Method for extracting useful components from algae

Abstract

Provided is a method for extracting useful components from algae, characterized by comprising the following processes (1a) and (2a), or (2b) and (1b), process (1a): extracting a first useful component from dried algae using a non-polar solvent; process (2a): extracting a second useful component from the residue after extraction in process (1a) using a solvent containing a polar solvent; process (2b): extracting a second useful component from dried algae using a solvent containing a polar solvent; and process (1b): extracting a first useful component from the residue after extraction in process (2b) using a non-polar solvent.

Description

Technical Field

[0001] This invention relates to a method for extracting useful components from algae. This method enables the separate extraction of useful components such as triglycerides and carotenoids from those such as omega-3 fatty acids and oxylipins. Background Technology

[0002] Nannochloropsis (hereinafter, sometimes referred to as "micro-chondria"), a marine algae with high oil production capacity and ultrafine structure, is a species containing various useful components. Regarding triglycerides (TAGs), which have attracted considerable attention as biofuels and food oils, reports have indicated lipid accumulation under nutrient-deficient conditions, accumulation of TAGs as storage lipids, and an increased proportion of saturated fatty acids (Biotechnol. Bioeng. 102, (2009) 100-112., Frontiers in Microbiology (2015) https: / / doi.org / 10.3389 / fmicb.2015.00912). The TAGs of Nannochloropsis contain a large amount of palmitic acid (C16:0), making it advantageous as a feedstock for biodiesel. On the other hand, *Microcystis globulus* also contains a large amount of eicosapentaenoic acid (C20:5, EPA), an omega-3 fatty acid (The Plant Journal (2008) 54, 621-639). Omega-3 fatty acids in *Microcystis globulus* are mainly found in membrane lipids and are important for various functions in animals, belonging to the category of polyunsaturated fatty acids (Frontiers in Microbiology (2015) https: / / doi.org / 10.3389 / fmicb.2015.00912). Eicosapentaenoic acid (C20:5, EPA) and DHA, as omega-3 fatty acids, are found in fatty seafood such as fish (e.g., salmon, tuna, trout) and crustaceans (e.g., crab, mussels, oysters). The omega-3 fatty acids in such seafood are not synthesized in the fish or crustaceans, but rather are substances synthesized in the algae ingested as food and accumulated in the seafood. With declining fish catches, marine algae have attracted attention as a new source of omega-3 fatty acids. Furthermore, oxidized lipids, which are EPA metabolites, can be generated from *Microcystis globulus*, which possesses EPA within its cells (Phytochemistry 102 (2014) 152-161). These oxidized lipids have been reported to exhibit broad anti-inflammatory activity (Phytochemistry 102 (2014) 152-161), and a method for extracting hydroxyeic acid (HEPE) from krill and shrimp has been reported (Japanese Patent Application Publication No. 2015-163607). In addition, *Microcystis globulus* is a naturally occurring pigment found in nature and also contains carotenoids known to have antioxidant properties. While carotenoids are abundant in green and yellow vegetables such as tomatoes and pumpkins, microalgae also accumulate various carotenoids. For example, Euglena contains carotenoid pigments such as diatomaceoustin, and brown algae contain carotenoid pigments such as fucoxanthin.Carotenoid accumulation has also been reported in the genus *Microcystis* (Algal Research volume 3 (2014) 36-43).

[0003] Although *Microcystis globulus* contains various useful components, no method has been developed to separate and extract these components. Lipid extraction from algae widely utilizes extraction methods using a chloroform / methanol / water mixture (Non-Patent Literature 1-4). In chloroform / methanol / water extraction, membrane lipids and storage lipids are extracted uniformly, necessitating further separation of the useful components.

[0004] Existing technical documents

[0005] Non-patent literature

[0006] Non-patent literature 1: J Biol Chem 226 (1957) 497-509

[0007] Non-patent literature 2: Can J Biochem Physiol 37 (1959) 911-917

[0008] Non-patent literature 3: Eur J Biochem 230 (1995) 987-993

[0009] Non-patent literature 4: BMC Biotechnology 11:7 (2011) doi: 10.1186 / 1472-6750-11-7. Summary of the Invention

[0010] The problem that the invention aims to solve

[0011] If the useful components contained in algae can be extracted separately, the recycling process can be made more efficient. This invention is proposed in this context, with the aim of providing a means for the separate extraction of useful components from algae.

[0012] Methods for solving problems

[0013] In order to solve the above-mentioned problems, the inventors have repeatedly conducted in-depth research and found that by using two organic solvents, namely non-polar and polar, in two stages, useful components such as TAG and carotenoids can be extracted separately from useful components such as ω3 fatty acids and oxidized lipids. Based on this understanding, the present invention was completed.

[0014] That is, the present invention provides the following [1] to [7].

[0015] [1] A method for extracting useful components from algae, characterized in that it comprises the following steps (1a) and (2a), or steps (2b) and (1b), Step (1a): Using a non-polar solvent, the first useful component is extracted from dried algae. Step (2a): Using a solvent containing a polar solvent, extract the second useful component from the residue after extraction in step (1a). Step (2b): Using a solvent containing a polar solvent, extract the second useful component from the dried algae. Step (1b): Using a non-polar solvent, extract the first useful component from the residue after extraction in step (2b).

[0016] [2] The method according to [1] is characterized by comprising step (1a) and step (2a).

[0017] [3] The method according to [1] or [2] is characterized in that the first useful component is triacylglycerol and / or carotenoid, and the second useful component is ω3 fatty acid and / or oxidized lipids.

[0018] [4] The method according to [1] or [2] is characterized in that the nonpolar solvent is hexane, and the solvent containing the polar solvent is ethanol or a mixture of ethanol and hexane.

[0019] [5] The method according to [1] or [2] is characterized in that the algae are algae containing triacylglycerols and containing ω3 fatty acids and oxidized lipids in the membrane lipids.

[0020] [6] The method according to [1] or [2] is characterized in that the algae are algae belonging to the genera *Nannochloropsis*, *Microchloropsis*, *Monodus*, *Fistulifera*, or *Phaeodactylum*.

