Method for extracting useful component of algae

Abstract

Provided is a method for extracting a useful component from algae, the method being characterized by comprising the following steps (1a) and (2a) or the following steps (2b) and (1b). (1a) is a step for extracting a first useful component from dried algae by using a non-polar solvent. (2a) is a step for extracting a second useful component from the residue after the extraction in the step (1a) by using a solvent including a polar solvent. (2b) is a step for extracting a second useful component from dried algae by using a solvent including a polar solvent. (1b) is a step for extracting a first useful component from the residue after the extraction in the step (2b) by using a non-polar solvent.

Description

Algae useful component extraction method

[0001] The present invention relates to a method for extracting useful components from algae, which enables the separate extraction of useful components such as triacylglycerols and carotenoids from useful components such as ω-3 fatty acids and oxylipins.

[0002] Nannochloropsis (hereafter referred to as "Nannochloropsis"), a marine algae with high oil production potential, contains a variety of useful compounds. Triacylglycerols (TAGs), which are attracting attention as potential sources of biofuels and edible oils, have been reported to accumulate lipids under nutrient-deficient conditions, including the accumulation of storage lipids (TAGs), and the increase in the proportion of saturated fatty acids (Biotechnol. Bioeng. 102, (2009) 100-112., Frontiers in Microbiology (2015) https: / / doi.org / 10.3389 / fmicb.2015.00912). Nannochloropsis TAGs contain a high content of palmitic acid (C16:0), making them an advantageous feedstock for biodiesel production. On the other hand, Nannochloropsis also contains a large amount of the ω-3 fatty acid eicosapentaenoic acid (EPA) (The Plant Journal (2008) 54, 621-639). ω-3 fatty acids are found primarily in membrane lipids in Nannochloropsis and are important for various functions in animals. They belong to the polyunsaturated fatty acid family (Frontiers in Microbiology (2015) https: / / doi.org / 10.3389 / fmicb.2015.00912). The ω-3 fatty acids eicosapentaenoic acid (EPA) and DHA are found in seafood such as fatty fish (e.g., salmon, tuna, and trout) and crustaceans (e.g., crab, mussels, and oysters). The ω-3 fatty acids in these seafoods are not synthesized by the fish or crustaceans themselves; rather, they are synthesized in the algae they consume as food and subsequently accumulate in the seafood. As fish catches decline, marine algae are attracting attention as a new source of omega-3 fatty acids. Furthermore, Nannochloropsis, which contains EPA, can produce oxylipins, which are metabolic products of EPA (Phytochemistry 102 (2014) 152-161).These oxylipins have been reported to exhibit a wide range of anti-inflammatory activities (Phytochemistry 102 (2014) 152-161), and a method for extracting hydroxyeicosapentaenoic acids (HEPEs) from krill and shrimp has been reported (JP Patent Publication No. 2015-163607). Nannochloropsis also contains carotenoids, a natural pigment found in nature and known to have antioxidant properties. While carotenoids are abundant in green and yellow vegetables such as tomatoes and pumpkins, microalgae have also been reported to accumulate various carotenoids. For example, Euglena contains diatoxanthin, and brown algae contain fucoxanthin, a carotenoid pigment. Accumulation of carotenoids has also been reported in the genus Nannochloropsis (Algal Research Volume 3 (2014) 36-43).

[0003] Although Nannochloropsis contains such a variety of useful components, no method has been developed to separate and extract each of these components. A method using a chloroform-methanol-water mixture is widely used to extract lipids from algae (Non-Patent Documents 1-4), but membrane lipids and storage lipids are extracted together with this method, necessitating further separation of the useful components.

[0004] J Biol Chem 226 (1957) 497-509Can J Biochem Physiol 37 (1959) 911-917Eur J Biochem 230 (1995) 987-993BMC Biotechnology 11:7 (2011) doi: 10.1186 / 1472-6750-11-7.

[0005] If the useful components contained in algae could be separated and extracted, the recovery process could be made more efficient. The present invention was made against this background, and its object is to provide a means for separating and extracting the useful components from algae.

[0006] As a result of extensive research to solve the above problems, the inventors discovered that by using two types of organic solvents, one non-polar and one polar, in two stages, it is possible to extract useful components such as TAGs and carotenoids separately from useful components such as omega-3 fatty acids and oxylipins. Based on this finding, they have completed the present invention.

[0007] That is, the present invention provides the following [1] to [7]: [1] A method for extracting useful components from algae, comprising the following steps (1a) and (2a), or steps (2b) and (1b): (1a) extracting a first useful component from dried algae using a non-polar solvent, (2a) extracting a second useful component from the residue after extraction in step (1a) using a solvent containing a polar solvent, (2b) extracting the second useful component from dried algae using a solvent containing a polar solvent, and (1b) extracting the first useful component from the residue after extraction in step (2b) using a non-polar solvent.

[0008] [2] The method according to [1], comprising steps (1a) and (2a).

[0009] [3] The method according to [1] or [2], characterized in that the first useful component is triacylglycerol and / or carotenoid, and the second useful component is omega-3 fatty acid and / or oxylipin.

[0010] [4] The method according to [1] or [2], wherein the nonpolar solvent is hexane, and the solvent containing a polar solvent is ethanol or a mixed solvent of ethanol and hexane.

[0011] [5] The method according to [1] or [2], characterized in that the algae contain triacylglycerol and contain omega-3 fatty acids and oxylipins in their membrane lipids.

[0012] [6] The method according to [1] or [2], characterized in that the algae belong to the genus Nannochloropsis, Microchloropsis, Monodus, Fistulifera, or Phaeodactylum.

[0013] [7] The method according to [1] or [2], characterized in that the first useful component is triacylglycerol and / or carotenoid, the second useful component is ω3 fatty acid and / or oxylipin, the non-polar solvent is hexane, the solvent containing a polar solvent is ethanol or a mixed solvent of hexane and ethanol, and the algae is algae belonging to the genus Nannochloropsis.

