Methods for Producing Oxylipin-Containing Compositions

Abstract

To provide means for improving the yield of oxylipin from oxylipin-containing material.SOLUTION: A gel of crushed oxylipin-containing material is extracted with a solvent.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing an oxylipin-containing composition. [Background technology]

[0002] Oxylipins are substances found in a wide variety of organisms, including microorganisms (e.g., algae), plants, and animals, and are known to have various physiological activities. Oxylipins are produced from fatty acids as raw materials. A known method for producing oxylipins is to extract crushed algae of the genus Nannochloropsis with water (Patent Document 1). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Publication No. 2021-136959 Summary of the Invention [Problem to be solved by the invention]

[0004] The present inventors first discovered that the yield of oxylipins decreased when crushed algae was coagulated by adding citric acid to separate the aqueous extract from crushed algae described in Patent Document 1. Specifically, the inventors found that the amount of 15-HEPE (a type of oxylipin) contained in the aqueous extract obtained by coagulating crushed algae was approximately 10% of the amount of 15-HEPE contained in the crushed algae before aqueous extraction. Therefore, the objective of the present invention was to provide a method for producing oxylipins without using a process for flocculating crushed algae. [Means for solving the problem]

[0005] As a result of extensive research into the above-mentioned problems, the present inventors have found that by gelling crushed algae and subjecting the gelled material to solvent extraction, it is possible to obtain an oxylipin-containing composition without causing the crushed algae to aggregate. The present invention is based on these findings.

[0006] That is, the present invention relates to the following [1] to

[19] . [1] A method for producing an oxylipin-containing composition, comprising: A manufacturing method comprising a step of extracting a gel of crushed oxylipin-containing material with a solvent. [2] The manufacturing method described in [1] above, wherein the gel is an alginate gel or an agarose gel. [3] The manufacturing method described in [1] above, wherein the gel is an alginate gel. [4] The method of producing according to [1] above, wherein the content of crushed oxylipin-containing material in the gel is 5 to 30% (w / v). [5] The manufacturing method described in [1] above, wherein the gel has a thread-like or spherical shape. [6] The manufacturing method described in [1] above, further comprising a step of preparing a gel of crushed oxylipin-containing material before the extraction step. [7] The manufacturing method described in [6] above, in which a gel is prepared by adding dropwise a gel stock solution containing crushed oxylipin-containing material and a gelling agent to an aqueous solution of a polyvalent metal ion compound. [8] The manufacturing method described in [7] above, wherein the gelling agent is sodium alginate. [9] The method according to [7] above, wherein the polyvalent metal ion compound is calcium chloride.

[10] The method according to [9] above, wherein the calcium chloride concentration in the aqueous calcium chloride solution is 2.5 to 20% (w / v).

[11] The manufacturing method described in [1] above, wherein the oxylipin-containing material is a plant (excluding those classified as algae).

[12] The manufacturing method described in [1] above, wherein the oxylipin-containing material is algae.

[13] The manufacturing method described in [1] above, wherein the oxylipin-containing material is algae of the genus Nannochloropsis.

[14] The production method according to [1] above, wherein the oxylipin is selected from the group consisting of 8-HEPE, 11-HEPE, 15-HEPE, 18-HEPE, 11-HETE, and 15-HETE.

[15] The solvent is selected from the group consisting of water, alcohol or an aqueous solution thereof, and hydrocarbon; and The method for production described in [1] above, wherein the oxylipin is selected from the group consisting of 8-HEPE, 15-HEPE, and 18-HEPE.

[16] The solvent is water, and The method for production described in [1] above, wherein the oxylipin is selected from the group consisting of 8-HEPE, 15-HEPE, and 18-HEPE.

[17] The solvent is water, and The method for production described in [1] above, wherein the oxylipin is 15-HEPE.

[18] The production method described in [1] above, wherein the extraction is carried out continuously.

[19] The production method described in [1] above, further comprising a step of subjecting the extract to column purification at pH 2.5 to 5.0. [Effects of the Invention]

[0007] As will be shown in the examples below, according to the present invention, an oxylipin-containing composition can be produced from crushed algae without agglomeration. Thus, the present invention provides a means for producing oxylipins that has advantages not available in the prior art. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 1 shows the results of Example 2 (effect of debris content in gel and gel shape on extraction). [Figure 2] FIG. 2 shows the results of Example 3 (study of the concentration of calcium chloride solution during gelation). DETAILED DESCRIPTION OF THE INVENTION

