Preparation method of unsaturated fatty acid

Abstract

The invention provides a preparation method of unsaturated fatty acid, which obviously reduces the residual amount of dioxin in grease through tromethamine treatment, effectively separates EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid) through magnesium acetate precipitation, and improves the cycle use frequency of silver nitrate cycle use liquid through stannous chloride treatment and alkaline alumina treatment. And the unsaturated fatty acid separation capability of the silver nitrate recycling liquid is maintained.

Description

Technical Field

[0001] The present invention relates to the technical field of fatty acid separation, and particularly to a method for preparing unsaturated fatty acids. Background Art

[0002] Unsaturated fatty acids include monounsaturated fatty acids and polyunsaturated fatty acids. Polyunsaturated fatty acids, especially DHA (docosahexaenoic acid) and EPA (eicosapentaenoic acid), play important roles in human health. DHA is crucial for brain development, cognitive function, and vision protection, and is suitable for pregnant women, infants, and those in need of improving brain health. EPA has anti-inflammatory and cardiovascular protection effects and is suitable for patients with cardiovascular diseases and inflammatory diseases. The applicable populations of DHA and EPA are not completely the same. Therefore, effectively separating DHA and EPA is of great significance in meeting specific health needs, optimizing dosage and ratio, and improving bioavailability. However, due to the highly similar chemical structures and physical properties of DHA and EPA, it is difficult to effectively separate DHA and EPA by the existing low-temperature crystallization method and solvent extraction method.

[0003] Dioxins are a class of chlorine-containing organic pollutants with extremely strong toxicity and environmental persistence. They have high chemical stability and lipophilicity, are difficult to degrade, and easily accumulate in adipose tissue and oils, posing a serious threat to human health and the ecosystem. Microalgae crude oil is rich in various fatty acids, especially DHA, and is an ideal raw material for preparing high-purity DHA. However, due to the aggravation of environmental pollution, microalgae absorb dioxins from water bodies and enrich and accumulate dioxins in the lipid part (crude oil) of microalgae. Due to the lipophilicity of dioxins, the traditional adsorption method and extraction method have poor effects on removing dioxins from microalgae crude oil.

[0004] The silver nitrate complex method is a method for separating unsaturated fatty acids based on the property that double bonds in unsaturated fatty acids form complexes with silver ions. Since the separation effect is poor when silver nitrate is reused and the price of silver nitrate is expensive, the cost of this method is relatively high, which limits the application of the silver nitrate complex method.

[0005] Therefore, it is of great value to establish a method for preparing unsaturated fatty acids that can effectively separate DHA and EPA, effectively reduce dioxin residues, and reuse silver nitrate solution. Summary of the Invention

[0006] In order to solve the above problems, the present invention provides a method for preparing unsaturated fatty acids, which includes the following steps:

[0007] Step S1, saponification reaction: adding a sodium hydroxide solution to microalgae crude oil for saponification reaction, and the reaction solution is oil A;

[0008] Step S2, primary acidification: While stirring, add a citric acid solution to Oil A, adjust the pH value of Oil A to 2 - 3, let it stand for layering, and take the oil phase to prepare Oil B;

[0009] Step S3, tromethamine treatment: Add a tromethamine solution to Oil B, disperse evenly and then extract. After extraction, take the aqueous phase to prepare Solution C;

[0010] Step S4, secondary acidification: While stirring, add a citric acid solution to Solution C, adjust the pH value of Solution C to 2 - 3, let it stand for layering, and take the oil phase to prepare Oil D;

[0011] Step S5, urea inclusion reaction: Dissolve urea in ethanol to prepare a urea solution; add Oil D to the urea solution for the inclusion reaction; filter and collect the filtrate, wash the filtrate with purified water, and take the oil phase to prepare Oil E;

[0012] Step S6, magnesium acetate precipitation: Disperse magnesium acetate in acetone to prepare Solution F; stir and mix Oil E with Solution F, then let it stand for the precipitation reaction; filter to remove the precipitate, collect the filtrate, and prepare Oil G;

[0013] Step S7, esterification reaction: Add n - hexane, ethanol, and sodium hydroxide to Oil G for the esterification reaction;

[0014] Step S8, vacuum distillation: Place the esterification reaction solution from Step S7 in a distiller for vacuum distillation, collect the heavy fraction to prepare Oil H;

[0015] Step S9, stannous chloride treatment: Add stannous chloride to Oil H for the reduction reaction to prepare Oil I;

[0016] Step S10, basic alumina treatment: Add basic alumina to Oil I to adsorb free fatty acids, then filter and collect the filtrate to prepare Oil J;

[0017] Step S11, silver nitrate complexation reaction: Add an aqueous silver nitrate solution to Oil J for the complexation reaction, then let it stand for layering, and collect the lower aqueous phase; add cyclohexane to the aqueous phase to extract the oil, then let it stand for layering, collect the upper layer to prepare Oil K, and collect the lower layer to prepare the silver nitrate recycling solution;

[0018] Step S12, molecular fractionation: Place Oil K in a molecular fractionator for fractionation, and collect the distillate and the heavy fraction respectively. The distillate is ethyl unsaturated fatty acid.

[0019] As a preferred embodiment, the method for preparing the unsaturated fatty acid further includes Step S13 and Step S14;

[0020] In step S13, the heavy fraction is treated with immobilized lipase: The bottom heavy fraction of the tower is added with immobilized lipase and glycerol for an enzymatic reaction; the lipase is removed by filtration to produce oil L;

[0021] In step S14, molecular fractionation: Oil L is fractionated in a molecular fractionator, and the distillate is collected to produce very long-chain polyunsaturated fatty acid ethyl ester.

[0022] As a preferred solution, in step S1, the reaction temperature is 60°C to 70°C, the reaction time is 1 hour to 3 hours; the sodium hydroxide solution is an aqueous sodium hydroxide solution with a mass fraction of 20% to 30%; the mass ratio of the microalgae crude oil to the sodium hydroxide solution is 1:(2 - 4).

[0023] As a preferred solution, in step S2, the mass concentration of the citric acid solution is 50%, and the stirring time is 20 minutes to 40 minutes.

[0024] As a preferred solution, in step S3, the tromethamine solution is an aqueous tromethamine solution with a mass fraction of 10% to 30%; the mass ratio of oil B to the tromethamine solution is 1:(2 - 4); the extraction time is 3 hours to 5 hours.

[0025] As a preferred solution, in step S4, the mass concentration of the citric acid solution is 50%, and the stirring time is 20 minutes to 40 minutes.

[0026] As a preferred solution, in step S5, the clathration reaction temperature is 5°C to 7°C, the clathration reaction time is 8 hours to 10 hours; the mass ratio of oil D to the urea solution is 1:(3 - 5).

[0027] As a preferred solution, in step S6, the temperature of the precipitation reaction is -15°C to -25°C, the precipitation reaction time is 10 hours to 20 hours; the mass ratio of acetone to magnesium acetate is 1:(0.04 - 0.06).

[0028] As a preferred solution, in step S7, the mass ratio of oil G to n-hexane is 1:(0.2 - 0.4); the mass ratio of oil G to ethanol is 1:(3 - 6); the mass ratio of oil G to sodium hydroxide is 1:(0.01 - 0.03); the esterification reaction temperature is 80°C to 90°C; the esterification reaction time is 5 hours to 7 hours.

[0029] As a preferred solution, in step S8, the temperature of the vacuum distillation is 60°C to 100°C, the vacuum distillation pressure is 5 Pa to 10 Pa, and the vacuum distillation time is 3 hours to 5 hours.

[0030] As a preferred embodiment, the mass ratio of stannous chloride to oil H in step S9 is 1:(100-200); the reduction reaction temperature is 20° C.-30° C., and the reduction reaction time is 40 minutes-60 minutes.

