Method for extracting lipophilic useful substance from moist cells of green alga genus coccomyxa

Abstract

To provide a method for extracting a lipophilic useful substance synthesized by green alga genus Coccomyxa in a simple, inexpensive, and efficient manner.SOLUTION: A method for extracting lipophilic useful substances such as oils and fats, comprising: a pretreatment step of heat-treating cells of green alga belonging to genus Coccomyxa at a temperature of 50°C or higher for 5 minutes or longer; and a solvent extraction step of extracting lipophilic useful substances such as oils and fats from the cells heat-treated in the pretreatment step by using an organic solvent.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a technique for easily and efficiently extracting useful lipophilic substances from wet cells of the green algae Coccomyxa genus. [Background technology]

[0002] While the production of oils and fats using microalgae and the production of biofuels using those oils as raw materials have attracted much attention, there is still no clear outlook for commercialization. The main reason for this is the large amount of labor and energy required in each process, from cultivation to oil extraction. As a result, the cost of biofuel production is very high, the total CO2 emissions (lifecycle GHG emissions) during biofuel production increase, and the energy balance ratio [(energy produced by biofuel production) / (energy input into biofuel production)] is less than 1.

[0003] Many microalgae, including green algae, accumulate lipids primarily composed of triglycerides. Extracting these lipids using nonpolar solvents such as hexane requires drying the algal biomass prior to extraction. However, the energy required to evaporate water (heat of evaporation) is enormous, and drying significantly worsens the energy balance ratio in biofuel production. For example, when microalgae cells are harvested from a culture solution using a centrifuge, a slurry-like biomass is obtained, but the weight of the water contained therein is generally more than five times the weight of the lipid. In this case, the energy required to dry this slurry is calculated to be more than one-third of the energy contained in the lipid. For these reasons, research into extracting lipids from wet algal biomass has become increasingly active.

[0004] While methods such as oil extraction are used to extract oil from oilseeds, the hard cell walls of microalgae make extraction difficult. Therefore, other methods, such as oil extraction using organic solvents, must be developed. Soybean oil extraction using organic solvents was already commercialized before the war. Meanwhile, organic solvent extraction from microalgae remains in the development stage. Other methods, such as supercritical CO2 and ionic liquids, have also been proposed. Research has also begun on oil extraction using deep-eutectic solvents and switching solvents, which have properties similar to ionic liquids but are less expensive than them, but there is still a long way to go before they can be put to practical use (Non-Patent Document 1).

[0005] Because many microalgae with high oil productivity are green algae, numerous papers have been published on the organic solvent extraction of oils contained in green algae. However, the efficiency of oil extraction from wet cells is generally low, which is believed to be due to the water-containing biological material, particularly the cell wall, preventing the penetration of organic solvents into the cells. The need for pretreatment to remove this barrier has become widely recognized, and various pretreatment methods have been attempted. Typical pretreatment methods include physical cell disruption methods such as ultrasonic disruption, microwave irradiation, homogenization, and freeze-thawing. However, these methods require significant energy input and capital investment for disruption. Meanwhile, cell wall weakening using enzymes has also been studied, but enzymes are expensive and unsuitable for biofuel production, which has a low unit price. Therefore, chemical pretreatment methods, such as cell disruption using acids, alkalis, and surfactants, have also been studied, but these methods often fail to produce significant results (Non-Patent Document 2).

[0006] On the other hand, only a few papers have reported oil extraction methods using organic solvents without pretreatment. For example, Yang et al. were able to recover almost 100% of the oil from wet cells of the green alga Picochlorum by extracting them with ethanol as a solvent at room temperature (Non-Patent Document 3). Later, it was reported that a similar method could be used to extract oil with high efficiency not only from Picochlorum but also from Chlorella (Non-Patent Document 4).

[0007] However, this method cannot be applied to all green algae. For example, when extracting oil from Scenedesmus almeriensis, the extraction efficiency was about 20% when extracted with ethanol at room temperature for 24 hours (Non-Patent Document 5).

[0008] Similarly, when oil was extracted from Auxenochlorella protothecoides using a mixed solvent of ethanol:hexane (2.6:1) over a day and night, only 4% or less of the dry weight of the algae could be extracted. On the other hand, by heating the algae at 70°C for 5 minutes or more, the amount of oil extracted increased to approximately 35% of the dry weight of the algae. Although this report did not calculate the oil extraction efficiency, since the oil content of Auxenochlorella is approximately 50% at most, the oil extraction efficiency is thought to be 70% or more (Non-Patent Document 6).

[0009] One possible reason for the large differences in the efficiency of oil and fat solvent extraction among green algae is differences in cell wall structure. Analysis of cell wall structure using an electron microscope (Non-Patent Document 7) and analysis of the sugars that make up the cell wall (Non-Patent Document 8) have shown that the cell wall structure of the Chlorella genus varies from strain to strain. However, because classification of green algae in papers published before 2000 was often inaccurate, it cannot be denied that strains classified as Chlorella may actually be from other genera. While systematic research into how the cell wall structure of green algae differs among genera and species is desirable, it is currently difficult to predict cell wall structure or the efficiency of oil and fat extraction using organic solvents from the classification name.

[0010] Green algae produce not only oils and fats but also many other useful substances, including carotenoids. However, when these useful substances are extracted with organic solvents without pretreatment such as cell disruption, the yield is very low (Non-Patent Document 9). The present inventors have been studying the production of oils and carotenoids using two strains of the green algae genus Coccomyxa, the Obi and KJ strains. However, it has been difficult to efficiently extract oils and carotenoids from the wet cells of these strains without cell disruption. For example, the Bligh & Dyer method (Non-Patent Document 10), which uses a mixed solvent of chloroform and methanol, is the most common method for extracting total lipids from biological materials. However, when this method was used to extract oil from the wet cells of the Obi strain, only 28.6 ± 9.0% of the total oil was extracted. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] de Maria, PD (2017). Ionic liquids, switchable solvents, and eutectic mixtures. In The application of green solvents in separation processes (pp. 139-154). Elsevier. [Non-patent document 2] Dong, T., Knoshaug, EP, Pienkos, PT, & Laurens, LM (2016). Lipid recovery from wet oleaginous microbial biomass for biofuel production: a critical review. Applied Energy, 177, 879-895. [Non-patent document 3] Yang, F., Xiang, W., Sun, X., Wu, H., Li, T., & Long, L. (2014). A novel lipid extraction method from wet microalga Picochlorum sp. at room temperature. Marine drugs, 12(3), 1258-1270.

