaction at 60℃ for 2.5 h to obtain the hydrolysis product; centrifuge the hydrolysis product at 2000 r / min for 20 min to obtain the hydrolysis supernatant.

[0090] S2: The hydrolysate supernatant from step S1 or other steps is distilled to obtain a light phase and a heavy phase; the distillation temperature is 195℃.

[0091] S3: Using the heavy phase obtained from distillation as a substrate, add 2.5% water by mass of the heavy phase and 3% Lipozyme TL100 L enzyme by mass of the heavy phase for hydrolysis; During hydrolysis: set the stirring rate to 250 r / min, the reaction temperature to 55℃, and the reaction time to 3 h to obtain the hydrolysis product; centrifuge the aqueous phase of the hydrolysis product (centrifuge at 2000 r / min for 20 min) to obtain the hydrolysis supernatant.

[0092] S4: Repeat steps S2 and S3 twice (N=6). This involves a total of seven rounds of enzymatic hydrolysis and distillation to separate the light phase. All collected light phases are then mixed to obtain a light phase mixture.

[0093] S5: Add 25% glycerol and 6% lipase SMG1 (by weight of the light phase mixture) to the light phase mixture, place it in a reaction vessel, set the stirring speed to 250 r / min, and carry out the enzymatic esterification reaction at 60℃ for 2.5 h; after the reaction is completed, centrifuge (centrifuge at 2000 r / min for 20 min) to obtain the esterification supernatant; The esterified supernatant is then distilled; the distillation process includes primary distillation and secondary distillation, and the light phase obtained from the secondary distillation is the diglyceride oil product. The first-stage distillation was set at a distillation temperature of 160℃ and a vacuum degree of 6Pa. Two-stage distillation, with a distillation temperature of 240℃ and a vacuum of 0.8Pa.

[0094] Comparative Example 1 The production process for preparing diglyceride oil includes the following steps: (1) Take 10 kg of corn oil, add 30% of the weight of corn oil glycerol and 5% of the weight of corn oil lipase SMG1, place them in a reaction vessel, set the stirring speed to 250 r / min, and carry out the enzymatic esterification reaction at 60℃ for 3 h.

[0095] (2) After the reaction is complete, centrifuge (centrifuge at 4000 r / min for 10 min) to obtain the esterified supernatant; The esterified supernatant is then distilled; the distillation process includes primary distillation and secondary distillation, and the light phase obtained from the secondary distillation is the diglyceride oil product. In the first stage of distillation, the distillation temperature is set at 160℃ and the vacuum degree is less than 10Pa (controlled between 6 and 9Pa). Two-stage distillation, with the distillation temperature set at 260℃ and the vacuum degree less than 2Pa (controlled between 0.5 and 1.5Pa).

[0096] Comparative Example 2 The production process for preparing diglyceride oil includes the following steps: (1) Take 10 kg of corn oil, add 1% of Lipozyme TL100 L enzyme and 2% of water by weight of corn oil into a reaction vessel, set the stirring speed to 200 r / min, the reaction temperature to 55℃ and the reaction time to 4 h, and obtain the hydrolysis product. Centrifuge the hydrolysis product at 4000 r / min for 10 min to obtain the hydrolysis supernatant. (2) The hydrolyzed supernatant obtained in step (1) is distilled at a temperature of 190°C to obtain a light phase and a heavy phase.

[0097] (3) Add 20% of the light phase mass of glycerol and 2% of the light phase mass of lipase SMG1 to the collected light phase, place it in a reaction vessel, set the stirring speed to 200 r / min, and carry out the enzymatic esterification reaction at 60℃ for 3 h; after the reaction is completed, centrifuge (centrifuge at 4000 r / min for 10 min) to obtain the esterification supernatant. The esterified supernatant is then distilled; the distillation process includes primary distillation and secondary distillation, and the light phase obtained from the secondary distillation is the diglyceride oil product. In the first stage of distillation, the distillation temperature is set at 160℃ and the vacuum degree is less than 10Pa (controlled between 6 and 9Pa). Two-stage distillation, with the distillation temperature set at 260℃ and the vacuum degree less than 2Pa (controlled between 0.5 and 1.5Pa).

