Preparation method and application of high alpha-linolenic acid

Abstract

The invention provides a preparation method and application of high alpha-linolenic acid, and relates to the technical field of medicines. Comprising the following steps: selecting purple perilla seeds, cleaning, drying, mechanically grinding in a protective atmosphere, and filtering to obtain purple perilla seed crude oil; carrying out degumming, deacidification, decoloration and dewaxing treatment on the crude perilla seed oil in a protective atmosphere to obtain primarily enriched crude alpha-linolenic acid oil; and respectively carrying out supercritical CO2 extraction, enzymatic molecular distillation and preparative chromatographic separation on the preliminarily enriched perilla seed oil to obtain the high alpha-linolenic acid. According to the method, purple perilla seeds are subjected to multi-stage treatment, extraction and refinement are performed layer by layer through three steps of supercritical CO2 extraction, enzymatic molecular distillation and preparative chromatographic separation, free fatty acid in purple perilla seed crude oil can be effectively removed, and alpha-linolenic acid is enriched to obtain high alpha-linolenic acid with the purity higher than 96%. The method is suitable for mass production in the medicine field.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a preparation method of high α-linolenic acid and application thereof. Background Art

[0002] α-Linolenic acid (ALA), found in green plants, is a member of the N-3 (also known as ω-3) unsaturated fatty acid family. It is essential for the human body and beneficial in preventing and treating coronary heart disease, diabetes, rheumatoid arthritis, dermatitis, depression, schizophrenia, dementia, allergies, asthma, kidney disease, and chronic obstructive pulmonary disease. A deficiency in ALA can disrupt lipid metabolism, leading to symptoms such as decreased immunity, forgetfulness, fatigue, vision loss, and atherosclerosis. A deficiency in ALA, particularly in infants and adolescents, can severely impact their intellectual and visual development. Once in the body, ALA is metabolized by dehydrogenases and carbon chain elongation enzymes to form EPA and DHA, precursors of prostaglandins, exhibiting a wide range of physiological activities and significant pharmacological effects.

[0003] Existing methods for enriching α-linolenic acid to high-α-linolenic acid include urea inclusion, molecular distillation, low-temperature crystallization, and chromatography. Currently, the urea inclusion method is the predominant method of choice in industry due to its relatively low cost. However, during use, not all α-linolenic acid is included, and a certain proportion of α-linolenic acid is always mixed into the mother liquor. This also limits the separation of α-linolenic acid and linoleic acid, which are both polyunsaturated fatty acids, resulting in a low final yield. Furthermore, the process requires the use of large amounts of organic solvents such as methanol and ethanol, posing significant challenges in solvent recovery and wastewater treatment.

[0004] Chinese patent CN105331648A discloses a high-purity α-linolenic acid monoglyceride and its preparation method, which uses the specific catalytic function of lipase to enrich α-linolenic acid into monoglyceride. However, the specific catalytic function of lipase alone produces a mixture of α-linolenic acid and free fatty acid (FFA) mixture, resulting in a low purity of α-linolenic acid.

[0005] In addition, since α-linolenic acid is a polyunsaturated fatty acid containing three double bonds, the hydrogen atoms on the methylene groups adjacent to the double bonds are very active and can be easily damaged by oxygen, light, high temperature, metal ions (such as Fe 2+ ,Cu 2+ ) and other factors trigger free radical chain reactions, leading to oxidative deterioration and the production of harmful substances such as peroxides, aldehydes, and ketones, which affect product quality and safety. Therefore, ensuring that α-linolenic acid is not oxidized during the enrichment process is a major challenge in the entire production process. Summary of the Invention

[0006] The present invention aims to provide a method for preparing high α-linolenic acid. The method comprises the following steps: multi-stage treatment of perilla seeds, and layer-by-layer extraction and refining through supercritical CO2 extraction, enzymatic molecular distillation, and preparative chromatography separation. The method can effectively remove free fatty acids from perilla seed crude oil and enrich α-linolenic acid to obtain high α-linolenic acid with a purity higher than 96%. The method is suitable for batch production in the pharmaceutical field.

[0007] The present invention solves the technical problem by adopting the following technical solutions.

[0008] On the one hand, the present invention provides a method for preparing high α-linolenic acid, characterized in that it comprises the following steps:

[0009] S1: Raw material pretreatment: Perilla seeds are selected, cleaned, dried, mechanically ground under a protective atmosphere, and then filtered to obtain perilla seed crude oil;

[0010] S2: crude oil refining, degumming, deacidification, decolorization, and dewaxing the perilla seed crude oil under a protective atmosphere to obtain a crude oil with preliminary enrichment of α-linolenic acid;

[0011] S3: The preliminarily enriched perilla seed oil is subjected to supercritical CO2 extraction, enzymatic molecular distillation, and preparative chromatography separation to obtain high α-linolenic acid.

