Bacillus velezensis SCB88 strain suitable for starfish liquid fertilizer and composition comprising the same

Abstract

The present invention relates to a Bacillus velezensis SCB88 strain having excellent growth ability and maturation-promoting function in a starfish liquid fertilizer environment, and a composition containing the same. The above-mentioned SCB88 strain is a strain isolated from a specific environment and, when compared to other Bacillus velezensis strains (SCB64, SCB92) belonging to the same species, exhibits excellent proliferation ability, improved maturity, and increased fertilizer components (N, P, K) within starfish liquid fertilizer. In particular, this strain significantly increases the number of viable cells after inoculation into liquid fertilizer, improves the maturity to the 'completed maturity' stage, and significantly increases the content of major components such as nitrogen, phosphorus, and potassium, thereby demonstrating a very high potential for use as a functional probiotic. Furthermore, by experimentally proving that functional characteristics can vary depending on the source of isolation of the strain even within the same species, this invention emphasizes the importance of strain selection and source in the development of microorganism-based liquid fertilizer or fertilizer compositions.

Description

Technology Field

[0001] The present invention relates to a Bacillus velezensis SCB88 strain applicable to starfish-based liquid fertilizers and a composition containing the same. Background Technology

[0003] Starfish are marine organisms with high potential for use as eco-friendly agricultural resources, as they are rich in various organic and inorganic minerals such as amino acids, proteins, calcium, magnesium, and organic sulfur. In particular, attempts to agriculturally recycle starfish—which cause ecological disturbance by mass-producing in marine ecosystems—by composting or liquidizing them are a very important task in terms of resource circulation.

[0004] However, when raw starfish or their extracts are processed into liquid fertilizer alone, problems such as poor composting due to high protein and high salt environments, odor generation, and contamination by pathogenic microorganisms occur, limiting their application in actual farms. In particular, incompletely composted liquid fertilizer can lead to inhibited crop growth and the risk of soil contamination, thus requiring technology that induces stable composting and ensures biological safety.

[0005] Meanwhile, microorganisms possess endospore formation, high tolerance enabling survival in high-temperature, high-salinity, and dry environments, and various plant growth-promoting (PGP) functions; as such, they are currently utilized in various fields, including biofertilizers, probiotics, and soil conditioners.

[0006] However, research on microorganisms capable of stably establishing and proliferating while withstanding environmental stresses (salinity, protein decomposition products, etc.) even when used in combination with starfish-based liquid fertilizers, and simultaneously enhancing the maturity and fertilizer component content of the liquid fertilizers, is very limited, and there is a need to discover microbial resources that can be widely utilized industrially.

[0007] Accordingly, the development of high-functional microbial strains that exhibit excellent viability and colonization ability even in starfish liquid fertilizer environments, promote decomposition, and contribute to crop growth can have a significant ripple effect across resource recycling agriculture, the eco-friendly liquid fertilizer market, and the functional fertilizer industry as a whole. Prior art literature

[0009] Registered Patent No. 10-2345317 (December 27, 2021) The problem to be solved

[0010] Due to complex biological constraints—such as the high salinity environment of conventional starfish liquid fertilizer, as well as antimicrobial substances like saponins contained in the starfish itself, proteolytic enzyme inhibitors, nutritional imbalances, and pH instability—the strains included in existing starfish liquid fertilizers failed to survive and proliferate, resulting in a lack of substantial effects on composting and fertilizer component production.

[0011] In the present invention, Bacillus velezensis ( Bacillus velezensis The invention provides a Bacillus velezensis strain in which the SCB88 strain promotes plant growth, survives and proliferates stably when inoculated into starfish liquid fertilizer, and can improve the maturity and fertilizer content (NPK) of the liquid fertilizer.

[0012] In the present invention, the above Bacillus velezensis ( Bacillus velezensis It was confirmed that plant growth / development is promoted when treated with the SCB88 strain, and the present invention was completed after adding the strain to starfish liquid fertilizer and culturing it, resulting in passing the maturity test and an increase in fertilizer components.