[0014] This specification includes part or all of the contents as disclosed in the specification and / or drawings of Japanese Patent Application No. 2024-011901, which is a priority document of the present application.

[0015] The present invention provides a novel method for extracting useful components from algae, which enables the separate extraction of useful components such as TAG and carotenoids from those such as ω-3 fatty acids and oxylipins.

[0016] FIG. 1 is a diagram showing an outline of the two-stage extraction method. FIG. 2 is a diagram showing the appearance of the extract under each condition. FIG. 3 is a diagram showing the composition of fatty acids contained in each extract. FIG. 4 is a diagram showing the TAG contained in each extract. The white spots indicated by the arrows are TAG. FIG. 5 is a diagram showing the composition of fatty acids contained in each TAG. FIG. 6 is a diagram showing the components contained in dried algal cells. FIG. 7 is a diagram showing the pigments contained in each extract. FIG. 8 is a diagram showing the amount of oxylipins contained in the extraction solvent in the second stage. FIG. 9 is a diagram showing an outline of the procedure in Example 2. FIG. 10 is a diagram showing the appearance of the extract under each condition. FIG. 11 is a diagram showing the TAG contained in each extract. The white spots indicated by the arrows are TAG.

[0017] The present invention will be described in detail below. The method for extracting useful components from algae of the present invention is characterized by comprising (1) a step of extracting a first useful component using a nonpolar solvent and (2) a step of extracting a second useful component using a solvent containing a polar solvent. The order of steps (1) and (2) is not particularly limited, and step (1) or step (2) may be performed first. That is, the method of the present invention may comprise the following steps (1a) and (2a), or may comprise steps (2b) and (1b).

[0018] In step (1a), a first useful component is extracted from the dried algae using a non-polar solvent.

[0019] Examples of non-polar solvents include hexane, cyclohexane, and dichloromethane. Among these, hexane is preferred. The use of hexane allows for almost complete recovery of TAG and β-carotene contained in algae (FIGS. 4 and 7), enabling efficient extraction of these components. Note that "hexane" in the present invention may be composed solely of normal hexane, or may be a solvent containing normal hexane as the main component with small amounts of other substances (e.g., methylcyclopentane, methylpentane, etc.).

[0020] The first useful component is not particularly limited as long as it can be extracted with a non-polar solvent, but TAG and carotenoids are preferred. Only one of these components may be extracted, or both may be extracted. Examples of carotenoids include β-carotene, α-carotene, γ-carotene, ε-carotene, lutein, zeaxanthin, fucoxanthin, diatoxanthin, and diadinoxanthin.

[0021] The algae used are not particularly limited as long as useful components can be extracted from them, but algae belonging to the genus Nannochloropsis, Microchloropsis, Monodus, Fistulifera, or Phaeodactylum are preferred, and algae belonging to the genus Nannochloropsis are particularly preferred. Examples of algae belonging to the genus Nannochloropsis include Nannochloropsis oceanica, Nannochloropsis oculata, 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 solaris, Fistulifera pelliculosa, and Fistulifera saprophila. Examples of algae belonging to the genus Phaeodactylum include Phaeodactylum tricornutum. The algae used preferably contain triacylglycerol and contain ω-3 fatty acids and oxylipins in their membrane lipids.Examples of such algae include algae belonging to the genus Monodus (e.g., Monodus subterraneus), algae belonging to the genus Fistulifera (e.g., Fistulifera solaris, Fistulifera pelliculosa, Fistulifera saprophila), algae belonging to the genus Microchloropsis (e.g., Microchloropsis gaditana, Microchloropsis salina), and algae belonging to the genus Nannochloropsis (e.g., Nannochloropsis oceanica, Nannochloropsis oculata, Nannochloropsis granulata, Nannochloropsis australis, Nannochloropsis limnetica, Nannochloropsis maritima).

[0022] When using algae belonging to the genus Nannochloropsis as the algae, those cultured in a normal medium may be used, but those cultured in a phosphorus-deficient medium or a nitrogen-deficient medium and allowed to accumulate TAG may also be used. Accumulation of TAG in a phosphorus-deficient medium can be carried out, for example, as described in the Examples below or in WO 2015 / 137449. Accumulation of TAG in a nitrogen-deficient medium can be carried out, for example, as described in WO 2015 / 137449.

[0023] Algae can be dried using conventional methods. Extraction of useful components from dried algae can be performed, for example, by a method comprising the steps of: 1) adding a solvent to the dried algae; 2) allowing a certain period of time to elapse; and 3) recovering the supernatant. The amount of solvent added is not particularly limited; however, 3 to 100 mL of solvent is preferably added per 100 mg of dried algae, and 15 to 45 mL is more preferred. The time allowed to elapse after solvent addition is not particularly limited; however, 0.3 to 24 hours is preferred, and 0.5 to 18 hours is more preferred. Supernatant recovery can be performed using conventional methods, such as centrifugation. Steps 1) to 3) above are typically repeated two or more times, and the resulting supernatants are combined to form an extract of useful components. The number of repetitions is sufficient; however, 3 to 7 times is preferred, and 4 to 6 times is more preferred. Since the extract contains components other than the useful components, the useful components may be isolated using chromatography or other methods.

[0024] In step (2a), a second useful component is extracted from the residue after extraction in step (1a) using a solvent containing a polar solvent.

