Classifications

• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K10/00—Animal feeding-stuffs
  • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
  • A61K31/19—Carboxylic acids, e.g. valproic acid
  • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
  • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/68—Protozoa, e.g. flagella, amoebas, sporozoans, plasmodium or toxoplasma
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/20—Hypnotics; Sedatives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/12—Antihypertensives
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/12—Unicellular algae; Culture media therefor

Definitions

• the present invention relates to heterotrophic microalgae, uses thereof, and methods for increasing ⁇ -aminobutyric acid in heterotrophic microalgae.
• GABA Gamma-aminobutyric acid
• ⁇ -Aminobutyric acid is widely distributed in nature and is biosynthesized from glutamic acid in vivo. In the mammalian brain, ⁇ -aminobutyric acid acts as an inhibitory neurotransmitter, improving brain function, relaxing, stress-reducing, and lowering blood pressure, and also has the ability to improve sleep quality. It has been reported that
• Patent Document 1 listed below discloses microalgae belonging to the family Pavlovaceae (strain OPMS30543 and strain OPMS30543X) containing ⁇ -aminobutyric acid.
• Patent Document 2 listed below discloses an algae belonging to the class Ideyucogonaceae that contains ⁇ -aminobutyric acid.
• Patent Document 3 listed below discloses Idyukogome cells containing ⁇ -aminobutyric acid.
• heterotrophic microalgae Some microalgae exhibit heterotrophic properties (heterotrophic microalgae).
• heterotrophic microalgae refers to microalgae that can be cultured and proliferated without light irradiation by providing an organic carbon source, rather than through photosynthetic culture, which requires light irradiation. It is an algae with special properties.
• the main purpose of the present invention is to provide heterotrophic microalgae with a high content of ⁇ -aminobutyric acid. Further, the main object of the present invention is to provide a method for increasing the content of ⁇ -aminobutyric acid in heterotrophic microalgae.
• heterotrophic microalgae take up ⁇ -aminobutyric acid contained in the culture medium and accumulate it within the algal bodies.
• the present inventors established a technique for increasing ⁇ -aminobutyric acid in heterotrophic microalgae and a technique for obtaining heterotrophic microalgae containing a large amount of ⁇ -aminobutyric acid.
• the present inventors also discovered a method for recovering ⁇ -aminobutyric acid contained in a culture medium using heterotrophic microalgae.
• the present invention has been completed as a result of these findings.
• the present invention provides heterotrophic microalgae having a content of ⁇ -aminobutyric acid of 100 mg or more per 100 g of dry algal bodies.
• the heterotrophic microalgae may be microalgae belonging to Thraustochytriales.
• the heterotrophic microalgae may be microalgae belonging to the genus Aurantiochytrium.
• the present invention also provides foods, medicines, or feeds containing the heterotrophic microalgae.
• the present invention also provides a method for increasing the content of ⁇ -aminobutyric acid in heterotrophic microalgae, which comprises culturing the heterotrophic microalgae in a medium containing ⁇ -aminobutyric acid.
• the present invention also provides a method for recovering ⁇ -aminobutyric acid from a medium, which includes the steps of culturing heterotrophic microalgae in a medium containing ⁇ -aminobutyric acid, and collecting the heterotrophic microalgae.
• the present invention comprises the steps of preparing a medium using a composition containing ⁇ -aminobutyric acid, culturing heterotrophic microalgae in the medium, and collecting the heterotrophic microalgae.
• the present invention also provides a method for producing food, medicine, or feed comprising using heterotrophic microalgae cultured in a medium containing ⁇ -aminobutyric acid.
• the present invention provides heterotrophic microalgae with a high content of ⁇ -aminobutyric acid.
• the present invention also provides a method for increasing the content of ⁇ -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 within this specification.
• the content of ⁇ -aminobutyric acid is 100 mg or more per 100 g of dry algae.
• the content of the ⁇ -aminobutyric acid per 100 g of dry algae is, 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 mm or more, 300 mg or more, 350 mg or more, It may be 400 mg or more, 450 mg or more, 500 mg or more, or 550 mg or more.
• the heterotrophic microalgae according to the present embodiment can be obtained by culturing the heterotrophic microalgae in a medium containing ⁇ -aminobutyric acid, and can be obtained by culturing the heterotrophic microalgae in a medium not containing at least ⁇ -aminobutyric acid.
• the content of ⁇ -aminobutyric acid in the algae is higher than that of heterotrophic microalgae. That is, the heterotrophic microalgae according to the present embodiment is a heterotrophic microalgae containing a high content of ⁇ -aminobutyric acid.
• the medium that does not contain preferably is a medium that does not contain ⁇ -aminobutyric acid and/or L-glutamic acid, and preferably contains ⁇ -aminobutyric acid and/or L-glutamic acid of 1 mg/L or less. desirable.
• the content of ⁇ -aminobutyric acid in the algae of the heterotrophic microalgae according to the present embodiment is, for example, 1.1 times or more, 1.3 times or more the content of ⁇ -aminobutyric acid in the algae of the control. 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.
• heterotrophic microalgae examples include microalgae belonging to Thraustochytriales or Labyrinthulales, and one or more types from these groups can be used.
• microalgae belonging to the above-mentioned thraustochytrium examples include Aurantiochytrium, Schizochytrium, Thraustochytrium, Parietichytrium, or Ulkenia. Microalgae belonging to this group can be mentioned, and one type or two or more types from these groups can be used.
