Greenhouse gas production inhibitor, composition for suppressing greenhouse gas production, feed composition for suppressing greenhouse gas production, food composition for suppressing greenhouse gas production, pharmaceutical composition for suppressing greenhouse gas production, method for suppressing greenhouse gas production, propionic acid production promoter, composition for promoting propionic acid production, feed composition for promoting propionic acid production, food composition for promoting propionic acid production, pharmaceutical composition for promoting propionic acid production, and method for promoting propionic acid production

Abstract

This greenhouse gas production inhibitor contains microalgae belonging to the genus Coccomyxa.

Description

Greenhouse gas production inhibitor, composition for inhibiting greenhouse gas production, feed composition for inhibiting greenhouse gas production, food composition for inhibiting greenhouse gas production, pharmaceutical composition for inhibiting greenhouse gas production, method for inhibiting greenhouse gas production, propionic acid production promoter, composition for promoting propionic acid production, feed composition for promoting propionic acid production, food composition for promoting propionic acid production, pharmaceutical composition for promoting propionic acid production, and method for promoting propionic acid production

[0001] The present disclosure relates to a greenhouse gas production inhibitor, a composition for inhibiting greenhouse gas production, a feed composition for inhibiting greenhouse gas production, a food composition for inhibiting greenhouse gas production, a pharmaceutical composition for inhibiting greenhouse gas production, a method for inhibiting greenhouse gas production, a propionic acid production promoter, a composition for promoting propionic acid production, a feed composition for promoting propionic acid production, a food composition for promoting propionic acid production, a pharmaceutical composition for promoting propionic acid production, and a method for promoting propionic acid production.

[0002] Greenhouse gases are one cause of global warming. Approximately 15% of greenhouse gas emissions worldwide are attributed to the livestock industry, and of that 15%, approximately 40% is said to be emitted by ruminants. Ruminants, in particular, break down the food they eat and, through the interaction of the decomposition products with microorganisms such as methanogens in their digestive tracts, generate greenhouse gases such as methane, which are then released outside the body, i.e., into the atmosphere. Emission of greenhouse gases into the atmosphere can be considered the release of the energy source taken in as food as greenhouse gases. Therefore, reducing greenhouse gas emissions from ruminants also leads to improved ruminant productivity. Therefore, reducing greenhouse gas emissions such as methane emitted by animals is desirable in order to mitigate global warming and improve animal productivity.

[0003] For example, Non-Patent Document 1 discloses that methane production was suppressed in an in vitro test by Euglena gracilis, which belongs to the phylum Euglenozoa. Non-Patent Document 2 discloses that methane production was suppressed in an in vitro test by Chlorella vulgaris, which belongs to the family Chlorellaceae, order Chlorellales, class Trebouxiophyceae, phylum Chlorophyta.

[0004] Non-patent document 1: E. Ahmed, K. Suzuki, T. Nishida, Micro- and Macro-Algae Combination as a Novel Alternative Ruminant Feed with Methane-Mitigation Potential. Animals 13, 796 (2023) Non-patent document 2: AE Kholif, GA Gouda, TA Morsy, OH Matloup, SM Sallam, AK Patra, Associative effects between Chlorella vulgaris microalgae and Moringa oleifera leaf silage used at different levels decreased in vitro ruminal greenhouse gas production and altered ruminal fermentation. Environmental Science and Pollution Research, 30:6001-6020 (2023)

[0005] However, there is a further demand for new methods that can suppress greenhouse gas production. The present disclosure has been made in view of the above, and provides a greenhouse gas production inhibitor, a composition for suppressing greenhouse gas production, a feed composition for suppressing greenhouse gas production, a food composition for suppressing greenhouse gas production, a pharmaceutical composition for suppressing greenhouse gas production, and a method for suppressing greenhouse gas production, all of which are capable of suppressing greenhouse gas production, as well as a propionic acid production promoter, a composition for promoting propionic acid production, a feed composition for promoting propionic acid production, a food composition for promoting propionic acid production, a pharmaceutical composition for promoting propionic acid production, and a method for promoting propionic acid production, all of which are capable of promoting propionic acid production.

[0006] Specific means for solving the above problems include the following aspects: <1> A greenhouse gas production inhibitor containing microalgae belonging to the genus Coccomyxa. <2> The greenhouse gas production inhibitor according to <1>, in which the microalgae belonging to the genus Coccomyxa are at least one of nitrogen-sufficient microalgae belonging to the genus Coccomyxa and nitrogen-deficient microalgae belonging to the genus Coccomyxa. <3> The greenhouse gas production inhibitor according to <1> or <2>, in which the microalgae belonging to the genus Coccomyxa include at least one selected from the group consisting of algal bodies, dried powder, and extracts of microalgae belonging to the genus Coccomyxa. <4> The greenhouse gas production inhibitor according to any one of <1> to <3>, wherein the microalgae belonging to the genus Coccomyxa include at least one species selected from the group consisting of Coccomyxa sp. KJ strain or a mutant thereof, Coccomyxa sp. Obi strain or a mutant thereof, and Coccomyxa sp. N1 strain or a mutant thereof. <5> A composition for inhibiting greenhouse gas production, comprising the greenhouse gas production inhibitor according to any one of <1> to <4>. <6> A feed composition for inhibiting greenhouse gas production, comprising the greenhouse gas production inhibitor according to any one of <1> to <4> or the composition for inhibiting greenhouse gas production according to <5>. <7> A food composition for inhibiting greenhouse gas production, comprising the greenhouse gas production inhibitor according to any one of <1> to <4> or the composition for inhibiting greenhouse gas production according to <5>. <8> A pharmaceutical composition for inhibiting greenhouse gas production, comprising the greenhouse gas production inhibitor according to any one of <1> to <4> or the composition for inhibiting greenhouse gas production according to <5>. <9> A method for inhibiting greenhouse gas production in a subject, comprising administering microalgae belonging to the genus Coccomyxa to the subject. <10> The method for inhibiting greenhouse gas production according to <9>, wherein the subject is a ruminant. <11> The method for inhibiting greenhouse gas production according to <9> or <10>, wherein microalgae belonging to the genus Coccomyxa are administered in an amount of 0.1% to 5.0% dry matter based on the total amount of feed. <12> A propionic acid production promoter, comprising microalgae belonging to the genus Coccomyxa. <13> A composition for promoting propionic acid production, comprising the propionic acid production promoter according to <12>.<14> A feed composition for promoting propionic acid production, comprising the propionic acid production promoter according to <12> or the composition for promoting propionic acid production according to <13>. <15> A food composition for promoting propionic acid production, comprising the propionic acid production promoter according to <12> or the composition for promoting propionic acid production according to <13>. <16> A pharmaceutical composition for promoting propionic acid production, comprising the propionic acid production promoter according to <12> or the composition for promoting propionic acid production according to <13>. <17> A method for promoting propionic acid production in a subject, comprising administering to the subject microalgae belonging to the genus Coccomyxa.

