Preparation method of lipase for preparing diester oil

Abstract

The invention relates to a preparation method of lipase for preparing diester oil. The preparation method is characterized by comprising the following steps: (1) preparing a lipase solution, namely performing high-density fermentation on a lipase strain; (2) preparing the immobilized lipase: mixing the lipase liquid obtained by high-density fermentation with a resin carrier, heating and adsorbing in a shaking table, immobilizing for a certain time, cleaning with pure water, filtering, and drying to obtain the immobilized lipase; (3) catalytic reaction: mixing edible oil, glycerol and immobilized lipase, and heating to react for a certain time to prepare crude diester oil; (4) purifying the crude diester oil by an ethanol multiple extraction temperature control method, and removing fatty acid and monoglyceride; and carrying out vacuum drying to remove ethanol and water to obtain the diester oil with high diglyceride content. The invention provides a brand-new purification method, namely an ethanol extraction method, for purifying diglyceride oil by improving the stability and the use frequency of enzyme through self-made immobilized lipase. Compared with a traditional molecular distillation method, the method is higher in efficiency and mainly has the advantages that purification equipment is simple and easy to obtain, low-temperature conditions are adopted in the whole process, the single purification material amount is large, and the extraction time is short.

Description

Technical Field

[0001] The invention relates to the technical field of chemical industry, in particular to a method for preparing lipase for preparing diester oil. Background Art

[0002] Enzymatic glycerolysis reaction is preferred for the preparation of diglycerides because the raw materials in the system are simple and easy to obtain, the reaction conditions are mild, and the environmental pollution is small. Traditional enzymatic glycerolysis reaction, due to the high purchase cost of immobilized enzymes, needs to add excessive glycerol in the reaction system, has the problems of large substrate viscosity, insufficient reaction, slow reaction, etc., which is unfavorable for industrial production. The present invention adopts homemade immobilized enzymes to carry out glycerolysis reaction to reduce the cost of enzymes, and obtains crude diester oil with high diester content, which is further purified to obtain pure diester oil. The diester oil obtained by enzymatic hydrolysis contains more fatty acids and monoglycerides. The diester oil purification process currently used in industrial production is molecular distillation. The equipment cost required for this process is high, the energy consumption is high, and the purification rate is low, so that the diester oil yield is extremely low and the price is expensive. The present invention provides a diester oil purification method suitable for industrial production, which can greatly reduce production costs and improve purification efficiency. Summary of the invention

[0003] The object of the present invention is to provide a method for preparing lipase for preparing diester oil, so as to solve the problems raised in the above background technology.

[0004] The technical solution of the present invention is as follows:

[0005] A method for preparing lipase for preparing diester oil comprises the following steps:

[0006] Step (1) preparing lipase solution: high-density fermentation of lipase strains;

[0007] Step (2) preparing immobilized lipase: mixing the lipase solution obtained by high-density fermentation with a resin carrier, heating and adsorbing in a shaker, fixing for a certain period of time, washing with pure water, filtering and drying to obtain immobilized lipase;

[0008] Step (3) catalytic reaction: edible oil, glycerol, and immobilized lipase are mixed and heated for a certain period of time to prepare crude diester oil;

[0009] Step (4) purifying the crude diester oil by multiple ethanol extraction and temperature control to remove fatty acids and monoglycerides; vacuum drying to remove ethanol and water to obtain a diester oil with a high diglyceride content.

[0010] The lipase strain described in step (1) has a lipase sequence derived from Candida antarctica lipase B, and the expression strain is Pichia pastoris.

[0011] The resin carrier in step (2) comprises one of a macroporous adsorption resin, an epoxy resin, and an ion exchange resin.

[0012] The resin in step (2) needs to be activated before immobilization, and the steps are as follows: soaking in 95% ethanol solution for 24 hours (2 to 3 cm above the resin), washing with pure water several times until there is no alcohol smell, and then filtering; soaking in 5% hydrochloric acid solution for 4 hours (2 to 3 cm above the resin), washing with pure water several times until the filtrate is neutral; soaking in 5% sodium hydroxide solution for 4 hours (2 to 3 cm above the resin), washing with pure water several times until the filtrate is neutral; soaking in PBS pH 7.0 solution for 4 hours (2 to 3 cm above the resin), soaking and washing with PBS pH 7.0 solution several times until the filtrate is neutral, and placing in a 4°C refrigerator for standby use.

