Novel Bacillus megatherium HyangYak-01 strain and its uses

Abstract

This invention relates to a novel Bacillus megatherium HyangYak-01 strain and its applications. According to one embodiment of the novel Bacillus megatherium HyangYak-01 strain, it exhibits effects such as increasing the diversity of microbiome species in soils used for crop cultivation or improving their physicochemical properties, and promoting crop growth or the biosynthesis of physiologically active substances. Therefore, it can be applied to a variety of uses, such as improving soil conditions, increasing crop cultivation efficiency, or as a fertilizer to enhance crop efficacy.

Description

[Technical Field]

[0001] This application claims priority to Republic of Korea Patent Application No. 10-2023-0041710, filed on 30 March 2023, and Republic of Korea Patent Application No. 10-2023-0124308, filed on 18 September 2023, and the entirety of the above specification is a reference to this application.

[0002] Regarding novel bacterial strains and their applications. [Background technology]

[0003] Recently, the excessive use of organic and chemical fertilizers has led to numerous problems, including soil contamination, continuous cropping problems, soil nutrient imbalances, and pest and disease outbreaks, resulting in side effects such as reduced crop quality and decreased farmers' incomes. Nitrogen (N), an essential plant nutrient, has been supplied through the use of such chemical fertilizers, but the excessive use of chemical fertilizers to maintain even higher yields contradicts the principles of sustainable agriculture, which requires high productivity with low inputs, and also causes environmental problems.

[0004] To address these problems, biological methods that minimize the burden on the agricultural environment and ecosystems while ensuring safe food are being actively researched as alternatives to chemical fertilizers. As a result, interest in and the need for microbial fertilizers that can promote crop growth are increasing.

[0005] Such microbial fertilizers have the advantage of not causing environmental problems such as soil contamination due to salt accumulation, are excellent at maintaining ecosystem stability, and have the benefit of sustainably improving crop productivity. With increasing interest in environmentally friendly and sustainable agriculture, research on agriculturally beneficial microorganisms is steadily being conducted.

[0006] Currently, a wide variety of agricultural microorganisms are commercially available, but there is little or no verification of their precise crop-benefiting activity. Furthermore, given the decreasing number of currently available chemical pesticides and fertilizers, there is a need to develop verified agricultural microorganism formulations that can serve as alternatives.

[0007] As a result, the inventors excavated a novel Bacillus megatherium HyangYak-01 strain, confirmed the strain's remarkably superior effects in increasing the diversity of microbiome species and improving the physicochemical properties of soil in crop cultivation areas, and in promoting crop growth and biosynthesis of physiologically active substances. The inventors also confirmed the remarkably superior skin condition improvement and anti-inflammatory effects of Centella asiatica cultivated with the strain, thereby completing the present invention. [Overview of the project] [Problems that the invention aims to solve]

[0008] One embodiment is to provide a novel strain of Bacillus megaterium HyangYak-01 (also known as Priestia megaterium HyangYak-01).

[0009] Another embodiment is to provide a microbial preparation comprising the bacterial strain, its lysate or culture, or an extract of the bacterial strain, its lysate or culture.

[0010] Another embodiment is to provide a microbial fertilizer containing the microbial preparation.

[0011] Another embodiment is to provide a method for cultivating plants using the aforementioned strain, microbial preparation, or microbial fertilizer.

[0012] Another embodiment is to provide plants cultivated by the method described above.

[0013] Another embodiment provides a cosmetic composition for improving skin condition, a topical skin preparation composition, or a food composition containing Centella asiatica cultivated by the method described above or an extract thereof.

[0014] Another embodiment provides a pharmaceutical composition for the prevention or treatment of inflammatory skin diseases, comprising Centella asiatica cultivated by the above method or an extract thereof as an active ingredient.

[0015] Another embodiment provides a method for improving the skin condition of an individual or preventing, improving, or treating an inflammatory skin disease in an individual, comprising the step of administering an effective amount of the composition to the individual in need.

[0016] Another aspect is to provide a use for producing the bacterial strain, its lysate or culture, or an extract of the bacterial strain, its lysate or culture. [Means for solving the problem]

[0017] One embodiment provides a strain of Bacillus megaterium.

[0018] The scientific name Priestia megaterium can be used as the same name as the aforementioned Bacillus megaterium. Therefore, the aforementioned strain can also be referred to as the Priestia megaterium strain.

[0019] The strain is also isolated. As used herein, the term "isolated" means that it is not in its natural state and can be artificially separated and utilized from nature or the like. The strain is also isolated from the soil. Specifically, the strain is isolated by a method of pure separation and culture of colonies cultured by inoculating a continuously diluted (serial dilution) rhizosphere soil of a crop cultivation field into a TSB (Tryptic Soy Broth) agar medium or an R2A (Reasoner's 2A) agar medium.

[0020] The strain also belongs to the genus Bacillus (Bacillus sp.).

[0021] The strain also contains 16S rRNA containing the nucleotide sequence of SEQ ID NO: 1. Specifically, the strain also contains 16S rRNA containing a nucleotide sequence having a sequence identity of about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, about 99.5% or more, or about 99.9% or more with SEQ ID NO: 1. The strain also contains 16S rRNA having the nucleotide sequence of SEQ ID NO: 1.

[0022] As used herein, the term "sequence identity" means the degree of identity of amino acid residues or bases between sequences after aligning the two sequences to maximize their match in a specific comparison region. Sequence identity can be confirmed by methods known in the art. The percentage of the sequence identity is determined using known sequence comparison programs, and examples include BLASTN (NCBI), CLC Main Workbench (CLC bio), MegAlignTM (DNASTAR Inc), etc.

[0023] The Bacillus megaterium strain is also the strain deposited under the accession number KCCM13074P. The strain deposited under the above accession number is also the Bacillus megaterium HyangYak-01 (alias: Priestia megaterium HyangYak-01) strain.

[0024] The strain also has an effect of improving soil conditions, promoting plant growth, or promoting the biosynthesis of plant bioactive substances. Specifically, the plant is a plant cultivated for human use purposes, that is, a crop, but is not limited thereto. More specifically, the plant is a plant cultivated in a dry environment (e.g., fields, gardens, flower pots, mountains, etc.) (e.g., horticultural plants, field crops, mountain crops, etc.), submerged or flooded cultivated crops (e.g., paddy crops such as rice), hydroponic crops, and aquatic plants, and is also one or more selected from the group consisting of all monocotyledonous and dicotyledonous plants, or all herbaceous and woody plants. For example, the plant is also one or more selected from the group consisting of Centella asiatica, Angelica gigas, Platycodon grandiflorus, Ophiopogon japonicus, Cynanchum wilfordii, Eleusine coracana, and Glycyrrhiza glabra, but is not limited thereto.

[0025] The term "Centella asiatica" used in this specification refers to a perennial creeping medicinal plant mainly inhabiting South Asia, which grows in groups in the low wetlands of Jeju Island and the south-central region of Korea. The main morphological characteristics of the Centella asiatica include that the main stem extends horizontally, and there are about two degenerated scaly leaves near the nodes where roots emerge. The leaves are densely arranged at the nodes, kidney-shaped, with a diameter of about 2 - 5 cm, the surface is shiny, there are blunt serrations on the edge, and the length of the leaf stalk is about 4 - 20 cm, etc., but is not limited thereto.

[0026] The term "Angelica gigas" used in this specification refers to a perennial herb belonging to the Apiaceae family, distributed in Korea, China, Japan, and other regions. For reference, it is also known as Korean Angelica or Sin Gan Chae. The main morphological characteristics of Angelica gigas include a height of approximately 1-2m, a purplish stem, and purplish flowers, but these are not the only characteristics.

[0027] The term "bellflower (scientific name: Platycodon grandiflorum)" as used in this specification refers to a perennial herb of the genus Platycodon in the family Campanulaceae. It is also called Kikyo, and other kanji names include Oshobo, Meibasai, and Doraji. The main morphological characteristics of the aforementioned bellflower include a height of approximately 40-100 cm, a thick root, a stem that branches into single or multiple branches, a white sap that oozes out when the main stem is cut, and alternate or opposite leaves that are oblong-ovate, approximately 5-12 cm long, narrow at both ends, and have serrated edges, but are not limited to these characteristics.

[0028] The term "Codonopsis lanceolata" as used in this specification refers to a perennial herbaceous plant belonging to the family Campanulaceae, native to Korea, and also distributed in Japan, China, Manchuria, and Ussuri. It is also known as Shasam or Shanhailuo. The main morphological characteristics of Codonopsis lanceolata include, but are not limited to, its length of approximately 2-5 m, its leaves which are long and oval in shape, with about four leaves clustered closely together at the tip of short branches, approximately 3-10 cm long and 1.5-4 cm wide, with smooth margins and a green upper surface but a powdery white underside, its flowers which are light green on the outside and have brown spots on the inside, its roots which are thick like those of a bellflower, and the fact that a white sap is released when the vine is cut.

[0029] The term "Kawarasaiko (scientific name: Potentilla chinensis)" as used herein refers to a perennial herbaceous plant belonging to the rose family, also known as wilting vegetable or Dongnokupuru. The main morphological characteristics of Kawarasaiko include, but are not limited to, growing to a height of approximately 30-60 cm, having thick roots, pinnately compound leaves that are dark green on the upper surface and densely hairy and white on the underside, and yellow flowers.

[0030] The term "Hikiokoshi (scientific name: Isodon japonicus)" as used herein refers to a perennial herb belonging to the Lamiaceae family of the Lamiales order of dicotyledonous plants. The main morphological characteristics of Hikiokoshi include, but are not limited to, a height of approximately 50-100 cm, the presence of an underground rhizome, an erect stem with square ridges and downward-pointing hairs, and numerous branches; opposite leaves that are broadly ovate, approximately 6-15 cm long and 3.5-8 cm wide, with serrated edges, tapering sharply at the base to form a petiole wing; and pale purple flowers that bloom in a panicle.

[0031] The term "licorice (scientific name: Glycyrrhiza uralensis)" as used in this specification refers to a perennial herb belonging to the Fabaceae family of the Rosales order of dicotyledonous plants. The main morphological characteristics of licorice include, but are not limited to, the following: the roots are reddish-brown and penetrate deep into the ground; the stems are angular, erect to about 1 m in height, densely covered with white hairs that give them a grayish-white appearance, and have scattered glandular dots; the leaves are alternate, odd-pinnately compound, with about 7 to 17 leaflets each, ovate, pointed at the tip, about 2 to 5 cm long and 1 to 3 cm wide, with white hairs and glandular dots on both sides and no serrations; and the flowers are about 1.4 to 2.5 cm long, purple, and borne in racemes in the leaf axils.