[0098] Comparative Example 3 The production process for preparing diglyceride oil includes the following steps: Take 10 kg of corn oil, add 40% glycerol (by weight of corn oil) and 5% Lipase G50 (by weight of corn oil), place in a reaction vessel, set the stirring speed to 300 r / min, and carry out the enzymatic esterification reaction at 55℃ for 3 h. After the reaction is completed, centrifuge (centrifuge at 4000 r / min for 10 min) to obtain the esterification supernatant; the esterification supernatant is then distilled; the distillation process includes primary distillation and secondary distillation, and the light phase obtained from the secondary distillation is the diglyceride oil product; The first-stage distillation is set at a distillation temperature of 170℃ and a vacuum degree of less than 5Pa (controlled between 1 and 4Pa). The secondary distillation is set at a distillation temperature of 280℃ and a vacuum degree of less than 0.5Pa (controlled between 0.1 and 0.4Pa).

[0099] Comparative Example 4 The production process for preparing diglyceride oil includes the following steps: (1) Take 10 kg of corn oil, add 1% of Lipozyme TL100 L enzyme and 2% of water by weight of corn oil into a reaction vessel, set the stirring speed to 200 r / min, react at 55℃ for 4 h to obtain hydrolysis product, centrifuge the hydrolysis product at 4000 r / min for 10 min to obtain supernatant. (2) Add 20% of the supernatant mass of glycerol and 2% of the supernatant mass of lipase SMG1 to the supernatant obtained in step (1), place it in a reaction vessel, set the stirring speed to 200 r / min, and carry out the enzymatic esterification reaction at 60℃ for 3 h. After the reaction is completed, centrifuge (centrifuge at 4000 r / min for 10 min). The supernatant obtained is the diglyceride oil product.

[0100] Comparative Example 5 The one-step process for preparing diglyceride oil includes the following steps: In step S5, the distillation process is a single-stage distillation, and the heavy phase after the single-stage distillation is the diglyceride oil product; the single-stage distillation is set at a distillation temperature of 260°C and a vacuum degree of 1 Pa. The remaining conditions are the same as in Example 1.

[0101] The diglyceride oil products of Examples 1 to 5 and Comparative Examples 1 to 5 were tested, and the test results are summarized in Table 1.

[0102] Table 1

[0103] As can be seen from the comparison of the experimental data of the above embodiments and comparative examples, the embodiments of this application effectively convert vegetable oil through enzymatic cyclic hydrolysis. The light phase collected after molecular distillation is then subjected to a highly efficient enzymatic esterification reaction with glycerol, and further purified by two-stage molecular distillation. This significantly increases the content of diglycerides in the product to over 90%, with a maximum of 98.83%, while effectively controlling the acid value and by-product content.

[0104] In contrast, the comparative examples that did not undergo sufficient enzymatic hydrolysis or lacked effective purification steps had significantly lower diglyceride content than the examples in this application.

[0105] It should be noted that, in this document, the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, or article that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, or article.

[0106] The various embodiments in this specification are described in a related manner. The same or similar parts between the various embodiments can be referred to each other. Each embodiment focuses on describing the differences from other embodiments.

[0107] The above are merely embodiments of this application and are not intended to limit the scope of protection of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of protection of this application.

Claims

1. A one-step process for preparing diglyceride oil, characterized in that, Includes the following steps: S1: Mix vegetable oil and water, add hydrolytic enzyme to carry out enzymatic hydrolysis, and then separate to obtain the hydrolysis supernatant; S2: The hydrolysate supernatant is distilled to obtain a light phase and a heavy phase; S3: Using the aforementioned heavy phase as a substrate, add hydrolytic enzyme and water to continue the enzymatic hydrolysis reaction, and obtain the hydrolysis supernatant after separation; S4: Repeat steps S2 and S3 to complete N distillation processes and N enzymatic hydrolysis reactions, collect the light phase obtained from each distillation process, and obtain a light phase mixture. S5: The light phase mixture from step S4 is mixed with a protic solvent and lipase to carry out an enzymatic esterification reaction. After separation, the esterification supernatant is obtained. The esterification supernatant is then distilled to obtain diglyceride oil.

