Heterotrophic microalgae and uses thereof as well as method for increasing γ-aminobutyric acid in heterotrophic microalgae

Abstract

To provide heterotrophic microalgae with a high content of γ-aminobutyric acid, and to provide a method for increasing the content of γ-aminobutyric acid in heterotrophic microalgae.SOLUTION: The present invention provides heterotrophic microalgae having a γ-aminobutyric acid content of 100 mg or more per 100 g of dry algae. The heterotrophic microalgae may be microalgae belonging to the Thraustochytriales or the Aurantiochytrium genus. The present invention also provides a food, medicine, or feed containing the heterotrophic microalgae. The present invention also provides a method for increasing the γ-aminobutyric acid content in heterotrophic microalgae, comprising a step of culturing the heterotrophic microalgae in a medium containing γ-aminobutyric acid.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to heterotrophic microalgae and uses thereof, as well as a method for increasing γ-aminobutyric acid in heterotrophic microalgae. [Background technology]

[0002] γ-Aminobutyric acid (GABA) is a type of water-soluble amino acid that does not form proteins. γ-Aminobutyric acid is widely distributed in nature and is biosynthesized from glutamic acid in the body. In the mammalian brain, γ-aminobutyric acid acts as an inhibitory neurotransmitter and has been reported to improve brain function, relax, reduce stress, and lower blood pressure, as well as improve the quality of sleep.

[0003] To date, there have been several reports on microalgae containing γ-aminobutyric acid. For example, Patent Document 1 below discloses microalgae of the Pavlovaceae family that contain γ-aminobutyric acid (OPMS30543 strain and OPMS30543X strain). Patent Document 2 below discloses algae belonging to the Polytrichum class that contain γ-aminobutyric acid. Patent Document 3 below discloses Polytrichum cells that contain γ-aminobutyric acid.

[0004] Some microalgae exhibit heterotrophic properties (heterotrophic microalgae). Here, "heterotrophic microalgae" refers to algae that can be cultured and grown without light exposure by providing an organic carbon source, rather than relying on photosynthetic culture, which requires light exposure to grow them through photosynthesis. To date, there have been no reports of microalgae that exhibit heterotrophic properties and contain a large amount of γ-aminobutyric acid. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2021-13313 A [Patent Document 2] Special Publication No. 2020-72698 [Patent Document 3] Special Publication No. 2014-524248 [Patent Document 4] Special Publication No. 10-072590 [Patent Document 5] Patent Gazette No. 7218023

Non-licensed documents

[0006]

Non-patent document 1

Non-patent document 2

Non-patent document 3

[0007] The present invention is primarily intended to provide heterotrophic microalgae having a high content of γ-aminobutyric acid, and a method for increasing the content of γ-aminobutyric acid in heterotrophic microalgae. [Means for solving the problem]

[0008] The present inventors conducted extensive research to solve the above problems and discovered that heterotrophic microalgae take up γ-aminobutyric acid contained in a culture medium and accumulate it within the algae. As a result, the present inventors established a technique for increasing γ-aminobutyric acid in heterotrophic microalgae and a technique for obtaining heterotrophic microalgae that contain a large amount of γ-aminobutyric acid. The present inventors also discovered a method for recovering γ-aminobutyric acid contained in a culture medium using heterotrophic microalgae. The present invention was completed as a result of these findings.

[0009] That is, the present invention provides heterotrophic microalgae having a γ-aminobutyric acid content of 100 mg or more per 100 g of dry algae. The heterotrophic microalgae may be microalgae belonging to the Thraustochytriales family. The heterotrophic microalgae may be microalgae belonging to the genus Aurantiochytrium. The present invention also provides a food, a medicine, or a feed containing the heterotrophic microalgae. The present invention also provides a method for increasing the content of γ-aminobutyric acid in heterotrophic microalgae, comprising the step of culturing the heterotrophic microalgae in a medium containing γ-aminobutyric acid. The present invention also provides a method for recovering γ-aminobutyric acid from a culture medium, the method comprising the steps of culturing heterotrophic microalgae in a culture medium containing γ-aminobutyric acid, and recovering the heterotrophic microalgae. The present invention also provides a method for recovering γ-aminobutyric acid from a composition, the method comprising the steps of preparing a medium using a composition containing γ-aminobutyric acid, culturing heterotrophic microalgae in the medium, and recovering the heterotrophic microalgae. The present invention also provides a method for producing a food, a medicine, or a feed, comprising using a heterotrophic microalgae cultured in a medium containing γ-aminobutyric acid. Effect of the Invention

[0010] The present invention provides heterotrophic microalgae having a high content of γ-aminobutyric acid. The present invention also provides a method for increasing the content of γ-aminobutyric acid in heterotrophic microalgae. Note that the effects of the present invention are not limited to the effects described herein, and may be any of the effects described in this specification. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Preferred embodiments for carrying out the present invention will be described below. The embodiments described below are representative embodiments of the present invention, and the scope of the present invention is not limited to these embodiments.

[0012] 1. Heterotrophic microalgae with high γ-aminobutyric acid content

[0013] In the heterotrophic microalgae according to one embodiment of the present invention, the content of γ-aminobutyric acid is 100 mg or more per 100 g of dry algae. The content of γ-aminobutyric acid may be, for example, 120 mg or more, 140 mg or more, 160 mg or more, 180 mg or more, 200 mg or more, 220 mg or more, 240 mg or more, 260 mg or more, 280 mg or more, 300 mg or more, 350 mg or more, 400 mg or more, 450 mg or more, 500 mg or more, or 550 mg or more per 100 g of dry algae.

[0014] The heterotrophic microalgae according to the present embodiment are obtained by culturing heterotrophic microalgae in a medium containing γ-aminobutyric acid, and have a higher content of γ-aminobutyric acid in the algal bodies than heterotrophic microalgae obtained by culturing in a medium not containing at least γ-aminobutyric acid. That is, the heterotrophic microalgae according to the present embodiment are heterotrophic microalgae with a high content of γ-aminobutyric acid. The medium not containing γ-aminobutyric acid is preferably a medium not containing γ-aminobutyric acid and / or L-glutamic acid, and is preferably a medium containing 1 mg / L or less of γ-aminobutyric acid and / or L-glutamic acid.

[0015] As used herein, "high in γ-aminobutyric acid" and "high in γ-aminobutyric acid" refer to a high content of γ-aminobutyric acid in the algal cells compared to the content of γ-aminobutyric acid in the algal cells of heterotrophic microalgae (control) obtained by culturing in a medium not containing γ-aminobutyric acid. When comparing a sample with a control, the samples may be cultured under the same culture conditions except for the concentrations of γ-aminobutyric acid and / or L-glutamic acid in the medium, and the "high in γ-aminobutyric acid" and "high in γ-aminobutyric acid" of the sample may be determined. The content of γ-aminobutyric acid in the algal cells of the heterotrophic microalgae of this embodiment may be, for example, 1.1 times or more, 1.3 times or more, 1.5 times or more, 1.8 times or more, 2.0 times or more, 2.3 times or more, 2.5 times or more, 2.8 times or more, 3.0 times or more, 3.5 times or more, 4.0 times or more, 4.5 times or more, 5.0 times or more, 5.5 times or more, or 6.0 times or more of the content of γ-aminobutyric acid in the algal cells of the control.

[0016] Examples of the heterotrophic microalgae include microalgae belonging to the Thraustochytriales or Labyrinthuleas, and one or more species from these groups can be used.

[0017] Examples of the microalgae belonging to the Thraustochytrium group include microalgae belonging to the genera Aurantiochytrium, Schizochytrium, Thraustochytrium, Parietichytrium, and Ulkenia, and one or more types from these groups can be used.

[0018] Examples of the microalgae belonging to the Labyrinthula genus include microalgae belonging to the genus Labyrinthula.

[0019] Examples of the microalgae belonging to the genus Aurantiochytrium include microalgae belonging to Aurantiochytrium limacinum or Aurantiochytrium mangrovei. Examples of strains of Aurantiochytrium limacinum include SR-21, 4W-1b, NIES3737, ATCC MYA-1381, mh0186, and F29-b. Examples of strains of Aurantiochytrium mangrovei include 18W-13a, RCC893, MP2, BL10, and FB3, and one or more of these strains can be used.

