A method for improving the purity of nervonic acid

Abstract

The present invention provides a method for improving the purity of nervonic acid, which belongs to the technical field of separation and purification, and comprises the following steps: (1) preparing pretreated rapeseed powder; (2) extracting rapeseed oil by supercritical carbon dioxide extraction; (3) mixing rapeseed oil, water and emulsifier evenly, ultrasonicating in an ice-water bath for 10-15 minutes, and obtaining an emulsion; (4) adding the emulsion to a culture medium to obtain a fermentation substrate, adding mixed bacteria to the fermentation substrate, shaking, and culturing in a constant temperature shaker for 40-45 hours; obtaining a culture solution; (5) preparing a mixture containing nervonic acid; (6) preparing a crude nervonic acid; (7) mixing the crude nervonic acid and ethanol in a mass ratio of 1: (4-6) to obtain a mixture, subjecting the mixture to gradient crystallization, and after the crystallization is completed, separating by suction filtration, and drying the filter cake to obtain nervonic acid. The nervonic acid prepared by the method of the present invention has high purity and high yield.

Description

Technical Field

[0001] The present invention belongs to the technical field of separation and purification, and specifically relates to a method for improving the purity of nervonic acid. Background Art

[0002] Nerve acid, scientific name: tetracosyl-cis-1,5-enoic acid, is an unsaturated fatty acid with 24 carbon atoms and 1 double bond found in nerve tissue and fish oil. Nerve acid is a component of cerebrosides, and research suggests that it plays an important role in the development and function of the nervous system, especially in the formation of myelin, a lipid layer surrounding nerve axons that helps speed up the conduction of nerve impulses.

[0003] Among plant resources, neuraminic acid is only found in a few woody oils such as Acer truncatum and Xanthoceras sorbifolia oil, and none of them has the value of large-scale industrial production. Rapeseed oil has a higher content of unsaturated fatty acids than Acer truncatum and Xanthoceras sorbifolia oils, is rich in bioactive substances, and is currently one of the main raw materials for the preparation of neuraminic acid.

[0004] At present, most of the existing technologies improve the production of nervonic acid from the aspect of improving the purity of the product. The invention patent with application number CN201810588314.6 discloses a method for separating and purifying nervonic acid. When the crude nervonic acid is obtained, the unsaponifiable matter is removed by fixed bed adsorption, and then the sample solution is pumped into the chromatographic injector at 80-120atm to accelerate the separation of nervonic acid in the crude nervonic acid, and the crude nervonic acid is subjected to gradient elution, and the nervonic acid in the crude nervonic acid is effectively separated and purified from other substances such as erucic acid, lignin acid, behenic acid, etc., which solves the technical problems of low separation purity and long production cycle in the production process of existing nervonic acid. However, this method still cannot solve the problem that the nervonic acid itself in the rapeseed oil raw material cannot meet the market demand, and the purity and yield of this method still need to be further improved. Summary of the invention

[0005] The purpose of the present invention is to provide a method for improving the purity of nervonic acid.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] A method for improving the purity of nervonic acid, comprising the following steps:

[0008] (1) crushing rapeseed, drying, sieving, and taking powder with a particle size between 40 and 60 mesh as pretreated rapeseed powder;

[0009] (2) loading the pretreated rapeseed powder into the extraction kettle, sealing the extraction kettle, performing supercritical carbon dioxide extraction, collecting rapeseed oil every 25-30 minutes, extracting for a total of 140-150 minutes, and collecting rapeseed oil from the separation kettle at a separation pressure of 5.2-5.5 MPa and a separation temperature of 50-55° C.;

[0010] (3) Evenly mix rapeseed oil, water and emulsifier in a mass ratio of 2: (1.7-1.9): (0.6-0.8), and perform ultrasonic treatment in an ice-water bath for 10-15 minutes to obtain an emulsion;

[0011] (4) adding the emulsion to the culture medium to obtain a fermentation substrate, adding the mixed bacteria to the fermentation substrate, shaking well, and culturing in a constant temperature shaker at 28-30° C. and 300-400 r / min for 40-45 hours to obtain a culture solution;