• microalgae belonging to the genus Labyrinthula examples include microalgae belonging to the genus Labyrinthula.
• microalgae belonging to the genus Aurantiochytrium examples include microalgae belonging to Aurantiochytrium limacinum or Aurantiochytrium mangrovei.
• strains of Aurantiochytrium rimassinum include SR-21 strain, 4W-1b strain, NIES3737 strain, ATCC MYA-1381 strain, mh0186 strain, and F29-b strain.
• strains of Aurantiochytrium mangrove bay include 18W-13a strain, RCC893 strain, MP2 strain, BL10 strain, and FB3 strain, and one or more types from these groups can be used.
• the SR-21 strain was submitted to the National Institute of Biotechnology and Biotechnology, Agency of Industrial Science and Technology (currently the National Institute of Industrial Science and Technology, National Institute of Technology and Product Evaluation, Biotechnology Center, Patent Organism Depositary (NITE-IPOD)) under the name "Marine Bacteria SR21 Strain”. It was deposited on March 6, 1994, with the accession number FERM BP-5034, and it was deposited with the Fermentation Research Institute, Inc. on March 17, 1995, with the accession number IFO. 32693 (paragraph [0035] of Patent Document 4 (Japanese Unexamined Patent Publication No.
• Non-Patent Document 1 https://www.atcc.org
• Non-Patent Document 2 https://www.ncbi.nlm.nih.gov
• MYA-1381 strain the date of registration is listed as June 28, 2011.
• strain SR-21 was originally classified into the genus Schizochytrium, but is now classified into the genus Auranthiochytrium (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.
• the heterotrophic microalgae according to this embodiment has a high content of ⁇ -aminobutyric acid, for example, by ingesting, administering, or applying the heterotrophic microalgae, it has an effect on improving brain function, a relaxing effect, and reducing stress. It is expected to have effects such as blood pressure lowering effect, collagen production promoting effect, epidermal cell proliferation effect, and hyaluronic acid synthesis promoting effect. Therefore, the heterotrophic microalgae according to the present embodiment is preferably used in various compositions (e.g., foods, medicines, external skin preparations, feeds, etc.), such as products that are orally ingested by animals (including humans). is more preferable, and more preferably food, medicine, or feed. That is, the heterotrophic microalgae according to the present embodiment is preferably a heterotrophic microalgae for food, medicine, or feed.
• various compositions e.g., foods, medicines, external skin preparations, feeds, etc
• the above heterotrophic microalgae containing high ⁇ -aminobutyric acid can be obtained by the method for producing heterotrophic microalgae according to one embodiment of the present invention.
• the method for producing heterotrophic microalgae is a method for producing heterotrophic microalgae in which the content of ⁇ -aminobutyric acid is 100 mg or more per 100 g of dry algae. This is a method for producing heterotrophic microalgae containing high butyric acid content.
• the method for producing heterotrophic microalgae includes a step of culturing heterotrophic microalgae in a medium containing ⁇ -aminobutyric acid (hereinafter also referred to as "cultivation step").
• a step of culturing heterotrophic microalgae in a medium containing ⁇ -aminobutyric acid hereinafter also referred to as "cultivation step”.
• the culture step it is also possible to convert L-glutamic acid in the medium to ⁇ -aminobutyric acid by the heterotrophic microalgae, and to accumulate ⁇ -aminobutyric acid within the microalgae. Furthermore, when performing the culture step, it is also possible to accumulate ⁇ -aminobutyric acid and/or to convert glutamic acid to ⁇ -aminobutyric acid and to accumulate the converted ⁇ -aminobutyric acid.
• heterotrophic microalgae having a higher content of ⁇ -aminobutyric acid than heterotrophic microalgae cultured in a medium containing no ⁇ -aminobutyric acid and/or glutamic acid can be cultured.
• glutamic acid is preferably L-glutamic acid.
• the method for producing ⁇ -aminobutyric acid-rich heterotrophic microalgae may include a step of preparing a medium containing ⁇ -aminobutyric acid (hereinafter also referred to as "medium preparation step").
• the medium used in the above culture step may be prepared in the medium preparation step.
• the medium may be a medium containing ⁇ -aminobutyric acid and/or glutamic acid, and a medium containing at least ⁇ -aminobutyric acid is more preferable because it is easier to obtain heterotrophic microalgae with a high content of ⁇ -aminobutyric acid. .
• the content of ⁇ -aminobutyric acid in the medium used in the above culture step is, 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. /L or more, 80mg/L or more, 90mg/L or more, or 100mg/L or more.
• the content of glutamic acid in the medium used in the above culture step is, 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.
• the content of ⁇ -aminobutyric acid and glutamic acid in the medium used in the culture step may be the sum of the above-mentioned ⁇ -aminobutyric acid content and glutamic acid content in an appropriate combination.
• the medium containing ⁇ -aminobutyric acid and/or glutamic acid is, for example, a composition containing ⁇ -aminobutyric acid and/or glutamic acid, or a composition containing ⁇ -aminobutyric acid and/or glutamic acid alone, and a heterotrophic microorganism. It can be prepared using ingredients necessary for culturing algae. Therefore, the medium preparation step may be a step of preparing a medium containing ⁇ -aminobutyric acid using a composition containing ⁇ -aminobutyric acid or ⁇ -aminobutyric acid.
• the content of ⁇ -aminobutyric acid in the medium can be adjusted within the numerical range described above.
• a medium containing glutamic acid can be prepared using, for example, a composition containing glutamic acid or glutamic acid alone, and components necessary 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.
