Method for producing useful substance and production apparatus for producing useful substance

Abstract

The present disclosure provides a method for producing a useful substance using algae. The method comprises a step for obtaining an algae culture by culturing algae under light irradiation, and a step for isolating the useful substance from the algae culture.

Description

Method for producing useful substances and production apparatus for producing useful substances

[0001] The present disclosure relates to a method for producing useful substances and a production apparatus for producing useful substances.

[0002] Microalgae have the ability to synthesize various useful substances and are attracting attention in various industrial fields as sustainable biological resources. These useful substances include carnosine, citrulline, creatine, etc.

[0003] Carnosine exhibits an antioxidant effect in the body and has an anti-aging effect, so there is a high commercial demand as a health-related substance (Non-Patent Document 1). In addition, citrulline has a blood flow improving effect, and creatine has an energy supply function to muscles, and both are known as health-related substances that contribute to the improvement of exercise performance (Non-Patent Documents 2 and 3).

[0004] In order to industrially utilize these useful substances, technologies for supplying them at a lower cost and stably are required. For this reason, the development of technologies for improving the content of useful substances in production organisms is important.

[0005] Conventionally, bacteria of the genus Klebsiella, which are heterotrophic organisms, have been used for the production of these useful substances. However, in this method, organic substances are required for culturing, and a special culture apparatus is required when performing anaerobic culture. In addition, when utilizing gene recombination technology, dedicated experimental facilities and management systems are required, and these are factors that affect production costs. [[ID=XVII]]

[0006] Under such a background, it is expected to realize a more efficient and economical method for producing useful substances.

[0007] Reddy VP, Garrett MR, Perry G, Smith MA. Carnosine: a versatile antioxidant and antiglycating agent. Sci Aging Knowledge Environ. 2005 May 4;2005(18):pe12.Allerton, TD; Proctor, DN; Stephens, JM; Dugas, TR; Spielmann, G.; Irving, BA l-Citrulline Supplementation: Impact on Cardiometabolic Health. Nutrients 2018, 10, 921. Kreider, RB Effects of creatine supplementation on performance and training adaptations. Mol Cell Biochem 244, 89-94 (2003).

[0008] This disclosure is made to solve the above-mentioned problems and aims to provide a method for producing useful substances and a manufacturing apparatus for producing useful substances.

[0009] One aspect of the present disclosure is a method for producing a useful substance using algae, comprising the steps of culturing algae under light irradiation to obtain an algal culture, and isolating the useful substance from the algal culture. One aspect of the present disclosure is a production apparatus for producing a useful substance using algae, comprising a culture section for culturing algae and an isolation section for isolating the useful substance from the algal culture, wherein the culture section has a capacity of 30 μmol photon / m³ 2 / s or more, 100μmol photon / m 2 Light irradiation at light intensity of less than / s, and 200 μmol photon / m 2 / s or more, 800μmol photon / m 2 The manufacturing apparatus includes a light irradiation device capable of irradiating with light at least one of the following light intensities: / s or less, and the isolation unit includes a chromatography apparatus capable of isolating water-soluble useful substances and lipid-soluble useful substances.

[0010] According to this disclosure, a method for producing a useful substance and a manufacturing apparatus for producing a useful substance can be provided.

[0011] Figure 1 shows the change in cell density in a Chaetoceros gracilis culture from the start of culture to day 10. The black circles represent conditions under high light (250 μmol photon / m²). 2 The cell density in culture under normal light conditions (50 μmol photon / m² / s) is shown as shown by the white circles (50 μmol photon / m² / s). 2 This shows the cell density in culture at 2 / s. Figure 2 is an overall configuration diagram showing one embodiment of the apparatus for producing the useful substance of this disclosure.

[0012] The following describes non-limiting embodiments of this disclosure. This disclosure is not limited to the embodiments described below.

[0013] <Method for Producing Useful Substances> According to one embodiment of the present disclosure, a method for producing useful substances using algae is provided, comprising the steps of culturing algae under light irradiation to obtain an algal culture, and isolating the useful substance from the algal culture. In the method for producing useful substances of this embodiment, the useful substance produced can be controlled simply by adjusting the amount of culture light, without the need for specialized techniques such as genetic modification, organic substrates, or equipment for anaerobic culture.

[0014] The types of algae in this disclosure are not limited. The algae may be non-sessile algae. The algae may be, for example, unicellular algae or microalgae. Examples of algae that may be used include any of the following classifications: Aurantiochytrium, Chlamydomonas, Chlorella, red algae Schizoan, Spirulina, Botryococcus, Euglena, haptophytes, Prasinophytes, green algae, brown algae, red algae, cyanobacteria, diatoms, yellow-green algae, golden algae, dinoflagellates, and seaweed. More specific examples of algae include algae of the genera Chaetoceros, Isochrysis, Pavlova, Pyramimonas, and Tisochrysis, among which algae of the genus Chaetoceros, such as Chaetoceros gracilis, may be used. The algae in this disclosure may be a single species of algae or a population containing multiple species of algae. For example, a population of the same species of algae with genetic variation may be used. The algae of this disclosure may be algae derived from isolated algae. In the production methods of the embodiments, the algae may include algae of the genus Chaetoceros.

[0015] In this disclosure, "useful substance" refers to a substance that has usefulness as an ingredient in pharmaceuticals, food additives, supplements, etc., as well as usefulness in industry, agriculture, and other industrial applications. A useful substance may be a substance that has commercial value in an isolated state. A useful substance in this disclosure may include low molecular weight compounds. The molecular weight of a "low molecular weight compound" in this disclosure may be 1000 g / mol or less, 900 g / mol or less, or 800 g / mol or less. A "low molecular weight compound" may also be an organic compound. A "low molecular weight compound" in this disclosure may be a compound with a molecular weight of 800 g / mol or less, or an organic compound with a molecular weight of 800 g / mol or less.