[0007] <18> Use of microalgae belonging to the genus Coccomyxa in a greenhouse gas production inhibitor. <19> Use of microalgae belonging to the genus Coccomyxa according to <18>, wherein the microalgae belonging to the genus Coccomyxa are at least one of nitrogen-sufficient microalgae belonging to the genus Coccomyxa and nitrogen-deficient microalgae belonging to the genus Coccomyxa. <20> Use of microalgae belonging to the genus Coccomyxa in a propionic acid production promoter.

[0008] <21> Use of microalgae belonging to the genus Coccomyxa in the production of a greenhouse gas production inhibitor. <22> Use of microalgae belonging to the genus Coccomyxa according to <21>, wherein the microalgae belonging to the genus Coccomyxa are at least one of nitrogen-sufficient microalgae belonging to the genus Coccomyxa and nitrogen-deficient microalgae belonging to the genus Coccomyxa. <23> Use of microalgae belonging to the genus Coccomyxa in the production of a propionic acid production promoter.

[0009] According to the present disclosure, there are provided a greenhouse gas production inhibitor capable of suppressing greenhouse gas production, a composition for inhibiting greenhouse gas production, a feed composition for inhibiting greenhouse gas production, a food composition for inhibiting greenhouse gas production, a pharmaceutical composition for inhibiting greenhouse gas production, and a method for inhibiting greenhouse gas production, as well as a propionic acid production promoter capable of promoting propionic acid production, a composition for promoting propionic acid production, a feed composition for promoting propionic acid production, a food composition for promoting propionic acid production, a pharmaceutical composition for promoting propionic acid production, and a method for promoting propionic acid production.

[0010] Figure 1 is a bar graph showing methane production. Figure 2 is a bar graph showing methane production per dry matter decomposition amount.

[0011] An embodiment of the present disclosure will be described in detail below. However, the present disclosure is not limited to the following embodiment. In the following disclosure, components (including element steps, etc.) are not essential unless otherwise specified. The same applies to numerical values ​​and their ranges, and do not limit the present disclosure. In the present disclosure, numerical ranges indicated using "to" include the numerical values ​​before and after "to" as the lower and upper limits, respectively. In numerical ranges described in stages in the present disclosure, the upper or lower limit of one numerical range may be replaced with the upper or lower limit of another numerical range described in stages. Furthermore, in numerical ranges described in the present disclosure, the upper or lower limit of that numerical range may be replaced with a value shown in the examples. In the present disclosure, when multiple substances corresponding to each component are present in the composition, the content of each component in the composition means the total content of the multiple substances present in the composition, unless otherwise specified.

[0012] <Greenhouse Gas Production Inhibitor> The greenhouse gas production inhibitor of the present disclosure contains microalgae belonging to the genus Coccomyxa.

[0013] A greenhouse gas production inhibitor containing microalgae belonging to the genus Coccomyxa can inhibit the production of greenhouse gases. The action of the greenhouse gas production inhibitor disclosed herein is unclear, but is presumed to be as follows: Starch, cellulose, hemicellulose, and the like contained in food ingested by ruminants are broken down into sugars such as glucose by the degradative enzymes of microorganisms present in the rumen. Microorganisms use these sugars to produce volatile fatty acids and other substances that serve as energy sources for ruminants, while also producing hydrogen and carbon dioxide in the process. The main methanogens that inhabit the rumen of ruminants use the hydrogen and carbon dioxide to produce methane. It is believed that microalgae belonging to the genus Coccomyxa inhibit the production of greenhouse gases such as methane by directly or indirectly inhibiting the activity of the microorganisms and methanogens. The present disclosure is not limited in any way to the presumed mechanism described above.

[0014] <Microalgae Belonging to the Genus Coccomyxa> The greenhouse gas production inhibitor of the present disclosure contains microalgae belonging to the genus Coccomyxa. The microalgae belonging to the genus Coccomyxa may be at least one of nitrogen-sufficient microalgae belonging to the genus Coccomyxa and nitrogen-deficient microalgae belonging to the genus Coccomyxa, may be nitrogen-sufficient and nitrogen-deficient microalgae belonging to the genus Coccomyxa, or may be nitrogen-sufficient or nitrogen-deficient microalgae belonging to the genus Coccomyxa. From the viewpoint of further suppressing greenhouse gas emissions, the microalgae belonging to the genus Coccomyxa are preferably nitrogen-sufficient microalgae belonging to the genus Coccomyxa.

[0015] Nitrogen-sufficient microalgae of the genus Coccomyxa refer to microalgae of the genus Coccomyxa obtained by culturing in a medium containing a sufficient amount of nitrogen. Specifically, nitrogen-sufficient microalgae of the genus Coccomyxa refer to microalgae of the genus Coccomyxa obtained by culturing in a medium having an N / DM (intracellular nitrogen content (on a dry mass basis)) of 8% by mass or more.

[0016] In N / DM, N means the total mass of nitrogen from the nitrogen source added to the culture medium per unit volume, and DM means the mass of microalgae in a dry state contained in the culture solution per unit volume.

[0017] N can be determined from the nitrogen source added to the culture medium and its mass. DM can be determined, for example, from the absorbance of the culture solution. Specifically, the absorbance of the culture solution at a wavelength of 720 nm is measured. A calibration curve is previously determined to show the relationship between the absorbance of the culture solution and the mass of microalgae powder obtained by removing water from the culture solution and drying it per unit volume. DM can be determined from the measured absorbance based on this calibration curve.

[0018] Nitrogen-sufficient microalgae belonging to the genus Coccomyxa are cultured in a medium containing a sufficient amount of nitrogen source, i.e., in a nutrient-rich environment. In other words, nitrogen-sufficient microalgae belonging to the genus Coccomyxa can also be said to be microalgae belonging to the genus Coccomyxa in the growth phase. Microalgae belonging to the genus Coccomyxa hardly accumulate oils such as triglycerides intracellularly under conditions of active growth. In nitrogen-sufficient microalgae belonging to the genus Coccomyxa, the mass of oil within the algae body relative to the mass of the algae body is preferably 0% to 2% by mass, more preferably 0% to 1% by mass, and even more preferably 0% to 0.5% by mass.

[0019] [Nitrogen-deficient type] On the other hand, nitrogen-deficient microalgae belonging to the genus Coccomyxa refer to microalgae belonging to the genus Coccomyxa obtained by culturing in a medium that is not sufficiently rich in nitrogen sources, specifically, microalgae belonging to the genus Coccomyxa obtained by culturing in a medium with an N / DM ratio of less than 8% by mass.

[0020] Nitrogen-deficient microalgae belonging to the genus Coccomyxa are cultured in a medium lacking a sufficient nitrogen source, i.e., in a harsh, nutrient-deficient environment. Under stressful conditions resulting from nutrient deficiency, microalgae belonging to the genus Coccomyxa exhibit slowed cell growth and accumulate oils such as triglycerides within the cells. In other words, nitrogen-deficient microalgae belonging to the genus Coccomyxa can also be considered microalgae belonging to the genus Coccomyxa in the oil accumulation phase. In nitrogen-deficient microalgae belonging to the genus Coccomyxa, the mass of oil within the algae relative to the mass of the algae is preferably 2% to 80% by mass. The mass of oil within the algae relative to the mass of the algae may be 2% to 40% by mass, 2% to 20% by mass, or 20% to 40% by mass.