Non-patent Document 4

Non-patent Document 5

Non-patent Document 6

Non-patent Document 7

[0012] The development of a simple, inexpensive, and highly efficient method for extracting lipophilic useful substances such as oils and fats synthesized by eukaryotic microalgae is an important factor for realizing the commercial production of products using these compounds as raw materials. Therefore, the object of the present invention is to provide a simple, inexpensive, and highly efficient method for extracting lipophilic useful substances synthesized by the green algae of the genus Coccomyxa. [Means for solving the problem]

[0013] As a result of intensive research to solve the above problems, we discovered a method for efficiently extracting lipophilic useful compounds from the wet cells of Coccomyxa, and completed the present invention without using highly toxic solvents such as chloroform.

[0014] That is, the present invention includes the following. [1] A method for extracting lipophilic useful substances, comprising a "pretreatment step" of heat-treating cells of green algae belonging to the genus Coccomyxa at a temperature of 50°C or higher for 5 minutes or more, and a "solvent extraction step" of extracting lipophilic useful substances from the cells heat-treated in the pretreatment step using an organic solvent. [2] The method according to [1], wherein in the solvent extraction step, the water content of the cells heat-treated in the pretreatment step is 1 to 100 times the dry weight of the cells by mass. [3] The method according to [1] or [2], wherein in the solvent extraction step, the temperature of the organic solvent is 40 to 85°C and the extraction time is 5 minutes or longer. [4] The method according to any one of [1] to [3], wherein the organic solvent is a polar solvent selected from the group consisting of ethanol, isopropanol, acetone, diethyl ether, and a mixture of two or more thereof. [5] The method according to any one of [1] to [3], wherein the organic solvent is a mixture of a polar solvent selected from the group consisting of ethanol, isopropanol, acetone, diethyl ether, and a mixture of two or more thereof and a non-polar solvent. [6] The method according to [5], wherein the non-polar solvent is hexane. [7] The method according to any one of [1] to [6], wherein the amount of organic solvent used in one solvent extraction step is 5 to 100 times the dry weight of the cells in mass ratio. [8] The method according to any one of [1] to [7], wherein the solvent extraction step is repeated one or more times. [Effects of the Invention]

[0015] According to the present invention, it is now possible to extract useful lipophilic substances such as oils and fats from Coccomyxa wet cells simply, inexpensively, and efficiently, which is expected to reduce the cost of industrial use of lipophilic compounds synthesized by Coccomyxa cells. [Brief explanation of the drawings]

[0016] [Figure 1] This diagram shows a bench-scale process for extracting lipophilic useful substances such as fats and carotenoids, characterized by a heat treatment prior to the addition of a solvent. Pretreatment: Pretreatment involves heating cells in an aqueous solution at 50°C or higher for a certain period of time, increasing the permeability of polar organic solvents into the cells. Solvent extraction (first stage): Lipophilic useful substances present inside the pretreated cells are extracted by the solvent in a polar organic solvent. Solvent extraction (second stage): Cells containing lipophilic useful substances not extracted in the first stage are transferred to a new polar organic solvent, extracting the remaining lipophilic organic substances. Oil recovery: Adding an appropriate amount of a nonpolar organic solvent such as hexane and water to the aqueous extract causes phase separation, with most of the lipophilic useful substances being distributed in the nonpolar organic solvent. The nonpolar organic solvent phase containing the oil is recovered, and the solvent is removed using an appropriate evaporator to recover the oil. On a plant scale, a dehydrating agent such as zeolite and a solvent recycling process using distillation are required. [Figure 2] FIG. 1 shows the procedure for a standard fat and oil extraction experiment. [Figure 3]This photograph shows fluorescent staining of KJ strain cells with Calcofluor White. 10 mL of KJ strain culture was centrifuged, and the resulting cell pellet was suspended in 2 mL of water. This suspension was dispensed into two microtubes, 1 mL each. One microtube was incubated at 70°C for 1 hour (pretreated), while the other was left at room temperature (untreated). After 1 hour, both tubes were centrifuged and suspended in 1 mL of 0.85% (w / w) NaCl solution. The cells were then centrifuged again and suspended in 1 mL of Calcofluor White solution (1 g / L in H2O, Sigma-Aldrich, catalog number 18909) and left at room temperature for 10 minutes. A small amount of the cell suspension prepared in this manner was placed on a glass slide, covered with a cover glass, and observed under a fluorescence microscope using blue light as excitation light. A: Microscopic image of cells without pretreatment. Red indicates chlorophyll fluorescence. A very small number of cells emit blue-white fluorescence emitted by calcofluor white. B: Microscopic image of pretreated cells. Almost all cells emit blue-white fluorescence emitted by calcofluor white. A very small number of cells do not emit fluorescence from calcofluor white, but emit red fluorescence. [Figure 4] This graph shows the relationship between pretreatment temperature and oil extraction efficiency. A. KJ strain slurry was pretreated for 1 hour at five different temperatures: 25, 40, 50, 60, and 70°C. 5 mL of ethanol was then added as an extraction solvent, and extraction was performed at 70°C for 1 hour (first-stage extraction). Second-stage extraction was then performed under the same conditions. The oil extraction efficiency for the first-stage extraction is shown in the white bar (I). The oil extraction efficiency when the oils obtained from the first and second-stage extractions are combined is shown in the gray bar (I + II). B. KJ strain slurry was pretreated for 30 minutes at three different temperatures: 60, 70, and 80°C. 7 mL of ethanol was then added as an extraction solvent, and extraction was performed at 70°C for 1 hour (first-stage extraction). Second-stage extraction was then performed under the same conditions. The oil extraction efficiency for the first-stage extraction is shown in the white bar (I). The gray bar graph (I+II) shows the oil extraction efficiency when the oils obtained in the first and second extraction stages are added together. [Figure 5]This is a graph showing the relationship between moisture content and fat / oil extraction efficiency. Slurries obtained from culture solutions independently cultured for 10 to 14 days were pretreated at 70°C for 1 hour. The weight of water contained in each slurry was then determined using the method described in Example 2. Next, various weights of acetone (Graph A) or ethanol (Graph B) were added to the slurry to perform primary extraction. The horizontal axis of the graph shows the value [solvent weight / water weight] obtained by dividing the [weight of the organic solvent (acetone or ethanol) used] by the [weight of water contained in the slurry]. Meanwhile, the vertical axis shows the fat / oil extraction efficiency obtained when performing primary extraction on slurries with different [solvent weight / water weight] values. [Figure 6] This photograph shows the correlation between oil extraction efficiency and carotenoid extraction yield. In this experiment, a slurry of KJ strain cultured for 14 days was pretreated under different conditions, and then oil extraction was performed under different conditions. The upper photograph in this figure shows the degree of coloration of the initial extract. The lower photograph shows the oil extraction conditions for each sample and the initial oil extraction efficiency. Nitrogen was depleted in the medium on the 14th day of KJ strain culture, resulting in the decomposition of intracellular chlorophyll and the concurrent accumulation of oil. Carotenoids, such as fat-soluble compounds like lutein, were also extracted during the oil extraction, resulting in a yellow extract. A strong correlation was observed between the intensity of this coloration and the oil extraction efficiency. [Figure 7] This graph shows the solubility and miscibility of a ternary system of ethanol, water, and fat. 1 mL of olive oil and 0.5, 1, 2, or 5 mL of water were added to a 100 mL test tube. The horizontal axis of the graph represents the volume of water added. Ethanol was then added to the olive oil and water mixture, and the volume of ethanol required to completely dissolve the olive oil was recorded. The vertical axis represents the volume of ethanol required to completely dissolve the olive oil. At 25°C, nearly 70 mL of ethanol was required to dissolve the olive oil. Also, 1 mL of olive oil mixed with 1 mL of water did not completely dissolve even after adding 90 mL of ethanol. At 70°C, however, the olive oil dissolved with a smaller amount of ethanol. As the amount of water present increased, the amount of ethanol required to dissolve the olive oil increased. DETAILED DESCRIPTION OF THE INVENTION

