A green extraction and separation method and device for microbial oil

Abstract

The application discloses a kind of green extraction separation methods and devices of microbial oil, belong to the extraction field of microbial oil.The present application can continuously carry out cell disruption treatment to bacteria liquid in large quantities using high-pressure pulsed electric field, processing time is short, and it can also effectively avoid the oxidation of unsaturated fatty acids.In addition, fatty acid-based deep eutectic solvent and ethanol solvent are used for co-extraction, and the hydrophilic ethanol solvent is selected to carry out the hydrophilic material wrapped outside the oil inside the cell, so that the oil is exposed, and the hydrophobic fatty acid-based deep eutectic solvent can carry out the oil inside the cell more completely.The microbial oil is extracted by fatty acid-based deep eutectic solvent, ethanol and weak base solution, which can be recycled, thereby effectively reducing the cost and energy consumption.The present application also provides a kind of microbial oil extraction separation device, which can realize efficient extraction of microbial oil and efficient recovery of extraction solvent, thereby shortening the process flow, reducing equipment investment and other problems.

Description

TECHNICAL FIELD

[0001] The present application relates to a green extraction and separation method and device of microbial oil, belonging to the field of microbial oil extraction. BACKGROUND

[0002] Microbial oil is accumulated by oleaginous microorganisms such as yeast, fungi, and algae under nitrogen stress. For example, the oil content of Yarrowia lipolytica cells can reach more than 80%. On the one hand, compared with plant oil, the rapid growth and low cost of microorganisms are their inherent advantages, and the microbial oil obtained after extraction is more pure and has fewer impurities. On the other hand, synthetic biology can be used to modify microorganisms to produce functional lipids with higher value. However, due to the accumulation of intracellular oil in oleaginous microorganisms under harsh conditions to protect their growth and development, these microorganisms often have thick cell barriers, making it difficult to extract intracellular oil directly with solvents. Therefore, certain means must be used to break the microbial cells to obtain a large amount of microbial oil.

[0003] Currently, the main methods for extracting microbial oil are solvent extraction and solvent-free extraction, with solvent extraction being the main method. Solvent extraction uses lipophilic organic solvents such as chloroform, petroleum ether, dichloromethane, n-hexane, and acetone to extract microbial oil. The chloroform-methanol extraction method is considered the gold standard for oil extraction, with an extraction rate of more than 85%. However, traditional organic solvent extraction methods use organic solvents that are toxic and volatile, and the microbial oil obtained has high solvent residue, which seriously affects the biological safety of microbial oil. Although some researchers have developed methods for extracting microbial oil using low-toxicity polar solvents, such as CN113684088A, which uses a polar solvent with an Et(30) value of 38 or higher to extract microbial oil. However, this method requires strict control of the water content of the fermented broth and the subsequent extraction process is too complicated, requiring multiple steps and high-temperature treatment, which can lead to a decrease in the quality of microbial oil.

[0004] Non-solvent extraction, which does not require the addition of non-fermentation liquid system solvents, uses physical and / or enzymatic methods to break down the cell wall and separate the microbial oil. For example, CN112500918B first performs ultramicrofracture on dry microbial cells, then rehydrates the dry microbial cells for enzymatic cell wall disruption, and finally obtains microbial oil through phase separation, avoiding the problems of solvent residue and solvent toxicity caused by the addition of organic solvents. However, the non-solvent method requires the addition of multiple biological enzymes to break down the microbial cell wall, resulting in high production costs. The enzymatic hydrolysis process required for different microbial cell walls differs greatly, and the process steps are complex and require strict control of the enzymatic hydrolysis conditions to avoid loss of enzyme activity.

[0005] In order to solve the above-mentioned problems, it is urgent to find a safe and simple green extraction method of microbial oil. SUMMARY

[0006] Since the existing microbial oil extraction solvent is mostly non-biologically friendly organic solvent, there are safety problems such as solvent residue and biological toxicity, which limits the use of microbial oil as a food raw material to some extent.

[0007] In order to solve the above-mentioned problems of microbial oil extraction technology, the first technical solution provided by the present application is a green extraction method of microbial oil, and the specific technical solution is as follows:

[0008] A green extraction method of microbial oil, comprising the following steps:

[0009] S1 separates the wet microbial cells from the fermentation broth of the oil-producing microorganism, adds an ethanol aqueous solution to the wet microbial cells, and obtains an ethanol aqueous resolubilized microbial solution;

[0010] S2 places the ethanol aqueous resolubilized microbial solution in step S1 in a high-voltage pulse electric field to break the cells and obtain a mixed solution containing microbial oil;

[0011] S3 adds a fatty acid-based deep eutectic solvent to the mixed solution containing microbial oil in step S2 and mixes thoroughly, performs ultrasonic treatment, and realizes three-phase separation of hydrophobic phase, hydrophilic phase and solid phase;

[0012] The hydrophobic phase is a mixed solution composed of microbial oil and a fatty acid-based deep eutectic solvent;

[0013] The hydrophilic phase is an ethanol aqueous solution;

[0014] The solid phase is wet microbial cells and insoluble impurities;

[0015] S4 adds a weak alkaline aqueous solution to the hydrophobic phase of step S3 to cause the polarity of the hydrophobic fatty acid-based deep eutectic solvent to switch to generate a hydrophilic fatty acid salt, thereby separating the hydrophobic microbial oil and obtaining a mixed solution containing the hydrophilic fatty acid salt and ethanol.

[0016] In some embodiments, in step S1, the volume fraction of the ethanol aqueous solution is 75% or more.

[0017] In some embodiments, in step S1, the volume ratio of the wet microbial cells to the ethanol aqueous solution is 1:(3-5).

[0018] In some embodiments, in step S2, the supply voltage of the high-voltage pulse electric field is 0.25-1.5KV / cm, the pulse width is 5-10μs, and the pulse number is 10-100 times.

[0019] In some embodiments, in step S3, the fatty acid-based deep eutectic solvent is a mixture of hydrogen bond acceptors and hydrogen bond donors, the hydrogen bond acceptors are any one or two of caproic acid (C6), caprylic acid (C8), and pelargonic acid (C9); and the hydrogen bond donors are any one of capric acid (C10), lauric acid (C12), and myristic acid (C14).

[0020] In some embodiments, in step S3, the volume ratio of the fatty acid-based deep eutectic solvent to the aqueous ethanol solution is 1:(0.5-2).

[0021] In some embodiments, the ultrasonic treatment is performed under the following conditions: a frequency of 40-70 kHz, a power of 300-500 W, a treatment time of 15-20 min, and an extraction temperature of 25-30°C.