[0016] The useful substances in the embodiments are 2-oxoglutaric acid, 4-hydroxyphenethyl alcohol, 7-amino-4-hydroxy-2-naphthalenesulfonic acid, CTP (cytidine triphosphate), GTP (guanosine triphosphate), guanine, N-acetylglucosamine 6-phosphate, N-methyltryptamine, N6-methyllysine, Nω-methylarginine, pyroglutamine, ribavirin, thymidine, γ-Glu-Cys (γ-glutamylcysteine), 6-keto-prostaglandin E1, and acylcarnitine. (18:0) (Acylcarnitine (18:0)), cholesterolone, cycloartenol, lanosterol, fucoxanthin, linolenic acid, γ-linolenic acid, N-oleoylglycine, nobiletin, palmitoleic acid, palmitoylcholine, prostaglandin D2, 11β-prostaglandin E2, 13,14-dihydro-15-keto-prostaglandin E2, 15-keto-prostaglandin F2α (15-keto-prostaglandin F2α), prostaglandin E2 (prostaglandin E2), stearidonicacid (stearidonic acid), taurochenodeoxycholic acid, 1-methyladenosine, 2-aminoisobutyric acid, 2-aminobutyric acid, adenosine, Ala-Ala (alanylalanine), allantoin, ascorbate 2-sulfate, Asp (aspartic acid), butyrylcarnitine, citric acid, citrulline, cysteine ​​glutathione disulfide, decanoic acid, galacturonic acid, glucuronic acid, Gln (glutamine), Glu-Glu (glutamyl glutamic acid), gluconic acid (gluconic acid), glutathione (GSSG), Gly-Leu (glycylleucine), glyceric acid (glyceric acid), Ile (isoleucine), Leu (leucine), malic acid (malic acid), myristic acid (myristic acid), myristoleic acid acid (myristoleic acid), N-acetylputrescine, N-acetylthreonine, N-methylputrescine, cadaverine, N1,N8-diacetylspermidine, N1-acetylspermidine, N5-ethylglutamine, N8-acetylspermidine, NAD+ (nicotinamide adenine dinucleotide), NMN (nicotinamide mononucleotide), Nα-succinylarginine, ophthalmicacid (ophthalmic acid), p-toluic acid (para-toluic acid), o-toluic acid (ortho-toluic acid), m-toluic acid (meta-toluic acid), phosphorylcholine (phosphorylcholine), prostaglandin E2 (prostaglandin E2), retinoic acid (retinoic acid), Thr (threonine), Trp (tryptophan), Tyr (tyrosine), Val (valine), β-Ala (β-alanine), γ-Glu-Asn (γ-glutamyl asparagine), γ-Glu-Val (γ-glutamyl valine), 2,6-dihydroxybenzoic acid (2,6-dihydroxybenzoic acid), 3-deoxy-D-manno-2-octulosonic acid (3-deoxy-D-manno-2-octulosonic acid), 4-methyl-5-thiazoleethanol (4-methyl-5-thiazoleethanol), 6,8-thioctic acid (6,8-thioctic acid), benzoic acid, carnosine, creatine, creatinine, deoxyxylulose 5-phosphate, dihydroorotic acid, GABA (gamma-aminobutyric acid), hexanoic acid, 4-methylpentanoic acid, N-formylaspartic acid, N-lactoylethanolamine, N6-formyllysine, S-lactoylglutathione, S-methylcysteine, tartaric acid, tridecanoic acid, Tyr-Arg (tyrosylarginine), β-hydroxyisovaleric acid acid (β-hydroxyisovaleric acid), 2-hydroxyvaleric acid (2-hydroxyvaleric acid), 2-hydroxyisovalericacid (2-hydroxyisovaleric acid), γ-Glu-Glu (γ-glutamyl glutamic acid), 3-hydroxydodecanoic acid (3-hydroxydodecanoic acid), AEA (20:3) (acylethanolamide (20:3)), cholesterol sulfate (cholesterol sulfate), fatty acid (12:0) (fatty acid (12:0)), glucosylceramide (d18:1 / 24:1) (glucosylceramide (d18:1 / 24:1)), 1-myristoyl-glycero-3-phosphocholine (1-myristoyl-glycero-3-phosphocholine), 1-palmitoyl-glycero-3-phosphoethanolamine (1-palmitoyl-glycero-3-phosphoethanolamine), LPE (18:1) (lysophosphatidylethanolamine (18:1)), LPG The compounds may be one or more of (18:1) (lysophosphatidylglycerol (18:1)) and piperine, or may contain one or more of these. As will be understood by those skilled in the art, in these compounds, notations such as "n:m" (e.g., 20:3, 12:0, etc.) indicate the number of carbon atoms and unsaturated bonds in the fatty acid portion of the compound. Specifically, n represents the total number of carbon atoms constituting the fatty acid portion, and m represents the number of carbon-carbon double bonds (unsaturated bonds) in the molecule.

[0017] The method for producing the useful substance of the embodiment includes the step of culturing algae under light irradiation to obtain an algal culture. At least one of natural light and artificial light can be used for the light irradiation.

[0018] When using natural light, sunlight can be directly utilized to cultivate algae. When using sunlight, in order to adjust the light intensity, shading can be performed using a shading sheet as needed. The shading sheet can be, for example, a sheet made of synthetic resin such as polyethylene, polypropylene, polyvinyl chloride, or a metal mesh, etc. The light shielding rate can be selected, for example, from the range of 20 - 90%. The shading by the shading sheet may be performed throughout the entire cultivation period, or only during a specific period. For example, shading can be performed in summer with a large amount of solar radiation, and the shading can be removed in winter with a small amount of solar radiation. Thereby, an appropriate light intensity can be maintained throughout the year.

[0019] When using artificial light, light sources such as LEDs, fluorescent lamps, metal halide lamps, etc. can be used. The wavelength of the artificial light source may be selected to include a wavelength range suitable for the photosynthesis of algae (for example, 400 nm - 700 nm). In the step of obtaining the algal culture in the embodiment, it may include light irradiation of 30 μmol photon / m 2 / s or more, 40 μmol photon / m 2 / s or more, or 50 μmol photon / m 2 / s or more. Also, in the step of obtaining the algal culture in the embodiment, it may include light irradiation of 800 μmol photon / m 2 / s or less, 600 μmol photon / m 2 / s or less, or 400 μmol photon / m 2 / s or less. In the step of obtaining the algal culture in the method for producing a useful substance of the embodiment, the light intensity is 30 μmol photon / m 2 / s or more, 40 μmol photon / m 2 / s or more, or 50 μmol photon / m 2 / s or more, and is under normal light conditions of 100 μmol photon / m 2 / s or less or 75 μmol photon / m 2 / s or less, or 200 μmol photon / m 2 / s or more or 250 μmol photon / m 2 / s or higher and 800 μmol photon / m 2 / s or less, 600 μmol photon / m 2 / s or less, or 400 μmol photon / m 2 It can be controlled under strong light conditions of less than / s. In particular, approximately 50 μmol photon / m 2 Under normal light conditions of / s, and approximately 250 μmol photons / m² 2 Experimental studies have confirmed that high light conditions of / s are suitable for the production of many useful substances. These light intensity ranges can be achieved by adjusting the light intensity using a light-shielding sheet when using natural light, or by adjusting the output of the light source when using artificial light. In the process of obtaining algal cultures according to the embodiment, the light intensity range can be defined as a measurement taken on the surface of the culture tank or the culture.