[0020] The SR-21 strain was deposited on March 6, 1994 at the National Institute of Bioscience and Human-Technology (currently the National Institute of Technology and Evaluation, Biotechnology Center, Patent Organism Depositary (NITE-IPOD)) under the name "Marine Bacteria SR21 strain" and was given the accession number FERM BP-5034, and was also deposited on March 17, 1995 at the Institute for Fermentation, Foundation, given the accession number IFO 32693 (Patent Document 4 (JP Patent Publication No. 10-072590) paragraph

[0035] ). The SR-21 strain is also available from ATCC as ATCC MYA-1381 strain (strain designation SR21 [IFO 32693]) (Non-Patent Document 1: https: / / www.atcc.org; Non-Patent Document 2: https: / / www.ncbi.nlm.nih.gov), and Non-Patent Document 2 states that the registration date for this MYA-1381 strain is June 28, 2011. In addition, the SR-21 strain was originally classified into the genus Schizochytrium, but is now classified into the genus Aurantiochytrium (see Non-Patent Document 3: Mycoscience (2007) 48:199-211, etc.). ·NITE-IPOD: International Patent Organism Depositary (IPOD), Biological Resource Center, National Institute of Technology and Evaluation(NITE), #120, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba 2920818, Japan. ·ATCC:American Type Culture Collection, 10801 University Boulevard,Manassas, VA 20110-2209,USA.

[0021] Since the heterotrophic microalgae according to this embodiment has a high content of γ-aminobutyric acid, for example, by ingesting, administering, or applying the heterotrophic microalgae, it is expected that brain function improving effects, relaxation effects, stress reducing effects, blood pressure lowering effects, collagen production promoting effects, epidermal cell proliferation effects, hyaluronic acid synthesis promoting effects, etc. can be obtained. Therefore, the heterotrophic microalgae according to this embodiment are preferably used for various compositions (e.g., food, medicines, skin topicals, feed, etc.), and more preferably for articles that are orally ingested by animals (including humans), and more preferably for food, medicine, or feed. That is, the heterotrophic microalgae according to this embodiment are preferably heterotrophic microalgae for food, medicine, or feed.

[0022] 2. Method for producing heterotrophic microalgae with high gamma-aminobutyric acid content

[0023] The heterotrophic microalgae with a high content of γ-aminobutyric acid can be obtained by a method for producing heterotrophic microalgae according to one embodiment of the present invention. Specifically, the method for producing heterotrophic microalgae is a method for producing heterotrophic microalgae having a γ-aminobutyric acid content of 100 mg or more per 100 g of dry algae, i.e., a method for producing heterotrophic microalgae with a high content of γ-aminobutyric acid.

[0024] The method for producing heterotrophic microalgae according to the present embodiment includes a step of culturing heterotrophic microalgae in a medium containing γ-aminobutyric acid (hereinafter also referred to as a "culturing step"). By carrying out the culturing step, γ-aminobutyric acid in the medium can be taken up by the heterotrophic microalgae and accumulated. This allows the content of γ-aminobutyric acid in the heterotrophic microalgae to be increased. Furthermore, by carrying out the culturing step, it is also possible to convert L-glutamic acid in the medium to γ-aminobutyric acid by the heterotrophic microalgae and accumulate γ-aminobutyric acid in the microalgae. In addition, when carrying out the culturing step, it is also possible to accumulate γ-aminobutyric acid and / or convert glutamic acid to γ-aminobutyric acid and accumulate the converted γ-aminobutyric acid. Therefore, by the culturing step, heterotrophic microalgae having a higher content of γ-aminobutyric acid than heterotrophic microalgae cultured in a medium not containing γ-aminobutyric acid and / or glutamic acid can be cultured. The glutamic acid is preferably L-glutamic acid.

[0025] 2-1. Cultivation of heterotrophic microalgae

[0026] 2-1-1. Culture medium

[0027] The method for producing heterotrophic microalgae with a high content of γ-aminobutyric acid according to this embodiment may include a step of preparing a medium containing γ-aminobutyric acid (hereinafter also referred to as a "medium preparation step"). The medium used in the above-mentioned culturing step may be prepared in the medium preparation step. The medium may be a medium containing γ-aminobutyric acid and / or glutamic acid, and a medium containing at least γ-aminobutyric acid is more preferable because it makes it easier to obtain heterotrophic microalgae with a high content of γ-aminobutyric acid.

[0028] The content of γ-aminobutyric acid in the medium used in the above culture process may be, for example, 10 mg / L or more, 20 mg / L or more, 30 mg / L or more, 40 mg / L or more, 50 mg / L or more, 60 mg / L or more, 70 mg / L or more, 80 mg / L or more, 90 mg / L or more, or 100 mg / L or more. In addition, the glutamic acid content in the medium used in the above culture step may be, for example, 10 mg / L or more, 20 mg / L or more, 30 mg / L or more, 40 mg / L or more, 50 mg / L or more, 60 mg / L or more, 70 mg / L or more, 80 mg / L or more, 90 mg / L or more, or 100 mg / L or more. Furthermore, the contents of γ-aminobutyric acid and glutamic acid in the medium used in the above culture step may be the total value of an appropriate combination of the above-mentioned γ-aminobutyric acid contents and glutamic acid contents.

[0029] In the medium preparation step, the medium containing γ-aminobutyric acid and / or glutamic acid can be prepared, for example, using a composition containing γ-aminobutyric acid and / or glutamic acid, or γ-aminobutyric acid alone and / or glutamic acid alone, and a component required for culturing heterotrophic microalgae. Therefore, the medium preparation step may be a step of preparing a medium containing γ-aminobutyric acid using a composition containing γ-aminobutyric acid or γ-aminobutyric acid. By adjusting the blending amount of the composition containing γ-aminobutyric acid or γ-aminobutyric acid alone, the content of γ-aminobutyric acid in the medium can be adjusted to the above-mentioned numerical range. In addition, the medium containing glutamic acid can be prepared, for example, using a composition containing glutamic acid or glutamic acid alone, and a component required for culturing heterotrophic microalgae. Therefore, the medium preparation step may be a step of preparing a medium containing glutamic acid using a composition containing glutamic acid or glutamic acid. In addition, a medium containing both γ-aminobutyric acid and glutamic acid may be prepared.

[0030] The composition containing γ-aminobutyric acid may be, for example, fermentation residue and fermentation residue such as shochu lees, awamori distillation lees, and pickles, and waste liquid and waste food in the food manufacturing process using a food containing γ-aminobutyric acid, as long as γ-aminobutyric acid is present as a type of water-soluble amino acid. The composition containing glutamic acid may be, for example, waste liquid and waste food in the food manufacturing process when producing glutamic acid or polyglutamic acid, or waste liquid and waste food in the food manufacturing process using a food containing glutamic acid, as long as glutamic acid is present as a type of water-soluble amino acid. Glutamic acid is contained in kelp; vegetables such as tomatoes; fermented foods such as cheese, and fermentation residue such as the above-mentioned shochu distillation lees and awamori distillation lees. These compositions containing γ-aminobutyric acid and / or glutamic acid can be used as materials for culture media used in the cultivation of heterotrophic microalgae. It is preferable to use at least a composition containing γ-aminobutyric acid as a medium material, since this makes it easier to obtain heterotrophic microalgae with a high content of γ-aminobutyric acid.

[0031] The present inventors focused on residues generated after fermentation in sake production, such as awamori distillation lees (hereinafter also referred to as "sake production residues"), as a composition containing γ-aminobutyric acid and / or glutamic acid to be used in preparing a medium. Awamori distillation lees are generated during the production process of awamori, and some of them are used as vinegar mash, livestock feed, or fertilizer for agricultural crops, but if they cannot be disposed of, they are treated as industrial waste. In order to reduce the amount of waste of sake production residues such as awamori distillation lees, new methods of utilizing sake production residues such as awamori distillation lees are currently being sought. The present inventors considered using sake production residues such as awamori distillation lees as a material for a medium. As a result, the present inventors found that components derived from the liquid of sake production residues such as awamori distillation lees contain γ-aminobutyric acid, and that components derived from the liquid of sake production residues such as awamori distillation lees are suitable for the above-mentioned culture process. The above-mentioned medium preparation step will be described below by taking as an example the case of preparing a medium containing components derived from the liquid in the residue of liquor production, such as awamori distillation lees.

