Method for preparing diglyceride based on immobilized lipase

Abstract

The invention belongs to the technical field of diglyceride production, and particularly relates to a method for preparing diglyceride based on immobilized lipase, and the preparation method of the immobilized lipase comprises the following steps: A1, adding 2, 5-dihydroxy terephthalic acid, trimesic acid and iron powder into ethanol, and reacting to generate a framework material; a2, adding a framework material into ethanol, uniformly stirring to form a framework solution, dissolving lipase into water to form an enzyme solution, mixing the framework solution and the enzyme solution, and reacting to obtain a loading solution; and A3, putting the porous carrier into the loading liquid, then adding a cross-linking agent, and filtering to remove the liquid after reaction to obtain a solid product, namely the immobilized lipase. According to the invention, the framework material is used for providing a coordination point for the lipase, so that the lipase forms ordered spatial arrangement, and conformational deformation of the lipase in a crosslinking process is avoided, thereby improving the stability of the lipase, ensuring the activity and the service life of the generated immobilized enzyme catalyst, and improving the purity of the generated diglyceride.

Description

Technical Field

[0001] The invention belongs to the technical field of diglyceride production, and particularly relates to a method for preparing diglyceride based on immobilized lipase. Background Art

[0002] Diacylcerol is an important lipid compound with broad application prospects in the fields of food, medicine, chemistry, etc. The production process of diacylglycerol includes chemical synthesis and bioenzyme method. Both production processes can achieve industrial mass production. However, since the chemical synthesis method requires high temperature conditions, it will produce carcinogenic glycidol. With the development of the market, the requirements for food and medicine safety have increased. Therefore, the bioenzyme method is currently the main method for preparing diacylglycerol.

[0003] The bio-enzyme method is divided into liquid enzyme method and fixed enzyme method. The enzyme catalyst used in the liquid enzyme method is in liquid state. Liquid enzymes are easy to aggregate during use, which reduces the catalytic rate and has a short lifespan. The enzyme catalyst used in the fixed enzyme method is in solid state, which is easy to separate and disperse, thereby improving the preparation conversion rate of diglyceride. At the same time, it can be recycled and reused, reducing production costs.

[0004] At present, when lipase is immobilized, a cross-linking agent is generally used to cross-link the lipase and the carrier. However, during the cross-linking process, the lipase will aggregate or over-disperse due to uneven dispersion, resulting in deformed cross-linked network structure, ring structure or spatial defects, affecting the quality of the final enzyme catalyst, shortening the life of the enzyme catalyst and the purity of the generated diglyceride. Summary of the invention

[0005] In order to solve the problems existing in the above-mentioned prior art, the present invention provides a method for preparing diglyceride based on immobilized lipase. The present invention provides coordination sites for lipase through a framework material, so that the lipase forms an orderly spatial arrangement, avoids conformational deformation of the lipase during the cross-linking process, thereby improving the stability of the enzyme, ensuring the activity and life of the generated immobilized enzyme catalyst, and improving the purity of the generated diglyceride.

[0006] The specific technical solution adopted by the present invention is:

[0007] A method for preparing diglyceride based on immobilized lipase comprises the following steps:

[0008] S1, hydrolyzing the vegetable oil with a liquid hydrolase, and then performing sedimentation separation;

[0009] S2, taking out the upper layer product obtained by sedimentation separation in step S1 and performing molecular distillation to obtain heavy component a and light component a by stratification;

[0010] S3, the light component a enters the esterification tank, and then glycerol is added to carry out esterification reaction under the catalysis of immobilized lipase, and then sedimentation separation is carried out;

[0011] S4, taking out the upper layer product obtained by sedimentation separation in step S3 and performing molecular distillation to obtain heavy component b and light component b by stratification;

[0012] S5. Separate, purify and decolorize the heavy component b to obtain a finished diglyceride product.