[0022] In some embodiments, in step S4, the weakly basic aqueous solution is an aqueous solution prepared from any one of polyetheramine, ethylenediamine, propylenediamine, and diethylenetriamine; and the volume fraction of the solute in the weakly basic aqueous solution is 5-15%.

[0023] In some embodiments, in step S4, the weakly basic compound is added in an amount of 4-8 times the volume of the fatty acid-based deep eutectic solvent.

[0024] In some embodiments, step S5 is further included, in which the fatty acid-based deep eutectic solvent, ethanol, and weakly basic aqueous solution are recovered from the mixture of fatty acid salts and ethanol in step S4.

[0025] Further, the recovery method is as follows: the mixture of fatty acid salts and ethanol in step S4 is continuously subjected to CO2 bubbling until the volume of the hydrophobic phase, i.e., the fatty acid-based deep eutectic solvent, no longer changes, the hydrophobic phase is separated to obtain the fatty acid-based deep eutectic solvent, and a mixture of ethanol and carbonate is obtained in the hydrophilic phase; and the mixture of ethanol and carbonate is heated and distilled to recover ethanol, and the escaped CO2 is obtained as a weakly basic aqueous solution.

[0026] In some embodiments, the oleaginous microorganism is at least one of yeast, mold, and algae.

[0027] The yeast is selected from at least one of Yarrowia lipolytica, Rhodotorula giutinis, Rhodosporidium toruloides, Cryptococcus albiduns, Pichia pastoris and Lipomyces starkeyi.

[0028] The mold is selected from at least one of Mortierella alpina, Mortierella isabellina, Cunninghamella echinulata and Conidiobolus nanodes.

[0029] The algae is selected from at least one of Thraustochytriales, Schizochytrium, Chlorella zofingiensis, Nannochloropsis gaditana and Phaeodactylum tricornutum Bohlin.

[0030] In addition, to simplify the operation process of the present technology, the present application provides a second technical solution, which is a microbial oil extraction and separation device, and the specific technical solution is as follows:

[0031] The microbial oil extraction and separation device comprises a main body, wherein the main body comprises a pulsed electric field treatment chamber, a microbial oil extraction chamber and an extraction solvent separation chamber, the bottom of the pulsed electric field treatment chamber is communicated with the top of the microbial oil extraction chamber, and the bottom of the microbial oil extraction chamber is communicated with the top of the extraction solvent separation chamber.

[0032] The pulsed electric field treatment chamber is used for crushing the microbial cells by high-voltage pulsed electric field treatment.

[0033] The microbial oil extraction chamber is used for separating microbial oil from the product treated by the pulsed electric field treatment chamber.

[0034] The extraction solvent separation chamber is used for recovering fatty acid-based deep eutectic solvent, ethanol and weak alkaline aqueous solution from the byproduct of the microbial oil extraction chamber.

[0035] In some embodiments, the pulse electric field treatment chamber is provided with a discharger on the inner wall of the pulse electric field treatment chamber and a receiver electrically connected to the high-voltage pulse power supply and corresponding to the discharger.

[0036] Further, the receiver is provided with an inductive indicator for displaying the solution level in the pulse electric field treatment chamber.

[0037] In some embodiments, the pulse electric field treatment chamber is further provided with a first feeding port at the top of the pulse electric field treatment chamber, and the bottom of the pulse electric field treatment chamber is communicated with the microbial oil extraction chamber through at least one first material transfer channel.

[0038] In some embodiments, the microbial oil extraction chamber is provided with a stirrer for mixing the materials in the microbial oil extraction chamber and an ultrasonic generator electrically connected to the ultrasonic power supply and releasing ultrasonic waves into the microbial oil extraction chamber.

[0039] In some embodiments, the microbial oil extraction chamber is provided with a second feeding port at the top and a microbial oil discharge port at the bottom, and the bottom of the microbial oil extraction chamber is communicated with the extraction solvent separation chamber through at least one second material transfer channel.

[0040] In some embodiments, the microbial oil extraction chamber is further provided with a density sensor for displaying the solution density in the microbial oil extraction chamber.

[0041] In some embodiments, the extraction solvent separation chamber is divided into an upper layer and a lower layer, the upper layer is provided with one or more perforated plates, each perforated plate is connected with a liquid receiving tray below, each liquid receiving tray is funnel-shaped, and the lowermost center is provided with a liquid outlet, the liquid filtered by the perforated plate is collected on the liquid receiving tray and flows to the lower layer through the liquid outlet, the upper layer is communicated with an air inlet at the top and an air outlet at the bottom, and the lower layer is provided with two phase discharge ports at the bottom side.

[0042] In some embodiments, the bottom of the extraction solvent separation chamber is provided with a bottom valve.

[0043] In some embodiments, the main body further comprises a controller for controlling the circuit of the main body.

[0044] The third technical solution provided by the present application is the use of the method of the first technical solution or the device of the second technical solution in the preparation of a product containing microbial oil.

[0045] Compared with the prior art, the present application has the following advantages:

[0046] The high-voltage pulsed electric field adopted in the present application is one of the newly emerging non-thermal technologies in recent years, and its current main research is in the aspect of sterilization treatment of liquid food. The sterilization mechanism thereof is that microorganisms are caused to rupture under a transmembrane voltage, resulting in a large and free exchange of substances in the cell membrane and external substances, and finally cell death. The microorganism rupture ultimately pulverizes the cells, and makes it easy for the solvent to enter the cells and bring out the oil. The strong electric field environment generated by the high-voltage pulsed electric field can destroy the cell membrane and intracellular lipid of the oil-producing microorganism and promote the release of oil. Compared with the traditional physical rupture technology, the high-voltage pulsed electric field treatment technology can continuously and in large quantities treat the cell rupture of the bacterial solution, has low energy consumption, short processing time, and does not require water removal and other operation steps. In addition, the non-thermal rupture effect can effectively avoid the oxidation of unsaturated fatty acids.

[0047] One of the green extraction solvents adopted in the present application is a fatty acid-based deep eutectic solvent, that is, two or more fatty acids are mixed to obtain a freezing point significantly lower than the melting point of any of the component fatty acids. The main purpose thereof is to reduce the freezing point of long-chain fatty acids and introduce longer-chain fatty acids to obtain stronger hydrophobicity. For example, the melting temperature of the fatty acid-based deep eutectic solvent composed of caprylic acid (melting temperature 16.4℃, water solubility 0.68g / L) and lauric acid (melting temperature 43.5℃, insoluble in water) is 9℃, and the water solubility is 0.014g / L. Moreover, fatty acids are natural biological energy substances, which can effectively avoid the safety problems caused by the residue of traditional organic solvents.