[0032] In the present specification, the term "alcohol" is not particularly limited, but is preferably alcohol produced by fermenting raw materials using koji mold and yeast. Examples of the residues of alcohol production include slurry or solid lees generated in the production process of the alcohol. Examples of the residues of alcohol production include sake lees, awamori distillation lees, shochu distillation lees, etc., and one or more selected from these can be used. Among these, awamori distillation lees and / or shochu distillation lees, which are distillation lees, are preferred. The raw materials for alcohol are not particularly limited, and include raw materials (e.g., grains and potatoes) that are commonly used in the production of sake, awamori, shochu, etc., and more specific examples include grains such as rice, barley, wheat, buckwheat, soybeans, and corn; potatoes such as satsuma potatoes; sugarcane; chestnuts, etc., and one or more selected from these can be used.

[0033] "Awamori distillation lees" refers to the distillation residue generated in the production process of Awamori, and "shochu distillation lees" refers to the distillation residue generated in the production process of shochu. Furthermore, "sake lees" refers to the brewing residue generated in the production process of sake. There are no particular limitations on the state of the sake lees, but examples of the state of awamori distillation lees and shochu distillation lees include a slurry state, and examples of the state of sake lees include a solid state. Sake production residues such as awamori distillation lees include a liquid containing organic acids such as amino acids and citric acid, as well as minerals and vitamins, and a solid containing proteins, dietary fiber, and the like.

[0034] A culture medium containing components derived from liquids in alcohol production residues such as awamori distillation lees is sufficient as long as it contains at least components derived from liquids in alcohol production residues such as awamori distillation lees, and may or may not contain components derived from solids in alcohol production residues such as awamori distillation lees.

[0035] A medium containing components derived from the liquid in the residue of sake production such as awamori distillation lees can be prepared in the medium preparation step. In the medium preparation step, the residue of sake production such as awamori distillation lees may or may not be subjected to solid-liquid separation, for example.

[0036] First, as an example, the medium preparation step may be a step including solid-liquid separation of sake brewing residue such as awamori distillation lees. Specifically, the medium preparation step may be a step of preparing a medium containing a component derived from a liquid obtained by solid-liquid separation of sake brewing residue such as awamori distillation lees.

[0037] The solid-liquid separation may be carried out by known means such as centrifugation, sedimentation, filtration, squeezing, and using a commercially available solid-liquid separator.

[0038] The liquid obtained by solid-liquid separation may be used as it is. That is, in the medium preparation step, the liquid obtained by solid-liquid separation may be used to prepare a medium. For example, the liquid may be mixed with an additive described later to prepare a medium. Alternatively, the liquid obtained by solid-liquid separation may be subjected to at least one treatment to prepare a medium. For example, the liquid obtained by solid-liquid separation may be subjected to a drying treatment to prepare a dried product, and then the dried product may be mixed with an additive described later and water to prepare a medium.

[0039] The residue of liquor production, such as awamori distillation lees, which has been subjected to solid-liquid separation, may be used after being subjected to at least one treatment, for example, which may be selected from refrigeration, freezing, heating, concentration, sterilization, and drying.

[0040] Next, as another example, the medium preparation step may be a step that does not include solid-liquid separation of sake brewing residue such as awamori distillation lees. Specifically, the medium preparation step may be a step of using sake brewing residue such as awamori distillation lees without solid-liquid separation to prepare a medium containing a component derived from the liquid in the sake brewing residue such as awamori distillation lees.

[0041] The residue of sake production, such as awamori distillation lees, which is used without solid-liquid separation, may be, for example, residue of sake production, such as awamori distillation lees (distillation residue) produced in the distillation of aged mash, which has been subjected to at least one treatment other than solid-liquid separation. The at least one treatment may be selected from, for example, refrigeration, freezing, heating, concentration, sterilization, and drying. For example, the residue of sake production, such as awamori distillation lees, may be subjected to a drying treatment to produce a dried product, and then the dried product may be mixed with additives described below and water to prepare a medium.

[0042] The medium prepared in the medium preparation step may contain additives in addition to the components derived from the liquid in the residue of sake production, such as awamori distillation lees. That is, the medium preparation step may be a step of preparing a medium containing the components derived from the liquid in the residue of sake production, such as awamori distillation lees, and additives.

[0043] Examples of the additives include sugars, mineral components (including natural seawater and artificial seawater), organic acids, inorganic acids, organic bases, inorganic bases, vitamins, amino acids, peptides, and proteins. The types of additives contained in the medium may be appropriately selected by those skilled in the art depending on the type of heterotrophic microalgae to be cultured.

[0044] The sugars include, for example, at least one sugar selected from glucose, galactose, fructose, maltose, sucrose, lactose, oligosaccharides, and sugar alcohols (such as glycerol). The amount of sugar in the medium may be appropriately adjusted by those skilled in the art depending on the type of heterotrophic microalgae to be cultured.

[0045] Examples of the mineral components include sulfates such as potassium sulfate, magnesium sulfate, iron sulfate, ammonium sulfate, copper sulfate, nickel sulfate, and zinc sulfate; phosphates such as potassium phosphate; carbonates such as calcium carbonate; chlorides such as cobalt chloride, manganese chloride, sodium chloride, and calcium chloride; alkali metal oxides; molybdates such as sodium molybdate; selenites such as sodium selenite; halides such as potassium bromide and potassium iodide; natural sea salts; and artificial sea salts such as Red Sea Salt. The amount of mineral components in the medium may be appropriately adjusted by a person skilled in the art depending on the type of heterotrophic microalgae to be cultured.

[0046] In the medium preparation step, the pH of the medium containing components derived from the liquid in the residue of liquor production, such as awamori distillation lees, may be adjusted as necessary. For example, in the medium preparation step, the pH may be adjusted by adding an acid or a base before and / or after adding the additives. The pH may be appropriately adjusted by a person skilled in the art depending on the type of heterotrophic microalgae to be cultured.

[0047] In the medium preparation step, the medium containing components derived from the liquid in the residue of sake production, such as awamori distillation lees, is preferably sterilized. That is, the medium preparation step is preferably a step of preparing and sterilizing a medium containing components derived from the liquid in the residue of sake production, such as awamori distillation lees. The sterilization may be performed by known means, such as autoclave sterilization, filtration sterilization, boiling sterilization, and radiation sterilization.

[0048] For example, when solid parts of sake brewing residues such as awamori distillation lees remain in the medium, the solid parts in the medium may be partially or entirely solubilized by sterilizing the medium at high temperature. In this way, the sterilization treatment may also serve as a treatment for solubilizing the solid parts derived from sake brewing residues such as awamori distillation lees contained in the medium.

[0049] The medium containing the components derived from the liquid in the residue of sake production such as awamori distillation lees may be a liquid medium or a solid medium. When heterotrophic microalgae are mass-cultured, a liquid medium is more suitable than a solid medium.

[0050] Although the above has exemplified a medium containing a component derived from the liquid in the residue of alcohol production, such as awamori distillation lees, the medium used in the method for producing heterotrophic microalgae with a high content of γ-aminobutyric acid according to this embodiment is not limited to this, and any medium containing γ-aminobutyric acid may be used. Furthermore, although the medium preparation step has been mainly exemplified as a step of preparing a medium containing γ-aminobutyric acid using awamori distillation lees, the medium preparation step is not limited to this, and for example, the medium may be prepared using a composition containing γ-aminobutyric acid other than the residue of alcohol production, such as awamori distillation lees, or γ-aminobutyric acid.

[0051] 2-1-2.Culture conditions

[0052] The culture conditions in the culture step, such as the culture temperature, culture period, and culture method, may be any conditions that allow heterotrophic microalgae to be cultured. For example, the temperature is 5 to 40°C, preferably 10 to 35°C, more preferably 20 to 28°C, and even more preferably 25°C ± 1°C, and the culture is usually performed for 1 to 10 days, preferably 3 to 7 days, for example, 4 to 5 days, and may be performed by aeration agitation culture, shaking culture, or stationary culture. The culture may be started after adjusting the respective concentrations of γ-aminobutyric acid and / or glutamic acid in the medium, or the respective concentrations may be adjusted to a predetermined concentration during the culture step after the start of the culture. For example, in the culture step, the concentrations of these in the medium may be adjusted by feeding a composition containing γ-aminobutyric acid and / or glutamic acid into the medium after the start of the culture. Preferably, in the culture step, after the start of culture, the glutamic acid concentration in the medium is adjusted to a predetermined concentration while feeding a composition containing glutamic acid. The glutamic acid concentration in the medium at this time (converted to sodium glutamate) is not particularly limited, but is, for example, 0.001 to 50 g / L / hr.