[0025] The polar solvent-containing solvent may be any solvent containing a polar solvent, and may be a solvent consisting solely of a polar solvent or a mixed solvent of a polar solvent and a nonpolar solvent. Examples of polar solvents include ethanol, 1-butanol, 2-butanol, 1-propanol, 2-propanol, and acetone. Examples of nonpolar solvents to be mixed with a polar solvent include hexane, cyclohexane, and dichloromethane. Examples of mixed solvents of a polar solvent and a nonpolar solvent include a mixed solvent of ethanol and hexane, a mixed solvent of 1-butanol and hexane, a mixed solvent of 2-butanol and hexane, a mixed solvent of 1-propanol and hexane, a mixed solvent of 2-propanol and hexane, and a mixed solvent of acetone and hexane. The mixing ratio of the polar solvent to the nonpolar solvent is not particularly limited, but a mass ratio of polar solvent / nonpolar solvent of 30 / 70 to 70 / 30 is preferred, and a mass ratio of 40 / 60 to 60 / 40 is more preferred. Suitable solvents including polar solvents include ethanol, a mixed solvent of ethanol and hexane, a mixed solvent of 1-butanol and hexane, a mixed solvent of 2-butanol and hexane, a mixed solvent of 1-propanol and hexane, a mixed solvent of 2-propanol and hexane, and a mixed solvent of acetone and hexane.

[0026] The second useful component is not particularly limited as long as it can be extracted with a solvent containing a polar solvent, but ω3 fatty acids and oxylipins are preferred. Either one of these components may be extracted, or both may be extracted. ω3 fatty acids include EPA and DHA, and oxylipins include 15-hydroxyeicosapentaenoic acid, 15-hydroxyeicosatetraenoic acid, 13-hydroxy-9,11-octadecadienoic acid, 13-hydroxy-9,11,15-octadecatrienoic acid, 13-hydroxy-6,9,11-octadecatrienoic acid, 15-hydroxy-8,11,13-eicosatrienoic acid, 8-hydroxyeicosapentaenoic acid, 8-hydroxyeicosatetraenoic acid, and 18-hydroxyeicosapentaenoic acid.

[0027] Prior to the extraction in step (2a), it is desirable to remove any remaining nonpolar solvent from the residue after extraction in step (1a). Extraction of useful components from the residue after extraction in step (1a) can be carried out, for example, by a method comprising the steps of: 1) adding a solvent to the residue; 2) allowing a certain period of time to elapse; and 3) recovering the supernatant. The amount of solvent added is not particularly limited, but it is preferable to add 15 to 100 mL of solvent per 100 mg of residue, and more preferably 30 to 60 mL of solvent. The elapsed time after solvent addition is not particularly limited, but it is preferably 0.3 to 24 hours, and more preferably 0.5 to 18 hours. The supernatant can be recovered by conventional methods, such as centrifugation. Steps 1) to 3) above are typically repeated two or more times, and the combined supernatants are used as the extract of useful components. The number of repetitions is sufficient, but is preferably 2 to 6 times, and more preferably 3 to 4 times. Since the extract contains components other than the useful components, only the useful components may be separated by chromatography or the like.

[0028] In step (2b), a second useful component is extracted from the dried algae using a solvent containing a polar solvent. The polar solvent may be the same as the solvent used in step (2a), and the algae may be the same as the algae used in step (1a). The second useful component may be the same as the component used in step (2a).

[0029] In step (1b), a non-polar solvent is used to extract a first useful component from the residue after extraction in step (2b). The non-polar solvent may be the same as the solvent in step (1a). The first useful component may be the same as the component in step (1a).

[0030] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0031] [Example 1] Experimental Materials Nannochloropsis NIES-2145 (hereinafter referred to as "N. 2145") was used. This algae strain is available from the National Institute for Environmental Studies (http: / / www.nies.go.jp / ).

[0032] Experimental Procedure 1. Culture Conditions: For the cultivation of N. 2145, F2N medium was used as the standard liquid culture medium. 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 were dissolved in 100 mL of ion-exchanged water and stored at 4°C as f / 2 metal. 121.14 g of tris(hydroxymethyl)aminomethane was dissolved in 900 mL of ion-exchanged water, adjusted to pH 7.6 with HCl, and then adjusted to 1 L as 1 M Tris-HCl (pH 7.6) and stored at 4°C. NaNO3 7.5 mg, NH4Cl 26.745 mg, NaH2PO4・2H2O 3 mg, Vitamin B 12 0.25 μg of ATP, 0.25 μg of biotin, 50 μg of thiamine HCl, 0.5 mL of f / 2 metal, and 1 mL of 1M Tris-HCl (pH 7.6) were dissolved in 98.5 mL of artificial seawater, sterilized by filter, and used as F2N medium. Daigo Artificial Seawater SP (FUJIFILM) was used as the artificial seawater. This was used as a phosphorus-deficient medium by removing NaH2PO4 from F2N medium. Each liquid medium was used, and the concentration was 20-30 μmol photons / m 2 / sec, 23℃, 120 min -1 The cells were cultured in a rotating manner.

[0033] 2. Lipid Extraction: Cells in Standard Medium: After culturing for 20 days in standard medium, 850 mL of culture medium was centrifuged at 3500 x g for 5 minutes to precipitate the cultured cells. To remove sea salt, the cells were suspended in 50 mL of ion-exchanged water, and then centrifuged again at 3500 x g for 5 minutes to precipitate. The precipitate was suspended in 7 mL of ion-exchanged water, flattened, and dried at 60°C for 22 hours to yield 340 mg of dried algae. Three 65 mg samples of dried algae were prepared, and experiments were performed under the following conditions 1 to 3.