[0048] Another green extraction solvent adopted in the present application is an ethanol solvent. The safety of ethanol as a food-grade organic solvent is self-evident. The purpose of co-extraction of the fatty acid-based deep eutectic solvent and the ethanol solvent is to remove the hydrophilic substances in the cells of microorganisms, which wrap the oil droplets inside, so that the hydrophobic fatty acid-based deep eutectic solvent cannot bring out the oil from the cells. Therefore, the hydrophilic ethanol solvent is selected to bring out the hydrophilic substances wrapped outside the oil droplets, so that the oil is exposed. The hydrophobic fatty acid-based deep eutectic solvent can completely bring out the intracellular oil, and high-quality microbial oil is obtained. For example, the high-quality oil obtained from Yarrowia lipolytica has an acid value of ≤3mgKOH / g, a peroxide value of ≤0.25g / 100g, and a solvent residue of ≤20mg / kg.

[0049] The present application provides a microbial oil recycling extraction scheme using reusable extraction solvents. The reusable solvents include a fatty acid-based deep eutectic solvent, ethanol, and a weak alkali solution, and the recovery rates of the three solvents can reach 95.19%, 91.55%, and 97.88%, respectively. Based on the above-mentioned reusable solvent extraction of microbial oil, the production cost and energy consumption can be significantly reduced.

[0050] The present application provides a microbial oil extraction and separation device, which can realize efficient extraction of microbial oil and efficient recovery of extraction solvent, thereby shortening the process flow, reducing equipment investment and the like.

[0051] In summary, the microbial oil extraction and separation device and the biologically friendly green solvent adopted by the present application can realize efficient extraction of microbial oil and efficient recovery of extraction solvent. BRIEF DESCRIPTION OF DRAWINGS

[0052] Figure 1 Structure diagram of the microbial oil extraction and separation device.

[0053] Figure 2 Schematic diagram of the intermediate partition plate in the pulse electric field treatment chamber.

[0054] Figure 3 Schematic diagram of the operating system position.

[0055] Figure 4 Process flow diagram of the green microbial oil extraction process.

[0056] Figure 5 The fatty acid composition analysis diagram provided by the present application. DETAILED DESCRIPTION

[0057] The preferred embodiments of the present application are described below, and it should be understood that the embodiments are for better explaining the present application and are not used to limit the present application.

[0058] 1. Source of experimental materials:

[0059] n-octanoic acid (C8) (Shanghai Maikelin Biochemical Technology Co., Ltd.);

[0060] nonanoic acid (C9) (Shanghai Maikelin Biochemical Technology Co., Ltd.);

[0061] capric acid (C10) (Shanghai Titan Science and Technology Co., Ltd.);

[0062] lauric acid (C12) (Beijing Inokai Technology Co., Ltd.);

[0063] myristic acid (C14) (National Pharmaceutical Group Chemical Reagent Co., Ltd.);

[0064] polyether amine D-230 (Beijing Inokai Technology Co., Ltd.);

[0065] n-hexane (National Pharmaceutical Group Chemical Reagent Co., Ltd.);

[0066] chloroform (National Pharmaceutical Group Chemical Reagent Co., Ltd.);

[0067] petroleum ether 30-60℃ (National Pharmaceutical Group Chemical Reagent Co., Ltd.);

[0068] Anhydrous ethanol (National Pharmaceutical Group Chemical Reagent Co., Ltd.);

[0069] Oil-producing microorganism: Yarrowia lipolytica strain (strain number: po1g, donated by the Future Food Science Center, Food Synthetic Biology Research Room of Jiangnan University).

[0070] 2. Solvent configuration

[0071] Configuration of fatty acid-based deep eutectic solvent: preferably, four kinds of fatty acid-based deep eutectic solvents of C8-C12(3:1), C9-C12(2:1), C8-C14(4:1) and C9-C14(3:1) are configured, 5%, 10%, 15% polyetheramine D-230 aqueous solution, 75%, 85%, 95% ethanol solution, but not limited to the above solvents;

[0072] The raw material ratio of the configured fatty acid-based deep eutectic solvent is the molar ratio of the substances;

[0073] The mass fraction of the solute of the configured polyetheramine aqueous solution is 5%, 10%, 15%;

[0074] The volume fraction of the solute of the configured ethanol solution is 75%, 85%, 95%;

[0075] The water used in the above solvent configuration is secondary water.

[0076] Taking C8-C12(3:1) as an example, the preparation process of the fatty acid-based deep eutectic solvent is as follows:

[0077] Take two kinds of fatty acids with a molar ratio of 3:1, add solid fatty acid-lauric acid to a glass container containing liquid fatty acid-octanoic acid, magnetic stirring at 1000 rpm, 40°C water bath, and obtain the fatty acid-based deep eutectic solvent when the solution is clear and transparent.

[0078] Test method:

[0079] Microbial oil detection method:

[0080] The fatty acid composition detection method refers to the normalization method of GB 5009.168-2016, and is detected by gas chromatography.

[0081] The acid value detection method refers to the cold solvent indicator titration method of GB 5009.229-2016.

[0082] The peroxide value detection method refers to the titration method of GB 5009.227-2016.

[0083]

[0084] Example 1

[0085] This embodiment provides a microbial oil extraction and separation device, such as... Figures 1 to 3 As shown, the device includes a main body 1, which is equipped with a pulsed electric field treatment chamber 2, a microbial oil extraction chamber 11, an extraction solvent separation chamber 17, and a controller 25. The pulsed electric field treatment chamber 2 includes a first liquid inlet 3, a high-voltage pulse power supply 4, an induction indicator light 5, a receiver 6, a discharger 7, and a first material transfer channel 8. The microbial oil extraction chamber 11 includes an ultrasonic power supply 9, a second inlet 10, a stirrer 12, an ultrasonic generator 13, a density sensor 14, a second material transfer channel 15, and a microbial oil outlet 16. The extraction solvent separation chamber 17 includes an air inlet 18, a support column 19, two perforated plates 20, two liquid receiving plates 21, an exhaust port 22, a two-phase outlet 23, and a bottom valve 24.

[0086] The main body 1 consists of three layers from top to bottom, separated by steel coated with insulating material. The first layer is the pulse electric field processing chamber 2, which is divided into two sub-processing chambers by a middle partition. A high-voltage pulse power supply 4 is provided at the upper part of the outside of the pulse electric field processing chamber 2, and the high-voltage pulse power supply 4 is connected to the generators 7 at both ends of the processing chamber and the receivers 6 in the middle partition. The receivers are equipped with induction indicators 5 at the top, and two first material transfer channels 8 are provided at the bottom.

