Method for separating and purifying EPA and DHA from microbial oil and application thereof

Abstract

The invention relates to the technical field of deep processing of microbial oil, and discloses a method for separating and purifying EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid) from microbial oil and application of the method. The method comprises the following steps: (1) in the presence of compound lipase and an acidic material, carrying out hydrolysis reaction on microbial oil subjected to deoxidation treatment to obtain a first mixed material containing EPA and DHA; (2) sequentially carrying out first-stage fractionation treatment and second-stage fractionation treatment on a light-phase product obtained by separating the first mixed material through a first-stage fractionating tower and a second-stage fractionating tower to obtain a tower top light fraction and a tower bottom heavy fraction of the second-stage fractionating tower; (3) sequentially carrying out first-stage freezing fractionation and second-stage freezing fractionation on the tower top light fraction of the second-stage fractionating tower to obtain a liquid-phase mixture containing EPA (Eicosapentaenoic Acid); and (4) separating and purifying the liquid-phase mixture to obtain an EPA product. According to the method provided by the invention, the high-purity and high-yield EPA product can be separated from the microbial oil.

Description

Technical Field

[0001] This invention relates to the field of microbial oil processing technology, specifically to a method for separating and purifying EPA and DHA from microbial oils and its application. Background Technology

[0002] Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) play significant roles in cardiovascular health, anti-inflammatory responses, brain and optic nerve development, and metabolic syndrome intervention, and are therefore widely used in medical nutrition and dietary supplements.

[0003] Microalgae oils, with their advantages of not relying on marine fishery resources, stable composition, and scalable production, have become one of the important sources of EPA and DHA for industrial production. Because they align with sustainable development principles, they are increasingly favored by consumers, gradually replacing fish oil, and will play an even greater social and economic role in the future.

[0004] Among numerous microalgae, the genus *Schizochytrium* has become a major source of ω-3 long-chain polyunsaturated fatty acids through microbial fermentation due to its high biomass, high oil content, and rich ω-3 long-chain polyunsaturated fatty acids. Oils obtained from the fermentation of naturally occurring *Schizochytrium* strains typically have a high DHA content and a low EPA content. Currently, through domestication or mutagenesis techniques, several mutant strains with high economic value have been obtained, whose algal oil EPA content can be increased to 8-20%. However, relying solely on natural fermentation to obtain algal oil with higher EPA content has reached a bottleneck, necessitating the further development of green and efficient separation and purification processes.

[0005] In existing technologies, in order to realize the industrial application of EPA free fatty acids, their purity usually needs to reach more than 90%, especially in the fields of pharmaceuticals and high-end nutrition, where the industrial requirement is generally a purity of more than 95%. When the purity of EPA free fatty acids reaches or exceeds 98%, it is usually considered a high-end product, which places higher demands on process stability and separation efficiency.

[0006] Currently, the commonly used method for obtaining high-purity EPA is ethyl esterification followed by purification. This involves transesterification of ethanol with oils to produce fatty acid ethyl esters, which are then separated and purified by methods such as distillation, molecular distillation, and chromatography to obtain high-purity EPA ethyl esters, with high-purity DHA ethyl esters as a byproduct. These are then resynthesized with glycerol to form high-purity triglycerides. This type of high-purity product is usually called re-esterified triglyceride (rTG). The aforementioned separation and purification methods generally have the following shortcomings: Firstly, ethanol presents certain safety and environmental impact issues during production; secondly, rTG contains ethyl ester residues, limiting its application. Furthermore, EPA and DHA fatty acid ethyl esters and triglycerides differ in terms of absorption and metabolism. For example, patent CN103864614A discloses a process for separating and purifying DHA ethyl esters from microbially fermented oils, achieving a total DHA and DPA ethyl ester content of over 98%. However, ethyl ester products are difficult for the human body to digest and utilize due to safety and absorption issues.

[0007] Therefore, it is necessary to develop safer, more environmentally friendly, and more economical technologies for separating and purifying EPA and DHA, as well as to develop green products of high-purity EPA and DHA glycerides that meet the requirements of sustainable development and health.

[0008] Given the above background, enzymatic hydrolysis and re-separation and purification technology—that is, the technology of using lipase to catalyze the hydrolysis of triglycerides to obtain free fatty acids and then separating and purifying them—has become a promising new method due to its advantages such as mild reaction conditions and environmental friendliness. How to improve the hydrolysis efficiency (i.e., the hydrolysis reaction rate) and the hydrolysis rate (i.e., the degree of hydrolysis) is the key to this technology. However, the limited efficiency and degree of hydrolysis of traditional enzymatic hydrolysis restrict its application in large-scale production.

[0009] Furthermore, existing publicly available enzymatic hydrolysis processes primarily focus on fish oil systems, aiming to obtain DHA or EPA (e.g., CN110257446A and US20200370079A1). However, those skilled in the art know that algal oil and fish oil differ significantly in composition: algal oil has a higher content of polyunsaturated fatty acids, and its lipid system differs significantly from fish oil in terms of oil-water interface properties, molecular conformational flexibility, and enzyme-substrate interaction mechanisms. Therefore, directly applying fish oil enzymatic hydrolysis technology to algal oil systems often results in insufficient effective reaction interfaces and slow reaction kinetics, leading to low hydrolysis efficiency and ultimately low yields of the target product. In actual production, it is often necessary to extend the reaction time, increase the enzyme dosage, or intensify reaction conditions (such as increasing the temperature) to achieve a higher hydrolysis rate, which is not conducive to continuous or large-scale industrial production.