• a medium containing both ⁇ -aminobutyric acid and glutamic acid may be prepared.
• compositions containing ⁇ -aminobutyric acid can be used, for example, in fermentation residues and fermentation residues such as shochu lees, awamori distillation lees, and pickles, as well as waste liquids and waste foods in food manufacturing processes using foods containing ⁇ -aminobutyric acid. Any substance containing ⁇ -aminobutyric acid as a type of water-soluble amino acid may be used.
• Compositions containing glutamic acid contain glutamic acid as a type of water-soluble amino acid, for example, in food manufacturing processes when producing glutamic acid or polyglutamic acid, or waste liquids and waste foods in food manufacturing processes using foods containing glutamic acid. It suffices as long as it exists.
• glutamic acid is contained in kelp; vegetables such as tomatoes; fermented foods such as cheese; and fermentation residues such as the above-mentioned shochu distillation lees and awamori distillation lees.
• a composition containing ⁇ -aminobutyric acid and/or a composition containing glutamic acid can be used as a material for a medium used for culturing heterotrophic microalgae. It is preferable to use at least a composition containing ⁇ -aminobutyric acid as a material for the medium because it is easy to obtain heterotrophic microalgae containing a high content of ⁇ -aminobutyric acid.
• the present inventors have developed a composition containing ⁇ -aminobutyric acid and/or glutamic acid used when preparing a culture medium, using residues generated after fermentation in sake production (hereinafter also referred to as "sake production residue") such as awamori distillation lees. ).
• Awamori distillation lees are generated during the awamori manufacturing process, and some of it is used as moromi vinegar, livestock feed, or crop fertilizer, but if it cannot be completely removed, it is treated as industrial waste.
• new ways of utilizing alcoholic beverage manufacturing residues such as awamori distilled lees are currently being sought.
• the present inventors have investigated the use of sake manufacturing residues such as awamori distillation lees as a material for a culture medium. As a result, the present inventors found that ⁇ -aminobutyric acid was included in the component derived from the liquid of liquor production residue such as Awamori distillation lees, and that the component derived from the liquid of liquor production residue such as Awamori distillation lees was It was found that it is suitable for the culture process.
• the above-mentioned culture medium preparation process will be explained using as an example a case where a culture medium containing components derived from a liquid in sake manufacturing residue such as awamori distillation lees is prepared.
• Alcohol in this specification is not particularly limited, but preferably is alcohol produced by fermenting raw materials using Aspergillus oryzae and yeast.
• sake manufacturing residues include slurry-like or solid lees generated during the sake manufacturing process.
• the alcohol production residue include sake lees, awamori distillation lees, and shochu distillation lees, 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 sake are not particularly limited, and include raw materials commonly used in the production of sake, awamori, shochu, etc.
• grains, potatoes, etc. include, for example, rice. , barley, wheat, buckwheat, soybean, corn, etc.; potatoes such as sweet potato; sugarcane; chestnuts, etc., and one or more selected from these can be used.
• “Awamori distillation lees” is the distillation residue produced in the awamori manufacturing process
• “shochu distillation lees” is the distillation residue produced in the shochu production process.
• “sake lees” is brewing residue produced during the sake manufacturing process.
• the state of the sake lees is not particularly limited, but awamori distilled lees or shochu distilled lees can be in the form of a slurry, and sake lees can be in the form of a solid.
• Liquor manufacturing residue, such as awamori distilled sake lees includes a liquid containing amino acids and organic acids such as citric acid, minerals and vitamins, and a solid containing protein, dietary fiber, and the like.
• the medium containing components derived from the liquid in liquor manufacturing residue such as awamori distilled lees only needs to contain at least a component derived from the liquid in liquor manufacturing residue such as awamori distilled lees. It may or may not contain components derived from solids in the residue.
• a medium containing components derived from liquid in sake manufacturing residue such as awamori distillation lees can be prepared in the medium preparation process described above.
• sake production residues such as awamori distillation lees may or may not be subjected to solid-liquid separation, for example.
• the culture medium preparation step may be a step that includes solid-liquid separation of liquor manufacturing residue such as awamori distillation lees.
• the medium preparation step may be a step of preparing a medium containing components derived from a liquid obtained by solid-liquid separation of sake manufacturing residue such as awamori distillation lees.
• the above-mentioned solid-liquid separation may be performed by known means such as centrifugation, sedimentation, filtration, compression, and commercially available solid-liquid separators.
• the liquid obtained by solid-liquid separation may be used as is. That is, in the culture medium preparation step, the culture medium may be prepared using the liquid obtained by solid-liquid separation itself.
• a culture medium may be prepared by mixing the liquid with additives described below.
• a medium may be prepared using a liquid obtained by solid-liquid separation that has been subjected to at least one treatment.
• a liquid obtained by solid-liquid separation may be subjected to a drying process to prepare a dried product, and then the dried product may be mixed with an additive described later and water to prepare a medium.
• the liquor manufacturing residue such as awamori distillation lees that is separated into solid and liquid may be used after being subjected to at least one treatment, for example.
• the at least one treatment may be selected from, for example, refrigeration, freezing, heating, concentration, sterilization, and drying.
• the culture medium preparation step may be a step that does not include solid-liquid separation of liquor manufacturing residues such as awamori distillation lees.
• the culture medium preparation step is a step of preparing a medium containing components derived from the liquid in the liquor production residue such as the awamori distillation lees by using the liquor production residue such as the awamori distillation lees without solid-liquid separation. It may be.