[0012] Furthermore, in step S1, during the mechanical grinding process, phytic acid with a concentration of 0.01-0.05% needs to be added to the perilla seeds, and the mechanical grinding process needs to be carried out at 20-40°C; the filtration method is diatomaceous earth and / or ion membrane filtration.

[0013] Furthermore, in step S2, the degumming method adopts enzymatic degumming, the reaction time of the enzymatic degumming is 1-2 hours, and the reaction temperature is 40-60°C.

[0014] Furthermore, in step S2, the deacidification method is molecular distillation deacidification under a protective atmosphere, the distillation temperature is 120-150° C., the reaction time is 10-20 min, and the deacidified oil obtained after distillation is added with an adsorbent and rosemary extract, stirred for 20-40 min for decolorization, and then filtered to obtain the decolorized oil.

[0015] Furthermore, in step S2, the dewaxing method is to first slowly cool the decolorized oil to 5-10°C, keep it warm for 4-6 hours to crystallize it, further cool it to 0-4°C, keep it warm for 12-20 hours to enlarge the crystals; then compress and filter it at a temperature of 0-5°C to obtain liquid oil containing α-linolenic acid, and then immediately heat the liquid oil to 15-20°C to avoid secondary crystallization, thereby obtaining the above-mentioned α-linolenic acid crude oil.

[0016] Furthermore, in step S3, 5-10% ethanol and / or methanol-KOH solvent is first added to the above-mentioned α-linolenic acid crude oil, mixed and placed in a supercritical extraction kettle, and reacted for 1-3 hours under the conditions of pressure of 300-400 bar, temperature of 30-40°C, and CO2 flow rate of 20-40L / h to obtain α-linolenic acid primary essential oil.

[0017] Furthermore, in step S3, the above-mentioned α-linolenic acid primary essential oil is mixed at an oil-water ratio of 1:(0.5-0.8), and 2-5w% of immobilized lipase and 5-8w% of glycerol are added, and the mixture is stirred at 40-50°C for 6-8h to obtain an enzymatic hydrolyzate; the above-mentioned enzymatic hydrolyzate is then centrifuged, and the free fatty acid mixture after centrifugation is taken, ethanol is added at a ratio of 1:(1-1.5), and then a selective lipase with a mass fraction of 5-8w% is added, and the mixture is reacted at 30-40°C for 4-6h, and centrifuged again to obtain an α-linolenic acid-free fatty acid mixture;

[0018] Methanol is added to the above-mentioned α-linolenic acid-free fatty acid mixture at a ratio of 1:(1-3), and then concentrated sulfuric acid with a mass fraction of 2-5w% is added to carry out a catalytic reaction for 2-4 hours to obtain a fatty acid methyl ester mixture, which is then subjected to three short-path distillations to obtain α-linolenic acid secondary essential oil. A composite antioxidant is added to the α-linolenic acid secondary essential oil and preserved.

[0019] Furthermore, in step S3, first, n-hexane, acetonitrile, methanol and water are selected to prepare a solvent in a ratio of 8:5:5:1, the mixture is placed in a separatory funnel, shaken for 10-30 minutes, and statically separated into an upper phase stationary liquid and a lower phase mobile liquid. The two phases are then ultrasonically degassed for 20 minutes to separate the two phases, and injected into a preparative chromatography column instrument; then the above-mentioned α-linolenic acid secondary essential oil is injected into the upper phase stationary liquid to dissolve, and the instrument is started under the conditions of a set temperature of 20-25°C, a rotation speed of 1500-2200rpm, and a flow rate of 10-20ml / min. The sample components are injected and eluted in sequence, and the fractions rich in α-linolenic acid are collected; after collection, the fractions rich in α-linolenic acid are combined, water-bathed and concentrated under a protective atmosphere to obtain high α-linolenic acid, and then vitamin E is added to the high α-linolenic acid. After collection, the fractions are refrigerated and stored in the dark.

[0020] Furthermore, the protective atmosphere is nitrogen or argon, and the composite antioxidant is 0.01-0.02% by mass of tocopherol and 0.02-0.03% by mass of ascorbyl palmitate.

[0021] Another embodiment of the present invention provides an application of high α-linolenic acid, including preparing an oral emulsion rich in high α-linolenic acid, which contains high α-linolenic acid prepared by the preparation method provided in the embodiment of the present invention and is synthesized according to the following components by mass fraction: 10-20% high α-linolenic acid, 10-15% soy lecithin, 1-5% ethylene glycol, 0.1-2% tea polyphenols, 0.5-1% fragrance, and the balance is purified water.