[0012] (5) Centrifuge the culture solution at 7000-8000 r / min for 10-15 min, remove the supernatant, obtain a solid, add 10-15 times the mass of the solid potassium hydroxide-methanol solution to the solid, mix well, reflux at 80-85°C for 3-4 h, add 5-6 mol / L hydrochloric acid solution to adjust the pH to 5.5-6.0 after reflux, add n-hexane, oscillate and extract for 3-4 h, take out the upper organic phase obtained by extraction, blow with N2 to 1 / 15-1 / 10 of the original volume of the upper organic phase, and obtain a mixture containing nervonic acid;

[0013] (6) subjecting the mixture containing nervonic acid to molecular distillation at a temperature of 170-190° C. and a pressure of 6-12 Pa to obtain an intermediate product, which is a crude nervonic acid;

[0014] (7) Mix crude neuraminic acid and ethanol in a mass ratio of 1:(4-6) to obtain a mixture, subject the mixture to gradient crystallization, and after the crystallization is completed, separate the mixture by suction filtration, dry the filter cake, and obtain neuraminic acid.

[0015] Furthermore, the mixed bacteria in step (4) include Saccharomyces cerevisiae, Candida lipolytica, and Trichosporon moniliformis.

[0016] Saccharomyces cerevisiae was purchased from China Industrial Microbiological Culture Collection Center, strain number: CICC 32883.

[0017] Candida lipolytica was purchased from China Industrial Microbiological Culture Collection Center, strain number: CICC 32245.

[0018] Cutaneotrichosporon moniliiforme was purchased from China Industrial Microbiological Culture Collection Center, strain number: CICC 33107.

[0019] Furthermore, the amount of brewer's yeast is 10 6 -10 7 CFU / mL fermentation substrate; the amount of Candida lipolytica is 10 8 -10 9 CFU / mL fermentation substrate; the dosage of Neotrichomonas spp. was 10 7 -10 8 CFU / mL fermentation substrate.

[0020] The content of nervonic acid in rapeseed is not high in itself. Most of the existing technologies have been improved in the purification method, and the actual nervonic acid obtained is still limited. The present invention uses three kinds of bacteria to ferment rapeseed, which can increase the content of nervonic acid in rapeseed and provide more sources of nervonic acid for subsequent extraction and purification. In the prior art, microorganisms that can convert processed biomass into oil and store it in the cell are called oil-producing microorganisms. The present invention attempts to use Candida lipolytica to ferment rapeseed powder, but in the practical research and development process, it is found that the culture time of Candida lipolytica is long and the actual effect is not ideal. The present invention can increase the content of nervonic acid by compounding three kinds of yeast for fermentation. The analysis is that these three kinds of yeast can work synergistically, have different metabolic pathways and enzyme systems, and through compound fermentation, the advantages of each strain can be used to complement each other and jointly promote the synthesis of nervonic acid. For example, a certain yeast may be good at oil degradation, while other yeasts are excellent in the synthesis of long-chain fatty acids, and can produce more nervonic acid through fermentation.

[0021] Furthermore, in step (1), the mixture is dried in a drying oven at 65-70° C. until the moisture content by mass is 1.5-1.7%.

[0022] Furthermore, the extraction conditions in step (2) are: extraction temperature 40-44° C., extraction pressure 36-40 MPa, and carbon dioxide flow rate 25-30 L / h.

[0023] During the experiment, it was found that the conditions of supercritical carbon dioxide extraction used in the present invention can improve the amount of nervonic acid produced by the mixed bacteria. The analysis shows that different extraction conditions may affect the extraction efficiency of fatty acids, resulting in changes in the ratio of different fatty acids in the final rapeseed oil. The surface tension of rapeseed oil may change due to different extraction conditions, affecting its contact with the aqueous phase and emulsification state. The types and proportions of fatty acids in rapeseed oil directly affect the energy supply of microorganisms. Certain fatty acids or antioxidant components may participate in the metabolic regulation of microorganisms, affecting their metabolic pathways, and thus affecting the amount of nervonic acid produced by the mixed bacteria.

[0024] Furthermore, the emulsifier in step (3) is Tween 80.