[0053] When heterotrophic microalgae are cultured on a large scale, the culture step is preferably divided into a seed culture and a main culture, or into a seed culture, a preculture, and a main culture. Specifically, the culture step preferably includes a seed culture step of culturing heterotrophic microalgae in a medium for seed culture to obtain seed algae, and a main culture step of culturing the seed algae in a medium for main culture containing γ-aminobutyric acid (containing a component derived from a liquid in a residue of liquor production such as awamori distillation lees). Alternatively, the culture step preferably includes the above-mentioned seed culture step, a preculture step of culturing the seed algae in a medium for preculture containing γ-aminobutyric acid (containing a component derived from a liquid in a residue of liquor production such as awamori distillation lees), and a main culture step of adding a preculture medium to a medium for main culture containing γ-aminobutyric acid (containing a component derived from a liquid in a residue of liquor production such as awamori distillation lees) and culturing the preculture medium.

[0054] The medium for seed culture may be, for example, a medium known as a medium for culturing heterotrophic microalgae, or may be a medium containing a component derived from a liquid in a residue of liquor production, such as awamori distillation lees. For efficient mass culture, the medium for seed culture is preferably a medium known as a medium for culturing heterotrophic microalgae. When a medium containing γ-aminobutyric acid (e.g., a component derived from a liquid in a residue of liquor production, such as awamori distillation lees) is used as the medium for seed culture, the component composition of the medium for seed culture may be the same as or different from the medium for preculture or main culture. The component composition of the medium for preculture may be the same as or different from the medium for main culture.

[0055] Heterotrophic microalgae may be cultured in a culture device having a suitable cell culture means. The term "cell culture means" refers to a means having a function for culturing cells, such as a culture tank. The culture tank may have one or more devices selected from a stirring device, a vibration device, a temperature control device, a pH adjustment device, a turbidity measurement device, a light control device, a gas concentration measurement device such as O2 and CO2, and a pressure measurement device. The culture tank may be the same tank as the concentration / separation tank, or may be a tank separate from the concentration / separation tank. When the culture tank is a tank separate from the concentration / separation tank, the culture tank and the concentration / separation tank may be connected by a suitable means (e.g., a flow path).

[0056] 2-2. Recovery of heterotrophic microalgae

[0057] The method for producing heterotrophic microalgae with a high content of γ-aminobutyric acid according to this embodiment may further include a step of recovering the heterotrophic microalgae (hereinafter also referred to as a "recovery step"). Specifically, the recovery step is a step of recovering the heterotrophic microalgae cultured by the culture step described above.

[0058] In the recovery step, the heterotrophic microalgae may be recovered by separation from the medium, or may be recovered together with the medium. That is, the heterotrophic microalgae recovered in the recovery step may be heterotrophic microalgae separated from the medium, or may be a mixture of heterotrophic microalgae and the medium. When recovering heterotrophic microalgae separated from the medium, the recovery step may include a step of separating the cultured heterotrophic microalgae from the medium (hereinafter also referred to as a "separation step"). In the separation step, the medium and the heterotrophic microalgae may be separated by known means, such as centrifugation or filtration.

[0059] 2-3. Drying of heterotrophic microalgae

[0060] The method for producing heterotrophic microalgae with a high content of γ-aminobutyric acid according to this embodiment may further include a step of drying the heterotrophic microalgae (hereinafter also referred to as a "drying step"). In the drying step, the cultured heterotrophic microalgae are dried, and the heterotrophic microalgae become dried algae. The drying step may be performed, for example, after the recovery step.

[0061] The heterotrophic microalgae to be dried may be, for example, heterotrophic microalgae separated from a culture medium, or may be a mixture of heterotrophic microalgae and a culture medium. In order to perform drying efficiently, it is preferable that the heterotrophic microalgae to be dried are heterotrophic microalgae separated from a culture medium. That is, in a preferred embodiment, the recovery step includes the separation step, and the drying step is a step of drying the heterotrophic microalgae separated from the culture medium.

[0062] The drying may be performed by a known means, for example, at least one selected from heat drying, low-temperature drying, ventilation drying, reduced-pressure drying, freeze drying, spray drying, natural drying, and a commercially available drying device.

[0063] 2-4.Crushing heterotrophic microalgae

[0064] The method for producing heterotrophic microalgae with a high content of γ-aminobutyric acid according to this embodiment may include a step of crushing the heterotrophic microalgae (hereinafter also referred to as a "crushing step") in addition to the steps described above. The crushing step may be carried out, for example, after the recovery step and before the drying step, or after the drying step. The crushing step is preferably carried out after the drying step. Since the moisture content of the heterotrophic microalgae (dried algae bodies) after the drying step is reduced, they can be easily crushed, and can also be crushed to a small particle size. The crushing may be carried out by a known crushing means.

[0065] The production method according to this embodiment can obtain heterotrophic microalgae with a high content of γ-aminobutyric acid, and may be a production method for obtaining such microalgae. Incidentally, the present inventors previously proposed a technology for improving the odor and taste of heterotrophic microalgae themselves in Japanese Patent Application No. 2022-6913 (Patent Document 5: Japanese Patent Publication No. 7218023). As disclosed in Japanese Patent Application No. 2022-6913, the present inventors discovered that the odor and taste of heterotrophic microalgae can be improved by performing a step of culturing heterotrophic microalgae in a medium containing a component derived from the liquid of awamori distillery lees and a step of drying the cultured heterotrophic microalgae.

[0066] When it is desired to improve the smell and taste of the heterotrophic microalgae produced in this embodiment, for example, the application of the technology disclosed in Japanese Patent Application No. 2022-6913 may be considered. When applying this technology, the method for producing heterotrophic microalgae with a high content of γ-aminobutyric acid according to this embodiment may include, for example, a step of culturing heterotrophic microalgae in a medium containing a component derived from the liquid of the liquor production residue such as awamori distillation lees, and a step of drying the cultured heterotrophic microalgae. In addition, the production method may further include the above-mentioned crushing step. By performing the crushing step, the particle size of the obtained dried algae can be reduced. This makes it possible to improve the mouthfeel of an article containing the dried algae, or to improve the dispersibility when the dried algae is mixed with other raw materials. In addition, when producing the heterotrophic microalgae with a high content of γ-aminobutyric acid according to this embodiment, the production method described in Japanese Patent Application No. 2022-6913 (Patent Document 5: Japanese Patent Publication No. 7218023) may be applied.

[0067] 3. Foods, medicines, feed, etc. containing heterotrophic microalgae with a high content of γ-aminobutyric acid

[0068] The present invention can also provide a composition (e.g., food, medicine, feed, or skin topical agent) containing heterotrophic microalgae with a high content of γ-aminobutyric acid. Specifically, the heterotrophic microalgae with a high content of γ-aminobutyric acid are heterotrophic microalgae having a γ-aminobutyric acid content of 100 mg or more per 100 g of dry algae. Details of the heterotrophic microalgae are as described above in 1. and 2., and the same description also applies to this embodiment.

[0069] In this specification, the term "food" refers to an edible product that can be directly used for human consumption. Foods given to animals other than humans, such as feed (including bait), are not included in the "food" described in this specification.

[0070] Foods include, for example, general foods, foods for specific uses, and foods with health claims (foods for specific health uses, foods with nutrient functions, and foods with functional claims). General foods include so-called health foods, such as functional foods, nutritional supplements, health supplements, and supplements.

[0071] Examples of types of food include, but are not limited to, processed meat products, processed seafood products, processed dairy products, processed vegetables, processed fruit products, oily foods, luxury foods, seasonings, confectioneries, frozen foods, retort foods, canned foods, bottled foods, and instant foods.

[0072] Feed is food given to animals other than humans. Examples of feed include pet food, livestock feed, poultry feed, and fish feed. Examples of animals that can be fed with feed include, but are not limited to, livestock (cattle, pigs, chickens, horses, sheep, goats, etc.), fish, shellfish, and pets (dogs, cats, hamsters, rabbits, parakeets, tropical fish, reptiles, amphibians, insects, etc.).

