A method for preparing diglyceride oil by solvent-free enzymatic method

Abstract

The present invention discloses a method for preparing a diglyceride product by a solvent-free enzymatic method, and belongs to the field of oils and fats. The present invention pre-adsorbs glycerol by a cellulose adsorbent, combines a fixed bed reaction column to fix the adsorption material, and continuously feeds a glycerol oil mixture of a suitable concentration into a reaction system. At the same time, polar resin and lipase are added to the reactor, and the ability of the polar resin material to adsorb excess polar substrates in the system is utilized to protect the enzyme active center, effectively extend the service life of the lipase, and significantly reduce the cost of preparing diglyceride by enzymatic glycerolysis reaction. At the same time, the present invention achieves the purpose of reducing the reaction time and reducing the amount of enzyme added while ensuring a high diglyceride yield of the product by screening the adsorbent and controlling the water content of the reaction system. The diglyceride of the crude product prepared by the method of the present invention is >50wt%, and the diglyceride after molecular distillation is >85wt%, which has a certain industrial application prospect.

Description

Technical Field

[0001] The present invention relates to a technology for preparing diglyceride products by solvent-free enzymatic method, belonging to the technical field of oils and fats. Background Art

[0002] Diacylcerol (DAG) is a product obtained by esterification of glycerol with two molecules of fatty acids. It is also an intermediate product obtained by the metabolism of natural edible oil in the body. Diacylcerol is divided into 1,3-diacylcerol and 1,2-diacylcerol. In natural oils and fats, diacylglycerol is a trace component, and its content generally does not exceed 5%. In recent years, many studies have found that diacylglycerol, especially 1,3-diacylcerol, has a series of effects such as weight loss, lowering blood triglycerides and cholesterol, and lowering uric acid, and has a broad market development prospect and value. In Japan, it has passed the safety and effectiveness review of the Ministry of Labor and has been designated as a specific health food. In the United States, diacylglycerol has passed the food safety review and has been listed as a recognized safe food by the U.S. Food and Drug Administration (FAD).

[0003] In recent years, lipase has been widely used in the preparation of diacylglycerol. The enzymatic production methods of diacylglycerol are mainly divided into enzymatic esterification, enzymatic glycerolysis and enzymatic hydrolysis. The advantages of enzymatic production include low reaction temperature and low risk factor content, but there are still some problems in enzymatic preparation: (1) Due to the high viscosity of the reaction substrate, the catalytic efficiency of some lipases is low. (2) The reusability of lipase is poor, which increases the cost of lipase use and is not conducive to the industrial development of diacylglycerol oil. (3) Organic solvent-assisted enzymatic hydrolysis to prepare diacylglycerol can overcome the problem of poor solubility between oil and glycerol and improve the catalytic efficiency of lipase, but the toxicity of the solvent and the safety of operation limit its industrial application. Therefore, it is still necessary to find an efficient solvent-free enzymatic diacylglycerol production process to further improve the catalytic efficiency of lipase and the content of diacylglycerol in the product, and improve the enzymatic preparation method of diacylglycerol.

[0004] In recent years, lipase has been widely used in the preparation of diacylglycerol. The enzymatic production methods of diacylglycerol are mainly divided into enzymatic esterification, enzymatic glycerolysis and enzymatic hydrolysis. The advantages of enzymatic production include low reaction temperature and low risk factor content, but there are still some problems in enzymatic preparation: (1) Due to the high viscosity of the reaction substrate, the catalytic efficiency of some lipases is low. (2) The reusability of lipase is poor, which increases the cost of lipase use and is not conducive to the industrial development of diacylglycerol oil. (3) Organic solvent-assisted enzymatic hydrolysis to prepare diacylglycerol can overcome the problem of poor solubility between oil and glycerol and improve the catalytic efficiency of lipase, but the toxicity of the solvent and the safety of operation limit its industrial application. Therefore, it is still necessary to find an efficient solvent-free enzymatic diacylglycerol production process to further improve the catalytic efficiency of lipase and the content of diacylglycerol in the product, and improve the enzymatic preparation method of diacylglycerol. Summary of the invention

[0005] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0006] Therefore, the object of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing diglyceride oil by a solvent-free enzymatic method, the method comprising the following steps:

[0007] Step 1, mixing glycerol and cellulose adsorption material and letting them stand to pre-adsorb the glycerol, mixing the pre-adsorbed glycerol and oil of the same mass as the glycerol and wet-loading them into a fixed bed reaction column;

[0008] Step 2, mixing the oil, polar resin material and lipase in a reactor;

[0009] The modified group of the polar resin material is a tertiary amine group, a primary amine group or a sulfonic acid group, the average pore size of the material is 10nm to 30nm, and the amount of the polar resin material added is not less than 80wt% of the amount of the lipase added;

[0010] Step 3, connecting the reactor to the fixed reaction column, setting a filtering device at the connection to allow only oil and glycerol to flow, blocking the polar resin and lipase; allowing the oil and glycerol mixture in the fixed bed reaction column to continuously flow into the reactor and reflux to the fixed bed reaction column through an oil pump, and the polar resin and lipase only flow and react in the reactor; cyclic reaction, collecting the product in the reactor after the reaction is completed, and separating and purifying the oil of diglyceride.

[0011] In one embodiment, the tertiary amino polar resin material in step 2 includes ion exchange resin FPA-54 or ion exchange resin FPA-53; the primary amine polar resin material includes D301; and the sulfonic acid polar resin material includes AMBERLITE R120Na.

[0012] In one embodiment, the moisture content of the polar resin material is 5 wt % to 10 wt % of the weight of the adsorbent.

[0013] In one embodiment, the cellulose adsorption material in step 1 includes carboxymethyl cellulose or microcrystalline cellulose.

[0014] In one embodiment, the lipase immobilization carrier is a medium-polarity macroporous adsorption resin and a weak-base cation exchange resin.

[0015] In one embodiment, the lipase comprises one or more of NE-100, Novozyme 435, Lipozyme 435, Lipase PS IM, Lipase DF "Amano" IM, Lipase TL IM and Lipase RM IM.

[0016] In one embodiment, the lipase is produced by Candida antarctica, Rhizopus oryzae, Aspergillus niger, Candida rugosa, Burkholderia, and Pseudomonas.

[0017] In one embodiment, the separation and purification method is molecular distillation.

[0018] In one embodiment, the molecular distillation is a three-stage molecular distillation, and the specific conditions are: first stage 105°C-120°C, 50Pa; second stage 130°C-150°C, 10Pa; third stage 180°C-190°C, 1Pa.

[0019] In one embodiment, the lipase is added in an amount of 3 wt% to 7 wt% of the weight of the oil;

[0020] The reaction temperature is 45-60°C;

[0021] The molar ratio of all the oils and fats added in the reaction to glycerol is 1:(0.75-2), and the mass ratio of the cellulose adsorption material to glycerol is 1:(0.5-2).

[0022] Beneficial Effects

[0023] 1. The present invention selects a tertiary amino polar resin that can effectively protect the activity of lipase, and utilizes the resin's adsorption performance for glycerol to adsorb excess glycerol in the enzyme reaction system, thereby preventing glycerol from aggregating at the active site of lipase, effectively reducing the enzyme activity loss generated during the reaction, extending the service life of lipase for glycerol hydrolysis reaction, and reducing production costs.

[0024] 2. The present invention regulates the moisture content in the adsorbent and screens the optimal moisture content of the resin. While ensuring the material's adsorption performance for glycerol, it promotes the glycerol hydrolysis reaction, inhibits the occurrence of the hydrolysis reaction, reduces the generation of fatty acid by-product impurities, and maintains the stability of the diglyceride content in the product.

[0025] 3. The present invention reduces the loss of oil and fat caused by the adsorption product during the reaction process by using a cellulose-type adsorbent in combination with fixed-bed reaction column technology. Compared with adsorbents such as silica gel and diatomaceous earth, the cellulose adsorbent has a strong adsorption capacity for glycerol, but a weak adsorption capacity for diglyceride products, thereby effectively reducing the yield loss. Fixed-bed reaction column technology is used to reduce the relative movement of the adsorbent in the system, and the efficient recovery of the adsorbent is achieved while reducing product losses. The loading time is reduced by replacing the reaction column, which can effectively improve production efficiency. Cellulose adsorbents can be recycled multiple times, are easy to regenerate, and are green and environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The present invention is a schematic diagram of a device for preparing diglyceride by enzymatic method;

[0027] The components include 1 fixed bed reaction column, 2 cellulose adsorbent, 3 heat exchanger, 4 pump, 5 enzyme reactor, 6 polar resin material and 7 lipase.

