Bio-oil extraction method with improved oil recovery rate using cooling process

Abstract

The present invention relates to a bio-oil extraction method in which an oil recovery rate is improved by utilizing a cooling process. The bio-oil extraction method of the present invention, which adds a process of cooling a suspension of crushed cells of a culture solution containing microalgae, exhibits a high oil recovery rate. Since no other additives are added to increase the oil recovery rate, the oil can be recovered and the remaining by-products can be used as feed, making sustainable production possible. The recovered oil can be usefully utilized in feed compositions or food compositions, etc.

Description

[Technical Field]

[0001] Cross-Citation of Related Applications This application claims the benefit of priority based on Korean Patent Application No. 10-2022-0012556, filed January 27, 2022, and all contents disclosed in the documents of this Korean patent application are incorporated herein by reference.

[0002] The present invention relates to a method for extracting bio-oil using a cooling process, through which bio-oil can be extracted from microalgae with a high oil recovery rate. [Background technology]

[0003] Docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid with numerous benefits in preventing cardiovascular diseases such as high blood pressure, angina pectoris, and myocardial infarction, is generally considered an essential fatty acid because it cannot be synthesized in the human body and must be ingested. DHA is found in large amounts primarily in fish oils. It is commonly found in high concentrations in salmon, sardines, and tuna, and is often extracted from fish livers, with cod liver being the most widely used source worldwide. However, the production of DHA oil from fish oils has raised many issues. Overfishing has led to the destruction of marine ecosystems, and fish oils contain large amounts of organic pollutants such as mercury, heavy metals, and dioxins, which are marine environmental pollutants, raising persistent concerns about the risk of human exposure to harmful substances.

[0004] DHA, an omega-3 extracted from microalgae, solves the problems associated with fish oil-derived omega-3 DHA. Microorganisms belonging to the genera Thraustochytrium and Schizochytrium, which are marine microalgae, are capable of producing polyunsaturated fatty acids. The method for producing omega-3 polyunsaturated fatty acids (PUFAs) using microalgae was first described by Ellenbogen (Ellenbogen B. B et al., Comparative Biochemistry and Physiology, 29:805-811 (1969)). By cultivating pure microalgae under 100% clean conditions, DHA oil production poses no risk to the marine ecosystem. Furthermore, mass production through cultivation is possible, enabling stable supply of quality. Furthermore, DHA oil has attracted attention due to its higher content of unsaturated fatty acids than fish oil and its reduced risk of extracting impurities such as heavy metals, leading to a growing market.

[0005] The most common method for producing oil containing polyunsaturated fatty acids (PUFAs) from microalgae involves fermenting and cultivating microalgae containing the target substance in a reactor, recovering and drying them, and then extracting them using a solvent. However, organic solvent extraction requires explosion-proof equipment and increases production costs due to the solvent recovery process. There is also the risk of residual solvents remaining in the oil. To address these issues, there is a need for a method for extracting oil from cells without using organic solvents. Solvent-free extraction involves methods for obtaining oil without or with minimal use of organic solvents such as hexane, such as enzymatic hydrolysis and heat treatment. Solvent-free extraction typically utilizes the difference in specific gravity between the oil and other polar substances, such as centrifugation or natural maceration, to obtain oil. This method has the advantage of eliminating the use of organic solvents, which are harmful to the human body. However, there is a problem in that phospholipids derived from the outer wall of microorganisms, which can hinder oil separation from microalgae, act as emulsifiers, reducing oil recovery rates.

[0006] To solve this problem, there are methods such as adding acidic and basic chemicals to adjust the pH or adding large amounts of salt substances such as sodium chloride (US16 / 473805). However, these methods do not allow the by-products remaining after oil extraction to be used as animal feed and lead to rapid corrosion of the equipment. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] US Published Patent US2019 / 0323043A [Patent Document 2] US Patent Application US16 / 473805 [Non-patent literature]

[0008] [Non-Patent Document 1] Ellenbogen B. B et al, Comparative Biochemistry and Physiology, 29:805-811 (1969) Summary of the Invention [Problem to be solved by the invention]

[0009] One embodiment of the present application is 1) obtaining a cell lysate from a microalgae culture; 2) centrifuging the cell lysate to recover the supernatant; 3) cooling the supernatant; 4) heating the cooled supernatant; and 5) centrifuging the heated supernatant to recover the oil. [Means for solving the problem]

[0010] Each description and embodiment disclosed in this application may also be applied to each other description and embodiment. In other words, all combinations of the various elements disclosed in this application are within the scope of this application. Furthermore, the specific descriptions set forth below are not intended to limit the scope of this application. Furthermore, those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific aspects of this application described herein. Furthermore, such equivalents are intended to be encompassed by this application.

