Method for preparing 1, 3-dioleic acid-2-palmitic acid triglyceride by enzyme method

Abstract

The invention provides a method for preparing 1, 3-dioleic acid-2-palmitic acid triglyceride by an enzyme method, and relates to the technical field of preparation of the 1, 3-dioleic acid-2-palmitic acid triglyceride. The 1, 3-dioleic acid-2-palmitic acid triglyceride is prepared by mixing lard oil with cholesterol mass content lower than 0.05% and oleate, performing low-temperature high-pressure treatment, performing variable-pressure enzymolysis, separating lipase, and performing crystallization washing, thereby obtaining a final product. According to the method, the defects in the prior art are overcome, the high-purity 1, 3-dioleic acid-2-palmitic acid triglyceride can be prepared from the lard oil and the oleate, the lipase can be used for multiple times, the preparation efficiency of the 1, 3-dioleic acid-2-palmitic acid triglyceride is comprehensively improved, and the preparation cost is reduced.

Description

Technical Field

[0001] This invention relates to the field of preparation technology of 1,3-dioleoyl-2-palmitoylglycerol triglyceride, and specifically to an enzymatic method for preparing 1,3-dioleoyl-2-palmitoylglycerol triglyceride. Background Technology

[0002] 1,3-Dioleoyl-2-palmitoylglycerol (OPO) is a unique fatty acid molecule with a structure that mimics that of breast milk. It is a common additive in the food industry, especially in infant formula and health supplements.

[0003] There are two main methods for preparing OPO: chemical preparation and enzymatic preparation. Chemical preparation is complex and requires the use of a large number of harmful reagents, so it is rarely used. Enzymatic preparation has attracted much attention from researchers due to its advantages such as high specificity, mild catalytic conditions, and high purity of the product, and is currently the mainstream method for preparing OPO.

[0004] The existing patent application number 201110139857.8, entitled "A method for producing 1,3-dioleoyl-2-palmitoylglycerol triglyceride," uses lard as raw material to prepare OPO. It mainly promotes catalytic efficiency and increases the content of OPO in the subsequent process through temperature-variable enzymatic hydrolysis. However, in actual operation, it was found that the higher reaction temperature has an adverse effect on the stability of the enzyme, reduces the enzyme's lifespan, and increases energy consumption. Furthermore, due to the possible side reactions and acyl migration during the reaction process, it is difficult to guarantee the purity of the final OPO product. Therefore, it is crucial to improve the yield and purity of OPO. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides an enzymatic method for preparing 1,3-dioleoyl-2-palmitoyl glycerol triglyceride. This method utilizes lard and oleic acid esters to prepare high-purity 1,3-dioleoyl-2-palmitoyl glycerol triglyceride. The lipase can be reused multiple times, thereby comprehensively improving the preparation efficiency of 1,3-dioleoyl-2-palmitoyl glycerol triglyceride and reducing the preparation cost.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A method for preparing 1,3-dioleoyl-2-palmitoylglycerol triglyceride by enzymatic method, the method comprising the following steps: S1. Mix lard with oleic acid esters containing less than 0.05% cholesterol until well combined to obtain a mixture for later use. S2. Add the above mixture into a high-pressure reactor, adjust the temperature to 4-6℃ and the pressure to 10-15MPa, stir and process to obtain the pretreated material for later use. S3. Heat the pretreated material to 35-40℃, then add lipase, stir evenly, and continue heating to 50-60℃. Perform enzymatic hydrolysis treatment at pressures of 0.2-0.3 MPa, 0.8-1.0 MPa, 3-4 MPa, and 6-8 MPa in sequence, and control the oxygen concentration during enzymatic hydrolysis to be 10%-15%. S4. After enzymatic hydrolysis, vacuum filter to separate the lipase, then mix with anhydrous ethanol to separate and crystallize, and then wash to obtain the final product.

[0007] Preferably, in step S1, the ratio of lard with a cholesterol content of less than 0.05% to oleic acid esters is 1:3-5 by mass.

[0008] Preferably, the oleate in step S1 is at least one of methyl oleate, ethyl oleate, trioleate, butyl oleate, and isopropyl oleate.