[0028] Figure 2 The number of times the lipase is used for the glycerol adsorption operation comparison of the present invention;

[0029] In the figure, a is the diglyceride content of the crude product under the conditions of Example 1, and in the figure, b is the diglyceride content of the crude product under the conditions of Comparative Example 1.

[0030] Figure 3 This is a liquid chromatogram of glycerol adsorbed by cellulose adsorbent and ion exchange resin FPA54 as the resin protection material in Example 3 of the present invention. DETAILED DESCRIPTION

[0031] Examples of what is included in the claims

[0032] Example 1

[0033] A method for preparing a high-content diglyceride product by enzymatic method under solvent-free conditions comprises the following steps:

[0034] After mixing glycerol and cellulose adsorbent, let it stand for adsorption for 60 minutes and then wet-load it into a fixed bed reaction column. Refined rapeseed oil, ion exchange resin FPA-54 and lipase Lipase TL IM were mixed and cyclically reacted at 60°C for 8 hours, wherein the molar ratio of oil to glycerol was 1:1, the mass ratio of polar adsorbent to glycerol was 1:1, the enzyme addition amount was 5%, and the ion exchange resin FPA-54 addition amount was 3%. After the reaction was completed, the sample was taken out from the reactor, and the lipase and protective resin were collected. After cleaning the lipase and resin with tert-butanol, the refined rapeseed oil, lipase and resin were added to the reactor again, and a new fixed bed reaction column was replaced to start the reaction again. This was repeated 10 times, and samples were taken to analyze the diglyceride content in the crude product each time. The results are shown in Table 2. Figure 2 As shown in a.

[0035] After ten cycles of glycerolysis experiments, the diacylglycerol content of the crude product decreased from 48.88% to 39.51%.

[0036] Example 2

[0037] A method for preparing a high-content diglyceride product by enzymatic method under solvent-free conditions comprises the following steps:

[0038] After mixing glycerol and cellulose adsorbent, let it stand for 60 minutes of adsorption and then wet-coat it in a fixed bed reaction column. Polar resins with different groups were mixed with refined rapeseed oil and lipase Lipase TL IM, and the enzymatic reaction was cyclically carried out at 60°C for 8 hours, wherein the molar ratio of oil to glycerol was 1:1, the mass ratio of polar adsorbent to glycerol was 1:1, the enzyme addition amount was 5%, and the polar resin addition amount was 5%. After the reaction was completed, the sample was taken out from the reactor, and the lipase and protective resin were collected. After the lipase and resin were washed with tert-butyl alcohol, the refined rapeseed oil, lipase and resin were added to the reactor again, and a new fixed bed reaction column was replaced to start the reaction again. The reaction was repeated 10 times, and the content of diglyceride in the crude product of the tenth reaction was sampled and analyzed. The results are shown in Table 1.

[0039] Table 1 Effect of different protective resins on diglyceride content and yield in crude glycerol hydrolysis products

[0040]

[0041] From the results in Table 1, it can be seen that the group type of polar resin will also significantly affect the protection performance of lipase, among which tertiary amine resin has the best protection effect on lipase activity. After the lipase is recycled ten times, the content of diglycerol in the crude product is still as high as 43.54%. Primary amine and sulfonic acid modified resins also have a certain protective effect. The diglycerol yield after the lipase is recycled ten times also reaches 30.55% and 28.20%.