[0010] Furthermore, increasing the hydrolysis rate of algal oil systems through the above methods can also lead to a decrease in the stability and selectivity of target fatty acids. This is because polyunsaturated fatty acids in algal oil systems are structurally unstable and prone to side reactions such as oxidation, isomerization, excessive hydrolysis, or emulsification under prolonged or strong reaction conditions, resulting in the loss of EPA in subsequent phase separation, washing, and purification processes. As a result, although the hydrolysis rate is high, the actual recovery yield of EPA does not increase accordingly, or even decreases, ultimately making it difficult to balance product purity and yield, thus limiting the practical application value of this technology in the production of high-purity EPA.

[0011] Furthermore, the mixed fatty acids obtained from hydrolysis still require further separation and purification to obtain high-purity EPA and DHA. Currently used separation techniques include urea inclusion complexation, silver ion complexation, simulated moving bed chromatography, supercritical fluid extraction, and methods such as distillation and molecular distillation. However, these methods generally suffer from problems such as complex processes, high energy consumption, and large solvent usage in large-scale production, and often fail to achieve both high purity and high yield simultaneously: pursuing high purity often requires multiple separation operations, which are often accompanied by significant loss of target components, thereby reducing the overall yield; while focusing on high yield makes it difficult to guarantee product purity. The contradiction between the two further limits the economics and industrial feasibility of the overall process.

[0012] In conclusion, how to obtain high-purity EPA and DHA while maintaining a high product yield, and taking into account the economic efficiency, environmental friendliness, and scalability of the process, remains a key issue that urgently needs to be addressed in the current technological field. Summary of the Invention

[0013] The purpose of this invention is to solve the problem in the existing technology of separating and purifying EPA and DHA from microbial oils where high hydrolysis efficiency, high purity, and high yield cannot be achieved simultaneously.

[0014] To achieve the above objectives, a first aspect of the present invention provides a method for separating and purifying EPA and DHA from microbial lipids, the method comprising: (1) In the presence of a complex lipase and an acidic substance, the deoxygenated microbial oil is hydrolyzed to obtain a first mixture containing EPA and DHA; the complex lipase is a combination of lipase I and lipase II in a mass ratio of 1:0.1-0.8; lipase I is different from lipase II, and lipase II is selected from lipases derived from Candida albicans; the acidic substance is at least one of C3-C12 saturated fatty acids; (2) The light phase product obtained by separating the first mixture is subjected to first-stage fractionation and second-stage fractionation in a first-stage fractionation column and a second-stage fractionation column respectively to obtain the light distillate at the top and the heavy distillate at the bottom of the second-stage fractionation column; the light distillate at the top of the second-stage fractionation column includes at least 50 wt% EPA and the heavy distillate at the bottom includes at least 50 wt% DHA; the bottom temperature of the first-stage fractionation column is not lower than 170°C and the bottom temperature of the second-stage fractionation column is not lower than 190°C. (3) The light fraction at the top of the second-stage fractionation column is subjected to first-stage and second-stage freeze fractionation in sequence to obtain a liquid mixture containing EPA; the temperature of the second-stage freeze fractionation is not higher than -40℃; (4) The liquid mixture is separated and purified to obtain an EPA product with a purity of not less than 98% and a yield of not less than 70 wt%.

[0015] A second aspect of the invention provides the application of the method described in the first aspect in the preparation of triglycerides rich in EPA and DHA.

[0016] Compared with the prior art, the present invention has at least the following beneficial effects: 1. The method provided by this invention can efficiently separate and purify EPA and DHA from microbial oils rich in polyunsaturated fatty acids. In a system with complex interfacial properties, high enrichment of polyunsaturated fatty acids, and easy oxidation and degradation, it achieves a synergistic improvement effect of high hydrolysis efficiency, high purity and high yield. 2. This invention employs a strategy of synergistic addition of compound lipase and C3-C12 saturated fatty acids, which effectively increases the reaction rate while significantly enhancing the selective separation ability of EPA and DHA, thereby systematically improving the prominent problems of low hydrolysis rate and high loss of target product in traditional catalytic hydrolysis process. (3) The present invention adopts a combination of two-stage fractionation and two-stage freeze fractionation, which is significantly different from the traditional multi-stage molecular distillation or rectification method. It can not only significantly improve the separation efficiency, but also effectively avoid the generation of harmful substances such as trans fatty acids, chloropropanol esters, and oxidation products. Thus, while achieving high-purity EPA / DHA separation, it significantly improves the overall yield of the target product, achieving a synergistic unity of high purity, high yield and green safety. (4) The method provided by the present invention is safe, green and efficient, and is especially suitable for continuous and large-scale industrial production, with significant economic and social benefits. Detailed Implementation

[0017] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0018] The "C3-C12 saturated fatty acids" mentioned in this invention refer to saturated fatty acids with a total number of carbon atoms of 3-12. For example, the C3-C12 saturated fatty acids can be C3 saturated fatty acids, C4 saturated fatty acids, C5 saturated fatty acids, C6 saturated fatty acids, C7 saturated fatty acids, C8 saturated fatty acids, C9 saturated fatty acids, C10 saturated fatty acids, etc.