[0009] [Oxylipin] In the present invention, oxylipins refer to substances produced by living organisms using eicosapentaenoic acid (EPA) or arachidonic acid (AA) as raw materials. Oxylipins derived from eicosapentaenoic acid (EPA) include 8-hydroxyeicosapentaenoic acid (8-HEPE), 11-hydroxyeicosapentaenoic acid (11-HEPE), 15-hydroxyeicosapentaenoic acid (15-HEPE), 18-hydroxyeicosapentaenoic acid (18-HEPE), 17,18-dihydroxyeicosapentaenoic acid (resolvin E3), and prostaglandin G3 (PGG3). Examples of oxylipins derived from arachidonic acid (AA) include 11-hydroxyeicosatetraenoic acid (11-HETE), 15-hydroxyeicosatetraenoic acid (15-HETE), and prostaglandin G2 (PGG2). Among these, the present invention can be suitably applied to the production of 8-HEPE, 11-HEPE, 15-HEPE, 18-HEPE, 11-HETE, and 15-HETE. When the oxylipin-containing material contains multiple types of oxylipins, a composition containing the multiple types of oxylipins can be produced according to the present invention.

[0010] [Oxylipin-containing materials] In the present invention, any biologically derived material containing oxylipins can be used without particular limitation. Organisms that contain oxylipins include algae, microorganisms (excluding those classified as algae), plants (excluding those classified as algae), and animals. Examples of algae include algae of the genus Nannochloropsis, algae of the genus Chlorella, and algae of the genus Euglena. Examples of microorganisms (excluding those classified as algae) include Labyrinthula, Fungi, and yeasts. Examples of plants (excluding those classified as algae) include perilla, flax, and chia. Examples of animals include tuna, sardines, and krill. The oxylipin-containing material is preferably derived from algae or plants (excluding those classified as algae), more preferably from algae, and even more preferably from algae of the genus Nannochloropsis. Oxylipin-containing materials are available from microbial depositories, commercially available, or may be isolated from nature. The present invention can be practiced with one or more oxylipin-containing materials. The following is a detailed description of the algae of the genus Nannochloropsis.

[0011] [Nannochloropsis algae] Nannochloropsis is a unicellular alga belonging to the Heterokontophyta and Euthenophyceae classes. Nannochloropsis algae that can produce and accumulate oxylipins intracellularly can be used without particular limitation. Specific examples include Nannochloropsis oceanica, Nannochloropsis oculata, Nannochloropsis gaditana, Nannochloropsis salina, Nannochloropsis atomus, Nannochloropsis maculata, Nannochloropsis granulata, and Nannochloropsis sp. Among these, Nannochloropsis oceanica and Nannochloropsis gaditana are preferred, with Nannochloropsis oceanica being particularly preferred. Examples of Nannochloropsis oceanica include the NIES-2145 strain deposited at the National Institute for Environmental Studies' Microbial Culture Collection (NIES Collection), a national research and development agency, and the CCMP-1779 strain deposited at the National Center for Marine Algae and Microbiota (NCMA) (USA), with the NIES-2145 strain being preferred. Nannochloropsis algae are available from microorganism depositories and on the market, or may be isolated from nature.

[0012] Nannochloropsis algae can be cultured according to known methods. As for the culture medium, in addition to the ESM medium used in the examples described below, the NIES Collection website ([online], [searched February 21, 2023], Internet,<https: / / mcc.nies.go.jp / 02medium.html> ) "2. Media for marine and brackish water algae" can be used. The culture conditions can be set appropriately depending on the type of algae and the scale of the culture. Nannochloropsis oceanica is grown at a temperature of 20 to 25°C under white light or natural light (preferably with a photon density of 17 to 300 μmol / m 2 It is preferable to culture the cells under shaking or aeration at a temperature of 1000 K / s.

[0013] [Method for producing oxylipin-containing composition] In the present invention, an oxylipin-containing composition is produced by extracting a gel of crushed oxylipin-containing material (hereinafter also referred to as "crushed material gel") with a solvent. The following describes in detail "disruption of oxylipin-containing material," "preparation of disrupted material gel," and "solvent extraction of disrupted material gel."

[0014] [Crushing of oxylipin-containing materials] To increase the yield of oxylipins, the oxylipin-containing material is disrupted. The disruption means is not particularly limited as long as it can disrupt the oxylipin-containing material, and examples of the disruption means include a bead homogenizer, a polytron homogenizer, a pressure homogenizer, and an ultrasonic homogenizer. In the case of animal-derived materials, for example, the material can be roughly crushed together with a liquid medium in a blender, and then finely pulverized in a Polytron homogenizer to obtain a crushed material. In the case of a plant-derived material, for example, the material can be placed in a mortar together with zirconia beads and crushed with a pestle to obtain a crushed material. In the case of materials derived from algae, for example, algae cells recovered from a culture medium are suspended in a liquid medium and subjected to a disruption means to obtain a disrupted material. Examples of the liquid medium include water, alcohol, or a mixture of water and alcohol, with water (particularly pure water) being preferred in terms of safety during scale-up. Examples of alcohol include methanol and ethanol, with ethanol being preferred. The alcohol content in the mixture of water and alcohol is 20 to 80% by mass, preferably 40 to 60% by mass, based on the total mass of the mixture. 2-morpholinoethanesulfonic acid may be added to the liquid medium to adjust the pH.