[0021] Furthermore, the N / DM value can be adjusted by adjusting the culture conditions of microalgae belonging to the genus Coccomyxa (e.g., the components of the culture medium or the culture time, etc.). Therefore, in order to obtain the desired microalgae belonging to the genus Coccomyxa, it is also possible to culture microalgae belonging to the genus Coccomyxa by adjusting the N / DM.

[0022] [Algae bodies, dried powder, and extract] The microalgae belonging to the genus Coccomyxa preferably include at least one selected from the group consisting of algae bodies, dried powder, and extracts of microalgae belonging to the genus Coccomyxa.

[0023] (Algal Cells) Algal cells of microalgae belonging to the genus Coccomyxa can be obtained by culturing microalgae belonging to the genus Coccomyxa in a medium and recovering the grown microalgae belonging to the genus Coccomyxa after culturing. The method for culturing microalgae belonging to the genus Coccomyxa is not particularly limited. The medium for culturing microalgae belonging to the genus Coccomyxa may be a medium commonly used for culturing microalgae, and for example, any of known freshwater microalgae media containing various nutrients, trace metal salts, vitamins, etc., or media for marine microalgae can be used. A specific example of a medium is AF6 medium. The composition of AF6 medium (per 100 ml) is as follows:

[0024] NaNO ３14 mg NH ４ NO ３ 2.2 mg MgSO ４ ・7H ２ O 3mg KH ２ P.O. ４ 1 mg K ２ HPO ４ 0.5 mg CaCl ２ ・2H ２ O 1mg CaCO ３ 1mg Fe-citrate 0.2mg Citric acid 0.2mg Biotin 0.2μg Thiamine HCl 1μg Vitamin B ６ 0.1μg Vitamin B １２ 0.1μg Trace metals 0.5mL Distilled water 99.5mL

[0025] Examples of nutrient salts include NaNO ３ , KNO ３ , N.H. ４ Cl, and a nitrogen source such as urea, and K ２ HPO ４ , K.H. ２ P.O. ４ and phosphorus sources such as sodium glycerophosphate. Trace metals include iron, magnesium, manganese, calcium, and zinc. Vitamins include vitamin B １ and vitamin B １２ etc.

[0026] Culturing may be static culture, or, when the medium is a liquid medium, may be agitated culture. Culturing may be aerobic or anaerobic. For example, carbon dioxide may be supplied and agitated under aeration conditions. Culturing may be performed indoors using fluorescent light with a light-dark cycle, such as 12 hours of light and 12 hours of darkness, or continuous light may be used. Culturing may also be performed outdoors, i.e., the light-dark cycle, such as the duration of light irradiation and the duration of darkness, may be determined by natural sunlight. The culture conditions are not particularly limited as long as they do not adversely affect the growth of microalgae belonging to the genus Coccomyxa. For example, the culture conditions are preferably pH 2 to pH 9, and more preferably pH 3 to pH 7. The culture temperature is preferably 10°C to 40°C, and more preferably 15°C to 35°C.

[0027] Regarding the method for culturing microalgae belonging to the genus Coccomyxa, methods for culturing the genus Coccomyxa, Pseudococcomyxa, and Coccomyxa sp. KJ strain described in JP 2014-117202 A, JP 2015-15918 A, WO 2015 / 190116, or Satoh, A. et al., Characterization of the Lipid Accumulation in a New Microalgal Species, Pseudochoricystis ellipsoidea (Trebouxiophyceae) J. Jpn. Inst. Energy (2010) 89: 909-913. Can be appropriately used.

[0028] The algal cells of microalgae belonging to the genus Coccomyxa may be obtained as a culture solution containing the algal cells, as a concentrated solution of the algal cells, or as a paste.

[0029] (Dry Powder) The dry powder of microalgae belonging to the genus Coccomyxa can be obtained by subjecting algal bodies of microalgae belonging to the genus Coccomyxa to a drying process and a crushing (pulverizing) process. For the drying process, for example, drum drying, spray drying, or freeze drying can be used. For the crushing process, a bead-type crusher, homogenizer, French press, mixer / blender, or fine grinder can be used. The order of the drying process and the crushing process is not particularly limited. Alternatively, the drying process and the crushing process can be performed simultaneously using a device equipped with drying and crushing functions.

[0030] The particle size of the dry powder is not particularly limited. For example, it is preferable that the dry powder has an average particle size of 0.2 μm to 2 mm, preferably 0.4 μm to 400 μm. The average particle size can be measured, for example, using a laser diffraction / scattering particle size distribution analyzer.

[0031] (Extract) The extract of microalgae belonging to the genus Coccomyxa is preferably a substance extracted from dried powder of microalgae belonging to the genus Coccomyxa using water as a solvent. The water extract of microalgae belonging to the genus Coccomyxa can be obtained by drying the algae of microalgae belonging to the genus Coccomyxa, suspending and mixing the dried material in distilled water, and collecting the resulting supernatant after centrifugation. The collected supernatant may be used as the extract as is, or the collected supernatant may be dried and used as the extract. The drying process for the supernatant can be, for example, drum drying, spray drying, or freeze drying. The temperature of the water used for extraction may be as high as 120°C or as low as 10°C or below. It is not particularly limited as long as the components can be sufficiently extracted. It is preferably in the range of 10°C to 50°C, and more preferably 30°C to 40°C. The extraction time is preferably 1 hour to 100 hours. The extract of microalgae belonging to the genus Coccomyxa may be a substance extracted from a dry powder of microalgae belonging to the genus Coccomyxa using an organic solvent (e.g., acetone, ethanol, hexane, etc.) as a solvent.

[0032] [Genus Coccomyxa] Microalgae belonging to the genus Coccomyxa are not particularly limited as long as they are microalgae included in the genus Coccomyxa, and their species and strains are not particularly limited. The species of microalgae belonging to the genus Coccomyxa may be unclassified. The microalgae belonging to the genus Coccomyxa are preferably non-genetically modified microalgae belonging to the genus Coccomyxa, i.e., preferably natural microalgae belonging to the genus Coccomyxa.

[0033] The greenhouse gas production inhibitor of the present disclosure may contain multiple species of microalgae belonging to the genus Coccomyxa, or may contain multiple strains of microalgae belonging to the genus Coccomyxa.