[0042] Example 3

[0043] A method for preparing a high-content diglyceride product by enzymatic method under solvent-free conditions comprises the following steps:

[0044] After mixing glycerol with different polar adsorbents and standing for 60 minutes, wet column loading was carried out in a fixed bed reaction column. Refined rapeseed oil, ion exchange resin FPA-54 and lipase Lipase DF "Amano" IM were mixed and cyclically enzymatic reaction was carried out for 6 hours at 55°C, wherein the molar ratio of oil to glycerol was 1:1, the mass ratio of polar adsorbent to glycerol was 1:1, the enzyme addition amount was 5%, and the ion exchange resin FPA-54 addition amount was 3%. After the reaction, glycerol and lipase were separated, and the content of diglycerol and oil yield in the crude product were sampled and analyzed. The crude product was molecularly distilled, wherein the conditions of the three-stage molecular distillation were: 110°C, 50Pa for the first stage; 130°C, 10Pa for the second stage, and 180°C, 1Pa for the third stage. The diglycerol content in the molecular distillation product was sampled and analyzed, and the results are shown in Table 2.

[0045] Table 2 Effect of different adsorbents on diglyceride content and yield in crude glycerol hydrolysis products

[0046] Adsorbent type Microcrystalline Cellulose Carboxymethyl cellulose Silicone diatomite Activated carbon Diacylceride content in crude product, % 53.03 47.55 43.23 14.52 18.32 Oil yield of crude product, % 94.45 91.95 87.4 83.24 83.15 Diacylceride content after molecular distillation, % 90.67 84.37 81.12 36.49 42.71

[0047] From the results in Table 2, it can be seen that the difference in adsorbents affects the yield of diacylglycerol and the yield of glycerol hydrolysis reaction. Among them, using cellulose as an adsorbent can significantly reduce the loss of oil yield. When cellulose is used as a glycerol adsorbent, the diacylglycerol content in the crude product reaches 53.03%, and the product yield is 94.45%. The liquid chromatogram (HPLC-RID) of the crude product is as follows Figure 1 Cellulose can be recycled and reused multiple times through washing and drying, which effectively reduces the production cost of diglyceride and is conducive to the industrial development of diglyceride products.

[0048] Example 4

[0049] A method for preparing a high-content diglyceride product by enzymatic method under solvent-free conditions comprises the following steps:

[0050] After mixing glycerol and cellulose adsorbent, let it stand for 60 minutes of adsorption and then wet-column it into a fixed bed reaction column. Then, ion exchange resin FPA-54 with different moisture contents, refined soybean oil and lipase Lipozyme TL IM enzyme reactor were mixed and cyclically enzymatic reaction was carried out for 8 hours at 60°C, wherein the molar ratio of oil to glycerol was 1:2, the mass ratio of ion exchange resin oil FPA-54 to glycerol was 2:1, the enzyme addition amount was 5%, and the ion exchange resin FPA-54 addition amount was 3%. After the reaction, glycerol and lipase were separated, and the content of diglyceride and fatty acid content in the crude product were sampled and analyzed. The crude product was molecularly distilled, wherein the conditions of the three-stage molecular distillation were: 120°C, 50Pa for the first stage; 140°C, 10Pa for the second stage, and 185°C, 1Pa for the third stage. The results are shown in Table 3.

[0051] Table 3 Effect of tertiary amino resin moisture and glycerol hydrolysis time on diglycerol content in glycerol hydrolysis crude product

[0052] Resin moisture content, % 2 4 6 8 10 12 14 Diacylceride content in crude product, % 7.02 24.23 46.64 49.19 44.30 42.43 41.59 Fatty acid content in crude product, % 2.54 5.11 6.21 8.34 12.79 16.74 18.45 Diacylceride content after molecular distillation, % 10.23 48.56 81.15 91.43 89.82 85.66 86.27

[0053] From the results in Table 3, it can be seen that the difference in moisture content of the adsorbent significantly affects the content of diglyceride and fatty acid in the product. When the resin has less moisture, the internal pores collapse due to lack of water in the resin, the resin's adsorption capacity for glycerol is reduced, and it cannot play the function of slow-release glycerol, and the glycerol hydrolysis yield decreases. However, as the moisture content increases to more than 10%, the moisture content of the resin affects the esterification of fatty acids in the glycerol hydrolysis reaction of the system, and the hydrolysis reaction is promoted, resulting in a large increase in the fatty acid content in the crude product and a decrease in the content of diglyceride.