[0034] Condition 1 served as a control, and extraction was performed using the conventional chloroform-methanol mixture. 1.5 mL of chloroform and 3 mL of methanol were added to 65 mg of dried algae and left at room temperature for 1 hour, with suspension every 10 minutes. The mixture was centrifuged at 1000 × g for 5 minutes in a swing-type rotor, and 4.4 mL of the supernatant was collected as the first extraction. Another 1.5 mL of chloroform and 3 mL of methanol were added, and the mixture was left at room temperature for 30 minutes, with suspension every 10 minutes. The mixture was centrifuged at 1000 × g for 5 minutes in a swing-type rotor, and 5 mL of the supernatant was collected as the second extraction. Another 1.5 mL of chloroform and 3 mL of methanol were added, and the mixture was left at room temperature for 30 minutes, with suspension every 10 minutes. The mixture was centrifuged at 1000 × g for 5 minutes in a swing-type rotor, and 4.5 mL of the supernatant was collected as the third extraction. Another 3 mL of chloroform and 1.5 mL of methanol were added, and the mixture was left at room temperature for 30 minutes, with suspension every 10 minutes. The mixture was centrifuged at 1000 × g for 5 minutes in a swing-type rotor, and 4.5 mL of the supernatant was collected as the fourth extract. Finally, 3 mL of chloroform and 1.5 mL of methanol were added, and the mixture was left at room temperature for 30 minutes, suspending every 10 minutes. The mixture was centrifuged at 1000 × g for 5 minutes in a swing-type rotor, and 4.5 mL of the supernatant was collected as the fifth extract. The first to fifth extracts were combined, dried, and dissolved in chloroform:methanol (2:1) to a concentration of 25 mg / mL. The mixture was then stored at -20°C.

[0035] In condition 2, the first extraction was performed with hexane. After the precipitate was dried, the solvent was removed and the second extraction was performed with ethanol. 4.5 mL of hexane was added to 65 mg of dried algae and the mixture was left at room temperature for 1 hour, with suspension every 10 minutes. The mixture was centrifuged at 1000 × g for 5 minutes in a swing-type rotor. 4.5 mL of the supernatant was collected as the first extraction solution. 4.5 mL of hexane was added again and the mixture was left at room temperature for 30 minutes, with suspension every 10 minutes. The mixture was centrifuged at 1000 × g for 5 minutes in a swing-type rotor. 4.5 mL of the supernatant was collected as the second extraction solution. 4.5 mL of hexane was added again and the mixture was left at room temperature for 30 minutes, with suspension every 10 minutes. The mixture was centrifuged at 1000 × g for 5 minutes in a swing-type rotor. 4.5 mL of the supernatant was collected as the third extraction solution. 4.5 mL of hexane was added again and the mixture was left at room temperature for 30 minutes, with suspension every 10 minutes. The mixture was centrifuged at 1000 × g for 5 minutes in a swing-cage rotor, and 4.5 mL of the supernatant was collected as the fourth extraction. Finally, 4.5 mL of hexane was added, and the mixture was left at room temperature for 30 minutes, with suspension every 10 minutes. The mixture was centrifuged at 1000 × g for 5 minutes in a swing-cage rotor, and 4.5 mL of the supernatant was collected as the fifth extraction. The first to fifth extractions 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 the remaining hexane from the precipitate by drying, the second extraction was performed using ethanol. 9 mL of ethanol was added to the precipitate, and the mixture was left at room temperature for 18 hours. The mixture was centrifuged at 1000 × g for 5 minutes in a swing-cage rotor, and 9 mL of the supernatant was collected as the first extraction. Another 9 mL of ethanol was added, and the mixture was left at room temperature for 1 hour, with suspension every 10 minutes. The mixture was centrifuged at 1000 x g for 5 minutes in a swing-cage rotor, and 9 mL of the supernatant was collected as the second extraction solution. An additional 9 mL of ethanol was added, and the mixture was left at room temperature for 1 hour, with suspension every 10 minutes. The mixture was centrifuged at 1000 x g for 5 minutes in a swing-cage rotor, and 9 mL of the supernatant was collected as the third extraction solution. Finally, 9 mL of ethanol was added, and the mixture was left at room temperature for 1 hour, with suspension every 10 minutes. The mixture was centrifuged at 1000 x g for 5 minutes in a swing-cage rotor, and 9 mL of the supernatant was collected as the fourth extraction solution.The first to fourth extracts were combined, dried, dissolved in ethanol to a concentration of 25 mg / mL, and stored at -20°C as the second-stage extract.

[0036] In condition 3, the first step was extraction with hexane, as in condition 2. After the precipitate solvent was removed by drying, the second step was extraction with a hexane-ethanol mixture. The procedure was the same as condition 2, except that 6 mL of hexane and 3 mL of ethanol were used instead of 9 mL of ethanol.

[0037] Cells in phosphorus-deficient medium: 3 L of culture medium cultured for 7 days in phosphorus-deficient medium was centrifuged at 3500 x g for 5 minutes to precipitate the cultured cells. To remove sea salt, the cells were suspended in 50 mL of ion-exchanged water, and then centrifuged again at 3500 x g for 5 minutes to precipitate. The precipitate was suspended in 7 mL of ion-exchanged water, flattened, 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 performed under the following conditions 1 to 3.