[0014] To achieve the above objective, the objective of the present invention is to provide a Bacillus velezensis having colonization ability in starfish liquid fertilizer deposited as KACC 92641P ( Bacillus velezensis ) It is to provide the SCB88 strain.

[0015] Another objective of the present invention is to provide a composition for promoting plant growth comprising the above-mentioned SCB88 strain, a culture thereof, or a culture extract as an active ingredient.

[0016] Another objective of the present invention is to provide a method for promoting plant growth comprising the step of spraying or applying the plant growth-promoting composition to a plant body.

[0017] Another objective of the present invention is the above Bacillus velezensis ( Bacillus velezensis The present invention provides a method for preparing a composition for promoting plant growth, comprising the step of culturing the SCB88 strain.

[0018] Another objective of the present invention is the above Bacillus velezensis ( Bacillus velezensis The present invention provides a starfish liquid fertilizer composition for promoting plant growth, comprising the SCB88 strain, a culture thereof, or a culture extract as an active ingredient.

[0019] Another objective of the present invention is the above Bacillus velezensis ( Bacillus velezensis The present invention provides a probiotic composition for promoting plant growth, comprising the SCB88 strain, a culture thereof, or a culture extract as an active ingredient.

[0020] Another objective of the present invention is the above Bacillus velezensis ( Bacillus velezensis The present invention provides a composition for promoting microbial colonization in starfish liquid fertilizer, comprising the SCB88 strain, a culture thereof, or a culture extract thereof as an active ingredient.

[0021] Another objective of the present invention is the above Bacillus velezensis ( Bacillus velezensis The present invention provides a composition for promoting the maturation of starfish liquid fertilizer, comprising the SCB88 strain, a culture thereof, or a culture extract thereof as an active ingredient.

[0022] Another objective of the present invention is the above Bacillus velezensis ( Bacillus velezensisThe present invention provides a composition for enhancing the fertilizer component content of a starfish liquid fertilizer, comprising the SCB88 strain, a culture thereof, or a culture extract thereof as an active ingredient. means of solving the problem

[0024] The present specification will be described in more detail below.

[0025] Each description and embodiment disclosed in the present invention may be applied to other descriptions and embodiments thereof. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention should not be considered limited by the specific descriptions provided below.

[0026] Expressions such as “comprising” as used in this specification should be understood as open-ended terms implying the possibility of including other embodiments, unless specifically otherwise stated in the phrase or sentence containing such expression.

[0027] Terms and words used in the description and claims of the present invention shall not be interpreted as being limited to their ordinary or dictionary meanings, but shall be interpreted in a meaning and concept consistent with the technical spirit of the present invention, based on the principle that the inventor can appropriately define the concept of the terms to best describe his invention.

[0029] To achieve the above objective, the present invention relates to Bacillus velezensis having colonization ability in starfish liquid fertilizer deposited as KACC 92641P ( Bacillus velezensis ) Provides the SCB88 strain.

[0030] The inventors of the present invention isolated and identified microorganisms from wild kiwi sap soybean paste. In addition, as a result of analyzing the 16S rRNA gene sequence of the isolated and identified strain, it was confirmed that it has the sequence of SEQ ID NO. 3, and as a result of analyzing the similarity of the sequence and confirming the taxonomic position, it was found to be a standard strain of Bacillus velezensis ( Bacillus velezensis CR-502 Ｔ It was confirmed to be a novel strain belonging to Bacillus velezensis by showing 99.85% homology with ) (Fig. 1), and this was identified as Bacillus velezensis ( Bacillus velezensis It was named SCB88 (KACC 92641P).