[0009] Preferably, the stirring speed in step S2 is 400-600 r / min, and the processing time is 20-40 min.

[0010] The lipase in step S3 is an immobilized lipase, and the amount of lipase added is 8%-10% of the total mass of the pretreated material.

[0011] Preferably, in step S3, the enzymatic hydrolysis time is 10-20 min under a pressure of 0.2-0.3 MPa, 10-20 min under a pressure of 0.8-1.0 MPa, 1-2 h under a pressure of 3-4 MPa, and 40-60 min under a pressure of 6-8 MPa.

[0012] Preferably, in step S4, the method of separating and crystallizing by mixing anhydrous ethanol is to add 5-10 times the volume of anhydrous ethanol and then separate and crystallize at a temperature of 15-20°C.

[0013] Preferably, the washing method in step S4 is to add 5-8 times the amount of anhydrous ethanol, heat to 60-80°C, keep warm for 5-10 minutes, then cool down to 4-8°C, let stand for 15-20 minutes, separate the anhydrous ethanol, and then evaporate the remaining anhydrous ethanol under reduced pressure.

[0014] Preferably, the rate of heating to 60-80℃ is 5-10℃ / min, and the rate of cooling to 4-8℃ is 2-3℃ / min.

[0015] This invention provides an enzymatic method for preparing 1,3-dioleoyl-2-palmitoylglycerol triglyceride, which has the following advantages compared with the prior art: This invention facilitates subsequent enzymatic hydrolysis by pre-treating low-cholesterol lard and oleic acid esters under high pressure, thereby shortening the overall hydrolysis time and improving efficiency. Combined with subsequent pressure swing oxygen-controlled enzymatic hydrolysis, it further enhances product generation efficiency. Compared to temperature-controlled enzymatic hydrolysis in existing technologies, this invention effectively extends the lifespan of related lipases, saves preparation costs, and further improves the purity of subsequent 1,3-dioleoyl-2-palmitoylglycerol, thus comprehensively improving the efficiency and effectiveness of the preparation. Attached Figure Description

[0016] Figure 1 This is a schematic diagram showing the changes in the content of the final product POP under different ratios of lard and methyl oleate in embodiments of the present invention. Figure 2 This is a schematic diagram showing the change in the content of the final product POP under different oxygen concentrations during enzymatic hydrolysis in an embodiment of the present invention. Figure 3 This is a schematic diagram comparing the content of POP in the final product and the proportion of palmitic acid at the sn-2 position in POP under different treatment methods in Example 1 and Comparative Examples 1-5 of the present invention. Figure 4 This is a schematic diagram comparing the results of lipase recycling in embodiments of the present invention. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] The lipase used below was purchased from Guangzhou Daidi Biological Reagents Co., Ltd., and its product name is immobilized lipase Dizyme 435.

[0019] Lard with a cholesterol content of less than 0.05% was prepared in the laboratory, and the specific preparation method is as follows: (1) Select pork fat as raw material, test its crude fat content to be 85.2% and protein content to be 4.5%, mince it into meat paste, then add water of equal weight, mix and stir evenly and adjust the pH to 8-9 to obtain a mixture for later use; (2) Add 3% of the total mass of alkaline protease (800 U / g) to the above mixture, react at 50°C oil bath temperature for 3 h, then centrifuge and collect the upper oil phase; (3) Mix the upper oil phase with an equal mass of water, and add 4% of the total mass of the upper oil phase of β-cyclodextrin. Homogenize the mixture at 50°C in an oil bath to obtain a homogenized material. (4) Centrifuge the above homogenized material again, collect the upper layer of oil, and test its cholesterol content to be within 0.05% to obtain raw lard for use.