• Sake production residues such as awamori distillation lees that are used without solid-liquid separation are, for example, alcohol production residues such as awamori distillation lees (distillation residue) produced in the distillation of aged mash that are subjected to at least one treatment other than solid-liquid separation. It may be something that has been applied.
• the at least one treatment may be selected from, for example, refrigeration, freezing, heating, concentration, sterilization, and drying.
• a dry product may be prepared by drying sake manufacturing residue such as awamori distillation lees, and then a culture medium may be prepared by mixing the dry product with water and additives to be described later.
• the medium prepared in the medium preparation step may contain additives in addition to components derived from the liquid in the liquor manufacturing residue such as awamori distillation lees. That is, the medium preparation step may be a step of preparing a medium containing a component derived from a liquid in sake manufacturing residue such as awamori distillation lees, and an additive.
• additives examples include sugars, mineral components (including natural seawater and artificial seawater), organic acids, inorganic acids, organic bases, inorganic bases, vitamins, amino acids, peptides, and proteins.
• 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.
• saccharides include at least one saccharide selected from glucose, galactose, fructose, maltose, sucrose, lactose, oligosaccharides, and sugar alcohols (glycerol, etc.).
• the amount of saccharides in the medium may be adjusted as appropriate by those skilled in the art depending on the type of heterotrophic microalgae to be cultured.
• the above mineral components include, for example, 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, manganese chloride, sodium chloride, and calcium chloride; alkali metal oxides; molybdates such as sodium molybdate; selenites such as sodium selenite; potassium bromide and potassium iodide. At least one mineral component selected from halides; natural sea salt; artificial sea salt such as Red Sea Salt; The amount of mineral components in the medium may be appropriately adjusted by those skilled in the art depending on the type of heterotrophic microalgae to be cultured.
• sulfates such as potassium sulfate, magnesium sulfate, iron sulfate, ammonium sulfate
• the pH of the medium containing components derived from the liquid in the liquor manufacturing residue such as awamori distillation lees may be adjusted as necessary.
• an acid or a base may be added to adjust the pH before and/or after adding the above-mentioned additives.
• the pH may be adjusted as appropriate by those skilled in the art depending on the type of heterotrophic microalgae to be cultured.
• the culture medium preparation step the culture medium containing components derived from the liquid in sake production residues 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 a liquid in sake manufacturing residue such as awamori distillation lees. Such sterilization may be performed by known means such as autoclave sterilization, filter sterilization, boiling sterilization, and radiation sterilization.
• the sterilization process may also serve as a process to solubilize the solid portion derived from liquor manufacturing residues such as awamori distillation lees contained in the medium.
• the medium containing components derived from the liquid in liquor manufacturing residue such as awamori distillation lees may be a liquid medium or a solid medium.
• a liquid medium is more suitable than a solid medium.
• the culture medium containing components derived from the liquid in liquor production residue such as awamori distillation lees is the culture medium used in the method for producing heterotrophic microalgae with a high content of ⁇ -aminobutyric acid according to the present embodiment.
• Any medium containing ⁇ -aminobutyric acid may be used.
• the medium preparation process is mainly exemplified as a process of preparing a medium containing ⁇ -aminobutyric acid using awamori distillation lees, the medium preparation process is not limited to this.
• a medium may be prepared using a composition containing ⁇ -aminobutyric acid other than ⁇ -aminobutyric acid, or using ⁇ -aminobutyric acid.
• the culture conditions such as the culture temperature, culture period, and culture method in the culture step may be any conditions that allow the cultivation of heterotrophic microalgae.
• the temperature is 5 to 40°C, preferably 10 to 35°C, more preferably 20 to 28°C, even more preferably 25°C ⁇ 1°C, usually for 1 to 10 days, preferably 3 to 7 days, e.g.
• Culture is carried out for 4 to 5 days, and can be carried out by aeration agitation culture, shaking culture, or static culture.
• culture may be started after adjusting each concentration of ⁇ -aminobutyric acid and/or glutamic acid in the medium, but each of these concentrations may be adjusted to a predetermined concentration during the culture process after the start of culture.
• a composition containing ⁇ -aminobutyric acid and/or glutamic acid may be added to the medium to adjust their concentrations in the medium.
• the glutamic acid concentration in the medium is adjusted to a predetermined concentration while feeding a composition containing glutamic acid.
• Na in terms of Na is not particularly limited, but is, for example, 0.001 to 50 g/L/hr.
• the culturing step includes the above seed culturing step and before culturing the seed algae in a preculture medium containing ⁇ -aminobutyric acid (for example, a component derived from liquid in sake manufacturing residue such as awamori distillation lees).
• the medium for seed culture may be, for example, a medium known as a medium for culturing heterotrophic microalgae, or a medium containing components derived from liquid in sake manufacturing residue such as awamori distillation lees. good.
• the medium for seed culture is preferably a medium known as a medium for culturing heterotrophic microalgae.
• the component composition of the seed culture medium may be different from that used for pre-culture or main culture. It may be the same as the culture medium or may be different.
• the component composition of the medium for pre-culture may be the same as or different from that of the medium for main culture.
• Heterotrophic microalgae may be cultured in a culture device having suitable cell culture means.
• Cell culture means means means having a function for culturing cells, and is, for example, a culture tank.
• the culture tank may be one selected from a stirring device, a vibration device, a temperature control device, a pH control device, a turbidity measurement device, a light control device, a gas concentration measurement device such as O 2 or CO 2 , and a pressure measurement device. It may have multiple devices.