[0025] Furthermore, in step (4), the mass ratio of the emulsion to the culture medium is (3-5):100.

[0026] Furthermore, in the step (5), the mass percentage of potassium hydroxide in the potassium hydroxide-methanol solution is 8-10%.

[0027] Furthermore, the volume of n-hexane added is 1.2-1.5 times the volume of the potassium hydroxide-methanol solution.

[0028] Furthermore, the steps of gradient crystallization are: slowly controlling the temperature to 0°C, performing a cooling crystallization, separating by suction filtration after the crystallization is completed to obtain a primary filtrate, controlling the temperature of the primary filtrate to -5°C for a secondary cooling crystallization, separating by suction filtration after the crystallization is completed to obtain a secondary filtrate, and controlling the temperature of the secondary filtrate to -15°C for a third cooling crystallization.

[0029] The present invention can improve the purity of nervonic acid by using gradient crystallization. Due to the complex composition of the crude oleic acid product of the present invention, the solubility of oleic acid and other components at different temperatures is different by using the gradient crystallization of the present invention. At different temperatures, the solubility difference between oleic acid and impurities is fully utilized. Impurities can be removed more fully and gradually by gradient crystallization, thereby improving the purity of the product. At the same time, the recovery rate of nervonic acid is improved.

[0030] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0031] 1. The present invention proposes a method for effectively improving the purity of neuraminic acid, aiming to obtain high-purity neuraminic acid by optimizing the extraction and purification process. Neuroacid is an important unsaturated fatty acid that plays a key role in maintaining the health of the nervous system. The method of the present invention first uses supercritical carbon dioxide extraction technology to preliminarily extract crude products from natural raw materials rich in neuraminic acid, and then further purifies them using advanced technologies such as high performance liquid chromatography (HPLC) or molecular distillation. By precisely controlling the extraction conditions such as temperature, pressure, and selection of mobile phases, and combining the optimization of parameters in subsequent purification steps, the present invention successfully achieved a significant improvement in the purity of neuraminic acid. Experimental results show that the purity of the final product reached more than 98%, which is much higher than the level that can be achieved with the prior art.

[0032] 2. The method of the present invention is green and environmentally friendly, avoids the use of harmful solvents, and ensures the safety and biological activity of the product. This method not only provides a new way for the large-scale production of nervonic acid, but also lays a solid foundation for the development of related pharmaceutical and health care products industries.

[0033] 3. The present invention can increase the content of nervonic acid by compounding and fermenting three kinds of yeast.

[0034] 4. The present invention uses supercritical carbon dioxide extraction conditions to improve the amount of neuraminic acid produced by mixed bacteria.

[0035] 5. The present invention can improve the purity of nervonic acid by using gradient crystallization. DETAILED DESCRIPTION

[0036] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. 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.

[0037] The raw materials used in the following examples of the present invention are all commercially available products:

[0038] Saccharomyces cerevisiae was purchased from China Industrial Microbiological Culture Collection Center, strain number: CICC 32883.

[0039] Candida lipolytica was purchased from China Industrial Microbiological Culture Collection Center, strain number: CICC 32245.

[0040] Cutaneotrichosporon moniliiforme was purchased from China Industrial Microbiological Culture Collection Center, strain number: CICC 33107.

[0041] Culture medium: PDA culture medium, purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number R30024.

[0042] Example 1

[0043] This embodiment provides a method for improving the purity of nervonic acid, comprising the following steps:

[0044] (1) crushing rapeseed, drying in a drying oven at 68° C. to a moisture content of 1.6% by weight, sieving, and taking powder with a particle size of 40-60 mesh as pretreated rapeseed powder;

[0045] (2) The pretreated rapeseed powder was placed in an extraction kettle, the extraction kettle was sealed, and supercritical carbon dioxide extraction was performed under the following extraction conditions: extraction temperature 42°C, extraction pressure 38 MPa, and carbon dioxide flow rate 27 L / h; rapeseed oil was collected every 28 minutes, and the extraction was performed for a total of 145 minutes. The rapeseed oil was collected from the separation kettle at a separation pressure of 5.3 MPa and a separation temperature of 52°C;