[0022] Compared with the prior art, the embodiments of the present invention have at least the following advantages or beneficial effects:

[0023] 1. The present invention performs multi-stage processing on perilla seeds and extracts and refines them layer by layer through three steps: supercritical CO2 extraction, enzymatic molecular distillation, and preparative chromatography separation. The free fatty acids in the perilla seed crude oil can be effectively removed, and α-linolenic acid is enriched to obtain high α-linolenic acid with a purity higher than 96%, which is suitable for batch production in the pharmaceutical field.

[0024] 2. The present invention, by adding different antioxidants in various steps of production and preparation and operating under a protective atmosphere, can ensure that α-linolenic acid is not deteriorated due to oxidation throughout the entire process from raw material grinding to enrichment and preservation, thereby improving the quality and safety of the finished product of high α-linolenic acid. DETAILED DESCRIPTION

[0025] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0026] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to specific embodiments.

[0027] On the one hand, the present invention provides a method for preparing high α-linolenic acid, characterized in that it comprises the following steps:

[0028] S1: Raw material pretreatment: Perilla seeds are repeatedly washed at 10°C to remove impurities, then removed and air-dried, 0.01-0.05% phytic acid is added, and the seeds are placed in a grinding dish. The dish is then sealed and filled with nitrogen or argon, and fully ground at 20-40°C for 2-3 hours. After the seeds are completely ground, the residue is filtered out using diatomaceous earth and / or an ion membrane to obtain perilla seed crude oil;

[0029] S2: Crude oil refining, which needs to be carried out in a nitrogen-filled environment, includes:

[0030] First, the above-mentioned perilla seed crude oil is placed in a sealed environment filled with nitrogen, heated to 40-50°C, then 30-50ppm of phospholipase is added, and the pH is adjusted to 4.5-5.0 with 0.01-0.02% citric acid. After the reaction is allowed to stand for 1-2 hours, the temperature is raised to 75-80°C to inactivate the enzyme, and then centrifuged to obtain degummed oil. This step can remove phospholipids, colloids and trace amounts of Fe in the perilla seed crude oil. 2+ Mg 2+ ;

[0031] It should be noted that in the heating process, in order to avoid high temperature destroying the molecular structure of α-linolenic acid, the heating time should not be too long, and each heating time can be kept within 3-5 minutes. Citric acid can also be used as a metal chelating agent in this step to prevent high temperature catalytic oxidation of α-linolenic acid.

[0032] Furthermore, the degummed oil is placed in a short-range molecular distillation apparatus and distilled under a vacuum of less than 0.5 Pa and a distillation temperature of 120-150° C. This step evaporates some free fatty acids and low-boiling impurities into a condenser, and the α-linolenic acid mixed oil is collected as the distillate residue (deacidified oil). 1-2w% activated clay, 0.1-0.5w% activated carbon, and 0.1w% rosemary extract are then added to the deacidified oil and stirred for 20-40 minutes to adsorb pigments, phospholipids, polycyclic aromatic hydrocarbons, and odors in the deacidified oil. The rosemary extract protects the α-linolenic acid from oxidation during the stirring process. After stirring, the mixture is cooled to room temperature, and then the activated carbon and activated clay are filtered out through a plate and frame filter press. The resulting decolorized oil is then nitrogen-filled for protection.

[0033] It should be noted that in this step, since the distillation temperature is close to the heat resistance limit of α-linolenic acid, the distillation temperature cannot exceed 150°C, and the heating time needs to be precisely controlled, and each heating time cannot exceed 30 seconds.

[0034] The bleached oil is then subjected to a low-temperature treatment, slowly cooled (0.5°C / min) to 5-10°C and then maintained at this temperature for 4-6 hours to allow the bleached oil to crystallize. Once small crystals are observed, the temperature is further lowered to 0-4°C and maintained for 12-20 hours to allow the crystals to further grow. Once the crystals are in bulk, they are compressed and filtered using a plate-and-frame filter press with a cooling interlayer at 0-5°C. 0.1% diatomaceous earth may be used as a filter aid during the compression filtration process. The resulting filtrate is a liquid oil containing α-linolenic acid. Following compression filtration, the liquid oil is immediately heated to 15-20°C to prevent secondary crystallization caused by the low temperature, thereby obtaining crude α-linolenic acid oil. This step removes high-melting-point saturated fatty acid glycerides and waxes, significantly increasing the proportion of α-linolenic acid in the mixed oil.