[0013] Wherein, the preparation method of the immobilized lipase comprises the following steps:

[0014] A1. Add 2,5-dihydroxyterephthalic acid and mesitylene tribenzoic acid to ethanol, heat to 120-150°C for reaction for 4-8h, then add iron powder, cool to 80-100°C and continue to react for 4-8h to generate a framework material;

[0015] A2. Add the framework material to ethanol and stir evenly to form a framework liquid, then dissolve the lipase in water to form an enzyme liquid, mix the framework liquid and the enzyme liquid, heat to 50-80° C. and react for 4-6 hours to obtain a loading liquid;

[0016] A3. Place the porous carrier into the loading liquid, then add the cross-linking agent, stir for 12-20 hours, filter and remove the liquid, and the obtained solid product is the immobilized lipase.

[0017] Furthermore, in step S3, the light component a, glycerol and immobilized lipase are respectively 6-10 parts, 0.5-1 parts and 0.025-0.075 parts by mass.

[0018] Furthermore, the temperature of the esterification reaction in step S3 is 50-60° C., and the esterification time is 6-8 h.

[0019] Furthermore, the immobilized lipase in step S3 is loaded into an enzyme reaction column and placed in an esterification tank, and the reaction liquid formed by mixing the light component and glycerol is circulated through the reaction column.

[0020] Furthermore, the 2,5-dihydroxyterephthalic acid, tris-benzoic acid and iron powder in step A1 are respectively 3-3.5 parts, 9-10 parts and 5.5-6 parts by mass.

[0021] Furthermore, in step A2, the mass ratio of the framework material to the lipase is 10-15:1.

[0022] Furthermore, the lipase is derived from any one of Candida antarctica, Pseudomonas alcaligenes and Rhizomucor miehei.

[0023] Furthermore, the cross-linking agent includes any one of polyallylamine, glutaraldehyde, and terephthalaldehyde.

[0024] The beneficial effects of the present invention are:

[0025] The framework material is used in the present invention to provide coordination sites for lipase, so that the lipase forms an orderly spatial arrangement, avoiding conformational deformation of the lipase during the cross-linking process, thereby improving the stability of the enzyme, ensuring the activity of the generated immobilized enzyme catalyst, and improving the purity of the generated diglyceride.

[0026] At the same time, the framework material itself forms a binding effect on the lipase. Under the dual effects of the adsorption of the framework material and the chemical connection of the cross-linking agent, the fixation effect on the lipase is further improved, preventing the lipase on the enzyme catalyst from falling off, thereby increasing the number of times the enzyme catalyst is reused and increasing the service life of the enzyme catalyst. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the production process of diglyceride in the present invention;

[0028] Figure 2 This is the production process of decolorization in the present invention. DETAILED DESCRIPTION

[0029] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. 1. Specific embodiments

[0031] Preparation of diglycerides

[0032] The specific preparation steps of diglyceride are as follows:

[0033] S1, stirring the soybean oil with liquid hydrolase at 40°C for 6 hours to carry out hydrolysis reaction, and then separating by sedimentation after the reaction;

[0034] S2, taking out the upper layer product obtained by sedimentation separation in step S1, inputting it into a molecular distillation storage tank for molecular distillation, and layering to obtain a heavy component a and a light component a;

[0035] S3, the light component a enters the esterification tank, and then glycerol is added to carry out esterification reaction under the catalytic action of immobilized lipase. The esterification reaction temperature is 50°C, the esterification time is 8h, and then sedimentation separation is carried out;

[0036] The weight proportions of the light component a, glycerol and immobilized lipase are 10 parts, 1 part and 0.05 parts respectively;

[0037] S4, taking out the upper layer product obtained by sedimentation separation in step S3 and performing molecular distillation to obtain heavy component b and light component b by stratification;

[0038] S5. Separate, purify and decolorize the heavy component b to obtain a finished diglyceride product with a purity of ≥90%.

[0039] The heavy component a obtained in step S2 is also separated, purified and decolorized to obtain a low-purity diglyceride product with a purity of about 50%. The high-purity diglyceride product and the low-purity diglyceride product are sold separately to improve economic benefits. In addition, the decolorization process is an existing technology, see Figure 2 .