[0011] One embodiment of the present application is 1) obtaining a cell lysate from a microalgae culture; 2) centrifuging the cell lysate to recover the supernatant; 3) cooling the supernatant; 4) heating the cooled supernatant; and 5) centrifuging the heated supernatant to recover the oil.

[0012] As used herein, the term "microalgae" refers to microscopic algae that are too small to be seen with the naked eye, but only under a microscope. Microalgae include a wide variety of species, including strains that are unable to photosynthesize and grow solely heterotrophically. Because microalgae can fix carbon dioxide and have high protein and lipid content, they can be used as biomass for food, feed, or fuel production. Specifically, the lipid components contained in microalgae can be used as a feedstock for producing biodiesel oil, which is used in liquid fuels.

[0013] In this specification, the microalgae may be strains of the genus Schizochytrium, Thraustochytrium, Japonochytrium, Ulkenia, Crypthecodinium, or Haliphthoros, more preferably strains of the genus Schizochytrium or Thraustochytrium, and most preferably strains of the genus Schizochytrium.

[0014] The term "culture medium" used herein includes products produced by culturing microalgae and may be used in the same sense as the term "fermentation medium." Specifically, it may be a culture medium containing microalgae, or a culture filtrate obtained by removing microalgae from the culture medium, but is not limited thereto.

[0015] The microalgae culture solution can be produced by inoculating a microalgae culture medium with microalgae and using a culture method known in the art.

[0016] The term "disruption solution" as used herein refers to a solution containing the contents of disrupted cells, and can be obtained by disrupting a microalgae culture solution by treating it with an enzyme or by disrupting it using a physical method.

[0017] The term "supernatant" as used herein may refer to the liquid located at the top of the centrifuged liquid, and may be used in the same sense as "supernatant liquid." Furthermore, in this specification, the "supernatant" in the step of centrifuging a cell lysate obtained by treating microalgae in a culture medium with an enzyme or by physically lysing the microalgae may be used in the same sense as an "emulsified layer," "emulsion," or "disrupted suspension." Furthermore, the supernatant in the step of collecting the supernatant, cooling and / or heat-treating it, and then centrifuging the collected supernatant may contain "oil."

[0018] In step 1) of the present invention, the cell lysate may be a cell lysate obtained by treating a microalgae culture solution with an enzyme to lyse the cells.

[0019] The enzyme used to disrupt the microalgae culture liquid may be alcalase, protease, cellulase, pectinase, chitinase, lipase, β-glucanase, xylanase, mannanase, amylase, or a combination thereof, and is preferably alcalase, but is not limited thereto.

[0020] In step 1) of the present invention, the cell lysate may be obtained by lysing the cells using a physical method.

[0021] The term "physical method" refers to a method of crushing microalgae using equipment capable of destroying the cell walls of microalgae (i.e., a crusher), and may be a bead mill, French pressure, Braun homogenizer, or microfluidizer, preferably a bead mill or homogenizer, and more preferably a bead mill.

[0022] In the present invention, the centrifugation step 2) can be carried out at 3000 g to 4600 g, 3200 g to 4400 g, 3400 g to 4200 g, or 3600 g to 4000 g for 1 to 10 minutes, 3 to 8 minutes, or 4 to 6 minutes.

[0023] In the examples of the present invention, the oil recovery rate was compared depending on whether or not a centrifugation process was performed before the cooling process. As a result, it was confirmed that the oil recovery rate was low when the microalgae culture solution underwent a temperature adjustment process of cooling and heat treatment without a centrifugation process, but that the oil recovery rate increased significantly when the supernatant recovered from the centrifugation of the microalgae culture solution underwent a cooling and heat treatment process.

[0024] In the present invention, the cooling step 3) can be carried out at a temperature of 45°C or less, 40°C or less, 35°C or less, 30°C or less, 5°C to 45°C, 5°C to 40°C, or 5°C to 35°C.

[0025] The cooling step 3) can be carried out for 30 minutes or more, 40 minutes or more, 50 minutes or more, or 30 minutes to 4 hours, but is not limited thereto.