• the culture tank may be the same tank as the concentration/separation tank, or may be a separate tank 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 appropriate means (for example, a flow path).
• the method for producing ⁇ -aminobutyric acid-rich heterotrophic microalgae according to the present embodiment may further include a step of collecting the heterotrophic microalgae (hereinafter also referred to as "recovery step").
• the collection step is a step of collecting the heterotrophic microalgae cultured by the culture step described above.
• the heterotrophic microalgae may be collected separately from the medium, or may be collected together with the medium. That is, the heterotrophic microalgae collected in the collection step may be the heterotrophic microalgae separated from the medium, or may be a mixture of the heterotrophic microalgae and the medium.
• the recovery step may include a step of separating the cultured heterotrophic microalgae from the medium (hereinafter also referred to as "separation step"). In the separation step, the culture medium and the heterotrophic microalgae may be separated by known means such as centrifugation or filtration.
• the method for producing ⁇ -aminobutyric acid-rich heterotrophic microalgae according to the present embodiment may further include a step of drying the heterotrophic microalgae (hereinafter also referred to as "drying step").
• drying step the cultured heterotrophic microalgae is dried, and the heterotrophic microalgae becomes dried algal bodies.
• the drying step may be performed, for example, after the recovery step.
• the heterotrophic microalgae to be dried may be, for example, heterotrophic microalgae separated from the medium, or may be a mixture of heterotrophic microalgae and the medium.
• the heterotrophic microalgae to be dried is preferably heterotrophic microalgae separated from the culture medium. That is, in a preferred embodiment, the recovery step is a step including the separation step, and the drying step is a step of drying the heterotrophic microalgae separated from the culture medium.
• the above drying may be performed by known means.
• the drying may be performed by at least one selected from heat drying, low temperature drying, ventilation drying, reduced pressure drying, freeze drying, spray drying, natural drying, commercially available drying equipment, and the like.
• the method for producing heterotrophic microalgae with a high content of ⁇ -aminobutyric acid includes, in addition to the steps described above, a step of pulverizing heterotrophic microalgae (hereinafter also referred to as "pulverization step"). It's okay to be there.
• the pulverization step may be performed, for example, after the recovery step and before the drying step, or after the drying step.
• the grinding step is preferably performed after the drying step. Since the heterotrophic microalgae (dried algal bodies) after the drying process have a reduced water content, they can be easily pulverized and can also be pulverized to a small particle size.
• the pulverization may be performed by known pulverization means.
• heterotrophic microalgae containing a high content of ⁇ -aminobutyric acid can be obtained, and the production method may be used to obtain such microalgae.
• Patent Document 5 Japanese Patent No. 7218023.
• the present inventors have developed a process of culturing heterotrophic microalgae in a medium containing components derived from the liquid of awamori distillation lees, and It has been found that the odor and taste of heterotrophic microalgae can be improved by performing the step of drying the heterotrophic microalgae.
• the method for producing heterotrophic microalgae with a high content of ⁇ -aminobutyric acid is, for example, a method for producing heterotrophic microalgae containing a high content of ⁇ -aminobutyric acid in a medium containing components derived from a liquid of liquor manufacturing residue such as awamori distillation lees.
• the method may include the steps of culturing trophic microalgae and drying the cultured heterotrophic microalgae.
• the manufacturing method may further include the above-mentioned pulverization step.
• the crushing step By performing the crushing step, the particle size of the dried algae obtained can be reduced. Thereby, it is possible to improve the mouthfeel of an article containing dried algal bodies, and to improve the dispersibility when dry algal bodies are blended with other raw materials.
• Patent Document 5 Japanese Patent No. 7218023
• the production method described in Japanese Patent Application No. 2022-6913 Patent Document 5: Japanese Patent No. 7218023
• the present invention can also provide compositions (for example, foods, medicines, feeds, or external preparations for skin) containing heterotrophic microalgae with a high content of ⁇ -aminobutyric acid.
• the heterotrophic microalgae with high ⁇ -aminobutyric acid content is heterotrophic microalgae in which the content of ⁇ -aminobutyric acid is 100 mg or more per 100 g of dry algae. Details of the heterotrophic microalgae can be found in 1. above. and 2. The same explanation applies to this embodiment as well.
• food refers to food in an edible state that can be used directly for human consumption. Food given to non-human animals, such as feed (including fodder), does not fall under “food” as described herein.
• Foods include, for example, general foods, foods for specified uses, and foods with health claims (foods for specified health uses, foods with nutritional function claims, and foods with functional claims).
• General foods include so-called health foods, such as functional foods, nutritional supplements, health supplements, and supplements.
• Types of foods include, for example, processed meat products, processed seafood products, processed milk products, processed vegetable products, processed fruit products, oil and fat foods, luxury foods, seasonings, confectionery, frozen foods, retort foods, canned foods, and bottled foods. Examples include, but are not limited to, stuffed foods and instant foods.
• Feed is food given to animals other than humans.
• Feed includes, for example, pet food, livestock feed, poultry feed, and fish feed.
• animals to which feed is given include livestock (cows, pigs, chickens, horses, sheep, goats, etc.), fish, shellfish, and pets (dogs, cats, hamsters, rabbits, parakeets, tropical fish, reptiles, amphibians, insects, etc.), but are not limited to these.
• composition containing heterotrophic microalgae containing high ⁇ -aminobutyric acid content may be in the form of, for example, liquid, solid, semisolid, paste, granule, or powder. , capsule shape, etc., but are not particularly limited.