[0035] S3: extracting and refining the above-mentioned crude α-linolenic acid oil through supercritical CO2 extraction, enzymatic molecular distillation, and preparative chromatography separation layer by layer, further removing free fatty acids in the crude α-linolenic acid oil and enriching α-linolenic acid, including:

[0036] First, add 5-10% ethanol and / or methanol-KOH solvent to the above-mentioned α-linolenic acid crude oil, stir thoroughly for 30 minutes, mix evenly, and then put it into a supercritical extraction kettle. React for 1-3 hours under the conditions of pressure of 300-400 bar, temperature of 30-40°C, and CO2 flow rate of 20-40 L / h to obtain α-linolenic acid primary essential oil.

[0037] Furthermore, the above-mentioned α-linolenic acid primary essential oil is mixed in an oil-water ratio of 1: (0.5-0.8), and 2-5w% of immobilized lipase (e.g. 435) and 5-8w% glycerol, placing them in a constant temperature stirring reactor, mixing and stirring at 40-50°C for 6-8h, thereby hydrolyzing the α-linolenic acid primary essential oil to obtain an enzymatic hydrolyzate; then placing the above enzymatic hydrolyzate in a disc centrifuge for centrifugation, and then standing to separate the layers, the upper layer being a free fatty acid mixture, and the lower layer being glycerol and water; taking the free fatty acid mixture after centrifugation, adding ethanol in a molar ratio of 1: (1-1.5), and then adding 5-8w% of a selective lipase (such as RM IM), react at 30-40℃ for 4-6h, and centrifuge again. The upper layer is a mixture of α-linolenic acid and free fatty acids, and the lower layer is the separated saturated fatty acids;

[0038] Methanol is added to the α-linolenic acid-free fatty acid mixture at a ratio of 1:(1-3), and concentrated sulfuric acid with a mass fraction of 2-5w% is added for a catalytic reaction for 2-4 hours to obtain a fatty acid methyl ester mixture, which is then subjected to three short-path distillations using a continuous molecular distillation unit to obtain an α-linolenic acid secondary essential oil, and 0.01-0.02% by mass of tocopherol and 0.02-0.03% by mass of ascorbyl palmitate are added to the α-linolenic acid secondary essential oil and stored.

[0039] It should be noted that this step requires nitrogen blanketing throughout to prevent oxidation of α-linolenic acid. Adding 5-8w% glycerol can further inhibit α-linolenic acid esterification. In addition, in this step, the three short-path distillations include:

[0040] ① Distill at 70°C and 100 Pa for 10 min to remove water, residual methanol and short-chain fatty acid esters from the fatty acid methyl ester mixture;

[0041] ② Distill at 100°C and 10 Pa for 5 minutes to further remove methyl oleate and methyl linoleate from the fatty acid methyl ester mixture;

[0042] ③ Distill multiple times at 150 degrees Celsius and 0.5 Pa, each distillation for 30 seconds, to obtain α-linolenic acid secondary essential oil.

[0043] Furthermore, a solvent is first prepared by selecting n-hexane, acetonitrile, methanol and water in a ratio of 8:5:5:1, and the mixture is placed in a separatory funnel and shaken for 10-30 minutes. The two phases are separated into an upper phase stationary liquid and a lower phase mobile liquid, and then ultrasonic degassing is performed for 20 minutes to separate the two phases and inject them into a preparative chromatographic column instrument; then, the α-linolenic acid secondary essential oil is injected into the upper phase stationary liquid and dissolved, and the instrument is started under the conditions of a set temperature of 20-25°C, a rotation speed of 1500-2200 rpm, and a flow rate of 10-20 ml / min. The sample components are sequentially injected and eluted to collect the α-linolenic acid-rich fraction; after collection, the above-mentioned α-linolenic acid-rich fractions are combined, and under vacuum conditions with nitrogen blanketing, a 10% mass fraction of KOH-ethanol solution is first added, and hydrolysis is carried out at 60°C for 1 hour. Then, concentrated HCl is added to adjust the pH to 2.0, and then ether is added and shaken for 1 hour to extract the remaining free fatty acids, and finally anhydrous Na2SO4 is added. 4 , washed with water several times and concentrated to obtain high α-linolenic acid, then vitamin E is added to the high α-linolenic acid, and after collection, it is refrigerated and stored away from light.

[0044] Optionally, this step can also be scaled up for industrial production. For example, a dynamic axial compression column coupling system can be used to achieve a throughput of 1 kg / day, enabling batch production.

[0045] Another embodiment of the present invention provides an oral emulsion rich in high α-linolenic acid, which contains high α-linolenic acid prepared by the preparation method provided in the embodiment of the present invention and is synthesized according to the following components in mass fraction: 10-20% high α-linolenic acid, 10-15% soy lecithin, 1-5% ethylene glycol, 0.1-2% tea polyphenols, 0.5-1% fragrance, and the balance is purified water.