[0021] [7] The method according to [1] or [2] is characterized in that the first useful component is triglyceride and / or carotenoid, the second useful component is ω3 fatty acid and / or oxidized lipid, the nonpolar solvent is hexane, the solvent containing the polar solvent is ethanol or a mixture of hexane and ethanol, and the algae is an algae belonging to the genus Nannochloropsis.

[0022] This specification includes the contents described in the Japanese patent application, Japanese Patent Application No. 2024-011901, and / or the drawings, which form the basis of the priority claim of this application.

[0023] The effects of the invention

[0024] This invention provides a novel method for extracting useful components from algae. This method enables the separate extraction of useful components such as TAG and carotenoids from those such as ω3 fatty acids and oxidized lipids. Attached Figure Description

[0025] Figure 1 A diagram illustrating the general outline of the two-stage extraction method.

[0026] Figure 2 A graph showing the condition of the extract under various conditions.

[0027] Figure 3 A diagram showing the composition of fatty acids contained in each extract.

[0028] Figure 4 This diagram shows the tags contained in each extract. The white dots at the arrows represent tags.

[0029] Figure 5 A diagram showing the composition of fatty acids contained in each TAG.

[0030] Figure 6 A diagram showing the components contained in dried algae.

[0031] Figure 7 A diagram showing the pigments contained in each extract.

[0032] Figure 8 A graph showing the amount of oxidized lipids contained in the extraction solvent of stage 2.

[0033] Figure 9 A diagram illustrating a general outline of the operation of Example 2.

[0034] Figure 10 A graph showing the condition of the extract under various conditions.

[0035] Figure 11 This diagram shows the tags contained in each extract. The white dots at the arrows represent tags. Detailed Implementation

[0036] The present invention will now be described in detail.

[0037] The method for extracting useful components from algae according to the present invention is characterized by comprising a step of (1) extracting a first useful component using a non-polar solvent and a step of (2) extracting a second useful component using a solvent containing a polar solvent. The order of steps (1) and (2) is not particularly limited; step (1) may be performed first, or step (2) may be performed first. That is, the method of the present invention may include steps (1a) and (2a) as described below, or it may include steps (2b) and (1b).

[0038] In step (1a), a non-polar solvent is used to extract the first useful component from dried algae.

[0039] Examples of nonpolar solvents include hexane, cyclohexane, and dichloromethane. Hexane is preferred among these because it allows for the recovery of almost all of the TAG and β-carotene contained in algae. Figure 4 and Figure 7 This allows for the efficient extraction of these components. Furthermore, the "hexane" used in this invention can consist solely of n-hexane, or it can be a main component of n-hexane with small amounts of other substances (e.g., methylcyclopentane, methylpentane, etc.).

[0040] There are no particular limitations on the primary useful component as long as it can be extracted using a non-polar solvent; TAGs and carotenoids are preferred. Only one of these components can be extracted, or both can be extracted. Examples of carotenoids include β-carotene, α-carotene, γ-carotene, ε-carotene, lutein, zeaxanthin, fucoxanthin, diatomaceoustin, and dinoflagellin.

[0041] There are no particular restrictions on the algae used, as long as useful components can be extracted. Algae belonging to the genera *Nannochloropsis*, *Microchloropsis*, *Monodus*, *Fistulifera*, or *Phaeodactylum* are preferred, with algae belonging to the genus *Nannochloropsis* being particularly preferred. Examples of algae belonging to the genus *Nannochloropsis* include *Nannochloropsis oceanica*, *Nannochloropsisoculata*, *Nannochloropsis granulata*, *Nannochloropsis australis*, *Nannochloropsis limnetica*, and *Nannochloropsis maritima*. Examples of algae belonging to the genus *Microchloropsis* include *Microchloropsis gaditana* and *Microchloropsis salina*. Examples of algae belonging to the genus *Monodus* include *Monodus subterraneus*. Examples of algae belonging to the genus *Fistulifera* include *Fistulifera*. Examples of algae belonging to the genus *Phaeodactylum* include *Phaeodactylum tricornutum*, *Fistulifera pelliculosa*, and *Fistulifera saprophila*. Furthermore, the algae used are preferably those containing triglycerides and whose membrane lipids include ω3 fatty acids and oxidized lipids.Examples of such algae include those belonging to the genus *Monodus* (e.g., *Monodus subterraneus*), those belonging to the genus *Fistulifera* (e.g., *Fistulifera olaris*, *Fistulifera pelliculosa*, *Fistulifera aprophila*), those belonging to the genus *Microchloropsis* (e.g., *Microchloropsis gaditana*, *Microchloropsis salina*), and those belonging to the genus *Nannochloropsis* (e.g., *Nannochloropsis oceanica*, *Nannochloropsis oculata*, *Nannochloropsis granulata*, *Nannochloropsis australis*). (e.g., *Australis*, *Nannochloropsislimnetica*, *Nannochloropsis maritima*)

[0042] When using algae belonging to the genus *Nannochloropsis*, algae cultured in conventional media can be used, as well as algae cultured in phosphorus-deficient or nitrogen-deficient media that have accumulated TAG. The accumulation of TAG using phosphorus-deficient media can be performed, for example, according to the description in the examples described later, or according to International Publication No. 2015 / 137449. The accumulation of TAG using nitrogen-deficient media can be performed, for example, according to International Publication No. 2015 / 137449.

[0043] The drying of algae can be carried out using conventional methods. The extraction of useful components from the dried algae can be achieved, for example, by a method comprising: 1) adding a solvent to the dried algae; 2) allowing a certain time for extraction; and 3) recovering the supernatant. The amount of solvent added is not particularly limited, but 3 to 100 mL of solvent is preferred, and more preferably 15 to 45 mL, relative to 100 mg of dried algae. The time allowed after solvent addition is not particularly limited, but is preferably 0.3 to 24 hours, and more preferably 0.5 to 18 hours. The recovery of the supernatant can be carried out using conventional methods such as centrifugation. Steps 1) to 3) are typically repeated at least twice, and the resulting supernatants are combined as the extract of the useful components. The number of repetitions is only required to be at least twice, but is preferably 3 to 7 times, and more preferably 4 to 6 times. The extract also contains components other than the useful components, so only the useful components can be separated by chromatography or the like.