[0073] The form of the composition containing heterotrophic microalgae with a high content of γ-aminobutyric acid (e.g., food, medicine, feed, etc.) may be, for example, liquid, solid, semi-solid, paste, granule, powder, capsule, etc., but is not limited thereto.

[0074] Compositions containing heterotrophic microalgae with a high content of γ-aminobutyric acid according to this embodiment (e.g., foods, medicines, feed, topical skin preparations, oral compositions, etc.) have a high content of γ-aminobutyric acid, and therefore it is expected that the use of such foods, medicines, or feeds, through ingestion, administration, application, or the like, will result in effects such as improved brain function, relaxation, stress reduction, and blood pressure reduction.

[0075] Therefore, the heterotrophic microalgae with a high content of γ-aminobutyric acid used in this embodiment can be contained in a composition that has a brain function improving effect, a relaxing effect, a stress reducing effect, and a blood pressure lowering effect, and can be used to prevent, improve, or treat symptoms or diseases that can be prevented, improved, or treated with γ-aminobutyric acid. The present embodiment can also provide the heterotrophic microalgae with a high content of γ-aminobutyric acid or use thereof for use in the production of or for the production of various compositions or various pharmaceutical agents, such as compositions for improving brain function, compositions for relaxation, compositions for reducing stress, and compositions for lowering blood pressure. Furthermore, this embodiment can provide a method for preventing, improving or treating symptoms or diseases that can be prevented, improved or treated by γ-aminobutyric acid, using the heterotrophic microalgae with a high γ-aminobutyric acid content or a composition containing the same. In addition, the present embodiment can also provide the heterotrophic microalgae with a high content of γ-aminobutyric acid or a composition containing the same for preventing, improving or treating symptoms or diseases that can be prevented, improved or treated by γ-aminobutyric acid. Furthermore, the composition according to this embodiment may include foods, supplements, and the like that are based on the concept of preventing, improving, or treating the above-mentioned symptoms or diseases, or symptoms or diseases related thereto, and are labeled as such as necessary, and compositions for ingestion are preferred.

[0076] In this embodiment, "prevention" refers to preventing or delaying the onset of symptoms or disease in the subject, or reducing the risk of onset of symptoms or disease in the subject. In this technology, "improvement" refers to improving or maintaining the disease, symptoms, or condition in the subject; preventing or delaying deterioration; or reversing, preventing, or delaying progression. In addition, "non-therapeutic purposes" is a concept that does not include medical procedures, i.e., treatment of the human body by treatment. Examples include health promotion, beauty procedures, etc.

[0077] 4. Method for producing the composition containing heterotrophic microalgae with high γ-aminobutyric acid (e.g., food, medicine, feed, or skin topical agent, etc.)

[0078] The present invention also provides a method for producing a composition (e.g., food, pharmaceutical, feed, skin topical agent, etc.) containing heterotrophic microalgae with a high content of γ-aminobutyric acid. Specifically, the production method according to one embodiment of the present invention includes using heterotrophic microalgae (heterotrophic microalgae with a high content of γ-aminobutyric acid) cultured in a medium containing γ-aminobutyric acid.

[0079] The heterotrophic microalgae cultured in a medium containing γ-aminobutyric acid are preferably heterotrophic microalgae produced by a method for producing heterotrophic microalgae with a high content of γ-aminobutyric acid according to one embodiment of the present invention. That is, a method for producing a composition according to this embodiment (e.g., food, medicine, feed, topical skin preparation, etc.) may preferably include the steps described in the method for producing heterotrophic microalgae with a high content of γ-aminobutyric acid according to one embodiment of the present invention. Details of the steps are as described in 2 above.

[0080] The method for producing a composition according to the present embodiment includes using a heterotrophic microalgae with a high content of γ-aminobutyric acid and one or more components acceptable as a composition. The method for producing a food according to the present embodiment includes using a heterotrophic microalgae with a high content of γ-aminobutyric acid and one or more components acceptable as a food. The method for producing a pharmaceutical according to the present embodiment includes using a heterotrophic microalgae with a high content of γ-aminobutyric acid and one or more components acceptable as a pharmaceutical. The method for producing a feed according to the present embodiment includes using a heterotrophic microalgae with a high content of γ-aminobutyric acid and one or more components acceptable as a feed.

[0081] Details of the heterotrophic microalgae-containing composition with high γ-aminobutyric acid content (e.g., food, medicine, feed, etc.) produced by the production method of this embodiment are as described in 3. above, and this description also applies to this embodiment.

[0082] 5. Method for increasing the content of gamma-aminobutyric acid in heterotrophic microalgae

[0083] As described above, the content of γ-aminobutyric acid in heterotrophic microalgae can be increased by culturing the heterotrophic microalgae in a medium containing γ-aminobutyric acid. This is because the above-mentioned culturing allows the heterotrophic microalgae to take up and accumulate γ-aminobutyric acid in the medium. Therefore, the present invention also provides a method for increasing the content of γ-aminobutyric acid in heterotrophic microalgae (hereinafter also referred to as a "method for increasing γ-aminobutyric acid content").

[0084] A method for increasing the content of γ-aminobutyric acid according to one embodiment of the present invention includes a step of culturing heterotrophic microalgae in a medium containing γ-aminobutyric acid. This step is the same as the culturing step described in 2. above. Furthermore, the method for increasing the content of γ-aminobutyric acid according to this embodiment may include at least one step selected from a medium preparation step, a recovery step, and a drying step, in addition to the culturing step. Details of these steps are as described in 2. above, and this description also applies to this embodiment.

[0085] As used herein, "increasing the content of γ-aminobutyric acid" refers to increasing the content of γ-aminobutyric acid in heterotrophic microalgae by the above-mentioned culture step. The increase may be, for example, increasing the content of γ-aminobutyric acid in the heterotrophic microalgae before the above-mentioned culture step by 1.1 times or more, 1.3 times or more, 1.5 times or more, 1.8 times or more, 2.0 times or more, 2.3 times or more, 2.5 times or more, 2.8 times or more, 3.0 times or more, 3.5 times or more, 4.0 times or more, 4.5 times or more, 5.0 times or more, 5.5 times or more, or 6.0 times or more.

[0086] 6. Method for recovering γ-aminobutyric acid from culture medium

[0087] As described above, by culturing heterotrophic microalgae in a medium containing γ-aminobutyric acid, the γ-aminobutyric acid in the medium can be taken up and accumulated by the heterotrophic microalgae. By recovering the heterotrophic microalgae that have taken up the γ-aminobutyric acid in the medium, the γ-aminobutyric acid can be recovered from the medium. Thus, the present invention also provides a method for recovering γ-aminobutyric acid from a medium.

[0088] A method for recovering γ-aminobutyric acid from a culture medium according to one embodiment of the present invention includes a step of culturing heterotrophic microalgae in a culture medium containing γ-aminobutyric acid, and a step of recovering the heterotrophic microalgae. These steps are the same as the culturing step and the recovering step described in 2. above, respectively. Furthermore, the method for recovering γ-aminobutyric acid from a culture medium according to this embodiment may include a culture medium preparation step and / or a drying step in addition to the culturing step and the recovering step. Details of these steps are as described in 2. above, and the description also applies to this embodiment.

[0089] 7. Method for recovering gamma-aminobutyric acid from a composition

[0090] As described above, by culturing heterotrophic microalgae in a medium prepared using a composition containing γ-aminobutyric acid, the γ-aminobutyric acid in the medium can be taken up and accumulated by the heterotrophic microalgae. By recovering the heterotrophic microalgae that have taken up the γ-aminobutyric acid in the medium, the γ-aminobutyric acid contained in the composition can be recovered from the medium. Thus, the present invention also provides a method for recovering γ-aminobutyric acid from a composition.

[0091] A method for recovering γ-aminobutyric acid from a composition according to one embodiment of the present invention includes a step of preparing a medium using a composition containing γ-aminobutyric acid, a step of culturing heterotrophic microalgae in the medium, and a step of recovering the heterotrophic microalgae. These steps are the same as the medium preparation step, the culture step, and the recovery step described in 2. above, respectively. Furthermore, the method for recovering γ-aminobutyric acid from a composition according to this embodiment may include a drying step in addition to the medium preparation step, the culture step, and the recovery step. Details of the drying step are as described in 2. above, and the same description also applies to this embodiment.

