Method for preparing diglyceride oil by solvent-free enzymatic method

Abstract

The invention provides a method for preparing diglyceride oil by a solventless enzymatic method, which 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, and carrying out esterification reaction to obtain diglyceride; s3, separating the prepared diglyceride, adding the saturated fatty acid A and / or the saturated fatty acid B into the reaction system again to form a liquid eutectic mixture again, and repeating the step 2; wherein in the step S1, the saturated fatty acid A and the saturated fatty acid B are selected from C10-C20 alkanoic acid, in the step 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 DEG C. The method solves the problem that the high-melting-point saturated fatty acid is difficult to utilize in the existing technology for preparing the diglyceride oil by the solvent-free enzymatic method.

Description

Technical Field

[0001] The present invention relates to the field of oil processing, and in particular to a method for preparing diglyceride oil by a solvent-free enzymatic method. Background Art

[0002] Diacylceride (DAG) is a compound composed of two fatty acid molecules bound to a glycerol molecule. It can be obtained through the esterification reaction of glycerol with two fatty acids. While diacylglycerol levels are typically low in natural oils and fats, they can reach higher levels in certain vegetable oils, such as cottonseed oil. DAG offers numerous advantages, including safety, nutritional value, good processing suitability, and high human compatibility. It is a versatile additive widely used in the food, pharmaceutical, and chemical (cosmetic) industries.

[0003] When preparing DAG using existing solvent-free enzymatic methods, some saturated fatty acids have high melting points (solid at room temperature) and require heating to melt them into a liquid state before reacting with glycerol. Furthermore, since the reaction between fatty acids and glycerol is slow, the addition of lipase is required to accelerate the reaction. However, excessively high temperatures can easily inactivate the lipase in the reaction system. Therefore, unsaturated fatty acids that are liquid at room temperature are often used as substrates to prepare DAG in this field. Summary of the Invention

[0004] In view of this, the present invention provides a method for preparing diglyceride oil by a solvent-free enzymatic method to solve the problem that high-melting-point saturated fatty acids are difficult to utilize in the existing technology for preparing diglyceride oil by a solvent-free enzymatic method.

[0005] To achieve the above-mentioned object, the present invention provides a method for preparing diglyceride oil by a solvent-free enzymatic method, comprising the following steps: S1, mixing saturated fatty acids A and saturated fatty acids B to prepare a liquid eutectic mixture; S2, placing the eutectic mixture, glycerol and lipase in a reaction system, mixing them, and performing an esterification reaction to obtain diglyceride; S3, separating the prepared diglyceride, and re-adding saturated fatty acids A and / or saturated fatty acids B to the reaction system to re-form a liquid eutectic mixture, and repeating step 2; wherein, in S1, the saturated fatty acids A and saturated fatty acids B are both selected from C10-C20 alkanoic 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°C-78°C.

[0006] As an embodiment, in the above S1, the molar ratio of saturated fatty acid A to saturated fatty acid B is 1:(1-9).

[0007] As an embodiment, in the above S2, the mass ratio of the eutectic mixture to glycerol is 1:(1.5-3).

[0008] As an embodiment, in the above S2, the lipase is 2%-10% of the total mass of the eutectic mixture and glycerol.

[0009] As an embodiment, in the above S2, the lipase is selected from Candida rugosa lipase, Candida antarctica lipase (Novozym 435), Mucor miehei lipase (Lipozyme RM IM), Thermomyces lanuginosus lipase (Lipozyme TL IM) and Aspergillus oryzae lipase (AOL).

[0010] As an embodiment, in the above S2, the esterification reaction temperature is 40-75°C.

[0011] As an embodiment, in the above S2, the esterification reaction temperature is 50-60°C.

[0012] As an embodiment, in the above S2, the esterification reaction time is 6-24 hours.

[0013] As an embodiment, in the above S2, the esterification reaction time is 10-15 hours.

[0014] As an embodiment, in the above S2, during the esterification reaction, the reaction system is subjected to ultrasonic treatment, and the frequency of the ultrasonic wave is 500-1000 kHz.