• composition for example, food, medicine, feed, skin external preparation, oral composition, etc.
• heterotrophic microalgae containing high ⁇ -aminobutyric acid has a high content of ⁇ -aminobutyric acid. It is expected that by ingesting, administering, or applying it to foods, medicines, or feeds, effects such as improving brain function, relaxing effects, reducing stress, and lowering blood pressure can be obtained.
• the heterotrophic microalgae high in ⁇ -aminobutyric acid used in this embodiment can be included in a composition that has a brain function improving effect, a relaxing effect, a stress reducing effect, and a blood pressure lowering effect. It can be used for the prevention, amelioration, or treatment of symptoms or diseases that can be prevented, ameliorated, or treated by.
• the present embodiment describes the method used in or for the production of various compositions or various drugs such as compositions for improving brain function, compositions for relaxing, compositions for stress reduction, and compositions for lowering blood pressure. It is also possible to provide heterotrophic microalgae containing a high content of ⁇ -aminobutyric acid or uses thereof.
• the present embodiment provides a method for preventing and improving symptoms or diseases that can be prevented, ameliorated, or treated by ⁇ -aminobutyric acid, using the heterotrophic microalgae containing high ⁇ -aminobutyric acid or a composition containing the same. Or a method of treatment can be provided. Further, this embodiment provides the above heterotrophic microalgae containing high ⁇ -aminobutyric acid or a composition containing the same for preventing, improving or treating symptoms or diseases that can be prevented, improved or treated by ⁇ -aminobutyric acid. You can also provide things.
• the composition according to the present embodiment includes foods, supplements, etc. that are based on the concept of prevention, improvement, or treatment of the above-mentioned symptoms or diseases, symptoms or diseases related thereto, and are labeled accordingly as necessary. compositions for ingestion are preferred.
• prevention refers to preventing or delaying the onset of a symptom or disease in a subject, or reducing the risk of onset of a symptom or disease in a subject.
• improvement refers to improvement or maintenance of a disease, symptom, or condition in a subject; prevention or delay of deterioration; reversal, prevention, or delay of progression.
• non-therapeutic purpose is a concept that does not include medical treatment, that is, treatment of the human body through treatment. Examples include health promotion, beauty practices, etc.
• the present invention also provides a method for producing a composition (eg, food, medicine, feed, skin external preparation, etc.) containing heterotrophic microalgae with a high content of ⁇ -aminobutyric acid.
• a composition eg, food, medicine, feed, skin external preparation, etc.
• the production method according to an embodiment of the present invention uses heterotrophic microalgae (heterotrophic microalgae containing high ⁇ -aminobutyric acid) cultured in a medium containing ⁇ -aminobutyric acid. including.
• the heterotrophic microalgae cultured in the medium containing ⁇ -aminobutyric acid are preferably heterotrophic microalgae produced by the method for producing ⁇ -aminobutyric acid-rich heterotrophic microalgae according to an embodiment of the present invention. It is a trophic microalgae. That is, the method for producing the composition (for example, food, medicine, feed, skin external preparation, etc.) according to the present embodiment preferably uses the ⁇ -aminobutyric acid-rich heterotrophic microorganism according to the embodiment of the present invention. The method may include the steps described in the method for producing algae. Details of the process are described in 2. above. As explained in .
• ⁇ -aminobutyric acid-rich heterotrophic microalgae-containing composition for example, food, medicine, feed, etc.
• the present invention also provides a method for increasing the content of ⁇ -aminobutyric acid in heterotrophic microalgae (hereinafter also referred to as "method for increasing ⁇ -aminobutyric acid content").
• a method for increasing ⁇ -aminobutyric acid content according to an embodiment of the present invention includes the step of culturing heterotrophic microalgae in a medium containing ⁇ -aminobutyric acid.
• the process is as described in 2. above. This is the same culture process as explained in .
• the method for increasing ⁇ -aminobutyric acid content according to the present embodiment may include at least one step selected from a culture medium preparation step, a collection step, and a drying step, in addition to the culturing step. Details of these steps can be found in 2. above. The same explanation applies to this embodiment as well.
• increasing the content of ⁇ -aminobutyric acid refers to increasing the content of ⁇ -aminobutyric acid in the heterotrophic microalgae through the above culture step.
• the increase is, 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. It may be increased by 0 times or more, 5.5 times or more, or 6.0 times or more.
• the present invention also provides a method for recovering ⁇ -aminobutyric acid from a culture medium.
• a method for recovering ⁇ -aminobutyric acid from a medium according to an embodiment of the present invention includes the steps of culturing heterotrophic microalgae in a medium containing ⁇ -aminobutyric acid, and collecting the heterotrophic microalgae. and, including. These steps are respectively described in 2. above. This is the same as the culture step and recovery step explained in . Furthermore, the method for recovering ⁇ -aminobutyric acid from a culture medium according to the present embodiment may include a culture medium preparation process and/or a drying process in addition to the culture process and recovery process. Details of these steps can be found in 2. above. The same description applies to this embodiment as well.
• the present invention also provides a method for recovering ⁇ -aminobutyric acid from a composition.
• a method for recovering ⁇ -aminobutyric acid from a composition according to an embodiment of the present invention includes the steps of preparing a medium using a composition containing ⁇ -aminobutyric acid, and culturing heterotrophic microalgae in the medium. and collecting the heterotrophic microalgae. These steps are respectively described in 2. above. This is the same as the culture medium preparation step, culture step, and collection step described in . Furthermore, the method for recovering ⁇ -aminobutyric acid from the composition according to the present embodiment may include a drying process in addition to the medium preparation process, culture process, and recovery process. For details of the drying process, see 2. above. The same explanation applies to this embodiment as well.
