CN118086414B - A method for preparing a high-content EPA and low-content DHA fatty acid product by enzymatic method - Google Patents

Abstract

The present invention discloses a method for preparing a high-content eicosapentaenoic acid (EPA) and low-content docosahexaenoic acid (DHA) fatty acid product by an enzymatic method. The present invention uses EPA-rich oil as a raw material, utilizes the difference in the degree of difficulty of combining 1,2-propylene glycol/tert-butyl alcohol with different fatty acids, improves the selectivity of the enzyme, and simultaneously regulates the balance of the enzymatic hydrolysis reaction of the system to improve the difference in the content between EPA and DHA in the fatty acid. The present invention controls the water content of the reaction system to ensure a certain degree of hydrolysis reaction, and jointly regulates the balance of the hydrolysis reaction with 1,2-propylene glycol/tert-butyl alcohol to ensure the product yield. The lipase used in the present invention has a high degree of reusability, can significantly reduce production costs, and meet the requirements of large-scale production. EPA>70wt%, DHA<4wt% prepared by the method of the present invention.

Description

Method for preparing fatty acid product with high EPA content and low DHA content by using enzymatic method

Technical Field

The invention relates to a method for preparing a fatty acid product with high content of eicosapentaenoic acid (EPA) and low content of docosahexaenoic acid (DHA) by an enzyme method.

Background

N-3 polyunsaturated fatty acids are nutrients involved in many physiological processes in the body and play an important role in metabolism in the human body. EPA and DHA are the most common long-chain n-3 polyunsaturated fatty acids and have wide physiological effects, wherein EPA is used as a 'vascular scavenger', can reduce blood lipid and cholesterol at will, prevent cardiovascular diseases and thrombosis, and is commonly used for middle-aged and elderly people. DHA is used as brain gold, can enhance memory and promote the development of brain and vision system of infants, and is commonly used for infants.

The EPA and DHA are mainly derived from marine organisms such as tuna, sardine, etc. For EPA and DHA from different sources, the content of EPA and DHA also has certain difference, but the relative content difference of EPA and DHA is lower. For example, the tuna oil contains 6% EPA, 22% DHA, 16% sardine oil EPA, 9% DHA, 22% Pteris multifida oil EPA, 9% DHA, 7% EPA, 11% DHA, 10% EPA in cod, 10% DHA, 14% EPA in herring oil, and 8% DHA. Several studies have found that EPA alone has a varying degree of influence on the physiological function of organisms, even in contrast to the physiological function, with mixtures of EPA and DHA. Thus, obtaining EPA in high purity is of great importance.

The biggest challenge faced in preparing high purity EPA is that EPA tends to coexist with DHA in natural sources or concentrated fish oils. Because of the high degree of similarity in chemical structures of EPA and DHA, it is difficult to completely separate them using conventional separation techniques, which results in EPA purity that does not reach the desired level. In addition, some separation processes may introduce new impurities, cause oxidation of EPA or cause loss of EPA, further increasing the difficulty of preparing high purity, high quality EPA products.

EPA is an important n-3 series polyunsaturated fatty acid and has important effects on human body, but its acquisition route is limited. Although linolenic acid can be converted to EPA in humans, both the amount and rate of conversion are at low levels and must be obtained from food. Currently the EPA content in marine fish oils is generally below 20% and no targeted process for EPA production has been found in prior studies. For EPA acquisition with high purity, the most effective method is to prepare liquid phase, but the cost is too high, and the EPA produced is only used for the production of some special medicines, and the application area is narrow. The fatty acid of high concentration EPA produced by the enzyme method has the advantages of high efficiency, specificity, mild reaction condition, few byproducts, high enzyme recycling degree and the like, and has very high application value in the field of high added value structural fat production.

Disclosure of Invention

This section is intended to outline some aspects of embodiments of the application and to briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section as well as in the description of the application and in the title of the application, which may not be used to limit the scope of the application.

The present invention has been made in view of the above and/or problems occurring in the prior art.

Therefore, the invention aims to overcome the defects in the prior art and provide a method for preparing fatty acid products with high EPA content and low DHA content by an enzyme method.