[0087] The second layer is the microbial oil extraction chamber 11. The microbial oil extraction chamber 11 is equipped with a stirrer 12 in the middle, and ultrasonic generators 13 connected to ultrasonic power supplies 9 are set on the end plates. The second feeding port 10 is placed between the upper left two chamber plates. Density sensors 1 are set at both the upper and lower ends. The lower end is equipped with a second material transfer channel 15 and a microbial oil outlet 16.

[0088] The third layer is the extraction solvent separation chamber 17. In the middle of the upper layer of the extraction solvent separation chamber 17, a multi-layer perforated plate 20 is placed. Each perforated plate 20 is supported by a support column 19. A liquid receiving plate 21 is set under the support column 19 to collect the solution. An air inlet 18 is set at the upper left end of the extraction solvent separation chamber 17. An exhaust port 22 is set between the liquid receiving plate 21 and the perforated plate 20 at the right end. A two-phase discharge port 23 is set at the lower right end. A bottom valve 24 is set at the lower end of the main body 1.

[0089] Among them, the high-voltage pulse power supply 4, generator 7, and receiver 6 together form a high-voltage pulse electric field module, which is controlled by the pulse power supply; the induction indicator 5 is connected to the controller 25 to display the solution water level; the ultrasonic generator 13 and the ultrasonic power supply 9 constitute an ultrasonic module; the density sensor 14 is connected to the controller 25 to indicate changes in solution density; the controller 25 is used for all circuit control of the main body 1.

[0090] The microbial oil extraction and separation device provided in this embodiment is generally implemented as follows:

[0091] The user first turns on the controller 25 and the high-voltage pulse power supply 4. When the system and power supply display are stable, the user adds the ethanol-water reconstituted bacterial solution through the first feed port 3. When the bacterial solution fills the pulse electric field treatment chamber 2, the user triggers the sensor indicator 5 to indicate that the feeding should be stopped. Then, the user sets the pulse electric field parameters and starts the high-voltage pulse power supply 4 to form a high-voltage AC pulse electric field in the pulse electric field treatment chamber 2 for high-voltage pulse cell disruption. At the same time, the user adds a fatty acid-based eutectic solvent through the second feed port 10 in proportion. The user opens the air inlet 18 to fill the extraction solvent separation chamber 17 with carbon dioxide gas. After the cell disruption is completed, the user opens the first transfer channel 8, and the disrupted bacterial solution flows into the microbial oil extraction chamber. 11. Then turn on the ultrasonic power supply 9 and the stirrer 12. After the bacterial solution and the fatty acid-based eutectic solvent are fully mixed, add the weak alkaline compound aqueous solution through the second feed port 10 in proportion. Mix thoroughly again until the density sensors 14 at the top and bottom show different densities, that is, the solution separates into phases. The upper layer is the microbial oil phase and the lower layer is the hydrophilic salt solution. Open the exhaust port 22 to make the air intake > the air output. Then open the second transfer channel 15 to let the lower layer solution flow into the extraction solvent separation chamber 17. When the lower density sensor 14 senses the change in density, the second transfer channel 15 is automatically closed, so that the upper microbial oil phase flows out through the microbial oil outlet 16 and the microbial oil is collected. The hydrophilic salt solution entering the extraction solvent separation chamber 17 flows to the center of the perforated plate 20, then diffuses outwards from the center, and flows through the pores on the perforated plate 20 to the receiving plate. This process causes the gas and solution to mix thoroughly, and finally flows to the bottom layer of the extraction solvent separation chamber 17, where it stands and separates into two phases, and is discharged through the two-phase outlet 23.

[0092] Example 2

[0093] A green extraction method for microbial oils, such as Figure 4 As shown, it includes the following steps:

[0094] a. Take a certain amount of fermented Yeast lipolyticis fermentation broth after fermentation, centrifuge, remove the aqueous phase, obtain wet cells, add 5 times the amount of 75% ethanol solution to reconstitute, and then place it in a high-voltage pulse treatment chamber, set the treatment parameters, and carry out the high-voltage pulse electric field cell disruption process; the high-voltage pulse electric field treatment conditions are 1KV / cm, pulse width is 7μs, and pulse number is 60 times.

[0095] b. Add an equal volume of C8-C12 (3:1) fatty acid-based eutectic solvent to the ethanol-water bacterial culture after the disruption in step (a). Then place the mixture on a magnetic stirrer and mix continuously at 1000 rpm for 20 h. After that, centrifuge at 25 °C, 8000 rpm, for 10 min to separate the hydrophobic liquid phase.

[0096] c. To the hydrophobic liquid phase after the completion of the microbial oil extraction in step (b), add 10% polyetheramine D-230 aqueous solution equivalent to 1 mol equivalent of fatty acid-based deep eutectic solvent, mix thoroughly until the volume of the upper oil phase no longer changes. Separate the upper hydrophobic oil phase to obtain microbial oil. The yield of microbial oil is 84.75%, and the fatty acid composition analysis chart is shown in Figure 5 The lower layer is a hydrophilic mixture containing fatty acid salt and ethanol.

[0097] d. To the mixture containing fatty acid salt and ethanol obtained in step (c), pass sufficient CO2, and the fatty acid salt decomposes. Separate the hydrophobic phase (fatty acid-based deep eutectic solvent) when the volume of the hydrophobic phase no longer changes. The obtained hydrophilic phase is a hydrophilic phase solution containing ethanol and carbonate. The recovery rate of the fatty acid-based deep eutectic solvent is 94.65%

[0098] e. Distill the ethanol and carbonate hydrophilic phase solution obtained in step (d) by heating to recover ethanol and escape CO2 to obtain a weak alkaline aqueous solution. The recovery rate of ethanol is 90.50%, and the recovery rate of the weak alkaline aqueous solution is 97.50%.

[0099] According to the national standard, the acid value of the obtained microbial oil is 1.42 mgKOH / g, and the peroxide value is 0.15 g / 100g.

[0100] Example 3

[0101] A green extraction method of microbial oil, comprising the following steps:

[0102] a. Take a certain amount of Yarrowia lipolytica fermentation broth after fermentation, centrifuge, remove the water phase, obtain wet cells, add anhydrous ethanol solution equivalent to 5 times the wet cells, and then place it in a high-voltage pulse treatment chamber. Set the treatment parameters and perform high-voltage pulse electric field cell disruption process; the high-voltage pulse electric field treatment conditions are 0.25KV / cm, pulse width is 5μs, and pulse number is 10 times.