[0044] In step (2a), a second useful component is extracted from the residue after extraction in step (1a) using a solvent containing a polar solvent.

[0045] The solvent containing a polar solvent can be any solvent that contains a polar solvent, and can be a solvent composed solely of a polar solvent, or a mixture of a polar solvent and a non-polar solvent. Examples of polar solvents include ethanol, 1-butanol, 2-butanol, 1-propanol, 2-propanol, and acetone. Examples of non-polar solvents that can be mixed with a polar solvent include hexane, cyclohexane, and dichloromethane. Examples of mixtures of polar and non-polar solvents include mixtures of ethanol and hexane, 1-butanol and hexane, 2-butanol and hexane, 1-propanol and hexane, 2-propanol and hexane, and acetone and hexane. The mixing ratio of the polar solvent to the non-polar solvent is not particularly limited, but by mass ratio, it is preferably 30 / 70 to 70 / 30, more preferably 40 / 60 to 60 / 40. Suitable solvents that include polar solvents include ethanol, a mixture of ethanol and hexane, a mixture of 1-butanol and hexane, a mixture of 2-butanol and hexane, a mixture of 1-propanol and hexane, a mixture of 2-propanol and hexane, and a mixture of acetone and hexane.

[0046] The second useful component is not particularly limited as long as it can be extracted by a solvent containing a polar solvent, but ω3 fatty acids and oxidized lipids are preferred. Only one of these components can be extracted, or both can be extracted. Examples of ω3 fatty acids include EPA and DHA, and examples of oxidized lipids include 15-hydroxyeicosapoleonic acid, 15-hydroxyeicosaptraenoic acid, 13-hydroxy-9,11-octadecadienoic acid, 13-hydroxy-9,11,15-octadectrienoic acid, 13-hydroxy-6,9,11-octadectrienoic acid, 15-hydroxy-8,11,13-eicosaptraenoic acid, 8-hydroxyeicosapoleonic acid, 8-hydroxyeicosaptraenoic acid, and 18-hydroxyeicosapoleonic acid.

[0047] It is desirable to remove residual nonpolar solvents from the residue after extraction in step (1a) before extraction in step (2a). Extraction of the useful component from the residue after extraction in step (1a) can be performed, for example, by a method comprising: 1) adding solvent to the residue; 2) allowing a certain time; and 3) recovering the supernatant. The amount of solvent added is not particularly limited, but 15 to 100 mL of solvent is preferred, and 30 to 60 mL is more preferred, relative to 100 mg of residue. The time allowed after adding the solvent is not particularly limited, but 0.3 to 24 hours is preferred, and 0.5 to 18 hours is more preferred. Recovery of the supernatant can be performed using conventional methods such as centrifugation. Steps 1) to 3) are typically repeated at least twice, and the resulting supernatants are combined as the extract of the useful component. The number of repetitions is only required to be at least twice, but preferably 2 to 6 times, and more preferably 3 to 4 times. The extract also contains components other than the useful component, so only the useful component can be separated by chromatography or the like.

[0048] In step (2b), a second useful component is extracted from dried algae using a solvent containing a polar solvent. The solvent containing the polar solvent can be the same as the solvent used in step (2a), and the algae can be the same as the algae used in step (1a). Furthermore, the second useful component can be the same as the component used in step (2a).

[0049] In step (1b), a non-polar solvent is used to extract the first useful component from the residue following extraction in step (2b). The non-polar solvent can be the same as the solvent used in step (1a). The first useful component can be the same as the component from step (1a).

[0050] Example

[0051] The present invention will be further described in detail below through embodiments, but the present invention is not limited to these embodiments.

[0052] [Example 1]

[0053] Experimental materials

[0054] The algae *Nannochloropsis* NIES-2145 (hereinafter referred to as "N.2145") was used. This algal strain is available from the National Institute for Environmental Research (http: / / www.nies.go.jp / ).

[0055] Experimental Operation

[0056] 1. Cultivation conditions

[0057] F2N medium was used as the usual liquid culture medium in the cultivation of N.2145.

[0058] Dissolve 440 mg of Na₂EDTA·2H₂O, 316 mg of FeCl₃·6H₂O, 1.2 mg of CoSO₄·7H₂O, 2.1 mg of ZnSO₄·7H₂O, 18 mg of MnCl₂·4H₂O, 0.7 mg of CuSO₄·5H₂O, and 0.7 mg of Na₂MoO₄·2H₂O in 100 mL of ion-exchanged water and store as f / 2 metal at 4℃. Dissolve 121.14 g of tris(hydroxymethyl)aminomethane in 900 mL of ion-exchanged water, adjust the pH to 7.6 with HCl, and then adjust the volume to 1 L. Store as 1 M Tris-HCl (pH 7.6) at 4℃. Dissolve 7.5 mg of NaNO₃, 26.745 mg of NH₄Cl, 3 g of NaH₂PO₄·2H₂O, and vitamin B... 12 0.25 μg of biotin, 0.25 μg of thiamine HCl, 50 μg of f / 2 metal, 0.5 mL of 1M Tris-HCl (pH 7.6) were dissolved in 98.5 mL of artificial seawater. After sterilization by filtration, the solution was used as F2N medium. Daigo artificial seawater SP (FUJIFILM) was used. A phosphorus-deficient medium was prepared by removing NaH2PO4 from the F2N medium. Using various liquid media, measurements were performed at 20–30 μmol photons / m³. 2 / sec, 23℃, 120 min -1 Rotary culture.

[0059] 2. Lipid extraction

[0060] Cells in a typical culture medium

[0061] 850 mL of culture medium, which had been cultured for 20 days in normal medium, was centrifuged at 3500 × g for 5 minutes to precipitate the cells. To remove seawater salt, the cells were suspended in 50 mL of ion-exchange water and centrifuged again at 3500 × g for 5 minutes to precipitate. The precipitate was then suspended in 7 mL of ion-exchange water, spread out, and dried at 60°C for 22 hours to obtain 340 mg of dried algae. Three portions of dried algae, each 65 mg, were prepared, and experiments were conducted under conditions 1 to 3 as follows.