[0020] In the process of obtaining the algal culture according to the embodiment, it is also possible to use a combination of natural and artificial light. For example, by using natural light while adjusting the light intensity with a light-blocking sheet during the daytime when there is strong sunlight, and irradiating with artificial light at night, it is possible to achieve cultivation with appropriate light intensity continuously for 24 hours. During cultivation, the light intensity may be monitored using measuring instruments such as a quantum light meter, and adjustments may be made as needed.

[0021] The process of obtaining the algal culture according to the embodiment may include light irradiation for 48 hours or more, or 72 hours or more. Alternatively, the process of obtaining the algal culture according to the embodiment may include light irradiation for 360 hours or less, 300 hours or less, or 240 hours or less.

[0022] Algal cultivation can be carried out using culture media for algae known to those skilled in the art, seawater, other environmental water, diluted seawater, artificial seawater, mixtures thereof, or solutions with a partially common composition, depending on the properties of the control algae. The culture media for algae can be any aqueous solution in which natural algae can grow without particular limitations, or a specified aqueous medium for algae can be used. The medium may be an algal culture medium known to those skilled in the art, and may, for example, contain nutrients, carbon sources, rare metals, etc. Specific examples of the aqueous medium include IMK medium, SWM-3 medium, modified SWM-3 (mSWM-3) medium, modified media of these media, and media obtained by mixing these media or modified media. IMK medium contains 200mg / L NaNO3, 1.4mg / L Na2HPO4, 5mg / L K2HPO4, 2.68mg / L NH4Cl, 5.2mg / L Fe-EDTA, 0.332mg / L Mn-EDTA, 37.2mg / L Na2-EDTA, 0.023mg / L ZnSO4・7H2O, 0.014mg / L CoSO4・7H2O, 0.0073mg / L Na2MoO4・2H2O, 0.0025mg / L CuSO4・5H2O, 0.0017mg / L H2SeO3, 0.2mg / L Thiamin-HCl, 0.0015mg / L Biotin, 0.0015mg / L Vitamin B12 0.18mg / L A medium consisting of MnCl2・4H2O and the balance seawater. In particular, when culturing diatoms, 0.2 mM to 1 mM Na2SiO3 may be added to the IMK medium in addition to the above components.mSWM-3 medium contains 17 mg NaNO3, 1.56 mg NaH2PO4・2H2O, 5.68 mg Na2SiO3・9H2O, 1.12 mg Na2EDTA・2H2O, 0.084 mg Fe-EDTA, 0.0346 μg Na2SeO3, 1 ml P-1 metal solution (618.3 mg H3BO4, 69.25 mg MnCl2・4H2O, 5.45 mg ZnCl2, 238 μg CoCl2・6H2O, 100 ml distilled water), 0.2 μg Vitamin B12, 1 mL Vitamin mixture solution S3 (5 mg Thiamine HCl, 1 mg Nicotinic acid, 1 mg Calcium pantothenate, 0.1 mg p-Aminobenzoic acid, 0.01 mg Biotin, 50 mg Inositol, 0.02 mg Folic acid, 30 mg Thymine, 100 mL) The culture medium (pH 7.7-7.8) is prepared by mixing distilled water, 50 mg Tris(hydroxymethyl)aminomethane, and 98 mL of seawater. Algae are cultured under natural light or under light irradiation (e.g., 50 μmol photon m). -2 s -1 The process may also be carried out while irradiating with continuous white light. Furthermore, the algae culture may be performed by shaking, or while bubbling with air or sterile air.

[0023] The method for producing a useful substance according to the embodiment includes a step of isolating the useful substance from an algal culture. In this disclosure, "isolation" of a useful substance may mean separating and purifying the useful substance contained in the algal culture from the culture. The culture may be subjected to solid-liquid separation such as centrifugation, and depending on whether the useful substance is contained in the solid components such as cells in the culture or in the liquid components such as the culture medium, the supernatant or pellet may be subjected to subsequent isolation steps. The pellet containing cells may be washed with a suitable washing solution such as sterile artificial seawater, and then dissolved with a dissolving solution containing an organic solvent such as ethanol to extract the useful substance. At this time, cell disruption may be performed by physical means such as sonication.

[0024] Depending on the properties of the useful substance, water-soluble and lipid-soluble fractions can be separated. For example, using the Bligh-Dyer method, lipid-soluble and water-soluble components can be separated by two-layer partitioning of a chloroform / methanol / water system. Specifically, chloroform and methanol are added to the culture or cell lysate, and then an appropriate amount of water is added to separate it into two layers. In this case, lipid-soluble components may be partitioned into the lower layer (chloroform layer), and water-soluble components into the upper layer (water / methanol layer). Other lipid extraction methods such as the Folch method can also be used.

[0025] Each separated fraction can be subjected to treatments such as concentration and drying as necessary. Furthermore, the culture supernatant, cell-lysed sample, or each separated fraction may be pretreated with one or more of the following: stirring, centrifugation, filtration, ultrafiltration, deproteinization, and solid-phase extraction (SPE). In addition, separation and purification using chromatographic techniques such as liquid chromatography may be performed to isolate and purify the desired useful substances.

[0026] Pre-treated or unpre-treated samples can be separated by high-performance liquid chromatography (HPLC). Columns suitable for HPLC include reversed-phase columns such as C18 columns, normal-phase columns such as silica columns, and gel filtration columns. Those skilled in the art will understand that these columns can be appropriately selected depending on the target low-molecular-weight substances described herein. The mobile phase used in HPLC can be appropriately selected by those skilled in the art depending on the column and target substance. The target substance can be separated, for example, by gradient elution using an aqueous mobile phase and an organic solvent mobile phase with a reversed-phase column. The eluted useful substance can be fractionated, and after removing the solvent, it can be recovered as a dry powder. The purity of the recovered useful substance can be confirmed, for example, by analysis using a high-resolution mass spectrometer. If the purity does not reach the target value (e.g., 95% or higher), separation and purification by HPLC may be repeated.