[0019] As previously stated, a first aspect of the present invention provides a method for separating and purifying EPA and DHA from microbial lipids, the method comprising: (1) In the presence of a complex lipase and an acidic substance, the deoxygenated microbial oil is hydrolyzed to obtain a first mixture containing EPA and DHA; the complex lipase is a combination of lipase I and lipase II in a mass ratio of 1:0.1-0.8; lipase I is different from lipase II, and lipase II is selected from lipases derived from Candida albicans; the acidic substance is at least one of C3-C12 saturated fatty acids; (2) The light phase product obtained by separating the first mixture is subjected to first-stage fractionation and second-stage fractionation in a first-stage fractionation column and a second-stage fractionation column respectively to obtain the light distillate at the top and the heavy distillate at the bottom of the second-stage fractionation column; the light distillate at the top of the second-stage fractionation column includes at least 50 wt% EPA and the heavy distillate at the bottom includes at least 50 wt% DHA; the bottom temperature of the first-stage fractionation column is not lower than 170°C and the bottom temperature of the second-stage fractionation column is not lower than 190°C. (3) The light fraction at the top of the second-stage fractionation column is subjected to first-stage and second-stage freeze fractionation in sequence to obtain a liquid mixture containing EPA; the temperature of the second-stage freeze fractionation is not higher than -40℃; (4) The liquid mixture is separated and purified to obtain an EPA product with a purity of not less than 98% and a yield of not less than 70 wt%.

[0020] It should be noted that in this invention, the terms "first stage" and "second stage" in the first-stage fractionation tower and the second-stage fractionation tower are only used to distinguish that the material separation is carried out in two separate fractionation towers, and they do not limit the fractionation towers themselves; the terms "first stage" and "second stage" in the first-stage freeze fractionation and the second-stage freeze fractionation have the same interpretation.

[0021] In this invention, the first-stage fractionation tower and the second-stage fractionation tower refer to a device that separates components in a mixture based on the principle of gas-liquid equilibrium by utilizing the difference in volatility of the components; the first-stage fractionation treatment and the second-stage fractionation treatment refer to a process method that separates components in a mixture by subjecting the mixture to multiple partial vaporization and partial condensation processes in the fractionation tower.

[0022] In this invention, the first-stage freeze fractionation and the second-stage freeze fractionation refer to the process of selectively crystallizing and separating oil components to obtain high-melting-point components (solid phase) and low-melting-point components (liquid phase).

[0023] It should be noted that in this invention, in step (2), the bottom temperature of the first-stage fractionation column and the bottom temperature of the second-stage fractionation column refer to the temperature of the heavy fraction outlet.

[0024] This invention, through inventive research, has discovered that using the compound lipase provided by this invention to hydrolyze microbial oils rich in polyunsaturated fatty acids, with the specific addition of C3-C12 saturated fatty acids, significantly improves hydrolysis efficiency and degree while maintaining higher selectivity, reducing side reactions and over-hydrolysis. Furthermore, the combined separation method of two-stage fractionation and two-stage freeze-extraction ensures separation efficiency and avoids the generation of harmful substances. Thus, while achieving high-purity EPA / DHA separation, it significantly improves the overall yield of the target product, achieving a synergistic balance of high purity, high yield, and green safety.

[0025] Preferably, the microbial oil is derived from the genus Schizochytrium (…). Schizochytrium ) and / or genus *Cypripedium* ( Thraustochytrium Oil obtained through fermentation and culture.

[0026] Preferably, the composite lipase is a combination of lipase I and lipase II in a mass ratio of 1:0.25-0.5. The invention has found that this preferred embodiment achieves a better balance between high hydrolysis efficiency, high purity, and high yield.

[0027] In a preferred embodiment, the hydrolysis reaction is carried out in a hydrolyzer.

[0028] It should be noted that, in order to further improve the hydrolysis efficiency, the deoxygenated microbial oil can be selectively purified by acidification, degumming, neutralization, and water washing to remove various impurities.

[0029] More preferably, the lipase I is selected from at least one of the following lipases derived from porcine pancreas, Rhizopus oryzae, Rhizopus sylvae, Fusarium oxysporum, Thermophilus sparsely cottony, Candida sp. 99-125, Aspergillus oryzae, Rhizopus oryzae, Aspergillus niger, Fusarium oxysporum, Fusarium heterosporum, Fusarium oxysporum, and Candida antarctica.

[0030] Preferably, the C3-C12 saturated fatty acids are selected from butyric acid, propionic acid, caprylic acid, and decanoic acid.

[0031] More preferably, the saturated fatty acid of C3-C12 is caprylic acid. The inventors have discovered that in this complex microbial oil system with a high content of polyunsaturated fatty acids, when the saturated fatty acid of C3-C12 is caprylic acid, it has higher hydrolysis selectivity, which can significantly improve hydrolysis efficiency and degree of hydrolysis, thereby obtaining products with higher purity and yield.

[0032] More preferably, the amount of C3-C12 saturated fatty acids used is 0.1-2 parts by weight relative to 100 parts by weight of the deoxygenated microbial oil. The inventors have found that, under this preferred embodiment, a higher product yield can be obtained.

[0033] Preferably, in step (1), the deoxygenation treatment includes the following steps: In the presence of antioxidants, microbial oil raw materials are sprayed and contacted countercurrently with nitrogen to obtain microbial oil with an oxygen content of no more than 0.25% by volume.