In order to solve the technical problems, the invention provides the following technical scheme that the method for preparing the fatty acid product with high EPA content and low DHA content by using the enzyme method is characterized in that:

Mixing grease containing EPA and DHA, an alcohol solvent and water, and performing enzymatic reaction at 35-65 ℃ for 4-16 hours, wherein the mol ratio of the grease containing EPA and DHA to the alcohol solvent is 1 (11.5-46), and the oil-water ratio is 1 (1-5) in terms of w/w;

and after the reaction is finished, removing lipase and solvent, and further separating and purifying to obtain fatty acid products with EPA more than 70wt% and DHA less than 4 wt%.

As a preferable mode of the production method of the present invention, wherein the alcohol solvent is 1, 2-propanediol and/or t-butanol.

As a preferable scheme of the preparation method, the mol ratio of the grease containing EPA and DHA and the alcohol solvent is 1 (23-34.5), and the oil-water ratio is 1 (2-5) in terms of w/w.

As a preferable scheme of the preparation method, the reaction temperature is 35-45 ℃.

As a preferable scheme of the preparation method, the reaction time is 8-16 h.

As a preferred embodiment of the preparation process according to the invention, the EPA and DHA-containing oil comprises EPA >15 wt.% natural and/or synthetic lipids.

The preparation method of the synthetic lipid comprises the steps of carrying out enzymatic reaction for 4-12 hours under the condition that the ratio of non-glyceride to glycerin is greater than 3:1 and at the temperature of 40-80 ℃, and removing enzyme and unreacted substrates to obtain the synthetic lipid rich in EPA, wherein the pressure of a reaction system is below 1000 Pa.

The preparation method is a preferable scheme, wherein the method for separating the enzyme and the solvent is a centrifugal method, the upper layer after centrifugal layering is a crude product, the rotational speed of the centrifugal process is more than 3000r/min, and the time of the centrifugal process is 3-20 min.

The preparation method comprises the following steps of adding a crude product obtained through centrifugal separation into a sample injector, obtaining a heavy phase and a light phase through short-path distillation under certain pressure and temperature conditions, and collecting the light phase to obtain a fatty acid crude product containing EPA, wherein the temperature is 100-260 ℃, the pressure is 0.1-50 Pa, the feeding flow rate is 1-20 mL/min, and the scraper rotating speed is 100-500 r/min.

As a preferable scheme of the preparation method, the urea inclusion comprises the following steps of mixing the EPA-containing fatty acid crude product with urea in a certain proportion in 95% ethanol solution, heating and stirring until the whole mixture is a transparent homogeneous solution, and crystallizing at a certain temperature. The obtained crystals (urine-packed phase) and the liquid phase (non-urine-packed phase) are separated and then are continuously crystallized at a lower temperature, and finally the non-urine-packed phase is obtained by separation. Acidifying the non-urine-coated phase, adding water and n-hexane, extracting fatty acid, separating organic phase, and removing solvent under reduced pressure to obtain fatty acid product with high EPA content and low DHA content.

The preparation method is characterized in that the mass ratio of the EPA-containing crude fatty acid product to urea is 1:1-1:4, the first crystallization temperature is 0-8 ℃, the second crystallization temperature is-50-10 ℃, the pH after acidification is below 5, and the volume ratio of water to normal hexane is 1:2-2:1.

As a preferred embodiment of the production method of the present invention, the lipase is produced by Candida antarctica (CANDIDA ANTARCTICA), rhizopus oryzae (Rhizopus oryzae), aspergillus niger (Aspergillus niger), candida rugosa (Candida rugosa), burkholderia (Burkaholderia cepacia) and Pseudomonas.

As a preferred embodiment of the preparation method of the present invention, the Lipase comprises one or more of CAL-A, ET 2.0.0, novozyme 435, lipozyme 435, lipase TL 100L, lipase PS "Amano", LIPASE TL IM and LIPASE RM IM, preferably ET 2.0 and Lipase TL 100L.

The invention has the beneficial effects that:

1. The enzymatic reaction system utilizes the difference of the combination difficulty degree of the 1, 2-propylene glycol/tertiary butanol and different fatty acids, improves the selectivity of enzyme, and simultaneously regulates and controls the balance of enzymatic hydrolysis reaction of the system, and improves the difference of the content of EPA and DHA in the fatty acids;

2. the invention ensures the balance of the hydrolysis reaction with 1, 2-propylene glycol/tertiary butanol on the basis of ensuring the hydrolysis reaction to a certain extent by controlling the water content of the reaction system, thereby ensuring the product yield.

