A method for the solvent-free enzymatic production of diglyceride oil
By preparing a liquid eutectic mixture and reacting it with glycerol, the problem of high-melting-point saturated fatty acids being difficult to utilize in solvent-free enzymatic methods was solved, achieving efficient preparation of diglyceride oil, reducing production costs, and maintaining enzyme activity.
Patent Information
- Application Number
- CN202511224687.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-29
- Publication Date
- 2025-11-28
- Estimated Expiration
- 2045-08-29
AI Technical Summary
In existing solventless enzymatic methods for preparing diglyceride oil, high-melting-point saturated fatty acids are difficult to utilize, and high-temperature operation can easily lead to lipase inactivation, affecting production efficiency and cost.
The eutectic mixture technology is used to mix saturated fatty acid A and saturated fatty acid B to form a liquid eutectic mixture, which reacts with glycerol and lipase. The esterification reaction temperature is controlled at 35℃-78℃. The esterification reaction is carried out by separating and recycling saturated fatty acids to form a liquid eutectic mixture.
This method effectively utilizes high-melting-point saturated fatty acids at lower temperatures, maintains lipase activity, improves production efficiency, reduces costs, and increases diglyceride yield by controlling excessive glycerol to inhibit byproduct formation.
Smart Images
Figure CN120718969B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of oil processing, and specifically to a solvent-free enzymatic method for preparing diglyceride oil. Background Technology
[0002] Diacylglycerol (DAG) is a compound formed by the combination of two fatty acid molecules and a glycerol molecule. It can be obtained by esterification of glycerol with two fatty acids. The content of diglycerol in natural oils is usually low, but in some vegetable oils, such as cottonseed oil, the content can reach higher levels. DAG has many advantages, including safety, nutrition, good processing suitability, and high biocompatibility, making it a multifunctional additive with wide applications in the food, pharmaceutical, and chemical (cosmetic) industries.
[0003] In existing solvent-free enzymatic methods for preparing DAG, some saturated fatty acids have high melting points (they are solid at room temperature) and need to be heated to melt them into a liquid state before they can react with glycerol. Furthermore, since the reaction between fatty acids and glycerol is slow, lipase needs to be added to accelerate the reaction. However, excessively high temperatures can easily deactivate the lipase in the reaction system. Therefore, unsaturated fatty acids, which are liquid at room temperature, are often used as substrates for preparing DAG in this field. Summary of the Invention
[0004] In view of this, the present invention provides a solventless enzymatic method for preparing diglyceride oil, in order to solve the problem that high-melting-point saturated fatty acids are difficult to utilize in existing solventless enzymatic methods for preparing diglyceride oil.
[0005] To achieve the above objectives, the present invention provides a solventless enzymatic method for preparing diglyceride oil, comprising the following steps: S1, mixing saturated fatty acid A and saturated fatty acid B to prepare a liquid eutectic mixture; S2, mixing the eutectic mixture, glycerol, and lipase in a reaction system to carry out an esterification reaction to obtain diglyceride; S3, separating the prepared diglyceride, and adding saturated fatty acid A and / or saturated fatty acid B back into the reaction system to reform a liquid eutectic mixture, and repeating step 2; wherein, in S1, saturated fatty acid A and saturated fatty acid B are both selected from C10-C20 alkyl acids, in S2, the mass ratio of the eutectic mixture to the glycerol is 1:(1-5), and the temperature of the esterification reaction is 35℃-78℃.
[0006] In one embodiment, in S1 above, the molar ratio of saturated fatty acid A to saturated fatty acid B is 1:(1-9).
[0007] In one embodiment, in S2 above, the mass ratio of the eutectic mixture to glycerol is 1:(1.5-3).
[0008] In one embodiment, in S2 above, the lipase is 2%-10% of the total mass of the eutectic mixture and glycerol.
[0009] In one embodiment, in S2 above, the lipase is selected from Candida pleuropsis lipase, Candida antarcticis lipase (Novozym 435), Mucor miehei (Lipozyme RM IM), Thermomyces lanuginosus (Lipozyme TL IM), and Aspergillus oryzae (AOL).