[0013] The enzyme solution concentration in the immobilization system of step (2) is 4-10 mg / ml, preferably 8 mg / ml; the enzyme solution volume is 50 ml; the amount of activated wet resin added to the immobilization system is 1-4 g, preferably 2 g; the shaking table temperature is set to 20-30° C., preferably 25° C.; the rotation speed is set to 150-220 rpm, preferably 180 rpm; the fixing time is 2-12 h, preferably 4 h; the drying temperature is 20-40° C., preferably 30° C.

[0014] The edible oil in step (3) is triglyceride, preferably palm oil; the molar ratio of added palm oil to glycerol is 1-3:1, preferably 2:1; the mass ratio of added immobilized lipase to palm oil is 2%-5%, preferably 4%; the reaction temperature is set at 40-80°C, preferably 60°C; the reaction time is 4-12h, preferably 6h.

[0015] The ethanol concentration in step (4) is 60% to 70%, preferably 70%; the mass ratio of ethanol to crude diester oil is 5 to 1:1, preferably 3:1; the temperature is 40 to 60°C, preferably 50°C; and the number of extractions is 1 to 5 times, preferably 3 times.

[0016] During each extraction process in step (4), the mixing needs to be stirred for 5 to 20 minutes, preferably 10 minutes; the two phases need to be allowed to stand for stratification for 5 to 20 minutes, preferably 10 minutes.

[0017] The vacuum drying temperature in step (4) is 50-60°C, preferably 55°C; the drying time is 30min-2h, preferably 1h; the diglyceride content in the prepared diester oil is more than 50%, and the monoester content is less than 1.5%.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention fixes the lipase on the carrier by a physical adsorption method based on weak interactions such as van der Waals force, hydrogen bond, hydrophobic interaction, etc., so that the prepared immobilized lipase makes the active center of the enzyme protein not easily destroyed, thereby increasing the stability of the enzyme; the preparation method is simple and the cost is low; the carrier can be repeatedly used, which greatly reduces the production cost.

[0020] The present invention proposes an ethanol extraction method to purify crude diester oil. In the traditional molecular distillation method, the high temperature environment will lead to the production of glycidyl esters, which will decompose into harmful glycidol in the human body after consumption, so it is necessary to add a process for removing glycidyl esters in the later stage; molecular distillation equipment is expensive, energy-intensive, and has limited feed speed and volume, which makes the purification cost extremely high and the diester oil yield low. In the ethanol extraction method adopted by the present invention, ethanol is a friendly solvent, which is easy to obtain and can be recycled by rotary evaporation; the extraction tower equipment is simple to operate and is cheaper than the molecular distillation equipment; the temperature of the entire purification process is at a low temperature, which avoids the production of harmful substances such as glycidyl esters. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the high-efficiency size exclusion chromatography spectrum of Example 11. DETAILED DESCRIPTION

[0022] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] A method for preparing lipase for preparing diester oil comprises the following steps:

[0024] Step (1) preparing lipase solution: high-density fermentation of lipase strains;

[0025] Step (2) preparing immobilized lipase: mixing the lipase solution obtained by high-density fermentation with a resin carrier, heating and adsorbing in a shaker, fixing for a certain period of time, washing with pure water, filtering and drying to obtain immobilized lipase;

[0026] Step (3) catalytic reaction: edible oil, glycerol, and immobilized lipase are mixed and heated for a certain period of time to prepare crude diester oil;

[0027] Step (4) purifying the crude diester oil by multiple ethanol extraction and temperature control to remove fatty acids and monoglycerides; vacuum drying to remove ethanol and water to obtain a diester oil with a high diglyceride content.

[0028] The lipase strain described in step (1) has a lipase sequence derived from Candida antarctica lipase B, and the expression strain is Pichia pastoris.

[0029] The resin carrier in step (2) comprises one of a macroporous adsorption resin, an epoxy resin, and an ion exchange resin.

[0030] The resin in step (2) needs to be activated before immobilization, and the steps are as follows: soaking in 95% ethanol solution for 24 hours (2 to 3 cm above the resin), washing with pure water several times until there is no alcohol smell, and then filtering; soaking in 5% hydrochloric acid solution for 4 hours (2 to 3 cm above the resin), washing with pure water several times until the filtrate is neutral; soaking in 5% sodium hydroxide solution for 4 hours (2 to 3 cm above the resin), washing with pure water several times until the filtrate is neutral; soaking in PBS pH 7.0 solution for 4 hours (2 to 3 cm above the resin), soaking and washing with PBS pH 7.0 solution several times until the filtrate is neutral, and placing in a 4°C refrigerator for standby use.