[0031] The term "wild kiwi sap soybean paste" as used in this specification refers to a fermented food product made by adding sap collected from wild kiwi trees to traditional soybean paste and aging it. It is characterized by a harmony of the deep flavor of soybean paste with the subtle sweetness and antioxidant properties of wild kiwi sap. The main ingredients include meju, salt, wild kiwi sap, garlic, ginger, onion, red chili powder, etc., and may use rice syrup or plum extract as auxiliary ingredients as needed.

[0032] In addition, the term "colonization ability" as used herein refers to the ability of the SCB88 strain to survive within a specific environment or substrate (e.g., starfish liquid fertilizer), proliferate actively, adapt stably to the environment, and become dominant.

[0033] In particular, the present invention includes physiological characteristics in which the inoculated SCB88 strain exhibits a significant increase in viable cell count within a liquid fertilizer environment containing starfish components and / or high-salt conditions, adapts to the environment within a short period, and maintains quantitative proliferation.

[0034] Furthermore, in this specification, the expression "having colonization ability" indicates the ability of the microorganism to not merely survive, but to effectively establish itself and proliferate within a specific environment to exhibit functionality.

[0035] According to Example 4, the SCB88 strain was at an initial inoculation concentration of 1×10 6 7×10 at CFU / mL 2 days after inoculation 8 By multiplying to CFU / mL, the excellent colonization ability within starfish liquid fertilizer was objectively proven.

[0036] In addition, the above strain has the effect of promoting the maturation of starfish liquid fertilizer and increasing the fertilizer component content of the starfish liquid fertilizer.

[0037] The above strain contributes to the decomposition of organic matter, thereby shortening the maturation speed of the starfish liquid fertilizer and promoting maturation. In addition, the starfish liquid fertilizer inoculated with the above SCB88 strain recorded the highest values ​​of 0.25% nitrogen, 0.031% phosphorus, and 0.10% potassium, demonstrating an effect of increasing the content of nitrogen, phosphorus, and potassium, which are fertilizer components (see Example 5).

[0038] In addition, the strain has the effect of promoting plant growth. Specifically, the strain possesses microbial activities known to aid plant growth (starch degrading ability, protein degrading ability, ACC deaminase activity, phosphate solubilization ability, auxin production, siderophore production, etc.), and it was confirmed that it can grow under salt conditions of 10% NaCl and a wide temperature range (10 to 45℃) while maintaining a high survival rate.

[0039] As used in this specification, the terms "plant growth" or "plant growth" refer to a concept encompassing overall changes related to physiological and morphological growth, such as plant germination, differentiation and development of organs including roots, stems, branches, leaves, flowers, and fruits, cell proliferation, tissue elongation, increase in biomass (weight or size), shortening of the flowering period, fruit formation, and increased yield.

[0040] In addition, "growth promotion" or "growth promotion" refers to a phenomenon in which one or more of the above growth indicators are improved by treatment with the SCB88 strain or the composition of the present invention, and may include effects such as increased growth rate, increased yield, shortened growth period, and improved quality for specific plants.

[0041] For example, in Example 3, a composition containing the SCB88 strain induced a significant improvement in growth-related indicators, such as above-ground height and fresh weight, compared to the control group when applied to tomato plants.

[0042] Additionally, the above plants may be one or more selected from the group consisting of tomatoes, Chinese cabbage, lettuce, and kale, but are not limited thereto.

[0044] In another aspect, the present invention (the above Bacillus velezensis ( Bacillus velezensis ) Provides a composition for promoting plant growth comprising the SCB88 strain, a culture thereof, or a culture extract as an active ingredient.

[0045] In the present invention, the above Bacillus velezensis ( Bacillus velezensis Since the SCB88 strain has a plant growth-promoting effect, a composition for promoting plant growth can be provided using the strain, its culture, or culture extract.

[0046] The above plants may be one or more selected from the group consisting of tomatoes, Chinese cabbage, lettuce, and kale, but are not limited thereto.