[0046] (3) Rapeseed oil, water and Tween 80 in a mass ratio of 2:1.8:0.7 were mixed evenly, and ultrasonicated in an ice-water bath for 12 min to obtain an emulsion;

[0047] (4) Add the emulsion to the culture medium at a mass ratio of 3:100 to obtain a fermentation substrate, add the mixed bacteria to the fermentation substrate, shake well, and culture in a constant temperature shaker at 29°C and 350 r / min for 42 hours to obtain a culture solution; the mixed bacteria include Saccharomyces cerevisiae, Candida lipolytica, and Trichosporon sphenoides. The amount of Saccharomyces cerevisiae used is 10 6 CFU / mL fermentation substrate; the amount of Candida lipolytica is 10 9 CFU / mL fermentation substrate; the dosage of Neotrichomonas spp. was 10 7 CFU / mL fermentation substrate.

[0048] (5) The culture medium was centrifuged at 7500 r / min for 12 min, and the supernatant was removed to obtain a solid. A potassium hydroxide-methanol solution 13 times the mass of the solid was added to the solid, and the mass percentage of potassium hydroxide in the potassium hydroxide-methanol solution was 9%; after mixing, refluxed at 82°C for 3.5 h. After the reflux was completed, a 5.5 mol / L hydrochloric acid solution was added to adjust the pH to 5.8, and n-hexane was added, and the volume of n-hexane added was 1.4 times the volume of the potassium hydroxide-methanol solution; oscillation extraction was performed for 3.5 h, and the upper organic phase obtained by extraction was taken out, and N2 was blown to 1 / 12 of the original volume of the upper organic phase to obtain a mixture containing nervonic acid;

[0049] (6) subjecting the mixture containing nervonic acid to molecular distillation at a temperature of 180° C. and a pressure of 10 Pa to obtain an intermediate product, which is a crude nervonic acid;

[0050] (7) Mix crude neuraminic acid and ethanol in a mass ratio of 1:5 to obtain a mixture, and subject the mixture to gradient crystallization. The steps of the gradient crystallization are as follows: slowly control the temperature to 0°C, perform a cooling crystallization, separate by suction filtration after the crystallization is completed to obtain a primary filtrate, control the temperature of the primary filtrate to -5°C, perform a secondary cooling crystallization, and separate by suction filtration after the crystallization is completed to obtain a secondary filtrate. Control the temperature of the secondary filtrate to -15°C, perform a third cooling crystallization; after the crystallization is completed, separate by suction filtration, and dry the filter cake to obtain neuraminic acid.

[0051] Example 2

[0052] This embodiment provides a method for improving the purity of nervonic acid, comprising the following steps:

[0053] (1) crushing rapeseed, drying in a drying oven at 65° C. to a moisture content of 1.7% by weight, sieving, and taking powder with a particle size of 40-60 mesh as pretreated rapeseed powder;

[0054] (2) The pretreated rapeseed powder was placed in an extraction kettle, the extraction kettle was sealed, and supercritical carbon dioxide extraction was performed under the following extraction conditions: extraction temperature of 40°C, extraction pressure of 40 MPa, and carbon dioxide flow rate of 25 L / h; rapeseed oil was collected every 30 minutes, and the extraction was performed for a total of 140 minutes. The rapeseed oil was collected from the separation kettle at a separation pressure of 5.5 MPa and a separation temperature of 50°C;

[0055] (3) Rapeseed oil, water and Tween 80 in a mass ratio of 2:1.7:0.6 were mixed evenly, and ultrasonicated in an ice-water bath for 10 min to obtain an emulsion;

[0056] (4) Add the emulsion to the culture medium at a mass ratio of 3:100 to obtain a fermentation substrate, add the mixed bacteria to the fermentation substrate, shake well, and culture in a constant temperature shaker at 28°C and 400 r / min for 40 hours to obtain a culture solution; the mixed bacteria include Saccharomyces cerevisiae, Candida lipolytica, and Neotrichomonas spp. The amount of Saccharomyces cerevisiae used is 10 7 CFU / mL fermentation substrate; the amount of Candida lipolytica is 10 8 CFU / mL fermentation substrate; the dosage of Neotrichomonas spp. was 10 7 CFU / mL fermentation substrate.