[0027] Detection in HPLC can utilize detection methods known to those skilled in the art, such as mass spectrometry (MS), tandem mass spectrometry (MS / MS), ultraviolet-visible spectroscopy (UV-Vis), fluorescence spectroscopy (FL), and differential refractive index spectroscopy (ELS). Mass spectrometers used in MS and MS / MS include Fourier transform mass spectrometers (FT-MS) and time-of-flight mass spectrometers (TOF-MS). In particular, using high-resolution mass spectrometers such as FT-MS enables highly accurate metabolite identification with a mass error of ±10 ppm or less.

[0028] In one embodiment, the step of culturing algae is performed under normal light conditions (e.g., 30 μmol photon / m²). 2 / s or more, 40μmol photon / m 2 / s or more, or 50 μmol photon / m 2 / s or more, and 100 μmol photon / m 2 / s or less or 75 μmol photons / m² 2 Light intensity of less than / s), or strong light conditions (e.g., 200 μmol photon / m²) 2 / s or more, or 250 μmol photon / m 2 / s or more, and 800 μmol photon / m 2 / s or less, 600 μmol photon / m 2 / s or less, or 400 μmol photon / m 2 This includes light irradiation at intervals of 1 / s or less. It has been confirmed that the type or amount of water-soluble and lipid-soluble substances produced differs depending on the light irradiation conditions. Therefore, an appropriate light intensity can be selected according to the target product.

[0029] The step of obtaining the algal culture of the embodiment involves 30 μmol photon / m³ 2 / s or more, 40μmol photon / m 2 / s or more, or 50 μmol photon / m 2 / s or more, and 100 μmol photon / m 2 / s or less or 75 μmol photons / m² 2The method includes light irradiation at a light intensity of 1 / s or less, and the useful substance may include at least one of 2-oxoglutaric acid, 4-hydroxyphenethyl alcohol, 7-amino-4-hydroxy-2-naphthalenesulfonic acid, CTP, GTP, guanine, N-acetylglucosamine 6-phosphate, N-methyltryptamine, N6-methyllysine, Nω-methylarginine, pyroglutamine, ribavirin, thymidine, or γ-Glu-Cys. These compounds are water-soluble compounds that are usually favored for production under light conditions.

[0030] The step of obtaining the algal culture of the embodiment involves 30 μmol photon / m³ 2 / s or more, 40μmol photon / m 2 / s or more, or 50 μmol photon / m 2 / s or more, and 100 μmol photon / m 2 / s or less or 75 μmol photons / m² 2 The method includes light irradiation at a light intensity of 1 / s or less, and the useful substance may include at least one of 6-keto-prostaglandin E1, acylcarnitine (18:0), cholestenone, cycloartenol, lanosterol, fucoxanthin, linolenic acid, γ-linolenic acid, N-oleoylglycine, nobiletin, palmitoleic acid, palmitoylcholine, prostaglandin D2, 11β-prostaglandin E2, 13,14-dihydro-15-keto-prostaglandin E2, 15-keto-prostaglandin F2α, prostaglandin E2, stearidonic acid, or taurochenodeoxycholic acid. These compounds are lipophilic compounds that are usually favored for production under light conditions.

[0031] The step of obtaining the algal culture according to the embodiment involves a concentration of 200 μmol photon / m³. 2 / s or more, or 250 μmol photon / m 2 / s or more, and 800 μmol photon / m 2 / s or less, 600 μmol photon / m 2 / s or less, or 400 μmol photon / m 2including light irradiation at a light intensity of / s or less, wherein the useful substances are 1-methyladenosine, 2-aminoisobutyric acid, 2-aminobutyric acid, adenosine, Ala-Ala, allantoin, ascorbate 2-sulfate, Asp, butyrylcarnitine, citric acid, citrulline, cysteine glutathione disulfide, decanoic acid, galacturonic acid, glucuronic acid, Gln, Glu-Glu, gluconic acid, glutathione (GSSG), Gly-Leu, glyceric acid, Ile, Leu, malic acid, myristic acid, myristoleic acid, N-acetylputrescine, N-acetylthreonine, N-methylputrescine, cadaverine, N1,N8-diacetylspermidine, N1-acetylspermidine, N5-ethylglutamine, N8-acetylspermidine, NAD+, NMN, Nα-succinylarginine, ophthalmic acid, p-toluic acid, o-toluic acid, m-toluic acid, phosphorylcholine, prostaglandin E2, retinoic acid, Thr, Trp, Tyr, Val, β-Ala, γ-Glu-Asn, γ-Glu-Val, 2,6-dihydroxybenzoic acid, 3-deoxy-D-manno-2-octulosonic acid, 4-methyl-5-thiazoleethanol, 6,It may contain one or more of the following compounds: 8-thioctic acid, benzoic acid, carnosine, creatine, creatinine, deoxyxylulose, 5-phosphate, dihydroorotic acid, GABA, hexanoic acid, 4-methylpentanoic acid, N-formylaspartic acid, N-lactoylethanolamine, N6-formyllysine, S-lactoylglutathione, S-methylcysteine, tartaric acid, tridecanoic acid, Tyr-Arg, β-hydroxyisovaleric acid, 2-hydroxyvaleric acid, 2-hydroxyisovaleric acid, or γ-Glu-Glu. These compounds are water-soluble compounds that are favorably produced under strong light conditions.

[0032] The step of obtaining the algal culture according to the embodiment is 200 μmol photon / m². 2 / s or more, or 250 μmol photon / m 2 / s or more, and 800 μmol photon / m 2 / s or less, 600 μmol photon / m 2 / s or less, or 400 μmol photon / m 2 The process includes light irradiation at a light intensity of 1 / s or less, and the useful substance may include one or more of the following: 3-hydroxydodecanoic acid, AEA (20:3), cholesterol sulfate, fatty acid (12:0), glucosylceramide (d18:1 / 24:1), 1-myristoyl-glycero-3-phosphocholine, 1-palmitoyl-glycero-3-phosphoethanolamine, LPE (18:1), LPG (18:1), or piperine. These compounds are lipophilic compounds that are advantageous for production under strong light conditions.