[0015] The beneficial effects of the present invention are as follows: Saturated fatty acids A and B (C10-C20 alkanoic acids), both solid at room temperature, are mixed to form a liquid eutectic mixture. This mixture, formed by mixing two (or more) components, has a melting point lower than that of the individual components. The eutectic mixture, serving as one of the substrates for the esterification reaction, is mixed with a liquid substrate, glycerol (in excess relative to the eutectic mixture), increasing the substrate concentration and the contact area between the two substrates and the lipase, thereby boosting the reaction rate. The eutectic mixture allows the utilization of saturated fatty acids A and B at low temperatures without inactivating the lipase, reducing high-temperature operations and production costs while maintaining lipase activity and thus production efficiency. On the other hand, as the esterification reaction continues, one component of the eutectic mixture (saturated fatty acid A or saturated fatty acid B) preferentially reacts with glycerol to form diglyceride, and the remaining saturated fatty acid A and saturated fatty acid B are difficult to maintain a stable liquid form, and saturated fatty acid solids begin to precipitate. After the prepared diglyceride is separated, 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 re-form a liquid eutectic mixture, and then react with the excess glycerol substrate in the system, cyclically reacting and continuously generating diglyceride.

[0016] In summary, the method for preparing diglyceride oil by a solvent-free enzymatic method provided by the present invention solves the problem of difficulty in utilizing high-melting-point saturated fatty acids in the existing technology for preparing diglyceride oil by a solvent-free enzymatic method. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings, which constitute part of the present invention, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings: Figure 1 This is the process flow chart of the technical solution. DETAILED DESCRIPTION

[0018] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0019] Unless otherwise defined, technical or scientific terms used in the present invention shall have the same meaning as commonly understood by persons having ordinary skills in the field to which the present invention belongs.

[0020] As analyzed in the background technology of the present invention, the problem that high-melting-point saturated fatty acids are difficult to utilize in the prior art solvent-free enzymatic preparation of diglyceride oil. In order to solve the above problem, the present invention provides a method for preparing diglyceride oil by a solvent-free enzymatic method to solve the problem in the prior art that the esterification reaction temperature is too high when using high-melting-point saturated fatty acids and the high temperature makes lipase easily inactivated.

[0021] In one embodiment of the present invention, the present invention provides a method for preparing diglyceride oil by a solvent-free enzymatic method, comprising the following steps: S1, mixing saturated fatty acid A and saturated fatty acid B to prepare a liquid eutectic mixture; S2, placing the eutectic mixture, glycerol and lipase in a reaction system and mixing them, performing an esterification reaction to obtain diglyceride; S3, separating the prepared diglyceride, and re-adding saturated fatty acid A and / or saturated fatty acid B to the reaction system to re-form a liquid eutectic mixture, and repeating step 2; wherein, in S1, the saturated fatty acid A and the saturated fatty acid B are both selected from C10-C20 alkanoic acids, in S2, the mass ratio of the eutectic mixture to glycerol is 1:(1-5), the temperature of the esterification reaction is 35°C-78°C, and the specific process is as follows. Figure 1 shown.

[0022] A mixture formed by mixing two (or more) components is called a eutectic mixture. The melting temperature of this mixture is lower than the melting temperatures of the individual components. The lowest melting point that can be achieved by mixing two (or more) components is called the eutectic point. When the esterification reaction temperature drops to or below the eutectic point, the eutectic mixture precipitates simultaneously as a eutectic composition, with both saturated fatty acids A and B precipitating as solids. As the esterification reaction proceeds, the resulting diglyceride is separated. In S2, the mass ratio of the eutectic mixture to glycerol is controlled to be 1:(1-5), and the esterification reaction temperature is controlled to be between 35°C and 78°C. Depending on the reaction temperature, saturated fatty acids A and / or B can be added to the reaction system to reform the liquid eutectic mixture, which then reacts with the excess glycerol substrate in the system, cyclically reacting to produce diglyceride.