[0031] The enzyme solution concentration in the immobilization system of step (2) is 4-10 mg / ml, preferably 8 mg / ml; the enzyme solution volume is 50 ml; the amount of activated wet resin added to the immobilization system is 1-4 g, preferably 2 g; the shaking table temperature is set to 20-30° C., preferably 25° C.; the rotation speed is set to 150-220 rpm, preferably 180 rpm; the fixing time is 2-12 h, preferably 4 h; the drying temperature is 20-40° C., preferably 30° C.

[0032] The edible oil in step (3) is triglyceride, preferably palm oil; the molar ratio of added palm oil to glycerol is 1-3:1, preferably 2:1; the mass ratio of added immobilized lipase to palm oil is 2%-5%, preferably 4%; the reaction temperature is set at 40-80°C, preferably 60°C; the reaction time is 4-12h, preferably 6h.

[0033] The ethanol concentration in step (4) is 60% to 70%, preferably 70%; the mass ratio of ethanol to crude diester oil is 5 to 1:1, preferably 3:1; the temperature is 40 to 60°C, preferably 50°C; and the number of extractions is 1 to 5 times, preferably 3 times.

[0034] During each extraction process in step (4), the mixing needs to be stirred for 5 to 20 minutes, preferably 10 minutes; the two phases need to be allowed to stand for stratification for 5 to 20 minutes, preferably 10 minutes.

[0035] The vacuum drying temperature in step (4) is 50-60°C, preferably 55°C; the drying time is 30min-2h, preferably 1h; the diglyceride content in the prepared diester oil is more than 50%, and the monoester content is less than 1.5%.

[0036] Embodiment 1:

[0037] A lipase immobilization method and application, comprising the following steps:

[0038] (1) The gene sequence of lipase B from Candida antarctica was cloned into the pPICZαA secretion vector and transformed into GS115 Pichia pastoris competent cells to obtain a strain that stably expresses lipase. The strain was fermented at a high density, and the supernatant secreted was a lipase solution.

[0039] (2) Resin activation: Soak in 95% ethanol solution for 24 hours (2 to 3 cm above the resin), rinse with pure water several times until there is no alcohol smell, and then filter; soak in 5% hydrochloric acid solution for 4 hours (2 to 3 cm above the resin), rinse with pure water several times until the filtrate is neutral; soak in 5% sodium hydroxide solution for 4 hours (2 to 3 cm above the resin), rinse with pure water several times until the filtrate is neutral; soak in PBS pH 7.0 solution for 4 hours (2 to 3 cm above the resin), soak and rinse with PBS pH 7.0 solution several times until the filtrate is neutral, and place in a 4°C refrigerator for use.

[0040] (3) 50 ml of lipase solution with a final concentration of 8 mg / ml was mixed with 2 g of activated wet resin carrier and fixed at 25°C and 180 rpm for 4 h. After the immobilization, the mixture was filtered and washed three times with pure water. The water was dried at 30°C to prepare the immobilized lipase, which was placed in a 4°C refrigerator for later use.

[0041] (4) The catalytic reaction system is specifically as follows: 20 g palm oil, 1.4 g glycerol, 0.4 g immobilized lipase obtained in steps (1) to (3), 60° C., stirring reaction for 6 to 10 hours. The oil components were detected by high-performance size exclusion chromatography, and the results are shown in Table 1:

[0042] Table 1. Table of oil composition detection by high-efficiency size exclusion chromatography in Example 1

[0043] substance 6h 8h 10h fatty acid 2.13 2.27 2.18 Monoester 16.76 18.0 18.42 Diesters 43.0 46.07 47.84 Triesters 38.11 33.66 31.55

[0044] Embodiment 2:

[0045] A lipase immobilization method and application, comprising the following steps:

[0046] (1) Immobilized lipase prepared by the method of steps (1) to (3) in Example 1.