[0017] The method for extracting lipophilic useful substances according to the present invention (hereinafter referred to as "the method") comprises the steps of: A pretreatment step of heat-treating cells of green algae belonging to the genus Coccomyxa at a temperature of 50°C or higher for 5 minutes or more; a solvent extraction step in which lipophilic useful substances are extracted from the cells heat-treated in the pretreatment step using an organic solvent; It includes:

[0018] Examples of "green algae belonging to the genus Coccomyxa (eukaryotic microalgae belonging to the division Chlorophyta)" include, for example, Coccomyxa sp. KJ strain (sometimes referred to herein as "KJ strain of the green algae genus Coccomyxa") and Obi strain (sometimes referred to herein as "Coccomyxa Obi strain"), Coccomyxa elongata, Coccomyxa melkonianii, Coccomyxa simplex, Coccomyxa subellipsoidea, etc. The KJ strain was deposited on June 4, 2013, with the National Institute of Technology and Evaluation, International Patent Organism Depositary (NITE-IPOD) (Room 120, 2-5-8 Kazusa Kamatari, Kisarazu, Chiba Prefecture, 292-0818, Japan) under accession number FERM P-22254, and has been transferred to an international deposit under the Budapest Treaty under accession number FERM BP-22254. The Obi strain was deposited on February 15, 2005, with the National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary (Chuo-6, 1-1-1 Higashi, Tsukuba, Ibaraki Prefecture, 305-8566, Japan) under accession number FERM P-20401, and has been transferred to an international deposit under the Budapest Treaty under accession number FERM BP-10484. The Obi strain is available from the National Institute of Technology and Evaluation, Patent Organism Depositary (NITE-IPOD) (Room 120, 2-5-8 Kazusa Kamatari, Kisarazu, Chiba Prefecture, 292-0818, Japan).

[0019] In the present invention, examples of "lipophilic useful substances" include fats and oils such as triacylglycerol (triglyceride: TAG), carotenoids such as lutein, and chlorophyll.

[0020] (1) Pretreatment process In this method, first, a pretreatment step is carried out in which cells of green algae belonging to the genus Coccomyxa are heat-treated at a temperature of 50°C or higher for 5 minutes or longer.

[0021] Specifically, the cultured Coccomyxa cells are harvested by centrifugation, membrane filtration, or other methods, and then pretreated by incubating at 50°C or higher (preferably 60°C or higher, particularly preferably 70°C or higher, and 100°C or lower, preferably 90°C or lower, and particularly preferably 85°C or lower) for a certain period of time (5 minutes or more, preferably 30 minutes or more, particularly preferably 60 minutes or more, 360 minutes or less, preferably 240 minutes or less, and particularly preferably 120 minutes or less). During pretreatment, the dry weight concentration of cells in the cell suspension (hereinafter referred to as "biomass concentration") is not important. However, if pretreatment is performed at a low biomass concentration, it is necessary to concentrate the biomass concentration to, for example, 100 mg dry weight / mL or higher (400 mg dry weight / mL or lower) by centrifugation or other methods to minimize the amount of organic solvent used in the subsequent solvent extraction step. At this concentration, the cell suspension becomes a slurry. Hereinafter, the cells in the slurry and the surrounding water will be collectively referred to as the "slurry."

[0022] (2) Solvent extraction process In this method, a solvent extraction step is then carried out in which lipophilic useful substances are extracted from the cells heat-treated in the pretreatment step using an organic solvent.

[0023] Examples of organic solvents include polar solvents such as ethanol, isopropanol, acetone, diethyl ether, and mixtures of two or more thereof. Furthermore, a mixture of these polar solvents with a nonpolar solvent (e.g., hexane) may also be used as the organic solvent. The ratio of polar solvent to nonpolar solvent in the mixture may be, for example, 1:9, preferably 2:8, and particularly preferably 3:7.

[0024] In this step, the water content of the cells heat-treated in the pretreatment step may be 1 to 100 times, preferably 1 to 10 times, and particularly preferably 1 to 3 times the dry weight of the cells by mass. This water content allows the extraction efficiency of lipophilic useful substances to be maintained.

[0025] Furthermore, in this step, if the water content of the slurry is high, the efficiency of fat and oil extraction will be low if extracted at room temperature, so it is preferable to perform the extraction at a higher temperature. The temperature preferred for extraction (temperature of the organic solvent) can be determined by performing the experiment of Example 11 at a temperature of 40 to 85°C, but generally, the temperature of the organic solvent is above room temperature, for example, 40°C or above (preferably 50 to 85°C, particularly preferably 70 to 80°C), and the extraction time may be 5 minutes or longer (preferably 30 minutes or longer, particularly preferably 60 minutes or longer, 240 minutes or shorter, preferably 180 minutes or shorter, particularly preferably 120 minutes or shorter).

[0026] Furthermore, the amount of organic solvent used in this step varies depending on the moisture content of the material to be extracted. For example, when extraction is performed at 70°C, an oil / fat extraction efficiency of nearly 100% can be achieved by using an organic solvent in an amount 25 times or more by mass relative to the moisture content. Generally, the amount may be 5 to 200 times, preferably 20 to 200 times, and particularly preferably 50 to 100 times the dry cell weight by mass. This amount of organic solvent can maintain the extraction efficiency of lipophilic useful substances.