[0103] b. To the ethanol water cell solution after disruption in step (a), add C8-C12 (3:1) fatty acid-based deep eutectic solvent equivalent to the volume of the ethanol aqueous solution, then place the mixed solution on a magnetic stirrer at a speed of 1000 rpm, and mix continuously for 20h. After centrifugation at 25℃, 8000 rpm for 10 min, the hydrophobic liquid phase is separated.

[0104] c. To the hydrophobic liquid phase after the completion of the microbial oil extraction in step (b), add 10% polyetheramine D-230 aqueous solution equivalent to 1 mol equivalent of fatty acid-based deep eutectic solvent, mix thoroughly until the volume of the upper oil phase no longer changes. Separate the upper hydrophobic oil phase to obtain microbial oil. The yield of microbial oil is 79.64%. The lower layer is a mixture of hydrophilic fatty acid salt and ethanol. The separation and recovery process of fatty acid-based deep eutectic solvent, ethanol and weak alkaline solution is the same as steps (d), (e) in Example 1.

[0105] The recovery rates of fatty acid-based deep eutectic solvent, ethanol and weak alkaline solution are 93.21%, 90.34% and 96.56%, respectively.

[0106] According to the national standard, the acid value of the obtained microbial oil is 1.68 mgKOH / g, and the peroxide value is 0.20 g / 100g.

[0107] Example 4

[0108] A green extraction method of microbial oil, comprising the following steps:

[0109] a. Take a certain amount of Yarrowia lipolytica fermentation broth after fermentation, centrifuge, remove the water phase, obtain wet cells, add 85% ethanol solution equivalent to 5 times the wet cells, and then place it in a high-voltage pulse treatment chamber. Set the treatment parameters and perform high-voltage pulse electric field cell disruption process; the high-voltage pulse electric field treatment conditions are 1.5KV / cm, pulse width is 10μs, and pulse number is 100 times.

[0110] b. To the ethanol water cell solution after disruption in step (a), add C8-C12 (3:1) fatty acid-based deep eutectic solvent with the same volume as the ethanol aqueous solution, then place the mixed solution on a magnetic stirrer at a speed of 1000 rpm, mix for 20h, and then centrifuge at 25℃, 8000 rpm for 10 min to separate the hydrophobic liquid phase.

[0111] c. To the hydrophobic liquid phase after the completion of the microbial oil extraction in step (b), add 10% polyetheramine D-230 aqueous solution equivalent to 1 mol equivalent of fatty acid-based deep eutectic solvent, mix thoroughly until the volume of the upper oil phase no longer changes. Separate the upper hydrophobic oil phase to obtain microbial oil. The yield of microbial oil is 79.64%. The lower layer is a mixture of hydrophilic fatty acid salt and ethanol. The separation and recovery process of fatty acid-based deep eutectic solvent, ethanol and weak alkaline solution is the same as steps (d), (e) in Example 1.

[0112] The recovery rates of fatty acid-based deep eutectic solvent, ethanol and weak alkaline solution are 93.21%, 90.34% and 96.56%, respectively.

[0113] The recovery rates of fatty acid-based deep eutectic solvent, ethanol and weak alkaline solution are 94.66%, 88.78% and 97.88%, respectively.

[0114] According to the national standard, the acid value of the obtained microbial oil is 1.70 mgKOH / g, and the peroxide value is 0.22 g / 100g.

[0115] Example 5

[0116] A green extraction method of microbial oil, comprising the following steps:

[0117] a. Take a certain amount of Yarrowia lipolytica fermentation broth after fermentation, centrifuge, remove the water phase, obtain wet cells, add 75% ethanol solution equivalent to 5 times the wet cells, and then place in a high-voltage pulse treatment chamber, set the treatment parameters, and perform high-voltage pulse electric field cell disruption process; the high-voltage pulse electric field treatment conditions are 1.5KV / cm, pulse width is 10μs, and pulse number is 100 times.

[0118] b. Add C8-C14(4:1) fatty acid-based deep eutectic solvent to the ethanol aqueous cell solution after cell disruption in step (a), and then place the mixed solution on a magnetic stirrer at a speed of 1000 rpm, and continuously mix for 20h, and then centrifuge at 25℃, 8000 rpm, 10 min, and separate to obtain a hydrophobic liquid phase.

[0119] c. Add 15% polyetheramine D-230 aqueous solution equivalent to 1 mol equivalent of fatty acid-based deep eutectic solvent to the hydrophobic liquid phase after microbial oil extraction in step (b), mix thoroughly until the volume of the upper oil phase no longer changes. Separate the upper hydrophobic oil phase to obtain microbial oil. The yield of microbial oil is 75.45%. The lower layer is a mixture of hydrophilic fatty acid salt and ethanol.

[0120] The separation and recovery process of fatty acid-based deep eutectic solvent, ethanol and weak alkaline solution is the same as steps (d) and (e) in Example 1.

[0121] The recovery rates of fatty acid-based deep eutectic solvent, ethanol and weak alkaline solution are 93.62%, 90.34% and 95.62% respectively. According to the national standard, the acid value of the obtained microbial oil is 1.73 mgKOH / g, and the peroxide value is 0.24 g / 100g.

[0122] Example 6

[0123] A green extraction method of microbial oil, comprising the following steps:

[0124] a. Take a certain amount of Yarrowia lipolytica fermentation broth after fermentation is completed, centrifuge, remove the water phase, obtain wet cells, add 95% ethanol solution equivalent to 5 times the wet cells, then place in a high-voltage pulse treatment chamber, set the treatment parameters, and perform a high-voltage pulse electric field cell disruption process; the high-voltage pulse electric field treatment conditions are 1KV / cm, pulse width is 7μs, and pulse number is 60 times.

[0125] b. To the ethanol water cell liquid after disruption in step (a), add an equal volume of C8-C14 (4:1) fatty acid-based deep eutectic solvent to the ethanol water solution, then place the mixed solution on a magnetic stirrer at a speed of 1000 rpm, continue to mix for 20h, then centrifuge at 25℃, 8000 rpm for 10 min, and separate to obtain a hydrophobic liquid phase.

[0126] c. To the hydrophobic liquid phase after microbial oil extraction in step (b), add 10% polyetheramine D-230 aqueous solution equivalent to 1 mol equivalent of fatty acid-based deep eutectic solvent, mix thoroughly until the volume of the upper oil phase no longer changes. Separate the upper hydrophobic oil phase to obtain microbial oil. The yield of microbial oil is 78.55%. The lower layer is a mixture of hydrophilic fatty acid salt and ethanol.

[0127] The separation process of the fatty acid-based deep eutectic solvent, ethanol and weak alkaline solution is the same as steps (d) and (e) in Example 1.