[0038] Condition 1 served as a control, and extraction was performed using the conventional chloroform-methanol mixture. 3 mL of chloroform and 6 mL of methanol were added to 58 mg of dried algae, and the mixture was left at room temperature for 1 hour, with suspension every 10 minutes. The mixture was centrifuged at 1000 × g for 5 minutes in a swing-type rotor, and 9 mL of the supernatant was collected as the first extraction solution. 3 mL of chloroform and 6 mL of methanol were added, and the mixture was left at room temperature for 1 hour, with suspension every 10 minutes. The mixture was centrifuged at 1000 × g for 5 minutes in a swing-type rotor, and 9 mL of the supernatant was collected as the second extraction solution. 3 mL of chloroform and 6 mL of methanol were added, and the mixture was left at room temperature for 17 hours. The mixture was centrifuged at 1000 × g for 5 minutes in a swing-type rotor, and 9 mL of the supernatant was collected as the third extraction solution. 6 mL of chloroform and 3 mL of methanol were added, and the mixture was left at room temperature for 1 hour, with suspension every 10 minutes. The mixture was centrifuged at 1000 × g for 5 minutes in a swing-cage rotor, and 9 mL of the supernatant was collected as the fourth extraction solution. 6 mL of chloroform and 3 mL of methanol were then added, and the mixture was left at room temperature for 1 hour, with suspension every 10 minutes. The mixture was centrifuged at 1000 × g for 5 minutes in a swing-cage rotor, and 9 mL of the supernatant was collected as the fifth extraction solution. 6 mL of chloroform and 3 mL of methanol were then added, and the mixture was left at room temperature for 1 hour, with suspension every 10 minutes. The mixture was centrifuged at 1000 × g for 5 minutes in a swing-cage rotor, and 9 mL of the supernatant was collected as the sixth extraction solution. Finally, 6 mL of chloroform and 3 mL of methanol were added, and the mixture was left at room temperature for 1 hour, with suspension every 10 minutes. The mixture was centrifuged at 1000 × g for 5 minutes in a swing-cage rotor, and 9 mL of the supernatant was collected as the seventh extraction solution. To shorten the drying time, the mixture was separated into two layers. The lower chloroform layer was collected as the lipid extract. To separate the lipids, 5.4 mL of 1% (W / V) KCl and 3 mL of chloroform were added to the first to third extracts. 5.4 mL of 1% (W / V) KCl and 3 mL of methanol were added to the fourth to seventh extracts. After suspending, the mixture was centrifuged at 1000 × g for 5 minutes in a swing-out rotor, and the lower chloroform layer was collected as the lipid extract.The lipid extracts from the first to seventh times were combined, dried, dissolved in chloroform:methanol = 2:1 to a concentration of 25 mg / mL, and then stored at -20°C.

[0039] In condition 2, the first extraction was performed with hexane. After the precipitate was dried, the solvent was removed and the second extraction was performed with ethanol. 9 mL of hexane was added to 58 mg of dried algae and the mixture was left at room temperature for 1 hour, with suspension every 10 minutes. The mixture was centrifuged at 1000 × g for 5 minutes in a swing-cage rotor. 9 mL of the supernatant was collected as the first extraction solution. 9 mL of hexane was added again and the mixture was left at room temperature for 1 hour, with suspension every 10 minutes. The mixture was centrifuged at 1000 × g for 5 minutes in a swing-cage rotor. 9 mL of the supernatant was collected as the second extraction solution. 9 mL of hexane was added again and the mixture was left at room temperature for 1 hour, with suspension every 10 minutes. The mixture was centrifuged at 1000 × g for 5 minutes in a swing-cage rotor. 9 mL of the supernatant was collected as the third extraction solution. 9 mL of hexane was added again and the mixture was left at room temperature for 1 hour, with suspension every 10 minutes. The mixture was centrifuged at 1000 × g for 5 minutes in a swing-cage rotor. 9 mL of the supernatant was collected as the fourth extraction solution. An additional 9 mL of hexane was added, and the mixture was left at room temperature for 1 hour, with suspension every 10 minutes. The mixture was centrifuged at 1000 × g for 5 minutes in a swing-cage rotor, and 9 mL of the supernatant was collected as the fifth extraction. Another 9 mL of hexane was added, and the mixture was left at room temperature for 1 hour, with suspension every 10 minutes. The mixture was centrifuged at 1000 × g for 5 minutes in a swing-cage rotor, and 9 mL of the supernatant was collected as the sixth extraction. Finally, 9 mL of hexane was added, and the mixture was left at room temperature for 1 hour, with suspension every 10 minutes. The mixture was centrifuged at 1000 × g for 5 minutes in a swing-cage rotor, and 9 mL of the supernatant was collected as the seventh extraction. The first through seventh extractions were combined, dried, dissolved in hexane to a concentration of 25 mg / mL, and stored at -20°C as the first-step extract. The remaining hexane in the precipitate was removed by drying, and then the second extraction was performed with ethanol. 9 mL of ethanol was added to the precipitate, and the mixture was left at room temperature for 17 hours. The mixture was centrifuged at 1000 x g for 5 minutes in a swing-type rotor, and 9 mL of the supernatant was collected as the first extraction solution. An additional 9 mL of ethanol was added, and the mixture was left at room temperature for 1 hour, with suspension every 10 minutes. The mixture was centrifuged at 1000 x g for 5 minutes in a swing-type rotor, and 9 mL of the supernatant was collected as the second extraction solution. An additional 9 mL of ethanol was added, and the mixture was left at room temperature for 1 hour, with suspension every 10 minutes.The mixture was centrifuged at 1000 × g for 5 minutes in a swing rotor, and 9 mL of the supernatant was collected as the third extract. Finally, 9 mL of ethanol was added, and the mixture was left at room temperature for 1 hour, suspending every 10 minutes. The mixture was centrifuged at 1000 × g for 5 minutes in a swing rotor, and 9 mL of the supernatant was collected as the fourth extract. The first to fourth extracts were combined, dried, dissolved in ethanol to a concentration of 25 mg / mL, and stored at -20°C as the second extract.

[0040] In condition 3, the first step was extraction with hexane, as in condition 2. After the precipitate solvent was removed by drying, the second step was extraction with a hexane-ethanol mixture. The procedure was the same as condition 2, except that 6 mL of hexane and 3 mL of ethanol were used instead of 9 mL of ethanol.