[0062] In condition 1, as a control, extraction was performed using the previously used chloroform / methanol mixture. 65 mg of dried algae was added to 1.5 mL of chloroform and 3 mL of methanol, and the mixture was incubated at room temperature for 1 hour while being suspended every 10 minutes. The mixture was centrifuged at 1000×g for 5 minutes using a shaking rotor, and 4.4 mL of the supernatant was recovered as the first extract. 1.5 mL of chloroform and 3 mL of methanol were added again, and the mixture was incubated at room temperature for 30 minutes while being suspended every 10 minutes. The mixture was then centrifuged at 1000×g for 5 minutes using a shaking rotor, and 5 mL of the supernatant was recovered as the second extract. 1.5 mL of chloroform and 3 mL of methanol were added again, and the mixture was incubated at room temperature for 30 minutes while being suspended every 10 minutes. The mixture was then centrifuged at 1000×g for 5 minutes using a shaking rotor, and 4.5 mL of the supernatant was recovered as the third extract. 3 mL of chloroform and 1.5 mL of methanol were added again, and the mixture was incubated at room temperature for 30 minutes while being suspended every 10 minutes. Centrifuge at 1000×g for 5 minutes using a vibrating rotor, and collect 4.5 mL of the supernatant as the fourth extract. Finally, add 3 mL of chloroform and 1.5 mL of methanol, resuspending the extract every 10 minutes while allowing it to stand at room temperature for 30 minutes. Centrifuge at 1000×g for 5 minutes using a vibrating rotor, and collect 4.5 mL of the supernatant as the fifth extract. Combine the first through fifth extracts, dry them, and dissolve them in chloroform:methanol at a concentration of 25 mg / mL, then store at -20°C.

[0063] In condition 2, as the first stage, extraction was performed with hexane. After removing the solvent from the precipitate by drying, the second stage extraction was performed with ethanol. 4.5 mL of hexane was added to 65 mg of dried algae, and the mixture was incubated at room temperature for 1 hour while being suspended every 10 minutes. The mixture was centrifuged at 1000 × g for 5 minutes using a shaking rotor, and 4.5 mL of the supernatant was recovered as the first extract. Another 4.5 mL of hexane was added, and the mixture was incubated at room temperature for 30 minutes while being suspended every 10 minutes. The mixture was then centrifuged at 1000 × g for 5 minutes using a shaking rotor, and 4.5 mL of the supernatant was recovered as the second extract. Another 4.5 mL of hexane was added, and the mixture was incubated at room temperature for 30 minutes while being suspended every 10 minutes. The mixture was then centrifuged at 1000 × g for 5 minutes using a shaking rotor, and 4.5 mL of the supernatant was recovered as the third extract. Another 4.5 mL of hexane was added, and the mixture was incubated at room temperature for 30 minutes while being suspended every 10 minutes. Centrifuge at 1000×g for 5 minutes using a vibrating rotor, and recover 4.5 mL of the supernatant as the fourth extract. Finally, add 4.5 mL of hexane, and incubate at room temperature for 30 minutes while resuspending the extract every 10 minutes. Centrifuge at 1000×g for 5 minutes using a vibrating rotor, and recover 4.5 mL of the supernatant as the fifth extract. Combine the first to fifth extracts, dry them, dissolve them in hexane to a concentration of 25 mg / mL, and store as the first-stage extract at -20°C. After removing residual hexane from the precipitate by drying, perform the second-stage extraction with ethanol. Add 9 mL of ethanol to the precipitate and incubate at room temperature for 18 hours. Centrifuge at 1000×g for 5 minutes using a vibrating rotor, and recover 9 mL of the supernatant as the first extract. Add another 9 mL of ethanol, and incubate at room temperature for 1 hour while resuspending the extract every 10 minutes. Centrifuge at 1000×g for 5 minutes using a shaking rotor, and recover 9 mL of the supernatant as the second extract. Add 9 mL of ethanol, resuspend the extract every 10 minutes, and incubate at room temperature for 1 hour. Centrifuge at 1000×g for 5 minutes using a shaking rotor, and recover 9 mL of the supernatant as the third extract. Finally, add 9 mL of ethanol, resuspend the extract every 10 minutes, and incubate at room temperature for 1 hour. Centrifuge at 1000×g for 5 minutes using a shaking rotor, and recover 9 mL of the supernatant as the fourth extract. Combine the first through fourth extracts, dry them, dissolve them in ethanol to a concentration of 25 mg / mL, and store as the second-stage extract at -20°C.

[0064] In condition 3, as in condition 2, the first stage was performed with hexane extraction. After removing the solvent from the precipitate by drying, the second stage of extraction was carried out with a hexane / ethanol mixture. Instead of 9 mL of ethanol, 6 mL of hexane and 3 mL of ethanol were used; otherwise, the same procedures as in condition 2 were performed.

[0065] Cells in phosphorus-deficient culture medium

[0066] 3 L of culture medium, which had been cultured in phosphorus-deficient medium for 7 days, was centrifuged at 3500 × g for 5 minutes to precipitate the cells. To remove seawater salt, the cells were suspended in 50 mL of ion-exchange water and centrifuged again at 3500 × g for 5 minutes to precipitate. The precipitate was then suspended in 7 mL of ion-exchange water, spread out, and dried at 60 °C for 22 hours to obtain 302 mg of dried algae. Three 58 mg samples of dried algae were prepared and experiments were conducted under conditions 1 to 3.