[0033] <Manufacturing apparatus for producing useful substances> According to another embodiment of the present disclosure, a manufacturing apparatus for producing useful substances using algae comprises a culture section for culturing algae and an isolation section for isolating useful substances from the algal culture, wherein the culture section has a capacity of 30 μmol photon / m³ 2 / s or more, 100μmol photon / m 2 Light irradiation at light intensity of less than / s, and 200 μmol photon / m 2 / s or more, 800μmol photon / m 2 A production apparatus may be provided that includes a light irradiation device capable of irradiating with light at least one of light intensities of / s or less, and the isolation unit includes a chromatography apparatus capable of isolating water-soluble and lipid-soluble useful substances. In the embodiment, the production apparatus is 30 μmol photon / m³. 2 / s or more, 100μmol photon / m 2 Light irradiation at light intensity of less than / s, and 200 μmol photon / m 2 / s or more, 800μmol photon / m 2 The embodiment may include a light irradiation device capable of irradiating at a light intensity of less than or equal to / s. The manufacturing apparatus of this embodiment may be capable of culturing under both normal and strong light conditions in the same apparatus, and the optimal light irradiation conditions can be selected according to the target useful substance. Furthermore, by equipping the isolation section with a chromatography apparatus capable of isolating both water-soluble and lipid-soluble substances, it becomes possible to efficiently isolate a variety of useful substances produced according to the light irradiation conditions. For this reason, various useful substances can be efficiently manufactured without preparing multiple apparatuses. The elements of this embodiment (algae, useful substances, cultivation, light irradiation, isolation, etc.) may be described in the section on <Method for Manufacturing Useful Substances>.

[0034] The manufacturing apparatus of this embodiment comprises a culture section for cultivating algae and an isolation section for isolating useful substances from the algal culture. The culture section and the isolation section may be connected via a piping system.

[0035] The culture section of the embodiment includes a light irradiation device. The culture section may also include an environmental control device and a culture tank. The culture tank is a container for culturing algae and may be made of materials such as glass, stainless steel, or plastic. The culture tank may be equipped with an outlet for transferring the culture to an isolation section.

[0036] The light irradiation apparatus in this embodiment uses normal light conditions (30 μmol photon / m²). 2 / s or more, 40μmol photon / m 2 / s or more, or 50 μmol photon / m 2 / s or more, and 100 μmol photon / m 2 / s or less or 75 μmol photons / m² 2 (Light intensity of less than / s) and strong light conditions (200 μmol photon / m²) 2 / s or more, or 250 μmol photon / m 2 / s or more, and 800 μmol photon / m 2 / s or less, 600 μmol photon / m 2 / s or less, or 400 μmol photon / m 2 At least one light irradiation is possible with a light intensity of less than or equal to 1 / s. The light intensity may be adjustable stepwise or continuously within this range. The light irradiation device may include a light source such as an LED, fluorescent lamp, or metal halide lamp. The wavelength of the light emitted from the light source may include a wavelength range suitable for algal photosynthesis (e.g., 400nm-700nm). The light irradiation device may include a photodetector such as a photoquantum sensor and a photocontrol device that controls the output of the light source based on the detected light intensity. The photocontrol device may be capable of automatically adjusting the light intensity according to a pre-programmed light irradiation pattern.

[0037] The environmental control device of the embodiment may include one or more selected from the group consisting of a temperature control device, a pH control device, aeration device, culture medium supply device, and stirring device. The temperature control device may include a heater and / or cooler and be able to maintain the culture environment within a predetermined temperature range. The pH control device may include a pH measuring sensor and a reagent supply mechanism for adding an acid or alkaline solution.

[0038] The aeration device may include an air pump or compressor and a mass flow controller for controlling the gas flow rate. It may also include a HEPA filter or sterile filter for removing particulate matter from the gas being aerated. The culture medium supply device may include a liquid transfer pump for controlling the supply of fresh culture medium and the discharge of used culture medium, and a culture medium tank for holding fresh or used culture medium. The stirring device may include a mechanical impeller, a magnetic stirrer, or a bubble stirring mechanism.

[0039] These devices included in the environmental control unit can be integrally controlled by an environmental control device. The control device can automatically adjust culture parameters such as temperature, pH, aeration rate, culture medium supply rate, and stirring rate based on feedback signals from various sensors. The control device may also include functions for recording culture data, issuing alerts in case of abnormality detection, and remote monitoring.

[0040] The isolation unit of the embodiment includes a chromatography apparatus capable of isolating both water-soluble and lipid-soluble useful substances. This chromatography apparatus may be configured as an analytical and preparative high-performance liquid chromatography (HPLC) apparatus capable of handling both water-soluble and lipid-soluble useful substances in a single HPLC system.

[0041] An HPLC system may include a liquid delivery pump capable of delivering multiple mobile phases of different polarities at a rate of, for example, 0.1 mL / min to 10 mL / min, a sample injection device capable of injecting, for example, 1 μL to 1000 μL of sample, a column oven with temperature controllable in the range of, for example, 4°C to 80°C, and a detector. The liquid delivery pump may have a high-pressure gradient function that can mix solvents ranging from aqueous to organic solvent systems, for example, two to four solvents, in any ratio. Such a gradient function allows the HPLC system to function as a system capable of separating useful substances of different polarities, from water-soluble substances to lipid-soluble substances.

[0042] HPLC systems can be equipped with motorized valves that allow switching between multiple columns. For example, using 6-port, 10-port, or 12-port motorized valves, columns for water-soluble and lipid-soluble substances can be automatically switched. Furthermore, a column switching mechanism using a switching valve allows for column regeneration during analysis and switching between pre-columns and main columns. Additionally, column selection valves can be provided to sequentially switch between multiple columns arranged in parallel. These valves can be selected to be automatically controlled by a control computer.

[0043] For isolating water-soluble useful substances, the HPLC system may be equipped with an ODS column for reversed-phase chromatography to separate water-soluble substances with different polarities. Furthermore, the HPLC system may be equipped with cation exchange columns and anion exchange columns for ion exchange chromatography to separate ionic water-soluble substances. Additionally, the HPLC system may be equipped with a gel filtration column for size exclusion chromatography, for example, with an exclusion limit molecular weight of 1,000 to 1,000,000, to separate water-soluble substances with different molecular weights.