[0041] 3. Lipid Analysis 5 μl of lipid extract (equivalent to 0.125 mg) was added to 50 μl of 1 mM heneicosanoic acid and 500 μl of 1.5 M hydrochloric acid / methanol, suspended, and then allowed to stand at 85°C for 1 hour to methylate the fatty acids. 500 μl of hexane was added, suspended, and centrifuged at 1000 × g for 5 minutes in a swing-out rotor. The upper layer of hexane containing the methyl-esterified fatty acids was collected. 500 μl of hexane was added to the lower layer, suspended, and centrifuged at 1000 × g for 5 minutes in a swing-out rotor. The upper layer was collected. 500 μl of hexane was added to the lower layer, suspended, and centrifuged at 1000 × g for 5 minutes in a swing-out rotor. The upper layer was collected. The collected methyl-esterified fatty acids were dried, dissolved in 100 μl of hexane, and used as a gas chromatography sample. Triacylglycerols (TAGs) were isolated using thin-layer silica plates (TLC) and analyzed by gas chromatography. 5 μl (equivalent to 0.125 mg) of lipid extract was spotted onto a TLC plate and developed for 45 minutes with a mixture of 160 mL of hexane, 40 mL of diethyl ether, and 4 mL of acetic acid. 0.1 mg of triolein was spotted as a control for TAG mobility. TAGs were confirmed under UV irradiation using 0.001% primulin. The silica containing TAGs was scraped off, and the plate was suspended in 50 μl of 1 mM heneicosanoic acid and 500 μl of 1.5 M hydrochloric acid / methanol. The plate was then incubated at 85°C for 1 hour to methylate the fatty acids. 500 μl of hexane was added, suspended, and centrifuged at 1000 × g for 5 minutes in a swing-wheel rotor. The upper layer of hexane containing the methyl-esterified fatty acids was collected. 500 μL of hexane was added to the lower layer, and the suspension was centrifuged at 1000 × g for 5 minutes in a swing rotor. The upper layer was collected. 500 μL of hexane was added to the lower layer, and the suspension was centrifuged at 1000 × g for 5 minutes in a swing rotor. The upper layer was collected. The collected methyl-esterified fatty acids were dried and then dissolved in 100 μL of hexane to prepare a gas chromatography sample.Gas chromatography was performed using a Shimadzu GC-2030 equipped with an HR-SS-10 (inner diameter 0.25 mm, length 25 m) (SHINWA CHEMICAL INDUSTRIES, LTD.).

[0042] 4. Pigment Analysis: TLC analysis was performed to compare the types of pigments contained in the extracts. 10 μl (equivalent to 0.25 mg) of lipid extract was spotted onto a TLC plate and developed for 40 minutes in a mixture of 70 mL of petroleum ether and 30 mL of acetone. Pigments extracted from spinach with chloroform and methanol were used as a control.

[0043] 5. Oxylipin Analysis LC-MS was used to examine the oxylipins contained in the lipid extracts. The lipid extracts obtained from cells cultured in normal medium using ethanol in the second step of Condition 2 and the lipid extracts obtained from a hexane-ethanol mixture in the second step of Condition 3 were diluted 1000-fold, and the free 15-HEPE and 15-HETE contents in the solutions were measured using a Shimadzu LC-MS8050. A Phenomenex Kinetex C8 column (inner diameter 2.1 mm, length 150 mm, particle size 2.6 μm) was used.

[0044] Experimental Results 1. Lipid Extraction To compare the conventional extraction using a chloroform-methanol mixture with two-stage extraction, lipid extraction was performed under conditions 1 to 3 (Figure 1). Under condition 1, extraction was performed using the conventionally used chloroform-methanol mixture as a control. Under condition 2, extraction was performed with hexane in the first stage, and after the precipitation solvent was removed by drying, extraction was performed in the second stage with ethanol. Under condition 3, extraction was performed with hexane in the first stage, and after the precipitation solvent was removed by drying, extraction was performed in the second stage with a hexane-ethanol mixture. Two types of cells were used for lipid extraction: cells cultured in normal medium and cells cultured in phosphorus-deficient medium, which is more likely to accumulate TAG.

[0045] After 20 days of cultivation in standard medium, 340 mg of dried algae was obtained from 850 mL of culture medium. Three 65 mg samples of dried algae were prepared and tested under conditions 1 through 3. Under condition 1, a deep green extract was obtained in the first extraction, which gradually became lighter in the second and third extractions, with the fourth and fifth extractions being almost completely colorless (Figure 2, upper left). Under conditions 2 and 3, the first hexane extraction yielded a deep green extract in the first extraction, which did not gradually become lighter in the second through fifth extractions (Figure 2, center left). When the first hexane extraction was followed by a second extraction with a hexane-ethanol mixture, the color almost disappeared by the second extraction, but when extraction with ethanol was performed, the extract remained green even in the fourth extraction (Figure 2, lower left). The weight of each lipid recovered indicated that the two-step extraction yielded lipids comparable to those recovered by the previous chloroform-methanol extraction (Table 1).

[0046] After 7 days of cultivation in phosphorus-deficient medium, 302 mg of dried algae were obtained from 3 L of culture medium. Three 58 mg samples of dried algae were prepared and used for experiments under conditions 1 to 3. Because it was expected that TAG accumulation would increase in phosphorus-deficient medium, the amount of organic solvent used for extraction and the number of extractions were increased compared to normal medium. Under condition 1, a deep green extract was obtained in the first extraction, which gradually became lighter with the second and third extractions, with the extract remaining uncolored from the fourth extraction onward (Figure 2, upper right). Under conditions 2 and 3, the first hexane extraction yielded a deep green extract in the first extract and the third extraction (17 hours later), but the fourth extract did not gradually become lighter (Figure 2, middle right). When the first hexane extraction was followed by a second extraction with a hexane-ethanol mixture, the color almost disappeared by the second extraction, but when extracted with ethanol, the extract remained faintly green even after the fourth extraction (Figure 2, lower right). The weight of each recovered lipid showed that even in phosphorus-deficient cultured cells, where TAG accumulation increases, two-step extraction could recover lipids to the same extent as chloroform / methanol extraction (Table 1). It was also found that the lipid weight per dry algal mass was higher than in normal cultured cells. Furthermore, it was found that the amount of lipid recovered in the first step was higher than in normal cultured cells.