[0010] In one embodiment, the esterification reaction temperature in S2 above is 40-75°C.
[0011] In one embodiment, the esterification reaction temperature in S2 above is 50-60°C.
[0012] In one implementation, the esterification reaction time in S2 above is 6-24 hours.
[0013] In one embodiment, the esterification reaction time in S2 above is 10-15 hours.
[0014] As one implementation method, in S2 above, the reaction system is subjected to ultrasonic treatment during the esterification reaction, and the frequency of the ultrasonic waves is 500-1000kHz.
[0015] The beneficial effects of this invention are as follows:
[0016] A liquid eutectic mixture is prepared by mixing saturated fatty acid A and saturated fatty acid B (C10-C20 alkyl acids), which are solid at room temperature. The mixture formed by mixing two (or more) components is called an eutectic mixture, and its melting temperature is lower than that of each individual component. The eutectic mixture serves as one of the substrates for esterification reactions. It is mixed with another glycerol substrate (in excess relative to the eutectic mixture) in liquid form to increase the substrate concentration and simultaneously increase the contact area between the two substrates and the lipase, thereby increasing the reaction rate. On the one hand, by preparing the eutectic mixture, saturated fatty acid A and saturated fatty acid B can be utilized at lower temperatures and without lipase inactivation, reducing high-temperature operations and lowering production costs, while simultaneously ensuring lipase activity and thus production efficiency. On the other hand, as the esterification reaction continues, one component (saturated fatty acid A or saturated fatty acid B) in the eutectic mixture preferentially reacts with glycerol to generate diglycerides. The remaining saturated fatty acid A and saturated fatty acid B are difficult to maintain a stable liquid state, and solid saturated fatty acids begin to precipitate. After the diglycerides are separated and prepared, saturated fatty acid A and / or saturated fatty acid B can be added back to the reaction system according to the reaction temperature at this time to make it re-form a liquid eutectic mixture, which then reacts with the excess glycerol substrate in the system, and the esterification reaction is repeated to continuously generate diglycerides.
[0017] In summary, the solventless enzymatic method for preparing diglyceride oil provided by this invention solves the problem of the difficulty in utilizing high-melting-point saturated fatty acids in existing solventless enzymatic methods for preparing diglyceride oil. Attached Figure Description
[0018] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:
[0019] Figure 1 This is a process flow diagram of the technical solution. Detailed Implementation
[0020] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.
[0021] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.
[0022] As analyzed in the background section of this invention, the prior art has the problem that high-melting-point saturated fatty acids are difficult to utilize in the solventless enzymatic preparation of diglyceride oil. In order to solve the above problems, this invention provides a solventless enzymatic method for preparing diglyceride oil, which solves the problems of excessively high esterification reaction temperature and easy inactivation of lipase due to high temperature when using high-melting-point saturated fatty acids in the prior art.
[0023] In one embodiment of the present invention, a solvent-free enzymatic method for preparing diglyceride oil is provided, comprising the following steps: S1, mixing saturated fatty acid A and saturated fatty acid B to prepare a liquid eutectic mixture; S2, mixing the eutectic mixture, glycerol, and lipase in a reaction system to carry out an esterification reaction to obtain diglyceride; S3, separating the obtained diglyceride, and adding saturated fatty acid A and / or saturated fatty acid B back into the reaction system to reform a liquid eutectic mixture, and repeating step 2; wherein, in S1, saturated fatty acid A and saturated fatty acid B are both selected from C10-C20 alkyl acids; in S2, the mass ratio of the eutectic mixture to glycerol is 1:(1-5); the esterification reaction temperature is 35℃-78℃; the specific process is as follows. Figure 1 As shown.