[0044] For isolating lipid-soluble useful substances, an HPLC system may be equipped with silica gel columns for normal-phase chromatography for separating lipid-soluble substances with different polarities. Furthermore, an HPLC system may be equipped with phenyl-bonded columns and C8 columns for reverse-phase chromatography for separating aromatic and hydrophobic lipid-soluble substances. These columns can be selected from, for example, inner diameters of 2.1 mm to 20 mm and lengths of 50 mm to 250 mm. The column switching mechanism described above allows for switching between columns for water-soluble substances and columns for lipid-soluble substances within a single HPLC system.

[0045] An HPLC system may be equipped with multiple detectors capable of detecting both water-soluble and lipid-soluble useful substances. Specifically, it may include, for example, an ultraviolet-visible absorption detector capable of detecting in the wavelength range of 190 nm to 800 nm, a fluorescence detector capable of detecting in the excitation wavelength range of 200 nm to 900 nm and the fluorescence wavelength range of 200 nm to 900 nm, a differential refractive index detector capable of detection regardless of polarity, and a mass spectrometer capable of ionizing both water-soluble and lipid-soluble substances (for example, ionization methods such as ESI or APCI may be used). These detectors can be arranged in series, and a configuration that allows simultaneous detection by multiple detectors is possible.

[0046] The isolation unit of the embodiment may include a pretreatment device for separating water-soluble and lipid-soluble useful substances. The pretreatment device may include, for example, a centrifuge capable of centrifugation up to 10,000 × g, 15,000 × g, or 20,000 × g, a filtration device using, for example, a membrane filter with a pore size in the range of 0.22 μm to 10 μm, a concentration device using reduced pressure or a centrifugal evaporator, and a solvent extraction device. In particular, the isolation unit of the embodiment may include a two-phase partitioning device for efficiently separating water-soluble and lipid-soluble useful substances from a culture. The two-phase partitioning device can partition, for example, lipid-soluble components into the lower layer (chloroform layer) and water-soluble components into the upper layer (water / methanol layer) by two-phase partitioning based on a lipid extraction method such as the Bligh-Dyer method or the Folch method, for example, a chloroform / methanol / water system. After efficiently separating water-soluble and lipid-soluble useful substances by this two-phase partitioning, each fraction can be further purified by an appropriate HPLC system. The isolation unit may be equipped with a device for concentrating or drying each distributed fraction by nitrogen gas purging or freeze-drying.

[0047] These devices in the isolation section of the embodiment can be integrally controlled by a control computer. Furthermore, these devices in the isolation section of the embodiment can be configured to automatically perform the setting of analytical conditions, data acquisition, and peak analysis for water-soluble and lipid-soluble useful substances, respectively.

[0048] Examples of the present disclosure are described below, but the present disclosure is not limited to the examples described below.

[0049] [Example 1] Culture and metabolite analysis of Chaetoceros gracilis

[0050] Dissolve Daigo IMK medium for marine microalgae cultivation (manufactured by Shioya MS Co., Ltd.) in Daigo artificial seawater SP for marine microalgae, and add approximately 1 x 10⁶ Chaetoceros gracilis, known as an algae feed for aquaculture. 5 The solution was added at a concentration of cells / mL. Culture was performed in six flasks at 25°C under light conditions of 50 μmol photon / m². 2 / s (normal light) and 250 μmol photon / m2 Two conditions were set: / s (strong light), and three samples were cultured under each condition while shaking at 75 rpm.

[0051] Cell density increased from the start of culture to day 9 under both light conditions, and no significant difference in proliferation was observed (Figure 1).

[0052] On days 2, 4, and 9 after the start of culture, 40 mL of culture medium was collected, and cells were collected by centrifugation (16,000 g, 10 minutes). After removing the supernatant from the collected cells, the cells were resuspended in an equal volume of IMK medium, and the washing process of centrifugation was repeated twice. Finally, the supernatant was completely removed to obtain a cell pellet.

[0053] The obtained cell pellet was subjected to metabolite measurement and analysis using the following method.

[0054] Methanol solution was added to the prepared cell pellet of the water-soluble fraction, and the mixture was suspended and sonicated for 30 seconds. Milli-Q water containing an internal standard (0.5 μM) was added and mixed, then allowed to stand for 30 seconds. The extract was collected and centrifuged (2,300 × g, 4°C, 5 min). The obtained supernatant was transferred to an ultrafiltration tube (Ultrafree MC PLHCC, HMT, 5 kDa) and ultrafiltration was performed by centrifugation (9,100 × g, 4°C, 120 min). After drying the filtrate, it was redissolved in Milli-Q water to prepare the measurement sample.

[0055] An ethanol solution containing an internal standard (1 μM) was added to the prepared cell pellet of the lipid-soluble fraction, and sonication was performed for 30 seconds. After adding Milli-Q water, the mixture was stirred by sonication under ice cooling for 5 minutes, and the supernatant was collected by centrifugation (2,300 × g, 4°C, 5 min). After drying, this was redissolved in a 50% isopropanol aqueous solution (v / v) to prepare the measurement sample.

[0056] Metabolites were measured using CE-MS for the water-soluble fraction and LC-MS for the lipid-soluble fraction. CE-MS was performed according to the measurement method described in Japanese Patent No. 6106864, and LC-MS was performed using a Thermo Fisher Scientific Vanquish Flex UHPLC System and Orbitrap Exploris 240. An ODS column (2 × 50 mm, 2 μm) was used as the analytical column, and gradient analysis was performed using mobile phase A (water / 0.1% formic acid) and mobile phase B (isopropanol:acetonitrile:water = 65:30:5, 0.1% formic acid, 2 mM ammonium formate). Measurements were performed in both positive and negative modes.

[0057] Analysis of Measurement Data: The acquired measurement data was analyzed using MasterHands ver.2.20.0.1 developed by Keio University. Peaks with an S / N ratio of 3 or higher were automatically extracted, and the peak area values ​​were corrected using the area values ​​of the internal standard and the sample volume to convert them to relative area values. Metabolite identification was performed by matching peaks that met the conditions of a mass error of ±10 ppm and a retention time error of ±0.15 minutes with the metabolite library of Human Metabolome Technologies, Inc.

[0058] For 83 of the water-soluble substances, analysis was performed by adding a 20 μM internal standard, and the absolute quantitative value was calculated by comparing it with the peak area value of the internal standard.

[0059] Analysis of water-soluble metabolites revealed that 279 substances were detected in samples grown under normal light and 282 substances in samples grown under strong light at various points in time during the cultivation period. On day 9, which is the typical harvest time for algae used as aquaculture feed, 256 substances were detected under normal light and 269 substances under strong light.

