A method for enzymatic production of diglycerides in a deep eutectic solvent system

Abstract

The application discloses a method for preparing diglyceride by an enzymatic method in a eutectic solvent system, and belongs to the technical field of oil deep processing. First, a eutectic solvent is used as a reaction medium, glycerol and fatty acid are added and uniformly mixed; immobilized lipase is added, and esterification is carried out under the catalysis of the immobilized lipase; after the reaction is completed, the reaction mixture is subjected to centrifugal treatment, and the upper oil phase, the middle immobilized enzyme and the lower eutectic solvent are recovered in sequence, and the obtained upper oil phase is an esterification product rich in diglyceride. The method has the advantages that the separation of the reaction product and the eutectic solvent can be realized through simple centrifugation after the reaction is completed, the content of diglyceride in the product is high, the product is easy to separate and purify, the purity of diglyceride in the final product is high, the eutectic solvent can be recycled and reused, and the method has good economic benefits and industrial application prospects.

Description

Technical Field

[0001] This invention belongs to the field of deep processing technology of oils and fats, specifically relating to a method for preparing diglycerides enzymatically in a eutectic solvent system. Background Technology

[0002] Diacylglycerols are esters formed by the esterification of one molecule of glycerol with two molecules of fatty acids. Based on the positional distribution of fatty acids on the glycerol backbone during esterification, diglycerides can be classified into 1,3-diglycerides and 1,2-diglycerides. As a natural component of edible oils, diglycerides typically range from 0.8% to 9.5% in various edible oils. Studies have shown that diglycerides have a different metabolic pathway than triglycerides (the main component of common edible oils), resulting in unique physiological functions such as reducing postprandial serum triglyceride concentrations, inhibiting visceral, subcutaneous, and abdominal fat accumulation, reducing body weight, increasing serum high-density lipoprotein cholesterol concentrations, decreasing serum total cholesterol and low-density lipoprotein cholesterol concentrations, improving insulin sensitivity in individuals with insulin resistance, and delaying the progression of renal failure in diabetic patients. Since the diglyceride content in edible oils is usually below 9.5%, it is insufficient to effectively exert the physiological effects of diglycerides. Therefore, research on diglyceride preparation techniques is of great significance.

[0003] Enzymatic catalysis is an effective method for preparing diglycerides. Enzymatic hydrolysis, esterification, glycerolization, and alcoholysis are commonly used methods for preparing diglycerides. Compared with other methods, enzymatic esterification has the advantage of high diglyceride content in the product and is currently the most common method in the industrial production of diglycerides. Typically, sn-1,3 position-specific lipases such as Lipozyme RM and IM are used to catalyze esterification to prepare diglycerides because their sn-1,3 position specificity can effectively promote the formation of 1,3-diglycerides and effectively prevent the conversion of the generated diglycerides into triglycerides, thus resulting in a high diglyceride content in the esterification product. The key technical problems to be solved in the enzymatic esterification process for preparing diglycerides are how to promote sufficient contact between the reaction substrate, remove esterification byproducts, regulate the sn-1,3 position specificity of lipases, or inhibit the conversion of the generated diglycerides into triglycerides.

[0004] CN 104178530 A discloses a method for preparing diglycerides using a bubble reactor. By continuously introducing inert gas during the esterification reaction to form boiling bubbles, the substrate is brought into full contact, and water generated during the esterification process is removed, thus promoting the formation of diglycerides. The final product, after separation and purification, has a diglyceride content of approximately 90%. CN 105400837 A discloses a two-step enzymatic method for preparing diglycerides by moderate enzymatic hydrolysis and glycerol esterification. This method requires precise control of the fatty acid content in the hydrolysis product between 26 and 30 wt%, and the diglyceride content in the final product between 60% and 65%. CN 112322670 A discloses a two-step enzymatic method for preparing diglycerides by enzymatic hydrolysis and glycerol esterification. This method requires controlling the fatty acid content in the hydrolysis product to be greater than 45 wt%, the triglyceride content to be less than 22 wt%, and the diglyceride content in the final esterification product to be 50% to 56%.