[0034] More preferably, the deoxygenation treatment operation steps include: After mixing the microbial oil raw material and antioxidant, the mixture is introduced into the vacuum deaerator from the top via spraying. Simultaneously, nitrogen gas is introduced into the vacuum deaerator from the bottom via microporous aeration, allowing the microbial oil raw material to undergo continuous countercurrent nitrogen deaeration to remove oxygen and obtain an oxygen content of no more than 0.25%. Product % of microbial oil.

[0035] In a preferred embodiment, the antioxidant is selected from at least one of butylated hydroxyanisole, butylated hydroxytoluene, propyl gallate, tert-butylhydroquinone, tocopherol, ascorbic acid and its derivatives, rosemary extract, tea polyphenols, and phospholipids.

[0036] Preferably, in step (1), the hydrolysis reaction is carried out under stirring conditions, the conditions of which include: temperature of 40-60℃, time of 12-45h, and stirring speed of 200-600rpm.

[0037] In a preferred embodiment, the conditions for the hydrolysis reaction further include a pH value of 5-7 for the hydrolysis reaction system.

[0038] In a preferred embodiment, the hydrolysis reaction is carried out in the presence of water, and the water-to-oil mass ratio in the hydrolysis reaction is 0.5-1.5:1.

[0039] Preferably, the amount of the compound lipase used is 0.1-7 wt% of the mass of the microbial oil.

[0040] In some preferred embodiments, in step (2), the first-stage fractionation column and the second-stage fractionation column have the same or different structures, and each independently includes a distillation column and a thin-film evaporator arranged from top to bottom; the vapor outlet of the thin-film evaporator is connected to the vapor phase channel of the distillation column. The inventors have found that, under this preferred embodiment, the residence time of the separated material at high temperature can be shortened.

[0041] It should be noted that, taking the first-stage fractionation tower as an example, the flow path of the light phase product obtained from the separation of the first mixture in the first-stage fractionation tower includes: the light phase product is introduced into the thin-film evaporator through the liquid phase inlet for thin-film treatment and thermal evaporation; the vapor phase material formed by evaporation is introduced into the distillation tower through the vapor outlet and the vapor phase channel of the distillation tower for distillation separation and gas-liquid mass transfer, and the first-stage fractionation is carried out in this process. The light fraction obtained flows out from the light fraction outlet at the top of the distillation tower, and the heavy fraction flows out from the liquid phase outlet of the thin-film evaporator and is introduced into the second-stage fractionation tower for the second-stage fractionation; the material in the second-stage fractionation is processed in the distillation tower and the thin-film evaporator in the same way as the first-stage fractionation.

[0042] Preferably, in step (2), the operation of separating the light phase product from the first mixture is carried out in a sedimentation separator.

[0043] In a preferred embodiment, in step (2), the light phase product contains free fatty acids, monoglycerides, diglycerides and triglycerides.

[0044] In a preferred embodiment, the first mixture can also be separated to obtain a heavy phase product; the heavy phase product contains glycerol, water and lipase.

[0045] In a preferred embodiment, the separation of the first mixture can also yield an interfacial intermediate layer; the interfacial intermediate layer contains an emulsion layer of monoglycerides, diglycerides, lipase, and water.

[0046] Preferably, the conditions for the first-stage fractionation treatment include: feed temperature of 160-170℃, bottom temperature of 175-185℃, and pressure ≤30Pa.

[0047] More preferably, the conditions for the first-stage fractionation treatment also include: a feed flow rate of 15-20 kg / h and a scraping speed of 100-190 rpm for the thin-film evaporator.

[0048] It should be noted that the pressure in the first-stage fractionation process refers to the absolute pressure at the light fraction outlet at the top of the first-stage fractionation column.

[0049] Preferably, the conditions for the second-stage fractionation treatment include: feed temperature of 175-185℃, bottom temperature of 190-200℃, and pressure ≤30Pa.

[0050] More preferably, the conditions for the second-stage fractionation treatment also include: a feed flow rate of 5-15 kg / h and a scraping speed of 100-190 rpm for the thin-film evaporator.

[0051] It should be noted that the pressure in the second-stage fractionation process refers to the absolute pressure at the light fraction outlet at the top of the second-stage fractionation column.

[0052] In some preferred embodiments, the light fraction obtained from the first-stage fractionation process contains a mixture of C12-C20 fatty acids; the mixture of fatty acids mainly contains palmitic acid, octadecanoic acid (SDA) and stearic acid, and contains small amounts of myristic acid, oleic acid, linoleic acid, linolenic acid and EPA.

[0053] In some embodiments, the heavy fraction obtained from the first-stage fractionation process mainly contains DHA, EPA, and ARA, and contains small amounts of DPA, SDA, and palmitic acid.

[0054] Preferably, in step (3), the conditions for the first-stage freeze fractionation include a temperature of -5 to -35°C.

[0055] Preferably, the conditions for the second-stage cryogenic fractionation include a temperature of -40°C to -60°C.

[0056] Preferably, in step (3), the method further includes: subjecting the liquid phase material obtained by the first-stage freeze fractionation to a second-stage freeze fractionation to obtain a liquid phase mixture containing EPA.

[0057] In some embodiments, the liquid phase material obtained from the first-stage freeze fractionation contains DHA, ARA, and EPA.