[0092] The composition used in the medium preparation step of this embodiment may be, for example, awamori distillation residue, but is not particularly limited as long as it does not impair the effects of the present invention.

[0093] 8. The present invention may adopt the following configurations or technical features as appropriate. [1] Heterotrophic microalgae having a gamma-aminobutyric acid content of 100 mg or more per 100 g of dry algae. [2] The heterotrophic microalgae according to [1], wherein the heterotrophic microalgae is a microalgae belonging to the Thraustochytriales. [3] The heterotrophic microalgae according to [1] or [2], wherein the heterotrophic microalgae is a microalgae belonging to the genus Aurantiochytrium. [4] A composition comprising the heterotrophic microalgae according to any one of [1] to [3] above, which is preferably a food product, a medicine, or a feed.

[0094] [5] A method for producing heterotrophic microalgae or a method for producing a heterotrophic microalgae-containing composition, comprising a step of culturing heterotrophic microalgae in a medium containing γ-aminobutyric acid. The heterotrophic microalgae is preferably the heterotrophic microalgae described in [2] or [3] above. The produced heterotrophic microalgae is preferably a heterotrophic microalgae with a high content of γ-aminobutyric acid. The medium is a γ-aminobutyric acid-containing medium prepared and sterilized from a medium containing a component derived from a liquid in sake production residue. [6] The method according to [5], further comprising a step of recovering the heterotrophic microalgae after the culturing step. [7] The production method according to [5] or [6], comprising a step of preparing a medium using a composition containing γ-aminobutyric acid during or before the culture step. [8] preparing a medium using a composition containing γ-aminobutyric acid; Cultivating heterotrophic microalgae in the medium; A method for producing heterotrophic microalgae or a method for producing a heterotrophic microalgae-containing composition, comprising: [9] The production method according to any one of [5] to [8] above, for increasing the content of γ-aminobutyric acid in heterotrophic microalgae, for recovering γ-aminobutyric acid from a culture medium, or for producing a food, a medicine, or a feed.

[10] Any one of the production methods described in [1] to [9] above, wherein the medium is (i) a medium obtained by preparing and sterilizing a γ-aminobutyric acid-containing composition, or (ii) a medium obtained by preparing and sterilizing a medium containing a component derived from the sake brewing residue or a liquid in the sake brewing residue. The method may include a step of preparing and sterilizing a medium containing a component derived from the sake brewing residue or a liquid in the sake brewing residue during or before the culture step.

[0095]

[11] A method for producing a composition containing heterotrophic microalgae with a high γ-aminobutyric acid content, comprising using heterotrophic microalgae with a high γ-aminobutyric acid content obtained by the production method according to any one of [5] to

[10] above.

[12] A heterotrophic microalga with a high content of γ-aminobutyric acid, obtained by the production method according to any one of [5] to

[10] above, or use thereof.

[0096]

[13] A method for increasing the content of γ-aminobutyric acid in heterotrophic microalgae, comprising a step of culturing the heterotrophic microalgae in a medium containing γ-aminobutyric acid.

[14] A method for recovering γ-aminobutyric acid from a medium, comprising: culturing heterotrophic microalgae in a medium containing γ-aminobutyric acid; and recovering the heterotrophic microalgae.

[15] A method for recovering γ-aminobutyric acid from a composition, comprising the steps of: preparing a medium using a composition containing γ-aminobutyric acid; culturing heterotrophic microalgae in the medium; and recovering the heterotrophic microalgae.

[16] A composition (e.g., food, pharmaceutical, or feed) or a method for producing the composition, comprising using heterotrophic microalgae cultured in a medium containing γ-aminobutyric acid, or using a heterotrophic microalgae with a high content of γ-aminobutyric acid obtained by the production method according to any one of [5] to

[10] above. The cultured heterotrophic microalgae is preferably a heterotrophic microalgae with a high content of γ-aminobutyric acid.

[17] Any one of the methods described in

[13] to

[16] above, wherein the medium is a medium obtained by preparing and sterilizing a medium containing a component derived from the sake brewing residue or a liquid in the sake brewing residue. The method may include a step of preparing and sterilizing a medium containing a component derived from the sake brewing residue or a liquid in the sake brewing residue during or before the culture step.

[18] Any one of the methods described in

[13] to

[17] above, wherein the heterotrophic microalgae is the heterotrophic microalgae described in [2] or [3] above.

[0097]

[19] The heterotrophic microalgae according to any one of [1] to [4] above, or any one of the methods according to [5] to

[18] above, wherein the heterotrophic microalgae is used for one or more purposes selected from inhibitory neurotransmission, improvement of brain function, relaxation, stress reduction, lowering of blood pressure, and improvement of sleep quality.

[20] The composition according to [4] above or any one of the methods according to [5] to

[18] above, wherein the composition is used for one or more purposes selected from inhibitory neurotransmission, improvement of brain function, relaxation, stress relief, lowering blood pressure, reducing anxiety, and improving sleep quality.

[21] The composition according to

[19] or the method according to

[20] , wherein the improvement of brain function is prevention or improvement of one or more diseases or symptoms selected from hyperphagia, depression, cognitive dysfunction, etc. The cognitive dysfunction may be one or more selected from mild cognitive impairment, Alzheimer's disease, cognitive dysfunction due to aging (e.g., decline in memory, spatial cognition, etc.), etc. EXAMPLES

[0098] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0099] <Test Example 1>

[0100] (1) Preparation of dried algae of Example 1 Heterotrophic microalgae were cultured according to the following procedure to obtain dried algae.

[0101] a) Preparation of Awamori distillation lees supernatant medium Awamori distillation lees (distillation residue) obtained from an Awamori manufacturing plant were centrifuged at 6000×g for 10 minutes. The resulting liquid (supernatant) was adjusted to pH 6 to obtain an Awamori distillation lees supernatant medium.

[0102] b) Preparation of GTY medium 20 g of glucose, 5 g of yeast extract, 10 g of tryptone, and 16.7 g of red sea salt were added to 1 L of distilled water to obtain a GTY medium.

[0103] c) Seed culture Aurantiochytrium limacinum SR-21 strain (FERM BP-5034) provided by NITE (National Institute of Technology and Evaluation) was cultured with shaking in GTY medium for 3 days to obtain seed algae.

[0104] d) Main culture A 90L jar fermenter was charged with 30L of Awamori distillation lees supernatant medium, 30L of tap water, 1.2kg of glucose, and 1kg of red sea salt, and sterilized with steam at 121°C for 20 minutes. This resulted in a medium containing components derived from the liquid in the Awamori distillation lees (hereinafter also referred to as "Awamori distillation lees-containing medium"). The Awamori distillation lees-containing medium was seeded with GTY medium containing seed algae, and cultured under aerobic conditions for 24 hours with stirring.

[0105] e) Recovery and drying The medium after the main culture was centrifuged at 6000×g for 10 minutes to separate the cultured Aurantiochytrium limacinum (algae). The separated solid matter (algae) was collected and dried at 150° C. for 1 minute using a drum dryer to obtain the dried algae of Example 1.

[0106] (2) Preparation of dried algae bodies of Comparative Example 1 Aurantiochytrium limacinum was cultured under the same conditions as in (1) c) above to obtain algal bodies of Comparative Example 1. Thereafter, the procedure in (1) e) above was followed to obtain dried algal bodies of Comparative Example 1.

[0107] (3) Measurement of γ-aminobutyric acid content The content of γ-aminobutyric acid was measured for each of the medium with 0 hours of culture (main culture) in Example 1, the dried algae cells in Example 1, the medium with 0 hours of culture in Comparative Example 1, and the dried algae cells in Comparative Example 1. The measurement was performed using an automatic amino acid analyzer by the post-column derivatization method using a ninhydrin test solution.

[0108] The measurement results are shown in the following Table 1. Since the test of Example 1 was carried out twice, in Table 1, the culture medium and dried algae obtained in each test are indicated as "Example 1-1" and "Example 1-2".

[0109] [Table 1]

[0110] From the results in Table 1 above, it can be seen that the use of a medium containing awamori distillation lees can increase the γ-amino acid content in the algae compared to the use of GTY medium. This confirmed that the present invention can increase the γ-aminobutyric acid content in the algae. It was also confirmed that the present invention can produce algae with a high γ-aminobutyric acid content.