[0005] In summary, existing technologies mainly regulate the diglyceride content in the final product by controlling the degree of substrate contact, removing product inhibition, or combining enzymatic hydrolysis and esterification to regulate the degree of enzymatic hydrolysis. Summary of the Invention

[0006] To address the shortcomings of the existing technology, the present invention aims to provide a method for the enzymatic preparation of diglycerides in a eutectic solvent system. The product has a high content of diglycerides and low content of monoglycerides and triglycerides. Not only is product separation and subsequent separation and purification easy, but the solvent can also be reused repeatedly, which has good prospects for industrial application.

[0007] This invention is achieved through the following technical solution:

[0008] A method for the enzymatic preparation of diglycerides in a eutectic solvent system includes the following steps:

[0009] Step 1: Using a eutectic solvent as the reaction medium, add glycerol and fatty acids and homogenize; add immobilized lipase and carry out esterification reaction under the catalysis of immobilized lipase;

[0010] Step 2: After the reaction is completed, the reaction mixture is centrifuged to recover the upper oil phase, the middle immobilized enzyme, and the lower eutectic solvent in sequence. The upper oil phase is an esterification product rich in diglycerides.

[0011] Preferably, in step 1, the eutectic solvent is choline chloride-xylitol, betaine-xylitol, or choline chloride-glucose-water.

[0012] More preferably, when the eutectic solvent is choline chloride-xylitol, the molar ratio of choline chloride to xylitol is 1:(1-2); when the eutectic solvent is betaine-xylitol, the molar ratio of betaine to xylitol is 1:(1-2); when the eutectic solvent is choline chloride-glucose-water, the molar ratio of choline chloride, glucose and water is (3-1):1:(0-2).

[0013] Preferably, in step 1, the mass of the eutectic solvent accounts for 1 / 6 to 1 / 3 of the total mass of the esterification reaction system.

[0014] Preferably, in step 1, the homogenization speed is 5000-8000 rpm and the time is 1-2 min.

[0015] Preferably, in step 1, the fatty acid source is walnut oil, red walnut oil, purple walnut oil, flaxseed oil, olive oil, tea oil, peony seed oil, rapeseed oil, soybean oil, peanut oil, corn oil, marine fish oil, or algae oil.

[0016] Preferably, in step 1, the immobilized lipase is Novozym 435 or Lipozyme 435, and the amount added is 2% to 10% of the total substrate mass.

[0017] Preferably, in step 1, the molar ratio of glycerol to fatty acid is 2-6:1, the reaction temperature is 30-80℃, the reaction time is 2-6h, and the stirring speed during the reaction is 300-500rpm.

[0018] Preferably, in step 2, the centrifugation speed is 12000-15000 rpm and the time is 1-2 min.

[0019] Preferably, in step 2, the content of diglycerides in the obtained esterification reaction product is >82%, and the content of 1,3-diglycerides in the diglycerides is >77%; the content of monoglycerides in the obtained esterification reaction product is <5%, the content of fatty acids is <5%, and the content of triglycerides is 5% to 9%.

[0020] Compared with the prior art, the present invention has the following beneficial technical effects:

[0021] This invention discloses a method for the enzymatic preparation of diglycerides in a eutectic solvent system. The method uses a eutectic solvent as the reaction medium and prepares diglycerides under esterification catalyzed by immobilized lipase. On one hand, the eutectic solvent's regulatory effect on the active site of the immobilized lipase enhances the sn-1,3 position specificity of the immobilized lipase, promoting the conversion of free fatty acids and generated monoglycerides to 1,3-diglycerides, thereby promoting the accumulation of 1,3-diglycerides. On the other hand, the eutectic system inhibits the conversion of 1,3-diglycerides to 1,2-diglycerides in the product, thus inhibiting the formation of triglycerides. Under the combined effect of these two aspects, the prepared product has a high diglyceride content and low monoglyceride and triglyceride content; after separation and purification, the final product has a diglyceride content of over 90%. This invention is simple to operate. After the reaction is completed, the product can be separated from the eutectic solvent by simple centrifugation. The product has a high content of diglycerides and low content of monoglycerides and triglycerides. Subsequent separation and purification of diglycerides is easy. The purified diglycerides have high purity, and the eutectic solvent can be recycled and reused. It has good economic benefits and industrial application prospects.

[0022] Furthermore, the eutectic solvent is choline chloride-xylitol, betaine-xylitol, or choline chloride-glucose-water, and the molar ratio of each component of the three eutectic solvents is 1:1-2, 1:1-2, and 3-1:1:0-2, respectively. This can effectively regulate the catalytic active center of the immobilized lipase used, improve the sn-1,3 position specificity of the immobilized enzyme, and effectively inhibit the conversion of 1,3-diglyceride to 1,2-diglyceride generated during the reaction.