3. The invention adopts the enzymatic method to prepare the fatty acid product with high EPA content and low DHA content, and has mild reaction conditions, high efficiency, high specificity and environmental protection;

4. The invention adopts grease rich in EPA as raw material, can obtain fatty acid products with high EPA content and low DHA content, EPA is more than 70wt% and DHA is less than 4wt%;

5. The lipase used in the invention has high bioavailability, can obviously reduce the production cost and meets the requirement of large-scale production.

Drawings

FIG. 1 is a gas phase (GC) chromatogram of a fish oil feedstock used in example 1 of the present invention, which is data on fatty acid composition and content of the fish oil feedstock.

FIG. 2 is a GC chromatogram of a fatty acid product (after urea inclusion) prepared in example 1 of the present invention, showing the composition and content of a non-urea-coated fatty acid product obtained after urea inclusion.

FIG. 3 is a liquid chromatogram (HPLC-RID) of a crude EPA fatty acid product prepared by the enzymatic reaction of the present invention, wherein a is the lipid composition of the crude enzymatic reaction product obtained in example 2 using ET 2.0 as a catalyst, b is the lipid composition of the crude enzymatic reaction product obtained in comparative example 1, and c is the lipid composition of the crude enzymatic reaction product obtained in comparative example 2.

FIG. 4 is a GC chromatogram of the crude EPA fatty acid product prepared by molecular distillation in the enzymatic reaction of the present invention, wherein a is the composition and content of the crude fatty acid product obtained by molecular distillation of the lipid obtained by the catalyst ET 2.0 in example 2, and b is the composition and content of the crude fatty acid product obtained by molecular distillation of the lipid obtained by the condition of comparative example 1.

Detailed Description

In order that the above-recited objects, features and advantages of the present invention will become more apparent, a more particular description of the invention will be rendered by reference to specific embodiments thereof.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, it should be apparent that the described embodiments are merely some, but not all, embodiments of the present invention. The present invention may be embodied in other forms than those described herein, and those skilled in the art will readily appreciate that the present invention is not limited to the specific embodiments disclosed below without departing from the spirit or scope of the present invention.

Further, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic can be included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.

The raw materials and reagents used in the invention are refined fish oil rich in grease, the refined fish oil is purchased from Sanono biotechnology Co., norway, the organic reagents with the product model of OMAX 1812, 1, 2-propanediol, ethanol, methanol, isopropanol, tertiary butanol and the like are purchased from national pharmaceutical group chemical reagent Co., ltd, and ET 2.0, TL 100L and CAL-A lipase are purchased from tin-free Uppy biotechnology Co.

Example 1:

30g of fish oil, 60g of water and 50g of 1, 2-propylene glycol are added into a 250mL conical flask, ET 2.0 lipase is added, the addition amount of the lipase is 6.67 percent (based on the mass fraction of the fish oil), and after the mixture is uniformly mixed, the mixture is magnetically stirred for 300r/min for reaction for 8 hours at 45 ℃. After the reaction is finished, centrifuging for 5min at 5000r/min to obtain supernatant, and removing lipase and solvent. The oil phase obtained was subjected to molecular distillation (temperature 190 ℃ C., pressure 7Pa, feed flow rate 10mL/min, scraper rotation rate 300 r/min) to obtain crude fatty acid product. The crude product is subjected to urea inclusion (15 g of crude fatty acid product, 25g of urea, 100mL of 95% ethanol, and after uniform mixing, 4 ℃ inclusion for 8 hours, separation, and supernatant inclusion for 12 hours at minus 30 ℃) to obtain the fatty acid product with high EPA content and low DHA content. Lipid composition was measured by HPLC-RID and free fatty acid content was calculated. The composition and content of fatty acids were checked by GC.

The free fatty acid content in the different lipids is shown in Table 1, and the EPA and DHA content in the free fatty acids of the different lipids is shown in Table 2.

FIG. 1 shows the fatty acid composition and content of the fish oil raw material of example 1, and FIG. 2 shows the fatty acid composition and content of the non-urea-coated fatty acid product obtained after urea inclusion of example 1.