[0032] The aforementioned soil condition improvement may include increasing the diversity of microbial species in the soil microbiome or improving the physicochemical properties of the soil.

[0033] The physiologically active substances of the aforementioned plants may include one or more selected from the group consisting of chlorophyll, madecassic acid, and asiatic acid.

[0034] As used in this specification, the term "microbiome" refers to an ecological environment composed of microorganisms.

[0035] As used herein, the term "physiochemical property of soil" refers to the physical or chemical properties of soil (e.g., pH, amount of organic matter, amount of available phosphorus, amount of exchangeable cations, electrical conductivity, etc.), and as used herein, the term "improvement of soil physicochemical properties" refers to the improvement of the physical or chemical properties of soil to a state more suitable for plant cultivation or growth. Specifically, the improvement of soil physicochemical properties includes: maintaining the soil pH at a slightly acidic (pH 6.0-6.5) or mildly acidic (pH 6.5-7.0); promoting the decomposition of organic matter in the soil; inducing an increase in the amount of exchangeable cations in the soil; and raising the soil's electrical conductivity to a normal level (approximately 2 dsm). -1 Below or approximately 1-2 dsm -1 ) to maintain; and the amount of available phosphorus in the soil to normal levels (approximately 400-500 mg / kg) -1 ) may include one or more selected from the group consisting of;

[0036] As used herein, the term "growth" refers to an increase in the number of cells that make up an organism, resulting in an increase in the size or weight of the organism.

[0037] As used herein, the term "biologically active substance" refers to an organic substance or inorganic ion that affects the life processes of living organisms even in very small amounts.

[0038] As used herein, the term "biosynthesis" refers to the metabolic process by which organic substances are synthesized within a living organism through the action of cells.

[0039] In one example, it was confirmed that the microbiome species diversity significantly increased in crop cultivation soil treated with the Bacillus megatherium HyangYak-01 strain compared to untreated soil. Therefore, when the strain is applied to soil, the soil becomes more vigorous for crop cultivation due to increased microbiome species diversity, which can promote the growth of crops grown in that soil.

[0040] According to one example, in crop cultivation soil treated with the Bacillus megatherium HyangYak-01 strain, the physicochemical properties of the soil were further improved compared to untreated soil (maintaining the soil pH at a slightly acidic or mildly acidic level; promoting the decomposition of organic matter in the soil; increasing the amount of exchangeable cations in the soil; and maintaining normal levels of electrical conductivity and available phosphorus in the soil), making it more vigorous for growing crops, and as a result, the growth of crops grown in said soil may be further promoted.

[0041] In one example, when plants were cultivated after being treated with the Bacillus megatherium HyangYak-01 strain, it was confirmed that root length, root weight (both live weight and dry weight), leaf length, leaf weight (both live weight and dry weight), leaf width, leaf number, and chlorophyll content all increased significantly compared to an untreated control group. Therefore, the strain may have the effect of significantly promoting or improving plant growth (for example, one or more selected from root length, root weight, leaf length, leaf weight, leaf width, leaf number, and chlorophyll content).

[0042] In one example, when Centella asiatica was cultivated after being treated with the Bacillus megatherium HyangYak-01 strain, it was confirmed that the content of one or more substances selected from the group consisting of chlorophyll, madecasic acid, and asiatic acid in the Centella asiatica was significantly increased compared to the untreated control group. Therefore, the strain may exert an effect of improving the physiologically active substances of plants (for example, one or more substances selected from the group consisting of chlorophyll, madecasic acid, and asiatic acid), that is, an effect of promoting the biosynthesis of physiologically active substances in plants.

[0043] The aforementioned strain also possesses one or more of the following activities: indole-3-acetic acid (IAA) production activity; nitrogen fixation activity; phosphate solubilization activity; urea hydrolase activity; and denitrification activity.

[0044] As used herein, the term "nitrogen fixation" refers to the process of converting nitrogen gas molecules (N) in the air into nitrogen compounds, including ammonia (NH), that can be used by plant cells.

[0045] As used herein, the term "phosphate solubilization" refers to the process of making poorly soluble phosphates more readily available to plants and microorganisms through H2PO4. - HPO4 2- This refers to the process of converting substances into ionic forms such as ions.

[0046] As used herein, the term "urease activity" refers to activity that exhibits the same effect as urease, that is, activity that hydrolyzes urea. Specifically, it refers to the activity that hydrolyzes urea by the production of urease by a bacterial strain. Therefore, the term "urease activity" can be used interchangeably with terms such as "urea hydrolysis activity" and "urease production activity."

[0047] As used herein, the term "denitrification" refers to the process of reducing nitrate nitrogen to nitrogen gas (N2).

[0048] The aforementioned strain also possesses one or more genes selected from the group consisting of genes involved in crop growth promoting activity, such as genes related to root growth promoting hormone production, genes related to nitrogen fixation, genes related to phosphate solubilization, genes related to urea hydrolase, and genes related to denitridation. In one example, the aforementioned strain also possesses one or more genes selected from the group consisting of gatA, puuPER, aldH, ytnP, phoP, phoR, phoH, nifS, narT, nirCQ, norRMG, ureGFECBDARI, and ureDGFECBA.

[0049] (blank)

[0050] Another embodiment provides a lysate or culture of the Bacillus megatherium strain, or an extract of the strain, lysate, or culture.

[0051] The specific details of the aforementioned strain are as described above.

[0052] In this specification, the term “lysate” is used interchangeably with “solubil” and means the product obtained by disrupting the cell walls of a bacterial strain by chemical or physical force. The lysate may include the lysate itself, its concentrate, or its freeze-dried form.

[0053] In this specification, the term "culture" is used interchangeably with "culture medium," "supernatant," "conditional culture medium," or "adjusted medium," and refers to the entire culture medium containing the Bacillus megatherium strain, its metabolites, and excess nutrients, obtained by culturing the strain for a certain period of time in a medium that supplies nutrients for the growth and survival of the Bacillus megatherium strain in a test tube. Furthermore, "culture" refers to the culture medium obtained by removing the bacterial cells from the bacterial cell culture medium obtained by culturing the strain. On the other hand, the liquid from which the bacterial cells have been removed from the culture medium is also called the "supernatant," and can be obtained by letting the culture medium stand for a certain period of time and taking only the upper liquid after removing the portion that has settled to the bottom, by removing the bacterial cells through filtration, or by centrifuging the culture medium to remove the lower precipitate and taking only the upper liquid. The term "bacterial cells" refers to the bacterial strain of the present invention itself, and includes the bacterial strain itself that has been isolated and selected from a sample, or the bacterial strain that has been cultured and isolated from the culture medium. The aforementioned bacterial cells can be obtained by centrifugating the culture medium and taking the portion that has settled to the bottom, or, since they settle to the bottom layer of the culture medium due to gravity, by letting it stand for a certain period of time and then removing the upper liquid.

[0054] The culture may include the culture medium itself obtained by culturing the bacterial strain, its concentrate or freeze-dried form, or the culture supernatant obtained by removing the bacterial strain from the culture medium, its concentrate or freeze-dried form.

[0055] The culture medium and conditions for culturing the Bacillus megatherium strain can be appropriately selected or modified by a person with ordinary knowledge. For example, the culture solution may be obtained by culturing in a medium (e.g., R2A medium) at a temperature of approximately 20-40°C (e.g., approximately 30°C) for a certain period of time, for example, approximately 30-80, 40-80, or 48-72 hours.

[0056] In one embodiment, the culture supernatant of the bacterial strain is obtained by removing the bacterial cells from the culture solution of the bacterial strain by centrifuging or filtering it.

[0057] In other specific examples, the concentrate is obtained either from the bacterial strain culture solution itself, or by centrifuging or filtering the culture solution and then concentrating the resulting supernatant.

[0058] In this specification, the term “extract from a strain, lysate, or culture” means an extract obtained from the strain, lysate, or culture, or a concentrate thereof, and may include extracts, diluted or concentrated extracts, dried extracts obtained by drying the extract, or crude or purified products thereof, or fractions obtained by fractionating them.

[0059] The aforementioned bacterial strain, its lysate or culture, or an extract of the aforementioned bacterial strain, lysate or culture, is also biologically pure. The aforementioned bacterial strain is also obtained by pure culture.

[0060] Of the terms or elements mentioned in the “lysate, culture, or extract” section, those mentioned in the description relating to “bacterial strain” are understood to be the same as those mentioned in the description relating to “bacterial strain” above.

[0061] (blank)

[0062] Further embodiments provide uses for the Bacillus megatherium strain, its lysate or culture, or extracts of the strain, lysate or culture.

[0063] Further embodiments provide a microbial preparation containing the Bacillus megatherium strain, its lysate or culture, or an extract of the strain, lysate or culture as an active ingredient, and uses thereof.

[0064] Another embodiment provides a microbial fertilizer containing the microbial preparation as an active ingredient and its uses.

[0065] In the aforementioned uses, or in microbial preparations or microbial fertilizers, the Bacillus megatherium strain is also the Bacillus megatherium strain according to the aforementioned embodiment. Therefore, the strain is also the Bacillus megatherium HyangYak-01 strain.

[0066] The uses of the aforementioned strain, its crushed liquid or culture, or an extract of the aforementioned strain, crushed liquid or culture; or the aforementioned microbial preparation or microbial fertilizer may include improving soil conditions, promoting plant growth, or promoting the biosynthesis of physiologically active substances in plants. Furthermore, the aforementioned strain, its crushed liquid or culture, or an extract of the aforementioned strain, crushed liquid or culture; or the aforementioned microbial preparation or microbial fertilizer may have uses for cultivating plants, including crops (for example, Centella asiatica). The specific details of the aforementioned plants are as described above.

[0067] The form of the microbial formulation is not particularly limited, but may be provided as a microbial pesticide or microbial fertilizer, and more preferably as a seed coating agent, soil conditioner, compost accelerator, foliar spray, or irrigation spray. Specifically, the microbial formulation may be formulated for the aforementioned uses by conventional methods and manufactured in dry powder form or liquid fertilizer form. Specifically, the microbial formulation may be manufactured in liquid fertilizer form and used in powder form with the addition of a filler, or it may be formulated into granules, but is not particularly limited to such formulation. Preferably, it may be formulated as a biofertilizer to replace chemical fertilizers, that is, it may be formulated as a biofertilizer to overcome limitations in environmentally friendly organic farming where the supply of chemical fertilizers is limited.