[0023] Furthermore, the amount of eutectic solvent used is 1 / 6 to 1 / 3 of the total mass of the esterification reaction system, which not only promotes the efficient accumulation of diglycerides, but also facilitates the separation of esterification products.

[0024] Furthermore, the homogenization speed is 5000-8000 rpm and the time is 1-2 min, which can ensure that glycerol and fatty acids are uniformly distributed in the eutectic solvent system, thereby providing a larger reaction interface for the catalysis of immobilized lipase.

[0025] Furthermore, the immobilized lipase used is Novozym 435 or Lipozyme 435, and the amount added is 2% to 10% of the total substrate mass, which can effectively promote the accumulation of diglycerides and ensure the economy of the reaction.

[0026] Furthermore, the esterification reaction conditions can ensure a high fatty acid conversion rate and diglyceride yield, while effectively inhibiting the conversion of diglycerides to triglycerides. Attached Figure Description

[0027] Figure 1 This is a flowchart illustrating the technical process of the present invention. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to specific embodiments. These descriptions are for illustrative purposes only and not for limiting the scope of the invention. Unless otherwise stated, all percentages are by weight.

[0029] like Figure 1 The diagram shows the technical flow of this invention. The eutectic solvent used in the esterification reaction regulates the catalytic active site of the immobilized lipase and inhibits the conversion of 1,3-diglyceride to 1,2-diglyceride. This increases the conversion of fatty acids and esterified monoglycerides to 1,3-diglyceride, while inhibiting the conversion of 1,2-diglyceride to triglyceride.

[0030] Example 1

[0031] 50 g of eutectic solvent (choline chloride-xylitol, molar ratio 1:1) was added to a 1 L reaction flask, followed by 250 g of a mixture of glycerol and walnut oil fatty acids (molar ratio 1:4). The mixture was homogenized at 5000 rpm for 2 min. The reaction mixture was then heated to 30 °C, and 10 g of immobilized lipase Lipozyme 435 was added. Esterification was carried out at 300 rpm. After 6 h of reaction, the reaction mixture was centrifuged at 15000 rpm for 1 min, and the upper oil phase, lower enzyme layer, and eutectic solvent layer were recovered separately. HPLC-RID analysis of the upper oil phase product revealed a diglyceride content of 83.11%, including 77.56% 1,3-diglycerides, 4.68% monoglycerides, 4.89% fatty acids, and 7.32% triglycerides. The above products were separated and purified by molecular distillation, and the final product contained 91.42% diglycerides. New reaction substrates (glycerol and walnut oil fatty acids) were added to the recovered eutectic solvent and immobilized lipase, and the reaction was repeated 50 times. The composition of the product was not significantly different from that of the first batch of reaction products.

[0032] Example 2

[0033] 100 g of eutectic solvent (choline chloride-xylitol, molar ratio 1:2) was added to a 1 L reaction flask, followed by 200 g of a mixture of glycerol and red walnut oil fatty acids (molar ratio 1:6). The mixture was homogenized at 8000 rpm for 1 min. The reaction mixture was then heated to 80 °C, and 20 g of immobilized lipase Novozym 435 was added. Esterification was carried out at 500 rpm. After 2 h of reaction, the reaction mixture was centrifuged at 12000 rpm for 2 min, and the upper oil phase, lower enzyme layer, and eutectic solvent layer were recovered separately. HPLC-RID analysis of the upper oil phase product revealed a diglyceride content of 85.63%, including 79.71% 1,3-diglycerides, 4.33% monoglycerides, 3.54% fatty acids, and 6.50% triglycerides. The above products were separated and purified by molecular distillation, and the final product contained 92.53% diglycerides. New reaction substrates (glycerol and red walnut oil fatty acids) were added to the recovered eutectic solvent and immobilized lipase, and the reaction was repeated 50 times. The composition of the product was not significantly different from that of the first batch.