[0046] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0047] Example 1

[0048] In this embodiment, according to the above experimental method, it is specifically manifested as follows:

[0049] 1) Selecting perilla seeds, repeatedly washing and removing impurities at 10° C., then fishing out, air-drying, and weighing, then adding 0.02% phytic acid, placing them together in a grinding dish, then sealing the dish, filling it with nitrogen, and grinding it thoroughly at 20° C. for 2 hours. After it is completely ground, filtering out the residue with an ion membrane to obtain perilla seed crude oil;

[0050] The above-mentioned crude perilla seed oil was placed in a sealed environment filled with nitrogen, heated to 40°C, then 30ppm of phospholipase was added, and the pH was adjusted to 5.0 with 0.01% citric acid. After the reaction was allowed to stand for 1 hour, the temperature was raised to 75°C, the heating time was 3 minutes each time, the enzyme was inactivated, and then centrifuged to obtain degummed oil. This step can remove phospholipids, colloids and trace amounts of Fe in the crude perilla seed oil. 2+ Mg 2+ ;

[0051] 2) The degummed oil is placed in a short-range molecular distillation apparatus and distilled under a vacuum of 0.4 Pa and a distillation temperature of 120° C. This step evaporates some free fatty acids and low-boiling impurities into a condenser, and the α-linolenic acid mixed oil is collected as the distillate residue (deacidified oil). 1w% activated clay, 0.1w% activated carbon, and 0.1w% rosemary extract are then added to the deacidified oil and stirred for 30 minutes to absorb pigments, phospholipids, polycyclic aromatic hydrocarbons, and odors in the deacidified oil. The rosemary extract protects the α-linolenic acid from oxidation during the stirring process. After stirring, the mixture is cooled to room temperature, and then the activated carbon and activated clay are filtered out through a plate and frame filter press. The resulting decolorized oil is then nitrogen-filled for protection.

[0052] In this step, the distillation temperature is 140°C, and the heating time needs to be precisely controlled, and each heating time cannot exceed 30 seconds.

[0053] 3) The bleached oil is subjected to a low-temperature treatment, slowly cooling to 5°C at a rate of 0.5°C / min and then maintaining this temperature for 4 hours to allow the bleached oil to crystallize. Once small crystals are observed, the temperature is further lowered to 0°C and maintained for 12 hours to allow the crystals to further grow. Once the crystals are in bulk, they are compressed and filtered at 3°C ​​using a plate-and-frame filter press with a cooling interlayer. 0.1% diatomaceous earth can be used as a filter aid during the compression filtration process. The resulting filtrate is a liquid oil containing α-linolenic acid. After the compression filtration is complete, the liquid oil is immediately heated to 20°C to prevent secondary crystallization caused by the low temperature, thereby obtaining crude α-linolenic acid oil. This step removes high-melting-point saturated fatty acid glycerides and waxes, significantly increasing the proportion of α-linolenic acid in the mixed oil.

[0054] 4) First, add 5% ethanol to the above-mentioned α-linolenic acid crude oil, stir it thoroughly for 30 minutes, mix it evenly, put it into a supercritical extraction kettle, and react it for 1 hour under the conditions of pressure of 300 bar, temperature of 30°C, and CO2 flow rate of 20L / h to obtain α-linolenic acid primary essential oil. Then, according to the Soxhlet extraction method (GB / T 5009.6-2016), accurately weigh the mass of the α-linolenic acid primary essential oil, and then calculate it according to the formula

[0055]

[0056] The extraction rate of perilla seed oil was calculated, and the results are shown in Table 1;

[0057] 5) The above-mentioned α-linolenic acid primary essential oil was mixed in an oil-water ratio of 1:0.5, and 2% by weight of immobilized lipase (e.g. 435) and 5w% glycerol, placing them in a constant temperature stirring reactor, mixing and stirring at 40°C for 6 hours, in this way, the α-linolenic acid primary essential oil is hydrolyzed to obtain an enzymatic hydrolyzate; then the above enzymatic hydrolyzate is placed in a disc centrifuge for centrifugation, and then allowed to stand for stratification, the upper layer is a free fatty acid mixture, and the lower layer is glycerol and water; the free fatty acid mixture after centrifugation is taken, ethanol is added in a molar ratio of 1:1, and then a selective lipase (such as RM IM), react at 30℃ for 4h, and centrifuge again. The upper layer is a mixture of α-linolenic acid and free fatty acids, and the lower layer is the separated saturated fatty acids;