[0020] Example 1: Preparation of 1,3-dioleoyl-2-palmitoylglycerol triglyceride: (1) Mix the above raw materials, lard and methyl oleate, at a mass ratio of 1:4 and stir evenly. Then place them in a high-pressure reactor, adjust the temperature to 5℃ and the pressure to 12MPa, and stir at a speed of 500r / min for 30min to obtain the pretreated material for later use. (2) Heat the above pretreated material to 35°C, then add 9% of the total mass of the pretreated material with lipase, stir evenly, and continue to heat to 55°C. Adjust the pressure to 0.3 MPa and enzymatically hydrolyze for 15 min. Then adjust the pressure to 1 MPa and enzymatically hydrolyze for 15 min. Adjust the pressure to 3 MPa and enzymatically hydrolyze for 1.5 h. Then adjust the pressure to 7 MPa and enzymatically hydrolyze for 50 min. The oxygen concentration in the device is controlled at 12% throughout the entire enzymatic hydrolysis process. (3) After the enzymatic hydrolysis is completed, vacuum filter is used to separate the lipase and obtain the enzymatic hydrolysis product. Mix the enzymatic hydrolysis product with 8 times the volume of anhydrous ethanol, control the temperature at 18℃ for separation and crystallization, then filter to remove the filtrate, add 6 times the volume of anhydrous ethanol, heat to 70℃ at a heating rate of 5-10℃ / min, keep warm for 8min, then cool to 6℃ at a cooling rate of 2-3℃ / min, continue to stand for crystallization for 20min, separate the anhydrous ethanol, and evaporate the remaining solvent to obtain the final product.

[0021] 1. Following the method described above, the content of 1,3-dioleoyl-2-palmitoylglycerol (POP) in the final product was determined under different mass ratios of raw lard to methyl oleate. The specific results are shown in Table 1 below. Figure 1 As shown: Table 1 As shown in the table above, when the mass ratio of raw lard to methyl oleate is less than 1:3, the POP content in the final product is below 70%. However, when the mass ratio of raw lard to methyl oleate is greater than 1:5, the POP content fluctuates less compared to when the mass ratio of raw lard to methyl oleate is 1:5. Based on the requirements of increasing POP content and reducing costs, a mass ratio of raw lard to methyl oleate of 1:3-5 is more suitable.

[0022] 2. Following the method described above, the effect of different oxygen concentrations during enzymatic hydrolysis on the POP content in the final product was detected, as shown in Table 2 below. Figure 2 As shown: Table 2 As shown in the table above, when the oxygen concentration is between 10% and 15% during enzymatic hydrolysis, the POP content in the final product is relatively high.

[0023] Comparative Example 1: Preparation of 1,3-dioleoyl-2-palmitoylglycerol triglyceride: (1) Mix the above raw materials, lard and methyl oleate, at a mass ratio of 1:4 and stir evenly to obtain the pretreated material for later use; (2) Heat the above pretreated material to 35°C, then add 9% of the total mass of the pretreated material with lipase, stir evenly, and continue to heat to 55°C. Adjust the pressure to 0.3 MPa and enzymatically hydrolyze for 15 min. Then adjust the pressure to 1 MPa and enzymatically hydrolyze for 15 min. Adjust the pressure to 3 MPa and enzymatically hydrolyze for 1.5 h. Then adjust the pressure to 7 MPa and enzymatically hydrolyze for 50 min. The oxygen concentration in the device is controlled at 10%-15% throughout the entire enzymatic hydrolysis process. (3) After the enzymatic hydrolysis is completed, vacuum filter is used to separate the lipase and obtain the enzymatic hydrolysis product. Mix the enzymatic hydrolysis product with 8 times the volume of anhydrous ethanol, control the temperature at 18℃ for separation and crystallization, then filter to remove the filtrate, add 6 times the volume of anhydrous ethanol, heat to 70℃ at a heating rate of 5-10℃ / min, keep warm for 8min, then cool to 6℃ at a cooling rate of 2-3℃ / min, continue to stand for crystallization for 20min, separate the anhydrous ethanol, and evaporate the remaining solvent to obtain the final product.