[0034] Example 3

[0035] 75 g of eutectic solvent (betaine-xylitol, molar ratio 1:2) was added to a 1 L reaction flask, followed by 225 g of a mixture of glycerol and olive oil fatty acids (molar ratio 1:2). The mixture was homogenized at 5000 rpm for 2 min. The reaction mixture was then heated to 60 °C, and 11.25 g of immobilized lipase Novozym 435 was added. Esterification was carried out at 400 rpm. After 4 h of reaction, the reaction mixture was centrifuged at 15000 rpm for 1 min, and the upper oil phase, lower enzyme layer, and eutectic solvent layer were recovered separately. HPLC-RID analysis of the upper oil phase product revealed a diglyceride content of 87.75%, of which 1,3-diglycerides accounted for 80.89%, monoglycerides accounted for 4.02%, fatty acids accounted for 3.06%, and triglycerides accounted for 5.17%. The above products were separated and purified by molecular distillation, and the final product contained 93.92% diglycerides. New reaction substrates (glycerol and olive oil fatty acids) were added to the recovered eutectic solvent and immobilized lipase, and the reaction was repeated 50 times. The composition of the product was not significantly different from that of the first batch.

[0036] Example 4

[0037] 60 g of eutectic solvent (betaine-xylitol, molar ratio 1:1) was added to a 1 L reaction flask, followed by 240 g of a mixture of glycerol and linseed oil fatty acids (molar ratio 1:5). The mixture was homogenized at 8000 rpm for 1 min. The reaction mixture was then heated to 55 °C, and 14.4 g of immobilized lipase Lipozyme 435 was added. Esterification was carried out at 350 rpm. After 3 h of reaction, the reaction mixture was centrifuged at 12000 rpm for 2 min, and the upper oil phase, lower enzyme layer, and eutectic solvent layer were recovered separately. HPLC-RID analysis of the upper oil phase product revealed a diglyceride content of 85.22%, including 78.67% 1,3-diglycerides, 4.43% monoglycerides, 3.78% fatty acids, and 6.57% triglycerides. The above products were separated and purified by molecular distillation, and the final product contained 92.14% diglycerides. New reaction substrates (glycerol and linseed oil fatty acids) were added to the recovered eutectic solvent and immobilized lipase, and the reaction was repeated 50 times. The composition of the product was not significantly different from that of the first batch.

[0038] Example 5

[0039] 75 g of eutectic solvent (choline chloride-glucose, molar ratio 3:1) was added to a 1 L reaction flask, followed by 225 g of a mixture of glycerol and soybean oil fatty acids (molar ratio 1:3). The mixture was homogenized at 8000 rpm for 1 min. The reaction mixture was then heated to 70 °C, and 4.5 g of immobilized lipase Novozym 435 was added. Esterification was carried out at 450 rpm. After 6 h of reaction, the reaction mixture was centrifuged at 15000 rpm for 1 min, and the upper oil phase, lower enzyme layer, and eutectic solvent layer were recovered separately. HPLC-RID analysis of the upper oil phase product revealed a diglyceride content of 82.26%, including 77.25% 1,3-diglycerides, 4.53% monoglycerides, 4.97% fatty acids, and 7.24% triglycerides. The above products were separated and purified by molecular distillation, and the final product contained 90.14% diglycerides. New reaction substrates (glycerol and soybean oil fatty acids) were added to the recovered eutectic solvent and immobilized lipase, and the reaction was repeated 50 times. The composition of the product was not significantly different from that of the first batch.

[0040] Example 6

[0041] 100 g of eutectic solvent (choline chloride-glucose-water, molar ratio 2:1:2) was added to a 1 L reaction flask, followed by 200 g of a mixture of glycerol and peanut oil fatty acids (molar ratio 1:4). The mixture was homogenized at 5000 rpm for 2 min. The reaction mixture was then heated to 50 °C, and 10 g of immobilized lipase Novozym435 was added. Esterification was carried out at 500 rpm. After 3 h of reaction, the reaction mixture was centrifuged at 15000 rpm for 1 min, and the upper oil phase, lower enzyme layer, and eutectic solvent layer were recovered separately. HPLC-RID analysis of the upper oil phase product revealed a diglyceride content of 89.47%, including 82.59% 1,3-diglycerides, 2.87% monoglycerides, 2.56% fatty acids, and 5.10% triglycerides. The above products were separated and purified by molecular distillation, and the final product contained 94.02% diglycerides. New reaction substrates (glycerol and peanut oil fatty acids) were added to the recovered eutectic solvent and immobilized lipase, and the reaction was repeated 50 times. The composition of the product was not significantly different from that of the first batch.