[0015] [Preparation of crushed gel] In the present invention, the crushed material containing oxylipins is gelled. The gelling means is not particularly limited as long as it does not damage the oxylipins contained in the disrupted material, and any known means can be used. In a preferred embodiment, the gel is prepared by adding dropwise a gel stock solution containing crushed oxylipin-containing material and a gelling agent to an aqueous solution of a polyvalent metal ion compound.

[0016] Known gelling agents can be used, including sodium alginate, agarose (agar), gelatin, and glucomannan. The gelling agent is preferably sodium alginate or agarose, more preferably sodium alginate, in other words, the homogenate gel is preferably alginate gel or agarose gel, more preferably alginate gel.

[0017] The polyvalent metal ion compound is used to form a gel having sufficient strength to withstand subsequent washing treatments and the like after preparation. Examples of polyvalent metal ions include calcium ions and magnesium ions. Examples of polyvalent metal ion compounds include calcium chloride, calcium lactate, and magnesium chloride. The polyvalent metal ion compound is preferably calcium chloride or calcium lactate, more preferably calcium chloride. The concentration of the polyvalent metal ion compound in the aqueous solution (mass (w) of the polyvalent metal ion compound / volume (v) of the aqueous solution) is preferably 0.625 to 20% (w / v), more preferably 2.5 to 20% (w / v), from the viewpoint of preparing a gel with excellent strength.

[0018] In addition, when an oxylipin-containing material has a high calcium ion concentration, an alginate gel may be formed instantly upon mixing with sodium alginate, making it impossible to prepare a gel of the desired shape. In this case, sodium bicarbonate or sodium carbonate may be added to reduce the calcium ion concentration in the oxylipin-containing material according to the reaction described in Equation 1 or Equation 2. Formula 1: CaCl2+2NaHCO3→CaCO3↓+2NaCl+H2O Formula 2: CaCl2+Na2CO3→CaCO3↓+2NaCl

[0019] The content of the disrupted material in the gel (mass (w) of disrupted material / volume (v) of gel) is preferably 5 to 30% (w / v), more preferably 7 to 27% (w / v). A disrupted material content within this range can increase the extraction rate of oxylipins. The content of the crushed material in the gel can also be understood as the content of the crushed material in the above-mentioned gel stock solution.

[0020] The shape of the gel is not particularly limited. Examples of the shape of the gel include threads, spheres, and sheets. The gel is preferably in the form of a thread or sphere from the viewpoint of contact efficiency with the extraction solvent.

[0021] [Solvent extraction of crushed gel] The solvent can be any substance capable of dissolving oxylipins, without any particular limitations. The solvent includes water and organic solvents. Among these, water capable of dissolving multiple types of oxylipins is preferred. Alternatively, organic solvents may be used to selectively extract oxylipins corresponding to their polarity.

[0022] As the water, tap water, ion-exchanged water, pure water, ultrapure water, distilled water, etc. can be used without any particular limitation, but pure water or ultrapure water is preferred in terms of the purity of the extract. Pure water preferably has an electrical conductivity of 1 μS / cm or less. Ultrapure water preferably has an electrical conductivity of 0.06 μS / cm or less and a TOC of 5 ppb or less. Pure water and ultrapure water can be produced using commercially available production equipment (e.g., Milli-Q (registered trademark)).

[0023] The organic solvent may be an alcohol, a hydrocarbon, an ester, or the like. The organic solvent is preferably an alcohol or a hydrocarbon. The number of carbon atoms in the alcohol is preferably 1 to 4, more preferably 1 to 2. Examples of the alcohol include ethanol and methanol, with ethanol being preferred. The number of carbon atoms in the hydrocarbon is preferably 5 to 11, more preferably 6 to 8. Examples of the hydrocarbon include hexane and octane, with hexane being preferred. The number of carbon atoms in the alcohol constituting the ester is preferably 1 to 4, more preferably 1 to 2. The number of carbon atoms in the acid constituting the ester is preferably 1 to 4, more preferably 2 to 3. Examples of the ester include ethyl acetate and methyl acetate, with ethyl acetate being preferred.