[0034] In the present disclosure, the microalgae belonging to the genus Coccomyxa preferably include at least one species selected from the group consisting of Coccomyxa sp. KJ strain or a mutant thereof, Coccomyxa sp. Obi strain or a mutant thereof, and Coccomyxa sp. N1 strain or a mutant thereof, more preferably at least one species selected from the group consisting of Coccomyxa sp. KJ strain or a mutant thereof, Coccomyxa sp. Obi strain or a mutant thereof, and Coccomyxa sp. N1 strain or a mutant thereof, and even more preferably Coccomyxa sp. KJ strain or a mutant thereof. The Coccomyxa sp. KJ strain was deposited on June 4, 2013, at the National Institute of Technology and Evaluation, Biotechnology Center, International Patent Organism Depositary (NITE-IPOD) (Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture) under the accession number FERM P-22254, and transferred to international deposit under the provisions of the Petapore Treaty under the accession number FERM BP-22254 on June 2, 2015. The Coccomyxa sp. Obi strain was deposited on February 15, 2005, at the National Institute of Technology and Evaluation, Biotechnology Center, International Patent Organism Depositary (NITE-IPOD) (Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture) under accession number FERM P-10484, and was transferred to international deposit under the provisions of the Petapore Treaty under accession number FERM BP-10484 on January 18, 2006. The Coccomyxa sp. N1 strain has been internationally deposited with the National Institute of Technology and Evaluation, Biotechnology Center, International Patent Organism Depositary (NITE-IPOD) (Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture) under the provisions of the Petapore Convention on January 18, 2006, under the accession number FERM BP-10485.In addition, Coccomyxa sp. KJ strain, Coccomyxa sp. Obi strain, and Coccomyxa sp. N1 strain were previously classified in the genus Pseudococcomyxa, but are now classified in the genus Coccomyxa.

[0035] The mutant strains of Coccomyxa sp. KJ strain, Coccomyxa sp. Obi strain, and Coccomyxa sp. N1 strain can be obtained by irradiation with ultraviolet rays, X-rays, gamma rays, etc., mutagen treatment, heavy beam irradiation, genetic engineering (introduction of foreign genes, gene disruption, gene modification by genome editing, etc.), etc. The method for obtaining the mutant strains and their characteristics, etc. are not particularly limited, as long as mutant strains that can suppress greenhouse gas production can be obtained.

[0036] [Uses] The greenhouse gas production inhibitor of the present disclosure can be used as feed or feed additives, foods or food additives, pharmaceuticals or quasi-drugs, cosmetics, miscellaneous goods, etc. The greenhouse gas production inhibitor of the present disclosure is preferably used as feed or feed additives.

[0037] As mentioned above, suppressing greenhouse gas emissions from the target animals also leads to improved productivity of the animals, so the greenhouse gas production inhibitor of the present disclosure can also be used to improve animal productivity.

[0038] [Production Method] A method for producing the greenhouse gas production inhibitor of the present disclosure includes culturing the microalgae belonging to the genus Coccomyxa of the present disclosure.

[0039] <Composition for Inhibiting Greenhouse Gas Production> The composition for inhibiting greenhouse gas production according to the present disclosure contains the greenhouse gas production inhibitor according to the present disclosure. That is, the composition for inhibiting greenhouse gas production may contain any component other than the greenhouse gas production inhibitor. For example, the composition for inhibiting greenhouse gas production may contain a substance that has a known greenhouse gas production inhibitory effect. The content of the greenhouse gas production inhibitor in the composition for inhibiting greenhouse gas production is not particularly limited.

[0040] The composition for inhibiting greenhouse gas production can be used, for example, as feed or feed additives, food or food additives, medicines or quasi-drugs, cosmetics, miscellaneous goods, or the like.

[0041] <Feed Composition for Suppressing Greenhouse Gas Production> The feed composition for suppressing greenhouse gas production according to the present disclosure contains the greenhouse gas production inhibitor or the composition for suppressing greenhouse gas production according to the present disclosure. Examples of feed compositions for suppressing greenhouse gas production containing the greenhouse gas production inhibitor or the composition for suppressing greenhouse gas production include regular feed (such as livestock feed and pet feed) and nutritional supplements (such as livestock nutritional supplements and pet nutritional supplements) that contain the greenhouse gas production inhibitor or the composition for suppressing greenhouse gas production. Examples of feed compositions for suppressing greenhouse gas production include roughage and concentrated feed that contain the greenhouse gas production inhibitor or the composition for suppressing greenhouse gas production. Examples of roughage include hay (grass, legume, and wild hay, etc.), straw (rice straw and wheat straw, etc.), grains (pea husks, etc.), green grasses (grass, legume, and wild grass, etc.), silage (grass silage, corn silage, dried corn silage, and ear corn silage, etc.), root vegetables (turnips, radishes, carrots, sweet potatoes, etc.), and leafy vegetables (radish leaves and vegetable scraps, etc.). Examples of concentrated feed include grains (wheat, corn, barnyard millet, foxtail millet, brown rice, etc.), legumes (soybean, pea, kidney bean, etc.), brans (rice bran, wheat bran, wheat bran, etc.), oilseed cakes (soybean meal, cottonseed meal, rapeseed meal, linseed meal, peanut meal, etc.), and manufacturing residues (brewer's lees, tofu cake, juice cake, etc.). The feed composition for inhibiting greenhouse gas production of the present disclosure can also be used as a feed additive.

[0042] The feed composition for inhibiting greenhouse gas production can be formed by a conventional method. The feed composition for inhibiting greenhouse gas production can be provided in an orally ingestible form such as powder, granules, mash, pellets, crumbles, flakes, or liquid. It can also be provided in an orally ingestible form such as a mixture of two or more of the above forms, for example, a mixture of pellets and flakes, or a mixture of mash and pellets.

[0043] The feed composition for inhibiting greenhouse gas production may contain additives that are commonly used in the manufacture of feed. Examples of additives include emulsifiers, antioxidants, antifungal agents, binders, flavoring agents, adjusters, flavoring agents, synthetic antibacterial agents, antibiotics, enzymes, probiotics, organic acids, various amino acids, various vitamins (vitamin A, vitamin B １ , Vitamin B ２ , Vitamin B ６ , vitamin C, vitamin D, vitamin E, and vitamin K, etc.), various minerals (magnesium, zinc, iron, sodium, potassium, and selenium, etc.), and color enhancers.

[0044] <Food Composition for Inhibiting Greenhouse Gas Production> The food composition for inhibiting greenhouse gas production of the present disclosure contains the greenhouse gas production inhibitor or the greenhouse gas production inhibitor composition of the present disclosure. Examples of food compositions for inhibiting greenhouse gas production containing the greenhouse gas production inhibitor or the greenhouse gas production inhibitor composition include foods (cereals, vegetables, fruits, meat, fish, etc.), various processed foods (processed agricultural foods, processed livestock foods, processed seafood foods, oils and fats, seasonings, etc.), sweets and desserts (gummy candies, jellies, gum, chocolate, cookies, candies, caramels, Japanese sweets, snacks, etc.), beverages (milk, lactic acid drinks, soft drinks, fruit juice drinks, vegetable juice drinks, tea drinks, coffee drinks, alcoholic drinks, sports drinks, carbonated drinks, etc.), nutritional supplements (supplements and nutritional drinks, etc.), foods with functional claims, foods with nutrient function claims, and foods for specified health uses, etc. The food composition for inhibiting greenhouse gas production of the present disclosure can also be used as a food additive.