[0031] As a preferred embodiment, the mass ratio of the solvent I to the alkaline alumina in step S10 is 1:(0.1-0.2); the adsorption temperature is 50°C-70°C; the adsorption time is 20 minutes-30 minutes; and the alkaline alumina is prepared by soaking, washing and drying alumina with sodium hydroxide.

[0032] As a preferred embodiment, the mass fraction of the silver nitrate aqueous solution in step S11 is 50% to 60%; the mass ratio of the oil J to the silver nitrate aqueous solution is 1:(4 to 6); the complex reaction temperature is 8°C to 10°C; the complex reaction time is 20 minutes to 30 minutes; the extraction temperature is 40°C to 60°C; and the extraction time is 30 minutes to 40 minutes.

[0033] As a preferred solution, the feed flow rate of oil K during fractionation in step S12 is 100 kg / h / m 2 ~120kg / h / m 2 The distillation temperature is 150℃~180℃, and the distillation pressure is 20Pa~50Pa.

[0034] As a preferred embodiment, the immobilized lipase in step S13 is Lipozyme 435; the reaction temperature is 60°C to 80°C, the reaction time is 30 hours to 40 hours; and the mass ratio of the bottom heavy fraction to the lipase Lipozyme 435 is 20:(1-2).

[0035] As a preferred solution, the feed flow rate of oil L during fractionation in step S14 is 60 kg / h / m 2 ~80kg / h / m 2 The distillation temperature is 170℃~180℃, and the distillation pressure is 10Pa~20Pa.

[0036] The second aspect of the present invention provides an unsaturated fatty acid, which is prepared by the above-mentioned preparation method.

[0037] Through the above technical solutions, the present invention produces the following technical effects:

[0038] (1) The dioxin residue in oil and fat was significantly reduced by tromethamine treatment.

[0039] (2) EPA and DHA are effectively separated by magnesium acetate precipitation.

[0040] (3) By treating with stannous chloride and basic alumina, the number of times of recycling the silver nitrate recycling solution is increased, and the ability of the silver nitrate recycling solution to separate unsaturated fatty acids is maintained. Detailed implementation manners

[0041] To make the above objects, features, and advantages of the present invention more obvious and understandable, the applicant explains and analyzes through test examples and embodiments.

[0042] Definition explanations in this application:

[0043] Fatty acids in this application include free fatty acids, esters of fatty acids, and fatty acid chain derivatives.

[0044] Unsaturated fatty acids include monounsaturated fatty acids and polyunsaturated fatty acids. Unsaturated fatty acids generally refer to free fatty acids, esters of fatty acids, and fatty acid chain derivatives.

[0045] Polyunsaturated fatty acids refer to fatty acids with two or more double bonds in the molecule.

[0046] Very long-chain polyunsaturated fatty acids are unsaturated fatty acids with 24 or more carbon atoms.

[0047] Long-chain polyunsaturated fatty acids are unsaturated fatty acids with 20 or more carbon atoms.

[0048] EPA is eicosapentaenoic acid, and DHA is docosahexaenoic acid.

[0049] TEQ (Toxic Equivalent Quantity) is a standardized method for evaluating the toxicity of dioxin-like compounds (including polychlorinated dibenzo-p-dioxins (PCDDs)). Since there are many types of dioxin-like compounds and their toxicity differences are huge, TEQ simplifies the toxicity assessment and comparison by standardizing the toxicity of various compounds relative to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

[0050] As common knowledge, in each step of this application, the oil and fat can be washed with purified water to remove water-soluble substances such as metal ions and short-chain fatty acids.

[0051] Test Example 1, Removal of Dioxin by Tromethamine Treatment Test:

[0052] 1. Preparation of test samples:

[0053] Preparation of oil and fat D1:

[0054] Saponification reaction: Sodium hydroxide solution is added to microalgae crude oil for saponification reaction, and the reaction solution is oil A; the reaction temperature is 60 °C, and the reaction time is 1 hour; the sodium hydroxide solution is an aqueous sodium hydroxide solution with a mass fraction of 20%; the mass ratio of the microalgae crude oil to the sodium hydroxide solution is 1:2;

[0055] Primary acidification: While stirring, citric acid solution is added to oil A to adjust the pH value of oil A to 2, and the oil phase is taken by static separation to prepare oil B; the mass concentration of the citric acid solution is 50%; the stirring time is 20 minutes;

[0056] Tromethamine treatment: Tromethamine solution is added to oil B, and after being dispersed evenly, extraction is carried out. After extraction, the aqueous phase is taken to prepare solution C; the tromethamine solution is an aqueous tromethamine solution with a mass fraction of 10%; the mass ratio of oil B to the tromethamine solution is 1:2; the extraction time is 3 hours;

[0057] Secondary acidification: While stirring, citric acid solution is added to solution C to adjust the pH value of solution C to 2, and the oil phase is taken by static separation to prepare oil D1; the mass concentration of the citric acid solution is 50%; the stirring time is 20 minutes.

[0058] Preparation of oil D2:

[0059] Saponification reaction: Sodium hydroxide solution is added to microalgae crude oil for saponification reaction, and the reaction solution is oil A; the reaction temperature is 60 °C, and the reaction time is 1 hour; the sodium hydroxide solution is an aqueous sodium hydroxide solution with a mass fraction of 20%; the mass ratio of the microalgae crude oil to the sodium hydroxide solution is 1:2;

[0060] Primary acidification: While stirring, citric acid solution is added to oil A to adjust the pH value of oil A to 2, and the oil phase is taken by static separation to prepare oil B; the mass concentration of the citric acid solution is 50%; the stirring time is 20 minutes;

[0061] Purified water treatment: Purified water is added to oil B, and after being dispersed evenly, extraction is carried out. After extraction, the oil phase is taken to prepare oil C; the mass ratio of oil B to the purified water is 1:2; the extraction time is 3 hours;

[0062] Secondary acidification: While stirring, citric acid solution is added to oil C to adjust the pH value of oil C to 2, and the oil phase is taken by static separation to prepare oil D2; the mass concentration of the citric acid solution is 50%; the stirring time is 20 minutes.

[0063] 2. Detection items:

[0064] Measure the dioxin content of Oil A, Oil D1, and Oil D2 according to the literature "Dioxins and polychlorinated biphenyls in fish oil dietary supplements and licensed medicines" (Food Surveillance Information Sheet, Vol. 106, June 1997, MAFF, London).

[0065] Take the mass percentage of Oil D to Oil A as the oil yield.

[0066] 3. Results and analysis:

[0067] The test results are shown in the following table. The results indicate that the treatment with tromethamine can significantly reduce the dioxin residue in the oil.

[0068] The amino group (-NH2) of tromethamine, as a basic group, can react with the carboxyl group (-COOH) of free fatty acids to form tromethamine-fatty acid salts and water. Compared with fatty acids, the solubility of tromethamine-fatty acid salts in water increases significantly. The inventor utilizes this property to extract tromethamine-fatty acid salts from the oil phase into the water phase. Due to the lipophilic property of dioxins, dioxins remain in the oil phase, thus achieving the separation of fatty acids and dioxins and reducing the dioxin residue in fatty acids. While reducing the dioxin residue in the oil, the oil yield of the present invention does not decrease significantly.

[0069] Since dioxins are insoluble in water, using a similar process for purified water treatment cannot effectively remove the dioxin residue in the oil.

[0070] The acidification step of the present invention is necessary to ensure that the fatty acid is in its acid form (COOH) so that it can combine with tromethamine.

[0071] Table 1 Test results of tromethamine treatment

[0072] Test Example 2, magnesium acetate precipitation test:

[0073] 1. Preparation of test samples:

[0074] Prepare Oil G:

[0075] Urea inclusion reaction: Dissolve urea in ethanol to make a urea solution; add Oil D1 to the urea solution for inclusion reaction; filter and collect the filtrate, wash the filtrate with purified water, and take the oil phase to make Oil E; the temperature of the inclusion reaction is 5°C, the inclusion reaction time is 8 hours; the mass ratio of Oil D1 to the urea solution is 1:3;

[0076] Magnesium acetate precipitation: Disperse magnesium acetate in acetone to prepare solution F; stir and mix oil E with solution F, then let it stand for precipitation reaction; filter to remove the precipitate and collect the filtrate to prepare oil G; the temperature of the precipitation reaction is -15°C, and the time of the precipitation reaction is 10 hours; the mass ratio of acetone to magnesium acetate is 1:0.04.