[0047] 2. Lipid Analysis Gas chromatography was performed to analyze the fatty acids contained in lipids. Figure 3 shows that in normal culture, the proportion of palmitic acid (C16:0) was lower in the second-stage ethanol extraction or hexane / ethanol extraction compared to the chloroform / methanol extraction. Compared to the ethanol extraction, the hexane / ethanol extraction had a lower proportion of C16:0 and a higher proportion of eicosapentaenoic acid (C20:5, EPA). In phosphorus-deficient culture, the proportion of EPA was lower in the first-stage hexane extraction compared to the chloroform / methanol extraction, while the second-stage ethanol extraction or hexane / ethanol extraction had a higher proportion of EPA and lower proportions of C16:0 and palmitoleic acid (C16:1). It is known that the proportion of arachidonic acid (C20:4) and EPA in TAGs is lower in Nannochloropsis than in membrane lipids (Frontiers in Microbiology (2015) https: / / doi.org / 10.3389 / fmicb.2015.00912, Plant Physiol. 171,(2016),2469-2482). The lower proportion of EPA in the first stage was thought to be due to the high content of TAGs in the hexane extract. Therefore, TLC and gas chromatography were performed to analyze the TAGs contained in each extract. The results in Figure 4 indicate that the entire amount of TAG was recovered in the first hexane extraction in both normal and phosphorus-deficient cultures, while the second extract contained no TAG. Furthermore, the amount of TAG per lipid was higher in the hexane extract than in the chloroform-methanol extract. The fatty acid components of each TAG are shown in Figure 5. Due to the low amount of TAG in the chloroform-methanol extract from normal culture, EPA was not detected. Therefore, the proportion of C16:0 was higher than previously reported, but in the hexane extraction, EPA was 6%, which is the same as the fatty acid components contained in TAG after conventional cultivation. In phosphorus-starved cultivation, the proportion of EPA was particularly low at 1%.The results of the lipid analysis above revealed that the first-stage hexane extraction can extract TAGs at the same level or higher than chloroform / methanol extraction, that the second-stage extract does not contain TAGs, and that hexane extraction followed by ethanol extraction or hexane / ethanol extraction can extract lipids at the same level as chloroform / methanol extraction (Figure 6).

[0048] 3. Pigment Analysis Each extract was spotted on a TLC plate and developed using petroleum ether and acetone. Results indicated that β-carotene was present only in the hexane extract from the first stage of normal culture, whereas it was absent in the ethanol or hexane-ethanol extracts from the second stage (Figure 7, left). Four novel carotenoid-like spots (indicated by arrows with circled numbers 1, 2, 4, and 5) with mobilities not seen in spinach were also observed. Although β-carotene was present in the hexane extract from phosphorus-deficient culture, it was present in lower amounts than in normal culture (Figure 7, right). A new carotenoid-like spot (indicated by arrow with circled number 3) was also observed, which was not observed in normal culture. These results demonstrate that the two-stage extraction method using nonpolar and polar solvents of the present invention can recover all β-carotene in the first stage extract, separately recover novel carotenoids with different properties, and control the accumulation of carotenoids during culture.

[0049] 4. Oxylipin Analysis LC-MS was used to investigate which extracts contain oxylipins, which are metabolites of EPA, when using a two-step extraction method. When hexane extraction is followed by ethanol extraction or hexane / ethanol extraction, it was found that the second-step extract contained large amounts of free hydroxyeicosapentaenoic acid (15-HEPE) and hydroxyeicosatetraenoic acid (15-HETE), which have an affinity for polar solvents (Figure 8). It was found that ethanol extraction yielded more oxylipins than hexane / ethanol extraction.

[0050] 5. Summary The above results demonstrate that the present invention, which involves two-stage extraction using a nonpolar and polar solvent after drying the algae, can fully recover TAG and β-carotene using the nonpolar solvent in the first stage. There are two options for the second stage extraction solvent: using a polar solvent allows for efficient recovery of free oxylipins, while using a mixed solvent of a nonpolar and polar solvent reduces the recovery rate of free oxylipins but increases the recovery rate of EPA contained in membrane lipids. Thus, the present invention is an effective method for separating and recovering useful components from algae according to the purpose.

[0051] [Example 2] Experimental Materials The same experimental materials as those used in Example 1 were used.

[0052] Experimental Procedure: Lipid Extraction. Condition 4 was performed using cells cultured in standard medium, with the extraction steps reversed from Condition 2. Specifically, in Condition 4, the first extraction was performed with ethanol, followed by drying to remove the precipitated solvent. The second extraction was performed with hexane. 4.5 mL of ethanol was added to 65 mg of dried algae and the mixture was left at room temperature for 1 hour, with suspension every 10 minutes. The mixture was centrifuged at 1000 × g for 5 minutes in a swing-out rotor. 4.5 mL of the supernatant was collected as the first extraction. Another 4.5 mL of ethanol was added and the mixture was left at room temperature for 30 minutes, with suspension every 10 minutes. The mixture was centrifuged at 1000 × g for 5 minutes in a swing-out rotor. 4.5 mL of the supernatant was collected as the second extraction. Another 4.5 mL of ethanol was added and the mixture was left at room temperature for 30 minutes, with suspension every 10 minutes. The mixture was centrifuged at 1000 × g for 5 minutes in a swing-out rotor. 4.5 mL of the supernatant was collected as the third extraction. An additional 4.5 mL of ethanol was added, and the mixture was left at room temperature for 30 minutes, with suspension every 10 minutes. The mixture was centrifuged at 1000 × g for 5 minutes in a swing-cage rotor, and 4.5 mL of the supernatant was collected as the fourth extraction. Finally, 4.5 mL of ethanol was added, and the mixture was left at room temperature for 30 minutes, with suspension every 10 minutes. The mixture was centrifuged at 1000 × g for 5 minutes in a swing-cage rotor, and 4.5 mL of the supernatant was collected as the fifth extraction. The first to fifth extractions 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 the remaining ethanol from the precipitate by drying, a second extraction was performed using hexane. 9 mL of hexane was added to the precipitate, and the mixture was left at room temperature for 18 hours. The mixture was centrifuged at 1000 × g for 5 minutes in a swing-cage rotor, and 9 mL of the supernatant was collected as the first extraction. Another 9 mL of hexane was added, and the mixture was left at room temperature for 1 hour, with suspension every 10 minutes. The mixture was centrifuged at 1000 x g for 5 minutes in a swing-type rotor, and 9 mL of the supernatant was collected as the second extraction solution. An additional 9 mL of hexane was added, and the mixture was left at room temperature for 1 hour, with suspension every 10 minutes. The mixture was centrifuged at 1000 x g for 5 minutes in a swing-type rotor, and 9 mL of the supernatant was collected as the third extraction solution. Finally, 9 mL of hexane was added, and the mixture was left at room temperature for 1 hour, with suspension every 10 minutes.The mixture was centrifuged at 1000 × g for 5 minutes in a swing rotor, and 9 mL of the supernatant was collected as the fourth extract. The first to fourth extracts were combined, dried, dissolved in hexane to a concentration of 25 mg / mL, and then stored at -20°C as the second-stage extract. For comparison with condition 4, the same procedures as in Example 1 were also carried out under conditions 1 and 2.