[0045] The food composition for inhibiting greenhouse gas production can be formed by a conventional method. The food composition for inhibiting greenhouse gas production can be provided in an orally ingestible form such as a powder, granules, a solid molding, a tablet, a paste, or a liquid.

[0046] The food composition for inhibiting greenhouse gas production may contain additives commonly used in food production, such as fats and oils, emulsifiers, stabilizers, antioxidants, preservatives, thickeners, flavorings, coloring agents, pH adjusters, sweeteners, enzymes, organic acids, dietary fiber, various nutrients, various amino acids, and various vitamins (vitamin A, vitamin B １ , Vitamin B ２ , Vitamin B ６ , vitamin C, vitamin D, vitamin E, and vitamin K, etc.), various minerals (magnesium, zinc, iron, sodium, potassium, and selenium, etc.), and flavoring components (citric acid and malic acid, etc.).

[0047] When the food composition for inhibiting greenhouse gas production is a nutritional supplement (such as a supplement or nutritional drink), a food with functional claims, a food with nutrient function claims, or a food for specified health uses, examples of the dosage form thereof include tablets, powders, fine granules, granules, pills, chewable tablets, rapidly disintegrating tablets, capsules, syrups, suspensions, injections, topical preparations (ointments, creams, lotions, liquids, gels, poultices, plasters, tapes, aerosols, etc.), and suppositories.

[0048] When the food composition for inhibiting greenhouse gas production is a nutritional supplement (such as a supplement or nutritional drink), a food with functional claims, a food with nutrient function claims, or a food for specified health uses, examples of additives contained therein include excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, antiseptics, thickeners, binders, lubricants, flavoring agents, colorants, pH adjusters, sweeteners, coating agents, and physiological saline.

[0049] Examples of excipients that can be used include lactose, starch, dextrin, crystalline cellulose, sorbitol, D-mannitol, silicon dioxide, calcium stearate, and sucrose. Examples of disintegrants that can be used include starch, carboxymethylcellulose, and calcium carbonate. Examples of buffers that can be used include phosphates, citrates, and acetates. Examples of emulsifiers that can be used include gum arabic, sodium alginate, and tragacanth. Examples of suspending agents that can be used include glycerin monostearate, aluminum monostearate, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, and sodium lauryl sulfate.

[0050] As the soothing agent, benzyl alcohol, chlorobutanol, sorbitol, etc. can be used. As the stabilizer, propylene glycol, ascorbic acid, etc. can be used. As the preservative, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, etc. can be used. As the antiseptic, benzalkonium chloride, parahydroxybenzoic acid, chlorobutanol, etc. can be used.

[0051] <Pharmaceutical Composition for Inhibiting Greenhouse Gas Production> The pharmaceutical composition for inhibiting greenhouse gas production according to the present disclosure contains the greenhouse gas production inhibitor or the composition for inhibiting greenhouse gas production according to the present disclosure. The pharmaceutical composition for inhibiting greenhouse gas production may contain additives commonly used in the preparation of pharmaceuticals. Examples of additives include excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, antiseptics, thickeners, binders, lubricants, flavoring agents, coloring agents, pH adjusters, sweeteners, coating agents, and physiological saline.

[0052] Examples of excipients that can be used include lactose, starch, dextrin, crystalline cellulose, sorbitol, D-mannitol, silicon dioxide, calcium stearate, and sucrose. Examples of disintegrants that can be used include starch, carboxymethylcellulose, and calcium carbonate. Examples of buffers that can be used include phosphates, citrates, and acetates. Examples of emulsifiers that can be used include gum arabic, sodium alginate, and tragacanth. Examples of suspending agents that can be used include glycerin monostearate, aluminum monostearate, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, and sodium lauryl sulfate.

[0053] As the soothing agent, benzyl alcohol, chlorobutanol, sorbitol, etc. can be used. As the stabilizer, propylene glycol, ascorbic acid, etc. can be used. As the preservative, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, etc. can be used. As the antiseptic, benzalkonium chloride, parahydroxybenzoic acid, chlorobutanol, etc. can be used.

[0054] The pharmaceutical composition for inhibiting greenhouse gas production can be formulated according to a conventional method. The dosage form of the pharmaceutical composition for inhibiting greenhouse gas production is not particularly limited, and examples thereof include tablets, powders, fine granules, granules, pills, chewable tablets, rapidly disintegrating tablets, capsules, syrups, suspensions, injections, topical preparations (ointments, creams, lotions, liquids, gels, poultices, plasters, tapes, aerosols, etc.), and suppositories.

[0055] Depending on the dosage form, the pharmaceutical composition for inhibiting greenhouse gas production may be administered to a subject by oral administration or parenteral administration (local injection, intravenous, intraarterial, subcutaneous, intradermal, intramuscular, or intraperitoneal injection, transdermal, intranasal, or transmucosal administration, etc.). Systemic administration and local administration may also be indicated depending on the subject. These administration routes are not mutually exclusive, and any two or more of them may be used in combination.

[0056] <Method for Suppressing Greenhouse Gas Production> The method for suppressing greenhouse gas production according to the present disclosure includes administering microalgae belonging to the genus Coccomyxa to a subject.

[0057] The method for inhibiting greenhouse gas production of the present disclosure may be administered in any form: the microalgae belonging to the genus Coccomyxa of the present disclosure; the greenhouse gas production inhibitor of the present disclosure; the composition for inhibiting greenhouse gas production of the present disclosure; the feed composition for inhibiting greenhouse gas production of the present disclosure; the food composition for inhibiting greenhouse gas production of the present disclosure; or the pharmaceutical composition for inhibiting greenhouse gas production of the present disclosure.

[0058] <Subject> The subject to which the microalgae belonging to the genus Coccomyxa of the present disclosure, the greenhouse gas production inhibitor of the present disclosure, the composition for inhibiting greenhouse gas production of the present disclosure, the feed composition for inhibiting greenhouse gas production of the present disclosure, the food composition for inhibiting greenhouse gas production of the present disclosure, and the pharmaceutical composition for inhibiting greenhouse gas production of the present disclosure are administered is not particularly limited, but is preferably an animal, more preferably a mammal, even more preferably a subject other than humans, particularly preferably a herbivore, and most preferably a ruminant. Ruminant animals include animals belonging to the suborder Bovidae (such as cattle, yaks, buffalo, sheep, and goats), animals belonging to the suborder Cervidae (such as reindeer and red deer), family Giraffidae (such as giraffes and okapi), and animals belonging to the suborder Camelidae (such as dromedaries, llamas, and alpacas), with animals belonging to the suborder Bovidae being preferred, and cattle being more preferred.

[0059] In the microalgae belonging to the genus Coccomyxa disclosed herein, the greenhouse gas production inhibitor disclosed herein, the composition for inhibiting greenhouse gas production disclosed herein, the feed composition for inhibiting greenhouse gas production disclosed herein, the food composition for inhibiting greenhouse gas production disclosed herein, and the pharmaceutical composition for inhibiting greenhouse gas production disclosed herein, the dosage of the microalgae belonging to the genus Coccomyxa is set so as to obtain the expected effect. The dosage is set taking into consideration the species, body weight, age, amount of food, or the amount of microorganisms or methanogens present in the body of the subject to be administered.