[0058] Methanol is added to the above-mentioned α-linolenic acid-free fatty acid mixture at a ratio of 1:1, and then concentrated sulfuric acid with a mass fraction of 2% by weight is added for a catalytic reaction for 2 hours to obtain a fatty acid methyl ester mixture, which is then subjected to three short-path distillations using a continuous molecular distillation unit to obtain an α-linolenic acid secondary essential oil, and 0.01-0.02% by mass of tocopherol and 0.02-0.03% by mass of ascorbyl palmitate are added to the α-linolenic acid secondary essential oil and stored;

[0059] In this step, three short-path distillations are performed, respectively:

[0060] ① Distill at 70°C and 100 Pa for 10 min to remove water, residual methanol and short-chain fatty acid esters from the fatty acid methyl ester mixture;

[0061] ② Distill at 100°C and 10 Pa for 5 minutes to further remove methyl oleate and methyl linoleate from the fatty acid methyl ester mixture;

[0062] ③ Distill multiple times at 150 degrees Celsius and 0.5 Pa, each distillation for 30 seconds, to obtain α-linolenic acid secondary essential oil.

[0063] 6) First, select n-hexane, acetonitrile, methanol and water in a ratio of 8:5:5:1 to prepare a solvent, mix and place in a separatory funnel, shake for 10 minutes, and let it stand to separate into an upper phase stationary liquid and a lower phase mobile liquid. Then, ultrasonic degassing is performed for 20 minutes to separate the two phases and inject them into a preparative chromatographic column instrument; then, α-linolenic acid secondary essential oil is injected into the upper phase stationary liquid to dissolve, and the instrument is started under the conditions of setting temperature of 20°C, speed of 1500 rpm, and flow rate of 10 ml / min. The sample components are injected and eluted in sequence to collect the α-linolenic acid-rich fraction; after collection, the above α-linolenic acid-rich fractions are combined, and under vacuum conditions with nitrogen blanketing, a 10% mass fraction of KOH-ethanol solution is first added, and hydrolysis is carried out at 60°C for 1 hour. Then, concentrated HCl is added to adjust the pH to 2.0, and then ether is added and shaken for 1 hour to extract the remaining free fatty acids, and finally anhydrous Na2SO 4 , washed with water several times and concentrated to obtain high α-linolenic acid, then vitamin E is added to the high α-linolenic acid, and after collection, it is refrigerated and stored away from light.

[0064] In this step, after the fractions rich in α-linolenic acid are collected, the time required for the reaction is calculated.

[0065] In addition, during the experiment, the α-linolenic acid primary essential oil, α-linolenic acid secondary essential oil, and high α-linolenic acid samples obtained in steps 4) to 6) were respectively used to determine the purity of α-linolenic acid using GC-FID (CP-Sil 88 column). The specific results are shown in Table 2.

[0066] Example 2

[0067] Steps 1) to 3) of this embodiment are the same as those of embodiment 1, and the only difference is that the experimental parameters of steps 4) to 6) are different, which are specifically as follows:

[0068] In step 4), the supercritical reactor was reacted for 1.5 h at a pressure of 320 bar, a temperature of 32° C., and a CO2 flow rate of 24 L / h;

[0069] In step 5), the selected immobilized lipase (e.g. 435) mass fraction of 3w%, glycerol 6w%, under the condition of 42 ° C stirring for 6.5h to obtain enzymatic hydrolyzate; the selected selective lipase (such as The mass fraction of RMIM was 6w%, and the reaction was carried out at 32°C for 4.5h;

[0070] In step 6), the temperature was set to 21° C., the rotation speed was 1700 rpm, and the flow rate was 12 ml / min, and the rest were the same.

[0071] Example 3

[0072] This embodiment is the same as the embodiment 1 in steps 1) to 3), and the only difference is that the experimental parameters of steps 4) to 6) are different, which are specifically as follows:

[0073] In step 4), the supercritical reactor was reacted for 2 h at a pressure of 340 bar, a temperature of 34° C., and a CO2 flow rate of 28 L / h;

[0074] In step 5), the selected immobilized lipase (e.g. 435) mass fraction of 4w%, glycerol 7w%, under the condition of 44 ° C for 7 h to obtain an enzymatic solution; the selected selective lipase (such as The mass fraction of RM IM) was 7w%, and the reaction was carried out at 34 °C for 5 h;

[0075] In step 6), the temperature was set to 22° C., the rotation speed was set to 1900 rpm, and the flow rate was set to 14 ml / min, and the rest were the same.