[0024] A mixture formed by mixing two (or more) components is called a eutectic mixture. This mixture melts at a temperature lower than the melting temperatures of its individual components. The lowest melting point achievable by mixing two (or more) components is called the eutectic point. When the esterification reaction temperature drops to or below the eutectic point temperature, the eutectic mixture precipitates simultaneously as a eutectic composition, i.e., saturated fatty acid A and saturated fatty acid B solids precipitate simultaneously. As the esterification reaction continues, the resulting diglyceride is separated. In S2, the mass ratio of the eutectic mixture to glycerol is controlled at 1:(1-5), and the esterification reaction temperature is 35℃-78℃. Based on this reaction temperature, saturated fatty acid A and / or saturated fatty acid B can be added back to the reaction system to reform a liquid eutectic mixture, which then reacts with excess glycerol substrate in the system, cyclically undergoing esterification and continuously generating diglycerides.
[0025] Specifically, in the technical solution of this invention, on the one hand, by preparing a eutectic mixture, saturated fatty acid A and saturated fatty acid B can be utilized at lower temperatures and without lipase inactivation, reducing high-temperature operation and production costs, while ensuring lipase activity and thus production efficiency. On the other hand, as the esterification reaction continues, saturated fatty acid A and / or saturated fatty acid B are added back to the reaction system to reform a liquid eutectic mixture, which then reacts with excess glycerol substrate in the system, cyclically undergoing esterification and continuously generating diglycerides. In S2, the mass ratio of the eutectic mixture to glycerol is controlled at 1:(1-5) to maximize the mixing of liquid saturated fatty acids and liquid glycerol in liquid form for the esterification reaction. This not only maximizes the reaction contact area between the two, but the excess glycerol also inhibits the formation of the byproduct triglycerides, pushing the reaction equilibrium towards the positive reaction direction. Furthermore, since glycerol is cheaper than saturated fatty acids, its excess use can increase the conversion rate of saturated fatty acids and reduce raw material waste. When the reaction finally stops, the remaining substance is mainly unreacted glycerol, which can be easily recovered and reused through molecular distillation. In this scheme, the temperature of the esterification reaction is controlled between 35℃ and 78℃. First, this avoids the reaction temperature from being too high and damaging the enzyme activity. Second, it avoids the generation of high-temperature byproducts, which would introduce impurities into the system and increase the number of separation operations and costs.
[0026] In summary, the solventless enzymatic method for preparing diglyceride oil provided by this invention solves the problem of the difficulty in utilizing high-melting-point saturated fatty acids in existing solventless enzymatic methods for preparing diglyceride oil.
[0027] In one embodiment of the present invention, in S1, the molar ratio of saturated fatty acid A to saturated fatty acid B is 1:(1-9). This molar ratio of saturated fatty acid A to saturated fatty acid B can be any value within the range of 1:(1-9), such as 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, etc. Different molar ratios of saturated fatty acid A and saturated fatty acid B can achieve different eutectic states, meaning the resulting eutectic mixtures have different eutectic points. Combining saturated fatty acid A and saturated fatty acid B in different molar ratios allows them to melt into a liquid state below their respective melting points. The molar ratio of saturated fatty acid A to saturated fatty acid B can be adjusted according to the optimal temperature (reaction temperature) of the enzyme-catalyzed reaction to ensure that the reaction system remains liquid when the reaction temperature is above the eutectic point temperature.
[0028] In one embodiment of the present invention, in S2, the mass ratio of the eutectic mixture to glycerol is 1:(1.5-3). The mass ratio of the eutectic mixture to glycerol can be any value within the range of 1:(1.5-3), such as 1:1.5, 1:1.6, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2.0, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, or 1:3. An excess of glycerol in the reaction system can promote the forward reaction, not only inhibiting the formation of triglycerides, but also making glycerol, as the main residue in the system when the reaction stops, easy to recover and reuse compared to saturated fatty acids.
[0029] In one embodiment of the present invention, in S2, the lipase is 2%-10% of the total mass of the eutectic mixture and glycerol. The lipase can be any value within the range of 2%-10%, such as 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the total mass of the eutectic mixture and glycerol. Using lipase within this range ensures that the lipase retains its biological activity before the lipid reaction ceases, allowing it to be reused to catalyze the esterification reaction.