[0024] Solvents are known materials and are readily available commercially or can be prepared. The solvent may be used alone or in combination of two or more kinds. The solvent may be a mixture of water and an organic solvent (for example, an aqueous solution of an alcohol, a mixture of ethyl acetate and water, etc.).

[0025] In one embodiment of the present invention, when the oxylipin is selected from the group consisting of 8-HEPE, 15-HEPE, and 18-HEPE, it is preferable to use a solvent selected from the group consisting of water, alcohol (preferably ethanol) or an aqueous solution thereof, and hydrocarbon (particularly hexane).

[0026] There are no particular limitations on the extraction means, as long as the crushed gel is mixed with a solvent. It is preferred to add the homogenate gel to the solvent, but the solvent may also be added to the homogenate gel. The extraction may be carried out batchwise, but from the viewpoint of improving the oxylipin yield, continuous extraction in which fresh solvent is supplied while the extract is being collected is preferred. The amount of solvent used is not particularly limited. For example, in the case of a crushed algae gel, the volume used is preferably 550 to 1200 times, and more preferably 600 to 800 times the wet mass (wet weight) of the algae used for crushing. In another embodiment, for example, in the case of a crushed algae gel, the volume used is preferably 50 to 1200 times, and more preferably 80 to 800 times, the wet weight of the algae contained in the gel. In yet another embodiment, for example, in the case of crushed algae gel, the volume used is preferably 10 to 400 times, more preferably 20 to 200 times the volume of the gel. When the crushed algae gel is continuously extracted, the solvent supply rate is, for example, 20 to 100 mL / min, and preferably 40 to 55 mL / min. It is preferable to carry out the extraction with stirring in terms of the extraction rate. The extraction temperature is not particularly limited, but is preferably carried out at 4 to 25°C in order to prevent decomposition of oxylipins. The extraction time is not particularly limited, but is, for example, 0.5 to 36 hours.

[0027] Although the present invention is not limited to a particular theory, it is believed that the presence of the gel suppresses the re-adsorption of oxylipins extracted into the solvent onto the crushed material (a phenomenon that leads to a decrease in yield), thereby improving the yield of oxylipins.

[0028] [Column purification of solvent extract] To increase the purity of oxylipins, it is preferable to subject the solvent extract to column purification. In the present invention, column purification techniques utilizing hydrophobic interactions can be used. For example, a solid-phase column (e.g., product name: STRATA-X, supplier: Shimadzu GLC) made of a polymer (e.g., styrene / divinylbenzene polymer) to which a functional group (e.g., N-vinylpyrrolidone) effective for retaining polar compounds has been introduced, or a solid-phase column packed with a synthetic adsorbent made of a porous styrene polymer (product name: SP850, supplier: Mitsubishi Chemical) can be suitably used. Column purification is preferably performed at a pH of 2.5 to 5.0 to maintain oxylipins in an undissociated state and strengthen their hydrophobic interaction with the solid-phase column. The pH can be adjusted by adding an acid (e.g., citric acid) or an alkali (e.g., sodium hydroxide). Column purification may be carried out multiple times.

[0029] [Oxylipin-containing composition] The disrupted gel is extracted with a solvent and optionally subjected to further column purification to obtain a composition containing oxylipins. For example, an oxylipin-containing composition containing 80 to 98% of the amount of 15-HEPE contained in the crushed algae material before extraction can be produced according to the present invention.

[0030] [Uses of oxylipins] Oxylipins obtained according to the present invention can be used for various purposes depending on their physiological activities. For example, 8-HEPE, which activates PPAR (peroxisome proliferator-activated receptor), is effective against lifestyle-related diseases and metabolic syndrome. [Example]

[0031] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0032] Nannochloropsis algae were used as the oxylipin-containing material.

[0033] Example 1: Solvent extraction of crushed algae gel [Cultivation of Nannochloropsis algae] The Nannochloropsis oceanica NIES-2145 strain was used, which was provided by the Microbial Culture Collection of the National Institute for Environmental Studies, National Research and Development Agency.

[0034] The culture medium used was ESM medium having the following composition:

[0035] TIFF0007799335000001.tif83143

[0036] 1 L of preculture solution of NIES-2145 strain (cell count: 1 × 10) was added to an 11 L transparent container containing 9 L of ESM medium. 9 cells / mL) was inoculated (cell count in the medium after inoculation: 1 × 10 8 cells / mL). Air (CO2 concentration: 2%) was blown into the transparent container (flow rate: 3 L / min) while white light was irradiated (light source: white LED, photon density: 300 μmol / m 2 / second), and culture was carried out at a liquid temperature of 20°C.