[0076] Example 4

[0077] Steps 1) to 3) of this embodiment are the same as those of embodiment 1, and the only difference is that the experimental parameters of steps 4) to 6) are different, which are specifically as follows:

[0078] In step 4), the supercritical reactor was reacted for 2.5 hours at a pressure of 360 bar, a temperature of 36° C., and a CO2 flow rate of 32 L / h;

[0079] In step 5), the selected immobilized lipase (e.g. 435) mass fraction of 5w%, glycerol 8w%, under the condition of 46 ° C stirring for 7.5h to obtain enzymatic hydrolyzate; the selected selective lipase (such as The mass fraction of RMIM was 8w%, and the reaction was carried out at 36°C for 5.5h;

[0080] In step 6), the temperature was set to 24° C., the rotation speed was set to 2100 rpm, and the flow rate was set to 16 ml / min, and the rest were the same.

[0081] Example 5

[0082] Steps 1) to 3) of this embodiment are the same as those of embodiment 1, and the only difference is that the experimental parameters of steps 4) to 6) are different, which are specifically as follows:

[0083] In step 4), the supercritical reactor was reacted for 3 hours at a pressure of 400 bar, a temperature of 40° C., and a CO2 flow rate of 40 L / h;

[0084] In step 5), the selected immobilized lipase (e.g. 435) mass fraction of 5w%, glycerol 8w%, under the condition of 50 ° C stirring for 8h to obtain enzymatic hydrolyzate; the selected selective lipase (such as The mass fraction of RM IM) was 8w%, and the reaction was carried out at 36 °C for 6 h;

[0085] In step 6), the temperature was set to 24° C., the rotation speed was set to 2100 rpm, and the flow rate was set to 16 ml / min, and the rest were the same.

[0086] Control Example

[0087] The difference between this comparative example and Examples 1-5 is that in step 6), a commercially available α-linolenic acid reagent with a purity of 96% was selected, and GC-FID (CP-Sil 88 column) was used to determine the purity of the α-linolenic acid.

[0088] Table 1 Extraction rate of perilla seed oil

[0089]

[0090]

[0091] It can be seen from Table 1 that during the supercritical CO2 extraction process, the extraction rate of perilla seed oil will first increase and then decrease with the increase of pressure, temperature, CO2 flow rate and reaction time, and it does not increase all the time. Therefore, the supercritical CO2 extraction reaction conditions are selected as follows: the pressure of the supercritical reactor is 360 bar, the temperature is 36°C, and the CO2 flow rate is 32 L / h, and the reaction time is 2.5 hours.

[0092] Table 2 Purity of α-linolenic acid

[0093]

[0094] As shown in Table 2, the purity of the synthesized high-α-linolenic acid reached over 96%, meeting the medical production standards. The parameters in the preparative chromatography separation process had little effect on the enrichment of α-linolenic acid.

[0095] Another embodiment of the present invention provides an oral emulsion rich in α-linolenic acid. The emulsion contains α-linolenic acid prepared by the method provided in the embodiments of the present invention and is synthesized according to the following components by weight: 10-20% α-linolenic acid, 10-15% soy lecithin, 1-5% ethylene glycol, 0.1-2% tea polyphenols, 0.5-1% fragrance, and the balance purified water. The specific preparation steps can be found in CN107441217A and are not further described here.

[0096] In summary, the present invention provides a preparation method and application of high α-linolenic acid. The method comprises multi-stage treatment of perilla seeds, and layer-by-layer extraction and refinement through supercritical CO2 extraction, enzymatic molecular distillation, and preparative chromatography separation. The method can effectively remove free fatty acids from perilla seed crude oil and enrich α-linolenic acid to obtain high α-linolenic acid with a purity exceeding 96%, which is suitable for mass production in the pharmaceutical field.

[0097] In addition, by adding different antioxidants in various steps of production and preparation and operating under a protective atmosphere, the present invention can ensure that α-linolenic acid is not deteriorated due to oxidation throughout the entire process from raw material grinding to enrichment and preservation, thereby improving the quality and safety of the finished product of high α-linolenic acid.

[0098] The embodiments described above are some, but not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but rather merely represents selected embodiments of the present invention. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are also within the scope of protection of the present invention.

Claims

1. A method for preparing high α-linolenic acid, characterized in that: The steps include: S1: Raw material pretreatment: Perilla seeds are selected, cleaned, dried, mechanically ground under a protective atmosphere, and then filtered to obtain perilla seed crude oil; S2: crude oil refining, degumming, deacidification, decolorization, and dewaxing the perilla seed crude oil under a protective atmosphere to obtain a crude oil with preliminary enrichment of α-linolenic acid; S3: The initially enriched α-linolenic acid crude oil is subjected to supercritical CO2 extraction, enzymatic molecular distillation, and preparative chromatography separation to obtain high α-linolenic acid.

2. The method for preparing high α-linolenic acid according to claim 1, wherein: In step S1, during the mechanical grinding process, phytic acid with a concentration of 0.01-0.05% needs to be added to the perilla seeds, and the mechanical grinding process needs to be carried out at 20-40°C; the filtration method is diatomaceous earth and / or ion membrane filtration.