[0047] The composition for promoting plant growth according to the present invention can be utilized as a plant growth / growth-promoting agent. The agent may include, for example, fertilizers, seed coating agents, soil conditioners, liquid drenching agents, foliar sprays, or probiotics. The agent according to the present invention can be manufactured in various forms, and for stable formulation, it can be manufactured in various forms such as wettable powders, emulsions, granules, liquids, suspensions, capsules, dispersible granules, and tablets.

[0048] In the present invention, the term "strain" includes a fragment of the ruptured cell wall, dead cells, or dried cells of a strain. Centrifugation or filtration may be performed to remove the culture medium from the culture solution and recover only the concentrated cells, and these steps may be performed as needed by a person skilled in the art. The concentrated cells may be preserved by freezing or freeze-drying according to conventional methods so as not to lose their activity.

[0049] The Bacillus velezensis SCB88 strain of the present invention can be cultured in large quantities by conventional culture methods for microorganisms of the genus Bacillus. As the culture medium, a medium composed of a carbon source, a nitrogen source, vitamins, and minerals can be used.

[0050] The culture medium for the above-mentioned culture may include both solid and liquid media. For example, solid media such as Reasoner's 2A agar (R2A), nutrient agar (NA), tryptic soy agar (TSA), and potato dextrose agar (PDA) may be used, and liquid media may include, but are not limited to, nutrient broth (NB), King's medium B broth (KB), peptone sucrose broth (PSB), or tryptic soy broth (TSB) containing the strain. NB medium may be used as a preferred medium. Culture may be performed under normal culture conditions.

[0051] In the present invention, the term "culture" includes the culture medium itself obtained by culturing a strain according to the present invention in a suitable liquid medium, a filtrate (filtrate or supernatant obtained by centrifugation) from which the strain has been removed by filtering or centrifuging the culture medium, and a cell lysate obtained by ultrasonically treating the culture medium or treating the culture medium with a lysozyme.

[0052] In the present invention, the term "extract of a culture" means that the culture is extracted using various organic solvents to include various physiologically active substances, and is not limited thereto, but the extraction may be performed using an organic solvent selected from the group consisting of C1-C4 alcohols (e.g., methanol, ethanol, butanol, etc.), acetone, and ethyl acetate, either alone or sequentially using two or more types.

[0054] In another aspect, the present invention provides a method for promoting plant growth comprising the step of spraying or applying the plant growth-promoting composition to a plant body.

[0055] The above Bacillus velezensis of the present invention Bacillus velezensis Plant growth can be promoted by the step of spraying or applying a composition for promoting plant growth containing the SCB88 strain to a plant body.

[0056] The above plant body may be one or more plants selected from the group consisting of tomato, Chinese cabbage, lettuce, and kale, but is not limited thereto.

[0057] As used herein, "application" refers to the act of applying a composition containing an active ingredient by applying it directly to the surface of a plant (e.g., leaves, stems, etc.). For example, application using a brush, sponge, roller, or manual applicator is included.

[0058] As used in this specification, "spraying" refers to the application of a composition containing an active ingredient by spreading it widely over a plant or soil using a spray, atomizer, irrigation device, etc., and includes both foliar spray and irrigation methods.

[0059] Therefore, the expression "application or spraying" encompasses all physical delivery methods in which the composition of the present invention is applied directly or indirectly to a plant body.

[0060] The plant growth-promoting composition comprising the SCB88 strain of the present invention can be applied to plants through the application or spraying step defined above, and accordingly, can induce growth-promoting effects such as promoting crop growth, enhancing physiological activity, and increasing productivity.

[0062] In another aspect, the present invention (the above Bacillus velezensis ( Bacillus velezensis A method for preparing a composition for promoting plant growth is provided, comprising the step of culturing the SCB88 strain.

[0063] The above Bacillus velezensis ( Bacillus velezensis By culturing the SCB88 strain, it is possible to produce a composition for promoting plant growth, and it can be formulated in the form described above.

[0064] In addition, the method for culturing the Bacillus velezensis SCB88 strain and preparing the composition may utilize any method known in the art and is not specifically limited to a particular method.