• composition used in the culture medium preparation step of this embodiment may be, for example, awamori distillation lees, but is not particularly limited as long as it does not impair the effects of the present invention.
• the present invention can employ the following configurations or technical features as appropriate.
• - [1] Heterotrophic microalgae having a content of ⁇ -aminobutyric acid of 100 mg or more per 100 g of dry algae.
• -[2] The heterotrophic microalgae according to [1] above, wherein the heterotrophic microalgae is a microalgae belonging to Thraustochytriales.
• -[3] The heterotrophic microalgae according to [1] or [2] above, wherein the heterotrophic microalgae is a microalgae belonging to the genus Aurantiochytrium.
• the heterotrophic microalgae is preferably the heterotrophic microalgae described in [2] or [3] above.
• the produced heterotrophic microalgae is preferably a heterotrophic microalgae containing a high content of ⁇ -aminobutyric acid.
• the medium is a medium containing ⁇ -aminobutyric acid, which is obtained by preparing and sterilizing a medium containing components derived from liquid in sake manufacturing residue.
• ⁇ [9] For increasing the content of ⁇ -aminobutyric acid in heterotrophic microalgae, or for recovering ⁇ -aminobutyric acid from a culture medium, or for producing food, medicine, or feed. , the manufacturing method according to any one of [5] to [8] above.
• the medium is (i) a medium obtained by adjusting and sterilizing a ⁇ -aminobutyric acid-containing composition, or (ii) a component derived from liquor production residue or a liquid in liquor production residue.
• the method may include a step of adjusting and sterilizing a medium containing liquor production residue or a component derived from a liquid in the liquor production residue.
• ⁇ [11] ⁇ -aminobutyric acid-rich heterotrophic microalgae obtained by the production method according to any one of [5] to [10] above, using ⁇ -aminobutyric acid-rich heterotrophic microalgae A method for producing a composition containing microalgae. - [12] Heterotrophic microalgae high in ⁇ -aminobutyric acid content obtained by the production method according to any one of [5] to [10] above, or use thereof.
• a method for increasing the content of ⁇ -aminobutyric acid in heterotrophic microalgae which includes the step of culturing the heterotrophic microalgae in a medium containing ⁇ -aminobutyric acid.
• a method for recovering ⁇ -aminobutyric acid from a medium comprising the steps of culturing heterotrophic microalgae in a medium containing ⁇ -aminobutyric acid, and collecting the heterotrophic microalgae.
• a method of recovering ⁇ -aminobutyric acid from a composition comprising: ⁇ [16] ⁇ - obtained by using heterotrophic microalgae cultured in a medium containing ⁇ -aminobutyric acid, or by the production method described in any one of [5] to [10] above.
• a composition for example, food, medicine, feed, etc.
• a method for producing the composition which comprises using heterotrophic microalgae containing high aminobutyric acid content.
• the cultured heterotrophic microalgae is preferably a heterotrophic microalgae containing a high content of ⁇ -aminobutyric acid.
• the medium is a medium obtained by adjusting and sterilizing a medium containing liquor manufacturing residue or a component derived from a liquid in the liquor manufacturing residue.
• the method may include a step of adjusting and sterilizing a medium containing liquor production residue or a component derived from a liquid in the liquor production residue.
• the heterotrophic microalgae is used for one or more types selected from inhibitory neurotransmission, brain function improvement, relaxation, stress reduction, blood pressure reduction, and sleep quality improvement, The heterotrophic microalgae according to any one of [1] to [4] above, or the method according to any one of [5] to [18] above.
• the composition is used for one or more selected from inhibitory neurotransmission, improvement of brain function, relaxation, stress reduction, reduction of blood pressure, reduction of anxiety, and improvement of sleep quality. The composition according to [4] above, or the method according to any one of [5] to [18] above.
• the cognitive dysfunction include one or more selected from mild cognitive dysfunction, Alzheimer's disease, cognitive dysfunction due to aging (eg, decline in memory, spatial cognition, etc.), and the like.
• Example 1 Production of dried algal bodies in Example 1 Heterotrophic microalgae were cultured according to the following procedure to obtain dried algal bodies.
• Example 1 The measurement results are shown in Table 1 below.
• Example 1-1 the culture medium and dried algal bodies obtained in each test were written as "Example 1-1" and "Example 1-2".
• Example 2 Heterotrophic microalgae were cultured according to the following procedure to obtain dried algae.
• the content of ⁇ -aminobutyric acid per 100g of dry algae was 278mg, so approximately 84.5% of the decrease in ⁇ -aminobutyric acid in the medium was caused by dry algae. It is considered that the amount was taken into the body and accumulated (278/329 ⁇ 100 ⁇ 84.5 [%]).
• the stopper was a breathable silicone rubber open-cell sponge plug.
• the culture medium used for flask culture is a medium containing 25% shochu distilled lees
• the medium is a medium containing 50 mL of shochu distilled lees supernatant medium, 150 mL of water, 4 g of glucose, and 3.34 g of red sea salt.
• "%" of 25% shochu lees containing medium is "volume of shochu distilled lees supernatant medium (mL)/[total volume of shochu distilled lees supernatant medium (mL) and water volume (mL)"] (mL)] x 100 (%).