[0060] Of the 247 substances detected under both light conditions, water-soluble substances showing differences in quantity are summarized in Tables 1-1 and 1-2. As shown in Tables 1-1 and 1-2, 5 substances showed significantly higher relative amounts under normal light, and 46 substances showed significantly higher relative amounts under strong light. Of these substances, 3 substances showed significantly higher absolute amounts under normal light, and 16 substances showed significantly higher absolute amounts under strong light. In addition, 9 substances were detected only under normal light, and 22 substances were detected only under strong light.

[0061] Regarding lipid-soluble metabolites, 84 substances were detected in samples under normal light and 77 substances in samples under strong light, at some point during the culture period. On day 9, 77 substances were detected under normal light and 72 substances under strong light.

[0062] Table 2 summarizes the lipid-soluble substances that showed differences in quantity among the 67 substances detected under both light conditions. As shown in Table 2, three substances showed significantly higher relative amounts under normal light, and five substances showed significantly higher relative amounts under strong light. In addition, 10 substances were detected only under normal light, and 5 substances were detected only under strong light.

[0063] These detected substances were found to include numerous health-related beneficial substances, as shown in Table 3. These results reveal that specific beneficial substances accumulate within the cells of Chaetoceros gracilis as it proliferates, and that their composition fluctuates depending on the growth stage or the amount of culture light.

[0064] This embodiment demonstrates that the amount of useful substances produced in algal cells can be increased simply by adjusting the light intensity during cultivation, without changing the culture medium components or introducing special experimental techniques or equipment. For example, it is possible to simultaneously increase the amounts of multiple useful substances, such as carnosine and creatine, under specific light intensity conditions. Furthermore, since adjusting the light intensity does not cause a significant change in cell growth, it is possible to increase the productivity of useful substances while maintaining a constant cell harvest. In addition, for algae cultivated in environments where light intensity changes seasonally, such as outdoors, it becomes possible to predict the amount of useful substances present in the supernatant by referring to the light intensity during cultivation and harvesting. The above results exemplify how the method disclosed herein enables the efficient and economical production of useful substances using algae.

[0065] This disclosure includes the following embodiments: (1) A method for producing a useful substance using algae, comprising the steps of: culturing algae under light irradiation to obtain an algal culture; and isolating the useful substance from the algal culture. (2) The method for producing a useful substance according to 1, wherein the step of obtaining the algal culture includes light irradiation for 24 hours or more and 360 hours or less. (3) The step of obtaining the algal culture includes 30 μmol photon / m 2 / s or more, 800μmol photon / m 2 A method for producing a useful substance according to item 1 or 2, comprising light irradiation at a light intensity of 30 μmol photon / m² or less. (Item 4) The step of obtaining the culture of the algae is to provide a method for producing a useful substance according to item 1 or 2, comprising light irradiation at a light intensity of 30 μmol photon / m² or less. 2 / s or more, 100μmol photon / m 2A method for producing a useful substance according to any one of claims 1 to 3, comprising light irradiation at a light intensity of 30 μmol photon / m² or less, wherein the useful substance comprises at least one of 2-oxoglutaric acid, 4-hydroxyphenethyl alcohol, 7-amino-4-hydroxy-2-naphthalenesulfonic acid, CTP, GTP, guanine, N-acetylglucosamine 6-phosphate, N-methyltryptamine, N6-methyllysine, Nω-methylarginine, pyroglutamine, ribavirin, thymidine, or γ-Glu-Cys. (Clause 5) The step of obtaining the culture of the algae comprises 30 μmol photon / m² 2 / s or more, 100μmol photon / m 2 A method for producing a useful substance according to any one of claims 1 to 3, comprising light irradiation at a light intensity of 200 μmol photon / m² or less, wherein the useful substance comprises at least one of 6-keto-prostaglandin E1, acylcarnitine (18:0), cholestenone, cycloartenol, lanosterol, fucoxanthin, linolenic acid, γ-linolenic acid, N-oleoylglycine, nobiletin, palmitoleic acid, palmitoylcholine, prostaglandin D2, 11β-prostaglandin E2, 13,14-dihydro-15-keto-prostaglandin E2, 15-keto-prostaglandin F2α, prostaglandin E2, stearidonic acid, or taurochenodeoxycholic acid. (Clause 6) The step of obtaining the culture of the algae comprises 200 μmol photon / m² 2 / s or more, 800μmol photon / m 2including light irradiation at a light intensity of / s, wherein the useful substances are 1-methyladenosine, 2-aminoisobutyric acid, 2-aminobutyric acid, adenosine, Ala-Ala, allantoin, ascorbate 2-sulfate, Asp, butyrylcarnitine, citric acid, citrulline, cysteine glutathione disulfide, decanoic acid, galacturonic acid, glucuronic acid, Gln, Glu-Glu, gluconic acid, glutathione (GSSG), Gly-Leu, glyceric acid, Ile, Leu, malic acid, myristic acid, myristoleic acid, N-acetylputrescine, N-acetylthreonine, N-methylputrescine, cadaverine, N1,N8-diacetylspermidine, N1-acetylspermidine, N5-ethylglutamine, N8-acetylspermidine, NAD+, NMN, Nα-succinylarginine, ophthalmic acid, p-toluic acid, o-toluic acid, m-toluic acid, phosphorylcholine, prostaglandin E2, retinoic acid, Thr, Trp, Tyr, Val, β-Ala, γ-Glu-Asn, γ-Glu-Val, 2,6-dihydroxybenzoic acid, 3-deoxy-D-manno-2-octulosonic acid, 4-methyl-5-thiazoleethanol, 6,A method for producing a useful substance according to any one of claims 1 to 3, comprising one or more of 8-thioctic acid, benzoic acid, carnosine, creatine, creatinine, deoxyxylulose 5-phosphate, dihydroorotic acid, GABA, hexanoic acid, 4-methylpentanoic acid, N-formylaspartic acid, N-lactoylethanolamine, N6-formyllysine, S-lactoylglutathione, S-methylcysteine, tartaric acid, tridecanoic acid, Tyr-Arg, β-hydroxyisovaleric acid, 2-hydroxyvaleric acid, 2-hydroxyisovaleric acid, or γ-Glu-Glu. (Clause 7) The step of obtaining the culture of the algae includes light irradiation at a light intensity of 200 μmol photon / m 2 / s or more and 800 μmol photon / m 2 / s or less. The useful substance includes one or more of 3-hydroxydodecanoic acid, AEA (20:3), cholesterol sulfate, fatty acid (12:0), glucosylceramide (d18:1 / 24:1), 1-myristoyl-glycero-3-phosphocholine, 1-palmitoyl-glycero-3-phosphoethanolamine, LPE (18:1), LPG (18:l), or piperine. A method for producing a useful substance according to any one of claims 1 to 3. (Clause 8) A production apparatus for producing a useful substance using algae, comprising a culture unit for culturing algae and an isolation unit for isolating a useful substance from the culture of the algae. The culture unit includes light irradiation at a light intensity of 30 μmol photon / m 2 / s or more and 100 μmol photon / m 2 / s or less, and light irradiation at a light intensity of 200 μmol photon / m 2 / s or more and 800 μmol photon / m 2A manufacturing apparatus comprising a light irradiation device capable of irradiating with light at least one of light intensities of / s or less, wherein the isolation unit comprises a chromatography apparatus capable of isolating water-soluble useful substances and lipid-soluble useful substances.