[0066] In another aspect, the present invention (the above Bacillus velezensis ( Bacillus velezensis ) Provides a starfish liquid fertilizer composition for promoting plant growth, comprising the SCB88 strain, a culture thereof, or a culture extract as an active ingredient.

[0067] The aforementioned starfish is a resource containing various organic and inorganic minerals such as amino acids, proteins, calcium, magnesium, and organic sulfur, and when manufactured in the form of liquid fertilizer, it can supply basic nutrients that aid in crop growth. However, starfish liquid fertilizer has the disadvantage that the colonization and proliferation of general microorganisms are inhibited due to complex biological constraints, such as high-salinity environments, antimicrobial substances like saponins contained in the starfish itself, protein-degrading enzyme inhibitors, nutritional imbalances, and pH instability. In fact, commercially available starfish liquid fertilizer products are sold with very low levels of viable bacteria and almost no microbial activity.

[0068] On the other hand, the SCB88 strain used in the present invention possesses excellent colonization and proliferation capabilities even under high-salinity conditions in a starfish liquid fertilizer environment (see Example 4), and in fact, after 48 hours of culture, the viable cell count was 7×10 8 It has been proven to be a useful microorganism capable of effectively surviving and functioning within liquid fertilizer, increasing up to CFU / mL.

[0069] Accordingly, a liquid fertilizer prepared by inoculating the above-mentioned SCB88 strain into a substrate containing a starfish extract or a starfish-derived component can be applied as a starfish liquid fertilizer composition for promoting plant growth.

[0070] The composition of the present invention can be manufactured in the form of a liquid, powder, or granule, and can be utilized in various ways, such as as an agricultural probiotic, a compost fermentation aid, or a functional fertilizer additive.

[0072] In another aspect, the present invention (the above Bacillus velezensis ( Bacillus velezensis ) Provides a probiotic composition for promoting plant growth comprising the SCB88 strain, a culture thereof, or a culture extract as an active ingredient.

[0073] The above composition can be utilized as a probiotic composition because it utilizes the characteristics of the SCB88 strain, which exhibits excellent viability and proliferation even in high-salinity or organic matter-concentrated environments, to promote crop growth, improve the environment of beneficial microorganisms in the soil, and enhance the availability of nutrients such as nitrogen, phosphorus, and potassium.

[0074] The probiotic composition of the present invention can be prepared in a liquid, powder, granule, or encapsulated form and can be applied in various ways, such as by treating the soil around the roots of crops, foliar spraying, or seed coating. Since this probiotic composition can promote crop growth while reducing the use of pesticides or chemical fertilizers, it can be effectively utilized in the fields of eco-friendly agriculture, organic farming, or the cultivation of high-value specialty crops.

[0076] In another aspect, the present invention provides a composition for promoting microbial colonization in starfish liquid fertilizer, comprising a Bacillus velezensis SCB88 strain, a culture thereof, or a culture extract thereof as an active ingredient.

[0077] The SCB88 strain exhibits excellent viability and proliferation ability even in environments where it is difficult for microorganisms to colonize within the starfish liquid fertilizer composition of the present invention, thereby improving the disadvantages of existing commercially available liquid fertilizers, such as low viable cell counts and insufficient microbial activity.

[0078] The SCB88 strain used in the present invention possesses excellent colonization and proliferation capabilities even under high-salinity conditions in a starfish liquid fertilizer environment (see Example 4), and in practice, after 48 hours of culture, the viable cell count was 7×10 8 It was proven that the microbial population could increase up to CFU / mL and effectively survive and function within the liquid fertilizer. Consequently, this composition has the effect of significantly improving the microbial colonization ability within starfish liquid fertilizer.

[0080] In another aspect, the present invention (Bacillus velezensis) Bacillus velezensis) Provides a composition for promoting the maturation of starfish liquid fertilizer comprising the SCB88 strain, a culture thereof, or a culture extract as an active ingredient.