[0027] It is also preferable to repeat this process at least once (preferably at least twice, but not more than three times, preferably twice). For example, after the first extraction, the organic solvent suspension is centrifuged to separate the organic solvent (supernatant) from the cell extract residue (precipitate), and the organic solvent (supernatant) is transferred to a separate test tube (first extract recovery). Meanwhile, for the second extraction, the cell extract residue is suspended in the same type of fresh organic solvent as in the first extraction, and extraction is performed under the same conditions as in the first extraction (second extraction of lipophilic useful substances). Next, this organic solvent suspension is centrifuged to separate the organic solvent (supernatant) from the cell extract residue (precipitate), and the organic solvent (supernatant) is transferred to a separate test tube (second extract recovery).

[0028] When the pretreated slurry is suspended in the polar organic solvent or a mixture of a polar organic solvent and a non-polar solvent and lipophilic useful substance extraction is performed, almost 100% of the lipophilic useful substances contained in the wet cells can be extracted (lipophilic useful substance extraction efficiency = 100%). On the other hand, if this pretreatment is not performed, the extraction efficiency is low at around 20%. Furthermore, even when the lipophilic useful substances in the pretreated wet cells are extracted using only a non-polar solvent, the lipophilic useful substance extraction efficiency is low at around 30%.

[0029] In other words, this method enables the extraction of lipophilic useful substances such as oils and fats with nearly 100% extraction efficiency, without using physical, chemical, or enzymatic cell disruption methods, but only by mild heat treatment and the addition of organic solvents.

[0030] (3) Lipophilic useful substance recovery process The method may optionally include a lipophilic useful substance recovery step. After the solvent extraction step, the lipophilic useful substances can be recovered by drying the extract using an evaporator, etc. Alternatively, the extract can be separated into a water-containing layer and a non-polar solvent (e.g., hexane) by adding water and a non-polar solvent, and then the non-polar solvent can be evaporated to recover the lipophilic useful substances. [Example]

[0031] The present invention will be described in detail below based on the results shown in Examples, but the technical scope of the present invention is not limited to these Examples.

[0032] In this example, "oils and fats" includes triacylglycerols (triglycerides: TAGs).

[0033] [Example 1] Cultivation of the green alga Coccomyxa and measurement of biomass dry weight and oil content The KJ strain of the green alga Coccomyxa sp. (hereafter referred to as KJ strain) was cultured in a 300 mL round-bottom Roux bottle containing 200 mL of A7 / 2 medium (Hayakawa, J., Sato, E., Takagi, S., Ide, Y., Imamura, S., Koike, H., ... & Harayama, S. (2023). A truncated antenna mutant of the unicellular green alga Coccomyxa sp. strain Obi shows better biomass productivity than the wild-type strain under higher irradiance, at higher cell density, and in greater depth of culture. Algal Research, 74, 103216). The incubator was placed in a plant culture incubator at 25°C, and CO2 was supplied to the medium by bubbling air containing 1% (v / v) CO2. The light source used was a fluorescent lamp for plant growth (40 watts, FL40S·FR·P, Panasonic), and the light energy density on the surface of the incubator was approximately 150 μmol m -2 s -1The light was adjusted so that the cell density was 0.1, and the culture was continued for 10 to 14 days. These strains began to accumulate lipids intracellularly when the OD750 exceeded 7, and the lipid content (weight of lipid contained per dry weight of cells) reached a maximum when the OD750 exceeded 10. As lipid accumulation progressed, intracellular chlorophyll decomposed, and the culture medium, which had been green, turned yellow due to carotenoids by the 14th day after the start of culture.

[0034] The dry weight concentration of cells (mg / mL) and the oil / fat concentration (mg / mL) in the culture medium were measured as follows. 10 mL of culture medium was placed in a pre-weighed 15 mL centrifuge tube (weight: a mg), and the cells were collected by centrifugation and removal of the supernatant. The tube was then placed in a freeze-dryer to dry the cells. The tube containing the dried cells was then weighed (weight: b mg). From this value, the dry weight of biomass present in 1 mL of culture medium was calculated as [(b - a) / 10] mg. Next, a portion of the dried biomass was transferred to a pre-weighed short NMR sample tube (outer diameter: 5 mm, length: 20 mm, New Era Enterprises, Vineland, NJ, USA) (weight: c mg) and weighed (weight: d mg). The dry weight of biomass in the short NMR sample tube [(d - c) mg] was measured. The tube was then inserted into an Oxford Instruments MQC pulsed nuclear magnetic resonance spectrometer. The biomass dry weight (dc) mg was entered into the instrument, and the total fat weight (e mg) of the short-wave NMR sample was measured to determine the fat content (f%) of the sample. Note that the MQC pulsed nuclear magnetic resonance spectrometer only displays the fat content (f%). From the fat content thus obtained, the fat concentration of the culture medium, i.e., the fat content per mL of culture medium, [(b - a) × f] / (10 × 100) mg, can be calculated. Table 1 shows an example of a measurement. The biomass dry weight concentration of the culture medium and the fat content of the cells vary from culture to culture, so measurements must be performed each time.

[0035] [Table 1]

[0036] [Example 2] Overview of the method for extracting oil from KJ strain wet cells using organic solvents The relationship between oil extraction conditions and oil extraction efficiency was investigated as follows. 25 mL of KJ strain culture medium was dispensed into multiple 50 mL centrifuge tubes, and cells were recovered as a pellet by centrifugation (1,000–4,000 rpm, 10 or 20 minutes). This pellet, or a suspension of this pellet in an appropriate volume (e.g., 5 mL) of water, was used in the oil extraction experiment. The oil extraction method of the present invention consists of three steps: (i) pretreatment of wet cells by incubating them at a temperature of 50°C or higher for a certain period of time; (ii) organic solvent extraction of oil from the pretreated cells; and (iii) recovery of oil by removing the solvent from the oil extract (Figure 1).

[0037] FIG. 1 shows the fat extraction process, which is characterized by a heat treatment prior to the addition of the solvent: Pretreatment: When extracting oils and fats accumulated in wet cells using organic solvents, it is important to pretreat the cells in aqueous solution by heating them to above 50°C; Solvent extraction: A polar solvent such as ethanol or a mixture of polar and non-polar solvents is used to extract the oils and fats from the pretreated cell concentrate (slurry). If the slurry contains very little water, oil and fat extraction is possible at room temperature, but if the water content is high, extraction at room temperature is difficult and extraction is performed at, for example, 70°C. Oil recovery: After extraction, the oil can be recovered by drying the extract using an evaporator or similar. Alternatively, as shown in the "Oil Recovery" box in Figure 1, adding water and a non-polar solvent to the extract separates it into a water-containing layer and a non-polar solvent containing the oil, and then evaporating the non-polar solvent allows the solvent to evaporate more quickly and recover the oil. There are many other possible methods for oil recovery and for recovering and reusing used solvents.