[0057] (5) The culture medium was centrifuged at 70,000 r / min for 15 min, and the supernatant was removed to obtain a solid. A potassium hydroxide-methanol solution 10 times the mass of the solid was added to the solid, and the mass percentage of potassium hydroxide in the potassium hydroxide-methanol solution was 10%; after mixing, refluxed at 80°C for 4 h. After the reflux was completed, 5 mol / L hydrochloric acid solution was added to adjust the pH to 6.0, and n-hexane was added, and the volume of n-hexane added was 1.2 times the volume of the potassium hydroxide-methanol solution; oscillation extraction was performed for 4 h, and the upper organic phase obtained by extraction was taken out, and N2 was blown to 1 / 10 of the original volume of the upper organic phase to obtain a mixture containing nervonic acid;

[0058] (6) subjecting the mixture containing nervonic acid to molecular distillation at a temperature of 170° C. and a pressure of 12 Pa to obtain an intermediate product, which is a crude nervonic acid;

[0059] (7) Mix crude neuraminic acid and ethanol in a mass ratio of 1:6 to obtain a mixture, and subject the mixture to gradient crystallization. The steps of the gradient crystallization are as follows: slowly control the temperature to 0°C, perform a cooling crystallization, separate by suction filtration after the crystallization is completed to obtain a primary filtrate, control the temperature of the primary filtrate to -5°C, perform a secondary cooling crystallization, and separate by suction filtration after the crystallization is completed to obtain a secondary filtrate. Control the temperature of the secondary filtrate to -15°C, perform a third cooling crystallization; after the crystallization is completed, separate by suction filtration, and dry the filter cake to obtain neuraminic acid.

[0060] Comparative Example 1

[0061] The difference between this comparative example and Example 1 is that no fermentation culture is performed.

[0062] A method for improving the purity of nervonic acid, comprising the following steps:

[0063] (1) crushing rapeseed, drying in a drying oven at 68° C. to a moisture content of 1.6% by weight, sieving, and taking powder with a particle size of 40-60 mesh as pretreated rapeseed powder;

[0064] (2) The pretreated rapeseed powder was placed in an extraction kettle, the extraction kettle was sealed, and supercritical carbon dioxide extraction was performed under the following extraction conditions: extraction temperature 42°C, extraction pressure 38 MPa, and carbon dioxide flow rate 27 L / h; rapeseed oil was collected every 28 minutes, and the extraction was performed for a total of 145 minutes. The rapeseed oil was collected from the separation kettle at a separation pressure of 5.3 MPa and a separation temperature of 52°C;

[0065] (3) hydrolyzing the rapeseed oil at a temperature of 250°C and a pressure of 5 MPa. After the hydrolysis is completed, transferring the hydrolyzate into a separatory funnel for separation, and taking the upper layer of liquid as a mixture containing nervonic acid;

[0066] (4) subjecting the mixture containing nervonic acid to molecular distillation at a temperature of 180° C. and a pressure of 10 Pa to obtain an intermediate product, which is a crude nervonic acid;

[0067] (5) Mix crude neuraminic acid and ethanol in a mass ratio of 1:5 to obtain a mixture, and subject the mixture to gradient crystallization. The steps of the gradient crystallization are as follows: slowly control the temperature to 0°C, perform a cooling crystallization, separate by suction filtration after the crystallization is completed to obtain a primary filtrate, control the temperature of the primary filtrate to -5°C, perform a secondary cooling crystallization, and separate by suction filtration after the crystallization is completed to obtain a secondary filtrate. Control the temperature of the secondary filtrate to -15°C, perform a third cooling crystallization; after the crystallization is completed, separate by suction filtration, and dry the filter cake to obtain neuraminic acid.

[0068] Comparative Example 2

[0069] The difference between this comparative example and Example 1 is that the mixed bacteria is replaced by Candida lipolytica. The dosage of Candida lipolytica is 1.011×10 9 CFU / mL fermentation substrate.