[0068] The microbial preparation can be manufactured by adding additives such as excipients, bulking agents, and nutrients to a bacterial strain, its lysate or culture, or an extract of the bacterial strain, its lysate or culture. In this case, one or more additives selected from the group consisting of polycarboxylates, sodium ligninsulfonate, calcium ligninsulfonate, sodium dialkylsulfosuccinate, sodium alkylarylsulfonate, polyoxyethylene alkylphenyl ether, sodium polyphosphate, polyoxyethylene alkylaryl phosphate ester, polyoxyethylene alkylaryl ether, polyoxyethylene alkylaryl polymer, polyoxyalkylone alkylphenyl ether, polyoxyethylene nonylphenyl ether, sodium sulfonate, naphthalene formaldehyde, Triton 100, and Tween 80 may be used as fillers and nutrients, one or more selected from the group consisting of skim milk powder (culture medium), soy flour, rice, wheat, loess, diatomaceous earth, bentonite, dextrin, glucose, and starch may be used as fillers and nutrients, and one or more selected from the group consisting of bentonite, talc, diamondite, kaolin, and calcium carbonate may be used as disintegrants.

[0069] In this specification, the term "microbial fertilizer" refers to a product that produces a fertilizer-like effect by pure culturing microorganisms that have a beneficial effect on the growth of plants such as crops, mixing them with seeds, and then sowing the seeds or inoculating them directly into the soil.

[0070] The microbial fertilizer may have a dosage form selected from the group consisting of seed coating agents, seed dipping agents, soil conditioners, compost accelerators, foliar sprays, and irrigation sprays, and may be manufactured or formulated for the aforementioned uses by conventional methods known in the art.

[0071] The microbial fertilizer contains a carrier or excipient used in conventional plant pesticides, and preferably, bentonite, surfactants, talc, zeolite, etc., can be used as the carrier or excipient.

[0072] Of the terms or elements referred to as “uses, microbial preparations, or microbial fertilizers” above, those referred to in the description relating to “bacterial strains” or “lysates, cultures, or extracts” are understood to be the same as those referred to in the description relating to “bacterial strains” or “lysates, cultures, or extracts” above.

[0073] (blank)

[0074] Another embodiment provides a method for cultivating plants, comprising the step of treating one or more of the following with the Bacillus megatherium strain, microbial preparation, or microbial fertilizer: soil, plants, and plant seeds.

[0075] The aforementioned method is also intended for improving soil conditions, promoting plant growth, or facilitating the biosynthesis of physiologically active substances in plants.

[0076] Therefore, further embodiments provide a method for improving soil conditions, promoting plant growth, or promoting the biosynthesis of physiologically active substances in plants, comprising the step of treating one or more of the following with the Bacillus megatherium strain, microbial preparation, or microbial fertilizer: soil, plants, and plant seeds.

[0077] The aforementioned plants are one or more species selected from the group consisting of Centella asiatica, Angelica acutiloba, Platycodon grandiflorus, Codonopsis lanceolata, Bupleurum falcatum, Isodon japonicus, and Glycyrrhiza uralensis, but are not limited to these, and the specific contents of the aforementioned plants are as described above.

[0078] The treatment method in the aforementioned treatment step can be carried out by commonly used methods, namely, spraying (e.g., misting, atomization, powder application, granule application, water surface application, continuous application, etc.), soil application (e.g., mixing, irrigation, sowing, etc.), surface application (e.g., coating, smearing, covering, etc.), immersion, poisoning, fumigation, etc., but is not limited thereto. In one embodiment, in the aforementioned method, the treatment of plant seeds with the aforementioned strain, microbial preparation, or microbial fertilizer can be carried out by mixing the aforementioned strain, microbial preparation, or microbial fertilizer with the plant seeds and then sowing them.

[0079] The amount of Bacillus megatherium strain, microbial preparation, or microbial fertilizer used can be appropriately determined based on its formulation, purpose, application method, application location, application area, cultivation yield, soil condition, plant characteristics, or condition. For example, the effective amount of microorganisms treated by the above method is calculated based on the cultivated land area (m²). 2 ) Approximately 1 to 1 x 10 100 The microbial population (or CFU (colony forming unit)) may be, but is not limited to, a certain number of microorganisms.

[0080] Another embodiment provides plants cultivated by the method described above.

[0081] The aforementioned plants are one or more species selected from the group consisting of Centella asiatica, Angelica acutiloba, Platycodon grandiflorus, Codonopsis lanceolata, Bupleurum falcatum, Isodon japonicus, and Glycyrrhiza uralensis, but are not limited to these, and the specific contents of the aforementioned plants are as described above.

[0082] In one example, plants cultivated by the method described above exhibit further enhanced growth compared to plants not cultivated by the method described above (for example, plants cultivated without treatment with the Bacillus megatherium strain, microbial preparation, or microbial fertilizer), resulting in a further improvement in growth levels (e.g., an increase in one or more levels selected from root length, root weight, leaf length, leaf weight, leaf width, leaf number, and chlorophyll content), or further enhanced biosynthesis of physiologically active substances, resulting in a further improvement in the content of physiologically active substances (e.g., one or more selected from the group consisting of chlorophyll, madecasic acid, and asiatic acid).

[0083] According to one example, in the case of Centella asiatica cultivated by the method described above, the chlorophyll content increases by approximately 5-70, 5-35, 10-60, 10-30, 20-60, 20-30, or 30-60% compared to Centella asiatica cultivated without treatment with the Bacillus megatherium strain, microbial preparation, or microbial fertilizer, or the madecasic acid content increases by approximately 1-50, 5-50, or 10-5 The asiatic acid content increases by 0, 30-50, 1-40, 5-40, 10-40, 30-40, 1-30, 5-30, or 10-30%, or the asiatic acid content increases by approximately 1-60, 5-60, 10-60, 30-60, 1-50, 5-50, 10-50, 30-50, 1-40, 5-40, 10-40, 30-40, 1-30, 5-30, or 10-30%.

[0084] Any terms or elements referred to in the aforementioned “Method or Plant” that are referred to in the descriptions of “Strain,” “Liquid, Culture, or Extract,” or “Use, Microbial Preparation, or Microbial Fertilizer,” are understood to be the same as those referred to in the aforementioned descriptions of “Strain,” “Liquid, Culture, or Extract,” or “Use, Microbial Preparation, or Microbial Fertilizer.”

[0085] (blank)

[0086] Another embodiment provides uses for Centella asiatica or an extract thereof cultivated by the above method.

[0087] Further embodiments provide cosmetic compositions, topical skin preparation compositions, or food compositions containing Centella asiatica or an extract thereof cultivated by the above method as an active ingredient, and uses thereof.

[0088] The aforementioned uses may include improving skin condition. This improvement in skin condition may also include strengthening the skin barrier or reducing inflammation (or suppressing (or alleviating) skin inflammation).

[0089] The Centella asiatica extract (Centella asiatica extract) includes the extract itself and all dosage forms that can be formed using the extract, such as the extract obtained by extracting Centella asiatica, diluted or concentrated solutions of the extract, dried products obtained by drying the extract, preparations or purified products of the extract, or mixtures thereof.

[0090] The Centella asiatica extract (Centella asiatica extract) can be extracted from natural, hybrid, or variety plants of Centella asiatica, and can also be extracted from plant tissue cultures.

[0091] The method for extracting Centella asiatica to produce the Centella asiatica extract is not particularly limited and can be performed by methods commonly used in the art. Non-limiting examples of the extraction method include hot water extraction, ultrasonic extraction, filtration, reflux extraction, etc., which can be performed alone or in combination of two or more methods. The type of extraction solvent used for the extraction is not particularly limited and any solvent known in the art can be used. Non-limiting examples of the extraction solvent include, but are not limited to, one or more selected from the group consisting of water, C1-C4 anhydrous or lower alcohols, a mixed solvent of water and lower alcohol, acetone, 1,3-butylene glycol, ethyl acetate, chloroform, ethanol, etc., which can be used alone or in combination of two or more. More specifically in the present invention, ethanol (e.g., about 60-100, 70-100, 60-80, or 70% ethanol) can be used as the extraction solvent. A solvent extract can be produced by extracting Centella asiatica once or more times using the aforementioned solvent, and a dry extract can be produced by vacuum distillation of the solvent extract followed by freeze-drying or spray-drying. Furthermore, the extract obtained by heat extraction or cold extraction can be used as is, or dried by freeze-drying, hot-air drying, spray-drying, etc., to remove suspended solid particles, for example by filtering the particles using nylon or by cryofiltration, in order to remove them.

[0092] According to one example, Centella asiatica cultivated by the method described above, or its extract or composition, may exhibit one or more of the following characteristics: increased expression induction of skin barrier strengthening factors such as filaggrin (FLG), claudin-1 (CLD1), or a combination thereof; and inhibition of expression induction of proinflammatory cytokines, which are pro-inflammatory factors.

[0093] In this specification, the term “proinflammatory cytokine” may mean a cytokine that induces or promotes inflammation, or thereby exacerbates a disease.

[0094] The aforementioned inflammatory cytokines may be, but are not limited to, interleukin-1 alpha (IL-1α), interleukin-1 beta (IL-1β), or a combination thereof.

[0095] Therefore, the Centella asiatica or its extract, or the composition thereof, can significantly increase the expression of skin barrier strengthening factors or significantly suppress the expression of inflammatory cytokines that induce inflammation (or skin inflammation), thereby exhibiting excellent skin condition improvement effects (e.g., skin barrier strengthening effects) or anti-inflammatory (or anti-inflammatory or anti-inflammatory (or alleviating) skin inflammation) effects.

[0096] In this specification, the term "expression" means gene expression, and refers to all or part of the process by which the genetic information constituting DNA, i.e., genes, are formed into the diverse proteins that make up an organism. For example, such gene expression includes the expression of DNA, RNA (e.g., mRNA), proteins, or combinations thereof, and more specifically, may include the transcription of DNA into RNA, the translation of RNA into proteins, or combinations thereof. For example, in this specification, the term "increased expression" means an increase in the expression of a protein, or the DNA or RNA (e.g., mRNA) that codes for it.

[0097] The aforementioned "strengthening of the skin barrier" means all actions that improve the function of the skin barrier, which is located on the outermost layer of the skin and prevents moisture and nutrient loss, or all actions that suppress or restore damage to the skin barrier function.

[0098] The aforementioned damage to the skin barrier function means all changes that appear on the skin as a result of a decrease or damage to the function of the skin barrier. These may include, for example, increased wrinkles, dryness, dermatitis, atopic dermatitis, allergic dermatitis, and acne.

[0099] The term "anti-inflammatory" refers to all actions that suppress or inhibit the inflammatory response.