[0026] The time for the cooling step 3) may vary depending on the step 1) of obtaining the cell lysate. Specifically, when the cell lysate in step 1) is obtained by enzymatic treatment, the cooling step 3) may be performed for 1 hour to 5 hours, 1 hour 30 minutes to 4 hours 30 minutes, 2 hours to 4 hours, or 2 hours 30 minutes to 3 hours 30 minutes, but is not limited to these. When the cell lysate in step 1) is obtained using a physical method, the cooling step 3) may be performed for 30 minutes to 3 hours 30 minutes, 30 minutes to 3 hours, 30 minutes to 2 hours 30 minutes, 30 minutes to 2 hours, or 30 minutes to 1 hour 30 minutes, but is not limited to these.

[0027] In the present invention, the heating step 4) can be carried out at a temperature of 50°C or higher, 55°C or higher, 60°C or higher, 50°C to 75°C, 50°C to 70°C, 50°C to 65°C, 55°C to 75°C, 55°C to 70°C, or 55°C to 65°C.

[0028] The heating step 4) can be carried out for 30 minutes or more, 40 minutes or more, 50 minutes or more, or 30 minutes to 4 hours, but is not limited thereto.

[0029] The method for extracting oil from a microalgae culture solution of the present invention can improve the oil recovery rate.

[0030] As used herein, the term "oil" is used interchangeably with "bio-oil" and refers to all types of oil contained in microalgae, including, for example, polyunsaturated fatty acids (e.g., omega-3 unsaturated fatty acids and omega-6 unsaturated fatty acids), saturated fatty acids such as lauric acid, myristic acid, palmitic acid, stearic acid, and arachidic acid, phospholipids, and steroid compounds.

[0031] As used herein, the term "oil recovery rate" refers to the amount of oil that can be extracted from the oil content in the cells, i.e., the crude fat content, when oil is extracted through experiments.

[0032] When oil is extracted using the method for extracting oil from a microalgae culture solution of the present invention, the oil recovery rate can be 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more based on the total weight of crude fat in the microalgae.

[0033] In the examples of the present invention, it was confirmed that the oil recovery rate can be significantly increased by centrifuging the cell lysate of microalgae, followed by cooling and heat treatment. Therefore, the method of extracting oil of the present invention, which includes the steps of centrifuging, cooling and heating the cell lysate obtained from a microalgae culture medium, increases the oil recovery rate and does not require the addition of other additives. Therefore, the oil can be recovered and the remaining by-products can be used as animal feed, making it a useful method that enables sustainable production. [Effects of the Invention]

[0034] The bio-oil extraction method of the present invention, which includes a step of cooling a suspension of disrupted cells of a culture solution containing microalgae, exhibits a high oil recovery rate. Since no other additives are added to increase the oil recovery rate, the oil can be recovered and the remaining by-products can be used as feed, enabling sustainable production. The recovered oil can be usefully used in feed compositions or food compositions, etc. DETAILED DESCRIPTION OF THE INVENTION

[0035] The present invention will be described in more detail with reference to the following examples. However, these examples are intended to illustrate one or more specific examples, and the scope of the present invention is not limited to these examples.

[0036] Example 1. Extraction of bio-oil from cell lysate using enzyme treatment Example 1-1. Obtaining cell lysate from microalgae whose cell walls have been disrupted by enzyme treatment A microalgae strain, Schizochytrium sp., was cultured in a 5 L fermenter for 60 hours, with glucose as a carbon source being supplied at 35% of the total culture medium.

[0037] For the purpose of seed culture, sterilized MJW02 medium was used and cultured in a 500 mL flask at 30°C and 150 rpm for approximately 20 hours.

[0038] The flask containing the seed culture was dispensed and inoculated into a 5 L fermenter, and culture was carried out in sterilized MJW02 medium under the conditions of 30°C, 500 rpm, and 1.5 vvm to obtain a culture solution.

[0039] After heating the culture medium to 60°C, Alcalase (Alcalase 2.4FG, Novozymes) was added at 0.2% (w / w) based on the weight of the culture medium, and the mixture was stirred at 60°C for 3 hours to prepare a cell lysate in which the outer cell walls had been lysed.

[0040] Example 1-2: Comparison of oil recovery rate with and without cooling process The cell lysate obtained in Example 1-1 was centrifuged at 3800 g for 5 minutes, and the supernatant was collected using a dropper or pipette, and it was confirmed whether or not the oil recovery rate was improved depending on whether or not cooling treatment was performed.