[0081] As used in this specification, the term "maturation" refers to the process in which organic matter within compost, liquid fertilizer, or other organic materials is decomposed and stabilized by the action of microorganisms, and in this process, the organic matter is converted into a state that is not harmful to plants.

[0082] More specifically, composting involves a fermentation or maturation process in which organic high-molecular substances (e.g., proteins, carbohydrates, fats, etc.) are decomposed by microorganisms and converted into low-molecular organic acids, inorganic salts, ammonia, carbon dioxide, and other substances. During this process, pathogenic microorganisms, harmful gases, and toxic substances are removed, the availability and absorbability of nutrients increase, and environmental indicators such as odor, salinity, and pH are improved to a stable range.

[0083] Accordingly, the term "completion of maturation" in this specification means a state in which the starfish liquid fertilizer is sufficiently stabilized so as not to be harmful to plant growth, contains nutritional components suitable for crop growth, and satisfies the conditions for agricultural use.

[0084] The composition of the present invention contributed to shortening the maturation speed of starfish liquid fertilizer and changing the maturity status from immature to fully matured by the SCB88 strain contributing to the decomposition of organic matter (see Example 5).

[0085] Through the action of the above SCB88 strain, volatile organic acids and harmful gases are reduced, and organic matter is converted into a stabilized form, reaching a state suitable for crop growth, thereby allowing it to be safely utilized in agricultural fields.

[0087] In another aspect, the present invention (Bacillus velezensis) Bacillus velezensis) Provides a composition for enhancing the fertilizer component content of a starfish liquid fertilizer, comprising the SCB88 strain, a culture thereof, or a culture extract thereof as an active ingredient.

[0088] The above SCB88 strain can simultaneously promote the maturation of starfish liquid fertilizer and induce a significant increase in the fertilizer component content (NPK) within the liquid fertilizer.

[0089] According to the experimental results, the SCB88 inoculated group recorded the highest values ​​of nitrogen 0.25%, phosphorus 0.031%, and potassium 0.10%, proving that it can be used as a compound fertilizer to increase crop productivity.

[0090] Therefore, the composition of the present invention can be applied as a premium compound fertilizer composition having a triple effect of improving liquid fertilizer quality, enhancing crop growth, and fortifying nutrients.

[0091] The composition for promoting plant growth of the present invention, as well as the starfish liquid fertilizer composition for promoting plant growth, the probiotic composition for promoting plant growth, the composition for promoting microbial colonization in the starfish liquid fertilizer, the composition for promoting the maturation of the starfish liquid fertilizer, and the composition for increasing fertilizer component content, can all be formulated into various forms of preparations such as fertilizers, seed coating agents, soil conditioners, liquid drenching agents, foliar sprays, and probiotics. In addition, the compositions can be manufactured into various formulations, such as wettable powders, emulsions, granules, liquids, suspensions, capsules, dispersible granules, and tablets, to improve stability and ease of use. Effects of the invention

[0093] Since treating plants with the Bacillus velezensis SCB88 strain of the present invention promotes plant growth, the SCB88 strain can be effectively utilized as an eco-friendly microbial agent. Furthermore, because it exhibits superior survival and proliferation capabilities, increased maturity, and enhanced fertilizer component (NPK) content within starfish liquid fertilizer, it can be used as a composition for promoting plant growth, enhancing microbial colonization within starfish liquid fertilizer, accelerating the maturity of starfish liquid fertilizer, and increasing the fertilizer component content of starfish liquid fertilizer. Brief explanation of the drawing

[0095] Figure 1 shows the results of phylogenetic analysis performed with other Bacillus strains that are highly related by analyzing the 16S rRNA sequence of the SCB88 strain. Figure 2 is a graph comparing the height and fresh weight of tomato plants treated with the SCB88 strain in Example 3, untreated plants, and control plants treated with microorganisms belonging to the same genus, velezensis. Specific details for implementing the invention

[0096] Hereinafter, the present invention will be described in detail with reference to examples and the like to aid in understanding the invention. However, the embodiments according to the present invention may be modified in various different forms, and the scope of the present invention should not be interpreted as being limited to the following embodiments. The embodiments of the present invention are provided to more completely explain the invention to those with average knowledge in the art.