[0070] Comparative Example 3

[0071] The difference between this comparative example and Example 1 is that the mixed bacteria include Saccharomyces cerevisiae, Candida lipolytica, and Trichosporon sphenoides. The amount of Saccharomyces cerevisiae used is 10 9 CFU / mL fermentation substrate; the amount of Candida lipolytica is 10 7 CFU / mL fermentation substrate; the dosage of Neotrichomonas spp. was 10 6 CFU / mL fermentation substrate.

[0072] Comparative Example 4

[0073] The difference between this comparative example and Example 1 is that the mixed bacteria include Mortierella lutea, Candida lipolytica, and Mortierella lutea. The dosage of Mortierella lutea is 10 6 CFU / mL fermentation substrate; the amount of Candida lipolytica is 10 9 CFU / mL fermentation substrate; the dosage of Neotrichomonas spp. was 107 CFU / mL fermentation substrate. Mortierella flavum (GDMCC 3.241): purchased from Guangdong Microbial Culture Collection Center; Mortierella flavum (BNCC 336207): purchased from Beijing Beina Chuanglian Biotechnology Research Institute.

[0074] Comparative Example 5

[0075] The difference between this comparative example and Example 1 is that the extraction conditions are: extraction temperature 35° C., extraction pressure 45 MPa, and carbon dioxide flow rate 20 L / h.

[0076] Comparative Example 6

[0077] The difference between this comparative example and Example 1 is that the steps of gradient crystallization are: slowly controlling the temperature to 10°C, performing a cooling crystallization, and after the crystallization is completed, separating by suction filtration to obtain a primary filtrate, and controlling the temperature of the primary filtrate to -15°C for a secondary cooling crystallization.

[0078] Performance Testing

[0079] 1. Use gas chromatography to detect the content of nervonic acid in the mixture containing nervonic acid prepared in Example 1-2 and Comparative Example 1-6. The content of nervonic acid = the mass of nervonic acid / the mass of the mixture containing nervonic acid × 100%.

[0080] 2. The nervonic acid prepared in Example 1-2 and Comparative Example 1-6 was detected by gas chromatography, and the purity and yield were calculated. Purity = the mass of nervonic acid detected by gas chromatography / the mass of nervonic acid prepared in Example 1-3 and Comparative Example 1-6 × 100%. Yield = the mass of nervonic acid detected by gas chromatography / the mass of rapeseed × 100%.

[0081] The test results are shown in Table 1.

[0082] Table 1 Performance test results

[0083] Nerve acid content% purity% Recovery rate% Example 1 10.43 99.3 1.23 Example 2 10.36 99.1 1.20 Comparative Example 1 Not detected 98.5 0.86 Comparative Example 2 9.65 98.2 0.98 Comparative Example 3 10.28 98.7 1.19 Comparative Example 4 10.07 98.1 1.04 Comparative Example 5 10.43 98.4 1.16 Comparative Example 6 10.43 98.3 1.17

[0084] It can be seen from the above performance test results that the purity of the nervonic acid prepared in Example 1-2 is high, and the amount of nervonic acid produced per unit mass of rapeseed is significantly increased.

[0085] However, the comparative examples are significantly worse than the embodiments in terms of corresponding performance tests because they do not adopt the necessary technical solutions. In comparative example 1, nervonic acid is not fermented and cultured, and fatty acids are prepared by the traditional hydrolysis method and then purified. It can be found that the final unit mass of nervonic acid produced by rapeseed is significantly lower than that of the present invention, indicating that the fermentation of the present invention can increase the content of nervonic acid in the mixed fatty acid, and the subsequent further purification effect is better, the impurities are removed more cleanly, and the loss of nervonic acid is smaller. In comparative examples 2-4, the strains and strain concentrations are changed, and it can be found that the content of nervonic acid decreases, indicating the synergistic effect of specific strains. At the same time, the purity and yield also decrease, indicating that the fermentation product will affect the subsequent purification effect. In comparative example 5, the extraction conditions are different, the composition of the extract obtained is different, which affects the subsequent purification and the purity decreases. In comparative example 6, the recrystallization method is different, the purity decreases, and the recovery rate also decreases. The above experimental results further prove the importance of the technical solution defined in the present invention for its technical effect.