[0053] Lipid Analysis To examine the TAG contained in the extracted lipids, TLC development was carried out in the same manner as in Example 1.

[0054] Experimental Results: Lipid Extraction To investigate whether extraction was possible even if the order of the extraction steps in Condition 2 shown in Example 1 was reversed, lipid extraction was performed under Conditions 1, 2, and 4 (Figure 9). As in Example 1, under Condition 1, extraction was performed using a conventionally used chloroform-methanol mixture as a control. Under Condition 2, extraction was performed with hexane as the first step, the precipitate solvent was removed by drying, and then extraction was performed with ethanol as the second step. Under Condition 4, extraction was performed with ethanol as the first step, the precipitate solvent was removed by drying, and then extraction was performed with hexane as the second step. Cells cultured in normal medium were used for lipid extraction.

[0055] Under Condition 1, as in Example 1, a deep green extract was obtained in the first extraction, which gradually became lighter in the second and third extractions, and the extracts from the fourth and fifth extractions were almost completely colorless (Figure 10, top).

[0056] In the first hexane extraction under condition 2, a dark green extract was obtained in the first extraction, as in Example 1, and the extracts from the second to fourth extractions did not gradually become lighter (Figure 10, center right). When the first hexane extraction was followed by ethanol extraction in the second extraction, the extract remained green even in the fourth extraction, as in Example 1 (Figure 10, bottom right). In the first ethanol extraction under condition 4, a dark green extract was obtained in the first extraction, and the extracts from the second to fifth extractions did not gradually become lighter (Figure 10, center left). When the first ethanol extraction was followed by hexane extraction in the second extraction, a light green extract was obtained in the first extraction, and the color almost disappeared by the second extraction (Figure 10, bottom left). The hexane extract from the second extraction under condition 4 was lighter green than the hexane extract from the first extraction under condition 2. This suggests that the hexane extract from the second extraction under condition 4 contains fewer pigment components than the hexane extract from the first extraction under condition 2, and that TAG was recovered more efficiently. Furthermore, based on the weight of each lipid recovered, it was found that a certain amount of lipid could be recovered even if the order of the two-stage extraction was reversed (Table 2).

[0057] Lipid Analysis To analyze the TAG contained in each extract, TLC analysis was performed in the same manner as in Example 1. The results in Figure 11 show that under condition 2, the entire amount of TAG could be recovered in the first hexane extraction, as in Example 1, and that no TAG was contained in the second ethanol extract. Under condition 4, in which the order of the two extraction steps was reversed, some TAG was contained in the first ethanol extract, but most of the TAG could be recovered in the second hexane extract, and it was revealed that the amount of TAG contained per lipid was greater than that in conventional chloroform-methanol extraction.

[0058] The results of the lipid analysis above suggest that it is possible to reverse the order of the solvents used in the first and second steps. When the order is reversed, the second extraction results in less pigment contamination, and it has become clear that TAG can be recovered more efficiently than with conventional chloroform / methanol extraction.

[0059] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

[0060] The TAG extractable by the present invention can be used as fuel or food, and the ω-3 fatty acids, carotenoids, and oxylipins can be used as food or medicine. Therefore, the present invention can be used in industrial fields related to fuel, food, medicine, etc.

Claims

1. A method for extracting useful components from algae, comprising the following steps (1a) and (2a), or (2b) and (1b): (1a) extracting a first useful component from dried algae using a non-polar solvent and recovering the supernatant; (2a) extracting a second useful component from the residue after extraction in step (1a) using a solvent containing a polar solvent; (2b) extracting a second useful component from the dried algae using a solvent containing a polar solvent and recovering the supernatant; (1b) A step of extracting a first useful component from the residue after extraction in step (2b) using a non-polar solvent.

2. 2. The method according to claim 1, comprising steps (1a) and (2a).

3. 3. The method according to claim 1 or 2, wherein the first useful component is triacylglycerol and / or carotenoid, and the second useful component is omega-3 fatty acid and / or oxylipin.

4. 3. The method according to claim 1, wherein the non-polar solvent is hexane, and the solvent containing a polar solvent is ethanol or a mixed solvent of ethanol and hexane.

5. The method according to claim 1 or 2, characterized in that the algae contain triacylglycerol and contain omega-3 fatty acids and oxylipins in their membrane lipids.

6. 3. The method according to claim 1, wherein the algae are algae belonging to the genus Nannochloropsis, Microchloropsis, Monodus, Fistulifera, or Phaeodactylum.

7. 3. The method according to claim 1 or 2, wherein the first useful component is triacylglycerol and / or carotenoid, the second useful component is omega-3 fatty acid and / or oxylipin, the non-polar solvent is hexane, the solvent containing a polar solvent is ethanol or a mixed solvent of hexane and ethanol, and the algae is algae belonging to the genus Nannochloropsis.