[0040] Preparation of immobilized lipase

[0041] Example 1

[0042] A1. Add 3.2 parts of 2,5-dihydroxyterephthalic acid and 10 parts of tris-benzoic acid to ethanol by mass, heat to 140°C for reaction for 6 hours, then add 5.6 parts of iron powder, cool to 90°C and continue to react for 6 hours to generate a framework material;

[0043] A2. Take a framework material and lipase in a mass ratio of 12:1, add the framework material to ethanol and stir evenly to form a framework liquid, then dissolve the lipase in water to form an enzyme liquid, mix the framework liquid and the enzyme liquid, heat to 60°C for 5 hours, and react to obtain a load liquid;

[0044] A3. Put a macroporous adsorption resin into the loading liquid, wherein the particle size of the macroporous adsorption resin is 200 μm and the pore size is 0.2 μm, then add glutaraldehyde, stir at a speed of 80 rpm for 18 hours, filter and remove the liquid, and the obtained solid product is the immobilized lipase.

[0045] Example 2

[0046] A1. Add 3 parts of 2,5-dihydroxyterephthalic acid and 9 parts of mesitylene tribenzoic acid to ethanol by mass, heat to 120°C for reaction for 8 hours, then add 5.5 parts of iron powder, cool to 80°C and continue to react for 8 hours to generate a framework material;

[0047] A2, taking a framework material and lipase in a mass ratio of 10:1, adding the framework material to ethanol and stirring evenly to form a framework liquid, then dissolving the lipase in water to form an enzyme liquid, mixing the framework liquid and the enzyme liquid, heating to 50°C for 6 hours, and reacting to obtain a load liquid;

[0048] A3. Put a macroporous adsorption resin into the loading liquid, wherein the particle size of the macroporous adsorption resin is 200 μm and the pore size is 0.2 μm, then add glutaraldehyde, stir at a speed of 80 rpm for 18 hours, filter and remove the liquid, and the obtained solid product is the immobilized lipase.

[0049] Example 3

[0050] A1. Add 3.5 parts of 2,5-dihydroxyterephthalic acid and 10 parts of tris-benzoic acid to ethanol by mass, heat to 120°C for reaction for 8 hours, then add 6 parts of iron powder, cool to 80°C and continue to react for 8 hours to generate a framework material;

[0051] A2, taking a framework material and lipase in a mass ratio of 15:1, adding the framework material to ethanol and stirring evenly to form a framework liquid, then dissolving the lipase in water to form an enzyme liquid, mixing the framework liquid and the enzyme liquid, heating to 80°C for 4 hours, and reacting to obtain a load liquid;

[0052] A3. Put a macroporous adsorption resin into the loading liquid, wherein the particle size of the macroporous adsorption resin is 200 μm and the pore size is 0.2 μm, then add glutaraldehyde, stir at a speed of 80 rpm for 18 hours, filter and remove the liquid, and the obtained solid product is the immobilized lipase.

[0053] Comparative Example 1

[0054] The difference between Comparative Example 1 and Example 1 is that in Comparative Example 1, the lipase is directly cross-linked with the porous carrier, and the specific steps are as follows:

[0055] A1. Dissolve lipase in water to form enzyme solution;

[0056] A3. Put a macroporous adsorption resin into the enzyme solution. The particle size of the macroporous adsorption resin is 200 μm and the pore size is 0.2 μm. Then add glutaraldehyde. Stir at a speed of 80 rpm for 18 hours. Filter to remove the liquid. The obtained solid product is the immobilized lipase.

[0057] 2. Performance Test

[0058] Preparation of diglycerides

[0059] According to the preparation steps of diglyceride in the present invention, the immobilized lipase prepared in Examples 1-3 and Comparative Example 1 was used to prepare diglyceride, which was repeated multiple times. Then, the purity of the diglyceride prepared each time was measured. The test results are shown in Table 1.