[0060] The dosage may be, for example, 0.01% to 10% dry matter of microalgae belonging to the genus Coccomyxa relative to the total amount of the diet, more preferably 0.1% to 5.0% dry matter of microalgae belonging to the genus Coccomyxa relative to the total amount of the diet, and even more preferably 0.3% to 1.5% dry matter of microalgae belonging to the genus Coccomyxa relative to the total amount of the diet. In this disclosure, "% dry matter" refers to the ratio (%) of the mass of the solid matter excluding moisture (i.e., dry matter). In other words, "administering 0.01% dry matter of microalgae belonging to the genus Coccomyxa relative to the total amount of the diet" means administering 0.01% by mass of microalgae belonging to the genus Coccomyxa relative to the total mass of the diet converted to dry matter. As described above, the microalgae belonging to the genus Coccomyxa of the present disclosure can achieve the effect of suppressing greenhouse gas production even at small dosages.

[0061] The above-mentioned dose may be administered in multiple doses per day. The administration schedule may be, for example, once to several times a day, once every two days, or once every three days. When creating the administration schedule, the condition of the subject and the duration of the effect of the microalgae belonging to the genus Coccomyxa may be taken into consideration.

[0062] The timing of administration is not particularly limited as long as it does not impair the effects of the present disclosure. The microalgae belonging to the genus Coccomyxa according to the present disclosure, the greenhouse gas production inhibitor according to the present disclosure, the composition for inhibiting greenhouse gas production according to the present disclosure, the feed composition for inhibiting greenhouse gas production according to the present disclosure, the food composition for inhibiting greenhouse gas production according to the present disclosure, and the pharmaceutical composition for inhibiting greenhouse gas production according to the present disclosure may be administered continuously from immediately after birth, to an adult, or for only a partial period, or administration may be continued and discontinued repeatedly for any period.

[0063] The dosage may be adjusted, for example, according to the growth stage of the target. The N / DM value of the microalgae belonging to the genus Coccomyxa to be administered may also be adjusted according to the growth stage of the target.

[0064] <Propionic Acid Production Promoter> The propionic acid production promoter of the present disclosure contains the microalgae belonging to the genus Coccomyxa of the present disclosure.

[0065] A propionic acid production promoter containing microalgae belonging to the genus Coccomyxa can promote the production of propionic acid. The action of the propionic acid production promoter of the present disclosure is unclear, but is presumed as follows: As described above, food ingested by an animal is decomposed by microorganisms living in the rumen, producing carbon dioxide and hydrogen in the process. Microalgae belonging to the genus Coccomyxa directly or indirectly inhibit the activity of methanogens, thereby preventing them from utilizing this carbon dioxide and hydrogen. This makes hydrogen more readily available in the reaction system that produces propionic acid. Therefore, it is believed that microalgae belonging to the genus Coccomyxa can promote the production of propionic acid. The present disclosure is not limited in any way to the above-mentioned presumed mechanism.

[0066] In the propionic acid production promoter of the present disclosure, the description of the microalgae belonging to the genus Coccomyxa is the same as the description of the microalgae belonging to the genus Coccomyxa in the greenhouse gas production inhibitor of the present disclosure, including definitions, examples, preferred embodiments, etc.

[0067] The method for producing the propionic acid production promoter of the present disclosure includes culturing the microalgae belonging to the genus Coccomyxa of the present disclosure.

[0068] <Composition for Promoting Propionic Acid Production> The composition for promoting propionic acid production of the present disclosure contains the propionic acid production promoter of the present disclosure. That is, the composition for promoting propionic acid production may contain any component other than the propionic acid production promoter. For example, the composition for promoting propionic acid production may contain a known substance having a propionic acid production promoting effect. The content of the propionic acid production promoter in the composition for promoting propionic acid production is not particularly limited.

[0069] The composition for promoting propionic acid production can be used, for example, as feed or feed additives, food or food additives, pharmaceuticals or quasi-drugs, cosmetics, or miscellaneous goods.

[0070] <Feed composition for promoting propionic acid production> The feed composition for promoting propionic acid production of the present disclosure contains the propionic acid production promoter of the present disclosure or the composition for promoting propionic acid production of the present disclosure. For explanations regarding the feed, molding method, shape, additives, etc. of the feed composition for promoting propionic acid production, please refer to the explanations for the feed composition for suppressing greenhouse gas production of the present disclosure, including definitions, examples, preferred embodiments, etc.

[0071] <Food Composition for Propionic Acid Production Promotion> The food composition for promoting propionic acid production of the present disclosure contains the propionic acid production promoter of the present disclosure or the composition for promoting propionic acid production of the present disclosure. For explanations regarding the food, molding method, shape, additives, etc. of the food composition for promoting propionic acid production, please refer to the explanations for the food composition for suppressing greenhouse gas production of the present disclosure, including definitions, examples, preferred embodiments, etc.

[0072] <Pharmaceutical Composition for Propionic Acid Production> The pharmaceutical composition for promoting propionic acid production of the present disclosure contains the propionic acid production promoter of the present disclosure or the composition for promoting propionic acid production of the present disclosure. For explanations regarding additives, formulation methods, dosage forms, administration methods, etc., in the pharmaceutical composition for promoting propionic acid production, please refer to the explanations for the pharmaceutical composition for inhibiting greenhouse gas production of the present disclosure, including definitions, examples, preferred embodiments, etc.

[0073] <<Method for Promoting Propionic Acid Production>> The method for promoting propionic acid production of the present disclosure includes administering microalgae belonging to the genus Coccomyxa to a subject. In the method for promoting propionic acid production of the present disclosure, the description regarding the subject to be administered, the dosage, etc., including definitions, examples, and preferred embodiments, is the same as the description regarding the subject to be administered, the dosage, etc., in the method for suppressing greenhouse gas production of the present disclosure.

[0074] The present disclosure will be described in more detail below with reference to examples, but the present disclosure is not limited to the following examples as long as it does not deviate from the gist of the disclosure. Unless otherwise specified, "%" is based on mass.

[0075] Example 1 Microalgae Belonging to the Genus Coccomyxa (Coccomyxa sp. Strain KJ) A dry powder of Coccomyxa sp. strain KJ was prepared as a dry powder of microalgae belonging to the genus Coccomyxa. Specifically, the dry powder was obtained by culturing the strain using the culture method described in JP 2014-117202 A. The nitrogen-sufficient Coccomyxa sp. strain KJ was cultured in a medium with an N / DM ratio of 8% by mass or more. The nitrogen-deficient Coccomyxa sp. strain KJ was cultured in a medium with an N / DM ratio of less than 8% by mass. Specifically, the culture was initially initiated in a medium with an N / DM ratio of 8% by mass or more, and a portion of the Coccomyxa sp. strain KJ in the active growth phase was centrifuged and collected to obtain a nitrogen-sufficient dry powder. The remaining part was then continued to be cultured as is, and when the nitrogen in the medium became depleted and cell growth gradually slowed down, the remaining Coccomyxa sp. KJ strain was centrifuged to obtain a nitrogen-deficient dry powder.