[0077] 2. Detection items:

[0078] Detect the mass concentrations of EPA and DHA in oil D1 respectively, calculate the total mass of EPA and DHA in oil D1, and the mass ratio of DHA to EPA.

[0079] Detect the mass concentrations of EPA and DHA in oil G respectively, calculate the total mass of EPA and DHA in oil G, and the mass ratio of DHA to EPA.

[0080] Detect the mass concentrations of EPA and DHA in the precipitate respectively, calculate the total mass of EPA and DHA in the precipitate, and the mass ratio of DHA to EPA. The precipitate is dispersed in water, acidified, and the oil phase is taken for determination.

[0081] The detection methods for the mass concentrations of EPA and DHA are all existing technologies and will not be elaborated here.

[0082] 3. Results and analysis:

[0083] The test results are shown in the following table. The results show that magnesium acetate precipitation can effectively separate EPA and DHA.

[0084] The applicant unexpectedly found in the experiment that the magnesium salts of EPA and DHA have different solubilities in acetone. The solubility of EPA-magnesium salt in acetone is lower, and the solubility of DHA-magnesium salt in acetone is higher. At low temperatures, this solubility difference is more obvious. The applicant utilized the solubility difference of EPA-magnesium salt and DHA-magnesium salt in acetone to precipitate EPA-magnesium salt from the solvent, achieving the separation of EPA and DHA, thereby preparing DHA and EPA with higher purity.

[0085] Table 2 Test results of magnesium acetate precipitation

[0086] Sample Mass ratio of DHA and EPA Oil D 8:1 Oil G 30:1 Precipitate 0.04:1

[0087] Test Example 3, Recycling test of silver nitrate solution:

[0088] 1. Preparation of test samples:

[0089] Prepare ethyl unsaturated fatty acid Ⅰ:

[0090] Esterification reaction: Add n-hexane, ethanol, and sodium hydroxide to oil G for esterification reaction; the mass ratio of oil G to n-hexane is 1:0.2; the mass ratio of oil G to ethanol is 1:3; the mass ratio of oil G to sodium hydroxide is 1:0.01; the esterification reaction temperature is 80 °C; the esterification reaction time is 5 hours;

[0091] Vacuum distillation: Place the esterification reaction solution in a distiller for vacuum distillation, collect the heavy fraction, and make oil H; the temperature of the vacuum distillation is 60 °C, the vacuum distillation pressure is 5 Pa, and the vacuum distillation time is 3 hours;

[0092] Treatment with stannous chloride: Add stannous chloride to oil H for reduction reaction to make oil I; the mass ratio of stannous chloride to oil H is 1:100; the reduction reaction temperature is 20 °C, and the reduction reaction time is 40 minutes;

[0093] Treatment with basic alumina: Add basic alumina to oil I to adsorb free fatty acids, then filter and collect the filtrate to make oil J; the mass ratio of oil I to basic alumina is 1:0.1; the adsorption temperature is 50 °C; the adsorption time is 20 minutes; the basic alumina is made by soaking alumina in sodium hydroxide, washing, and drying;

[0094] Silver nitrate complexation reaction: Add an aqueous silver nitrate solution to oil J for complexation reaction, then let it stand for stratification and collect the lower aqueous phase; add cyclohexane to the aqueous phase to extract the oil, then let it stand for stratification, collect the upper layer to make oil KⅠ, and collect the lower layer to make the silver nitrate recycling solution; the mass fraction of the aqueous silver nitrate solution is 50%; the mass ratio of oil J to the aqueous silver nitrate solution is 1:4; the complexation reaction temperature is 8 °C; the complexation reaction time is 20 minutes; the extraction temperature is 40 °C; the extraction time is 30 minutes;

[0095] Molecular fractionation: Place oil KⅠ in a molecular fractionator for fractionation, and collect the distillate and the heavy fraction respectively. The distillate is unsaturated fatty acid ethyl ester Ⅰ; the flow rate of oil D during fractionation is 100 kg / h / m 2 , the fractionation temperature is 150 °C, and the fractionation pressure is 20 Pa.

[0096] Repeat the preparation of unsaturated fatty acid ethyl ester Ⅰ 40 times, and reuse the silver nitrate recycling solution 40 times. Each time, adjust the mass fraction of silver nitrate in the silver nitrate recycling solution to 50%.

[0097] Preparation of unsaturated fatty acid ethyl ester Ⅱ:

[0098] Esterification reaction: Add n-hexane, ethanol, and sodium hydroxide to oil G for esterification reaction; the mass ratio of oil G to n-hexane is 1:0.2; the mass ratio of oil G to ethanol is 1:3; the mass ratio of oil G to sodium hydroxide is 1:0.01; the esterification reaction temperature is 80 °C; the esterification reaction time is 5 hours;

[0099] Vacuum distillation: Place the esterification reaction solution in a distiller for vacuum distillation, collect the heavy fraction, and make it into oil H; the temperature of the vacuum distillation is 60 °C, the vacuum distillation pressure is 5 Pa, and the vacuum distillation time is 3 hours;

[0100] Tin(II) chloride treatment: Add tin(II) chloride to oil H for reduction reaction to make oil I; the mass ratio of tin(II) chloride to oil H is 1:100; the reduction reaction temperature is 20 °C, and the reduction reaction time is 40 minutes;

[0101] Aluminum oxide treatment: Add aluminum oxide to oil I to adsorb free fatty acids, then filter and collect the filtrate to make oil J; the mass ratio of oil I to aluminum oxide is 1:0.1; the adsorption temperature is 50 °C; the adsorption time is 20 minutes;

[0102] Silver nitrate complexation reaction: Add an aqueous silver nitrate solution to oil J for complexation reaction, then let it stand for stratification and collect the lower aqueous phase; add cyclohexane to the aqueous phase to extract the oil, then let it stand for stratification, collect the upper layer to make oil KⅡ, and collect the lower layer to make the silver nitrate recycling solution; the mass fraction of the aqueous silver nitrate solution is 50%; the mass ratio of oil J to the aqueous silver nitrate solution is 1:4; the complexation reaction temperature is 8 °C; the complexation reaction time is 20 minutes; the extraction temperature is 40 °C; the extraction time is 30 minutes;

[0103] Molecular fractionation: Place oil KⅡ in a molecular fractionator for fractionation, and collect the distillate and heavy fraction respectively. The distillate is unsaturated fatty acid ethyl ester Ⅱ; the flow rate of oil D during fractionation is 100 kg / h / m 2 , the fractionation temperature is 150 °C, and the fractionation pressure is 20 Pa.

[0104] Repeat the preparation of unsaturated fatty acid ethyl ester Ⅱ 40 times, and repeat the use of the silver nitrate recycling solution 40 times. Each time, adjust the mass fraction of silver nitrate in the silver nitrate recycling solution to 50%.