3. The method for preparing high α-linolenic acid according to claim 1, characterized in that: In step S2, the degumming method adopts enzymatic degumming, the reaction time of the enzymatic degumming is 1-2 hours, and the reaction temperature is 40-60°C.

4. The method for preparing high α-linolenic acid according to claim 3, characterized in that: In step S2, the deacidification method is to perform molecular distillation deacidification under a protective atmosphere, the distillation temperature is 120-150° C., the reaction time is 10-20 min, and the deacidified oil obtained after distillation is added with an adsorbent and rosemary extract, stirred for 20-40 min for decolorization, and then filtered to obtain the decolorized oil.

5. The method for preparing high α-linolenic acid according to claim 4, characterized in that: In step S2, the dewaxing method is to first slowly cool the decolorized oil to 5-10°C, keep it warm for 4-6 hours to crystallize it, further cool it to 0-4°C, keep it warm for 12-20 hours to enlarge the crystals; then compress and filter it at a temperature of 0-5°C to obtain liquid oil containing α-linolenic acid, and then immediately heat the liquid oil to 15-20°C to avoid secondary crystallization, thereby obtaining the crude α-linolenic acid oil.

6. The method for preparing high α-linolenic acid according to claim 5, characterized in that: In step S3, 5-10% ethanol and / or methanol-KOH solvent is first added to the α-linolenic acid crude oil, mixed and placed in a supercritical extraction kettle, and reacted for 1-3 hours under the conditions of pressure of 300-400 bar, temperature of 30-40°C, and CO2 flow rate of 20-40L / h to obtain α-linolenic acid primary essential oil.

7. The method for preparing high α-linolenic acid according to claim 6, characterized in that: In step S3, the α-linolenic acid primary essential oil is mixed at an oil-water ratio of 1:(0.5-0.8), 2-5w% of immobilized lipase and 5-8w% of glycerol are added, and the mixture is stirred at 40-50° C. for 6-8 hours to obtain an enzymatic hydrolyzate; the enzymatic hydrolyzate is then centrifuged, and the free fatty acid mixture after centrifugation is obtained. Ethanol is added at a ratio of 1:(1-1.5), and then 5-8w% of selective lipase is added. The mixture is reacted at 30-40° C. for 4-6 hours, and centrifuged again to obtain an α-linolenic acid-free fatty acid mixture; Methanol is added to the α-linolenic acid-free fatty acid mixture at a ratio of 1:(1-3), and concentrated sulfuric acid with a mass fraction of 2-5w% is added to carry out a catalytic reaction for 2-4 hours to obtain a fatty acid methyl ester mixture, which is then subjected to three short-path distillations to obtain an α-linolenic acid secondary essential oil. A composite antioxidant is added to the α-linolenic acid secondary essential oil and stored.

8. The method for preparing high α-linolenic acid according to claim 7, characterized in that: In step S3, a solvent is first prepared by selecting n-hexane, acetonitrile, methanol, and water in a ratio of 8:5:5:1, mixing the mixture and placing it in a separatory funnel. The mixture is shaken for 10-30 minutes, and then separated into an upper phase stationary liquid and a lower phase mobile liquid. The two phases are then ultrasonically degassed for 20 minutes to separate the two phases, and then injected into a preparative chromatographic column instrument. Subsequently, the α-linolenic acid secondary essential oil is injected into the upper phase stationary liquid to dissolve, and the instrument is started under the conditions of a set temperature of 20-25° C., a rotation speed of 1500-2200 rpm, and a flow rate of 10-20 ml / min. The sample components are sequentially injected and eluted, and the fraction rich in α-linolenic acid is collected. After the collection is completed, the fractions rich in α-linolenic acid are combined, placed in a water bath and concentrated under a protective atmosphere to obtain high α-linolenic acid. Vitamin E is then added to the high α-linolenic acid, and the fractions are collected and stored in a dark and refrigerated place.

9. The method for preparing high α-linolenic acid according to claim 8, characterized in that: The protective atmosphere is nitrogen or argon, and the composite antioxidant is 0.01-0.02% tocopherol and 0.02-0.03% ascorbyl palmitate in mass fraction.

10. An application of high α-linolenic acid, characterized in that: The high α-linolenic acid prepared by the preparation method according to claim 8 is used to prepare an oral emulsion rich in high α-linolenic acid; The oral emulsion is synthesized according to the following components in mass fraction: 10-20% high α-linolenic acid, 10-15% soybean lecithin, 1-5% ethylene glycol, 0.1-2% tea polyphenols, 0.5-1% aromatics, and the balance is purified water.

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