[0023] Specifically, in the technical solution of the present invention, on the one hand, by preparing a eutectic mixture, the saturated fatty acid A and saturated fatty acid B can be utilized when the lipase is relatively low and not inactivated, reducing high temperature operation, reducing production costs, and ensuring that the lipase activity is simultaneously guaranteed to ensure production efficiency. On the other hand, as the esterification reaction is continuously carried out, saturated fatty acid A and / or saturated fatty acid B are added to the reaction system to form a liquid eutectic mixture, and then react with the excessive glycerol substrate in the system, cyclic esterification occurs and diglyceride is continuously generated. In S2, the mass ratio of the eutectic mixture and glycerol is controlled to be 1: (1-5), to the greatest extent ensure that the liquid saturated fatty acids and liquid glycerol are mixed together in the form of liquid to carry out esterification, not only to the greatest extent ensure the reaction contact area between the two, but also excessive glycerol can suppress the generation of by-product triglyceride, promote the reaction equilibrium to move towards the direction of the positive reaction; and the price of glycerol is lower than the price of saturated fatty acids, excessive use can improve the saturated fatty acid conversion rate, reduce waste of raw materials, and when the final reaction stops, the remaining material is mainly unreacted glycerol, which is easy to recycle and reuse by molecular distillation. In this scheme, the temperature of the esterification reaction is controlled at 35°C-78°C, firstly to avoid the reaction temperature being too high to destroy the enzyme activity, and secondly to avoid the generation of high-temperature by-products, which introduce impurities into the system and increase the number and cost of separation operations.

[0024] In summary, the method for preparing diglyceride oil by a solvent-free enzymatic method provided by the present invention solves the problem of difficulty in utilizing high-melting-point saturated fatty acids in the existing technology for preparing diglyceride oil by a solvent-free enzymatic method.

[0025] 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). The 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, or 1:9. Different molar ratios between saturated fatty acid A and saturated fatty acid B can achieve different eutectic states, i.e., the resulting eutectic mixtures have different eutectic points. Combining saturated fatty acids A and B in different molar ratios can melt into a liquid state at temperatures below their own melting points. The molar ratio between saturated fatty acid A and saturated fatty acid B can be adjusted based on the optimal temperature (reaction temperature) of the enzyme-catalyzed reaction to ensure that the reaction system exhibits a liquid state when the reaction temperature is above the eutectic point.

[0026] In one embodiment of the present invention, in S2, the mass ratio of the eutectic mixture to glycerol is 1: (1.5-3). Wherein, the mass ratio of the eutectic mixture to glycerol can be 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, 1: 3, etc. Any point value within the range of 1: (1.5-3). Excess glycerol in the reaction system can help promote the forward direction of the catalytic reaction, not only can it inhibit the formation of triglycerides, but when the reaction stops, glycerol is the main residue in the system, and the recovery operation is simple, which is easier to recycle and reuse than saturated fatty acids.

[0027] In one embodiment of the present invention, in S2, the lipase is present in an amount of 2%-10% of the total mass of the eutectic mixture and glycerol. The lipase can be present in an amount 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. This amount of lipase ensures that the lipase remains biologically active before the fat reaction ceases, allowing for repeated use in catalyzing the esterification reaction.

[0028] In one embodiment of the present invention, the lipase in S2 is selected from Candida rugosa lipase, Candida antarctica lipase (Novozym 435), Mucor miehei lipase (Lipozyme RM IM), Thermomyces lanuginosus lipase (Lipozyme TL IM), and Aspergillus oryzae lipase (AOL). The selected lipase can accurately recognize the hydroxyl structure in the glycerol substrate and preferentially catalyze the formation of 1,3-diacylglycerol (1,3-DAG) with glycerol rather than random esterification, thereby avoiding the formation of isomers such as 1,2-DAG, thereby improving the biological activity of the target product.

[0029] In one embodiment of the present invention, in the above S2, the esterification reaction temperature is 40-75°C.

[0030] In one embodiment of the present invention, in the above S2, the esterification reaction temperature is 50-60°C.

[0031] Specifically, controlling the esterification reaction temperature effectively ensures the activity of the lipase, helps to increase the reaction speed, and improves the production efficiency of diglyceride. Among them, the above-mentioned esterification reaction temperature can be any point value in the range of 40-75°C, such as 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, etc., which will not be repeated here. The preferred heating temperature is any point value in the range of 50-60°C. It should also be noted that the temperature should not be too low to avoid reaching the eutectic point of the eutectic mixture, so that the eutectic mixture precipitates in the form of crystals, reducing the substrate concentration of the esterification reaction. At the same time, the temperature should not be too high to prevent the lipase from losing its catalytic activity.

[0032] In one embodiment of the present invention, in the above S2, the esterification reaction time is 6-24 hours.

[0033] In one embodiment of the present invention, in the above S2, the esterification reaction time is 10-15 hours.

[0034] Specifically, in one embodiment of the present invention, in the above S2, during the esterification reaction, the reaction system is subjected to ultrasonic treatment, and the frequency of the ultrasonic wave is 500-1000 kHz. Ultrasonic treatment helps to accelerate the esterification reaction.