[0067] In condition 1, as a control, extraction was performed using a previously used chloroform / methanol mixture. 58 mg of dried algae was added to 3 mL of chloroform and 6 mL of methanol, and the mixture was incubated at room temperature for 1 hour while being suspended every 10 minutes. The mixture was centrifuged at 1000×g for 5 minutes using a shaking rotor, and 9 mL of the supernatant was recovered as the first extract. 3 mL of chloroform and 6 mL of methanol were added again, and the mixture was incubated at room temperature for 1 hour while being suspended every 10 minutes. The mixture was centrifuged at 1000×g for 5 minutes using a shaking rotor, and 9 mL of the supernatant was recovered as the second extract. 3 mL of chloroform and 6 mL of methanol were added again, and the mixture was incubated at room temperature for 17 hours. The mixture was centrifuged at 1000×g for 5 minutes using a shaking rotor, and 9 mL of the supernatant was recovered as the third extract. 6 mL of chloroform and 3 mL of methanol were added again, and the mixture was incubated at room temperature for 1 hour while being suspended every 10 minutes. The mixture was centrifuged at 1000×g for 5 minutes using a shaking rotor, and 9 mL of the supernatant was recovered as the fourth extract. 6 mL of chloroform and 3 mL of methanol were added, and the mixture was incubated at room temperature for 1 hour while being suspended every 10 minutes. The mixture was centrifuged at 1000×g for 5 minutes using a shaking rotor, and 9 mL of the supernatant was recovered as the 5th extraction. 6 mL of chloroform and 3 mL of methanol were added, and the mixture was incubated at room temperature for 1 hour while being suspended every 10 minutes. The mixture was centrifuged at 1000×g for 5 minutes using a shaking rotor, and 9 mL of the supernatant was recovered as the 6th extraction. Finally, 6 mL of chloroform and 3 mL of methanol were added, and the mixture was incubated at room temperature for 1 hour while being suspended every 10 minutes. The mixture was centrifuged at 1000×g for 5 minutes using a shaking rotor, and 9 mL of the supernatant was recovered as the 7th extraction. To shorten the drying time, a two-layer separation was performed. The lower chloroform layer was recovered as the lipid extract. For the two-layer separation, 5.4 mL of 1% (w / v) KCl and 3 mL of chloroform were added to the first to third extractions. 5.4 mL of 1% (w / v) KCl and 3 mL of methanol were added to the 4th through 7th extracts. After resuscitation, the extracts were centrifuged at 1000 × g for 5 minutes using a gyratory rotor, and the lower chloroform layer was recovered as the lipid extract. The lipid extracts from the 1st to 7th extracts were combined, dried, and dissolved in chloroform:methanol at a concentration of 25 mg / mL in a 2:1 ratio, then stored at -20°C.

[0068] In condition 2, as the first stage, extraction was performed with hexane. After removing the solvent from the precipitate by drying, the second stage extraction was performed with ethanol. 9 mL of hexane was added to 58 mg of dried algae, and the mixture was suspended at room temperature for 1 hour while being resuspended every 10 minutes. The mixture was centrifuged at 1000 × g for 5 minutes using a shaking rotor, and 9 mL of the supernatant was recovered as the first extract. Another 9 mL of hexane was added, and the mixture was suspended at room temperature for 1 hour while being resuspended every 10 minutes. The mixture was centrifuged at 1000 × g for 5 minutes using a shaking rotor, and 9 mL of the supernatant was recovered as the second extract. Another 9 mL of hexane was added, and the mixture was suspended at room temperature for 1 hour while being resuspended every 10 minutes. The mixture was centrifuged at 1000 × g for 5 minutes using a shaking rotor, and 9 mL of the supernatant was recovered as the third extract. Another 9 mL of hexane was added, and the mixture was suspended at room temperature for 1 hour while being resuspended every 10 minutes. Centrifuge at 1000×g for 5 minutes using a shaking rotor, and collect 9 mL of the supernatant as the fourth extract. Add 9 mL of hexane, and suspend at room temperature for 1 hour while resuspending the extract every 10 minutes. Centrifuge at 1000×g for 5 minutes using a shaking rotor, and collect 9 mL of the supernatant as the fifth extract. Add 9 mL of hexane, and suspend at room temperature for 1 hour while resuspending the extract every 10 minutes. Centrifuge at 1000×g for 5 minutes using a shaking rotor, and collect 9 mL of the supernatant as the sixth extract. Finally, add 9 mL of hexane, and suspend at room temperature for 1 hour while resuspending the extract every 10 minutes. Centrifuge at 1000×g for 5 minutes using a shaking rotor, and collect 9 mL of the supernatant as the seventh extract. Combine the extracts from the first to the seventh extraction, dry them, dissolve them in hexane to a concentration of 25 mg / mL, and store as the first-stage extract at -20°C. After removing residual hexane from the precipitate by drying, a second stage of extraction was performed using ethanol. 9 mL of ethanol was added to the precipitate and the mixture was allowed to stand at room temperature for 17 hours. The mixture was centrifuged at 1000×g for 5 minutes using a shaking rotor, and 9 mL of the supernatant was recovered as the first extract. Another 9 mL of ethanol was added, and the mixture was allowed to stand at room temperature for 1 hour while being resuspended every 10 minutes. The mixture was centrifuged at 1000×g for 5 minutes using a shaking rotor, and 9 mL of the supernatant was recovered as the second extract. Another 9 mL of ethanol was added, and the mixture was allowed to stand at room temperature for 1 hour while being resuspended every 10 minutes. The mixture was centrifuged at 1000×g for 5 minutes using a shaking rotor, and 9 mL of the supernatant was recovered as the third extract. Finally, 9 mL of ethanol was added, and the mixture was allowed to stand at room temperature for 1 hour while being resuspended every 10 minutes. The mixture was centrifuged at 1000×g for 5 minutes using a shaking rotor, and 9 mL of the supernatant was recovered as the fourth extract.The extracts from the first to fourth extractions were combined, dried, dissolved in ethanol at a concentration of 25 mg / mL, and stored at -20°C as the second-stage extract.

[0069] In condition 3, similarly to condition 2, as the first stage, extraction was performed with hexane. After removing the solvent from the precipitate by drying, the second stage of extraction was carried out with a hexane / ethanol mixture. Instead of 9 mL of ethanol, 6 mL of hexane and 3 mL of ethanol were used; otherwise, the same procedures as in condition 2 were performed.