[0041] Specifically, the microalgae culture of Example 1-1 was treated according to the process sequences and conditions 1-A, 1-B, 1-C, 1-D, 1-E, and 1-F shown in Table 1 below, and the oil recovery rates were compared. The heating and cooling rates were adjusted to ±0.5°C / min, and after reaching the set temperature, the culture was heated and cooled for the time specified by the process conditions. The oil recovery rate was calculated using the following formula by measuring the ratio of the recovered oil to the weight of fat in the culture medium introduced. The weight of the recovered oil was measured after the supernatant after the final centrifugation step was recovered using a dropper or pipette. Oil recovery rate (%) = recovered oil weight / (culture solution weight * solids %) x 100

[0042] [Table 1]

[0043] As a result, as shown in Table 1, when the supernatant was not heated or cooled (Case 1-A), the oil recovery rate was 0%, and when it was heated at 60°C for about 1 hour and then immediately centrifuged (Case 1-B), the oil recovery rate was 20%. When it was heated at a higher temperature of 75°C for 1 hour (Case 1-C), the oil recovery rate was 22%, confirming that the oil recovery rate did not improve.

[0044] In contrast, when the supernatant was cooled at 30°C for 3 hours and then heated at 60°C for 1 hour (Case 1-D), or when it was cooled at 25°C for 3 hours and then heated at 60°C for 1 hour (Case 1-E), a high oil recovery rate of 88% was observed. It was confirmed that the oil recovery rate increased significantly if the supernatant was cooled and then heated again.

[0045] However, in cases such as Case 1-F, which only underwent a cooling process without heat treatment, the oil recovery rate was rather low at 8%. This is thought to be because the saturated fatty acids contained in the oil extracted from microalgae are solid at room temperature and are difficult to recover by centrifugation, resulting in a low extraction yield.

[0046] Example 1-3: Comparison of oil recovery rates with and without a centrifugal separation process before cooling treatment The following experiment was conducted to compare the oil recovery rate between the presence and absence of a centrifugation process before cooling the microalgae cell lysate, in which the cells were lysed through enzyme treatment.

[0047] Specifically, the microalgae cell lysate of Example 1-1 was treated according to the process sequences and conditions of 2-A and 2-B shown in Table 2 below, and the oil recovery rates were compared. The heating and cooling rates were adjusted to ±0.5°C / min, and after reaching the set temperature, the samples were heated and cooled for the time specified by the process conditions. The oil recovery rate was measured in the same manner as in Example 1-2.

[0048] [Table 2]

[0049] As a result, as shown in Table 2, when enzyme-treated microalgae cell lysate was subjected to the temperature adjustment processes of cooling and heating without centrifugation, the oil recovery rate was low at 9% (Case 2-A). In contrast, when the supernatant recovered from centrifugation was subjected to the same temperature adjustment process as Case 2-A, the oil recovery rate was confirmed to be significantly higher at 92% (Case 2-B). This indicates that when enzyme-treated microalgae cell lysate is used directly without centrifugation, oil recovery is difficult even after the temperature adjustment process. However, when using the supernatant, which contains a mixture of microalgae cell outer walls and oil through the centrifugation process, the oil recovery rate was confirmed to be significantly higher after the temperature adjustment processes of cooling and heating.

[0050] Example 2. Extraction of bio-oil from cell lysate using physical treatment Example 2-1. Obtaining a microalgae cell lysate by physically disrupting the cell walls A microalgae strain, Schizochytrium sp., was cultured in a 5 L fermenter for 60 hours, with glucose as a carbon source being supplied as 35% of the total culture medium.

[0051] For the purpose of seed culture, sterilized MJW02 medium was used and cultured in a 500 mL flask at 30°C and 150 rpm for approximately 20 hours.

[0052] The flask containing the seed culture was dispensed and inoculated into a 5 L fermenter, and culture was carried out in sterilized MJW02 medium under the conditions of 30°C, 500 rpm, and 1.5 vvm to obtain a culture solution.

[0053] After the cultivation, the microalgae culture solution was placed in a bead mill (Dyno-mill ECM-MULTI LAB) and filled to approximately 65% ​​of the reactor with 0.8 mm beads. The reactor was maintained at 30°C and one pass was carried out at a rotation speed of 3600 rpm and a culture solution injection rate of 0.15 kg / min, yielding a cell disruption solution with a cell disruption rate of 90% or more, a biomass concentration of 100 g / L or more, a pH of 7, and a temperature of 30°C.

[0054] Example 2-2. Comparison of oil recovery rate with and without cooling process Using the microalgae cell lysate obtained by physical treatment in Example 2-1, it was confirmed whether or not the oil recovery rate was improved by cooling treatment.