[0030] In one embodiment of the present invention, in S2, the lipase is selected from Candida albicans lipase, Candida antarcticis lipase (Novozym 435), Mucor miehei (Lipozyme RM IM), Thermomyces lanuginosus (Lipozyme TL IM), and Aspergillus oryzae (AOL). The selected lipase can precisely recognize the hydroxyl structure in the glycerol substrate, preferentially catalyzing the reaction of fatty acids with glycerol to form 1,3-diglyceride (1,3-DAG), rather than random esterification, avoiding the formation of isomers such as 1,2-DAG, thereby improving the bioactivity of the target product.
[0031] In one embodiment of the present invention, the esterification reaction temperature in S2 is 40-75°C.
[0032] In one embodiment of the present invention, the esterification reaction temperature in S2 is 50-60°C.
[0033] Specifically, controlling the esterification reaction temperature effectively ensures the activity of lipase, which helps to increase the reaction rate and improve the production efficiency of diglycerides. The esterification reaction temperature can be any value within the range of 40-75℃, such as 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, and 75℃. These values will not be elaborated further here, but the preferred heating temperature is any value within the range of 50-60℃. It is important to note that the temperature should not be too low, to avoid reaching the eutectic point of the eutectic mixture, which would cause the eutectic mixture to precipitate in crystal form, reducing the substrate concentration for the esterification reaction. At the same time, the temperature should not be too high, to prevent the lipase from losing its catalytic activity.
[0034] In one embodiment of the present invention, the esterification reaction time in S2 is 6-24 hours.
[0035] In one embodiment of the present invention, the esterification reaction time in S2 is 10-15 h.
[0036] Specifically, in one embodiment of the present invention, in S2 above, during the esterification reaction, the reaction system is subjected to ultrasonic treatment at a frequency of 500-1000 kHz. Ultrasonic treatment helps to accelerate the esterification reaction.
[0037] The present invention will be further described in detail below with reference to specific embodiments, which should not be construed as limiting the scope of protection claimed by the present invention. Furthermore, in the following embodiments, unless otherwise specified, the reagents and equipment used are commercially available. If specific processing conditions and methods are not explicitly described in the later embodiments, conditions and methods known in the art can be used for processing. Example 1
[0038] S1. Weigh out 107.7g of hexadecanoic acid (C) according to the molar ratio of saturated fatty acid A to saturated fatty acid B of 3:7. 16 H 32 O2) and 132.5g of decadecanoic acid (C 10 H 20 O2) is mixed to prepare a liquid eutectic mixture with a eutectic point of 23°C; wherein, the melting point of hexadecanoic acid is 61-62.5°C and the melting point of decadecanoic acid is 27-32°C.
[0039] S2. The eutectic mixture, 400g of glycerol and 5% of lipase (Novozym 435) were placed in the reaction system and mixed to carry out the esterification reaction. The reaction temperature was 50℃ and the reaction time was 20h to obtain the product diglyceride. The mass ratio of the eutectic mixture to glycerol was 1:1.67.
[0040] S3. Separate the prepared diglyceride and add a eutectic mixture of hexadecanoic acid and decadecanoic acid in a molar ratio of 3:7 back into the reaction system (the amount added should be consistent with the weight of the separated diglyceride), and repeat step 2.
[0041] Since the esterification reaction temperature is higher than the eutectic point of the eutectic mixture, no saturated fatty acid solids precipitate out, and the reaction system remains liquid. The addition of the eutectic mixture to S3 is beneficial to promote the forward reaction. Example 2
[0042] The difference from Example 1 is that, in S1, 107.7g of hexadecanoic acid (C) was weighed according to a molar ratio of saturated fatty acid A to saturated fatty acid B of 42:58. 16 H 32 O2) and 132.5g of tetradecanoic acid (C 14 H 28 O2) is mixed to prepare a liquid eutectic mixture with a eutectic point of 44.7℃; wherein, the melting point of hexadecanoic acid is 61-62.5℃ and the melting point of tetradecanoic acid is 52-54℃. Example 3
[0043] The difference from Example 1 is that in S2, the reaction temperature of the esterification reaction is 60°C. Example 4
[0044] The difference from Example 1 is that in S2, the esterification reaction system is subjected to ultrasonic treatment with an ultrasonic frequency of 800 kHz and an ultrasonic reaction time of 7 h. Example 5
[0045] The difference from Example 1 is that in S2, the mass ratio of the eutectic mixture to glycerol is 1:3. Example 6
[0046] The difference from Example 1 is that in S2, the mass ratio of the eutectic mixture to glycerol is 1:5.