[0070] 3. Lipid analysis

[0071] 50 μl of 1 mM docosanoic acid and 500 μl of 1.5 M hydrochloric acid / methanol were added to 5 μl of lipid extract (equivalent to 0.125 mg). After resuscitation, the mixture was allowed to stand at 85 °C for 1 hour to methylate the fatty acids. 500 μl of hexane was added, and the mixture was resuspended. The mixture was then centrifuged at 1000 × g for 5 minutes using a shaking rotor to recover the upper layer containing the methylated fatty acids. Another 500 μl of hexane was added to the lower layer, and the mixture was resuspended. The upper layer was then centrifuged at 1000 × g for 5 minutes using a shaking rotor to recover the upper layer. The recovered methylated fatty acids were dried and dissolved in 100 μl of hexane for gas chromatography. Triglycerides (TAG) were separated and purified using thin-layer silica (TLC) and analyzed by gas chromatography. 5 μl of the lipid extract (equivalent to 0.125 mg) was spotted onto a TLC plate and developed for 45 min with a mixture of 160 mL hexane, 40 mL diethyl ether, and 4 mL acetic acid. 0.1 mg of trioleic acid ester was spotted as a control for TAG migration. TAG was confirmed using 0.001% primrose yellow under UV irradiation. The silica containing the TAG was removed, and after resuscitation with 50 μl of 1 mM docosanoic acid and 500 μl of 1.5 M hydrochloric acid / methanol, the mixture was allowed to stand at 85 °C for 1 h to methylate the fatty acids. After resuscitation with 500 μl of hexane, the mixture was centrifuged at 1000 × g for 5 min using a shaking rotor to recover the supernatant hexane containing the methylated fatty acids. The lower layer was resuspended by adding 500 μl of hexane, and then centrifuged at 1000 × g for 5 minutes using a shaking rotor to recover the upper layer. The recovered methyl esterified fatty acids were dried and dissolved in 100 μl of hexane as the gas chromatography sample. Gas chromatography was performed using a Shimadzu GC-2030 HR-SS-10 (0.25 mm inner diameter, 25 m length) (SHINWA CHEMICAL INDUSTRIES, LTD.).

[0072] 4. Pigment Analysis

[0073] TLC analysis was performed to compare the types of pigments contained in the extracts. 10 μl of the lipid extract (equivalent to 0.25 mg) was spotted onto a TLC plate and developed for 40 min with a mixture of 70 mL petroleum ether and 30 mL acetone. Pigments obtained from spinach by chloroform / methanol extraction were used as controls.

[0074] 5. Analysis of oxidized lipids

[0075] LC-MS was used to investigate the oxidized lipids contained in the lipid extracts. Lipid extracts under Condition 2 (using cells cultured in standard medium with stage 2 ethanol) and Condition 3 (using a stage 2 hexane / ethanol mixture) were diluted 1000-fold. The contents of free 15-HEPE and 15-HETE in the solutions were determined using a Shimadzu LC-MS 8050. A Phenomenex Kinetex C8 column (inner diameter 2.1 mm, length 150 mm, particle size 2.6 μm) was used.

[0076] Experimental results

[0077] 1. Lipid extraction

[0078] To compare the extraction methods using chloroform / methanol mixtures to date with two-stage extraction, lipid extraction was performed using methods under conditions 1–3. Figure 1 In condition 1, as a control, extraction was performed using a conventional chloroform / methanol mixture. In condition 2, as the first stage, extraction was performed with hexane, and after drying to remove the solvent from the precipitate, a second stage extraction was performed with ethanol. In condition 3, as the first stage, extraction was performed with hexane, and after drying to remove the solvent from the precipitate, a second stage extraction was performed with a hexane / ethanol mixture. Two cell types were used for lipid extraction: cells cultured in conventional media and cells cultured in a phosphorus-deficient medium, which facilitates TAG accumulation.

[0079] 340 mg of dried algae was obtained from 850 mL of culture medium that had been cultured for 20 days in normal medium. Three portions of dried algae, each 65 mg, were prepared, and experiments were conducted under conditions 1 through 3. In condition 1, the first extraction yielded a dark green extract; the second and third extractions progressively lightened the color; and the fourth and fifth extractions were almost uncolored. Figure 2 (Top left). In the first stage of hexane extraction under conditions 2 and 3, a dark green extract was obtained from the first extract, and the extracts from the second to fifth extractions did not gradually lighten in color. Figure 2 (Left-middle). If a hexane extraction is performed in the first stage followed by a hexane / ethanol mixture extraction in the second stage, the coloring essentially disappears after the second extraction. However, if ethanol extraction is used, the extract remains green even after the fourth extraction. Figure 2 (Bottom left). As can be seen from the recovered lipid weights, lipids can also be recovered to a similar extent as those recovered by the chloroform / methanol extraction to date (Table 1).

[0080] 302 mg of dried algae was obtained from 3 L of culture medium cultured in phosphorus-deficient medium for 7 days. Three portions of dried algae, each 58 mg, were prepared, and experiments were conducted under conditions 1 through 3. The expected increase in TAG accumulation in the phosphorus-deficient medium led to a greater amount of organic solvent and more extractions than with conventional media. In condition 1, a dark green extract was obtained from the first extraction, which gradually lightened in the second and third extractions, and the extracts from the fourth and subsequent extractions remained uncolored. Figure 2 (Top right). In the first stage of hexane extraction under conditions 2 and 3, a dark green extract was obtained from the first extract and the third extract after 17 hours, and it did not gradually lighten in color from the fourth extract onwards. Figure 2 (Right center). If a hexane extraction is performed in the first stage followed by a hexane / ethanol mixture extraction in the second stage, the coloring essentially disappears in the second extraction. However, if ethanol extraction is used, even in the fourth extraction, the extract remains slightly stained green. Figure 2 (Bottom right). The recovered lipid weights show that even phosphorus-deficient cultured cells with increased TAG accumulation can recover lipids to the same extent as chloroform / methanol extraction through a two-stage extraction process (Table 1). Furthermore, the lipid weight per unit weight relative to dried algae is greater than that of normally cultured cells. Further comparison with normally cultured cells shows that the amount of lipids recovered in the first stage is greater.