[0060] Table 1

[0061] 1st time 2nd time 3rd 4th 5th 6th 7th 8th Example 1 96.51 96.01 95.74 95.53 95.14 94.73 94.21 93.52 Example 2 95.40 95.20 94.92 94.68 94.39 94.04 93.62 93.05 Example 3 95.00 94.86 94.63 94.44 94.07 93.67 93.18 92.51 Comparative Example 1 93.13 92.15 90.68 88.31 - - - -

[0062] As can be seen from Table 1, when the catalyst prepared in Examples 1-3 was used for the 4th time, the purity of diglyceride was above 94%, and when it was used for the 8th time, the purity of diglyceride was above 92.5%. It is conservatively estimated that the immobilized enzyme catalyst in the present invention can be used more than 10 times.

[0063] While the catalyst prepared in Comparative Example 1 was used, although the diglyceride prepared initially had a high purity, after being used four times, the purity of the diglyceride was less than 90%, which did not meet the preparation requirements and needed to be replaced.

[0064] The catalyst in the present invention has a good degree of bonding with the carrier. This is because the lipase is bound by the framework material, and the framework material and the porous carrier form a stable combination, thereby improving the fixation of the lipase and effectively preventing the lipase from falling off. At the same time, since the stable conformation of the lipase during the cross-linking reaction is guaranteed, the activity of the lipase is also improved, and the purity of diglyceride is further improved.

Claims

1. A method for preparing diglyceride based on immobilized lipase, characterized in that: The following steps are involved: S1, hydrolyzing the vegetable oil with a liquid hydrolase, and then performing sedimentation separation; S2, taking out the upper layer product obtained by sedimentation separation in step S1 and performing molecular distillation to obtain heavy component a and light component a by stratification; S3, the light component a enters the esterification tank, and then glycerol is added to carry out esterification reaction under the catalysis of immobilized lipase, and then sedimentation separation is carried out; S4, taking out the upper layer product obtained by sedimentation separation in step S3 and performing molecular distillation to obtain heavy component b and light component b by stratification; S5, separating, purifying and decolorizing the heavy component b to obtain a diglyceride finished product; Wherein, the preparation method of the immobilized lipase comprises the following steps: A1. Add 2,5-dihydroxyterephthalic acid and mesitylene tribenzoic acid to ethanol, heat to 120-150°C for reaction for 4-8h, then add iron powder, cool to 80-100°C and continue to react for 4-8h to generate a framework material; A2. Add the framework material to ethanol and stir evenly to form a framework liquid, then dissolve the lipase in water to form an enzyme liquid, mix the framework liquid and the enzyme liquid, heat to 50-80° C. and react for 4-6 hours to obtain a loading liquid; A3. Place the porous carrier into the loading liquid, then add the cross-linking agent, stir for 12-20 hours, filter and remove the liquid, and the obtained solid product is the immobilized lipase.

2. The method for preparing diglyceride based on immobilized lipase according to claim 1, characterized in that: In step S3, the light component a, glycerol and immobilized lipase are respectively 6-10 parts, 0.5-1 parts and 0.025-0.075 parts by mass.

3. The method for preparing diglyceride based on immobilized lipase according to claim 1, characterized in that: The temperature of the esterification reaction in step S3 is 50-60° C., and the esterification time is 6-8 hours.

4. The method for preparing diglyceride based on immobilized lipase according to claim 1, characterized in that: In step S3, the immobilized lipase is loaded into an enzyme reaction column and placed in an esterification tank, and the reaction liquid formed by mixing the light component and glycerol is circulated through the reaction column.

5. The method for preparing diglyceride based on immobilized lipase according to claim 1, characterized in that: The 2,5-dihydroxyterephthalic acid, mesitylene-benzoic acid and iron powder in step A1 are respectively 3-3.5 parts, 9-10 parts and 5.5-6 parts by mass.

6. The method for preparing diglyceride based on immobilized lipase according to claim 1, characterized in that: The mass ratio of the framework material to the lipase in step A2 is 10-15:

1.

7. The method for preparing diglyceride based on immobilized lipase according to claim 1, characterized in that: The lipase is derived from any one of Candida antarctica, Pseudomonas alcaligenes and Rhizomucor miehei.

8. The method for preparing diglyceride based on immobilized lipase according to claim 1, characterized in that: The cross-linking agent includes any one of polyallylamine, glutaraldehyde and terephthalaldehyde.

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