[0105] Preparation of unsaturated fatty acid ethyl ester Ⅲ:

[0106] Esterification reaction: Add n-hexane, ethanol, and sodium hydroxide to oil G for esterification reaction; the mass ratio of oil G to n-hexane is 1:0.2; the mass ratio of oil G to ethanol is 1:3; the mass ratio of oil G to sodium hydroxide is 1:0.01; the esterification reaction temperature is 80 °C; the esterification reaction time is 5 hours;

[0107] Vacuum distillation: Place the esterification reaction solution in a distiller for vacuum distillation, collect the heavy fraction, and make oil H; the temperature of the vacuum distillation is 60 °C, the vacuum distillation pressure is 5 Pa, and the vacuum distillation time is 3 hours;

[0108] Treatment with stannous chloride: Add stannous chloride to oil H for reduction reaction to make oil I; the mass ratio of stannous chloride to oil H is 1:100; the reduction reaction temperature is 20 °C, and the reduction reaction time is 40 minutes;

[0109] Silver nitrate complexation reaction: Add an aqueous silver nitrate solution to oil I for complexation reaction, then let it stand for stratification, and collect the lower aqueous phase; add cyclohexane to the aqueous phase to extract the oil, then let it stand for stratification, collect the upper layer to make oil KⅢ, and collect the lower layer to make the silver nitrate recycling solution; the mass fraction of the aqueous silver nitrate solution is 50%; the mass ratio of oil I to the aqueous silver nitrate solution is 1:4; the complexation reaction temperature is 8 °C; the complexation reaction time is 20 minutes; the extraction temperature is 40 °C; the extraction time is 30 minutes;

[0110] Molecular fractionation: Place oil KⅢ in a molecular fractionator for fractionation, and collect the distillate and heavy fraction respectively. The distillate is ethyl unsaturated fatty acid Ⅲ; the flow rate of oil D during fractionation is 100 kg / h / m 2 , the fractionation temperature is 150 °C, and the fractionation pressure is 20 Pa.

[0111] Repeat the preparation of ethyl unsaturated fatty acid Ⅲ 40 times, and reuse the silver nitrate recycling solution 40 times. Each time, adjust the mass fraction of silver nitrate in the silver nitrate recycling solution to 50%.

[0112] Preparation of ethyl unsaturated fatty acid Ⅳ:

[0113] Esterification reaction: Add n-hexane, ethanol, and sodium hydroxide to oil G for esterification reaction; the mass ratio of oil G to n-hexane is 1:0.2; the mass ratio of oil G to ethanol is 1:3; the mass ratio of oil G to sodium hydroxide is 1:0.01; the esterification reaction temperature is 80 °C; the esterification reaction time is 5 hours;

[0114] Vacuum distillation: Place the esterification reaction solution in a distiller for vacuum distillation, collect the heavy fraction, and make it into oil H; the temperature of the vacuum distillation is 60 °C, the vacuum distillation pressure is 5 Pa, and the vacuum distillation time is 3 hours;

[0115] Silver nitrate complexation reaction: Add an aqueous silver nitrate solution to oil H for complexation reaction, then let it stand for stratification, and collect the lower aqueous phase; add cyclohexane to the aqueous phase to extract the oil, then let it stand for stratification, collect the upper layer to make oil KⅣ, and collect the lower layer to make the silver nitrate recycling solution; the mass fraction of the aqueous silver nitrate solution is 50%; the mass ratio of oil H to the aqueous silver nitrate solution is 1:4; the temperature of the complexation reaction is 8 °C; the complexation reaction time is 20 minutes; the extraction temperature is 40 °C; the extraction time is 30 minutes;

[0116] Molecular fractionation: Place oil KⅣ in a molecular fractionator for fractionation, and collect the distillate and the heavy fraction respectively. The distillate is unsaturated fatty acid ethyl ester Ⅳ; the flow rate of oil D during fractionation is 100 kg / h / m 2 , the fractionation temperature is 150 °C, and the fractionation pressure is 20 Pa.

[0117] Repeat the preparation of unsaturated fatty acid ethyl ester Ⅳ 40 times, and repeat the use of the silver nitrate recycling solution 40 times. Each time, adjust the mass fraction of silver nitrate in the silver nitrate recycling solution to 50%.

[0118] 2. Detection items:

[0119] Use gas chromatography to determine the DHA mass fraction of unsaturated fatty acid ethyl ester Ⅰ, unsaturated fatty acid ethyl ester Ⅱ, unsaturated fatty acid ethyl ester Ⅲ, and unsaturated fatty acid ethyl ester Ⅳ prepared for the 1st, 10th, 20th, 30th, and 40th times.

[0120] 3. Results and analysis:

[0121] In this experiment, the DHA mass fraction in the starting oil G of the raw material was 74%. The detection results of the DHA mass fraction in the unsaturated fatty acid ethyl ester are shown in the following table. The results show that: (1) When stannous chloride treatment is combined with basic alumina treatment, the silver nitrate recycling solution can still maintain a very high ability to separate unsaturated fatty acids after being reused 40 times; (2) Compared with alumina treatment, basic alumina treatment has a better effect on increasing the number of times the silver nitrate recycling solution can be used; (3) Using stannous chloride treatment, basic alumina treatment, and alumina treatment alone can increase the number of times the silver nitrate recycling solution can be used; (4) Using stannous chloride treatment alone can increase the DHA mass fraction of unsaturated fatty acid ethyl ester.

[0122] Table 3 Determination results of DHA mass fraction in the silver nitrate recycling experiment

[0123]

[0124]

[0125] Autoxidation of oils and fats produces peroxides. The peroxy bond (-O-O-) in peroxides breaks, forming a complex with Ag + and affecting the complexation reaction between silver nitrate and unsaturated fatty acids. Treatment with stannous chloride can reduce the peroxides in oils and fats and lower the peroxide value. On the one hand, it reduces the impact of peroxides on the complexation reaction between silver nitrate and unsaturated fatty acids, increasing the mass fraction of DHA in unsaturated fatty acids; on the other hand, it reduces the impact of peroxides on the quality of the silver nitrate circulating solution, increasing the number of times the silver nitrate solution can be recycled.

[0126] Compared with alumina, basic alumina has more abundant surface hydroxyl groups (-OH) and basic sites, which can interact with the carboxyl groups (-COOH) of free fatty acids, thereby improving the effect of adsorbing free fatty acids, reducing the free fatty acids in the recycled silver nitrate solution, and increasing the number of times the recycled silver nitrate solution can be used.

[0127] Test Example 4, Peroxide Value Test:

[0128] 1. Preparation of test samples:

[0129] Preparation of ethyl unsaturated fatty acid Ⅳ-1:

[0130] According to the method for preparing ethyl unsaturated fatty acid Ⅳ in Test Example 3, before adding the aqueous silver nitrate solution, adjust the peroxide value of oil H to 1 meq / kg to prepare ethyl unsaturated fatty acid Ⅳ-1. Repeat the preparation of ethyl unsaturated fatty acid Ⅳ-1 10 times, and each time adjust the mass fraction of the recycled silver nitrate solution to 50%, and the recycled silver nitrate solution is reused 10 times.

[0131] Preparation of ethyl unsaturated fatty acid Ⅳ-3:

[0132] According to the method for preparing ethyl unsaturated fatty acid Ⅳ in Test Example 3, before adding the aqueous silver nitrate solution, adjust the peroxide value of oil H to 3 meq / kg to prepare ethyl unsaturated fatty acid Ⅳ-3. Repeat the preparation of ethyl unsaturated fatty acid Ⅳ-3 10 times, and each time adjust the mass fraction of the recycled silver nitrate solution to 50%, and the recycled silver nitrate solution is reused 10 times.

[0133] Preparation of ethyl unsaturated fatty acid Ⅳ-5:

[0134] According to the method for preparing ethyl unsaturated fatty acid Ⅳ in Test Example 3, before adding the aqueous silver nitrate solution, the peroxide value of oil H was adjusted to 5 meq / kg to prepare ethyl unsaturated fatty acid Ⅳ-5. Ethyl unsaturated fatty acid Ⅳ-5 was prepared 10 times repeatedly. Each time, the mass fraction of the recycled silver nitrate solution was adjusted to 50%, and the recycled silver nitrate solution was reused 10 times.

[0135] Preparation of ethyl unsaturated fatty acid Ⅳ-10:

[0136] According to the method for preparing ethyl unsaturated fatty acid Ⅳ in Test Example 3, before adding the aqueous silver nitrate solution, the peroxide value of oil H was adjusted to 10 meq / kg to prepare ethyl unsaturated fatty acid Ⅳ-10. Ethyl unsaturated fatty acid Ⅳ-10 was prepared 10 times repeatedly. Each time, the mass fraction of the recycled silver nitrate solution was adjusted to 50%, and the recycled silver nitrate solution was reused 10 times.