[0035] Below in conjunction with specific embodiment, the present invention is described in further detail, these embodiments should not be construed as limiting the scope of protection of the present invention.In addition, in the following examples, unless otherwise stated, the reagents and equipment used are all commercially available.If in the following examples, concrete treatment conditions and treatment process are not clearly described, then conditions and methods well known in the art can be adopted to process. Example 1

[0036] S1, weigh 107.7g of hexadecanoic acid (C 16 H 32 O2) and 132.5 g of decanoic acid (C 10 H 20 O2) to prepare a liquid eutectic mixture, the eutectic point of which is 23°C; wherein the melting point of hexadecanoic acid is 61-62.5°C, and the melting point of decanoic acid is 27-32°C; S2. The eutectic mixture, 400 g of glycerol, and 5% of lipase (Novozym 435) were placed in a reaction system and mixed to perform an esterification reaction at a temperature of 50° C. for 20 h to obtain a product, diglyceride, wherein the mass ratio of the eutectic mixture to the glycerol was 1:1.67; S3, separating the prepared diglyceride, and adding a eutectic mixture of hexadecanoic acid and decanoic acid in a molar ratio of 3:7 to the reaction system (the amount added is consistent with the weight of the separated diglyceride), and repeating step 2; Since the esterification reaction temperature is higher than the eutectic point of the low-melting mixture, no saturated fatty acid solid is precipitated and the reaction system is still liquid. The new addition of the low-melting mixture to S3 is beneficial to promote the forward progress of the reaction. Example 2

[0037] The difference from Example 1 is that in S1, 107.7 g of hexadecanoic acid (C 16 H 32 O2) and 132.5 g of tetradecanoic acid (C 14 H 28 O2) to prepare a liquid eutectic mixture, the eutectic point of the eutectic mixture is 44.7°C; wherein the melting point of hexadecanoic acid is 61-62.5°C, and the melting point of tetradecanoic acid is 52-54°C. Example 3

[0038] The difference from Example 1 is that in S2, the reaction temperature of the esterification reaction is 60°C. Example 4

[0039] The difference from Example 1 is that in S2, the esterification reaction system is subjected to ultrasonic treatment, the frequency of the ultrasonic wave is 800 kHz, and the ultrasonic reaction time is 7 hours. Example 5

[0040] The difference from Example 1 is that in S2, the mass ratio of the eutectic mixture to glycerol is 1:3. Example 6

[0041] The difference from Example 1 is that in S2, the mass ratio of the eutectic mixture to glycerol is 1:5 Comparative Example 1 The difference from Example 1 is that in S1, only 240.2 g 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.

[0042] Comparative Example 2 The difference from Example 1 is that in S1, only 240.2 g of hexadecanoic acid (C 16 H 32 O2); in S2, the reaction temperature of the esterification reaction is 90°C.

[0043] Comparative Example 3 The difference from Example 1 is that in S1, only 240.2 g of hexadecanoic acid (C 16 H 32 O2); in S2, the reaction temperature of the esterification reaction is 120°C.

[0044] The products (containing diglyceride) obtained after the reaction in the examples and comparative examples were molecularly distilled in three stages under the following conditions: first stage at 108°C and 50 Pa; second stage at 135°C and 10 Pa; and third stage at 182°C and 1 Pa.

[0045] The specific test results are shown in Table 1.