[0128] The recovery rates of the fatty acid-based deep eutectic solvent, ethanol and weak alkaline solution are 93.77%, 89.43% and 94.92% respectively. According to the national standard, the acid value of the obtained microbial oil is 1.82 mgKOH / g, and the peroxide value is 0.21 g / 100g.

[0129] Example 7

[0130] A green extraction method of microbial oil, comprising the following steps:

[0131] a. Take a certain amount of Yarrowia lipolytica fermentation broth after fermentation is completed, centrifuge, remove the water phase, obtain wet cells, add 85% ethanol solution equivalent to 5 times the wet cells, then place in a high-voltage pulse treatment chamber, set the treatment parameters, and perform a high-voltage pulse electric field cell disruption process; the high-voltage pulse electric field treatment conditions are 1KV / cm, pulse width is 0.25KV / cm, pulse width is 5μs, and pulse number is 10 times.

[0132] b. To the ethanol aqueous microbial solution after broken in step (a), add an equal volume of C9-C12 (2:1) fatty acid-based deep eutectic solvent to the ethanol aqueous solution, then place the mixed solution on a magnetic stirrer, mix for 20 h at a speed of 1000 rpm, and then centrifuge at 25°C, 8000 rpm for 10 min to separate the hydrophobic liquid phase.

[0133] c. To the hydrophobic liquid phase after the extraction of microbial oil in step (b), add an equivalent of 5% polyetheramine D-230 aqueous solution to 1 mol equivalent of fatty acid-based deep eutectic solvent, mix thoroughly until the volume of the upper oil phase no longer changes. Separate the upper hydrophobic oil phase to obtain microbial oil. The yield of microbial oil is 76.63%. The lower layer is a mixture of hydrophilic fatty acid salt and ethanol.

[0134] The separation process of fatty acid-based deep eutectic solvent, ethanol and weak alkaline solution is the same as steps (d) and (e) in Example 1.

[0135] The recovery rates of fatty acid-based deep eutectic solvent, ethanol and weak alkaline solution are 94.53%, 88.44% and 95.63%, respectively.

[0136] According to the national standard, the acid value of the obtained microbial oil is 1.67 mgKOH / g, and the peroxide value is 0.19 g / 100g.

[0137] Example 8

[0138] A green extraction method of microbial oil, comprising the following steps:

[0139] a. Take a certain amount of Yarrowia lipolytica fermentation broth after fermentation, centrifuge, remove the water phase, obtain wet microbial cells, add an equivalent of 75% ethanol solution to 5 times the wet microbial cells, and then place it in a high-voltage pulse treatment chamber. Set the treatment parameters to perform high-voltage pulse electric field cell disruption process; the high-voltage pulse electric field treatment conditions are 1KV / cm, pulse width is 1KV / cm, pulse width is 7μs, and pulse number is 60 times.

[0140] b. To the ethanol aqueous microbial solution after broken in step (a), add an equal volume of C9-C12 (2:1) fatty acid-based deep eutectic solvent to the ethanol aqueous solution, then place the mixed solution on a magnetic stirrer, mix for 20 h at a speed of 1000 rpm, and then centrifuge at 25°C, 8000 rpm for 10 min to separate the hydrophobic liquid phase.

[0141] c, to the hydrophobic liquid phase after the completion of the microbial oil extraction in step (b), add 15% polyether amine D-230 aqueous solution equivalent to 1 mol equivalent of fatty acid based deep eutectic solvent, mix thoroughly until the volume of the upper oil phase no longer changes. Separate the upper layer of hydrophobic oil phase to obtain microbial oil. The yield of microbial oil is 77.67%. The lower layer is a mixture of hydrophilic fatty acid salt and ethanol.

[0142] The separation process of fatty acid based deep eutectic solvent, ethanol and weak alkaline solution is the same as steps (d) and (e) in Example 1. The recovery rates of fatty acid based deep eutectic solvent, ethanol and weak alkaline solution are 95.19%, 90.64% and 96.45%, respectively.

[0143] According to the national standard, the acid value of the obtained microbial oil is 1.44 mgKOH / g, and the peroxide value is 0.23 g / 100g.

[0144] Example 9

[0145] A green extraction method of microbial oil, comprising the following steps:

[0146] a, take a certain amount of Yarrowia lipolytica fermentation broth after fermentation, centrifuge, remove the water phase, obtain wet cells, add 75% ethanol solution equivalent to 5 times the wet cells, and then place it in a high voltage pulse treatment chamber, set the treatment parameters, and carry out high voltage pulse electric field cell disruption process; the high voltage pulse electric field treatment conditions are 1KV / cm, pulse width is 1KV / cm, pulse width is 7μs, and pulse number is 60 times.

[0147] b, to the ethanol aqueous cell solution after disruption in step (a), add C8-C14 (4:1) fatty acid based deep eutectic solvent with the same volume as the ethanol aqueous solution, then place the mixed solution on a magnetic stirrer, mix for 20h at a speed of 1000rpm, and then centrifuge at 8000rpm for 10min at 25℃ to separate the hydrophobic liquid phase.

[0148] c, to the hydrophobic liquid phase after the completion of the microbial oil extraction in step (b), add 15% polyether amine D-230 aqueous solution equivalent to 1 mol equivalent of fatty acid based deep eutectic solvent, mix thoroughly until the volume of the upper oil phase no longer changes. Separate the upper layer of hydrophobic oil phase to obtain microbial oil. The yield of microbial oil is 77.67%. The lower layer is a mixture of hydrophilic fatty acid salt and ethanol.

[0149] The separation process of fatty acid based deep eutectic solvent, ethanol and weak alkaline solution is the same as steps (d) and (e) in Example 1. The recovery rates of fatty acid based deep eutectic solvent, ethanol and weak alkaline solution are 95.19%, 90.64% and 96.45%, respectively.

[0150] The recovery rates of fatty acid-based deep eutectic solvents, ethanol and weak alkaline solution were 94.53%, 91.12% and 90.45% respectively. According to the national standard, the acid value of the obtained microbial oil was 1.52 mgKOH / g, and the peroxide value was 0.24 g / 100g.

[0151] Comparative Example 1

[0152] A method for extracting microbial oil, comprising the following steps:

[0153] a. A certain amount of Yarrowia lipolytica fermentation broth after fermentation was placed in a high-voltage pulse treatment chamber, and the treatment parameters were set to perform a high-voltage pulse electric field cell disruption process; the high-voltage pulse electric field treatment conditions were 1.5KV / cm, pulse width was 10μs, and pulse number was 100 times.