[0137] Preparation of ethyl unsaturated fatty acid Ⅳ-20:

[0138] According to the method for preparing ethyl unsaturated fatty acid Ⅳ in Test Example 3, before adding the aqueous silver nitrate solution, the peroxide value of oil H was adjusted to 20 meq / kg to prepare ethyl unsaturated fatty acid Ⅳ-20. Ethyl unsaturated fatty acid Ⅳ-20 was prepared 10 times repeatedly. Each time, the mass fraction of the recycled silver nitrate solution was adjusted to 50%, and the recycled silver nitrate solution was reused 10 times.

[0139] 2. Detection items:

[0140] The DHA mass fraction of ethyl unsaturated fatty acid Ⅳ-1, ethyl unsaturated fatty acid Ⅳ-3, ethyl unsaturated fatty acid Ⅳ-5, ethyl unsaturated fatty acid Ⅳ-10, and ethyl unsaturated fatty acid Ⅳ-20 in the first and tenth times was determined by gas chromatography.

[0141] The peroxide value of oil H was determined by the iodometric method.

[0142] 3. Results and analysis:

[0143] The applicant unexpectedly found in the experiment that when the peroxide value of the oil during the silver nitrate complexation reaction was controlled below 5 meq / kg, the number of times the recycled silver nitrate solution could be used could be extended; when the recycled silver nitrate solution was used 10 times, the DHA mass fraction of the ethyl unsaturated fatty acid was still not less than 99.5%. When the peroxide value during the silver nitrate complexation reaction was controlled above 5 meq / kg, when the recycled silver nitrate solution was used 10 times, the DHA mass fraction of the ethyl unsaturated fatty acid was lower than 90%, which had a greater impact on the number of times the recycled silver nitrate solution could be used.

[0144] Table 4 Determination results of DHA mass fraction in the peroxide value test

[0145]

[0146] Test Example 5, Acid Value Test:

[0147] 1. Preparation of test samples:

[0148] Prepare ethyl unsaturated fatty acid ester Ⅳ-0.1:

[0149] According to the method for preparing ethyl unsaturated fatty acid ester Ⅳ in Test Example 3, before adding the silver nitrate aqueous solution, adjust the acid value of oil H to 0.1 meq / g to prepare ethyl unsaturated fatty acid ester Ⅳ-0.1. Repeat the preparation of ethyl unsaturated fatty acid ester Ⅳ-0.1 ten times. Each time, adjust the mass fraction of the recycled silver nitrate solution to 50%, and the recycled silver nitrate solution is reused ten times.

[0150] Prepare ethyl unsaturated fatty acid ester Ⅳ-0.2:

[0151] According to the method for preparing ethyl unsaturated fatty acid ester Ⅳ in Test Example 3, before adding the silver nitrate aqueous solution, adjust the acid value of oil H to 0.2 meq / g to prepare ethyl unsaturated fatty acid ester Ⅳ-0.2. Repeat the preparation of ethyl unsaturated fatty acid ester Ⅳ-0.2 ten times. Each time, adjust the mass fraction of the recycled silver nitrate solution to 50%, and the recycled silver nitrate solution is reused ten times.

[0152] Prepare ethyl unsaturated fatty acid ester Ⅳ-0.5:

[0153] According to the method for preparing ethyl unsaturated fatty acid ester Ⅳ in Test Example 3, before adding the silver nitrate aqueous solution, adjust the acid value of oil H to 0.5 meq / g to prepare ethyl unsaturated fatty acid ester Ⅳ-0.5. Repeat the preparation of ethyl unsaturated fatty acid ester Ⅳ-0.5 ten times. Each time, adjust the mass fraction of the recycled silver nitrate solution to 50%, and the recycled silver nitrate solution is reused ten times.

[0154] Prepare ethyl unsaturated fatty acid ester Ⅳ-0.9:

[0155] According to the method for preparing ethyl unsaturated fatty acid ester Ⅳ in Test Example 3, before adding the silver nitrate aqueous solution, adjust the acid value of oil H to 0.9 meq / g to prepare ethyl unsaturated fatty acid ester Ⅳ-0.9. Repeat the preparation of ethyl unsaturated fatty acid ester Ⅳ-0.9 ten times. Each time, adjust the mass fraction of the recycled silver nitrate solution to 50%, and the recycled silver nitrate solution is reused ten times.

[0156] 2. Detection items:

[0157] The DHA mass fractions of ethyl esters of unsaturated fatty acids Ⅳ-0.1, ethyl esters of unsaturated fatty acids Ⅳ-0.2, ethyl esters of unsaturated fatty acids Ⅳ-0.5, and ethyl esters of unsaturated fatty acids Ⅳ-0.9 in the 1st and 10th times were determined by gas chromatography.

[0158] The acid value was determined by potassium hydroxide titration.

[0159] 3. Results and analysis:

[0160] The applicant unexpectedly found in the experiment that when the acid value of the oil and fat during the silver nitrate complexation reaction was controlled below 0.2 meq / g, the number of times the recycled silver nitrate solution could be used could be extended; after the recycled silver nitrate solution was used 10 times, the DHA mass fraction of the ethyl esters of unsaturated fatty acids was still not lower than 99.7%. When the acid value during the silver nitrate complexation reaction was higher than 0.2 meq / kg, after the recycled silver nitrate solution was used 10 times, the DHA mass fraction of the ethyl esters of unsaturated fatty acids was lower than 90%, which had a greater impact on the number of times the recycled silver nitrate solution could be used.

[0161] Table 5 Results of the determination of the DHA mass fraction in the acid value test

[0162]

[0163]

[0164] The reason why the acid value of the oil and fat affects the number of times the recycled silver nitrate solution can be used is not clear at present. The applicant speculated that the carboxyl group (-COOH) of the free fatty acid has a certain polarity and may coordinate with silver nitrate, interfering with the complexation reaction.

[0165] Test Example 6, Immobilized Lipase Treatment Test:

[0166] 1. Preparation of test samples:

[0167] Preparation of ethyl esters of very long chain polyunsaturated fatty acids 1:

[0168] Treatment of the bottom heavy fraction with immobilized lipase: The bottom heavy fraction was added with immobilized lipase Lipozyme 435 and glycerol for transesterification reaction; the reaction temperature was 60 °C and the reaction time was 30 hours; the mass ratio of the bottom heavy fraction to lipase Lipozyme 435 was 20:1; the lipase was removed by filtration to obtain oil and fat L;

[0169] Molecular fractionation: The oil and fat L was fractionated in a molecular fractionator, and the distillate was collected to obtain ethyl esters of very long chain polyunsaturated fatty acids 1; the flow rate of the oil and fat L during fractionation was 60 kg / h / m 2 , the fractionation temperature was 170 °C, and the fractionation pressure was 10 Pa.

[0170] Preparation of ethyl esters of very long-chain polyunsaturated fatty acids 2: The bottom heavy fraction was directly subjected to molecular fractionation without treatment with immobilized lipase.

[0171] 2. Detection items:

[0172] Measure the mass fraction of polyunsaturated fatty acid ethyl esters with 24 or more carbon atoms in the heavy fraction, ethyl esters of very long-chain polyunsaturated fatty acids 1, and ethyl esters of very long-chain polyunsaturated fatty acids 2.

[0173] Measure the total cholesterol residue in the heavy fraction, ethyl esters of very long-chain polyunsaturated fatty acids 1, and ethyl esters of very long-chain polyunsaturated fatty acids 2 according to AOAC Official Method 994.1.

[0174] 3. Results and analysis:

[0175] The detection results are shown in the following table.