[0037] [Algae crushing] On the third day of cultivation, approximately 70 g (wet mass) of algal cells were collected by centrifugation and stored at -20°C. This procedure was repeated twice to obtain a total of 140 g of algal cells. 110 g of this was suspended in 2 mL of pure water per 1 g of wet mass of algal cells. The total volume of the suspension was approximately 330 mL. 10 mL of this suspension and 25 g of zirconia beads (particle size: 0.1 mm) were placed in a 50 mL crushing tube and crushed (2500 rpm for 1 minute) using a Multi-Beads Shocker (Yasui Kikai Co., Ltd.) to obtain crushed algal material.

[0038] [Solvent extraction of crushed algae gel] A gel stock solution containing crushed algae and sodium alginate (gelling agent) was added dropwise using a syringe to an aqueous solution of calcium chloride (a polyvalent metal ion compound) to prepare spherical gels. The calcium chloride concentration in the aqueous calcium chloride solution was 10% (w / v). The content of crushed algae in the gel was 24.4% (w / v). The crushed algae gel was added to an extraction tank containing pure water (extraction solvent) at a volume 18.2 times the wet weight of the crushed algae. Water extraction was performed at room temperature (approximately 20°C) for 24 hours while stirring with a vibrating mixer (80-100 rpm). During the water extraction, pure water (extraction solvent) was supplied to the extraction tank at a rate of 48-55 mL / min. The total amount of solvent used was 628-728 times the wet weight of the crushed algae. The total amount of solvent was also 628-728 times the wet weight of the algae contained in the gel. The total amount of solvent was also 168-192 times the volume of the gel. The liquid discharged from the extraction tank was adjusted to pH 3.5 to 4.5 by adding citric acid, and then subjected to subsequent column purification.

[0039] [Column purification of water extract] A column (internal diameter 3 cm x height 22.5 cm) packed with 150 mL of adsorbent (trade name: SP850, supplier: Mitsubishi Chemical) was used. The entire amount of the liquid discharged from the extraction tank was loaded onto the column, and then 450 mL of pure water was passed through the column over 15 minutes (first adsorbent washing). The adsorbent removed from the column was transferred to a 1 L bottle containing 450 mL of pure water and stirred with a stirrer for 10 minutes to remove any solid matter that had become mixed in the adsorbent (second washing). A 40% aqueous ethanol solution (450 mL) was added to the washed adsorbent, and the mixture was stirred with a stirrer for 10 minutes to remove impurities from the adsorbent (third washing). The adsorbent was again packed into a column (inner diameter 5 cm x height 50 cm), and 450 mL of 99.5% ethanol was passed through it. The eluate was collected every 50 mL, and nine fractions (50 mL x 9) were collected. 100 μL of each of the nine fractions was mixed with 900 μL of ethanol to dilute the solution 10-fold, and then subjected to liquid chromatography. 15-HEPE was detected in the fourth to ninth fractions in elution order. The 15-HEPE concentrations in the fourth to ninth fractions were as follows:

[0040] TIFF0007799335000002.tif41141

[0041] The total amount of 15-HEPE contained in the 4th to 9th fractions was 115.1 mg. The 15-HEPE yield (μg / gFW) per 1 g of algal body (wet weight (FW)) was 1046.36 μg / gFW.

[0042] Furthermore, the 15-HETE peak was identified in each chromatogram of the 4th to 9th fractions, and its concentration was measured.

[0043] TIFF0007799335000003.tif36123

[0044] The total amount of 15-HETE contained in the 4th to 9th fractions was 22.9 mg. The 15-HETE yield (μg / gFW) per 1 g of algal body (wet weight (FW)) was 208.18 μg / gFW.

[0045] The liquid chromatography conditions were as follows: Column: Kinetex C8 manufactured by Phenomenex (inner diameter: 2.1 mm, length: 150 mm, particle size: 2.6 μm) Injection volume: 10μL Column temperature: 40℃ Mobile phase A: 0.1% formic acid in water Mobile phase B: acetonitrile Gradient: Mobile phase A 90% → 5% Flow rate: 0.4mL / min Detection wavelength: 234 nm