[0100] Another embodiment provides a pharmaceutical composition for the prevention, improvement, or treatment of inflammatory skin diseases, comprising Centella asiatica cultivated by the above method or an extract thereof as an active ingredient.

[0101] The aforementioned inflammatory skin disease is one of the following selected conditions: dermatitis, atopic dermatitis, pruritus, eczematous skin disease, dry eczema, erythema, urticaria, psoriasis, drug eruption, and acne.

[0102] Centella asiatica cultivated by the above method or its extract, or the above-mentioned pharmaceutical composition, can prevent, improve, or treat inflammatory skin diseases by significantly suppressing the expression of inflammatory cytokines that induce inflammation (or skin inflammation).

[0103] In this specification, the term “prevention” includes suppressing the occurrence of a disease. In this specification, the term “treatment” includes suppressing, mitigating, or eliminating the development of a disease. In this specification, the term “improvement” means all actions relating to the parameters of alleviating or treating a condition, for example, all actions that at least reduce the severity of a symptom.

[0104] The composition is present in an amount of about 0.001% to 80% by weight relative to the total weight of the composition, for example, about 0.01% to 60% by weight, about 0.01% to 40% by weight, about 0.01% to 30% by weight, about 0.01% to 20% by weight, about 0.01% to 10% by weight, about 0.01% to 5% by weight, about 0.05% to 60% by weight, about 0.05% to 40% by weight, about 0.05% by weight The product may contain Centella asiatica or an extract cultivated by the aforementioned method in amounts of 30% by weight, approximately 0.05% to 20% by weight, approximately 0.05% to 10% by weight, approximately 0.05% to 5% by weight, approximately 0.1% to 60% by weight, approximately 0.1% to 40% by weight, approximately 0.1% to 30% by weight, approximately 0.1% to 20% by weight, approximately 0.1% to 10% by weight, or approximately 0.1% to 5% by weight. In this case, if the content of Centella asiatica or an extract cultivated by the aforementioned method falls outside the aforementioned range, the skin condition improving effect, such as the skin barrier strengthening effect or the anti-inflammatory effect, will be reduced or not fully exerted.

[0105] In this specification, the term "contains as an active ingredient" means that Centella asiatica cultivated by the method described above or its extract is added in an effective amount sufficient to produce the effects described above, and includes the addition of various components as auxiliary components for drug delivery and stabilization, and formulation into various forms.

[0106] The composition may be in a liquid state or a dry state. In one embodiment, the composition may also be in the form of a dry powder.

[0107] The drying method for producing the composition in a dry state is not particularly limited and may be a method commonly used in the industry. Non-limiting examples of such drying methods include air drying, natural drying, spray drying, and freeze-drying. These methods may be used individually or in combination of at least two methods.

[0108] The composition may contain an effective amount of additives sufficient to reduce the deterioration of Centella asiatica or its extract cultivated by the method described above. The additives may, but are not limited to, binders.

[0109] The composition may further comprise a carrier that is cosmetically, pharmaceutically, or food-grade acceptable. The composition may be formulated together with the carrier and provided as a cosmetic, pharmaceutical, food additive, or the like.

[0110] The cosmetic composition may further include, in addition to the active ingredients disclosed herein, ingredients commonly used in cosmetic compositions, functional additives, etc., such as antioxidants, stabilizers, solubilizers, surfactants, dispersants, emulsifiers, preservatives, vitamins, pigments, fragrances, and other common auxiliary agents, as well as carriers.

[0111] The cosmetic composition is not particularly limited to a specific dosage form, and the dosage form can be appropriately selected depending on the purpose. The cosmetic composition may also have, for example, a solubilizing agent type, an emulsifying agent type, or a dispersing agent type. The cosmetic composition may also have, but is not limited to, a softening lotion, a nourishing lotion, a massage cream, a nourishing cream, an essence, a pack, a gel, an ampoule, or a skin-adhesive type cosmetic dosage form.

[0112] The aforementioned cosmetic composition is also a cosmetic raw material composition used in the manufacture of cosmetics. Furthermore, the aforementioned cosmetic composition is a cosmetic composition having a final cosmetic dosage form.

[0113] The aforementioned topical skin preparation may have the dosage form of a cream, gel, ointment, skin emulsifier, skin suspension, transdermal patch, drug-containing bandage, lotion, or a combination thereof. The aforementioned topical skin preparation may be appropriately formulated as needed with ingredients commonly used in topical skin preparations such as cosmetics and pharmaceuticals, such as aqueous components, oily components, powder components, alcohols, humectants, thickeners, UV absorbers, whitening agents, preservatives, antioxidants, surfactants, fragrances, colorants, various skin nutrients, or combinations thereof. The aforementioned topical skin preparation may also appropriately contain chelating agents such as disodium edetate, trisodium edetate, trisodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid; drugs such as caffeine, tannins, verapamil, licorice extract, glabridin, quince fruit hot water extract, various herbal medicines, tocopherol acetate, glycyrrhizic acid, tranexamic acid, and their derivatives or salts; vitamin C, magnesium ascorbyl phosphate, ascorbyl glucoside, arbutin, and kojic acid; and sugars such as glucose, fructose, and trehalose.

[0114] The aforementioned food composition is also a functional food composition. In that case, it can be formulated into a dosage form of a typical functional food known in the art.

[0115] The aforementioned functional health food composition may use Centella asiatica or an extract thereof cultivated by the aforementioned method, or may be used in combination with other foods or food ingredients, and may be used appropriately in the usual manner. The amount of active ingredients mixed may be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment). There are no special restrictions on the types of functional health foods. Among the types of functional health foods, beverage compositions may include various flavorings, sweeteners, or natural carbohydrates as additional ingredients, like ordinary beverages. The natural carbohydrates include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; and polysaccharides such as dextrin and cyclodextrin; and sugar alcohols such as xylitol, sorbitol, and erythritol. As sweeteners, natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame may be used. The food composition may contain nutrients, vitamins, electrolytes, flavorings, colorings, pectin acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages, or combinations thereof. The food composition may also contain fruit pulp for the manufacture of natural fruit juices, fruit juice beverages, vegetable beverages, or combinations thereof.

[0116] The pharmaceutical composition may further comprise a pharmaceutically acceptable diluent or carrier. The diluent is lactose, corn starch, soybean oil, microcrystalline cellulose, mannitol, or a combination thereof. The carrier may also be an excipient, disintegrant, binder, lubricant, or a combination thereof. The excipient may also be microcrystalline cellulose, lactose, low-substituted hydroxycellulose, or a combination thereof. The disintegrant may be carboxymethylcellulose calcium, starch glycolate sodium, anhydrous calcium hydrogen phosphate, or a combination thereof. The binder may also be polyvinylpyrrolidone, low-substituted hydroxypropylcellulose, hydroxypropylcellulose, or a combination thereof. The lubricant may also be magnesium stearate, silicon dioxide, talc, or a combination thereof.

[0117] The aforementioned pharmaceutical composition may be formulated into oral or parenteral dosage forms. Oral dosage forms include granules, powders, liquids, tablets, capsules, and dried syrups. Parenteral dosage forms include injections and ointments.

[0118] Of the terms or elements mentioned in the “Uses or Compositions” section, those mentioned in the descriptions relating to “strains,” “lysates, cultures, or extracts,” “uses, microbial formulations, or microbial fertilizers,” or “methods or plants” are understood to be the same as those mentioned in the descriptions relating to “strains,” “lysates, cultures, or extracts,” “uses, microbial formulations, or microbial fertilizers,” or “methods or plants.”

[0119] (blank)

[0120] Another embodiment provides a method for preventing, improving, or treating the condition of an individual, comprising the step of administering an effective amount of the composition to an individual in need.

[0121] The aforementioned condition of the individual may also be a condition relating to the skin, such as a condition relating to damage to the skin barrier function or skin inflammation.

[0122] In this specification, the terms “administer,” “introduce,” and “implant” are used interchangeably and mean the placement of one embodiment of a composition into an individual by a method or route that results in at least partial localization of one embodiment of the composition to a desired site.

[0123] The drug may be administered by methods known to the art. It may be administered directly to the individual by any means, for example, intravenously, intramuscularly, orally, transdermally, mucousally, intranasally, intratracheally, or subcutaneously. The drug may be administered systemically or topically. The drug may include topical application to the skin.

[0124] The individual may also be a mammal, such as a human, or a non-human mammal (such as a cattle, horse, pig, dog, sheep, goat, or cat). The individual may also be an individual that requires prevention, improvement, or treatment of a skin condition, such as skin barrier strengthening or prevention, improvement, or treatment of skin inflammation.

[0125] The aforementioned administration also involves administering approximately 0.1 mg to 1,000 mg of the composition according to one embodiment per individual, for example, approximately 0.1 mg to 500 mg, approximately 0.1 mg to 100 mg, approximately 0.1 mg to 50 mg, approximately 0.1 mg to 25 mg, approximately 1 mg to 1,000 mg, approximately 1 mg to 500 mg, approximately 1 mg to 100 mg, approximately 1 mg to 50 mg, approximately 1 mg to 25 mg, approximately 5 mg to 1,000 mg, approximately 5 mg to 500 mg, approximately 5 mg to 100 mg, approximately 5 mg to 50 mg, approximately 5 mg to 25 mg, approximately 10 mg to 1,000 mg, approximately 10 mg to 500 mg, approximately 10 mg to 100 mg, approximately 10 mg to 50 mg, or approximately 10 mg to 25 mg. However, the dosage can be prescribed in various ways depending on factors such as the formulation method, administration method, patient's age, weight, sex, medical condition, diet, administration time, administration route, excretion rate, and response sensitivity, and a person skilled in the art can appropriately adjust the dosage considering such factors. The number of administrations can be once a day or two or more times a day within the range of clinically acceptable side effects, and the drug can be administered to one or more sites, daily or at intervals of 2 to 5 days, with a total administration period ranging from 1 to 30 days per treatment. If necessary, the same treatment can be repeated after an appropriate period. For non-human animals, the dosage can be the same as for humans per kg, or the dosage can be converted using, for example, the volume ratio of organs (such as the heart) of the target animal and humans (e.g., average value) and administered.