[0176] Compared with ethyl esters of very long-chain polyunsaturated fatty acids 2 without treatment with immobilized lipase, the mass fraction of polyunsaturated fatty acid ethyl esters with 24 or more carbon atoms in ethyl esters of very long-chain polyunsaturated fatty acids 1 was significantly increased. The difference in volatility between cholesterol and ethyl esters of very long-chain polyunsaturated fatty acids is small, and the separation difficulty during molecular fractionation is large. While the difference in volatility between cholesterol glyceride and ethyl esters of very long-chain polyunsaturated fatty acids is large, and the separation difficulty during molecular fractionation is small. Through the treatment conditions of immobilized lipase formulated in the present invention, free cholesterol can be converted into cholesterol glyceride, while ethyl esters of very long-chain polyunsaturated fatty acids rarely combine with glycerol and still remain in the ethyl ester state. Then, by utilizing the difference in volatility between cholesterol glyceride and ethyl esters of very long-chain polyunsaturated fatty acids, cholesterol glyceride is removed by molecular fractionation, while increasing the mass fraction of ethyl esters of very long-chain polyunsaturated fatty acids and reducing the total cholesterol residue.

[0177] The content of free cholesterol in the heavy fraction is relatively high, and its commercial value is relatively low, and it is generally discarded. However, such residues contain relatively more polyunsaturated fatty acids with 24 or more carbon atoms. Some studies have shown that very long-chain polyunsaturated fatty acids have important physiological functions and are beneficial to the functions of eyes and brain tissues as well as male fertility. High-purity very long-chain polyunsaturated fatty acids have important value for scientific research and dietary health.

[0178] Table 6 Results of the experiment on treatment with immobilized lipase

[0179]

[0180] Example 1:

[0181] Step S1, saponification reaction: Add sodium hydroxide solution to microalgae crude oil for saponification reaction, and the reaction solution is oil A; the reaction temperature is 60 °C and the reaction time is 1 hour; the sodium hydroxide solution is an aqueous sodium hydroxide solution with a mass fraction of 20%; the mass ratio of the microalgae crude oil to the sodium hydroxide solution is 1:2.

[0182] Step S2, primary acidification: While stirring, add citric acid solution to oil A to adjust the pH value of oil A to 2, let it stand for stratification and take the oil phase to make oil B; the mass concentration of the citric acid solution is 50%, and the stirring time is 20 minutes.

[0183] Step S3, tromethamine treatment: Add tromethamine solution to oil B, disperse evenly and then extract, take the aqueous phase after extraction to make solution C; the tromethamine solution is an aqueous tromethamine solution with a mass fraction of 10%; the mass ratio of oil B to the tromethamine solution is 1:2; the extraction time is 3 hours.

[0184] Step S4, secondary acidification: While stirring, add citric acid solution to solution C to adjust the pH value of solution C to 2, let it stand for stratification and take the oil phase to make oil D; the mass concentration of the citric acid solution is 50%, and the stirring time is 20 minutes.

[0185] Step S5, urea inclusion reaction: Dissolve urea in ethanol to make a urea solution; add oil D to the urea solution for inclusion reaction; filter and collect the filtrate, wash the filtrate with purified water, and take the oil phase to make oil E; the inclusion reaction temperature is 5 °C, the inclusion reaction time is 8 hours; the mass ratio of oil D to the urea solution is 1:3.

[0186] Step S6, magnesium acetate precipitation: Disperse magnesium acetate in acetone to make solution F; stir and mix oil E and solution F, then let it stand for precipitation reaction; filter to remove the precipitate, collect the filtrate to make oil G; the temperature of the precipitation reaction is -15 °C, the precipitation reaction time is 10 hours; the mass ratio of acetone to magnesium acetate is 1:0.04.

[0187] Step S7, esterification reaction: Add n-hexane, ethanol, and sodium hydroxide to oil G for esterification reaction; the mass ratio of oil G to n-hexane is 1:0.2; the mass ratio of oil G to ethanol is 1:3; the mass ratio of oil G to sodium hydroxide is 1:0.01; the esterification reaction temperature is 80 °C; the esterification reaction time is 5 hours.

[0188] Step S8, vacuum distillation: Place the esterification reaction solution in step S7 in a distiller for vacuum distillation, collect the heavy fraction to make oil H; the temperature of the vacuum distillation is 60 °C, the vacuum distillation pressure is 5 Pa, and the vacuum distillation time is 3 hours.

[0189] Step S9, stannous chloride treatment: Stannous chloride is added to oil H for a reduction reaction to produce oil I; the mass ratio of stannous chloride to oil H is 1:100; the reduction reaction temperature is 20°C, and the reduction reaction time is 40 minutes.

[0190] Step S10, basic alumina treatment: Basic alumina is added to oil I to adsorb free fatty acids, and then the filtrate is collected by filtration to produce oil J; the mass ratio of oil I to basic alumina is 1:0.1; the adsorption temperature is 50°C; the adsorption time is 20 minutes; the basic alumina is made by soaking alumina in sodium hydroxide, washing, and drying.

[0191] Step S11, silver nitrate complexation reaction: An aqueous silver nitrate solution is added to oil J for a complexation reaction, and then it is allowed to stand for stratification to collect the lower aqueous phase; cyclohexane is added to the aqueous phase to extract the oil, and then it is allowed to stand for stratification to collect the upper layer to produce oil K and the lower layer to produce a silver nitrate recycling solution; the mass fraction of the aqueous silver nitrate solution is 50%; the mass ratio of oil J to the aqueous silver nitrate solution is 1:4; the complexation reaction temperature is 8°C; the complexation reaction time is 20 minutes; the extraction temperature is 40°C; the extraction time is 30 minutes.

[0192] Step S12, molecular fractionation: Oil K is placed in a molecular fractionator for fractionation, and the distillate and heavy fraction are collected separately. The distillate is the ethyl ester of unsaturated fatty acids; the flow rate of oil K during fractionation is 100 kg / h / m 2 , the fractionation temperature is 150°C, and the fractionation pressure is 20 Pa.

[0193] Step S13, treatment of the heavy fraction with immobilized lipase: The bottom heavy fraction is added with immobilized lipase and glycerol for an enzymatic reaction; the lipase is removed by filtration to produce oil L; the immobilized lipase is Lipozyme 435; the reaction temperature is 60°C, and the reaction time is 30 hours; the mass ratio of the bottom heavy fraction to lipase Lipozyme 435 is 20:1.

[0194] Step S14, molecular fractionation: Oil L is placed in a molecular fractionator for fractionation, and the distillate is collected to produce the ethyl ester of very long chain polyunsaturated fatty acids; the flow rate of oil L during fractionation is 60 kg / h / m 2 , the fractionation temperature is 170°C, and the fractionation pressure is 10 Pa.

[0195] The preparation of ethyl ester of unsaturated fatty acids is repeated 40 times, and each time the mass fraction of the silver nitrate recycling solution is adjusted to 50%, and the silver nitrate recycling solution is reused 40 times.

[0196] Example 2:

[0197] Step S1, Saponification reaction: Add sodium hydroxide solution to microalgae crude oil for saponification reaction, and the reaction solution is oil A; the reaction temperature is 65 °C and the reaction time is 2 hours; the sodium hydroxide solution is an aqueous solution of sodium hydroxide with a mass fraction of 25%; the mass ratio of the microalgae crude oil to the sodium hydroxide solution is 1:3.

[0198] Step S2, First acidification: While stirring, add citric acid solution to oil A to adjust the pH value of oil A to 2.5, let it stand for stratification and take the oil phase to make oil B; the mass concentration of the citric acid solution is 50%, and the stirring time is 30 minutes.

[0199] Step S3, Tromethamine treatment: Add tromethamine solution to oil B, disperse evenly and then extract, take the aqueous phase after extraction to make solution C; the tromethamine solution is an aqueous solution of tromethamine with a mass fraction of 20%; the mass ratio of oil B to the tromethamine solution is 1:3; the extraction time is 4 hours.