[0046] Example 2: Effect of the content of debris in the gel and the gel shape on extraction The algal cells prepared in Example 1 (stored at −20° C.) were used as the oxylipin-containing material. 10 g (wet mass) of algae was suspended in 2 mL of pure water per 1 g of wet mass of algae. The total volume of the suspension was approximately 30 mL, and the algae concentration in the suspension was 33.3% (w / v). 10 mL of this suspension and 25 g of zirconia beads (particle size: 0.1 mm) were placed in a 50 mL crushing tube and crushed (2500 rpm for 1 minute) using a Multi-Beads Shocker (Yasui Kikai Co., Ltd.) to obtain crushed algae. The crushed algae were used to prepare the following three types of gel stock solutions. Gel stock solution 1 2.5% (w / v) sodium alginate was added to 10 mL of crushed algae to obtain gel stock solution 1 (crushed algae content: 26.67% (w / v), sodium alginate concentration: 0.5% (w / v)). Gel stock solution 2 5 mL of crushed algae was diluted 2-fold with 5 mL of pure water, and then 2.5% (w / v) sodium alginate was added to obtain gel stock solution 2 (crushed algae content: 13.33% (w / v), sodium alginate concentration: 0.5% (w / v)). Gel stock solution 3 2.5 mL of crushed algae was diluted 4-fold by adding 7.5 mL of pure water, and then 2.5% (w / v) sodium alginate was added to obtain gel stock solution 3 (crushed algae content: 6.67% (w / v), sodium alginate concentration: 0.5% (w / v)).

[0047] 5 mL of each gel stock solution was added dropwise using a syringe to an aqueous calcium chloride solution (calcium chloride concentration: 10% (w / v)) to prepare spherical gels. Furthermore, 5 mL of each gel stock solution was injected into an aqueous calcium chloride solution (calcium chloride concentration: 10% (w / v)) using a syringe to prepare a gel thread. Each gel was collected and washed in 25 mL of pure water. The physical properties of the six types of gels prepared are as follows: TIFF0007799335000004.tif41126

[0048] The gel from the crushed algae was placed in a 300 mL Erlenmeyer flask containing 150 mL of pure water (extraction solvent), and water extraction was carried out at 20°C for 8 hours while stirring with a shaker (100 rpm). The amount of solvent used was 30 times the wet mass (wet weight) of the algae used for disruption. The amount of solvent was also 30 times the volume of the gel. The amount of solvent relative to the wet weight (wet weight) of the algae contained in the gel was as follows for each type of gel: TIFF0007799335000005.tif47142 The extract was sampled every 2 hours, and the 15-HEPE concentration (μg / mL) in the extract was quantified by liquid chromatography. The results are shown in Figure 1. As shown in Figure 1, the extraction rate of 15-HEPE increased with increasing content of crushed algae in the gel. However, no clear difference in the extraction rate was observed depending on the gel shape.

[0049] Example 3: Study on the concentration of calcium chloride solution during gelation The algal cells prepared in Example 1 (stored at −20° C.) were used as the oxylipin-containing material. 10 g (wet mass) of algae was suspended in 2 mL of pure water per 1 g of wet mass of algae. The total volume of the suspension was approximately 30 mL. 10 mL of this suspension and 25 g of zirconia beads (particle size: 0.1 mm) were placed in a 50 mL crushing tube and crushed (at 2500 rpm for 1 minute) using a Multi-Beads Shocker (Yasui Kikai Co., Ltd.) to obtain crushed algae. 2.5% (w / v) sodium alginate was added to 20 mL of crushed algae to obtain a gel stock solution (crushed algae concentration: 26.67% (w / v), sodium alginate concentration: 0.5% (w / v)). 0.4 mL of the gel stock solution was dropped into wells containing 1.75 mL of one of 16 different calcium chloride solutions (calcium chloride concentrations: 0% (w / v), 0.010% (w / v), 0.020% (w / v), 0.0390% (w / v), 0.0781% (w / v), 0.1563% (w / v), 0.3125% (w / v), 0.625% (w / v), 1.25% (w / v), 2.5% (w / v), 3.8% (w / v), 5.0% (w / v), 6.3% (w / v), 7.5% (w / v), 8.8% (w / v), or 10% (w / v)). The crushed algae gelled under calcium chloride concentrations ranging from 0.0781 to 10% (w / v). Spherical gels were formed at calcium chloride concentrations of 0.625-10% (w / v). In particular, the spherical gels formed at calcium chloride concentrations of 2.5 to 10% (w / v) did not collapse even when subjected to the washing process (removing the calcium chloride aqueous solution from the well and adding 2 mL of pure water to wash the gel), and were strong enough to withstand subsequent solvent extraction.

[0050] Next, 0.4 mL of each of the seven types of spherical gels formed at calcium chloride concentrations of 2.5 to 10% (w / v) was placed in a test tube containing 10 mL of pure water (extraction solvent), and water extraction was carried out at 20°C for 8 hours while stirring with a shaker (100 to 120 rpm). The amount of solvent used was 25 times the wet mass (wet weight) of the algae used for disruption, 93.7 times the wet weight (wet weight) of the algae contained in the gel, and 25 times the volume of the gel. After 8 hours, the extract was sampled and the 15-HEPE concentration (μg / mL) in the extract was quantified by liquid chromatography. The results are shown in Figure 2. As shown in FIG. 2, 15-HEPE could be extracted using gels formed at calcium chloride concentrations of 2.5 to 10% (w / v).