[0047] (2) The catalytic reaction system is as follows: 20 g palm oil, 1.4 g glycerol, 0.6 g immobilized lipase, 60°C, stirring reaction for 6 to 10 hours. The oil components were detected by high-performance size exclusion chromatography, and the results are shown in Table 2:

[0048] Table 2. Table of oil composition detection by high-efficiency size exclusion chromatography in Example 2:

[0049] substance 6h 8h 10h fatty acid 3.62 3.41 4.22 Monoester 19.0 18.89 19.37 Diesters 45.85 48.73 49.15 Triesters 31.53 28.96 27.26

[0050] Embodiment three:

[0051] A lipase immobilization method and application, comprising the following steps:

[0052] (1) Immobilized lipase prepared by the method of steps (1) to (3) in Example 1.

[0053] (2) The catalytic reaction system is as follows: 20 g palm oil, 1.4 g glycerol, 0.8 g immobilized lipase, 60°C, stirring reaction for 6 to 10 hours. The oil components were detected by high-performance size exclusion chromatography, and the results are shown in Table 3:

[0054] Table 3. Detection of oil components by high-efficiency size exclusion chromatography in Example 3

[0055] substance 6h 8h 10h fatty acid 2.43 2.94 2.71 Monoester 18.97 19.23 19.52 Diesters 49.49 49.21 49.37 Triesters 29.1 28.63 28.41

[0056] Embodiment 4:

[0057] A lipase immobilization method and application, comprising the following steps:

[0058] (1) Immobilized lipase prepared by the method of steps (1) to (3) in Example 1.

[0059] (2) The catalytic reaction system is as follows: 1000 g palm oil, 70 g glycerol, 40 g immobilized lipase, 60° C., and stirred for 6 h. The oil components were detected by high-performance size exclusion chromatography, and the results are shown in Table 4:

[0060] Table 4. Detection of oil components by high-efficiency size exclusion chromatography in Example 4

[0061] substance 6h fatty acid 0.69 Monoester 19.48 Diesters 51.75 Triesters 28.05

[0062] Embodiment five:

[0063] A method for purifying diglyceride oil by ethanol extraction, comprising the following steps:

[0064] (1) The crude diester oil prepared in Example 4 was extracted and purified.

[0065] (2) Weigh 100 g of crude diester oil into a separatory funnel (with a heating jacket), add 300 g of 60% ethanol solution, mix thoroughly at 50° C., and allow to stand for stratification. Take the oil phase for high performance size exclusion chromatography to detect the oil components. The 60% ethanol solution has no significant extraction efficiency for fatty acids and monoglycerides. The results are shown in Table 5:

[0066] Table 5. Detection of oil components by high-performance size exclusion chromatography in Example 5

[0067] substance Before extraction After extraction fatty acid 0.69 0.68 Monoester 19.48 18.62 Diesters 51.75 52.15 Triesters 28.05 28.55

[0068] Embodiment six:

[0069] A method for purifying diglyceride oil by ethanol extraction, comprising the following steps:

[0070] (1) The crude diester oil prepared in Example 4 was extracted and purified.

[0071] (2) Weigh 100 g of crude diester oil into a separatory funnel (with a heating jacket), add 300 g of 70% ethanol solution, mix thoroughly at 50° C., and allow to stand for stratification. Take the oil phase for high performance size exclusion chromatography to detect the oil components. The 70% ethanol solution has a significant extraction efficiency for fatty acids and monoglycerides. The results are shown in Table 6:

[0072] Table 6. Detection of oil components by high-performance size exclusion chromatography in Example 6

[0073] substance Before extraction After extraction fatty acid 0.69 0.54 Monoester 19.48 10.65 Diesters 51.75 53.48 Triesters 28.05 35.33

[0074] Embodiment seven:

[0075] A method for purifying diglyceride oil by ethanol extraction, comprising the following steps:

[0076] (1) The crude diester oil prepared in Example 4 was extracted and purified.

[0077] (2) Weigh 100 g of crude diester oil into a separatory funnel (with a heating jacket), add 300 g of 80% ethanol solution, mix thoroughly at 50° C., and allow to stand for stratification. Take the oil phase for high performance size exclusion chromatography to detect the oil components. The 80% ethanol solution has a significant extraction efficiency for fatty acids and monoglycerides. The results are shown in Table 7:

[0078] Table 7. Detection of oil components by high-efficiency size exclusion chromatography in Example 7

[0079] substance Before extraction After extraction fatty acid 0.69 0.55 Monoester 19.48 8.16 Diesters 51.75 54.56 Triesters 28.05 36.73

[0080] Embodiment eight:

[0081] (1) The crude diester oil prepared in Example 4 was extracted and purified.