[0047] Comparative Example 1
[0048] The difference from Example 1 is that in S1, only 240.2g of hexadecanoic acid (C 16 H 32 O2), the esterification reaction temperature remains unchanged, and the esterification reaction temperature is lower than the melting point of hexadecanoic acid.
[0049] Comparative Example 2
[0050] The difference from Example 1 is that in S1, only 240.2g of hexadecanoic acid (C 16 H 32In O2 and S2, the reaction temperature for esterification is 90℃.
[0051] Comparative Example 3
[0052] The difference from Example 1 is that in S1, only 240.2g of hexadecanoic acid (C 16 H 32 In O2 and S2, the reaction temperature for esterification is 120℃.
[0053] The products (containing diglycerides) obtained after the reaction in the examples and comparative examples were subjected to molecular distillation. The molecular distillation was a three-stage molecular distillation, with the following conditions: first stage 108°C, 50 Pa; second stage 135°C, 10 Pa; and third stage 182°C, 1 Pa.
[0054] The specific test results are shown in Table 1.
[0055] Table 1
[0056] Example Diglyceride content % Triglyceride content % % of glycoside content Example 1 84.4 15.5 0.1 Example 2 82.7 17.1 0.2 Example 3 81.5 18.4 0.1 Example 4 84.8 15.1 0.1 Example 5 86.1 13.8 0.1 Example 6 87.4 12.5 0.1 Comparative Example 1 58.1 41.3 0.6 Comparative Example 2 63.2 36.1 0.7 Comparative Example 3 60.4. 38.5 1.1
[0057] Although combining saturated fatty acids A and B in different molar ratios allows high-melting-point saturated fatty acids to melt into a liquid state below their own melting points, Examples 1 and 2 show that when the proportion of high-melting-point saturated fatty acids is high, the eutectic point of the eutectic mixture also increases. Therefore, when the reaction needs to be carried out at a lower temperature, it is necessary to control the amount of high-melting-point saturated fatty acids in saturated fatty acids A and B as much as possible to match the eutectic point with the enzyme activity temperature. The test results in Table 1 show that controlling the amount of high-melting-point saturated fatty acids helps to improve the yield of diglycerides. Compared to Example 1, Example 3 increased the esterification reaction temperature. Although this temperature is higher than the eutectic point of the eutectic mixture, ensuring that saturated fatty acid A and saturated fatty acid B can mix with glycerol in a liquid state, it is also higher than the optimal temperature for lipase catalysis. This results in a decrease in lipase activity and catalytic efficiency, leading to a lower yield of the target product, diglyceride. Therefore, it is best to select the optimal temperature for lipase as the esterification reaction temperature, thereby adjusting the molar ratio between saturated fatty acid A and saturated fatty acid B to ensure that their eutectic points fall within the optimal temperature range for lipase. This invention mixes two saturated fatty acids that are solid at room temperature, forming a liquid eutectic mixture. The diglyceride content produced by the reaction with glycerol is not low. Furthermore, Example 4 adds ultrasonic treatment at a frequency of 800 kHz. Ultrasonic treatment helps to ensure uniform mixing and sufficient contact between saturated fatty acids, glycerol, and lipase, thus accelerating the esterification reaction. In Examples 5-6, the amount of glycerol is gradually increased compared to Example 1. An excess of glycerol in the reaction system not only promotes the forward catalytic reaction and increases the yield of diglyceride but also inhibits the formation of triglycerides. In Comparative Examples 1-3, only high-melting-point saturated fatty acids (hexadecanoic acid) and glycerol and lipase were used for the reaction. In Comparative Example 1, when the esterification reaction temperature was lower than the melting point of hexadecanoic acid, hexadecanoic acid reacted with glycerol and lipase in solid form. Even though the lipase catalytic activity was high at this time, the reaction was slow due to the small reaction contact area, and the yield of the target product was also very low, which did not meet the current production requirements. In Comparative Examples 2-3, the esterification reaction temperature was higher than the melting point of hexadecanoic acid. Although hexadecanoic acid could react with glycerol and lipase in liquid form and the reaction contact area was large, the catalytic activity of lipase was gradually lost at high temperature, the catalytic efficiency decreased, and the yield of the target product decreased. High-temperature operation not only increased production costs but also hindered the improvement of production efficiency.