[0126] Any terms or elements referred to in the “Methods for Prevention, Improvement, or Treatment” mentioned above, as referred to in the descriptions relating to “strains,” “lysates, cultures, or extracts,” “uses, microbial formulations, or microbial fertilizers,” “methods or plants,” or “uses or compositions,” are understood to be identical to those referred to in the descriptions relating to “strains,” “lysates, cultures, or extracts,” “uses, microbial formulations, or microbial fertilizers,” “methods or plants,” or “uses or compositions.” [Effects of the Invention]

[0127] According to one embodiment, the novel Bacillus megatherium HyangYak-01 strain exhibits effects such as increasing the diversity of microbiome species in soils used for crop cultivation, improving physicochemical properties, promoting crop growth, or accelerating the biosynthesis of physiologically active substances. Therefore, it can be applied to a variety of uses, such as improving soil conditions, increasing crop cultivation efficiency, or enhancing crop efficacy through fertilizers. Furthermore, according to one embodiment, Centella asiatica cultivated with the novel Bacillus megatherium HyangYak-01 strain or its extract shows a significant increase in the content of pharmacological components and exhibits effects such as improving the expression of factors related to strengthening the skin barrier and inhibiting the expression of inflammatory cytokines. Therefore, it can be applied to a variety of uses, such as improving skin conditions or preventing or treating skin inflammatory diseases. [Brief explanation of the drawing]

[0128] [Figure 1] Figure 1 shows the results of pure isolation of colonies of the HyangYak-01 strain.

[0129] [Figure 2A] Figures 2A and 2B show the results of the genome dielectric analysis of the HyangYak-01 strain. [Figure 2B] Same as above.

[0130] [Figure 3]Figure 3 shows the results of the analysis of the nitrogen fixation activity (A), phosphate solubilization activity (B), urea hydrolase activity (C), denitridation activity (D: denitridation activity against nitrite; E: denitridation activity against nitrate), and root growth promoting hormone production activity (F) of the HyangYak-01 strain.

[0131] [Figure 4] Figure 4 shows the results of visual observation of the leaves and roots of a crop (Centella asiatica) grown after being treated with the HyangYak-01 strain (Control: Crops grown without treatment with HyangYak-01 strain; Treat: Crops grown with treatment with HyangYak-01 strain).

[0132] [Figure 5] Figure 5 shows the results of measuring the growth of crops (Centella asiatica) grown after being treated with the HyangYak-01 strain (Control: Crops grown without treatment with HyangYak-01 strain; Treat: Crops grown after treatment with HyangYak-01 strain) (A: Root weight; B: Root length; C: Leaf weight; D: Leaf length; E: Leaf width; F: Chlorophyll content).

[0133] [Figure 6] Figure 6 shows the results of measuring the biological weight and dry weight of the roots of a crop (Angelica acutiloba) grown with treatment using the HyangYak-01 strain (control group: crop grown without treatment with HyangYak-01 strain; experimental group: crop grown with treatment using HyangYak-01 strain).

[0134] [Figure 7] Figure 7 shows the results of measuring the biological weight and dry weight of the roots of a crop (balloon flower) grown with treatment using the HyangYak-01 strain (control group: crop grown without treatment with the HyangYak-01 strain; experimental group: crop grown with treatment using the HyangYak-01 strain).

[0135] [Figure 8]Figure 8 shows the results of measuring the biological weight and dry weight of the roots of crops (Codonopsis lanceolata) grown after being treated with the HyangYak-01 strain (control group: crops grown without treatment with the HyangYak-01 strain; experimental group: crops grown after treatment with the HyangYak-01 strain).

[0136] [Figure 9] Figure 9 shows the results of measuring the biological weight and dry weight of the roots of crops (Bupleurum falcatum) grown after being treated with the HyangYak-01 strain (control group: crops grown without treatment with the HyangYak-01 strain; experimental group: crops grown after treatment with the HyangYak-01 strain).

[0137] [Figure 10] Figure 10 shows the results of measuring the biological weight and dry weight of the roots of crops (Isodon japonicus) grown with treatment using the HyangYak-01 strain (control group: crops grown without treatment using the HyangYak-01 strain; experimental group: crops grown with treatment using the HyangYak-01 strain).

[0138] [Figure 11] Figure 11 shows the results of measuring the biological weight and dry weight of the roots of crops (licorice) grown after being treated with the HyangYak-01 strain (control group: crops grown without treatment with the HyangYak-01 strain; experimental group: crops grown after treatment with the HyangYak-01 strain).

[0139] [Figure 12] Figure 12 shows the results of measuring the root mass of Centella asiatica crops grown after being treated with the HyangYak-01 strain and the KCTC 3007T strain, respectively (control group: crops grown without strain treatment; KCTC 3007T: crops grown with KCTC 3007T strain treatment; HyangYak-01: crops grown with HyangYak-01 strain treatment).

[0140] [Figure 13]Figure 13 shows the results of measuring the root-dry weight of crops (Centella asiatica) grown after being treated with the HyangYak-01 strain and the KCTC 3007T strain, respectively (control group: crops grown without strain treatment; KCTC 3007T: crops grown with KCTC 3007T strain treatment; HyangYak-01: crops grown with HyangYak-01 strain treatment).

[0141] [Figure 14] Figure 14 shows the results of measuring the leaf bioweight of Centella asiatica plants grown after being treated with the HyangYak-01 strain and the KCTC 3007T strain, respectively (control group: untreated crop; KCTC 3007T: crop treated with KCTC 3007T strain; HyangYak-01: crop treated with HyangYak-01 strain).

[0142] [Figure 15] Figure 15 shows the results of measuring the leaf-dry weight of crops (Centella asiatica) grown after being treated with the HyangYak-01 strain and the KCTC 3007T strain, respectively (control group: crops grown without strain treatment; KCTC 3007T: crops grown with KCTC 3007T strain treatment; HyangYak-01: crops grown with HyangYak-01 strain treatment).

[0143] [Figure 16] Figure 16 shows the results of measuring the number of leaves in crops (Centella asiatica) grown after treating them with the HyangYak-01 strain and the KCTC 3007T strain, respectively (control group: crops grown without strain treatment; KCTC 3007T: crops grown with KCTC 3007T strain treatment; HyangYak-01: crops grown with HyangYak-01 strain treatment).

[0144] [Figure 17A]Figure 17A shows the results of an analysis of microbiome changes in crop cultivation soil treated with the HyangYak-01 strain (analysis results of the Shannon index (A) and Observed OUT (B), which are factors of microbiome species diversity).

[0145] [Figure 17B] Figure 17B shows the results of the beta diversity analysis (C) performed based on PCoA1 and PCoA2 analysis (Control: untreated soil with HyangYak-01 strain; Treat: soil treated with HyangYak-01 strain).

[0146] [Figure 18] Figure 18 shows the LC-TOF / MS results of a qualitative analysis of the pharmacological component (madecasic acid and asiatic acid) content of Centella asiatica cultivated with HyangYak-01 strain (Control: Centella asiatica cultivated without treatment with HyangYak-01 strain; Treat: Centella asiatica cultivated with HyangYak-01 strain; y axis: intensity).

[0147] [Figure 19] Figure 19 shows the results of an analysis of the effect of Centella asiatica extract, prepared by extracting Centella asiatica cultivated with treatment of the HyangYak-01 strain, on the expression of skin barrier strengthening-related factors (FLG and CLD1) (None: group untreated with the test substance; RA: group treated with 1 μM Retinoic acid; Centella asiatica (-): group treated with extract from Centella asiatica cultivated without treatment of the HyangYak-01 strain; Centella asiatica (+): group treated with extract from Centella asiatica cultivated with the HyangYak-01 strain).

[0148] [Figure 20]Figure 20 shows the results of an analysis of the effect of Centella asiatica extract, produced by extracting Centella asiatica cultivated with the HyangYak-01 strain, on the expression of inflammatory cytokines (IL-1α and IL-1β) (None: normal cells untreated with the test substance; Poly I:C + rh IL-4: cells with induced inflammation untreated with the test substance; DEX: cells with induced inflammation treated with Dexamethasone 1 μM; HyangYak(-): cells with induced inflammation treated with extract from Centella asiatica cultivated without the HyangYak-01 strain; HyangYak(+): cells with induced inflammation treated with extract from Centella asiatica cultivated with the HyangYak-01 strain). [Modes for carrying out the invention]

[0149] The present invention will be described in more detail below through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

[0150] (blank) [Examples]

[0151] Example 1. Isolation and identification of bacterial strains

[0152] Bacillus megatherium strains were isolated and identified from rhizosphere soil.

[0153] Specifically, soil samples were taken from crops planted in the area of ​​11-37, Uigu-Gebong-gil, Uigu-eup, Gongju-si, Chungcheongnam-do. The obtained soil samples were serially diluted with sterile distilled water and streaked onto R2A (Reasoner's 2A) agar plates, and incubated at approximately 30°C for approximately 72 hours. Subsequently, white colonies were streaked from the bacterial colonies cultured on the agar plates, and single-species bacterial colonies were isolated as pure samples. The results are shown in Figure 1.

[0154] In addition, the isolated colonies were cultured, and genomic gene analysis and 16S rRNA gene sequencing analysis of the bacterial strains were performed to identify the isolated strains. Specifically, 16S rRNA gene sequencing was performed on colonies cultured in R2A agar medium by Macrogen, using the 27F (SEQ ID NO: AGA GTT TGA TCM TGG CTC AG) and 1492R (SEQ ID NO: GGT TAC CTT GTT ACG ACT TC) primer sets. The analyzed genetic information was identified using the Ezbiocloud database of CJbioscience (https: / / www.ezbiocloud.net / ).

[0155] As a result, the isolated strain was confirmed to be Bacillus megaterium, possessing a 16S rRNA gene sequence containing the base sequence of Sequence ID No. 1, and was named HyangYak-01. The Bacillus megaterium HyangYak-01 strain was deposited with the Korea Microbial Conservation Center and assigned deposit number KCCM13074P. The results of the genomic gene analysis of the Bacillus megaterium HyangYak-01 strain are shown in Figures 2A and 2B.

[0156] (blank)

[0157] Experimental Example 1. Activity Analysis of HyangYak-01 Strain

[0158] The root growth-promoting hormone (Indole-3-acetic acid: IAA) production activity, nitrogen fixation activity, phosphate solubilization activity, urease activity, and denitrification activity of the HyangYak-01 strain were analyzed.

[0159] Specifically, to confirm the ability of the HyangYak-01 strain to produce IAA (Inducing Auricular Activated Adjuvant), the HyangYak-01 strain was inoculated into R2A broth (+0.1% tryptophan) and cultured at approximately 30°C and 150 rpm for approximately 48-72 hours. The mixture was then centrifuged at approximately 7,000 rpm for approximately 20 minutes. After centrifugation, the obtained supernatant was mixed with Salkowski reagent (a mixture of 49 mL of 35% perchloric acid and 1 mL of 0.5 M ferric choloride) in a 1:1 ratio and reacted in the dark for approximately 30 minutes. The color change was then observed. If the reaction solution turned red, the strain could be determined to produce IAA.