[0038] The experimental procedure for verifying this extraction process is shown in Figure 2. For each oil extraction experiment, 25 mL of KJ strain culture was used, as described in Example 1 (culture medium sampling: Figure 2, Step 1), unless otherwise specified. The dry weight (g) of cells present in this culture medium and the oil content (h) were calculated using the dry weight biomass concentration (A) and oil concentration (B) of the culture medium, as shown in Table 1, as g = 25 × A and h = 25 × B. The culture medium was centrifuged in a pre-weighed 50 mL centrifuge tube weighing j mg to obtain a pellet of wet cells. If the weight of the centrifuge tube and pellet is k mg, the weight of the pellet (the sum of the dry weight of biomass and the weight of water in the pellet) is (k - j) mg. The weight of water in the pellet can be calculated by subtracting the dry weight (g) of cells present in 25 mL of culture medium from the weight of the pellet.

[0039] The wet cells in the centrifuge tube were heated for a certain period of time (pretreatment: Figure 2, step 2). Next, an organic solvent was added to the pretreated wet cells, and the cells were suspended and heated in a sealed state for a certain period of time (first stage oil extraction: Figure 2, step 3). The suspension was occasionally shaken while keeping warm. The standard conditions were pretreatment at 70°C for 1 hour, with 5 mL of acetone, ethanol, isopropanol, ethyl ether, or hexane added as the organic solvent. Extraction was also performed twice (first and second stages) at 70°C for 1 hour. These conditions were modified as necessary.

[0040] After the first stage of extraction, the organic solvent suspension was centrifuged to separate the organic solvent (supernatant) from the cell extract residue (precipitate), and the organic solvent (supernatant) was transferred to a round-bottom test tube (first stage extract recovery: Figure 2, Step 4). Meanwhile, the cell extract residue was suspended in the same solvent as in the first stage of extraction, and extraction was carried out under the same conditions as in the first stage (second stage oil extraction: Figure 2, Step 5). This suspension was occasionally shaken while keeping warm. After a certain period of time, the organic solvent suspension was centrifuged to separate the organic solvent (supernatant) from the cell extract residue (precipitate), and the organic solvent (supernatant) was transferred to a round-bottom test tube (second stage extract recovery: Figure 2, Step 6).

[0041] The first-stage extract and the second-stage extract in the round-bottom test tube were separately vacuum concentrators (CC-105 / MV-1100, Tomy Digital Biology, Tokyo, Japan) to evaporate the volatile organic solvent, yielding a dried product primarily composed of oil. Alternatively, the extract was mixed with an appropriate amount of water and hexane to transfer the oil to the hexane layer, and the hexane was then evaporated (oil recovery: Figure 2, Step 7). The weight of the dried product and the percentage of oil present in the dried product were quantified as follows: First, the dried product at the bottom of the round-bottom test tube was dissolved in approximately 100 μL of hexane, and this solution was transferred to a pre-weighed short-section NMR sample tube (this weight is defined as p mg). Approximately 100 μL of hexane was added to the round-bottom test tube again, and after rinsing the bottom of the round-bottom test tube, the solution was transferred to the same short-section NMR sample tube as above. This tube was then placed in a microtube with the lid open and dried using a centrifugal concentrator. After drying, the short NMR sample tube was reweighed (this weight was designated as q mg). The weight of the dried product extracted from 25 mL of culture medium was calculated as (q - p) mg. The tube was inserted into an Oxford Instruments MQC pulsed nuclear magnetic resonance spectrometer, and the lipid content (r%) of the dried product extracted with organic solvent was measured (oil content measurement: Figure 2, Step 8). From this lipid content, the weight of the lipid extracted from 25 mL of culture medium was calculated as (q - p) × (r / 100) mg. Furthermore, since the weight of the lipid originally contained in the 25 mL of culture medium was 25 × [(b - a) / 10] × (f / 100), the lipid extraction efficiency can be expressed as a percentage, S = [(q - p) × r] / {25 × [(b - a) / 10] × f}, or as a percentage, S multiplied by 100. The fat extraction efficiency in the latter stage extraction was also calculated using the same method. That is, it should be noted that the fat extraction efficiency in the latter stage extraction shown in the following examples is the ratio (percentage) of the weight of fat obtained in the latter stage extraction to the weight of fat before the first stage extraction, and is not the ratio to the weight of the remaining fat not extracted in the first stage extraction.

[0042] As shown in Figure 2 (Procedure for a Standard Oil Extraction Experiment), a standard oil extraction experiment consists of eight steps: Step 1: Culture medium recovery; Step 2: Pretreatment of the slurry obtained by centrifugation (kept at 50°C or higher for a certain period of time); Step 3: Primary oil extraction; Step 4: Primary extract recovery; Step 5: Secondary oil extraction; Step 6: Secondary extract recovery; Step 7: Oil recovery; and Step 8: Oil content measurement. Unless otherwise noted, the pretreatment conditions were 70°C for 1 hour. Unless otherwise noted, oil extraction was performed using 5 mL of organic solvent at 70°C for 1 hour.

[0043] [Example 3] Acetone extraction of oil from KJ strain wet cells Table 2A shows the results of pretreating the slurry at 70°C for 1 hour, followed by primary and secondary fat extractions at 70°C using 7 mL and 5 mL of acetone, respectively. Tables 2B and C show the results of fat extractions using the same slurry with 1 and 2 mL of water added.

[0044] The oil extraction efficiency in Table 2A was 82% for the first extraction stage and 94% when the second extraction stage was added. Experiments similar to those in Table 2A were performed seven times independently, and the oil extraction efficiency in the first stage was 89 ± 12%, and the oil extraction efficiency for the first and second extraction stages combined was 98 ± 11%.

[0045] In some experiments, the lipid extraction efficiency exceeded 100%. The MQC-pulsed NMR spectrometer used in this experiment detects lipids with high molecular mobility (i.e., long nuclear magnetic relaxation times), but does not detect lipids attached to stable structures within cells (Maitra, S., Dien, B., Long, SP, & Singh, V. (2021). Development and validation of time-domain 1H-NMR relaxometry correlation for high-throughput phenotyping method for lipid contents of lignocellulosic feedstocks. GCB Bioenergy, 13(7), 1179-1190.). However, lipids with low molecular mobility within cells may be detectable by MQC-pulsed NMR if the stable structures are dissolved by solvent extraction. In other words, when lipid content is measured using MQC-pulsed NMR, if 100% lipid is extracted from cells, the lipid extraction efficiency is expected to be slightly higher than 100%.