[0200] Step S4, Second acidification: While stirring, add citric acid solution to solution C to adjust the pH value of solution C to 2.5, let it stand for stratification and take the oil phase to make oil D; the mass concentration of the citric acid solution is 50%, and the stirring time is 30 minutes.

[0201] Step S5, Urea inclusion reaction: Dissolve urea in ethanol to make a urea solution; add oil D to the urea solution for inclusion reaction; filter and collect the filtrate, wash the filtrate with purified water, and take the oil phase to make oil E; the inclusion reaction temperature is 6 °C and the inclusion reaction time is 9 hours; the mass ratio of oil D to the urea solution is 1:4.

[0202] Step S6, Magnesium acetate precipitation: Disperse magnesium acetate in acetone to make solution F; stir and mix oil E and solution F, then let it stand for precipitation reaction; filter to remove the precipitate and collect the filtrate to make oil G; the temperature of the precipitation reaction is -20 °C and the precipitation reaction time is 15 hours; the mass ratio of acetone to magnesium acetate is 1:0.05.

[0203] Step S7, Esterification reaction: Add n-hexane, ethanol, and sodium hydroxide to oil G for esterification reaction; the mass ratio of oil G to n-hexane is 1:0.3; the mass ratio of oil G to ethanol is 1:5; the mass ratio of oil G to sodium hydroxide is 1:0.02; the esterification reaction temperature is 85 °C; the esterification reaction time is 6 hours.

[0204] Step S8, Vacuum distillation: Place the esterification reaction solution in step S7 in a distiller for vacuum distillation, collect the heavy fraction to make oil H; the temperature of the vacuum distillation is 80 °C, the vacuum distillation pressure is 7 Pa, and the vacuum distillation time is 4 hours.

[0205] Step S9, stannous chloride treatment: Add stannous chloride to oil and fat H for a reduction reaction to produce oil and fat I; the mass ratio of stannous chloride to oil and fat H is 1:150; the reduction reaction temperature is 25°C, and the reduction reaction time is 50 minutes.

[0206] Step S10, basic alumina treatment: Add basic alumina to oil and fat I to adsorb free fatty acids, then filter to collect the filtrate to produce oil and fat J; the mass ratio of oil and fat I to basic alumina is 1:0.15; the adsorption temperature is 60°C; the adsorption time is 25 minutes; the basic alumina is made by soaking alumina in sodium hydroxide, washing, and drying.

[0207] Step S11, silver nitrate complexation reaction: Add an aqueous silver nitrate solution to oil and fat J for a complexation reaction, then let it stand for stratification and collect the lower aqueous phase; add cyclohexane to the aqueous phase to extract the oil and fat, then let it stand for stratification, collect the upper layer to produce oil and fat K, and collect the lower layer to produce a silver nitrate recycling solution; the mass fraction of the aqueous silver nitrate solution is 50% - 60%; the mass ratio of oil and fat J to the aqueous silver nitrate solution is 1:5; the complexation reaction temperature is 9°C; the complexation reaction time is 25 minutes; the extraction temperature is 50°C; the extraction time is 35 minutes.

[0208] Step S12, molecular fractionation: Place oil and fat K in a molecular fractionator for fractionation, and collect the distillate and heavy fraction respectively. The distillate is ethyl unsaturated fatty acid; the flow rate of oil and fat K during fractionation is 110 kg / h / m 2 , the fractionation temperature is 160°C, and the fractionation pressure is 40 Pa.

[0209] Step S13, treatment of the heavy fraction with immobilized lipase: Add immobilized lipase and glycerol to the bottom heavy fraction for an enzymatic reaction; filter to remove the lipase to produce oil and fat L; the immobilized lipase is Lipozyme 435; the reaction temperature is 70°C, and the reaction time is 35 hours; the mass ratio of the bottom heavy fraction to lipase Lipozyme 435 is 20:1.5.

[0210] Step S14, molecular fractionation: Place oil and fat L in a molecular fractionator for fractionation, and collect the distillate to produce ethyl very long chain polyunsaturated fatty acid; the flow rate of oil and fat L during fractionation is 70 kg / h / m 2 , the fractionation temperature is 175°C, and the fractionation pressure is 15 Pa.

[0211] Repeat the preparation of ethyl unsaturated fatty acid 40 times, and adjust the mass fraction of the silver nitrate recycling solution to 50% each time. The silver nitrate recycling solution is reused 40 times.

[0212] Example 3:

[0213] Step S1, saponification reaction: Add sodium hydroxide solution to microalgae crude oil for saponification reaction, and the reaction solution is oil A; the reaction temperature is 70 °C, and the reaction time is 3 hours; the sodium hydroxide solution is an aqueous sodium hydroxide solution with a mass fraction of 30%; the mass ratio of the microalgae crude oil to the sodium hydroxide solution is 1:4.

[0214] Step S2, primary acidification: While stirring, add citric acid solution to oil A to adjust the pH value of oil A to 3, let it stand for stratification and take the oil phase to make oil B; the mass concentration of the citric acid solution is 50%, and the stirring time is 40 minutes.

[0215] Step S3, tromethamine treatment: Add tromethamine solution to oil B, disperse evenly and then extract, take the aqueous phase after extraction to make solution C; the tromethamine solution is an aqueous tromethamine solution with a mass fraction of 30%; the mass ratio of oil B to the tromethamine solution is 1:4; the extraction time is 5 hours.

[0216] Step S4, secondary acidification: While stirring, add citric acid solution to solution C to adjust the pH value of solution C to 3, let it stand for stratification and take the oil phase to make oil D; the mass concentration of the citric acid solution is 50%, and the stirring time is 40 minutes.

[0217] Step S5, urea inclusion reaction: Dissolve urea in ethanol to make a urea solution; add oil D to the urea solution for inclusion reaction; filter and collect the filtrate, wash the filtrate with purified water, and take the oil phase to make oil E; the inclusion reaction temperature is 7 °C, and the inclusion reaction time is 10 hours; the mass ratio of oil D to the urea solution is 1:5.

[0218] Step S6, magnesium acetate precipitation: Disperse magnesium acetate in acetone to make solution F; stir and mix oil E and solution F, then let it stand for precipitation reaction; filter to remove the precipitate and collect the filtrate to make oil G; the temperature of the precipitation reaction is -25 °C, and the precipitation reaction time is 20 hours; the mass ratio of acetone to magnesium acetate is 1:0.06.

[0219] Step S7, esterification reaction: Add n-hexane, ethanol, and sodium hydroxide to oil G for esterification reaction; the mass ratio of oil G to n-hexane is 1:0.4; the mass ratio of oil G to ethanol is 1:6; the mass ratio of oil G to sodium hydroxide is 1:0.03; the esterification reaction temperature is 90 °C; the esterification reaction time is 7 hours.

[0220] Step S8, vacuum distillation: Place the esterification reaction solution in step S7 in a distiller for vacuum distillation, collect the heavy distillate to make oil H; the temperature of the vacuum distillation is 100 °C, the vacuum distillation pressure is 10 Pa, and the vacuum distillation time is 5 hours.

[0221] Step S9, stannous chloride treatment: Stannous chloride is added to oil H for a reduction reaction to produce oil I; the mass ratio of stannous chloride to oil H is 1:200; the reduction reaction temperature is 30 °C, and the reduction reaction time is 60 minutes.

[0222] Step S10, basic alumina treatment: Basic alumina is added to oil I to adsorb free fatty acids, and then the filtrate is collected by filtration to produce oil J; the mass ratio of oil I to basic alumina is 1:0.2; the adsorption temperature is 70 °C; the adsorption time is 30 minutes; the basic alumina is made by soaking alumina in sodium hydroxide, washing, and drying.