[0076] <Preparation of culture medium> Rumen fluid was collected from two Holstein cows before feeding and filtered through quadruple gauze. Rumen fluid is a digestive fluid present in the rumen (first stomach) of ruminants. A large amount of feed is stored in the rumen, where it is decomposed by microorganisms living in the rumen. The filtered rumen fluid contains microorganisms such as methanogens.

[0077] Equal amounts of rumen fluid from the two animals were mixed at a ratio of artificial saliva to rumen fluid of 4:1 (v / v), and this was used as a culture medium. The culture medium was kept warm in a thermostatic chamber at 39°C and sprayed with carbon dioxide gas until use.

[0078] The composition of artificial saliva per liter is as follows: NaHCO ３ 9.8g KCl 0.57g CaCl ２ ・2H ２ O 0.053g Na ２ HPO ４ ・12H ２ O 9.3g NaCl 0.47g MgSO ４ 0.059g Cysteine ​​hydrochloride monohydrate 0.28g Resazurin 0.0010g

[0079] <Cultivation> The test plots were Comparative Example 1 and Examples 1 to 4 below. Three centrifuge tubes were prepared for each test plot, and evaluation was performed with n=3. To correct the measurement test described below, a centrifuge tube containing only the culture medium was also prepared as a blank. [Comparative Example 1] Substrate only [Example 1] Substrate + 0.5% dry matter amount of nitrogen-deficient Coccomyxa sp. KJ strain [Example 2] Substrate + 1.0% dry matter amount of nitrogen-deficient Coccomyxa sp. KJ strain [Example 3] Substrate + 0.5% dry matter amount of nitrogen-sufficient Coccomyxa sp. KJ strain [Example 4] Substrate + 1.0% dry matter amount of nitrogen-sufficient Coccomyxa sp. KJ strain

[0080] In Example 1, 0.5 g of dry corn silage powder sieved through a 1 mm mesh was used as the substrate. The dry matter (g) refers to the mass (g) of the solid matter excluding moisture (i.e., dry matter). Furthermore, as described above, the dry powder of the nitrogen-deficient Coccomyxa sp. KJ strain or the dry powder of the nitrogen-sufficient Coccomyxa sp. KJ strain was added at 0.5% or 1.0% dry matter of the substrate (i.e., 0.5% or 1.0% by mass of the substrate converted to dry matter).

[0081] 40 mL of the prepared culture medium was measured out and added to each test group of Comparative Example 1 and Examples 1 to 4, carbon dioxide gas was blown in, the container was sealed with a rubber stopper equipped with a three-way stopcock and a syringe, and the container was cultured at 39°C for 24 hours.

[0082] <Measurement of dry matter decomposition amount> After 24 hours of cultivation, the mixture was centrifuged at 500 × g for 5 minutes. The residue after centrifugation was placed in a fiber bag and dried at 105 ° C. for 40 hours, and the mass after drying was measured. The mass after drying was subtracted from the mass of the substrate before cultivation to calculate the dry matter decomposition amount.

[0083] <Measurement of gas production> During the culture, the gas production was confirmed from the scale on each syringe. The gases measured by this method include methane and carbon dioxide, which are greenhouse gases.

[0084] The results of measuring the amount of gas produced after 12 hours and 24 hours of culture are shown in Table 1. In Table 1, a, b, and c indicate that there is a significant difference between different symbols (P value < 0.05). After 12 hours and 24 hours of culture, the amount of gas produced in Examples 1 to 4 was lower than that in Comparative Example 1.

[0085]

[0086] <Measurement of methane production amount> The methane production amount was measured by gas chromatography (GC-2014, Shimadzu Corporation) equipped with a flame ionization detector (FID). The column used was an SH-Q-BOND PLOT Column (0.32 mm, 20 μm, 30 m). The measurement conditions were a column temperature of 220°C, an inlet temperature of 250°C, a detector temperature of 250°C, and a spirit ratio of 1:5. A standard gas (CH ４ Using a calibration curve prepared using a standard curve (25.1%), the methane concentration in each test plot was measured at 0-12 hours, 12-24 hours, and 0-24 hours of incubation. Methane production was calculated by multiplying the methane concentration by the gas production rate. Methane production per dry matter decomposition rate was calculated by dividing the methane production rate by the dry matter decomposition rate (IVDMD; In Vitro Dry Matter Degradability).

[0087] The results of the methane production measurements are shown in Table 2. In Table 2, a, b, and c indicate significant differences between different symbols (P value < 0.05). In all three periods of culture: 0 to 12 hours, 12 to 24 hours, and 0 to 24 hours, Examples 1 to 4 produced less methane than Comparative Example 1. Furthermore, the methane production per dry matter decomposition amount during 0 to 24 hours of culture was also lower in Examples 1 to 4 than in Comparative Example 1. In particular, in Example 3, the methane production per dry matter decomposition amount during 0 to 24 hours of culture decreased by approximately 48% compared to Comparative Example 1 (10.4 mL / IVDMD → 5.4 mL / IVDMD). In Example 4, the methane production per dry matter decomposition amount during 0 to 24 hours of culture decreased by approximately 41% compared to Comparative Example 1 (10.4 mL / IVDMD → 6.2 mL / IVDMD). That is, a significant methane production inhibitory effect was observed simply by adding a very small amount of microalgae belonging to the genus Coccomyxa, such as 0.5% or 1.0% of the dry matter amount relative to the substrate.

[0088]

[0089] <Measurement of Volatile Fatty Acids> The supernatant obtained by centrifugation in the above <Measurement of Dry Matter Decomposition Rate> was mixed with 25 (w / v)% metaphosphoric acid at a ratio of 5:1 (v / v) to obtain a mixture, which was then stored at -20°C. The mixture was thawed and centrifuged at 15,000 × g for 15 minutes, and 900 μL of the supernatant was mixed with 100 μL of 0.1 M tiglic acid aqueous solution. Volatile fatty acids were measured by the internal standard method using tiglic acid as the internal standard using a gas chromatograph (GC-2014, Shimadzu Corporation) equipped with an FID. A Restek Stabilwax column (30 m × 0.25 mm × 0.5 μm) was used. The measurement conditions were an inlet temperature of 200°C and a detector temperature of 250°C. The column temperature was initially 120°C and was increased to 230°C at 10°C / min. The spirit ratio was 1: 5. Furthermore, the contents (mol %) of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, and valeric acid in the volatile fatty acids were also determined.

[0090] The results of the measurement of volatile fatty acids are shown in Table 3. In Table 3, a, b, and c indicate that there is a significant difference between different symbols (P value < 0.05). In Examples 1 to 4, the amount of propionic acid produced was greater than in Comparative Example 1.