[0066] While this disclosure has been described with reference to several embodiments described above, it is not limited to the examples given in those embodiments. Various modifications can be made to the structure and details of this disclosure within the scope of this disclosure.

Claims

1. A method for producing a useful substance using algae, comprising the steps of: culturing algae under light irradiation to obtain an algal culture; and isolating the useful substance from the algal culture.

2. The method for producing a useful substance according to claim 1, wherein the step of obtaining the culture of the algae includes light irradiation for 48 hours or more and 360 hours or less.

3. The step of obtaining the culture of the aforementioned algae is 30 μmol photon / m². 2 / s or more, 800μmol photon / m 2 A method for producing a useful substance according to claim 1 or 2, comprising light irradiation at a light intensity of less than or equal to / s.

4. The step of obtaining the culture of the aforementioned algae involves a process of 200 μmol photon / m² 2 / s or more, 800μmol photon / m 2 including light irradiation at a light intensity of / s, wherein the useful substance is creatine, carnosine, citrulline, GABA, N5-ethylglutamine, citric acid, β-Ala, retinoic acid, allantoin, 1-methyladenosine, 2-aminoisobutyric acid, 2-aminobutyric acid, adenosine, Ala-Ala, ascorbate 2-sulfate, Asp, butyrylcarnitine,,, cysteine glutathione disulfide, decanoic acid, galacturonic acid, glucuronic acid, Gln, Glu-Glu, gluconic acid, glutathione (GSSG), Gly-Leu, glyceric acid, Ile, Leu, malic acid, myristic acid, myristoleic acid, N-acetylputrescine, N-acetylthreonine, N-methylputrescine, cadaverine, N1,N8-diacetylspermidine, N1-acetylspermidine,, N8-acetylspermidine, NAD+, NMN, Nα-succinylarginine, ophthalmic acid, p-toluic acid, o-toluic acid, m-toluic acid, phosphorylcholine, prostaglandin E2, Thr, Trp, Tyr, Val, γ-Glu-Asn, γ-Glu-Val, 2,6-dihydroxybenzoic acid, 3-deoxy-D-manno-2-octulosonic acid, 4-methyl-5-thiazoleethanol, 6,A method for producing a useful substance according to claim 1 or 2, comprising one or more of the following: 8-thioctic acid, benzoic acid, creatinine, deoxyxylulose 5-phosphate, dihydroorotic acid, hexanoic acid, 4-methylpentanoic acid, N-formylaspartic acid, N-lactoylethanolamine, N6-formyllysine, S-lactoylglutathione, S-methylcysteine, tartaric acid, tridecanoic acid, Tyr-Arg, β-hydroxyisovaleric acid, 2-hydroxyvaleric acid, 2-hydroxyisovaleric acid, or γ-Glu-Glu.

5. The step of obtaining the culture of the algae is 30 μmol photon / m². 2 / s or more, 100μmol photon / m 2 A method for producing a useful substance according to claim 1 or 2, comprising light irradiation at a light intensity of 0 / s or less, wherein the useful substance comprises at least one of 6-keto-prostaglandin E1, palmitoleic acid, nobiletin, cholesterolone, prostaglandin D2, linolenic acid, fucoxanthin, acylcarnitine (18:0), cycloartenol, lanosterol, γ-linolenic acid, N-oleoylglycine, palmitoylcholine, 11β-prostaglandin E2, 13,14-dihydro-15-keto-prostaglandin E2, 15-keto-prostaglandin F2α, prostaglandin E2, stearidonic acid, or taurochenodeoxycholic acid.

6. The step of obtaining the culture of the algae is 30 μmol photon / m². 2 / s or more, 100μmol photon / m 2 A method for producing a useful substance according to claim 1 or 2, comprising light irradiation at a light intensity of 0 / s or less, wherein the useful substance comprises at least one of ribavirin, pyroglutamine, 2-oxoglutaric acid, 4-hydroxyphenethyl alcohol, 7-amino-4-hydroxy-2-naphthalenesulfonic acid, CTP, GTP, guanine, N-acetylglucosamine 6-phosphate, N-methyltryptamine, N6-methyllysine, Nω-methylarginine, thymidine, or γ-Glu-Cys.

7. The step of obtaining the culture of the algae involves light irradiation at a light intensity of 200 μmol photon / m 2 / s or more and 800 μmol photon / m 2 / s or less, and the useful substance contains one or more of piperine, 3-hydroxydodecanoic acid, AEA (20:3), cholesterol sulfate, fatty acid (12:0), glucosylceramide (d18:1 / 24:1), 1-myristoyl-glycero-3-phosphocholine, 1-palmitoyl-glycero-3-phosphoethanolamine, LPE (18:1), or LPG (18:1). The method for producing the useful substance according to claim 1 or 2.

8. A manufacturing apparatus for producing useful substances using algae, comprising: a culture section for culturing algae; and an isolation section for isolating useful substances from the algal culture, wherein the culture section has a capacity of 30 μmol photon / m³ 2 / s or more, 100μmol photon / m 2 Light irradiation at light intensity of less than / s, and 200 μmol photon / m 2 / s or more, 800μmol photon / m 2 A manufacturing apparatus comprising a light irradiation device capable of irradiating with light at least one of light intensities of / s or less, wherein the isolation unit comprises a chromatography apparatus capable of isolating water-soluble useful substances and lipid-soluble useful substances.

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