[0082] (2) Weigh 100 g of crude diester oil into a separatory funnel (with a heating jacket), add 300 g of 70% ethanol solution, mix thoroughly at 50° C., and stand to separate the layers; separate the aqueous phase and the oil phase, add 300 g of 70% ethanol solution to the oil phase, mix thoroughly, and stand to separate the layers, take the oil phase and perform high performance size exclusion chromatography to detect the oil components. Two extractions with 70% ethanol solution have a significant effect on reducing fatty acids and monoglycerides. The results are shown in Table 8:

[0083] Table 8. Detection of oil components by high-efficiency size exclusion chromatography in Example 8

[0084] substance Before extraction After extraction fatty acid 0.69 0.45 Monoester 19.48 2.58 Diesters 51.75 55.45 Triesters 28.05 41.62

[0085] Embodiment nine:

[0086] (1) The crude diester oil prepared in Example 4 was extracted and purified.

[0087] (2) Weigh 100 g of crude diester oil into a separatory funnel (with a heating jacket), add 300 g of 80% ethanol solution, mix thoroughly at 50° C., and stand to separate the layers; separate the aqueous phase and the oil phase, add 300 g of 80% ethanol solution to the oil phase, mix thoroughly, and stand to separate the layers, and take the oil phase for high-performance size exclusion chromatography to detect the oil components. The two extractions with 80% ethanol solution have obvious extraction efficiency for fatty acids and monoglycerides, but will cause serious loss of diesters. The results are shown in Table 9:

[0088] Table 9. Detection of oil components by high-efficiency size exclusion chromatography in Example 9

[0089] substance Before extraction After extraction fatty acid 0.69 0.43 Monoester 19.48 1.95 Diesters 51.75 49.63 Triesters 28.05 47.99

[0090] Embodiment ten:

[0091] (1) The crude diester oil prepared in Example 4 was extracted and purified.

[0092] (2) Weigh 100 g of crude diester oil into a separatory funnel (with a heating jacket), add 300 g of 70% ethanol solution, mix thoroughly at 50° C., and stand for stratification; separate the aqueous phase and the oil phase, add 300 g of 70% ethanol solution to the oil phase, mix thoroughly, and stand for stratification; separate the aqueous phase and the oil phase, add 300 g of 70% ethanol solution to the oil phase, mix thoroughly, and stand for stratification, take the oil phase for high performance size exclusion chromatography to detect the oil components, and three extractions with 70% ethanol solution have a significant effect on reducing fatty acids and monoglycerides. The results are shown in Table 10:

[0093] Table 10. Detection of oil components by high-performance size exclusion chromatography in Example 10

[0094]

[0095]

[0096] Embodiment eleven:

[0097] (1) Immobilized lipase prepared by the method of steps (1) to (3) in Example 1.

[0098] (2) The catalytic reaction system is as follows: 2000 g palm oil, 140 g glycerol, 80 g immobilized lipase, 60° C., and stirring for 6 h.

[0099] (3) Weigh 2000 g of crude diester oil into a reactor, add 6000 g of 70% ethanol solution, mix thoroughly at 50° C., and stand for stratification; separate the aqueous phase and the oil phase, add 6000 g of 70% ethanol solution to the oil phase, mix thoroughly, and stand for stratification; separate the aqueous phase and the oil phase, add 6000 g of 70% ethanol solution to the oil phase, mix thoroughly, and stand for stratification, and take the oil phase for high performance size exclusion chromatography to detect the oil components. The results are shown in Table 11:

[0100] Table 11. Detection of oil components by high-performance size exclusion chromatography in Example 11

[0101] substance Proportion (%) fatty acid 0.36 Monoester 0.81 Diesters 56.90 Triesters 41.93

[0102] The above-described embodiments are only partial contents, and those skilled in the art may also obtain the same results by making modifications thereto, such as changes in the type of extractant, the concentration of the extractant, the extraction temperature, the number of extractions, the stirring time, the standing time, the repeated use of the extractant, etc. The changes made on these conditions are subject to the limitations of the present invention.