[0054] Example 5

[0055] A method for preparing a high-content diglyceride product by enzymatic method under solvent-free conditions comprises the following steps:

[0056] After the glycerol and cellulose adsorbent were mixed and allowed to stand for 60 minutes of adsorption, the column was wet loaded into a fixed bed reaction column. The ion exchange resin FPA-53, refined soybean oil and lipase Lipozyme RM IM were mixed, and the resin moisture content was 6%. The enzymatic reaction was carried out at 60°C for 8 hours, wherein the molar ratio of oil to glycerol was 1:1.5, the enzyme addition amount was 5%, and the resin addition amount was 3%. After the reaction, glycerol and lipase were separated, and the content of diglyceride in the crude product was sampled and analyzed. The crude product was molecularly distilled, wherein the conditions of the three-stage molecular distillation were: 125°C, 50Pa for the first stage; 140°C, 20Pa for the second stage, and 180°C, 3Pa for the third stage. The results are shown in Table 4.

[0057] Table 4 Effect of adsorbent to glycerol mass ratio on diglyceride content in crude glycerol hydrolysis product

[0058] Mass ratio of adsorbent to glycerol 0.5:1 2:1 1:1 2:1 2:1 2.5:1 Diacylceride content in crude product, % 30.12 36.73 42.69 44.74 41.08 35.62 Diacylceride content after molecular distillation, % 67.14 73.41 82.52 85.75 80.70 72.63

[0059] Example 6

[0060] After mixing glycerol and cellulose adsorbent, the mixture was allowed to stand for 60 minutes of adsorption and then wet-column-loaded to a fixed bed for reaction. Ion exchange resin FPA-53, refined soybean oil and different lipases were mixed, and the resin moisture content was 6%. The enzymatic reaction was carried out at 60°C for 8 hours, wherein the molar ratio of oil to glycerol was 1:1.5, the mass ratio of ion exchange resin oil FPA-54 to glycerol was 2:1, the enzyme addition amount was 5%, and the ion exchange resin FPA-53 addition amount was 3%. After the reaction, glycerol and lipase were separated, and the content of diglyceride in the crude product was sampled and analyzed. The crude product was molecularly distilled, wherein the conditions of the three-stage molecular distillation were: 110°C, 50Pa for the first stage; 130°C, 10Pa for the second stage, and 180°C, 1Pa for the third stage. The results are shown in Table 5.

[0061] Table 5 Effect of different lipases on diglyceride content in crude glycerol hydrolysis products

[0062]

[0063] From the results in Table 5, it can be seen that for lipases with polar materials as enzyme immobilization carriers, the pre-adsorption of glycerol can greatly improve the catalytic efficiency of enzymatic glycerolysis, among which Lipozyme RM IM has the highest catalytic efficiency, with the diglyceride content in the crude product reaching 52.57%, and the diglyceride content after molecular distillation reaching 88.70%. Pre-adsorption of glycerol by polar materials can effectively prevent polar substrates such as glycerol from gathering near the active site of lipase, causing enzyme activity loss.

[0064] Comparative Example 1

[0065] In step (2), no tertiary amine resin was added, and refined soybean oil and lipase Lipozyme RM IM were mixed in a reactor. The remaining steps were exactly the same as in Example 1. After 5 cycles of testing, the diglyceride content of the crude product was analyzed by sampling. The results are shown in Figure 2 As shown in b.

[0066] After five cycles of glycerol hydrolysis experiments, the diglyceride content of the crude product decreased from 37.02% to 11.37%. Compared with the diglyceride content of the crude product in Example 1, the tertiary amine resin effectively extended the number of times the lipase was used.

[0067] Comparative Example 2

[0068] In step (1), no adsorbent was used to pre-absorb glycerol, refined soybean oil, tertiary amine resin, glycerol and lipase Lipozyme TL IM were mixed in a reactor, and the remaining steps were exactly the same as in Example 6. The diglyceride content of the crude product was analyzed by sampling, and the results are shown in Table 6.

[0069] Comparative Example 3

[0070] In step (1), the fixed bed reaction column was not used, and the microcrystalline cellulose pre-adsorbed with glycerol, refined soybean oil, tertiary amine resin, glycerol and lipase Lipozyme TL IM were mixed in a reactor, and the remaining steps were exactly the same as those in Example 6. The diglyceride content of the crude product was analyzed by sampling, and the results are shown in Table 6.