[0055] Specifically, the physically treated microalgae cell lysate obtained in Example 2-1 was treated according to the process sequences and conditions shown in Table 3 below, 3-A, 3-B, 3-C, 3-D, 3-E, and 3-F, and the oil recovery rates were compared. A 500 ml double jacket was connected to a circulator (Julabo FP50-HE) to adjust the temperature, and the process solution was poured into the double jacket for the experiment. The heating and cooling rates were adjusted to ±1°C / min, and after reaching the set temperature, the solution was heated and cooled for the time specified in the process conditions shown in the table below. The oil recovery rate was measured in the same manner as in Example 1-2.

[0056] [Table 3]

[0057] As a result, as shown in Table 3, when the microalgae cell lysate was not subjected to heating or cooling treatment (Case 3-A), the oil recovery rate was low at 1.5%, and when it was heat-treated at 60°C for about 1 hour and then immediately centrifuged (Case 3-B), the oil recovery rate was 33.7%. When it was heat-treated at a higher temperature of 75°C for 1 hour (Case 3-C), the oil recovery rate was 32.3%, confirming that the oil recovery rate did not improve.

[0058] On the other hand, when microalgae cell lysate was cooled at 25°C for 1 hour and then heat-treated at 60°C for 1 hour (Case 3-D), and when microalgae cell lysate was cooled at 15°C for 1 hour and then heat-treated at 60°C for 1 hour (Case 3-E), high oil recovery rates of 76% and 75.8%, respectively, were observed. It was also confirmed that when microalgae cell lysate was cooled at 5°C for 1 hour (Case 3-F), a high oil recovery rate of 78.4% was observed. These results demonstrate that the oil recovery rate can be significantly increased by cooling microalgae cell lysate obtained by physical processing and then heat-treating it again.

[0059] Example 2-3. Comparison of oil recovery rates with and without a centrifugal separation process before cooling treatment The following experiment was conducted to compare the oil recovery rate between the presence and absence of a centrifugation process before cooling the microalgae cell lysate, in which the cells were lysed through physical treatment.

[0060] Specifically, the microalgae cell disruption process solution was obtained by disrupting the cell walls using physical treatment in the same manner as in Example 2-1, except that two passes were performed using a bead mill (Dyno-mill ECM-MULTI LAB) at a rotation speed of 2400 rpm and a culture medium injection rate of 0.15 kg / min. The resulting cell disruption process solution was placed in a 50 ml falcon, the temperature was adjusted using a Thermomix, and the mixture was stirred at 600 rpm for the experiment. Microalgae suspensions in which cells had been disrupted by a certain physical treatment before centrifugation were processed according to the process sequences and conditions shown in Table 1 below, 4-A and 4-B, and the oil recovery rates were compared. The heating and cooling rates were adjusted to ±1°C / min, and after reaching the set temperature, the samples were heated and cooled for the time specified in the process conditions below. The oil recovery rate was measured using the same method as in Example 1-2.

[0061] [Table 4]

[0062] As a result, as shown in Table 4, when microalgae cell lysate that had been processed using a bead mill was subjected to the temperature control processes of cooling and heating without centrifugation, the oil recovery rate was low at 48.3% (Case 4-A).In contrast, when the supernatant containing the cell wall material and oil recovered from the microalgae cell lysate that had been processed using a bead mill was centrifuged at 3800g for 5 minutes and subjected to the same cooling and heating processes as Case 4-A, the oil recovery rate increased to 95.4% (Case 4-B).

Claims

1. 1) obtaining a cell lysate from a culture of a strain of the genus Schizochytrium; 2) centrifuging the cell lysate to recover the supernatant; 3) cooling the supernatant; 4) heating the cooled supernatant; and 5) centrifuging the heated supernatant to recover the oil; The cooling step of 3) is carried out at a temperature of 5°C to 35°C; The heating step of 4) is carried out at a temperature of 55°C or higher. A method for extracting oil from a culture of a Schizochytrium strain.

2. 2. The method for extracting oil from a culture solution of a Schizochytrium strain according to claim 1, wherein the cell lysate in step 1) is obtained by lysing the cells through an enzymatic or physical treatment.

3. 3. The method for extracting oil from a microalgae culture solution according to claim 2, wherein the physical treatment involves physically disrupting the microalgae culture solution using a bead mill, a French pressure, a homogenizer, or a microfluidizer.

4. 2. The method for extracting oil from a culture broth of a Schizochytrium sp. strain according to claim 1, wherein the centrifugation in step 2) is performed at 3,000 to 4,600 g.

5. 2. The method for extracting oil from a culture broth of a Schizochytrium strain according to claim 1, wherein the cooling step (3) is carried out for 30 minutes to 4 hours.

6. 2. The method for extracting oil from a culture broth of a Schizochytrium strain according to claim 1, wherein the heating step (4) is carried out for 30 minutes to 4 hours.

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