• the total amount is also the volume (mL) of the medium.
• the ratio of the volume (mL) of the shochu distillation lees supernatant medium to the volume (mL) of water was adjusted to prepare 50%, 75%, and 100% shochu distillation lees containing media.
• the concentrations of glucose and red sea salt in the medium containing shochu distilled lees are the same as in the case of ⁇ 25%'', ⁇ glucose 4 g, red sea salt 3.34 g/volume'' even when the concentration of the shochu distilled lees supernatant medium is changed.
• the volume was adjusted to 200 mL.
• the 100% shochu distillation lees-containing medium has a volume of water (mL) of 0 mL and a volume of the shochu distillation lees supernatant medium (mL) of 200 mL per 200 mL of the total amount.
• Flask culture GTY medium containing seed algae was seeded in a medium containing shochu distillation lees, and cultured under aerobic conditions for 36 hours. The culture conditions at this time were 25°C and shaking at 110 strokes/min.
• Example 3-1 is a test example using a 25% shochu lees culture medium, and test examples in which the shochu lees supernatant medium content in the shochu lees culture medium was changed to 50%, 75%, and 100%, respectively. These were named Example 3-2, Example 3-3, and Example 3-4.
• Example 3 Collection and drying The collection and drying in Example 3 were carried out in Example 3-1, Example 3-2, and Example 3- according to the procedure of "(1) e)" of Test Example 1 above after flask culture. 3. Dried algal bodies of Examples 3-4 were obtained. The content of ⁇ -aminobutyric acid was measured in accordance with "(3) Measurement of the content of ⁇ -aminobutyric acid" in Test Example 1 above. The measurement results in Example 3 are shown in Table 3 below. DCW is an abbreviation for dry algae weight.
• the actual measured value/theoretical value of GABA within the algae is [actual measured value of GABA within the algae at the end of culture [mg/100g]/theoretical value of GABA within the algae assuming that all the reduced amount of GABA in the medium is collected in the algal body [ mg/100g]].
• the "medium” in “in the medium” in Table 3 refers to the "medium containing potato shochu distillation lees.”
• GABA in the culture medium could be incorporated into the algal bodies of heterotrophic microalgae even when using not only awamori distilled lees but also shochu distilled lees. From this, it is possible to use not only sake manufacturing residues (sake manufacturing lees, etc.) such as awamori distillation lees, shochu distillation lees, and sake lees, but also ⁇ -aminobutyric acid-containing compositions or ⁇ -aminobutyric acid-containing medium. It was confirmed that heterotrophic microalgae with high aminobutyric acid content could be obtained.
• ⁇ -aminobutyric acid could be contained in the algal bodies of heterotrophic microalgae. From this, it is considered that by adjusting the culture conditions such as the concentration of ⁇ -aminobutyric acid and/or glutamic acid in the medium, the upper limit of the amount accumulated within the algae can be adjusted as appropriate.
• ⁇ -aminobutyric acid could be contained within the algal bodies of heterotrophic microalgae to at least about 2, 3 or 5% by weight.
• a lees-containing medium was obtained.
• An aqueous solution of Na glutamate (sterilized by filtration) was added to the medium after the start of the culture during the culture under the conditions shown in Table 4.
• a glutamate Na aqueous solution (glutamate Na concentration 200 g/L) was added so that the glutamate Na concentration in the medium was 0.6 g/L/hr.
• the "medium” in “medium” in Table 4 refers to "medium containing awamori distillation lees.”
• L-glutamic acid in the culture medium is taken into the algal bodies of microalgae belonging to the Thraustochytrium family, particularly microalgae of the genus Aurantiochytrium, and is used for proliferation. It was thought that if more L-glutamic acid was taken into the algae than is used for growth, it would be converted to ⁇ -aminobutyric acid and accumulated within the algae.
• heterotrophic microalgae can produce ⁇ -aminobutyric acid from L-glutamic acid in their bodies and accumulate the produced ⁇ -aminobutyric acid in their bodies.
• heterotrophic microalgae containing a high content of ⁇ -aminobutyric acid can also be obtained by supplying L-glutamic acid in addition to ⁇ -aminobutyric acid to the culture medium.
• Auranthiochytrium strains (Auranthiochytrium M15d1 strain, Auranthiochytrium S28P strain) were grown in Awamori distillation lees-containing medium containing 50% Awamori distillation lees supernatant medium according to ⁇ Test Example 3>. (medium containing 50% awamori distiller's lees) and cultured in flasks. These two strains were collected and stored in Japan by the present inventors.
• the Awamori distiller's lees supernatant medium is a ⁇ -aminobutyric acid-containing composition (for culture medium) obtained according to the preparation of the Awamori distiller's lees supernatant medium of ⁇ Test Example 1>.
• the 50% awamori distiller's lees-containing medium used in Test Example 5 can be obtained by adjusting the concentration of the awamori distiller's lees supernatant medium according to ⁇ Test Example 3>.
• the "medium” in Table 5 refers to a medium containing 50% awamori distiller's lees.
• the content of ⁇ -aminobutyric acid was measured in the same manner as in ⁇ Test Example 1>.
• strains of the genus Auranthiochytrium other than strain SR21 collect GABA in the culture medium during cultivation and accumulate it in the algal bodies. It has been shown that microalgae belonging to the genus Aurantiochytrium, which is a heterotrophic microalgae, can have a high content of ⁇ -aminobutyric acid regardless of the algae strain, and that ⁇ -aminobutyric acid can be recovered from the culture medium.

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