[0091]

[0092] From the above, gas production and methane production were suppressed, while propionic acid production was promoted in Examples 1 to 4. In other words, by simply adding a very small amount of microalgae belonging to the genus Coccomyxa, such as 0.5% or 1.0% dry matter of the substrate, the effects of suppressing greenhouse gas production, suppressing methane production, and promoting propionic acid production were observed.

[0093] Example 2 Microalgae belonging to the genus Coccomyxa (Coccomyxa sp. KJ strain, Coccomyxa sp. Obi strain, and Coccomyxa sp. N1 strain) As dry powders of microalgae belonging to the genus Coccomyxa, dry powders of the nitrogen-sufficient Coccomyxa sp. KJ strain, the nitrogen-sufficient Coccomyxa sp. Obi strain, and the nitrogen-sufficient Coccomyxa sp. N1 strain were each prepared in the same manner as in Example 1 above.

[0094] <Preparation of Culture Solution> In the same manner as in Example 1 above, a culture solution containing microorganisms such as methanogens was prepared.

[0095] <Cultivation> The test plots were the following Comparative Example 2 and Examples 5 to 7. As in Example 1 above, evaluation was performed with n=4 for each test plot, and centrifuge tubes containing only the culture medium were also prepared as blanks. [Comparative Example 2] Substrate only [Example 5] Substrate + 0.5% dry matter amount of nitrogen-sufficient Coccomyxa sp. Obi strain [Example 6] Substrate + 0.5% dry matter amount of nitrogen-sufficient Coccomyxa sp. N1 strain [Example 7] Substrate + 0.5% dry matter amount of nitrogen-sufficient Coccomyxa sp. KJ strain

[0096] In Example 2, a 0.5 g dry matter mixture of dried corn silage powder and formulated feed (dry mass ratio: 3:2) sieved through a 1 mm mesh was used as the substrate. Furthermore, as described above, 0.5% dry matter of the dried powder of the nitrogen-sufficient Coccomyxa sp. Obi strain, the nitrogen-sufficient Coccomyxa sp. N1 strain, or the nitrogen-sufficient Coccomyxa sp. KJ strain was added to the substrate (i.e., 0.5% by mass of the substrate converted to dry matter).

[0097] 40 mL of the prepared culture medium was measured and added to each test group of Comparative Example 2 and Examples 5 to 7, and cultured at 39° C. for 24 hours in the same manner as in Example 1.

[0098] <Measurement of dry matter decomposition amount and decomposition rate> The dry matter decomposition amount was calculated, and further the decomposition rate (mass%) of the dry matter was calculated in the same manner as in Example 1. The results are shown in Table 4. In Table 4, a and b indicate that there is a significant difference between different symbols (P value < 0.05).

[0099]

[0100] <Measurement of methane production amount> In the same manner as in Example 1, the methane production amount after 24 hours of culture and the methane production amount per dry matter decomposition amount after 24 hours of culture were measured.

[0101] The results of measuring methane production are shown in Figure 1. In Figure 1, a and b indicate significant differences between different symbols (P value < 0.05). After 24 hours of culture, the methane production was significantly lower in Examples 5 to 7 than in Comparative Example 2. Compared to Comparative Example 2, the methane production after 24 hours of culture was reduced by 23.3% in Example 5, 24.6% in Example 6, and 26.5% in Example 7.

[0102] Furthermore, the results of the amount of methane produced per amount of dry matter decomposed after 24 hours of culture are shown in Figure 2. In Examples 5 to 7, the amount of methane produced was lower than in Comparative Example 2. Compared to Comparative Example 2, the amount of methane produced per amount of dry matter decomposed after 24 hours of culture was reduced by 20.0% in Example 5, 17.3% in Example 6, and 21.1% in Example 7. In other words, as long as the microalgae belonging to the genus Coccomyxa were added in an amount of just 0.5% dry matter relative to the substrate, a significant methane production inhibitory effect was observed, regardless of the type.

[0103] The disclosure of Japanese Patent Application No. 2023-142563, filed on September 1, 2023, is incorporated herein by reference in its entirety. All publications, patent applications, and technical standards mentioned herein are incorporated herein by reference to the same extent as if each individual publication, patent application, and technical standard was specifically and individually indicated to be incorporated by reference.

Claims

1. A greenhouse gas production inhibitor containing microalgae belonging to the genus Coccomyxa.

2. The greenhouse gas production inhibitor described in claim 1, wherein the microalgae belonging to the genus Coccomyxa are at least one of nitrogen-sufficient microalgae belonging to the genus Coccomyxa and nitrogen-deficient microalgae belonging to the genus Coccomyxa.

3. A greenhouse gas production inhibitor as described in claim 1 or claim 2, wherein the microalgae belonging to the genus Coccomyxa include at least one type selected from the group consisting of algae bodies, dried powder, and extracts of microalgae belonging to the genus Coccomyxa.

4. A greenhouse gas production inhibitor as described in claim 1 or claim 2, wherein the microalgae belonging to the genus Coccomyxa include at least one species selected from the group consisting of Coccomyxa sp. KJ strain or a mutant thereof, Coccomyxa sp. Obi strain or a mutant thereof, and Coccomyxa sp. N1 strain or a mutant thereof.

5. A composition for inhibiting greenhouse gas production, comprising the greenhouse gas production inhibitor according to claim 1.

6. A feed composition for inhibiting greenhouse gas production, comprising the greenhouse gas production inhibitor according to claim 1 or 2, or the composition for inhibiting greenhouse gas production according to claim 5.

7. A food composition for inhibiting greenhouse gas production, comprising the greenhouse gas production inhibitor according to claim 1 or 2, or the greenhouse gas production inhibitor composition according to claim 5.

8. A pharmaceutical composition for inhibiting greenhouse gas production, comprising the greenhouse gas production inhibitor according to claim 1 or 2, or the composition for inhibiting greenhouse gas production according to claim 5.

9. A method for suppressing greenhouse gas production in a subject, comprising administering to the subject microalgae belonging to the genus Coccomyxa.

10. The method for suppressing greenhouse gas production according to claim 9, wherein the subject is a ruminant.

11. A method for suppressing greenhouse gas production according to claim 9 or 10, wherein microalgae belonging to the genus Coccomyxa are administered in an amount of 0.1% to 5.0% dry matter relative to the total amount of feed.

12. A propionic acid production promoter containing microalgae belonging to the genus Coccomyxa.

13. A composition for promoting propionic acid production, comprising the propionic acid production promoter according to claim 12.

14. A feed composition for promoting propionic acid production, comprising the propionic acid production promoter according to claim 12 or the composition for promoting propionic acid production according to claim 13.

15. A food composition for promoting propionic acid production, comprising the propionic acid production promoter according to claim 12 or the composition for promoting propionic acid production according to claim 13.

16. A pharmaceutical composition for promoting propionic acid production, comprising the propionic acid production promoter according to claim 12 or the composition for promoting propionic acid production according to claim 13.

17. A method for promoting propionic acid production in a subject, comprising administering to the subject microalgae belonging to the genus Coccomyxa.

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