2. The production process according to claim 1, characterized in that, Step S1 satisfies at least one of the following conditions: (1) The hydrolytic enzyme is at least one of Lipozyme RMIM, Lipozyme TL100 L, and Palatase 20000 L; (2) Based on the mass of the vegetable oil, the mass of the hydrolytic enzyme added is 1% to 5%; (3) Based on the mass of the vegetable oil, the mass of water added is 1% to 3.5%; (4) The temperature of the enzymatic hydrolysis reaction is 55℃~65℃; (5) The time for the enzymatic hydrolysis reaction is 1h to 4h; (6) The vegetable oil includes at least one of corn oil, soybean oil, rapeseed oil, peanut oil, sunflower oil, olive oil and flaxseed oil.

3. The production process according to claim 1, characterized in that, In step S2, the distillation temperature is 180℃~200℃.

4. The production process according to claim 1, characterized in that, Step S3 satisfies at least one of the following conditions: (1) The hydrolytic enzyme is at least one of Lipozyme RMIM, Lipozyme TL100 L, and Palatase 20000 L; (2) Based on the mass of the heavy phase, the mass of the added hydrolytic enzyme is 1% to 5%; (3) Based on the mass of the heavy phase, the mass of water added is 1% to 3.5%; (4) The temperature of the enzymatic hydrolysis reaction is 55℃~65℃; (5) The time for the enzymatic hydrolysis reaction is 1h to 4h.

5. The production process according to claim 1, characterized in that, In step S4, N≥2; Preferably, 3 ≤ N ≤ 6.

6. The production process according to claim 1, characterized in that, Step S5 satisfies at least one of the following conditions: (1) The lipase is at least one of Lipase G50, Candida antarcticis lipase B, and lipase SMG1; (2) The protic solvent includes at least one of glycerol, ethylene glycol, and methanol; (3) Based on the mass of the light phase mixture, the mass of the protic solvent added is 15%~40%; (4) Based on the mass of the light phase mixture, the mass of the added lipase is 1% to 10%; (5) The temperature of the enzymatic esterification reaction is 55℃~65℃; (6) The time for the enzymatic esterification reaction is 1h to 4h; (7) The distillation process includes primary distillation and secondary distillation; The temperature of the first-stage distillation is 150℃~170℃, and the vacuum degree during the first-stage distillation is less than 10Pa. The temperature of the secondary distillation is 200℃~280℃, and the vacuum degree during the secondary distillation is less than 2Pa.

7. The production process according to claim 6, characterized in that, In step S5, the temperature of the first-stage distillation is 155℃~170℃, and the vacuum degree during the first-stage distillation is 1Pa to 9Pa. And / or, the temperature of the secondary distillation is 220℃~280℃, and the vacuum degree during the secondary distillation is 0.1Pa to 1.5Pa.

8. The production process according to claim 1, characterized in that, The separation described in steps S1, S3, and S5 is centrifugal separation, and the centrifugal rotation speed is 1000 rpm to 4000 rpm.

9. The production process according to claim 1, characterized in that, The diglyceride oil meets at least one of the following conditions: (1) The mass content of diglycerides in the diglyceride oil is ≥90%; (2) The oleic acid value of the diglyceride is 0.12 mg KOH / g to 0.17 mg KOH / g; (3) The triglyceride content in the diglyceride oil is ≤5.17% by mass; (4) The total residual solvent in the diglyceride oil is ≤10ppm.

10. The production process according to claim 9, characterized in that, The diglyceride oil meets at least one of the following conditions: (1) Preferably, the mass content of diglycerides in the diglyceride oil is 94.78%~98.83%; (2) The triglyceride content in the diglyceride oil is 1.14%~5.17% by mass; (3) The total solvent residue in the diglyceride oil was not detected.

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