[0051] Example 4: Examination of types of gelling agents In this example, a gel was prepared from crushed algae using sodium alginate or agarose (agar) as a gelling agent, and water extraction was performed to compare the yields of oxylipins.

[0052] (1) Preparation of gel stock solution containing crushed algae The algal cells prepared in Example 1 (stored at −20° C.) were used as the oxylipin-containing material. 3 g (wet mass) of algae was suspended in 6 mL of pure water. The total volume of the suspension was approximately 9 mL, and the algae concentration in the suspension was 33.3% (w / v). The entire suspension and 25 g of zirconia beads (particle size: 0.1 mm) were placed in a 50 mL crushing tube and crushed (2500 rpm for 1 minute) using a Multi-Beads Shocker (Yasui Kikai Co., Ltd.) to obtain crushed algae. This crushed algae was used for gelation and water extraction as described in (2) and (3) below.

[0053] (2) Gelation and water extraction using sodium alginate 4.8 mL of 0.625% (w / v) sodium alginate (39°C) was added to 1.2 mL of crushed algae and mixed to obtain 6 mL of gel stock solution (sodium alginate concentration: 0.5% (w / v)). 5 mL of the gel stock solution was poured into a round petri dish, and 50 mL of calcium chloride (a polyvalent metal ion compound) aqueous solution was poured on top to prepare a sheet gel. The calcium chloride concentration in the calcium chloride aqueous solution was 10% (w / v). The content of crushed algae in the sheet gel was 6.67% (w / v). The sheet gel was washed once with 50 mL of pure water, then placed in a 300 mL Erlenmeyer flask containing 150 mL of pure water (extraction solvent), and subjected to water extraction at 20°C for 4 hours while stirring with a shaker (100 rpm). The amount of solvent used was 450 times the wet mass (wet weight) of the algae contained in the gel, and 30 times the volume of the gel. The 15-HEPE concentration (μg / mL) in the extract was quantified by liquid chromatography. The results are shown in Table 1.

[0054] (3) Agarose gelation and water extraction 4.8 mL of 0.625% (w / v) agarose solution (39°C) was added to 1.2 mL of crushed algae and mixed to obtain 6 mL of gel stock solution (agarose concentration: 0.5% (w / v)). 5 mL of the gel stock solution was poured into a round petri dish and cooled at room temperature (approximately 20°C) to prepare a sheet-like gel. The content of crushed algae in the sheet-like gel was 6.67% (w / v). The sheet gel was placed in a 300 mL Erlenmeyer flask containing 150 mL of pure water (extraction solvent), and water extraction was carried out at 20°C for 4 hours while stirring with a shaker (100 rpm). The amount of solvent used was 450 times the wet mass (wet weight) of the algae contained in the gel, and 30 times the volume of the gel. The 15-HEPE concentration (μg / mL) in the extract was quantified by liquid chromatography. The results are shown in Table 1.

[0055] Table 1 TIFF0007799335000006.tif21140

[0056] As shown in Table 1, when agarose was used as a gelling agent, 15-HEPE could be extracted with water, just as with sodium alginate.

[0057] Example 5: Examination of gelation of crushed algae In this example, the same crushed algae was subjected to the extraction process (A) or (B) described below, and the extraction efficiency of oxylipins was compared. (A) Gelling of crushed algae and solvent extraction (Example) (B) Solvent extraction of crushed algae without gelation (Comparative Example)

[0058] (1) Preparation of crushed algae The algal cells prepared in Example 1 (stored at −20° C.) were used as the oxylipin-containing material. 71.71 g (wet mass) of algae was suspended in 143.42 mL of pure water, and the total volume of the suspension was approximately 205 mL. 30 mL of the suspension was dispensed into three 50 mL crushing tubes, each containing 10 mL of zirconia beads (particle size: 0.1 mm), and crushed using a Multi-Beads Shocker (Yasui Kikai Co., Ltd.) at 2500 rpm for 2 minutes. The crushing tube and zirconia beads were washed with 30 mL of pure water to recover 53 mL of crushed algae. This crushed algae was divided into 20 mL portions and subjected to the extraction steps (A) and (B) described below. Each 20 mL of crushed algae contained 3.33 g (wet mass) of algae.