[0046] Table 1 Example Diaceride content% Triglyceride content% Monoglyceride 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 Although the saturated fatty acid with a high melting point can be melted into a liquid state below its own melting point after combining saturated fatty acids A and saturated fatty acids B in different molar ratios, it can be seen from Examples 1 and 2 that when the proportion of saturated fatty acids with a high melting point is high, the eutectic point of the eutectic mixture will also increase. Therefore, when the reaction needs to be carried out at a lower temperature, it is necessary to control the amount of the high melting point saturated fatty acid in saturated fatty acid A and saturated fatty acid B as much as possible so that the eutectic point matches the enzyme activity temperature. According to the test results in Table 1, when the amount of the high melting point saturated fatty acid is controlled, it helps to increase the yield of diglycerides. Compared with Example 1, Example 3 increases the temperature of the esterification reaction. Although the temperature is higher than the eutectic point of the eutectic mixture, it is ensured that saturated fatty acids A and saturated fatty acids B can be mixed with glycerol in liquid form, but it is higher than the optimum temperature for lipase catalysis. The lipase activity decreases, the catalytic efficiency decreases, and the yield of the target product diglyceride decreases. Therefore, it is best to select the optimum temperature of lipase as the temperature of the esterification reaction, so as to adjust the molar ratio between saturated fatty acids A and saturated fatty acids B and ensure that its eutectic point falls within the optimum temperature of lipase. The present invention mixes two saturated fatty acids that are solid at room temperature to form a liquid eutectic mixture, and the diglyceride content generated by the reaction with glycerol is not low. At the same time, Example 4 increases the ultrasonic treatment with a frequency of 800kHz. The ultrasonic treatment helps to mix the saturated fatty acids, glycerol and lipase evenly, and the contact is sufficient, which helps to accelerate the esterification reaction. The amount of glycerol used in Examples 5-6 is gradually increased relative to Example 1. The excess glycerol in the reaction system can not only help to promote the catalytic reaction forward, increase the yield of diglyceride, but also inhibit the formation of triglycerides. In Comparative Examples 1-3, only a high-melting-point saturated fatty acid (hexadecanoic acid) was used to react with glycerol and lipase. In Comparative Example 1, when the esterification reaction temperature was lower than the melting point of hexadecanoic acid, hexadecanoic acid was in solid form and contacted with glycerol and lipase for reaction. Even though the catalytic activity of the lipase 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 be in liquid form and contacted with glycerol and lipase for reaction, and the reaction contact area was large, the catalytic activity of the lipase was gradually lost at high temperature, the catalytic efficiency was reduced, and the yield of the target product was reduced. High-temperature operation not only increased production costs, but was also not conducive to improving production efficiency.

[0047] The test results show that the present invention can effectively ensure the progress of the enzymatic reaction and improve the production efficiency and the content of the target product by controlling the eutectic point of the eutectic mixture within the range of lipase activity and using excess glycerol.

[0048] The foregoing description is merely a preferred embodiment of the present disclosure and is not intended to limit the present disclosure. Those skilled in the art will readily appreciate that various modifications and variations are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present disclosure shall be included within the scope of protection of the present disclosure.

Claims

1. A method for preparing diglyceride oil by a solvent-free enzymatic method, characterized in that: The following steps are involved: S1. Mixing saturated fatty acid A and saturated fatty acid B to prepare a liquid eutectic mixture; S2, placing the eutectic mixture, glycerol and lipase in a reaction system and mixing them to perform an esterification reaction to obtain diglyceride; S3, separating the prepared diglyceride, and re-adding the saturated fatty acid A and / or the saturated fatty acid B to the reaction system to re-form a liquid eutectic mixture, and repeating step 2; Wherein, in S1, the saturated fatty acid A and the saturated fatty acid B are both selected from C10-C20 alkanoic acids, in S2, the mass ratio of the low eutectic mixture to the glycerol is 1:(1-5), and the temperature of the esterification reaction is 35°C-78°C.

2. The method for preparing diglyceride oil by solvent-free enzymatic method according to claim 1, characterized in that: In S1, the molar ratio of the saturated fatty acid A to the saturated fatty acid B is 1:(1-9).

3. The method for preparing diglyceride oil by solvent-free enzymatic method according to claim 1, characterized in that: In S2, the mass ratio of the eutectic mixture to the glycerol is 1:(1.5-3).

4. The method for preparing diglyceride oil by solvent-free enzymatic method 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.

5. The method for preparing diglyceride oil by solvent-free enzymatic method 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.

6. The method for preparing diglyceride oil by solvent-free enzymatic method according to claim 1, characterized in that: In S2, the esterification reaction temperature is 40-75°C.

7. The method for preparing diglyceride oil by solvent-free enzymatic method according to claim 1, characterized in that: In S2, the esterification reaction temperature is 50-60°C.

8. The method for preparing diglyceride oil by solvent-free enzymatic method according to claim 1, characterized in that: In S2, the esterification reaction time is 6-24 hours.

9. The method for preparing diglyceride oil by solvent-free enzymatic method according to claim 1, characterized in that: In S2, the esterification reaction time is 10-15 hours.

10. The method for preparing diglyceride oil by solvent-free enzymatic method according to claim 1, characterized in that: In S2, during the esterification reaction, the reaction system is subjected to ultrasonic treatment, and the frequency of the ultrasonic wave is 500-1000 kHz.

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