[0111] <Test Example 2>

[0112] (1) Preparation of dried algae of Example 2 Heterotrophic microalgae were cultured according to the following procedure to obtain dried algae.

[0113] The medium was prepared and seed culture was carried out according to the procedures of (1) a) to c) in Test Example 1. Then, the following d) preculture and e) main culture were carried out.

[0114] d) Preculture A 90L jar fermenter was charged with 15L of Awamori distillation lees supernatant medium, 45L of tap water, 1.2kg of glucose, and 1kg of red sea salt, and sterilized with steam at 121℃ for 20 minutes. This resulted in an Awamori distillation lees-containing medium. The Awamori distillation lees-containing medium was seeded with GTY medium containing seed algae, and cultured under aerobic conditions for 24 hours with stirring.

[0115] e) Main culture A 600-L jar fermenter was charged with 150 L of Awamori distillation lees supernatant medium, 150 L of tap water, 12 kg of glucose, and 5 kg of red sea salt, and sterilized with steam at 121°C for 20 minutes. This resulted in an Awamori distillation lees-containing medium. 60 L of preculture medium was transferred to the Awamori distillation lees-containing medium, and agitation culture was carried out under aerobic conditions for 34 hours.

[0116] After the main culture, the dried algae of Example 2 was obtained according to the procedure of (1) e) of Test Example 1 above.

[0117] (2) Measurement of γ-aminobutyric acid content Using the method described in (3) of Test Example 1 above, the content of γ-aminobutyric acid was measured for the medium at the start of main culture, the medium at the end of main culture, and the dried algae of Example 2.

[0118] The measurement results are shown in Table 2 below.

[0119] [Table 2]

[0120] From the results in Table 2 above, it can be seen that the content of γ-aminobutyric acid per 1 L of medium was reduced by 79 mg (106-27=79 [mg / L]) after the main culture process. Assuming that all of the reduced γ-aminobutyric acid was taken up into the algae, the amount of dry algae obtained was 24 g per 1 L of medium, so the calculated content of γ-aminobutyric acid per 100 g of dry algae would be approximately 329 mg (79 / 24×100≒329 [mg / 100 g (dry algae)]). However, according to the actual measurement results, the content of γ-aminobutyric acid per 100 g of dry algae was 278 mg, so it is considered that approximately 84.5% of the reduced amount of γ-aminobutyric acid in the medium was taken up and accumulated in the dry algae (278 / 329×100≒84.5 [%]).

[0121] This confirmed that when heterotrophic microalgae are cultured in a medium containing γ-aminobutyric acid, the γ-aminobutyric acid in the medium is taken up by the heterotrophic microalgae, thereby increasing the content of γ-aminobutyric acid in the algae.

[0122] <Test Example 3>

[0123] (1) Preparation of dried algae of Example 3 Heterotrophic microalgae were cultured according to the following procedure to obtain dried algae.

[0124] a) Preparation of shochu distillery lees supernatant medium A shochu distillation lees supernatant medium (sweet potato shochu distillation lees supernatant medium) was obtained in accordance with “(1)a) Preparation of Awamori distillation lees supernatant medium” in Test Example 1 above, except that “Awamori distillation lees (residue)” was replaced with “sweet potato shochu distillation lees (residue)” obtained from a shochu manufacturing plant.

[0125] b) Preparation of GTY medium and c) Seed culture "b) Preparation of GTY medium" and "c) Seed culture" in Test Example 3 were carried out according to "(1)b)" and "(1)c)" in the above Test Example 1. The algae strain used was Aurantiochytrium limacinum SR-21 strain (FERM BP-5034). A 500 mL Erlenmeyer flask containing 200 mL of culture medium and a stopper was sterilized in an autoclave at 121°C for 20 minutes. This resulted in a medium containing components derived from the liquid in the shochu distillation lees (hereinafter also referred to as "shochu distillation lees-containing medium"). A breathable silicone rubber open-cell sponge plug was used as the stopper. When the culture medium used for flask culture is a 25% shochu distillation lees-containing medium, the medium contains 50 mL of shochu distillation lees supernatant medium, 150 mL of water, 4 g of glucose, and 3.34 g of red sea salt. The "%" of the 25% shochu distillation lees-containing medium can be calculated by "volume (mL) of shochu distillation lees supernatant medium / [total (mL) of volume (mL) of shochu distillation lees supernatant medium and volume (mL) of water]" x 100 (%). This total volume is also the volume (mL) of the medium. In addition, the ratio of the volume (mL) of the shochu distillation lees supernatant medium and the volume (mL) of water were adjusted to prepare 50%, 75%, and 100% shochu distillation lees-containing medium. The glucose and red sea salt concentrations in the shochu distillation lees-containing medium were adjusted to be the same as in the "25%" medium, "4 g glucose, 3.34 g red sea salt / volume 200 mL", even when the concentration of the shochu distillation lees supernatant medium was changed. Note that the 100% shochu distillation lees-containing medium has a volume (mL) of water of 0 mL and a volume (mL) of shochu distillation lees supernatant medium of 200 mL per the total amount of 200 mL.

[0126] d) Flask culture The GTY medium containing the seed algae was inoculated onto the medium containing shochu distillery lees and cultured for 36 hours under aerobic conditions at 25°C with shaking at 110 strokes / min.

[0127] A test example using a 25% shochu lees culture medium is designated as Example 3-1, and test examples in which the shochu lees supernatant medium content in the shochu lees culture medium was changed to 50%, 75%, and 100% are designated as Example 3-2, Example 3-3, and Example 3-4, respectively.

[0128] e) Recovery and drying In Example 3, the recovery and drying were carried out by following the procedure of "(1)e)" in Test Example 1 above, to obtain dried algal bodies of Examples 3-1, 3-2, 3-3, and 3-4 after flask cultivation. The content of γ-aminobutyric acid was measured according to "(3) Measurement of the content of γ-aminobutyric acid" in Test Example 1 above. The results of each measurement in Example 3 are shown in Table 3 below. DCW is an abbreviation for dry algal weight. The actual / theoretical values ​​of GABA in the algal body are values ​​calculated from [actual GABA value in the algal body at the end of the culture [mg / 100g] / theoretical GABA value in the algal body [mg / 100g] assuming that all the GABA lost in the medium is recovered in the algal body]. The "medium" in "medium" in Table 3 refers to the "medium containing sweet potato shochu distillery lees".

[0129] [Table 3]

[0130] From the results in Table 3 above, it was confirmed that GABA in the medium could be incorporated into the algae bodies of heterotrophic microalgae not only when awamori distillation lees was used, but also when shochu distillation lees was used. This confirmed that heterotrophic microalgae with a high content of γ-aminobutyric acid could be obtained not only from sake production residues (sake production lees, etc.) such as awamori distillation lees, shochu distillation lees, and sake lees, but also from compositions containing γ-aminobutyric acid or γ-aminobutyric acid-containing media. When a culture medium with a shochu distillery lees concentration of 50% or more was used, it was confirmed that more γ-aminobutyric acid than the total amount of γ-aminobutyric acid in the medium accumulated within the algae cells of heterotrophic microalgae, and the culture medium at the start of cultivation contained L-glutamic acid derived from the shochu distillery lees supernatant medium. This confirmed that γ-aminobutyric acid is produced from L-glutamic acid within the algae cells, and that the produced γ-aminobutyric acid accumulates within the algae cells. When a culture medium with a 50% shochu distillery lees concentration was used, the DCW obtained was high, and it is believed that the L-glutamic acid in the culture medium was mainly used for growth. Therefore, when a culture medium with a 50% shochu distillery lees concentration was used, it is believed that the amount of γ-aminobutyric acid derived from L-glutamic acid produced in the algae was low. On the other hand, when a culture medium with 75% shochu distillery lees was used, the DCW was low, and it is believed that the amount of γ-aminobutyric acid derived from L-glutamic acid produced in the algae was high. It was also confirmed that γ-aminobutyric acid can be contained in the algae of heterotrophic microalgae at about 1% by mass. This suggests that the upper limit of the amount of accumulation in the algae can be appropriately adjusted by adjusting the culture conditions, such as the concentration of γ-aminobutyric acid and / or glutamic acid in the medium. For example, it may be possible to contain γ-aminobutyric acid in the algae of heterotrophic microalgae at at least about 2, 3, or 5% by mass.