[0160] Furthermore, to confirm the nitrogen-fixing ability of the HyangYak-01 strain, Jensen's agar, which does not contain a nitrogen source, was used. Approximately 6 g of sucrose, approximately 0.2 g of MgSO4·7H2O, approximately 13.9 g of Na2HPO4, approximately 1.7 g of KH2PO4, approximately 2 g of NaCl, approximately 8 mg of FeCl3·6H2O, and approximately 1 mg of thiamin were dissolved in approximately 1 L of distilled water, and then approximately 15 g of agar powder was added and sterilized at approximately 121°C for approximately 15 minutes to prepare Jensen's agar medium. The HyangYak-01 strain was inoculated into the agar medium and cultured at approximately 30°C to confirm the nitrogen-fixing ability due to strain growth.

[0161] Furthermore, to confirm the phosphate solubilization ability of the HyangYak-01 strain, approximately 5g of Ca3(PO4)2, approximately 10g of sucrose, approximately 0.5g of yeast extract, approximately 0.27g of NH4NO3, approximately 0.2g of KCl, approximately 0.1g of MgSO4·7H2O, approximately 0.001g of MnSO4, and approximately 0.001g of FeSO4·7H2O were dissolved in approximately 1L of distilled water, and the pH was adjusted to 7.0. Subsequently, approximately 15g of agar powder was added and sterilized at approximately 121°C for approximately 15 minutes to prepare agar medium. The HyangYak-01 strain was inoculated into the agar medium and cultured at approximately 30°C, and the phosphate solubilization ability was confirmed by the presence or absence of clear zone formation.

[0162] Furthermore, to confirm the urea hydrolase activity of the HyangYak-01 strain, urea agar was used. Approximately 20g of urea, 1g of dextrose, 1g of peptone, 5g of NaCl, 2g of KH2PO4, and 122mg of phenol were dissolved in approximately 1L of distilled water, and then approximately 15g of agar powder was added and sterilized at approximately 121°C for approximately 15 minutes to prepare urea agar medium. The HyangYak-01 strain was inoculated into the agar medium and cultured at approximately 30°C, and the urea hydrolase activity was confirmed by the color change of the agar medium from yellow to pink.

[0163] Furthermore, to confirm the denitriding ability (nitrate:NO3-) of the HyangYak-01 strain, approximately 1.0g of NaNO3, approximately 1.0g of asparagine, approximately 5ml of bromothymol blue solution (containing approximately 1g of bromothymol blue, approximately 50ml of ethanol, and approximately 50ml of distilled water), approximately 8.5g of sodium citrate, approximately 1.0g of MgSO4·7H2O, approximately 0.05g of FeCl3·6H2O, approximately 1.0g of KH2PO4, and approximately 0.2g of CaCl2·2H2O were added to approximately 1L of distilled water, titrated to pH 7.0, and then approximately 15g of agar powder was added and sterilized at approximately 121°C for approximately 15 minutes to prepare an agar medium. The HyangYak-01 strain was inoculated into the agar medium and cultured at approximately 30°C, and the denitriding ability was confirmed by the formation of a clear zone in the blue medium as the strain grew.

[0164] Furthermore, to confirm the denitriding ability (nitrite:NO2-) of the HyangYak-01 strain, approximately 0.006 g of NaNO2, approximately 0.03 g of FeSO4·7H2O, approximately 1.0 g of K2HPO4, approximately 0.3 g of CaCl2·2H2O, approximately 0.3 g of NaCl, approximately 1.0 g of NaHCO3, and approximately 0.1 g of MgSO2·7H2O were dissolved in approximately 1.0 L of distilled water, and then approximately 15 g of agar powder was added. The mixture was sterilized at approximately 121°C for approximately 15 minutes to prepare an agar medium. The HyangYak-01 strain was inoculated into the agar medium and cultured at approximately 30°C. After culturing, a Griess test was performed (a test in which approximately 0.5 g of sulfanilic acid was dissolved in approximately 70 ml of boiling distilled water, cooled, and mixed with approximately 30 ml of acetic acid, then titrated with approximately 100 ml to prepare the first solution; 1-naphthylamine solution: approximately 0.5 g of 1-naphthylamine was dissolved in approximately 30 ml of acetic acid, and approximately 70 ml of distilled water was added to prepare the second solution; the first and second solutions were mixed, and the resulting solution was dispensed onto an agar plate on which the bacterial strain was cultured to check for the formation of a clear zone around the strain). The denitriding ability was confirmed by the formation of a clear zone around the strain.

[0165] As a result, as shown in Figure 3, it was confirmed that the solution obtained by reacting the supernatant of the HyangYak-01 strain culture with the Salkowski reagent turned red, indicating that the strain exhibits root growth-promoting hormone production activity. Furthermore, it was confirmed that the HyangYak-01 strain can grow even in Jensen's agar medium that does not contain a nitrogen source, indicating that the strain exhibits nitrogen fixation activity. In addition, when the HyangYak-01 strain was inoculated and cultured in agar medium containing Ca3(PO4)2, it was confirmed that a clear zone was generated around the strain as it grew, indicating that the strain exhibits phosphate solubilization activity. Furthermore, when the HyangYak-01 strain was inoculated and cultured in Urea agar medium, it was confirmed that the Urea agar medium changed color from yellow to pink as the strain grew, indicating that the strain exhibits urea hydrolase activity. Furthermore, the results of the denitridation activity confirmation test confirmed that when the HyangYak-01 strain grows on agar medium, a clear zone is generated around the strain, indicating that the strain exhibits denitridation activity.

[0166] (blank)

[0167] Experimental Example 2. Gene analysis of the crop growth promoting activity of the HyangYak-01 strain.

[0168] We analyzed the genes involved in the crop growth-promoting activity of the HyangYak-01 strain.

[0169] Specifically, to identify genes involved in crop growth promotion activity, DNA was extracted from HyangYak-01 strain cultured in R2A broth using the Wizard Genomic DNA Purification Kit (Promega). The extracted DNA was quality-checked using NanoDrop, and sequencing analysis was performed using Oxford Nanopore (Oxford Nanopore Technologies, Oxford, UK) and MGI DNBSEQ-G400 (MGI Tech, Shenzhen, China). The analyzed sequences were assembled using Flye de novo assemble (version: 2.9.1) and analyzed. The genes involved in the activity evaluated in Experimental Example 1 were identified using the prokka tool on the genetic information after whole-genome sequencing analysis was completed.

[0170] As a result, as shown in Table 1, it was confirmed that the HyangYak-01 strain possesses genes related to root growth-promoting hormone production, nitrogen fixation, phosphate solubilization, urea hydrolase, and denitridation, which are involved in crop growth-promoting activity. Through this, it was found that the HyangYak-01 strain exhibits root growth-promoting hormone production activity, nitrogen fixation activity, phosphate solubilization activity, urea hydrolase activity, and denitridation activity, thereby demonstrating crop growth-promoting activity.

[0171] [Table 1]

[0172] (blank)

[0173] Experimental Example 3. Confirmation of the crop growth promoting effect of the HyangYak-01 strain.

[0174] 3-1. Confirmation of the effect of HyangYak-01 strain on promoting the growth of Centella asiatica.

[0175] We analyzed the growth changes (specifically, changes in root length, root weight, leaf length, leaf weight, leaf width, and chlorophyll content) of crops treated with the HyangYak-01 strain to confirm the crop growth-promoting effect of the HyangYak-01 strain.

[0176] Specifically, Centella asiatica seedlings were planted in soil near 11-37, Gebong-gil, Igu-eup, Gongju-si, Chungcheongnam-do. Subsequently, the HyangYak-01 strain was cultured in R2A medium at approximately 30°C and 150 rpm for approximately 48-72 hours. The resulting culture solution was centrifuged at approximately 7,000 rpm for approximately 20 minutes, and the supernatant was removed to obtain only the fungal cells. The fungal cells were diluted 100-fold and treated in the soil where the Centella asiatica seedlings were planted. A control group was set up in which only water was treated instead of the strain. After that, the soil where the Centella asiatica seedlings were planted was watered at intervals of approximately 1-2 weeks, and open-field cultivation was carried out for approximately 3 months. After that, the harvested Centella asiatica were washed, the water was removed, and the root length, leaf length, and leaf width were measured. After removing the water, the root weight and leaf weight were measured using a scale. Chlorophyll was measured using a SPAD-502 Plus measuring instrument.

[0177] As a result, as shown in Figures 4 and 5, we confirmed that in Centella asiatica cultivated with the HyangYak-01 strain, root length, root weight, leaf length, leaf weight, leaf width, and chlorophyll content were all significantly increased compared to the untreated control group. Through this, we found that the HyangYak-01 strain has a remarkable effect in promoting the growth of crops (e.g., Centella asiatica).

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[0179] 3-2. Confirmation of the growth-promoting effect of the HyangYak-01 strain on various crops.

[0180] To determine whether the HyangYak-01 strain affects crops other than Centella asiatica, we analyzed the growth changes (specifically, root body weight and root dry weight) of various crops treated with the HyangYak-01 strain and confirmed the crop growth-promoting effect of the HyangYak-01 strain.

[0181] Specifically, the seedlings of Codonopsis lanceolata, Platycodon grandiflorum, Yam, Kawaratake, Hikioji, and licorice were planted in the soil adjacent to 11-37, Uijugye-bongil, Uiju-eup, Gongju-si, Chungnam. Then, the culture solution obtained by culturing the HyangYak-01 strain in an R2A medium at about 30°C and about 150 rpm for about 48 to 72 hours was centrifuged at about 7,000 rpm for about 20 minutes. After that, the supernatant was removed, and only the bacterial cells were obtained. The bacterial cells were diluted 100-fold and treated on the crop seedling planting soil. A group treated with only water was set as a control group instead of the strain. Then, water was given to the crop seedling planting soil at about 1- to 2-week intervals, and open-field cultivation was carried out for about 3 months. Thereafter, each harvested crop was washed, moisture was removed, the fresh weight of the roots was measured, and after drying, the dry weight of the roots was measured.

[0182] As a result, as shown in FIGS. 6 to 11, it was confirmed that the fresh weight and dry weight of the roots of Codonopsis lanceolata, Platycodon grandiflorum, Yam, Kawaratake, Hikioji, and licorice cultivated by treating with the HyangYak-01 strain were further increased compared to the untreated control group. Through this, it was found that the HyangYak-01 strain exhibits the effect of increasing the growth of various crops, not just tubers.