[0058] Test results show that by controlling the eutectic point of the eutectic mixture within the range of lipase activity and using excess glycerol, the present invention can effectively ensure the enzymatic reaction, improve production efficiency and target product content.
[0059] The above description is merely a preferred embodiment of this disclosure and is not intended to limit this disclosure. Various modifications and variations can be made to this disclosure by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this disclosure should be included within the scope of protection of this disclosure.
Claims
1. A process for the solvent-free, enzymatic production of a diglyceride oil, characterized in that, The method comprises the following steps: S1, mixing saturated fatty acid A and saturated fatty acid B to prepare a liquid eutectic mixture; S2, mixing the eutectic mixture, glycerol and lipase in a reaction system to perform esterification reaction to obtain diglyceride; S3, separating the prepared diglyceride, and adding the saturated fatty acid A and / or the saturated fatty acid B into the reaction system to form a liquid eutectic mixture again, and repeating step 2; In S1, the saturated fatty acid A and the saturated fatty acid B are both selected from C10-C20 alkanoic acid, and the molar ratio of the saturated fatty acid A to the saturated fatty acid B is 1:(1-9); in S2, the mass ratio of the eutectic mixture to the glycerol is 1:(1-5), and the esterification reaction temperature is 35-78℃. The combination of saturated fatty acid A and saturated fatty acid B with different molar ratios can be melted into a liquid state below the melting point of itself, and the molar ratio of saturated fatty acid A to saturated fatty acid B can be adjusted according to the optimum temperature of enzyme catalytic reaction, i.e. the reaction temperature, so that the reaction system is in a liquid state when the reaction temperature is higher than the eutectic point temperature.
2. The process for the solvent-free, enzymatic production of diglyceride oil according to claim 1, characterized in that, In S2, the mass ratio of the eutectic mixture to the glycerol is 1:(1.5-3).
3. The process for the solvent-free, enzymatic production of diglyceride oil according to claim 1, characterized in that, In S2, the lipase is 2-10% of the total mass of the eutectic mixture and the glycerol.
4. The process for the solvent-free, enzymatic production of diglyceride oil according to claim 1, characterized in that, In S2, the lipase is selected from Candida rugosa lipase, Candida antarctica lipase, Mucor miehei lipase, Thermomyces lanuginosus lipase and Aspergillus oryzae lipase.
5. The process for the solvent-free, enzymatic production of diglyceride oil according to claim 1, characterized in that, In S2, the esterification reaction temperature is 40-75℃.
6. The process for the solvent-free, enzymatic production of diglyceride oil according to claim 1, characterized in that, In S2, the esterification reaction temperature is 50-60℃.
7. The process for the solvent-free, enzymatic production of diglyceride oil according to claim 1, characterized in that, In S2, the esterification reaction time is 6-24h.
8. The process for the solvent-free, enzyme-based production of diglyceride oil according to claim 1, characterized in that, In S2, the esterification reaction time is 10-15h.
9. The process for the solvent-free, enzyme-based production of diglyceride oil according to claim 1, characterized in that, In S2, the reaction system is subjected to ultrasonic treatment during the esterification reaction, and the frequency of the ultrasonic wave is 500-1000kHz.