TABLE 1

TABLE 2

The free fatty acid content in the non-urine-coated product after inclusion of urea as shown in table 1 was as high as 99.69%, and the EPA and DHA content in the non-urine-coated product after inclusion of urea as shown in table 2 were 73.03% and 3.28%, respectively.

Example 2:

3g of fish oil, 6g of water and a certain amount of alcohol (the molar ratio of the alcohol to the oil is the same and is 23:1) are added into a 25mL conical flask, then ET 2.0 lipase is added, the addition amount of the lipase is 6.67 percent (based on the mass fraction of the fish oil), and after uniform mixing, the mixture is reacted for 8 hours under the condition of 45 ℃ under the magnetic stirring of 300 r/min. After the reaction is finished, centrifuging at 5000r/min for 5min, taking supernatant, removing lipase and solvent, detecting lipid composition in the reaction product by HPLC-RID, and calculating the content of free fatty acid. The glyceride and the fatty acid are separated by molecular distillation, and the composition and the content of the fatty acid in the obtained crude fatty acid product are detected by gas phase GC.

The influence of the alcohol type in the enzymatic reaction on the content of free fatty acid in the crude lipid product (without molecular distillation and urea inclusion treatment) is shown in Table 3, and the EPA and DHA content in the crude lipid product obtained by the enzymatic reaction after molecular distillation treatment in different alcohol systems is shown in Table 4. As can be seen from Table 3, the fatty acid content is lower in the methanol and ethanol system. The lipid product is mainly composed of methyl ester and ethyl ester, so that the requirement of the invention for producing fatty acid products is not met. The data in table 4 shows that the relative difference between EPA and DHA in the crude fatty acid product after molecular distillation of the experimental components of 1, 2-propanediol and t-butanol is more pronounced, the EPA content is 37-fold and 15-fold, respectively, of the DHA content, and the EPA content in the crude tertiary butanol group fatty acid product is up to 30.68%, which is suitable for further purification of EPA by subsequent processes.

FIG. 3a shows the lipid composition of the crude product of the enzymatic reaction obtained in example 2 using ET 2.0 as catalyst.

TABLE 3 Table 3

Type of alcohol	Methanol	Ethanol	1, 2-Propanediol	Isopropyl alcohol	Tert-butanol
						Free fatty acid content	3.37%	8.01%	33.17%	56.41%	67.42%

TABLE 4 Table 4

Type of alcohol	Methanol	Ethanol	1, 2-Propanediol	Isopropyl alcohol	Tert-butanol
						EPA content	24.26%	17.27%	22.24%	33.71%	30.68%
DHA content	5.95%	2.15%	0.60%	6.39%	2.02%

Example 3:

3g of fish oil, 5g of 1, 2-propylene glycol and 6g of water are added into a 25mL conical flask, lipase is added, the addition amount of the lipase is 6.67% (based on the mass fraction of the fish oil), and after uniform mixing, the mixture is reacted for 8 hours under the condition of 45 ℃ under the condition of magnetic stirring at 300 r/min. After the reaction is finished, centrifuging at 5000r/min for 5min, taking supernatant, removing lipase and solvent, detecting lipid composition in the reaction product by liquid phase HPLC-RID, and calculating the content of free fatty acid in the lipid. The glyceride and the fatty acid are separated by molecular distillation, and the composition and the content of the fatty acid in the obtained crude fatty acid product are detected by gas phase GC.

The influence of the enzyme type in the enzymatic reaction on the content of free fatty acid in the crude lipid product is shown in Table 5, and the content of EPA and DHA in the crude fatty acid product obtained by the enzymatic reaction after the molecular distillation treatment of the lipid under the catalysis of different lipases is shown in Table 6. The data in Table 6 show that the relative differences between EPA and DHA in the crude fatty acid products after molecular distillation of the ET 2.0 and TL 100L experimental components are very significant, and the EPA content is 37 times and 50 times that of DHA.