[0086] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for improving the purity of nervonic acid, characterized in that: The following steps are involved: (1) crushing rapeseed, drying, sieving, and taking powder with a particle size between 40 and 60 meshes as pretreated rapeseed powder; (2) loading the pretreated rapeseed powder into the extraction kettle, sealing the extraction kettle, performing supercritical carbon dioxide extraction, collecting rapeseed oil every 25-30 minutes, extracting for a total of 140-150 minutes, and collecting rapeseed oil from the separation kettle at a separation pressure of 5.2-5.5 MPa and a separation temperature of 50-55° C.; (3) Evenly mix rapeseed oil, water and emulsifier in a mass ratio of 2: (1.7-1.9): (0.6-0.8), and perform ultrasonic treatment in an ice-water bath for 10-15 minutes to obtain an emulsion; (4) adding the emulsion to the culture medium to obtain a fermentation substrate, adding the mixed bacteria to the fermentation substrate, shaking well, and culturing in a constant temperature shaker at 28-30° C. and 300-400 r / min for 40-45 hours to obtain a culture solution; (5) Centrifuge the culture solution at 7000-8000 r / min for 10-15 min, remove the supernatant, obtain a solid, add 10-15 times the mass of the solid potassium hydroxide-methanol solution to the solid, mix well, reflux at 80-85°C for 3-4 h, add 5-6 mol / L hydrochloric acid solution to adjust the pH to 5.5-6.0 after reflux, add n-hexane, oscillate and extract for 3-4 h, take out the upper organic phase obtained by extraction, blow with N2 to 1 / 15-1 / 10 of the original volume of the upper organic phase, and obtain a mixture containing nervonic acid; (6) subjecting the mixture containing nervonic acid to molecular distillation at a temperature of 170-190° C. and a pressure of 6-12 Pa to obtain an intermediate product, which is a crude nervonic acid; (7) Mixing crude nervonic acid and ethanol in a mass ratio of 1:(4-6) to obtain a mixture, subjecting the mixture to gradient crystallization, and after the crystallization is completed, separating by suction filtration, and drying the filter cake to obtain nervonic acid; The mixed bacteria in step (4) include Saccharomyces cerevisiae with strain number CICC 32883, Candida lipolytica with strain number CICC 32245, and Neotrichomonas moniliformis with strain number CICC 33107; The dosage of brewer's yeast is 10 6 -10 7 CFU / mL fermentation substrate; the amount of Candida lipolytica is 10 8 -10 9 CFU / mL fermentation substrate; the dosage of Neotrichomonas spp. was 10 7 -10 8 CFU / mL fermentation substrate.

2. The method for improving the purity of nervonic acid according to claim 1, characterized in that: In the step (1), the mixture is dried in a drying oven at 65-70° C. until the moisture content is 1.5-1.7%.

3. The method for improving the purity of nervonic acid according to claim 1, characterized in that: The extraction conditions in step (2) are: extraction temperature 40-44° C., extraction pressure 36-40 MPa, and carbon dioxide flow rate 25-30 L / h.

4. The method for improving the purity of nervonic acid according to claim 1, characterized in that: The emulsifier in step (3) is Tween 80.

5. The method for improving the purity of nervonic acid according to claim 1, characterized in that: The mass ratio of the emulsion to the culture medium in step (4) is (3-5):

100.

6. The method for improving the purity of nervonic acid according to claim 1, characterized in that: The mass percentage of potassium hydroxide in the potassium hydroxide-methanol solution in step (5) is 8-10%.

7. The method for improving the purity of nervonic acid according to claim 1, characterized in that: The volume of n-hexane added is 1.2-1.5 times the volume of the potassium hydroxide-methanol solution.

8. The method for improving the purity of nervonic acid according to claim 1, characterized in that: The steps of gradient crystallization are: controlling the temperature to 0°C, performing a cooling crystallization, separating by suction filtration after the crystallization is completed to obtain a primary filtrate, controlling the temperature of the primary filtrate to -5°C for a secondary cooling crystallization, separating by suction filtration after the crystallization is completed to obtain a secondary filtrate, and controlling the temperature of the secondary filtrate to -15°C for a third cooling crystallization.