[0071] Table 6 Physical and chemical properties of crude diglyceride oil

[0072] Diacylceride content, % Triglyceride content, % Monoglyceride content, % Fatty acid content, % Example 6 51.52 34.78 5.37 8.33 Comparative Example 2 8.36 85.84 1.46 4.34 Comparative Example 3 40.42 51.31 4.54 3.73

[0073] From the results in Table 6, it can be seen that in Comparative Example 2, when cellulose was not used for pre-adsorption of glycerol, the content of diglycerol in the product was only 8.36%. After adsorption with the adsorbent, the content of diglycerol in the product reached 40.42%. However, compared with the use of a fixed bed reaction column to fix the adsorption material, the contact between the glycerol carrier and the lipase was reduced, and the content of diglycerol in the crude product increased to 51.52%.

[0074] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the present invention.

Claims

1. A method for preparing diglyceride oil by a solvent-free enzymatic method, characterized in that: The method comprises the following steps: Step 1, mixing glycerol and cellulose adsorption material and letting them stand, pre-adsorbing the glycerol, mixing the pre-adsorbed glycerol and oil of the same mass as the glycerol, wet-loading the mixture, and adding the mixture to a fixed bed reaction column; the cellulose adsorption material is carboxymethyl cellulose or microcrystalline cellulose; Step 2, mixing the oil, polar resin material and lipase in a reactor; The modified group of the polar resin material is a tertiary amine group, a primary amine group or a sulfonic acid group, the average pore size of the polar resin material is 10nm to 30nm, and the amount of the polar resin material added is not less than 80wt% of the amount of lipase added; the tertiary amino polar resin material is an ion exchange resin FPA-54; the primary amine polar resin material is D301; and the sulfonic acid polar resin material is AMBERLITER120Na; Step 3, connecting the reactor to the fixed bed reaction column, setting a membrane at the connection to allow only oil and glycerol to flow, blocking the polar resin and lipase; allowing the oil and glycerol mixture in the fixed bed reaction column to continuously flow into the reactor and reflux to the fixed bed reaction column through an oil pump, and the polar resin and lipase only flow and react in the reactor; cyclic reaction, collecting the product in the reactor after the reaction is completed, and separating and purifying the oil of diglyceride.

2. The method for preparing diglyceride oil by a solvent-free enzymatic method as claimed in claim 1, characterized in that: The moisture content of the polar resin material is 5wt% to 10wt% of the weight of the adsorption material.

3. The method for preparing diglyceride oil by a solvent-free enzymatic method as claimed in claim 1, characterized in that: The lipase immobilization carrier is a medium polarity macroporous adsorption resin or a weak base cation exchange resin.

4. The method for preparing diglyceride oil by a solvent-free enzymatic method as claimed in claim 1, characterized in that: The lipase is one or more of NE-100, Novozyme 435, Lipozyme 435, Lipase PSIM, Lipase DF "Amano" IM, Lipase TLIM and Lipase RM IM.

5. The method for preparing diglyceride oil by a solvent-free enzymatic method as claimed in claim 1, characterized in that: The lipases are produced by Candida antarctica, Rhizopus oryzae, Aspergillus niger, Candida rugosa, Burkholderia and Pseudomonas.

6. The method for preparing diglyceride oil by a solvent-free enzymatic method as claimed in claim 1, characterized in that: The separation and purification method is molecular distillation.

7. The method for preparing diglyceride oil by a solvent-free enzymatic method as claimed in claim 6, characterized in that: The molecular distillation is a three-stage molecular distillation, and the specific conditions are: the first stage is 105℃~120℃, 50Pa; the second stage is 130℃~150℃, 10Pa; the third stage is 180℃~190℃, 1Pa.

8. The method for preparing diglyceride oil by a solvent-free enzymatic method as claimed in claim 1, characterized in that: The amount of lipase added is 3wt% to 7wt% of the weight of the oil; The reaction temperature is 45-60°C; The molar ratio of all the oils and fats added in the reaction to glycerol is 1:0.75-2, and the mass ratio of the cellulose adsorbent to glycerol is 1:0.5-2.

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