[0046] The results of Table 2A and similar experiments showed that nearly 100% of the oil could be extracted from the wet cells by pretreating at 70°C for 1 hour followed by two acetone extractions at 70°C for 1 hour. This means that oil can be efficiently extracted from the wet cells of the KJ strain by simply using mild heat treatment, without the need for the complicated pretreatment procedures used in many previous studies.

[0047] The sample slurry in Table 2A had a moisture content of 79% (water:dry biomass = 3.7:1). One or two mL of water was added to this slurry to prepare wet cell sample (B) with a water:dry biomass ratio of 20:1 and wet cell sample (C) with a water:dry biomass ratio of 37:1. Oil and fat were extracted using acetone. The oil extraction efficiency in the first extraction stage was very low for both samples (B) and (C), but it improved slightly in the second extraction stage. This is likely due to the transfer of some of the water contained in the sample to the solvent during the first extraction stage, resulting in a certain decrease in the sample's moisture content during the second extraction stage. As a result, the combined oil extraction efficiency for samples with moisture contents of 20 and 37 times their dry weight was 42% and 22%, respectively (Tables 2B and C). This indicates that a high water content in the slurry significantly reduces oil and fat extraction efficiency.

[0048] [Table 2]

[0049] [Example 4] Extraction of oil from wet cells using acetone, ethanol, hexane, and methanol as solvents Table 3 shows the results of extraction experiments similar to those in Table 2, using acetone, ethanol, hexane, and a mixture of ethanol and hexane as solvents. The solvent volume used for extraction was 5 mL for both the initial and subsequent extractions. The extraction efficiency in the initial extraction was generally high when polar solvents were used, but was low at 31% for extraction with hexane. On the other hand, the extraction efficiency was significantly improved when hexane was mixed with 30% or more ethanol by volume. When using a 90:10 volume ratio of hexane to ethanol, the extraction efficiency in the subsequent stages was higher than that in the initial stage. This is thought to be because 10% ethanol cannot completely remove all the water from the slurry, but some of the water was absorbed by the solvent during the initial extraction, improving the extraction efficiency in the subsequent extraction. In other words, when extracting oil from wet cells, the extraction efficiency was low when using a nonpolar solvent alone, and the presence of a polar solvent appropriate to the water content was important.

[0050] [Table 3]

[0051] The oil extraction efficiency was compared when using methanol as a solvent and when using ethanol as a solvent (Table 4). The solvent volume used for extraction was 4 mL for both the primary and secondary extractions. The extraction efficiency of methanol was lower than that of ethanol.

[0052] [Table 4]

[0053] [Example 5] Pretreatment is essential for achieving high oil extraction efficiency We investigated how the oil extraction efficiency differed depending on whether or not the cells were pretreated. As shown in Table 5, when oil was extracted from wet cells that had not been pretreated at 70°C for 1 hour using ethanol or acetone, the oil extraction efficiency was less than 20%. On the other hand, the oil extraction efficiency using acetone and ethanol from pretreated wet cells was 94% and 88%, respectively.

[0054] In the experiment shown in Table 5, the untreated sample was left at 70°C for 1 hour, while the pretreated sample was left at 70°C for 2 hours. To verify the possibility that the difference in the time spent at 70°C affected the oil extraction efficiency, ethanol was added without pretreatment and the sample was extracted for 2 hours. The oil extraction efficiency of this sample was not statistically different from that of the sample extracted with ethanol for 1 hour without pretreatment. This indicates that pretreatment with heat must be performed before adding the solvent (Table 6).

[0055] [Table 5]

[0056] [Table 6]

[0057] [Example 6] Pretreatment at 70°C for 1 hour changes the cell wall surface Calcofluor white (CFW) binds to polysaccharides and emits bluish-white fluorescence. However, when KJ cells were stained with CFW, only a few cells emitted fluorescence, and only red fluorescence derived from chlorophyll was observed (Figure 3A). In contrast, CFW fluorescence was observed in almost all cells pretreated at 70°C for 1 hour. This suggests that (i) KJ cells are surrounded by a barrier that is impermeable to CFW, and (ii) pretreatment at 70°C for 1 hour removes this barrier, allowing CFW to bind to polysaccharides such as cellulose present below the barrier.

[0058] The specific experiment shown in Figure 3 (fluorescent staining of KJ strain cells with CFW) was performed as follows. 1 mL of suspension of the KJ strain (untreated) and the KJ strain pretreated at 70°C for 1 hour was placed in a microtube and centrifuged to collect the cells as a pellet. The pellet was washed once with 0.85% (w / v) NaCl solution, and the cells were again collected as a pellet. 1 mL of CFW (Calcofluor solution 1 mg / mL, Sigma-Aldrich) was added to the pellet, mixed well, and left at room temperature for at least 10 minutes. Images were then examined through a blue-light excitation filter set (Blue Exciter Cube) on an Olympus fluorescence microscope. A: Microscopic image of untreated KJ strain cells. The majority of cells emit red fluorescence due to chlorophyll. B: Microscopic image of KJ strain cells heat-treated at 70°C for 1 hour. Most cells emit the whitish-blue fluorescence of CFW. The chlorophyll fluorescence appears to be quenched by the CFW fluorescence.

[0059] [Example 7] Relationship between pretreatment time at 70°C and oil extraction efficiency As shown in Tables 7 and 8, pretreatment at 70°C for 1 hour significantly improved the efficiency of fat extraction from KJ strain cells using acetone and ethanol. This result is consistent with the results in Tables 2 to 6. Therefore, to determine the pretreatment time required to achieve nearly 100% fat extraction efficiency, we varied the pretreatment time between 5 and 60 minutes and examined how the fat extraction efficiency changed.

[0060] The extraction efficiency decreased with decreasing pretreatment time, but the extraction efficiencies with acetone and ethanol after 5 minutes of pretreatment were 62% and 72%, respectively, compared with those after 60 minutes of pretreatment. The extraction efficiency after 30 minutes of pretreatment at 70°C was also lower than that after 60 minutes, but the difference was not statistically significant.

[0061] The extraction efficiency using isopropanol was also high, similar to that using acetone and ethanol. The extraction efficiency after 120 minutes of pretreatment at 70°C was higher than that after 60 minutes (Table 9). In other experiments (not shown), the extraction efficiency after 120 minutes tended to be higher than that after 60 minutes, but the difference did not exceed 20%.