[0183] (Blank)

[0184] 3-3. Comparative evaluation of the crop growth promotion effects between the HyangYak-01 strain and the standard strain

[0185] To comparatively evaluate the crop growth promotion effects between the HyangYak-01 strain and the standard strain, Priestia megaterium KCTC 3007, a standard strain of Priestia megaterium T was obtained through a transfer from the Korea Research Institute of Bioscience and Biotechnology Bioresource Center and utilized. The growth changes of crops (specifically, differences in fresh root weight, dry root weight, fresh leaf weight, dry leaf weight, and number of leaves) cultivated by treating with the HyangYak-01 strain and KCTC 3007 T strains were comparatively analyzed.

[0186] Specifically, after transplanting the Centella asiatica seedlings into flowerpots, the HyangYak-01 strain and KCTC 3007 T Each strain was cultured in R2A medium at approximately 30°C and 150 rpm for approximately 48-72 hours. The two resulting culture solutions were then centrifuged at approximately 7,000 rpm for approximately 20 minutes, and the supernatant was removed to obtain only the two types of bacterial cells. Each of the two bacterial cells was diluted 100-fold and treated with the respective independent Centella asiatica seedlings. A control group was set up in which only sterile distilled water was used instead of the bacterial cells. Thereafter, at one-week intervals, the independent Centella asiatica seedlings were treated with the HyangYak-01 bacterial cell and KCTC 3007. T The fungal cells and sterile distilled water were treated three times each, and cultivation was carried out for one month in a constant temperature and humidity chamber at approximately 26°C and 40-60% humidity. Afterwards, the harvested Centella asiatica were washed, the moisture was removed, and the weight of the living roots and living leaves was measured. After drying, the weight of the dried roots and dried leaves was measured. The number of leaves was also counted.

[0187] As a result, as shown in Figures 12 to 16, in the case of Centella asiatica cultivated with the HyangYak-01 strain, the sterilized distilled water treatment group (control group) and KCTC 3007 T Compared to the strain-treated group, we confirmed that root weight, dry root weight, leaf weight, dry leaf weight, and leaf number all increased further. This indicates that there are differences in crop growth promoting effects even among other strains of the same species, i.e., other strains belonging to Pristia megaterium, and that the HyangYak-01 strain exhibits an even superior crop growth promoting effect compared to other strains belonging to Pristia megaterium (e.g., the standard strain). This can be understood as a result of the HyangYak-01 strain having a difference in nucleotide sequence (i.e., a genetic difference) from other strains belonging to Pristia megaterium, due to the presence of 16S rRNA containing the nucleotide sequence of Sequence ID No. 1.

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[0189] Experimental Example 4. Confirmation of changes in soil microbiome in crop cultivation areas after treatment with the HyangYak-01 strain.

[0190] We analyzed changes in the microbiome of crop-cultivated soil treated with the HyangYak-01 strain.

[0191] Specifically, soil samples were taken from the experimental group treated with the HyangYak-01 strain and the control group not treated with the HyangYak-01 strain during the Centella asiatica cultivation stage of Experiment Example 3-1. DNA was extracted from these soil samples using DNeasy PowerSoil Pro Kits (QIAGEN). An amplicon library was created using the extracted DNA to analyze the bacterial community, which was performed through sequencing using Miseq equipment. Changes in the soil microbiome were analyzed using Qiime 2 on the sequenced data. Specifically, the Shannon index and Observed OUT values, which indicate species diversity, were derived to analyze changes in the species diversity of the soil microbiome. PCoA1 and PCoA2 were analyzed, and beta diversity analysis was performed to analyze changes in the soil microbiome community by grouping the experimental group and the control group based on the resulting values.

[0192] As a result, as shown in Figures 17A and 17B, the Shannon index and Observed OUT values ​​analyzed in soil samples from crop cultivation areas treated with the HyangYak-01 strain were significantly higher than those analyzed in control soil samples not treated with the HyangYak-01 strain. This indicates that when soil is treated with the HyangYak-01 strain, the species diversity of the microbiome increases significantly compared to soil that is not treated with the HyangYak-01 strain. Furthermore, the beta diversity analysis results showed that the microbiome is separated between soils depending on whether or not the soil is treated with the HyangYak-01 strain, resulting in significant community changes.

[0193] Through this experiment, it was found that when the HyangYak-01 strain is applied to soil, the soil undergoes community changes such as an increase in the species diversity of the microbiome, making it more vigorous for crop cultivation, and thereby potentially promoting the growth of crops grown in the soil.

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[0195] Experimental Example 5. Confirmation of the effect of HyangYak-01 strain treatment on improving the physicochemical properties of soil in crop cultivation areas.

[0196] We analyzed the changes in physicochemical properties of soil in crop cultivation areas treated with the HyangYak-01 strain.

[0197] Specifically, in the Centella asiatica cultivation and harvesting stage described in Experimental Example 3-1, soil samples were obtained by sampling approximately 1 kg each from the experimental group treated with the HyangYak-01 strain and the control group not treated with the HyangYak-01 strain (treated only with water instead of the strain). (Three samples were obtained for each group.) The physicochemical properties of these soil samples were analyzed. Specifically, the soil samples were prepared by naturally drying them in the shade for three days and then sieving them with a 2 mm sieve. pH, electrical conductivity (EC), effective phosphate content, organic matter content, and (exchangeable) cation (potassium ion, calcium ion, magnesium ion) content of the prepared soil samples were measured according to the soil chemical analysis method (NIAST, 2010). pH and EC were measured by extracting the soil solution using the 1:5 method. Organic matter content was measured by absorbance at 610 nm using the Walkley-Black method, and effective phosphate content was colorimetrically determined at 660 nm using the molybdenum blue method. Organic matter and effective phosphate content were measured using a UV-vis spectrometer (UV-2401PC, Shimadzu Corporation, Japan). For cation content, the soil samples were extracted with a 1N NH4OAc (pH 7.0) solution and analyzed using ICP (Intergra XL, GBC, Australia). Additionally, initial soil samples that had not been treated in any way before Centella asiatica cultivation were used as a control group, and the same analysis was performed on them.

[0198] As a result, as shown in Table 2, the pH of the soil after crop cultivation increased compared to the initial soil, and it was confirmed that the level of pH increase was even lower in soil treated with the HyangYak-01 strain and cultivated with crops compared to the control soil. Furthermore, the organic matter content of the soil decreased compared to the initial soil after crop cultivation, and it was confirmed that the organic matter content decreased even further in soil treated with the HyangYak-01 strain and cultivated with crops compared to the control soil. In addition, the effective phosphorus content of the soil increased compared to the initial soil after crop cultivation, and although the level of increase in effective phosphorus content was even lower in soil treated with the HyangYak-01 strain and cultivated with crops compared to the control soil, it was confirmed that this was within a normal level. Furthermore, while the exchangeable potassium ion content of soil after crop cultivation did not change significantly in soil treated with the HyangYak-01 strain compared to the initial soil, the exchangeable calcium ion and exchangeable magnesium ion content of soil after crop cultivation increased further in soil treated with the HyangYak-01 strain compared to the control soil compared to the initial soil. In addition, electrical conductivity increased slightly in soil treated with the HyangYak-01 strain compared to the initial soil, but this was confirmed to be within the normal range.

[0199] Through this experiment, it was found that soil pH is closely related to nutrient absorption, and that crops generally grow best in slightly acidic (pH 6.0-6.5) or mildly acidic (pH 6.5-7.0) soil. In the case of the HyangYak-01 strain, it was found that maintaining the pH of the soil in crop cultivation areas at slightly acidic or mildly acidic levels further improves the soil condition to a healthier state. Furthermore, it was found that the HyangYak-01 strain plays a major role in decomposing organic matter in crop cultivation soil, transforming it into a form usable by crops, thereby improving the soil condition to a state even more suitable for crop cultivation. Furthermore, the cation exchange capacity of soil (total amount of exchangeable cations) is an indicator of soil fertility and is involved in improving soil buffering capacity, nutrient retention, and supply. It is known that soils with a large cation exchange capacity are more fertile and contain more inorganic nutrients necessary for crop growth. In the case of the HyangYak-01 strain, it was found that it further improves soil fertility by increasing exchangeable cations such as calcium ions and magnesium ions in the soil. In addition, it was found that the HyangYak-01 strain does not significantly affect the electrical conductivity of the soil, maintaining it at a normal level, and also maintains the effective phosphorus content of the soil at a normal level. In summary, it was found that when the HyangYak-01 strain is applied to soil, the physicochemical properties of the soil are improved (soil pH is maintained at a slightly acidic or mildly acidic level; organic matter decomposition in the soil is promoted; the amount of exchangeable cations in the soil increases; and the electrical conductivity and available phosphorus content of the soil are maintained at normal levels), making the soil more vigorous for growing crops, and thereby potentially promoting the growth of crops grown in the soil.

[0200] [Table 2]

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[0202] Experimental Example 6. Confirmation of changes in pharmacological component content of Centella asiatica cultivated with HyangYak-01 strain.

[0203] We analyzed the changes in the pharmacological components (specifically, madecasic acid and asiatic acid) content of Centella asiatica cultivated with the HyangYak-01 strain.

[0204] Specifically, leaves were isolated from Centella asiatica cultivated with the HyangYak-01 strain obtained in Experimental Example 3-1, and the isolated leaves were extracted with approximately 70-100% ethanol for approximately 2-48 hours. The resulting extract was then filtered using Whatman paper to remove impurities, and the filtered solution was concentrated using a speedvac at approximately 45°C and approximately 5°C of reduced pressure (vacuum pressure) for approximately 3 hours. The concentrated substance was dissolved in methanol and then filtered using a 0.22 μm filter. The content of madecasic acid and asiatic acid in the filtered sample was qualitatively analyzed using LC-TOF / MS. The same analysis was performed on Centella asiatica leaves cultivated without treatment with the HyangYak-01 strain as a control group.

[0205] As a result, as shown in Figure 18, we confirmed that the content of madecasic acid and asiatic acid was significantly increased in Centella asiatica cultivated with the HyangYak-01 strain compared to the untreated control group. Through this, we found that the HyangYak-01 strain promotes the biosynthesis of physiologically active substances such as pharmacological components in crops (e.g., Centella asiatica).

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[0207] Experimental Example 7. Confirmation of the skin condition-improving efficacy of Centella asiatica cultivated with the HyangYak-01 strain.

[0208] We analyzed the skin condition improving effects (specifically, skin barrier strengthening effects) of Centella asiatica cultivated using the HyangYak-01 strain.