[0223] Step S11, silver nitrate complexation reaction: An aqueous silver nitrate solution is added to oil J for a complexation reaction, and then it is allowed to stand for liquid separation to collect the lower aqueous phase; cyclohexane is added to the aqueous phase to extract the oil, and then it is allowed to stand for liquid separation to collect the upper layer to produce oil K and the lower layer to produce a silver nitrate recycling solution; the mass fraction of the aqueous silver nitrate solution is 60%; the mass ratio of oil J to the aqueous silver nitrate solution is 1:6; the complexation reaction temperature is 10 °C; the complexation reaction time is 30 minutes; the extraction temperature is 60 °C; the extraction time is 40 minutes.

[0224] Step S12, molecular fractionation: Oil K is placed in a molecular fractionator for fractionation, and the distillate and heavy fraction are collected separately. The distillate is ethyl unsaturated fatty acid; the flow rate of oil K during fractionation is 120 kg / h / m 2 , the fractionation temperature is 180 °C, and the fractionation pressure is 50 Pa.

[0225] Step S13, immobilized lipase treatment of the heavy fraction: The bottom heavy fraction is added with immobilized lipase and glycerol for an enzymatic reaction; the lipase is removed by filtration to produce oil L; the immobilized lipase is Lipozyme 435; the reaction temperature is 80 °C, and the reaction time is 40 hours; the mass ratio of the bottom heavy fraction to lipase Lipozyme 435 is 20:2.

[0226] Step S14, molecular fractionation: Oil L is placed in a molecular fractionator for fractionation, and the distillate is collected to produce ethyl very long chain polyunsaturated fatty acid; the flow rate of oil L during fractionation is 80 kg / h / m 2 , the fractionation temperature is 180 °C, and the fractionation pressure is 20 Pa.

[0227] Ethyl unsaturated fatty acid is prepared 40 times repeatedly, and each time the mass fraction of the silver nitrate recycling solution is adjusted to 50%, and the silver nitrate recycling solution is reused 40 times.

[0228] The dioxin residues of ethyl esters of unsaturated fatty acids in Examples 1-3 were detected separately.

[0229] The total cholesterol residues and the total mass fraction of ethyl esters of very long-chain polyunsaturated fatty acids with 24 or more carbon atoms in Examples 1-3 were detected separately.

[0230] The DHA mass fraction of ethyl esters of unsaturated fatty acids prepared in the 1st, 10th, 20th, 30th, and 40th preparations of Examples 1-3 was determined by gas chromatography.

[0231] The test results are shown in the following table. The dioxin residues of ethyl esters of unsaturated fatty acids prepared by the technical solution of the present invention are extremely low, and the DHA mass fraction is relatively high; the total cholesterol residues of ethyl esters of very long-chain polyunsaturated fatty acids prepared are relatively low, and the total mass fraction of ethyl esters of polyunsaturated fatty acids with 24 or more carbon atoms is relatively high.

[0232] Under the conditions of the technical solution of the present invention, the recycled silver nitrate solution was recycled 40 times, and the quality of the recycled silver nitrate solution did not decrease significantly. When recycled 40 times, the DHA mass fraction of the prepared ethyl esters of unsaturated fatty acids was greater than 99.5%. It shows that the technical solution of the present invention can significantly extend the number of times of use of the recycled silver nitrate solution.

[0233] Table 7 Test results of dioxin residues of ethyl esters of unsaturated fatty acids in Examples 1-3

[0234] Example Sample Dioxin residue (TEQ ng / kg) Example 1 Polyunsaturated fatty acid ethyl ester 0.009 Example 2 Polyunsaturated fatty acid ethyl ester 0.008 Example 3 Polyunsaturated fatty acid ethyl ester 0.008

[0235] Table 8 Test results of ethyl esters of very long-chain polyunsaturated fatty acids in Examples 1-3

[0236]

[0237]

[0238] Table 9 Test results of DHA mass fraction of recycled silver nitrate in Examples 1-3

[0239]

Claims

1. A method for preparing unsaturated fatty acids, comprising the following steps: Step S1, saponification reaction: adding sodium hydroxide solution to the microalgae crude oil to carry out saponification reaction, and the reaction liquid is oil A; Step S2, primary acidification: while stirring, add citric acid solution to the oil A to adjust the pH value of the oil A to 2-3, let it stand for stratification and take the oil phase to prepare the oil B; Step S3, tromethamine treatment: adding tromethamine solution to oil B, dispersing evenly and then extracting, and taking the aqueous phase after extraction to prepare solution C; Step S4, secondary acidification: while stirring, add citric acid solution to solution C, adjust the pH value of solution C to 2-3, let stand to separate layers, take out the oil phase, and prepare oil D; Step S5, urea inclusion reaction: urea is dissolved in ethanol to prepare a urea solution; oil D is added to the urea solution to carry out an inclusion reaction; the filtrate is collected by filtration, the filtrate is washed with purified water, and the oil phase is taken to prepare oil E; Step S6, magnesium acetate precipitation: dispersing magnesium acetate in acetone to prepare solution F; stirring and mixing oil E and solution F, and standing to perform precipitation reaction; filtering to remove the precipitate, collecting the filtrate, and preparing oil G; Step S7, esterification reaction: adding n-hexane, ethanol and sodium hydroxide to the oil G to carry out esterification reaction; Step S8, vacuum distillation: placing the esterification reaction liquid of step S7 in a distiller for vacuum distillation, collecting the heavy fraction, and preparing oil H; Step S9, stannous chloride treatment: adding stannous chloride to the oil H for reduction reaction to prepare oil I; Step S10, alkaline alumina treatment: adding alkaline alumina to oil I to adsorb free fatty acids, and then filtering and collecting the filtrate to prepare oil J; Step S11, silver nitrate complex reaction: add silver nitrate aqueous solution to oil J for complex reaction, then stand for stratification, and collect the lower aqueous phase; add cyclohexane to the aqueous phase to extract the oil, then stand for stratification, collect the upper layer to make oil K, and collect the lower layer to make silver nitrate recycling liquid; Step S12, molecular fractionation: placing the oil K in a molecular fractionator for fractionation, and collecting the distillate and the heavy fraction respectively, wherein the distillate is the unsaturated fatty acid.

2. The preparation method according to claim 1, characterized in that: In step S1, the reaction temperature is 60° C. to 70° C., and the reaction time is 1 hour to 3 hours; the sodium hydroxide solution is a sodium hydroxide aqueous solution with a mass fraction of 20% to 30%; and the mass ratio of the microalgae crude oil to the sodium hydroxide solution is 1:(2 to 4).

3. The preparation method according to claim 2, characterized in that: The mass concentration of the citric acid solution in step S2 is 50%, and the stirring time is 20 minutes to 40 minutes.

4. The preparation method according to claim 3, characterized in that: The tromethamine solution in step S3 is a tromethamine aqueous solution with a mass fraction of 10% to 30%; the mass ratio of the oil B to the tromethamine solution is 1:(2 to 4); and the extraction time is 3 hours to 5 hours.

5. The preparation method according to claim 4, characterized in that: The mass concentration of the citric acid solution in step S4 is 50%, and the stirring time is 20 minutes to 40 minutes.

6. The preparation method according to claim 5, characterized in that: The inclusion reaction temperature in step S5 is 5° C. to 7° C., and the inclusion reaction time is 8 hours to 10 hours; the mass ratio of oil D to urea solution is 1:(3-5).

7. The preparation method according to claim 6, characterized in that: The temperature of the precipitation reaction in step S6 is -15°C to -25°C, and the time of the precipitation reaction is 10 hours to 20 hours; the mass ratio of acetone to magnesium acetate is 1:(0.04 to 0.06).

8. The preparation method according to claim 7, characterized in that: In step S7, the mass ratio of the oil G to n-hexane is 1:(0.2-0.4); the mass ratio of the oil G to ethanol is 1:(3-6); the mass ratio of the oil G to sodium hydroxide is 1:(0.01-0.03); the esterification reaction temperature is 80°C-90°C; and the esterification reaction time is 5 hours-7 hours.

9. An unsaturated fatty acid, characterized in that The unsaturated fatty acid is prepared by the method according to claim 1.

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