[0058] It should be noted that, unless otherwise specified, the present invention does not have special requirements for the specific operation method of cryo-fractionation, and those skilled in the art can choose to perform it conventionally.

[0059] Preferably, in step (4), the separation and purification are selected from high performance liquid chromatography separation.

[0060] It should be noted that the present invention does not limit the conditions of the liquid mixture in the high performance liquid chromatography separation and purification step (4). Those skilled in the art can choose to perform the conditions in a conventional manner. The present invention will not elaborate further here, and those skilled in the art should not understand it as a limitation of the present invention.

[0061] In a preferred embodiment, the method of the present invention further includes separating and purifying the heavy fraction at the bottom of the second-stage fractionation column obtained in step (2) to obtain a DHA product with a purity of not less than 90%.

[0062] It should be noted that the specific operational steps for separating and purifying the heavy fraction at the bottom of the second-stage fractionation column to obtain a DHA product with a purity of not less than 90% are not limited in this invention. Those skilled in the art can use known technical means in the field to carry out the process. This invention will not elaborate further here and should not be construed as a limitation of this invention.

[0063] As previously stated, a second aspect of the present invention provides the application of the method described in the first aspect in the preparation of triglycerides rich in EPA and DHA.

[0064] Preferably, the method of application includes: SI-1. In the presence of synthetic lipase, the EPA product or DHA product obtained by the method described in the first aspect is reacted with glycerol to obtain crude triglyceride. SI-2. The crude triglyceride product is subjected to adsorption decolorization, water washing, deodorization, and molecular distillation in sequence to obtain the finished triglyceride product.

[0065] It should be noted that in step S1, the present invention does not have any special requirements for the reactor and reaction operation steps of the synthesis reaction, nor does the present invention have any special requirements for the specific operation steps and conditions for refining crude triglycerides (including adsorption decolorization, water washing, deodorization, and molecular distillation). Those skilled in the art can make conventional choices.

[0066] Furthermore, in this invention, in the synthesis reaction of step S1, different triglyceride products can be obtained by designing different reaction ratios of EPA or DHA products with glycerol; alternatively, a 1,3-position selective fatty acid enzyme can be used to catalyze the reaction, first synthesizing 1,3-position diglycerides, and then using a synthetic lipase to further synthesize EPA or DHA, thereby synthesizing a structural lipid in which EPA or DHA has a higher proportion at the 2nd position of the triglyceride.

[0067] The present invention will be described in detail below through embodiments.

[0068] In the following examples, unless otherwise specified, all experimental instruments, reagents, and raw materials involved are commercially available products, and all reagents are analytical grade products.

[0069] In the following examples, acid value, saponification value, EPA and DHA content were determined by gas chromatography; It should be noted that in the following examples, the hydrolysis reaction time is the time when the acid value no longer increases, that is, the initial time when the hydrolysis rate reaches its highest value, and the hydrolysis rate no longer increases after the time is extended.

[0070] Example 1 Microbial oil raw materials: Schizochytrium ( Schizochytrium The oil obtained from microbial fermentation contains 10.45 g / 100 g of EPA, 39.13 g / 100 g of DHA, 0.2 mg KOH / g of acid value, 183 mg KOH / g of saponification value, and less than 0.1 wt% of moisture. The fatty acid composition is shown in Table 1. C3-C12 saturated fatty acids: caprylic acid; Lipase I: derived from Candida antarctica, purchased from Novozymes' Lipozyme® CALB L product; Lipase II: Lipase derived from Candida albicans (product L1754-5G purchased from Sigma-Aldrich). Deoxygenation treatment of microbial oil raw materials: 100 kg of microbial oil raw material and 0.01 kg of antioxidant (tocopherol) were mixed and introduced into the vacuum deaerator from the top by spraying. At the same time, nitrogen gas was introduced into the vacuum deaerator from the bottom by microporous aeration, so that the microbial oil raw material underwent continuous countercurrent nitrogen deaeration to remove oxygen from the microbial oil raw material, and microbial oil with an oxygen content of 0.25% by volume was obtained (the oxygen content was detected by measuring the oxygen volume fraction in the headspace gas of the sample container using a headspace oxygen analyzer). (1) The compound lipase, water, C3-C12 saturated fatty acids and deoxygenated microbial oil were mixed and hydrolyzed (temperature 45℃, time 39h, stirring speed 320rpm) to obtain the first mixture containing EPA and DHA. The complex lipase is a combination of lipase I and lipase II in a mass ratio of 1:0.46; The water-to-oil mass ratio is 1:1; The amount of compound lipase used was 0.5 wt% of the deoxygenated microbial oil. The amount of C3-C12 saturated fatty acids used is 1 part by weight relative to 100 parts by weight of deoxygenated microbial oil. (2) The light phase product obtained by sedimentation separation of the first mixture is subjected to first-stage fractionation and second-stage fractionation in a first-stage fractionation column and a second-stage fractionation column respectively to obtain the light fraction at the top of the second-stage fractionation column and the heavy fraction at the bottom of the column. The first-stage and second-stage fractionation columns have the same structure and each independently includes a distillation column and a thin-film evaporator arranged from top to bottom; the vapor outlet of the thin-film evaporator is connected to the vapor phase channel of the distillation column.