[0154] b. The Yarrowia lipolytica fermentation broth treated in step (a) was centrifuged at 4°C and 8000rpm for 10min, and the liquid phase was separated;

[0155] c. To the wet microbial cells obtained in step (b), 1:1 (v / v) chloroform-methanol solution equivalent to 5 times the dry microbial cells was added, and mixed uniformly on a magnetic stirrer at 1000rpm and room temperature for 30min. Repeat the centrifugation operation in step (b) to separate the lower chloroform phase, and then vacuumize at 35°C and rotary evaporate. When there is no solvent drop into the solvent collection bottle, microbial oil is obtained. The yield of microbial oil is 85.63%.

[0156] According to the national standard, the acid value of the obtained microbial oil was 1.67 mgKOH / g, and the peroxide value was 0.65 g / 100g.

[0157] Comparative Example 2

[0158] A method for extracting microbial oil, comprising the following steps:

[0159] a. A certain amount of Yarrowia lipolytica fermentation broth after fermentation was placed in a high-voltage pulse treatment chamber, and the treatment parameters were set to perform a high-voltage pulse electric field cell disruption process; the high-voltage pulse electric field treatment conditions were 1.5KV / cm, pulse width was 10μs, and pulse number was 100 times.

[0160] b. The Yarrowia lipolytica fermentation broth treated in step (a) was centrifuged at 4°C and 8000rpm for 10min, and the liquid phase was separated;

[0161] c、to the wet bacteria obtained in step (b) is added 1:1:1 (v / v / v) n-hexane-methanol-water solution equivalent to 5 times the dry bacteria, mixed on a magnetic stirrer at 1000 rpm at room temperature for 30 min, repeat the centrifugation operation in step (b), separate the upper n-hexane phase, and then vacuumize at 35℃, rotary evaporation until no solvent drops into the solvent collection bottle, to obtain microbial oil. The yield of microbial oil is 68.45%.

[0162] According to the national standard, the acid value of the obtained microbial oil is 1.66 mgKOH / g, and the peroxide value is 0.94 g / 100g.

[0163] Comparative Example 3

[0164] A method for extracting microbial oil, comprising the following steps:

[0165] a. A certain amount of Yarrowia lipolytica fermentation broth after fermentation is placed in a high-voltage pulse treatment chamber, and the treatment parameters are set to perform high-voltage pulse electric field cell disruption process; the high-voltage pulse electric field treatment conditions are 1.5KV / cm, pulse width is 10μs, and pulse number is 100 times.

[0166] b. The Yarrowia lipolytica fermentation broth treated in step (a) is centrifuged at 4℃ and 8000 rpm for 10 min, and then the liquid phase is separated;

[0167] c. To the wet bacteria obtained in step (b) is added 1:1 (v / v) petroleum ether-methanol solution equivalent to 5 times the dry bacteria, mixed on a magnetic stirrer at 1000 rpm at room temperature for 30 min, repeat the centrifugation operation in step (b), separate the upper petroleum ether phase, and then vacuumize at 35℃, rotary evaporation until no solvent drops into the solvent collection bottle, to obtain microbial oil. The yield of microbial oil is 78.44%.

[0168] According to the national standard, the acid value of the obtained microbial oil is 1.67 mgKOH / g, and the peroxide value is 0.79 g / 100g.

[0169] Comparative Example 4

[0170] A method for extracting microbial oil, comprising the following steps:

[0171] a. A certain amount of Yarrowia lipolytica fermentation broth after fermentation is centrifuged to remove the water phase and obtain wet bacteria, and then 75% ethanol solution equivalent to 5 times the wet bacteria is added for re-dissolution, and then placed in a microwave environment to set the treatment parameters for microwave cell disruption process; the microwave treatment conditions are power 350W and time 10min.

[0172] b. To the ethanol aqueous microbial solution after crushing in step (a), add an equal volume of C8-C14 (4:1) fatty acid-based deep eutectic solvent to the ethanol aqueous solution, and then place the mixed solution on a magnetic stirrer, continuously mix for 20 h at a speed of 1000 rpm, and then centrifuge at 25°C, 8000 rpm for 10 min to separate the hydrophobic liquid phase.

[0173] c. To the hydrophobic liquid phase after the completion of microbial oil extraction in step (b), add 10% polyetheramine D-230 aqueous solution equivalent to 1 mol equivalent of fatty acid-based deep eutectic solvent, mix thoroughly until the volume of the upper oil phase no longer changes. Separate the upper hydrophobic oil phase to obtain microbial oil. The yield of microbial oil is 70.23%. The lower layer is a mixture of hydrophilic fatty acid salt and ethanol.

[0174] The separation process of fatty acid-based deep eutectic solvent, ethanol and weak alkaline solution is the same as steps (d) and (e) in Example 1.

[0175] The recovery rates of fatty acid-based deep eutectic solvent, ethanol and weak alkaline solution are 94.22%, 91.55% and 90.67%, respectively.

[0176] According to the national standard, the acid value of the obtained microbial oil is 1.97 mgKOH / g, and the peroxide value is 0.98 g / 100g.

[0177] Comparative Example 5

[0178] A method for extracting microbial oil, comprising the following steps:

[0179] a. Take a certain amount of Yarrowia lipolytica fermentation broth after fermentation, centrifuge to remove the water phase, obtain wet microbial cells, add 75% ethanol solution equivalent to 5 times the wet microbial cells, and then place it in a high-pressure homogenizer, set the processing parameters, and perform high-pressure homogenization cell crushing process; the high-pressure homogenization treatment conditions are 40 MPa, and homogenization is performed twice.

[0180] b. To the ethanol aqueous microbial solution after crushing in step (a), add an equal volume of C8-C14 (4:1) fatty acid-based deep eutectic solvent to the ethanol aqueous solution, and then place the mixed solution on a magnetic stirrer, continuously mix for 20 h at a speed of 1000 rpm, and then centrifuge at 25°C, 8000 rpm for 10 min to separate the hydrophobic liquid phase.

[0181] c. To the hydrophobic liquid phase after the completion of microbial oil extraction in step (b), add 10% polyetheramine D-230 aqueous solution equivalent to 1 mol equivalent of fatty acid-based deep eutectic solvent, mix thoroughly until the volume of the upper oil phase no longer changes. Separate the upper hydrophobic oil phase to obtain microbial oil. The yield of microbial oil is 63.42%. The lower layer is a mixture of hydrophilic fatty acid salt and ethanol.

[0182] The separation process of fatty acid-based deep eutectic solvent, ethanol and weak alkaline solution is the same as steps (d) and (e) in Example 1.