[0042] Example 7

[0043] 50 g of eutectic solvent (choline chloride-glucose-water, molar ratio 1:1:1) was added to a 1 L reaction flask, followed by 250 g of a mixture of glycerol and algal oil fatty acids (molar ratio 1:5). The mixture was homogenized at 8000 rpm for 1 min. The reaction mixture was then heated to 55 °C, and 20 g of immobilized lipase Lipozyme 435 was added. Esterification was carried out at 500 rpm. After 2 h of reaction, the reaction mixture was centrifuged at 15000 rpm for 1 min, and the upper oil phase, lower enzyme layer, and eutectic solvent layer were recovered separately. HPLC-RID analysis of the upper oil phase product revealed a diglyceride content of 88.35%, including 81.76% 1,3-diglycerides, 2.99% monoglycerides, 2.84% fatty acids, and 5.82% triglycerides. The above products were separated and purified by molecular distillation, and the final product contained 93.16% diglycerides. New reaction substrates (glycerol and algal oil fatty acids) were added to the recovered eutectic solvent and immobilized lipase, and the reaction was repeated 50 times. The composition of the product was not significantly different from that of the first batch.

[0044] The table below compares the data on the reaction system, reactants, and products of each embodiment:

[0045]

[0046]

[0047] The above description is only a part of the embodiments of the present invention. Although some terms are used in the present invention, the possibility of using other terms is not excluded. The use of these terms is merely for the convenience of describing and explaining the essence of the present invention, and interpreting them as any kind of additional limitation would contradict the spirit of the present invention. The above description is only to further illustrate the content of the present invention through embodiments to facilitate easier understanding, but it does not mean that the embodiments of the present invention are limited to this. Any technical extension or re-creation based on the present invention is protected by the present invention.

Claims

1. A method for the enzymatic preparation of diglycerides in a eutectic solvent system, characterized in that, Includes the following steps: Step 1: Using a eutectic solvent as the reaction medium, add glycerol and fatty acids and homogenize; add immobilized lipase and carry out esterification reaction under the catalysis of immobilized lipase; Step 2: After the reaction is completed, the reaction mixture is centrifuged and the upper oil phase, the middle immobilized enzyme and the lower eutectic solvent are recovered in sequence. The upper oil phase obtained is the esterification product rich in diglycerides. The eutectic solvent is choline chloride-xylitol, betaine-xylitol, or choline chloride-glucose-water. When the eutectic solvent is choline chloride-xylitol, the molar ratio of choline chloride to xylitol is 1:(1-2); when the eutectic solvent is betaine-xylitol, the molar ratio of betaine to xylitol is 1:(1-2); when the eutectic solvent is choline chloride-glucose-water, the molar ratio of choline chloride, glucose, and water is (3-1):1:(0-2). The immobilized lipase is Novozym 435 or Lipozyme 435, and the amount added is 2% to 10% of the total substrate mass; In step 2, the content of diglycerides in the obtained esterification reaction product is >82%, and the content of 1,3-diglycerides in the diglycerides is >77%; the content of monoglycerides in the obtained esterification reaction product is <5%, the content of fatty acids is <5%, and the content of triglycerides is 5% to 9%.

2. The method for enzymatic preparation of diglycerides in a eutectic solvent system according to claim 1, characterized in that, In step 1, the mass of the eutectic solvent accounts for 1 / 6 to 1 / 3 of the total mass of the esterification reaction system.

3. The method for enzymatic preparation of diglycerides in a eutectic solvent system according to claim 1, characterized in that, In step 1, the homogenization speed is 5000-8000 rpm and the time is 1-2 min.

4. The method for enzymatic preparation of diglycerides in a eutectic solvent system according to claim 1, characterized in that, In step 1, the fatty acid source is walnut oil, red walnut oil, purple walnut oil, flaxseed oil, olive oil, tea oil, peony seed oil, rapeseed oil, soybean oil, peanut oil, corn oil, marine fish oil, or algae oil.

5. The method for enzymatic preparation of diglycerides in a eutectic solvent system according to claim 1, characterized in that, In step 1, the molar ratio of glycerol to fatty acids is 2-6:1, the reaction temperature is 30-80℃, the reaction time is 2-6 hours, and the stirring speed is 300-500 rpm.

6. The method for enzymatic preparation of diglycerides in a eutectic solvent system according to claim 1, characterized in that, In step 2, the centrifugation speed is 12000-15000 rpm and the time is 1-2 min.

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