[0071] The conditions for the first-stage fractionation process include: a pressure of 25 Pa, a feed flow rate of 15 kg / h for the thin-film evaporator, a scraper rotation speed of 150 rpm, a feed temperature of 165℃, and a bottom temperature of 180℃. The conditions for the second-stage fractionation process include: a pressure of 25 Pa, a feed flow rate of 5 kg / h for the thin-film evaporator, a scraper rotation speed of 150 rpm, a feed temperature of 180℃, and a bottom temperature of 190℃. (3) The light fraction at the top of the second-stage fractionation column is subjected to first-stage and second-stage freeze fractionation in sequence to obtain a liquid mixture containing EPA; The conditions for the first-stage freeze-fraction are: -30℃; the conditions for the second-stage freeze-fraction are: -60℃. (4) The liquid mixture is separated and purified by high performance liquid chromatography to obtain EPA product S1.

[0072] Example 2 This embodiment follows a similar process to Embodiment 1, except that the microbial oil raw material used in this embodiment is different. Microbial oil raw materials: Schizochytrium ( Schizochytrium The oil obtained from microbial fermentation contains 14.84 g / 100 g of EPA, 34.69 g / 100 g of DHA, an acid value of 0.3 mgKOH / g, a saponification value of 184.7 mg KOH / g, and a moisture content of less than 0.1%. The fatty acid composition is shown in Table 1.

[0073] Because the raw materials for microbial oils are different, the hydrolysis reaction time in this embodiment is 40 hours.

[0074] The remaining procedures and conditions are the same as in Example 1.

[0075] Obtain EPA product S2.

[0076] Example 3 Microbial oil raw materials: Same as in Example 1; C3-C12 saturated fatty acids: caprylic acid; Lipase I: derived from Aspergillus oryzae, purchased from Novozymes' Resinase® HT product; Lipase II: Same as in Example 1; Deoxygenation treatment of microbial oil raw materials: Example 1; (1) The compound lipase, water, C3-C12 saturated fatty acids and deoxygenated microbial oil were mixed and hydrolyzed (temperature 50℃, time 38h, stirring speed 320rpm) to obtain the first mixture containing EPA and DHA. The compound lipase is a combination of lipase I and lipase II in a mass ratio of 1:0.25; The water-to-oil mass ratio is 1.5:1; The amount of compound lipase used was 1 wt% of the deoxygenated microbial oil. The amount of C3-C12 saturated fatty acids used is 2 parts by weight relative to 100 parts by weight of the deoxygenated microbial oil. (2) The light phase product obtained by sedimentation separation of the first mixture is subjected to first-stage fractionation and second-stage fractionation in a first-stage fractionation column and a second-stage fractionation column respectively to obtain the light fraction at the top of the second-stage fractionation column and the heavy fraction at the bottom of the column. The first-stage and second-stage fractionation columns have the same structure and each independently includes a distillation column and a thin-film evaporator arranged from top to bottom; the vapor outlet of the thin-film evaporator is connected to the vapor phase channel of the distillation column.

[0077] The conditions for the first-stage fractionation process include: a pressure of 25 Pa, a feed flow rate of 20 kg / h for the thin-film evaporator, a scraper rotation speed of 100 rpm, a feed temperature of 170℃, and a bottom temperature of 185℃. The conditions for the second-stage fractionation process include: a pressure of 25 Pa, a feed flow rate of 15 kg / h for the thin-film evaporator, a scraper rotation speed of 190 rpm, a feed temperature of 185℃, and a bottom temperature of 200℃. (3) The light fraction at the top of the second-stage fractionation column is subjected to first-stage and second-stage freeze fractionation in sequence to obtain a liquid mixture containing EPA; The conditions for the first-stage freeze-fraction are: -20℃; the conditions for the second-stage freeze-fraction are: -55℃. (4) The liquid mixture is separated and purified by high performance liquid chromatography to obtain EPA product S3.

[0078] Example 4 This embodiment follows a similar process to Example 1. The difference is that the dosage of the compound lipase is controlled in the same way as in Example 1, but the compound lipase is a combination of lipase I and lipase II with a mass ratio of 1:0.6. The hydrolysis reaction takes 42 hours. The remaining procedures and conditions are the same as in Example 1.

[0079] Obtain EPA product S4.

[0080] Example 5 This embodiment uses a similar process to that of Example 1, except that in this embodiment, the saturated fatty acid of C3-C12 is butyric acid; The hydrolysis reaction took 43 hours. The remaining procedures and conditions are the same as in Example 1.

[0081] Obtain EPA product S5.

[0082] Example 6 This embodiment follows a similar process to that of Embodiment 1. The difference is that in this embodiment, the amount of caprylic acid used is 2.2 parts by weight relative to 100 parts by weight of the deoxygenated microbial oil. The hydrolysis reaction takes 40 hours. The remaining procedures and conditions are the same as in Example 1.

[0083] Obtain EPA product S6.

[0084] Comparative Example 1 Comparative Example 1 was carried out using a similar process to Example 1. The difference was that no acidic substance (octanoic acid) was added in the hydrolysis reaction of this comparative example, and the complex lipase in Example 1 was replaced with an equivalent amount of lipase II from Example 1 (that is, the amount of enzyme used in this comparative example was the same as in Example 1, but only a single enzyme derived from Candida pleuropsis was used). The hydrolysis reaction takes 70 hours. The remaining procedures and conditions are the same as in Example 1.