[0062] [Table 7]

[0063] [Table 8]

[0064] [Table 9]

[0065] [Example 8] Relationship between water content of pretreated cell suspension and oil extraction efficiency The results of Examples 6 and 7 suggest that treating KJ strain cells at 70°C for 5 minutes or longer removes the outer cell wall barrier that prevents the permeation of solvents such as CFW, acetone, and ethanol. Because the composition and structure of microalgae cell walls are largely unknown, it is difficult to predict the specific changes that occur. If the barrier "dissolves" into the water surrounding the cells upon heating, it is predicted that the greater the amount of surrounding water, the greater the effect of heating. Therefore, in addition to pretreating the slurry obtained by centrifuging 25 mL of culture medium, we also pretreated cells suspended in 5 mL of H2O. The pre-pretreatment slurry obtained by centrifugation contained 2.2 to 2.7 times the water content of the biomass dry weight. On the other hand, the latter cell suspension contained more than 60 times the water content of the biomass dry weight. After pretreatment at 70°C for 1 hour, 5 mL of ethanol was added to the slurry sample for oil extraction. On the other hand, the cell suspension to which 5 mL of water was added was centrifuged after pretreatment, the supernatant was removed, and ethanol was added to the resulting slurry for fat extraction. As shown in Table 10, the phenomenon of cells suspended in more water having a greater pretreatment effect was not observed. In other words, it was found that the fat extraction efficiency did not decrease even if the recovered slurry was pretreated as is, and it was found that highly efficient fat extraction was possible without adding water during pretreatment.

[0066] [Table 10]

[0067] [Example 9] Relationship between pretreatment temperature and oil extraction efficiency The oil extraction efficiency was investigated when pretreatment was performed at different temperatures, from room temperature (25°C) to 80°C. In panel A of Figure 4, pretreatment was performed at temperatures ranging from 25 to 70°C for 1 hour, followed by two rounds of extraction with 5 mL of ethanol at 70°C for 1 hour. In panel B of Figure 4, pretreatment was performed at temperatures ranging from 60 to 80°C for 30 minutes, followed by two rounds of extraction with 7 mL of ethanol at 70°C for 1 hour. Pretreatment at 40°C was completely ineffective, but at temperatures above 50°C, higher temperatures resulted in higher oil extraction efficiency.

[0068] Specifically, in the experiment shown in Figure 4 (relationship between pretreatment temperature and oil extraction efficiency), the slurry collected by centrifugation was placed at 25-70°C for 1 hour, followed by two rounds of extraction using 5 mL of ethanol at 70°C for 1 hour. Number of experiments: Pretreatment experiments at 25 and 40°C (n = 3); pretreatment experiments at 50, 60, and 70°C (n = 4). The slurry collected by centrifugation was also placed at 60-80°C for 30 minutes, followed by two rounds of extraction using 5 mL of ethanol at 70°C for 1 hour. n = 3. In Figure 4, bar I indicates the oil extraction efficiency in the first stage, and I + II indicates the combined oil extraction efficiency of the oils obtained in the first and second stages.

[0069] [Example 10] Relationship between moisture content and oil extraction efficiency As shown in Table 2 of Example 3, "Oil Extraction Using Acetone from Slurries with Different Water Contents," the oil extraction efficiency was significantly affected by the amount of water contained in the slurry. Therefore, experiments were conducted to more closely examine the relationship between the water content of the slurry and the oil extraction efficiency. First, slurries with different water contents were obtained by recovering cells from 25 mL of culture medium under different centrifugation conditions. When cells were recovered by centrifugation at 1,000 rpm for 10 minutes using a glass centrifuge tube, the resulting slurry contained nearly 300 mg of water. On the other hand, when the culture medium in a polypropylene centrifuge tube (HIMAC T9A31 tube) was centrifuged at 4,000 rpm for 20 minutes, the resulting slurry contained only 130 mg or less of water. In some cases, water was added to these slurries to increase the water content. The slurries prepared in this way were extracted with 1 to 12 mL of solvent, and the oil extraction efficiency in the first stage was measured. At the same time, the weight of the solvent used in the first extraction experiment was divided by the weight of the water in the slurry. The ratio of the solvent weight to the water weight, [Acetone / H2O (w / w)] or [ETOH / H2O (w / w)], indicates how many times the amount of solvent was used relative to the water content of the slurry. Figure 5 shows the relationship between the oil extraction efficiency and [Acetone / H2O (w / w)] or [ETOH / H2O (w / w)]. Figure 5A shows the results when acetone was used as the solvent, and Figure 5B shows the results when ethanol was used as the solvent. As can be seen from these figures, there is a clear positive correlation between the oil extraction efficiency and [Acetone / H2O (w / w)] or [ETOH / H2O (w / w)]. The correlation coefficient for Figure 5A is 0.64, and that for Figure 5B is 0.83. The somewhat weak correlation is thought to be due to experimental error and the influence of factors other than the solvent / water ratio on the oil extraction efficiency. In any case, it was experimentally confirmed that the oil extraction efficiency decreases when there is a lot of water, and that the oil extraction efficiency increases when the amount of solvent is increased. Furthermore, Figure 5 reveals that to extract 100% of the oil from wet cells at 70°C, it is necessary to use a solvent with a volume approximately 20 times the amount of water.

[0070] [Example 11] Correlation between oil and fat extraction efficiency and carotenoid extraction In all of the fat / oil extraction experiments up to Example 10, a strong correlation was observed between the fat / oil extraction efficiency and the degree of coloration of the extract. For example, the extract from cells on the 10th day after the start of culture was green due to chlorophyll, and a strong correlation was observed between the intensity of the green color and the fat / oil extraction efficiency. Furthermore, in cells on the 14th day after the start of culture, chlorophyll decomposition has progressed and carotenoids such as lutein have become the main pigment components. A strong correlation was also observed between the intensity of the yellow color of the extract from cells in this state and the fat / oil extraction efficiency. An example is shown in Figure 6.

[0071] It is known that some form of pretreatment is necessary for highly efficient extraction of chlorophyll and carotenoids from microalgae cells (Kim, DY, Vijayan, D., Praveenkumar, R., Han, JI, Lee, K., Park, JY, ... & Oh, YK (2016). Cell-wall disruption and lipid / astaxanthin extraction from microalgae: Chlorella and Haematococcus. Bioresource technology, 199, 300-310.). Figure 6 reveals that the conditions for high oil extraction efficiency are also good conditions for chlorophyll and carotenoid extraction, demonstrating that the present invention can also be used as a highly efficient extraction method for chlorophyll and carotenoid pigments.