[0131] <Test Example 4>

[0132] (1) Preparation of dried algae of Example 4 Heterotrophic microalgae were cultured according to the following procedure to obtain dried algae.

[0133] According to the procedures of (1) a) to c) in the above Test Example 1, the awamori distillation lees supernatant medium, the GTY medium, and the seed culture were prepared. Then, the 90 L jar culture of "(1) d) Main culture" in the above Test Example 2 was carried out under aerobic conditions. The algae strain used was Aurantiochytrium limacinum SR-21 strain (FERM BP-5034). The Awamori distillation lees-containing medium used in the 90 L jar culture was prepared so that the concentration (content) of the Awamori distillation lees supernatant medium in the Awamori distillation lees culture medium was 50%, and after sterilization, a 50% Awamori distillation lees-containing medium was obtained. After the start of the culture, a sodium glutamate aqueous solution (filter sterilized) was fed to the medium during the culture under the conditions shown in Table 4. A sodium glutamate aqueous solution (sodium glutamate concentration 200 g / L) was fed so that the sodium glutamate concentration in the medium was 0.6 g / L / hr. The "medium" in "medium" in Table 4 refers to the "Awamori distillation lees-containing medium."

[0134] [Table 4]

[0135] Although the amount of glutamic acid added was large, the DCW was low, and it was confirmed that L-glutamic acid was not being used effectively for growth. On the other hand, it was confirmed that more GABA than the total amount of GABA in the culture medium had accumulated in the algae. The L-glutamic acid in the culture medium is taken up into the algae of the Thraustochytrids, particularly Aurantiochytrium microalgae, and used for growth. If more L-glutamic acid than is used for growth is taken up into the algae, it is thought that it is converted to γ-aminobutyric acid and accumulated within the algae. This confirmed that heterotrophic microalgae can produce γ-aminobutyric acid from L-glutamic acid in their bodies and accumulate the produced γ-aminobutyric acid in their bodies. Thus, it was confirmed that heterotrophic microalgae with high γ-aminobutyric acid content can be obtained by supplying L-glutamic acid in addition to γ-aminobutyric acid to the culture medium.

[0136] <Test Example 5>

[0137] Two strains of Aurantiochytrium (Aurantiochytrium sp. M15d1 strain and Aurantiochytrium sp. S28P strain) were seeded in an Awamori distillation lees-containing medium (medium containing 50% Awamori distillation lees) containing 50% Awamori distillation lees supernatant medium according to <Test Example 3>, and flask culture was performed. These two strains were collected and stored in Japan by the present inventors. The Awamori distillation lees supernatant medium is a γ-aminobutyric acid-containing composition (for medium) obtained according to the preparation of the Awamori distillation lees supernatant medium in <Test Example 1>. The 50% awamori distillation lees-containing medium used in Test Example 5 can be obtained by adjusting the concentration of the awamori distillation lees supernatant medium according to Test Example 3. The "medium" in Table 5 refers to a medium containing 50% awamori distillation lees. The content of γ-aminobutyric acid was measured in the same manner as in <Test Example 1>.

[0138] [Table 5]

[0139] These test results showed that both Aurantiochytrium strains other than SR21 recovered GABA from the culture medium during cultivation and accumulated it within the algae. It was shown that microalgae belonging to the heterotrophic genus Aurantiochytrium can have a high content of γ-aminobutyric acid regardless of the algae strain, and that γ-aminobutyric acid can be recovered from the medium. It was also shown that using a medium containing γ-aminobutyric acid when culturing heterotrophic microalgae belonging to the genus Aurantiochytrium is beneficial for increasing the content of γ-aminobutyric acid in the microalgae.

Claims

1. A step of culturing heterotrophic Aurantiochytrium microalgae at 5 to 40°C in a culture medium containing waste liquid or waste food containing γ-aminobutyric acid generated in the food manufacturing process, A method for recovering γ-aminobutyric acid from the culture medium using the microalgae.

2. A method for recovering γ-aminobutyric acid from a culture medium using microalgae according to Claim 1, further comprising the step of recovering the microalgae in which γ-aminobutyric acid has accumulated in the algal cells during the cultivation step.

3. A method for recovering γ-aminobutyric acid using microalgae from the culture medium according to claim 1 or 2, wherein γ-aminobutyric acid is recovered by making the microalgae contain γ-aminobutyric acid from the culture medium in the culture step.

4. A method for recovering γ-aminobutyric acid from a culture medium according to claim 1 or 2, wherein the waste liquid or waste food is a fermentation residue or fermentation liquid containing γ-aminobutyric acid generated after fermentation in alcohol production, using the microalgae.

5. A method for recovering γ-aminobutyric acid from the culture medium according to claim 1 or 2 using microalgae, wherein the waste liquid or waste food is sake lees residue.

6. A method for recovering γ-aminobutyric acid from the culture medium according to claim 5, wherein the sake lees residue is one or more selected from sake lees, awamori distillation lees, and shochu distillation lees, using the microalgae.

7. A method for recovering γ-aminobutyric acid using microalgae from the culture medium according to claim 1 or 2, wherein the culture medium in the culturing step is a culture medium containing 8 mg / L or more of γ-aminobutyric acid.

8. A method for recovering γ-aminobutyric acid using microalgae from the culture medium according to claim 1 or 2, wherein the culture period in the culturing step is 1 to 10 days.

9. A method for recovering γ-aminobutyric acid from the culture medium according to claim 1 or 2, wherein the microalgae is Aurantiochytrium limacinum.

10. The method for recovering γ-aminobutyric acid from a culture medium according to Claim 2, wherein the microalgae recovered in the recovery step have a γ-aminobutyric acid content of 100 mg or more per 100 g of dried algae.

11. A step of preparing a culture medium using a composition containing waste liquid or discarded food containing γ-aminobutyric acid generated in the food manufacturing process, A step of culturing heterotrophic microalgae belonging to the genus Aurantiochytrium in the culture medium at 5 to 40°C, The process includes a step of recovering the microalgae that have accumulated γ-aminobutyric acid in their bodies during the cultivation process described above. A method for recovering γ-aminobutyric acid from a composition containing γ-aminobutyric acid using the microalgae.

12. A step comprising culturing heterotrophic Aurantiochytrium microalgae at 5 to 40°C in a culture medium containing waste liquid or waste food containing γ-aminobutyric acid generated in the food manufacturing process, A method for increasing the γ-aminobutyric acid content in the aforementioned microalgae to 100 mg or more per 100 g of dried algal tissue.

13. A method for producing a microalgae, which aims to produce a heterotrophic Aurantiochytrium microalgae with a high content of 100 mg or more of γ-aminobutyric acid per 100 g of dried algae, A step of culturing the microalgae at 5 to 40°C in a culture medium containing wastewater or discarded food containing γ-aminobutyric acid generated during the food manufacturing process, The process includes a step of recovering the microalgae having a γ-aminobutyric acid content of 100 mg or more per 100 g of dried algae, A method for producing the aforementioned microalgae.

14. The composition comprises including the microalgae or pulverized thereof recovered according to claim 2 or claim 13 in order to contain γ-aminobutyric acid, wherein the composition is a food, pharmaceutical, topical skin preparation or animal feed. A method for producing a composition containing γ-aminobutyric acid.

15. The microalgae or pulverized material recovered according to Claim 2 or Claim 13 is included in the food, pharmaceutical, topical skin preparation or feed in order to increase the γ-aminobutyric acid content in the food, pharmaceutical, topical skin preparation or feed. A method for producing food, pharmaceuticals, topical skin preparations, or animal feed containing γ-aminobutyric acid.

16. A method for using the microalgae or pulverized material recovered according to Claim 2 or Claim 13 in food, pharmaceuticals, topical skin preparations or animal feed in order to contain γ-aminobutyric acid.

17. A method for using heterotrophic Aurantiochytrium microalgae or a pulverized product thereof, which have a γ-aminobutyric acid content of 100 mg or more per 100 g of dried algae, in food, pharmaceuticals, topical skin preparations, or feed in order to increase the γ-aminobutyric acid content in food, pharmaceuticals, topical skin preparations, or feed.