[0085] Obtain EPA product DS1.

[0086] Comparative Example 2 This comparative example follows a similar procedure to Example 1, except that lipase II of the same mass is used to replace the compound lipase in Example 1 (that is, the amount of enzyme used in this comparative example is the same as in Example 1, but only a single enzyme derived from Candida albicans is used). The hydrolysis reaction takes 55 hours. The remaining procedures and conditions are the same as in Example 1.

[0087] Obtain EPA product DS2.

[0088] Comparative Example 3 This comparative example 1 was carried out using a similar process to Example 1, except that no acidic substance (octanoic acid) was added during the hydrolysis reaction of this comparative example. The hydrolysis reaction takes 58 hours. The remaining procedures and conditions are the same as in Example 1.

[0089] Obtain EPA product DS3.

[0090] Comparative Example 4 This comparative example was conducted using a similar procedure to Example 1. The difference was that the type of lipase II used in this comparative example was Lipozyme® TL 100L, a product of Novozymes, derived from Thermophilus spp. The hydrolysis reaction takes 52 hours. The remaining procedures and conditions are the same as in Example 1.

[0091] Obtain EPA product DS4.

[0092] Comparative Example 5 This comparative example was conducted using a similar procedure to Example 1. The difference was that the type of lipase II used in this comparative example was Palatase® 20000 L, derived from Mucormycium miheganus and purchased from Novozymes. The hydrolysis reaction takes 42 hours. The remaining procedures and conditions are the same as in Example 1.

[0093] Obtain EPA product DS5.

[0094] Comparative Example 6 This comparative example follows a similar procedure to Example 1, except that in this comparative example... The conditions for the first-stage fractionation process include: a pressure of 25 Pa, a feed flow rate of 15 kg / h for the thin-film evaporator, a scraper rotation speed of 150 rpm, a feed temperature of 150℃, and a bottom temperature of 165℃. The conditions for the second-stage fractionation process include: a pressure of 25 Pa, a feed flow rate of 5 kg / h for the thin-film evaporator, a scraper rotation speed of 150 rpm, a feed temperature of 170℃, and a bottom temperature of 180℃.

[0095] The remaining procedures and conditions are the same as in Example 1.

[0096] Obtain EPA product DS6.

[0097] Comparative Example 7 This comparative example follows a similar procedure to Example 1, except that in this comparative example... The conditions for the first-stage freeze fractionation are: a temperature of -10℃; the conditions for the second-stage freeze fractionation are: a temperature of -35℃.

[0098] The remaining procedures and conditions are the same as in Example 1.

[0099] Obtain EPA product DS7.

[0100] Comparative Example 8 This comparative example follows a similar procedure to Example 1. The difference is that in this comparative example, the type of lipase II is the same as that of lipase I, both derived from Candida antarctica and purchased from Novozymes' Lipozyme® CALBL product (that is, the amount of enzyme used in this comparative example is the same as in Example 1, but only a single enzyme derived from Candida antarctica is used).

[0101] The hydrolysis reaction takes 52 hours. The remaining procedures and conditions are the same as in Example 1.

[0102] Obtained EPA product DS8.

[0103] Comparative Example 9 This comparative example uses a similar process to Example 1. The difference is that, in this comparative example, the combination of two-stage fractionation and two-stage freeze extraction is not used. In this comparative example, the light phase product obtained by separating the first mixture is separated and purified into EPA and DHA according to step (2) in Example 2 of CN110257446A, and then separated and purified by high performance liquid chromatography.

[0104] The remaining procedures and conditions are the same as in Example 1.

[0105] Obtain EPA product DS9.

[0106] The fat composition of the microbial oil raw materials used in the above examples is shown in Table 1.

[0107] Table 1

[0108] Test Example 1 The hydrolysis rate, product yield, and purity of the above examples were tested. Wherein, oil loss rate % = (total fatty acid mass - mass of fatty acids obtained after hydrolysis) / total fatty acid mass × 100%; EPA yield % = EPA mass in EPA product / Total EPA mass in microbial oil feedstock × 100%; Hydrolysis rate % = Acid value / Saponification value × 100%; The results are shown in Table 2.

[0109] Table 2

[0110] Table 2 (continued)

[0111] As can be clearly seen from the results in Table 1, the method provided by this invention can efficiently separate and purify EPA and DHA from microbial oils rich in polyunsaturated fatty acids. In a system with complex interfacial properties, high enrichment of polyunsaturated fatty acids, and easy oxidation and degradation, it achieves a synergistic improvement effect of high hydrolysis efficiency, high purity and high yield.

[0112] Test Example 2 The EPA product obtained in Example 1 was then resynthesized to obtain EPA triglycerides, and the specific steps are as follows: Step 1: Under high vacuum conditions, a combination of a reaction vessel (with an internal immobilized enzyme bed) and an external column-shaped immobilized enzyme bed was used. An appropriate flow rate of external circulation spray was employed, with the external column-shaped immobilized enzyme bed and heat exchanger installed along the circulation route. The synthesis reaction used a batch reaction unit, with batch feeding and different types of lipases catalyzing the reaction in stages. In the first stage, 11 kg of purified EPA, 1 kg of glycerol, and 100 L of Lipozyme TL were added. The reaction temperature was 50℃, the stirring speed was 320 rpm, and the reaction lasted for 10 hours. In the second stage, Cal-A lipase was added, the reaction temperature was 50℃, the stirring speed was 320 rpm, and the reaction continued for 15 hours, finally yielding 10.3 kg of crude triglycerides.