[0209] Specifically, leaves were isolated from Centella asiatica cultivated using the HyangYak-01 strain obtained in Experimental Example 3-1. The isolated leaves were extracted using approximately 70% ethanol at room temperature for approximately 48-72 hours to obtain an extract solution. Subsequently, the extract solution was concentrated using a vacuum concentrator to obtain Centella asiatica extract. The efficacy of the Centella asiatica extract in improving skin condition was confirmed. Specifically, HaCaT cells, a human keratinocyte cell line, were cultured in DMEM medium (Dulbecco's modified Eagle's Medium, Gibco 1210-0038) containing approximately 10% fetal bovine serum. The culture was carried out in an incubator under conditions of approximately 37°C and approximately 5% CO2. The cultured cells were treated with the Centella asiatica extract at a concentration of approximately 1% (w / w), and cultured for an additional 24 hours. The relative expression levels of FLG (filaggrin) and CLD1 (claudin-1), skin barrier strengthening factors, relative to β-actin expression, were then analyzed in the cultured cells. Specifically, RNA was separated from the cultured cells using Trizole (RNA iso, DAKARA, Japan), then quantified at 260 nm using nanodrops. cDNA was then synthesized using an amplifier with approximately 2 μg of each RNA (C1000 Thermal Cycler, Bio-Rad, USA). The synthesized cDNA was used as a template and added along with primers and cDNA for the target genes, FLG and CLD1, respectively. A real-time polymerization chain reaction was performed using a real-time PCR machine (Step One Plus, Applied Biosystems, USA). The expression levels of FLG and CLD1 genes were finally analyzed after correction for β-actin expression. As control groups, the same analysis was performed on the following: a group treated with an extract of Centella asiatica cultivated without treatment of the HyangYak-01 strain (extraction method as described above), a group treated with retinoic acid (treated at a concentration of approximately 1 μM), and a group untreated with the test substance.

[0210] As a result, as shown in Figure 19, we confirmed that the Centella asiatica extract produced by extracting Centella asiatica cultivated with treatment of the HyangYak-01 strain significantly increased the expression of skin barrier strengthening-related factors such as FLG (filaggrin) and CLD1 (claudin-1) compared to the extract of Centella asiatica cultivated without treatment of the HyangYak-01 strain. Through this, we found that Centella asiatica cultivated with treatment of the HyangYak-01 strain, or its extract, exhibits excellent skin barrier strengthening efficacy and can be usefully used to improve skin condition.

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[0212] Experimental Example 8. Confirmation of the anti-inflammatory efficacy of Centella asiatica cultivated with the HyangYak-01 strain.

[0213] The anti-inflammatory effects of Centella asiatica cultivated with the HyangYak-01 strain were analyzed.

[0214] Specifically, leaves were isolated from Centella asiatica cultivated using the HyangYak-01 strain obtained in Experimental Example 3-1. The isolated leaves were extracted using approximately 70% ethanol at room temperature for approximately 48-72 hours to obtain an extract solution. Subsequently, the extract solution was concentrated using a vacuum concentrator to obtain Centella asiatica extract. The anti-inflammatory efficacy of the Centella asiatica extract was confirmed. Specifically, HaCaT cells, a human keratinocyte cell line, were cultured in DMEM medium (Dulbecco's modified Eagle's Medium, Gibco 1210-0038) containing approximately 10% fetal bovin serum. Culture was carried out in an incubator under conditions of approximately 37°C and approximately 5% CO2. The culture medium was changed every 3-4 days during the culture, and when the cells proliferated excessively, they were subcultured. Thereafter, the cultured cells were subcultured to approximately 5 x 10⁶ 5Each well was dispensed with the concentration per well and cultured for approximately 24 hours. After culturing, the cultured cells were washed with phosphate-buffered saline (PBS), and the washed cells were dispensed into each well containing DMEM medium without FBS. PolyI:C and IL-4 were added at concentrations of approximately 10 μg / ml and 10 ng / ml, respectively, to induce inflammation in the cells. Subsequently, the cells in which inflammation had been induced were treated with the Centella asiatica extract at a concentration of approximately 1% (w / w) and cultured for an additional 4 hours. The expression levels of the inflammatory cytokines IL-1α and IL-1β were then analyzed in the cultured cells. Specifically, RNA was separated from the cultured cells using Trizole (RNA iso, DAKARA, Japan), then the RNA was quantified at 260 nm using nanodrop, and cDNA was synthesized using an amplifier with approximately 2 μg of RNA (C1000 Thermal Cycler, Bio-Rad, USA). The synthesized cDNA was used as a template and added along with primers and cDNA for the target genes IL-1α and IL-1β, respectively. A real-time polymerase chain reaction was performed using a real-time PCR machine (Step One Plus, Applied Biosystems, USA), and the expression levels of IL-1α and IL-1β were analyzed. As a control group, the inflamed cells were treated with either an extract of Centella asiatica cultivated without treatment of the HyangYak-01 strain (extraction method as described above) or dexamethasone (treated at a concentration of approximately 1 μM), or no treatment with the test substance at all; and normal cells were also treated with no treatment with the test substance at all. The above analysis was performed on these groups as well.

[0215] As a result, as shown in Figure 20, we confirmed that the Centella asiatica extract produced by extracting Centella asiatica cultivated with treatment of the HyangYak-01 strain significantly suppressed the expression of proinflammatory cytokines such as interleukin-1 alpha (IL-1α) and interleukin-1 beta (IL-1β) in inflamed cells compared to the extract of Centella asiatica cultivated without treatment of the HyangYak-01 strain. Through this, we found that Centella asiatica cultivated with treatment of the HyangYak-01 strain or its extract exhibits excellent anti-inflammatory efficacy (i.e., anti-inflammatory efficacy), and therefore can be usefully used for the prevention, improvement, or treatment of inflammatory skin diseases (or inflammation in inflammatory skin diseases).

[0216] (blank)

[0217] In summary, the Bacillus megatherium HyangYak-01 strain was found to significantly increase the diversity of microbiome species in soils used for crop cultivation, improve physicochemical properties, and significantly promote crop growth. In particular, in the case of Centella asiatica cultivated with the strain, a significant increase in the content of pharmacological components was confirmed, indicating that the strain promotes the biosynthesis of physiologically active substances in crops (e.g., Centella asiatica).

[0218] In addition, we confirmed that the Centella asiatica extract produced by extracting Centella asiatica cultivated using the aforementioned strain significantly increased the expression of skin barrier strengthening factors and significantly suppressed the expression of inflammatory cytokines.

[0219] Therefore, it was found that the Bacillus megatherium HyangYak-01 strain exhibits excellent soil condition improvement effects, crop growth or biosynthesis of physiologically active substances, and that Centella asiatica cultivated with this strain or its extract exhibits excellent skin condition improvement effects (e.g., skin barrier strengthening effect) or anti-inflammatory effects.

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[0221] The above description merely illustrates the technical concept of the present invention, and a person with ordinary skill in the art to which the present invention belongs can make various modifications and variations as long as they do not deviate from the essential characteristics of the present invention.

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[0223] [Table 3]

Claims

1. Bacillus megaterium HyangYak-01 strain, deposited under deposit number KCCM13074P.

2. The strain according to claim 1, wherein the strain has a 16S rRNA containing a nucleotide sequence having 98% or more sequence identity with SEQ ID NO:

1.

3. The strain of bacteria according to claim 1, wherein the strain of bacteria has the effect of improving soil conditions, promoting plant growth, or promoting the biosynthesis of physiologically active substances in plants.

4. The strain according to claim 3, wherein the improvement of soil conditions includes increasing the diversity of microbial species in the soil microbiome or improving the physicochemical properties of the soil.

5. The strain described above has one or more of the following activities, as described in claim 1: (1) Root growth promoting hormone (Indole-3-acetic acid: IAA) production activity; (2) Nitrogen fixation activity; (3) Phosphate solubilization activity; (4) Urea hydrolase activity; and (5) Denitrification activity.

6. A microbial preparation comprising the bacterial strain described in claim 1, its lysate or culture, or an extract of the bacterial strain, lysate or culture.

7. The microbial preparation according to claim 6, wherein the microbial preparation is for improving soil conditions, promoting plant growth, or promoting the biosynthesis of physiologically active substances in plants.

8. A microbial fertilizer comprising the microbial preparation described in claim 6.

9. The microbial fertilizer according to claim 8, wherein the microbial fertilizer is for improving soil conditions, promoting plant growth, or promoting the biosynthesis of physiologically active substances in plants.

10. A method for cultivating plants, comprising the step of treating one or more of the following with a bacterial strain, microbial preparation, or microbial fertilizer described in any one of claims 1 to 9: soil, plants, and plant seeds.

11. The method according to claim 10, wherein the plant is Centella asiatica, Angelica acutiloba, Platycodon grandiflorus, Codonopsis lanceolata, Bupleurum falcatum, Isodon japonicus, or licorice.

12. A plant cultivated by the method described in claim 10.

13. A cosmetic composition for improving skin condition, comprising Centella asiatica or an extract thereof cultivated by the method described in claim 11.

14. The cosmetic composition according to claim 13, wherein the Centella asiatica has an improved content of one or more of chlorophyll, madecasic acid, and asiatic acid.

15. The cosmetic composition according to claim 13, wherein the Centella asiatica or its extract exhibits one or more of the following characteristics: (1) Induction of increased expression of filaggrin (FLG), claudin-1 (CLD1), or a combination thereof; and (2) Inhibition and induction of the expression of inflammatory cytokines (proinflammatory cytokines).

16. The cosmetic composition according to claim 13, wherein the improvement of the skin condition is strengthening of the skin barrier or anti-inflammatory.

17. A topical skin preparation composition comprising Centella asiatica cultivated by the method described in claim 11 or an extract thereof.

18. A food composition comprising Centella asiatica or an extract thereof cultivated by the method described in claim 11.

19. A pharmaceutical composition for the prevention or treatment of inflammatory skin diseases, comprising Centella asiatica cultivated by the method described in claim 11 or an extract thereof as an active ingredient.

20. The pharmaceutical composition according to claim 19, wherein the skin inflammatory disease is one selected from the group consisting of dermatitis, atopic dermatitis, pruritus, eczematous skin disease, dry eczema, erythema, urticaria, psoriasis, drug eruption, and acne.

21. A method for improving the skin condition of an individual or preventing or treating a skin inflammatory disease, comprising the step of administering an effective amount of Centella asiatica or an extract thereof cultivated by the method of claim 11 to the individual.

22. The deposit number KCCM13074P is for the Bacillus megaterium HyangYak-01 strain, its lysate or culture, or for use in the production of extracts of the said strain, lysate or culture.

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