[0059] (2) Solvent extraction (A) Solvent extraction of crushed algae by gelation (Example) A gel stock solution containing 20 mL of crushed algae and 5 mL of 2.5% (w / v) sodium alginate (gelling agent) was added dropwise using a syringe to 100 mL of an aqueous solution of calcium chloride (a polyvalent metal ion compound) to prepare spherical gels. The sodium alginate concentration in the gel stock solution was 0.5% (w / v). The calcium chloride concentration in the aqueous calcium chloride solution was 10% (w / v). The content of crushed algae in the gel was 13.33% (w / v). The gel of the crushed algae was washed with 100 mL of pure water. After washing, the gel of the crushed algae was placed in a 1000 mL reagent bottle containing 500 mL of pure water (extraction solvent), and water extraction was carried out at 20°C for 4 hours while stirring with a stirrer. The amount of solvent used was 150 times the wet mass of the algae contained in the gel, and 20 times the volume of the gel. The amount of 15-HEPE in the aqueous extract (500 mL) was determined by liquid chromatography. After the extraction was completed, the collected gel was poured into 250 mL of ethanol and allowed to stand at room temperature (approximately 20°C) for approximately 30 minutes to extract oxylipins, and the amount of 15-HEPE in the extract was quantified by liquid chromatography. The results are shown in Table 2.

[0060] (B) Solvent extraction of crushed algae without gelation (Comparative Example) 20 mL of the crushed algae material was placed in a 500 mL beaker containing 500 mL of pure water (extraction solvent), and the mixture was left to stand at 20°C for 4 hours to perform water extraction. Next, the algal crushed material was flocculated by adding citric acid (powder) to adjust the pH of the aqueous extract to 3.04, and the algal crushed material was separated into the algal crushed material and the filtrate using filter paper (pore size: 11 μm). The amount of 15-HEPE in the filtrate (500 mL) was determined by liquid chromatography. The filtered algae crushed material was placed together with the filter paper in 250 mL of ethanol and left to stand at room temperature (approximately 20°C) for approximately 30 minutes to extract oxylipins, and the amount of 15-HEPE in the extract was quantified by liquid chromatography. The results are shown in Table 2.

[0061] Table 2 TIFF0007799335000007.tif41146

[0062] In the comparative example in which water extraction was performed without gelling the crushed algae material, most of the 15-HEPE in the algae remained in the aggregates of the crushed algae material, and the water extraction efficiency was 8.63%. On the other hand, in the example in which the crushed algae material was gelled and extracted with water according to the present invention, no process for flocculating the crushed algae material was used, so most of the 15-HEPE in the algae was extracted into the water extract, and the water extraction efficiency was 92.98%. [Industrial Applicability]

[0063] The present invention can be used in the field of oxylipin production.

Claims

1. 1. A method for producing an oxylipin-containing composition, comprising: extracting a gel of the crushed oxylipin-containing material with a solvent; the gel is an alginate gel or an agarose gel, The method of manufacturing, wherein the solvent is water.

2. The method of claim 1, wherein the gel is an alginate gel.

3. The method according to claim 1, wherein the content of the crushed oxylipin-containing material in the gel is 5 to 30% (w / v).

4. The method according to claim 1, wherein the gel is in the form of threads or spheres.

5. The method of claim 1 further comprising the step of preparing a gel of the crushed oxylipin-containing material prior to the extraction step.

6. 6. The method according to claim 5, wherein the gel is prepared by adding dropwise a gel stock solution containing the crushed oxylipin-containing material and the gelling agent to an aqueous solution of a polyvalent metal ion compound.

7. The method of claim 6, wherein the gelling agent is sodium alginate.

8. 8. The method according to claim 7, wherein the polyvalent metal ion compound is calcium chloride.

9. The method according to claim 8, wherein the calcium chloride concentration in the aqueous calcium chloride solution is 2.5 to 20% (w / v).

10. The method according to claim 1, wherein the oxylipin-containing material is a plant (excluding those classified as algae).

11. The method of claim 1 , wherein the oxylipin-containing material is algae.

12. The method according to claim 1, wherein the oxylipin-containing material is algae of the genus Nannochloropsis.

13. 2. The method of claim 1, wherein the oxylipin is selected from the group consisting of 8-HEPE, 11-HEPE, 15-HEPE, 18-HEPE, 11-HETE, and 15-HETE.

14. The method of claim 1, wherein the oxylipin is selected from the group consisting of 8-HEPE, 15-HEPE, and 18-HEPE.

15. The manufacturing method described in claim 1, wherein the oxylipin is 15-HEPE.

16. 2. The process of claim 1, wherein the extraction is carried out continuously.

17. The method of claim 1, further comprising subjecting the extract to column purification at pH 2.5 to 5.0.

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