[0072] [Example 12] Solubility and miscibility in a ternary system of ethanol, water, and oil 1 mL of olive oil was placed in a 100 mL test tube, followed by the addition of 0, 0.5, 1, 2, and 5 mL of water. The water-oil mixture was then placed at 25°C or 70°C, and 0.5 mL of ethanol was added in increments until the oil droplets completely dissolved and the liquid became clear. If the olive oil did not completely dissolve after adding 90 mL of ethanol, the addition of ethanol was discontinued. Figure 7 shows the horizontal axis and the vertical axis, respectively, of the volume of water added to 1 mL of olive oil. At 25°C, adding 68.5 mL of ethanol to 1 mL of olive oil completely dissolved the olive oil. Furthermore, in the case of a mixture of 1 mL of olive oil and 1 mL of water, the olive oil did not completely dissolve even after adding more than 90 mL of ethanol. At 70°C, less than 5 mL of ethanol was required to dissolve 1 mL of olive oil. The presence of water increased the amount of ethanol required to dissolve olive oil. When 5 mL of water was present, the amount of ethanol required to completely dissolve olive oil was 85.5 mL. From this graph, it is predicted that to dissolve the oil from a slurry containing water into a solvent, a volume of about 20 to 25 times the volume of water, or a mass of about 16 to 20 times the mass of solvent, is required.

[0073] Figure 5 shows that when the weight ratio of solvent to water is around 20, the oil extraction efficiency is 100%, meaning that almost all of the oil is dissolved in the solvent. From this, the relationship between water content and oil extraction efficiency observed in Figure 5 can be interpreted as a pure reflection of physicochemical properties. Furthermore, the reason why the oil extraction efficiency was higher at higher temperatures than at room temperature can also be interpreted as a pure reflection of physicochemical properties.

[0074] It has been reported that when oil is extracted from oil-bearing plants using ethanol, the oil extraction efficiency decreases as the moisture content of the extracting material increases, whereas increasing the extraction temperature increases the oil extraction efficiency. These findings likely reflect observations and descriptions of the physicochemical properties related to solubility in the ternary ethanol-water-oil system (Sawada, MM, Venancio, LL, Toda, TA, and Rodrigues, CE, 2014. Effects of different alcoholic extraction conditions on soybean oil yield, fatty acid composition, and protein solubility of defatted meal. Food Research International, 62, pp. 662-670; Toda, TA, Sawada, MM, and Rodrigues, CE, 2016. Kinetics of soybean oil extraction using ethanol as solvent: Experimental data and modeling. Food and Bioproducts Processing, 98, pp. 1-10).

[0075] [Example 13] Extraction of oil from wet cells of Coccomyxa strain Obi using a diethyl ether / methanol mixture The results shown in Examples 1 to 9 demonstrate that pretreatment prior to the addition of solvent significantly increases the efficiency of oil extraction in solvent extraction of oils. To investigate whether this pretreatment is effective for other strains of the genus Coccomyxa, oil extraction from wet cells of the Coccomyxa Obi strain was examined with and without pretreatment.

[0076] A screw-cap test tube containing 10 mg of wet biomass (slurry) from Obi strains was incubated at room temperature or 80°C for 30 minutes. 0.5 mL of methanol and 2.0 mL of diethyl ether were added to the sample, the lid was tightly closed, and the tube was shaken vigorously up and down for 30 seconds. The tube was centrifuged (4000 rpm, 5 minutes, 25°C), and the organic solvent layer was aspirated with a Pasteur pipette and transferred to a vial. 0.5 mL of H2O and 0.5 mL of methanol were added to the lower layer and gently vortexed. 2 mL of diethyl ether was added, the lid was tightly closed, and the tube was shaken vigorously up and down for 30 seconds. The tube was centrifuged (4000 rpm, 5 minutes, 25°C), and the upper layer (ether layer) was aspirated with a Pasteur pipette and transferred to a vial. The volume of the ether extract was reduced by approximately 70% by blowing nitrogen gas through it, and the diethyl ether was completely evaporated in an oven at 105°C.

[0077] The oil extraction efficiency was calculated and found to be less than 10% from untreated wet cells, whereas the oil extraction efficiency from wet cells treated at 80°C for 30 minutes was 99.0 ± 3.7%.

[0078] [Example 14] Method and principle of oil extraction from Coccomyxa using organic solvents As explained above, the fat extraction method of the present invention consists of two steps: pretreatment (keeping wet cells at 50°C or higher for a certain period of time) and extraction (extraction of fats and oils using a polar solvent or a polar and non-polar solvent). It is believed that the pretreatment removes the cell surface barrier (the biochemical nature of which is currently unknown). Between 50 and 80°C, the higher the temperature, and between 5 and 120 minutes, the longer the treatment time. The removal effect was greater.

[0079] In the case of wet cells, extraction using only a non-polar solvent resulted in low extraction efficiency, but the inclusion of 10% or more of a polar solvent significantly increased extraction efficiency. When the moisture content of wet cells was low, a fairly high oil extraction efficiency could be achieved even at room temperature, but when the moisture content was high, high oil extraction efficiency could be ensured by raising the extraction temperature.

[0080] When a high oil extraction efficiency was obtained, the extract showed color due to chlorophyll and carotenoids. On the other hand, the color was not as strong in extracts with a low oil extraction efficiency. Since chlorophyll and carotenoids are also lipophilic compounds, it is not surprising that they were extracted more efficiently under the oil extraction conditions of the present invention. However, this observation clearly indicates that the present invention increases the extraction efficiency of not only oils but also other lipophilic compounds contained in cells. [Accession number]

[0081] FERM BP-22254 FERM BP-10484

Claims

1. A pretreatment step of heat-treating cells of green algae belonging to the genus Coccomyxa at a temperature of 50°C or higher for 5 minutes or more; a solvent extraction step in which lipophilic useful substances are extracted from the cells heat-treated in the pretreatment step using an organic solvent; A method for extracting lipophilic useful substances, comprising:

2. 2. The method according to claim 1, wherein in the solvent extraction step, the water content of the cells heat-treated in the pretreatment step is 1 to 100 times the dry weight of the cells in terms of mass ratio.

3. 2. The method according to claim 1, wherein in the solvent extraction step, the temperature of the organic solvent is 40 to 85°C and the extraction time is 5 minutes or longer.

4. 2. The method of claim 1, wherein the organic solvent is a polar solvent selected from the group consisting of ethanol, isopropanol, acetone, diethyl ether, and mixtures of two or more thereof.

5. 2. The method of claim 1, wherein the organic solvent is a mixture of a polar solvent and a non-polar solvent selected from the group consisting of ethanol, isopropanol, acetone, diethyl ether, and mixtures of two or more thereof.

6. 6. The method of claim 5, wherein the non-polar solvent is hexane.

7. 2. The method according to claim 1, wherein the amount of organic solvent used in each solvent extraction step is 5 to 100 times the dry weight of the cells in mass ratio.

8. 10. The method of claim 1, wherein the solvent extraction step is repeated one or more times.

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