[0024] Comparative Example 2: Preparation of 1,3-dioleoyl-2-palmitoylglycerol triglyceride: (1) Mix the above raw materials, lard and methyl oleate, at a mass ratio of 1:4 and stir evenly. Then place them in a high-pressure reactor, adjust the temperature to 5℃ and the pressure to 12MPa, and stir at a speed of 500r / min for 30min to obtain the pretreated material for later use. (2) Heat the above pretreated material to 35°C, then add 9% of the total mass of the pretreated material with lipase, stir evenly, and continue to heat to 55°C. Adjust the pressure to 0.3 MPa and perform enzymatic hydrolysis for 170 min. The oxygen concentration in the device is controlled at 10%-15% throughout the entire enzymatic hydrolysis process. (3) After the enzymatic hydrolysis is completed, vacuum filter is used to separate the lipase and obtain the enzymatic hydrolysis product. Mix the enzymatic hydrolysis product with 8 times the volume of anhydrous ethanol, control the temperature at 18℃ for separation and crystallization, then filter to remove the filtrate, add 6 times the volume of anhydrous ethanol, heat to 70℃ at a heating rate of 5-10℃ / min, keep warm for 8min, then cool to 6℃ at a cooling rate of 2-3℃ / min, continue to stand for crystallization for 20min, separate the anhydrous ethanol, and evaporate the remaining solvent to obtain the final product.

[0025] Comparative Example 3: Preparation of 1,3-dioleoyl-2-palmitoylglycerol triglyceride: (1) Mix the above raw materials, lard and methyl oleate, at a mass ratio of 1:4 and stir evenly. Then place them in a high-pressure reactor, adjust the temperature to 5℃ and the pressure to 12MPa, and stir at a speed of 500r / min for 30min to obtain the pretreated material for later use. (2) Heat the above pretreated material to 35°C, then add 9% of the total mass of the pretreated material with lipase, stir evenly, and continue to heat to 55°C. Adjust the pressure to 1 MPa and perform enzymatic hydrolysis for 170 min. The oxygen concentration in the device is controlled at 10%-15% throughout the entire enzymatic hydrolysis process. (3) After the enzymatic hydrolysis is completed, vacuum filter is used to separate the lipase and obtain the enzymatic hydrolysis product. Mix the enzymatic hydrolysis product with 8 times the volume of anhydrous ethanol, control the temperature at 18℃ for separation and crystallization, then filter to remove the filtrate, add 6 times the volume of anhydrous ethanol, heat to 70℃ at a heating rate of 5-10℃ / min, keep warm for 8min, then cool to 6℃ at a cooling rate of 2-3℃ / min, continue to stand for crystallization for 20min, separate the anhydrous ethanol, and evaporate the remaining solvent to obtain the final product.

[0026] Comparative Example 4: Preparation of 1,3-dioleoyl-2-palmitoylglycerol triglyceride: (1) Mix the above raw materials, lard and methyl oleate, at a mass ratio of 1:4 and stir evenly. Then place them in a high-pressure reactor, adjust the temperature to 5℃ and the pressure to 12MPa, and stir at a speed of 500r / min for 30min to obtain the pretreated material for later use. (2) Heat the above pretreated material to 35°C, then add 9% of the total mass of the pretreated material with lipase, stir evenly, and continue to heat to 55°C. Adjust the pressure to 3 MPa and perform enzymatic hydrolysis for 170 min. The oxygen concentration in the device is controlled at 10%-15% throughout the entire enzymatic hydrolysis process. (3) After the enzymatic hydrolysis is completed, vacuum filter is used to separate the lipase and obtain the enzymatic hydrolysis product. Mix the enzymatic hydrolysis product with 8 times the volume of anhydrous ethanol, control the temperature at 18℃ for separation and crystallization, then filter to remove the filtrate, add 6 times the volume of anhydrous ethanol, heat to 70℃ at a heating rate of 5-10℃ / min, keep warm for 8min, then cool to 6℃ at a cooling rate of 2-3℃ / min, continue to stand for crystallization for 20min, separate the anhydrous ethanol, and evaporate the remaining solvent to obtain the final product.