[0113] Step 2: The crude glycerol esters undergo low-temperature adsorption decolorization and two water washes to remove impurities such as chloride and iron ions, thereby minimizing the risk of generating harmful byproducts such as chloropropanol esters and glycidyl esters in subsequent processes. Then, deodorization and molecular distillation are performed to obtain the finished product. The final product weighs 6.72 kg, of which 6.41 kg is EPA.

[0114] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A method for separating and purifying EPA and DHA from microbial lipids, characterized in that, The method includes: (1) In the presence of a complex lipase and an acidic substance, the deoxygenated microbial oil is hydrolyzed to obtain a first mixture containing EPA and DHA; the complex lipase is a combination of lipase I and lipase II in a mass ratio of 1:0.1-0.8; lipase I is different from lipase II, and lipase II is selected from lipases derived from Candida albicans; the acidic substance is at least one of C3-C12 saturated fatty acids; (2) The light phase product obtained by separating the first mixture is subjected to first-stage fractionation and second-stage fractionation in a first-stage fractionation column and a second-stage fractionation column respectively to obtain the light distillate at the top and the heavy distillate at the bottom of the second-stage fractionation column; the light distillate at the top of the second-stage fractionation column includes at least 50 wt% EPA and the heavy distillate at the bottom includes at least 50 wt% DHA; the bottom temperature of the first-stage fractionation column is not lower than 170°C and the bottom temperature of the second-stage fractionation column is not lower than 190°C. (3) The light fraction at the top of the second-stage fractionation column is subjected to first-stage and second-stage freeze fractionation in sequence to obtain a liquid mixture containing EPA; the temperature of the second-stage freeze fractionation is not higher than -40℃; (4) The liquid mixture is separated and purified to obtain an EPA product with a purity of not less than 98% and a yield of not less than 70 wt%.

2. The method according to claim 1, characterized in that, The composite lipase is a combination of lipase I and lipase II in a mass ratio of 1:0.25-0.5; And / or, the lipase I is selected from at least one of the following lipases derived from porcine pancreas, Rhizopus oryzae, Rhizopus sylvae, Fusarium oxysporum, Thermophilus sparsely cottony, Candida sp. 99-125, Aspergillus oryzae, Rhizopus oryzae, Aspergillus niger, Fusarium oxysporum, Fusarium heterosporum, Fusarium oxysporum, and Candida antarctica.

3. The method according to claim 1 or 2, characterized in that, The C3-C12 saturated fatty acids are selected from butyric acid, propionic acid, caprylic acid, and decanoic acid; Preferably, the saturated fatty acid of C3-C12 is octanoic acid; More preferably, the amount of C3-C12 saturated fatty acids used is 0.1-2 parts by weight relative to 100 parts by weight of the deoxygenated microbial oil.

4. The method according to claim 1, characterized in that, In step (1), the deoxygenation treatment includes the following steps: In the presence of antioxidants, microbial oil raw materials are sprayed and contacted countercurrently with nitrogen to obtain microbial oil with an oxygen content of no more than 0.25% by volume.

5. The method according to any one of claims 1-3, characterized in that, In step (1), the hydrolysis reaction is carried out under stirring conditions, including a temperature of 40-60℃, a time of 12-45h, and a stirring speed of 200-600rpm. And / or, the hydrolysis reaction is also carried out in the presence of water, and the water-to-oil mass ratio in the hydrolysis reaction is 0.5-1.5:1; And / or, the amount of the complex lipase used is 0.1-7 wt% of the mass of the microbial oil.

6. The method according to any one of claims 1-4, characterized in that, In step (2), the first-stage distillation column and the second-stage distillation column have the same or different structures, and each independently includes a distillation column and a thin-film evaporator arranged from top to bottom; the vapor outlet of the thin-film evaporator is connected to the gas phase channel of the distillation column.

7. The method according to any one of claims 1-3, characterized in that, In step (2), the light phase product contains free fatty acids, monoglycerides, diglycerides and triglycerides; And / or, the conditions for the first-stage fractionation treatment include: feed temperature of 160-170℃, bottom temperature of 175-185℃, and pressure ≤30Pa; And / or, the conditions for the second-stage fractionation treatment include: feed temperature of 175-185℃, bottom temperature of 190-200℃, and pressure ≤30Pa.

8. The method according to any one of claims 1-3, characterized in that, In step (3), the conditions for the first-stage freeze fractionation include: a temperature of -5 to -35°C; And / or, the conditions for the second-stage cryogenic fractionation include a temperature of -40°C to -60°C.

9. The method according to any one of claims 1-7, characterized in that, In step (2), the method further includes: introducing the bottom heavy fraction of the first-stage fractionating column obtained from the first-stage fractionation treatment into the second-stage fractionating column for the second-stage fractionation treatment; And / or, in step (3), the method further includes: subjecting the liquid phase material obtained by the first-stage freeze fractionation to a second-stage freeze fractionation to obtain a liquid phase mixture containing EPA; And / or, in step (4), the separation and purification are selected from high performance liquid chromatography separation.

10. The use of the method according to any one of claims 1-9 in the preparation of triglycerides rich in EPA and DHA.

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