[0183] The recovery rates of fatty acid-based deep eutectic solvent, ethanol and weak alkaline solution are 94.18%, 91.44% and 90.67%, respectively.

[0184] According to the national standard, the acid value of the obtained microbial oil is 1.53 mgKOH / g, and the peroxide value is 0.24 g / 100g.

[0185] Table 1 Extraction rate of microbial oil of examples and comparative examples

[0186]

[0187] As can be seen from the above table, the fatty acid-based deep eutectic solvent and ethanol solvent can be used as a green extraction solvent for microbial oil, and the extraction efficiency is close to that of the traditional organic solvent extraction method. For example, the oil extraction rate of Example 1 is 84.75%, and the oil extraction rate of Comparative Example 1 is 85.63%, which is not much different. In addition, as compared with Comparative Examples 4 and 5 and Example 9, the microbial oil extraction rate of Example 9 using high-voltage pulsed electric field is higher. The green extraction method shown in the present application can be used as a substitute for the traditional organic solvent extraction method.

[0188] Although the present application has been disclosed with reference to the preferred embodiments above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application. Therefore, the protection scope of the present application should be defined by the claims.

Claims

1. A green extraction method for microbial oils, characterized in that, Includes the following steps: S1. Wet cells were separated from the fermentation broth of oil-producing microorganisms. An ethanol-water solution was added to the wet cells to obtain an ethanol-water reconstituted bacterial solution. S2 The ethanol-water reconstituted bacterial solution from step S1 is placed in a high-voltage pulsed electric field to disrupt the cells and obtain a mixture containing microbial lipids. S3. Add a fatty acid-based eutectic solvent to the mixture containing microbial oil in step S2 and mix thoroughly. Then, perform ultrasonic treatment to achieve three-phase separation of hydrophobic, hydrophilic and solid phases. The fatty acid-based eutectic solvent is a mixture of hydrogen bond acceptors and hydrogen bond donors, wherein the hydrogen bond acceptor is any one or two of hexanoic acid (C6), octanoic acid (C8), and nonanoic acid (C9); and the hydrogen bond donor is any one of decanoic acid (C10), lauric acid (C12), and myristic acid (C14). The volume ratio of the fatty acid-based eutectic solvent to the aqueous ethanol solution is 1:(0.5~2). The hydrophobic phase is a mixed solution composed of microbial oil and fatty acid-based eutectic solvent; The hydrophilic phase is an aqueous ethanol solution; The solid phase consists of wet bacterial cells and insoluble impurities; S4. Adding a weakly alkaline aqueous solution to the hydrophobic phase of step S3 causes the polarity of the hydrophobic fatty acid group eutectic solvent to switch, generating a hydrophilic fatty acid salt, thereby separating the hydrophobic microbial oil and obtaining a hydrophilic mixture containing fatty acid salt and ethanol; the weakly alkaline aqueous solution is an aqueous solution prepared from any one of the compounds polyetheramine, ethylenediamine, propylenediamine, and diethylenetriamine.

2. The method according to claim 1, characterized in that, In step S1, the volume fraction of the ethanol-water solution is 75% or higher; the volume ratio of the wet bacterial cells to the ethanol-water solution is 1:(3~5).

3. The method according to claim 1, characterized in that, In step S4, the volume fraction of the solute in the weakly alkaline aqueous solution is 5-15%. The amount of the weakly basic compound added is 4 to 8 times the volume of the fatty acid-based eutectic solvent.

4. The method according to claim 1, characterized in that, It also includes step S5, which involves recovering the fatty acid-based eutectic solvent, ethanol, and weakly alkaline aqueous solution from the fatty acid salt and ethanol mixture obtained in step S4.

5. A microbial oil extraction and separation apparatus using the method described in any one of claims 1 to 4, characterized in that, The system includes a main body (1), which includes a pulsed electric field treatment chamber (2), a microbial oil extraction chamber (11), and an extraction solvent separation chamber (17). The bottom of the pulsed electric field treatment chamber (2) is connected to the top of the microbial oil extraction chamber (11), and the bottom of the microbial oil extraction chamber (11) is connected to the top of the extraction solvent separation chamber (17). The pulsed electric field treatment chamber (2) is used to break down microbial cells using a high-voltage pulsed electric field; The microbial oil extraction chamber (11) is used to separate microbial oil from the product treated by the pulsed electric field treatment chamber (2); The extraction solvent separation chamber (17) is used to recover fatty acid-based eutectic solvent, ethanol, and weakly alkaline aqueous solution from the byproducts of the microbial oil extraction chamber (11). The microbial oil extraction chamber (11) is equipped with a stirrer (12) and an ultrasonic generator (13). The stirrer (12) is used to mix the materials in the microbial oil extraction chamber (11), and the ultrasonic generator (13) releases ultrasonic waves into the microbial oil extraction chamber (11) through an ultrasonic power supply (9) connected to it. The microbial oil extraction chamber (11) is provided with a second feed inlet (10) at the top and a microbial oil outlet (16) at the bottom. The bottom of the microbial oil extraction chamber (11) is connected to the extraction solvent separation chamber (17) through at least one second transfer channel (15). Density sensors (14) are provided at both the upper and lower ends of the microbial oil extraction chamber (11).

6. The apparatus according to claim 5, characterized in that, The pulse electric field processing chamber (2) is equipped with a discharger (7) and a receiver (6). The discharger (7) is located on the inner wall of the pulse electric field processing chamber (2) and releases an electrical signal to the pulse electric field processing chamber (2) through an electrical connection to a high-voltage pulse power supply (4) to form a pulse electric field. The receiver (6) is electrically connected to the high-voltage pulse power supply (4) and receives the electrical signal from the discharger (7). The pulse electric field treatment chamber (2) is also provided with a first feed inlet (3) at the top, and the bottom of the pulse electric field treatment chamber (2) is connected to the microbial oil extraction chamber (11) through at least one first transfer channel (8).

7. The apparatus according to claim 5, characterized in that, The extraction solvent separation chamber (17) is divided into an upper layer and a lower layer. The upper layer is provided with one or more perforated plates (20). Each perforated plate (20) is connected to a liquid receiving plate (21) below it. Each liquid receiving plate (21) is funnel-shaped and has a liquid outlet at the center. The liquid filtered out by the perforated plates (20) is collected on the liquid receiving plate (21) and flows down through the liquid outlet. The top of the upper layer is connected to an air inlet (18) and the bottom is connected to an exhaust outlet (22). The bottom side of the lower layer is provided with a two-phase discharge outlet (23).

8. The use of the method according to any one of claims 1 to 4 or the apparatus according to any one of claims 5 to 7 in the preparation of products containing microbial oils.

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