TABLE 5

Enzyme species	ET2.0	CAL-A	TL100L
				Free fatty acid content	33.17%	32.01%	30.13%

TABLE 6

Enzyme species	ET2.0	CAL-A	TL100L
				EPA content	22.24%	4.72%	23.59%
DHA content	0.60%	0.26%	0.47%

Example 4:

3g of fish oil, 1, 2-propylene glycol and 6g of water according to different oil-alcohol mole ratios are added into a 25mL conical flask, ET 2.0 is added, the enzyme addition amount is 6.67 percent (based on the mass fraction of the fish oil), and after uniform mixing, the reaction is carried out for 8 hours under the condition of 45 ℃ under the condition of magnetic stirring at 300 r/min. After the reaction is finished, centrifuging at 5000r/min for 5min, taking supernatant, removing lipase and solvent, detecting lipid composition in the reaction product by liquid phase HPLC-RID, and calculating the content of free fatty acid. The glyceride and the fatty acid are separated by molecular distillation, and the composition and the content of the fatty acid in the obtained crude fatty acid product are detected by gas phase GC.

The influence of the oil-alcohol ratio in the enzymatic reaction on the content of free fatty acid in the crude lipid product is shown in Table 7, and the content of EPA and DHA in the crude fatty acid product obtained by the enzymatic reaction after the molecular distillation treatment under the condition of different oil-alcohol ratios is shown in Table 8.

TABLE 7

TABLE 8

Oil to alcohol ratio	1:11.5	1:23	1:34.5	1:46
					EPA content	25.60%	22.24%	24.59%	23.32%
DHA content	2.40%	0.60%	1.61%	1.78%

Example 5:

3g of fish oil, 5g of 1, 2-propylene glycol and water according to different oil-water mass ratios are added into a 25mL conical flask, ET 2.0 is added, enzyme addition amount is 6.67 percent (based on the mass fraction of the fish oil), and after uniform mixing, magnetic stirring is carried out for 300r/min for reaction for 8 hours under the condition of 45 ℃. After the reaction is finished, centrifuging at 5000r/min for 5min, taking supernatant, removing lipase and solvent, detecting lipid composition in the reaction product by liquid phase HPLC-RID, and calculating the content of free fatty acid. The glyceride and the fatty acid are separated by molecular distillation, and the composition and the content of the fatty acid in the obtained crude fatty acid product are detected by gas phase GC.

The influence of the oil-water mass ratio in the enzymatic reaction on the content of free fatty acid in the crude lipid product is shown in Table 9, and the content of EPA and DHA in the crude fatty acid product obtained by the enzymatic reaction after the molecular distillation treatment under the condition of different oil-water mass ratios is shown in Table 10.

TABLE 9

Oil-water ratio	1:1	1:2	1:3	1:5
					Free fatty acid content	33.39%	33.17%	43.73%	46.17%

Table 10

Oil-water ratio	1:1	1:2	1:3	1:5
					EPA content	23.86%	22.24%	19.53%	21.53%
DHA content	3.61%	0.60%	0.91%	1.19%

Example 6:

3g of fish oil, 5g of 1, 2-propylene glycol and 6g of water are added into a 25mL conical flask, then ET 2.0 lipase is added, the addition amount of the lipase is 6.67 percent (based on the mass fraction of the fish oil), and after the mixture is uniformly mixed, the mixture is magnetically stirred for 300r/min for reaction for 8 hours under different temperature conditions. After the reaction is finished, centrifuging at 5000r/min for 5min, taking supernatant, removing lipase and solvent, detecting lipid composition in the reaction product by liquid phase HPLC-RID, and calculating the content of free fatty acid. The glyceride and the fatty acid are separated by molecular distillation, and the composition and the content of the fatty acid in the obtained crude fatty acid product are detected by gas phase GC.

The influence of the reaction temperature in the enzymatic reaction on the content of free fatty acid in the crude lipid product is shown in Table 11, and the content of EPA and DHA in the crude fatty acid product obtained by the enzymatic reaction after molecular distillation treatment under different reaction temperature conditions is shown in Table 12.