[0081]

[0082] 2. Lipid analysis

[0083] Gas chromatography was performed to analyze the fatty acids contained in the lipids. Figure 3As a result, in normal cultures, the proportion of palmitic acid (C16:0) was lower in the second-stage ethanol or hexane / ethanol extraction compared to chloroform / methanol extraction. The proportion of C16:0 was even lower and the proportion of eicosapentaenoic acid (C20:5, EPA) was higher in the hexane extraction compared to ethanol extraction. In phosphorus-deficient cultures, the proportion of EPA was lower in the first-stage hexane extraction compared to chloroform / methanol extraction, while the proportion of EPA was higher in the second-stage ethanol or hexane / ethanol extraction, and the proportions of C16:0 and palmitoleic acid (C16:1) were lower. It is known that in *Microcystis globulus*, the proportions of arachidonic acid (C20:4) and EPA in TAG are lower than those in membrane lipids (Frontiers in Microbiology (2015) https: / / doi.org / 10.3389 / fmicb.2015.00912, Plant Physiol. 171, (2016), 2469-2482). It can be assumed that the reduced EPA proportion in stage 1 is due to the high content of TAG in the hexane extract. Therefore, TLC and gas chromatography analyses were performed to analyze the TAG content in each extract. Figure 4 The results showed that both normal and phosphorus-deficient cultures could recover all TAGs through hexane extraction in the first stage, and the extract in the second stage contained no TAGs. Furthermore, it was determined that the amount of TAGs per unit lipid was greater than that extracted with chloroform / methanol compared to hexane extraction. The fatty acid composition of each TAG is shown in the figure. Figure 5 In the chloroform / methanol extraction of normal culture, EPA cannot be detected due to the low amount of TAG. Therefore, the C16:0 ratio is higher than previously reported, but in hexane extraction, the EPA content is 6%, similar to the fatty acid composition of TAG after normal culture. The EPA content is particularly low in phosphorus-deficient culture, decreasing to 1%. The above lipid analysis results clarify that TAG of the same degree or higher as in chloroform / methanol extraction can be extracted in the first stage of hexane extraction, while the extract in the second stage does not contain TAG. Lipids of the same degree as in chloroform / methanol extraction can be extracted by performing ethanol extraction or hexane / ethanol extraction after hexane extraction. Figure 6 ).

[0084] 3. Pigment Analysis

[0085] The extracts were spotted onto a TLC plate and developed using petroleum ether / acetone. The results showed that β-carotene was present only in the hexane extract from the first stage of normal culture, and not in the ethanol or hexane / ethanol extracts from the second stage. Figure 7(Left). Furthermore, four novel carotenoid-like spots with migration rates not observed in spinach were observed (indicated by circled arrows numbered 1, 2, 4, and 5). This indicates that while β-carotene is present in the hexane extract during phosphorus-deficient culture, the amount is significantly lower compared to normal culture. Figure 7 (Right). Furthermore, novel carotenoid spots not observed in normal culture were observed (indicated by arrows with circled numbers 3). These results demonstrate that the two-stage extraction method using non-polar and polar solvents of the present invention allows for the complete recovery of β-carotene from the extract in the first stage, the separate recovery of novel carotenoids with different properties, and the control of carotenoid accumulation through culture.

[0086] 4. Analysis of oxidized lipids

[0087] The presence of oxidized lipids, which are metabolites of EPA, in the extracts obtained using a two-stage extraction process was investigated using LC-MS. It was found that free hydroxyeicosapride (15-HEPE) and hydroxyeicosatetraenoic acid (15-HETE), which have affinity for polar solvents, were abundant in the second-stage extracts obtained by performing ethanol extraction or hexane / ethanol extraction following hexane extraction. Figure 8 From the data, it can be seen that ethanol extraction is more effective at recovering oxidized lipids compared to hexane / ethanol extraction.

[0088] 5. Summary

[0089] The results above demonstrate that in this invention, which involves a two-stage extraction process using non-polar and polar solvents after algal drying, TAG and β-carotene can be fully recovered using the non-polar solvent in the first stage. Two options are available for the extraction solvent in the second stage. Using a polar solvent allows for efficient recovery of free oxidized lipids, while using a mixture of non-polar and polar solvents results in a decreased recovery rate of free oxidized lipids but an increased recovery rate of EPA contained in membrane lipids. Thus, this invention is an effective method for the separate recovery of useful components from algae according to specific purposes.

[0090] [Example 2]

[0091] Experimental materials

[0092] The experimental materials used were the same as those used in Example 1.

[0093] Experimental Operation

[0094] Lipid extraction

[0095] Using cells cultured in standard culture medium, the extraction steps in Condition 2 were replaced, and the experiment was conducted as Condition 4. Specifically, in Condition 4, the first stage of extraction was performed with ethanol, and after removing the solvent from the precipitate by drying, the second stage of extraction was performed with hexane. 4.5 mL of ethanol was added to 65 mg of dried algae, and the mixture was incubated at room temperature for 1 hour while being suspended every 10 minutes. The mixture was centrifuged at 1000 × g for 5 minutes using a vibrating rotor, and 4.5 mL of the supernatant was recovered as the first extract. Another 4.5 mL of ethanol was added, and the mixture was incubated at room temperature for 30 minutes while being suspended every 10 minutes. The mixture was then centrifuged at 1000 × g for 5 minutes using a vibrating rotor, and 4.5 mL of the supernatant was recovered as the second extract. Another 4.5 mL of ethanol was added, and the mixture was incubated at room temperature for 30 minutes while being suspended every 10 minutes. The mixture was then centrifuged at 1000 × g for 5 minutes using a vibrating rotor, and 4.5 mL of the supernatant was recovered as the third extract. 4.5 mL of ethanol was added, and the mixture was incubated at room temperature for 30 minutes while resuspending the precipitate every 10 minutes. The mixture was centrifuged at 1000 × g for 5 minutes using a shaking rotor, and 4.5 mL of the supernatant was collected as the fourth extract. Finally, 4.5 mL of ethanol was added, and the mixture was incubated at room temperature for 30 minutes while resuspending the precipitate every 10 minutes. The mixture was centrifuged at 1000 × g for 5 minutes using a shaking rotor, and 4.5 mL of the supernatant was collected as the fifth extract. The extracts from the first to fifth stages were combined, dried, dissolved in hexane to a concentration of 25 mg / mL, and stored at -20°C as the first-stage extract. After removing residual ethanol from the precipitate by drying, the second-stage extraction was performed using hexane. 9 mL of hexane was added to the precipitate, and the mixture was incubated at room temperature for 18 hours. The mixture was centrifuged at 1000 × g for 5 minutes using a shaking rotor, and 9 mL of the supernatant was collected as the first extract. 9 mL of hexane was added, and the mixture was incubated at room temperature for 1 hour while resuspending the precipitate every 10 minutes. Centrifuge at 1000×g for 5 minutes using a gyratory rotor, and recover 9 mL of the supernatant as the second extract. Add 9 mL of hexane, and suspend at room temperature for 1 hour while resuspending the extract every 10 minutes. Centrifuge at 1000×g for 5 minutes using a gyratory rotor, and recover 9 mL of the supernatant as the third extract. Finally, add 9 mL of hexane, and suspend at room temperature for 1 hour while resuspending the extract every 10 minutes. Centrifuge at 1000×g for 5 minutes using a gyratory rotor, and recover 9 mL of the supernatant as the fourth extract. Combine the first to fourth extracts, dry them, dissolve them in hexane to a concentration of 25 mg / mL, and store as the second-stage extract at -20°C. For comparison with condition 4, conditions 1 and 2 were performed using the same procedures as in Example 1.