[0027] Comparative Example 5: Preparation of 1,3-dioleoyl-2-palmitoylglycerol triglyceride: (1) Mix the above raw materials, lard and methyl oleate, at a mass ratio of 1:4 and stir evenly. Then place them in a high-pressure reactor, adjust the temperature to 5℃ and the pressure to 12MPa, and stir at a speed of 500r / min for 30min to obtain the pretreated material for later use. (2) Heat the above pretreated material to 35°C, add 9% of the total mass of the pretreated material with lipase, stir evenly, and continue to heat to 55°C. Adjust the pressure to 7 MPa and perform enzymatic hydrolysis for 170 min. The oxygen concentration in the device is controlled at 10%-15% throughout the entire enzymatic hydrolysis process. (3) After the enzymatic hydrolysis is completed, vacuum filter is used to separate the lipase and obtain the enzymatic hydrolysis product. Mix the enzymatic hydrolysis product with 8 times the volume of anhydrous ethanol, control the temperature at 18℃ for separation and crystallization, then filter to remove the filtrate, add 6 times the volume of anhydrous ethanol, heat to 70℃ at a heating rate of 5-10℃ / min, keep warm for 8min, then cool to 6℃ at a cooling rate of 2-3℃ / min, continue to stand for crystallization for 20min, separate the anhydrous ethanol, and evaporate the remaining solvent to obtain the final product.

[0028] Detection: 1. The content of POP in the final products obtained in Example 1 and Comparative Examples 1-5 was detected, and the proportion of palmitic acid at the sn-2 position in POP was detected and calculated. The specific results are shown in Table 3 below. Figure 3 As shown: Table 3 The above tests show that the pressure swing regulation during enzymatic hydrolysis can effectively promote the POP content of the final product and further increase the proportion of palmitic acid at the sn-2 position.

[0029] 2. The lipase isolated in Example 1 was washed and reused (using the same method as in Example 1). The POP content of the final product was measured after 1, 2, 3, 4 and 5 uses of the lipase. The specific results are shown in Table 4 below: Table 4 As can be seen from the above tests, the method of Example 1, which involves repeated cycles of lipase, still has a good effect.

[0030] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing 1,3-dioleoyl-2-palmitoylglycerol triglyceride by enzymatic method, characterized in that, The method includes the following steps: S1. Mix lard with oleic acid esters containing less than 0.05% cholesterol until well combined to obtain a mixture for later use. S2. Add the above mixture into a high-pressure reactor, adjust the temperature to 4-6℃ and the pressure to 10-15MPa, stir and process to obtain the pretreated material for later use. S3. Heat the pretreated material to 35-40℃, then add lipase, stir evenly, and continue heating to 50-60℃. Perform enzymatic hydrolysis treatment at pressures of 0.2-0.3 MPa, 0.8-1.0 MPa, 3-4 MPa, and 6-8 MPa in sequence, and control the oxygen concentration during enzymatic hydrolysis to be 10%-15%. S4. After enzymatic hydrolysis, vacuum filter to separate the lipase, then mix with anhydrous ethanol to separate and crystallize, and then wash to obtain the final product.

2. The method according to claim 1, characterized in that: In step S1, the lard and oleic acid esters with a cholesterol content of less than 0.05% are used in a mass ratio of 1:3-5.

3. The method according to claim 1, characterized in that: In step S1, the oleate is at least one of methyl oleate, ethyl oleate, trioleate, butyl oleate, and isopropyl oleate.

4. The method according to claim 1, characterized in that: In step S2, the stirring speed is 400-600 r / min, and the processing time is 20-40 min.

5. The method according to claim 1, characterized in that: The lipase in step S3 is an immobilized lipase, and the amount of lipase added is 8%-10% of the total mass of the pretreated material.

6. The method according to claim 1, characterized in that: In step S3, the enzymatic hydrolysis time is 10-20 min under a pressure of 0.2-0.3 MPa, 10-20 min under a pressure of 0.8-1.0 MPa, 1-2 h under a pressure of 3-4 MPa, and 40-60 min under a pressure of 6-8 MPa.

7. The method according to claim 1, characterized in that: In step S4, the method for separating and crystallizing by mixing anhydrous ethanol is to add 5-10 times the volume of anhydrous ethanol and then separate and crystallize at a temperature of 15-20°C.

8. The method according to claim 1, characterized in that: The washing method in step S4 is to add 5-8 times the amount of anhydrous ethanol, heat to 60-80℃, keep warm for 5-10 minutes, then cool down to 4-8℃, let stand for 15-20 minutes, separate the anhydrous ethanol, and then evaporate the remaining anhydrous ethanol under reduced pressure.

9. The method according to claim 8, characterized in that: The rate of heating to 60-80℃ is 5-10℃ / min, and the rate of cooling to 4-8℃ is 2-3℃ / min.

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