TABLE 11

Temperature/°c	35	45	55	65
					Free fatty acid content	24.72%	33.17%	34.06%	38.44%

Table 12

Temperature/°c	35	45	55	65
					EPA content	19.12%	22.24%	20.07%	15.16%
DHA content	1.01%	0.60%	1.93%	1.65%

Example 7:

3g of fish oil, 5g of 1, 2-propylene glycol and 6g of water are added into a 25mL conical flask, then ET 2.0 lipase is added, the addition amount of the lipase is 6.67 percent (based on the mass fraction of the fish oil), and after the mixture is uniformly mixed, the mixture is magnetically stirred for 300r/min to react for different times under the condition of 45 ℃. After the reaction is finished, centrifuging at 5000r/min for 5min, taking supernatant, removing lipase and solvent, detecting lipid composition in the reaction product by liquid phase HPLC-RID, and calculating the content of free fatty acid. The glyceride and the fatty acid are separated by molecular distillation, and the composition and the content of the fatty acid in the obtained crude fatty acid product are detected by gas phase GC.

The influence of the reaction time in the enzymatic reaction on the content of free fatty acid in the crude lipid product is shown in Table 13, and the content of EPA and DHA in the crude fatty acid product obtained by subjecting the lipid obtained by the enzymatic reaction to molecular distillation treatment is shown in Table 14.

TABLE 13

Time/h	4	8	12	16
					Free fatty acid content	24.72%	33.17%	34.06%	38.44%

TABLE 14

Time of	4	8	12	16
					EPA content	20.81%	22.24%	23.15%	23.85%
DHA content	0.61%	0.60%	0.72%	1.58%

Comparative example 1:

the enzymatic system was not added with alcohol, and the other conditions were the same as in example 2.

FIG. 3b shows the lipid composition of the crude product of the enzymatic reaction under the conditions of comparative example 1, and FIG. 4b shows the composition and content of the crude fatty acid product obtained by subjecting the lipid obtained under the conditions of comparative example 1 to molecular distillation.

The obtained lipid crude product has a free fatty acid content of 52.18%, EPA and DHA contents of 20.83% and 2.84%, respectively, and EPA content is only 7.33 times of DHA content, and the relative difference is low, which is insufficient for the production of final fatty acid products.

Comparative example 2:

the enzymatic system was not added with water, and the other conditions were the same as in example 2.

FIG. 3c shows the lipid composition of the crude product of the enzymatic reaction under the conditions of comparative example 2.

The free fatty acid content of the resulting lipid crude product was only 0.14%.

It should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted without departing from the spirit and scope of the technical solution of the present invention, and it should be covered in the scope of the present invention.

Claims (8)

1. A method for preparing a fatty acid product with high EPA content and low DHA content by an enzymatic method is characterized by comprising the following steps of,

Mixing grease containing EPA and DHA, an alcohol solvent and water, and carrying out enzymatic reaction for 4-16 hours at 35-65 ℃, wherein the mol ratio of the grease containing EPA and DHA to the alcohol solvent is 1 (11.5-46), the oil-water ratio is 1 (1-5) in terms of w/w, and the alcohol solvent is 1, 2-propylene glycol and/or tert-butanol;

And (3) removing Lipase and solvent after the reaction is finished, and separating and purifying to obtain fatty acid products with EPA of more than 70 wt% and DHA of less than 4: 4 wt%, wherein the Lipase is one or more of ET 2.0 and Lipase TL 100L.

2. The method of claim 1, wherein the mole ratio of EPA and DHA-containing oil to alcohol solvent is 1 (23-34.5), and the oil-water ratio is 1 (2-5) in terms of w/w.

3. The method of claim 1, wherein the reaction temperature is 35-45 ℃.

4. The method of claim 1, wherein the reaction time is 8 to 16 hours.

5. The method of claim 1, wherein the EPA and DHA-containing oil comprises natural and/or synthetic lipids with EPA >15 wt%.

6. The method of claim 5, wherein the synthetic lipid is prepared by removing enzyme and unreacted substrate after lipase-catalyzed enzymatic reaction for 4-12 hours at a ratio of non-glyceride to glycerol of more than 3:1 and at a temperature of 40-80 ℃ to obtain EPA-enriched synthetic lipid;

wherein the pressure of the reaction system is below 1000 Pa.

7. The method of claim 1, wherein the separation of enzyme and solvent is performed by centrifugation, and the upper layer after centrifugation is the crude product;

the centrifugal speed is more than 3000 r/min, and the centrifugal time is 3-20 min.

8. The method of claim 1, wherein the further separation and purification method comprises two steps, namely molecular distillation and urea inclusion.