[0103] The present invention improves the stability and usage of the enzyme by self-made immobilized lipase, and proposes a new purification method, namely, ethanol extraction method, to purify diacylglycerol oil. This method is more efficient than the traditional molecular distillation method, mainly in that the purification equipment is simple and easy to obtain, the whole process is under low temperature conditions, the amount of single purification material is large, and the extraction time is short. Based on the extraction and purification method proposed by the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

Claims

1. A method for preparing lipase for preparing diester oil, characterized in that The steps include: Step (1) preparing lipase solution: high-density fermentation of lipase strains; Step (2) preparing immobilized lipase: mixing the lipase solution obtained by high-density fermentation with a resin carrier, heating and adsorbing in a shaker, fixing for a certain period of time, washing with pure water, filtering and drying to obtain immobilized lipase; Step (3) catalytic reaction: edible oil, glycerol, and immobilized lipase are mixed and heated for a certain period of time to prepare crude diester oil; Step (4) purifying the crude diester oil by multiple ethanol extraction and temperature control to remove fatty acids and monoglycerides; vacuum drying to remove ethanol and water to obtain a diester oil with a high diglyceride content.

2. The method for preparing a lipase unit for preparing diester oil according to claim 1, characterized in that The lipase strain described in step (1) has a lipase sequence derived from Candida antarctica lipase B, and the expression strain is Pichia pastoris.

3. The method for preparing a lipase unit for preparing diester oil according to claim 1, characterized in that The resin carrier in step (2) comprises one of a macroporous adsorption resin, an epoxy resin, and an ion exchange resin.

4. The method for preparing a lipase unit for preparing diester oil according to claim 1, characterized in that The resin in step (2) needs to be activated before immobilization, and the steps are as follows: soaking in 95% ethanol solution for 24 hours (2 to 3 cm above the resin), washing with pure water several times until there is no alcohol smell, and then filtering; soaking in 5% hydrochloric acid solution for 4 hours (2 to 3 cm above the resin), washing with pure water several times until the filtrate is neutral; soaking in 5% sodium hydroxide solution for 4 hours (2 to 3 cm above the resin), washing with pure water several times until the filtrate is neutral; soaking in PBS pH 7.0 solution for 4 hours (2 to 3 cm above the resin), soaking and washing with PBS pH 7.0 solution several times until the filtrate is neutral, and placing in a 4°C refrigerator for standby use.

5. The method for preparing a lipase unit for preparing diester oil according to claim 1, characterized in that The enzyme solution concentration in the immobilization system of step (2) is 4-10 mg / ml, preferably 8 mg / ml; the enzyme solution volume is 50 ml; the amount of activated wet resin added to the immobilization system is 1-4 g, preferably 2 g; the shaking table temperature is set to 20-30° C., preferably 25° C.; the rotation speed is set to 150-220 rpm, preferably 180 rpm; the fixing time is 2-12 h, preferably 4 h; the drying temperature is 20-40° C., preferably 30° C.

6. The method for preparing a lipase unit for preparing diester oil according to claim 1, characterized in that The edible oil in step (3) is triglyceride, preferably palm oil; the molar ratio of added palm oil to glycerol is 1-3:1, preferably 2:1; the mass ratio of added immobilized lipase to palm oil is 2%-5%, preferably 4%; the reaction temperature is set at 40-80°C, preferably 60°C; the reaction time is 4-12h, preferably 6h.

7. The method for preparing a lipase unit for preparing diester oil according to claim 1, characterized in that The ethanol concentration in step (4) is 60% to 80%, preferably 70%; the mass ratio of ethanol to crude diester oil is 5 to 1:1, preferably 3:1; the temperature is 40 to 60°C, preferably 50°C; and the number of extractions is 1 to 5 times, preferably 3 times.

8. The method for preparing a lipase unit for preparing diester oil according to claim 1, characterized in that During each extraction process in step (4), the mixing needs to be stirred for 5 to 20 minutes, preferably 10 minutes; the two phases need to be allowed to stand for stratification for 5 to 20 minutes, preferably 10 minutes.

9. The method for preparing a lipase unit for preparing diester oil according to claim 1, characterized in that The vacuum drying temperature in step (4) is 50-60°C, preferably 55°C; the drying time is 30min-2h, preferably 1h; the diglyceride content in the prepared diester oil is more than 50%, and the monoester content is less than 1.5%.

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