[0096] Lipid analysis

[0097] To investigate the TAG contained in the extracted lipids, TLC was performed using the same procedures as in Example 1.

[0098] Experimental results

[0099] Lipid extraction

[0100] To investigate whether extraction could still be performed even if the extraction procedure sequence of condition 2 shown in Example 1 was replaced, lipid extraction was performed using methods of conditions 1, 2, and 4. Figure 9 As in Example 1, extraction was performed using a conventional chloroform / methanol mixture as a control in Condition 1. In Condition 2, extraction was performed with hexane as the first stage, followed by a second stage extraction with ethanol after the solvent was removed by drying to remove the precipitate. In Condition 4, extraction was performed with ethanol as the first stage, followed by a second stage extraction with hexane after the solvent was removed by drying to remove the precipitate. Cells cultured in a standard culture medium were used in the lipid extraction.

[0101] In Condition 1, the same as in Example 1, a dark green extract was obtained through the first extraction; the second and third extractions progressively lightened the color; and the fourth and fifth extractions were almost uncolored. Figure 10 superior).

[0102] In the first stage of hexane extraction under condition 2, a dark green extract was obtained using the first extract, similar to that in Example 1, and the extracts from the second to fourth extractions did not progressively lighten in color. Figure 10 (Right center). If the hexane extraction in the first stage is followed by ethanol extraction in the second stage, then, as in Example 1, even in the fourth extraction, the extract is colored green ( Figure 10 (Bottom right). In the first stage of ethanol extraction under condition 4, a dark green extract is obtained from the first extract, and the extracts from the second to fifth extractions do not progressively lighten in color. Figure 10 (Left-middle). If hexane is used in the second stage after the first stage of ethanol extraction, a light green extract is obtained from the first extract, and the coloring almost disappears in the second extraction. Figure 10 (Bottom left). The hexane extract from stage 2 of condition 4 is a lighter green than the hexane extract from stage 1 of condition 2. Therefore, the hexane extract from stage 2 of condition 4 contains less pigment than the hexane extract from stage 1 of condition 2, and it is expected to recover TAG efficiently. In addition, the weights of the recovered lipids show that even if the extraction order of the two stages is changed, a certain amount of lipids can still be recovered (Table 2).

[0103]

[0104] Lipid analysis

[0105] To analyze the TAGs contained in each extract, TLC analysis was performed in the same manner as in Example 1. Figure 11 The results showed that, similar to Example 1 in Condition 2, all TAG could be recovered through the hexane extraction in the first stage, and no TAG was present in the ethanol extract in the second stage. It was clarified that in Condition 4, which replaced the two-stage extraction order, a portion of TAG was present in the ethanol extract in the first stage, but most of the TAG could be recovered through the hexane extraction in the second stage, and the amount of TAG per unit lipid was greater than that of the conventional chloroform / methanol extraction.

[0106] Based on the lipid analysis results above, it can be concluded that the solvents in the first and second stages can be used interchangeably. It was confirmed that with the change in order, less pigment contamination occurred during the second stage extraction, resulting in more efficient TAG recovery compared to the conventional chloroform / methanol extraction.

[0107] All publications, patents and patent applications cited in this specification are incorporated herein by reference directly.

[0108] Industry availability

[0109] The TAG extracted by this invention can be utilized as fuel and food, while ω3 fatty acids, carotenoids, and oxidized lipids can be utilized as food and pharmaceuticals. Therefore, this invention can be used in industries related to fuel, food, and pharmaceuticals.

Claims

1. A method for extracting useful components from algae, characterized in that, It includes the following steps (1a) and (2a), or steps (2b) and (1b). Step (1a): Using a non-polar solvent, the first useful component is extracted from dried algae. Step (2a): Using a solvent containing a polar solvent, extract the second useful component from the residue after extraction in step (1a). Step (2b): Using a solvent containing a polar solvent, extract the second useful component from the dried algae. Step (1b): Using a non-polar solvent, extract the first useful component from the residue after extraction in step (2b).

2. The method according to claim 1, characterized in that, It includes process (1a) and process (2a).

3. The method according to claim 1 or 2, characterized in that, The first useful component is triglycerides and / or carotenoids, and the second useful component is omega-3 fatty acids and / or oxidized lipids.

4. The method according to claim 1 or 2, characterized in that, The nonpolar solvent is hexane, and the solvent containing the polar solvent is ethanol, or a mixture of ethanol and hexane.

5. The method according to claim 1 or 2, characterized in that, Algae are algae that contain triglycerides and whose membrane lipids contain ω3 fatty acids and oxidized lipids.

6. The method according to claim 1 or 2, characterized in that, The algae belong to the genera *Nannochloropsis*, *Microchloropsis*, *Monodus*, *Fistulifera*, or *Phaeodactylum*.

7. The method according to claim 1 or 2, characterized in that, The first useful component is triglycerides and / or carotenoids, the second useful component is ω3 fatty acids and / or oxidized lipids, the nonpolar solvent is hexane, the solvent containing polar solvents is ethanol, or a mixture of hexane and ethanol, and the algae are algae belonging to the genus *Nannochloropsis*.

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