[0098] Example 1: Isolation of strain and identification of SCB88 strain

[0099] A strain was isolated from wild kiwi sap soybean paste produced in a household in Sacheon-si, Gyeongnam. The strain was plated on a solid medium and cultured at 37°C for 2 to 3 days, after which the colonies formed on the solid medium were used for strain identification.

[0100] The phylogenetic position and identification of the above-described isolated strain were determined using 16S rRNA sequencing analysis. Gene amplification was commissioned to Genotech (Daejeon, Korea), and the 16S rRNA gene was amplified using the universal primers 27F (AGAGTTTGATCMTGGCTCAG) of SEQ ID No. 1 and 1492R (GGTTACCTTGTTACGACTT) of SEQ ID No. 2. The amplification product was also sequenced using the company's 3100 Genetic Analyser (Applied Biosystems, USA). The obtained sequences were checked for sequence errors and spliced ​​using Seqman software (DNASTAR, USA).

[0101] The 16S rRNA nucleotide sequence of the strain isolated and identified in the present invention is shown in SEQ ID NO. 3. The nucleotide sequence similarity of the 16S rRNA gene was analyzed using EzTaxon (http: / / www.ezbiocloud.net / eztaxon). To construct a phylogenetic tree, the nucleotide sequences were aligned and their lengths were matched using SINA (https: / / www.arb-silva.de / aligner / ). The neighbor-joining algorithm of MAGA 6.0 was used, and the stability of the phylogenetic tree was verified by performing bootstrapping 1,000 times.

[0102] Phylogenetic analysis results show that the above strain is Bacillus velezensis ( Bacillus velezensis CR-502 Ｔ By confirming that it has 99.85% similarity to ), Bacillus velezensis ( Bacillus velezensis It was found that it is a novel strain belonging to ) (Fig. 1). Accordingly, the above strain is Bacillus velezensis ( Bacillus velezensis It was named the SCB88 strain.

[0103] In addition, the SCB64 strain was isolated from the rhizosphere soil of chili peppers in Yonghyeon-myeon, Sacheon-si, and the SCB92 strain was isolated from makgeolli produced by households. Phylogenetic analysis was performed after sequencing analysis, in the same manner as with the aforementioned SCB88 strain. The 16S rRNA sequences of the isolated and identified strains, SCB64 and SCB92, are shown in SEQ ID NOs. 4 and 5, respectively. As a result of the analysis, the SCB64 and SCB92 strains are Bacillus velezensis ( Bacillus velezensis CR-502 Ｔ By confirming that it has 99% and 99.85% similarity with ), Bacillus velezensis ( Bacillus velezensis It was found that they are novel strains belonging to ). Accordingly, the above strains were each Bacillus velezensis ( Bacillus velezensis ) SCB64, Bacillus velezensis( Bacillus velezensis It was named SCB92.

[0105] Example 2: Characterization of SCB88 strain and evaluation of plant growth-promoting enzyme activity

[0106] The separated and identified in Example 1 above Bacillus velezensis To compare and evaluate the characteristics of three strains (SCB64, SCB88, SCB92), these three strains were cultured for 24 hours and the following characteristics were evaluated.

[0108] 2-1. Starch Degradation Ability

[0109] Starch decomposition ability was evaluated using the following method.

[0110] A medium was prepared by mixing 10 g of soluble starch, 18.2 g of R2A agar, and 1000 ml of distilled water, and then autoclaved at 121°C for 15 minutes. The sterilized mixture was dispensed into Petri dishes, and cultured SCB88, SCB64, and SCB92 strains were streak-inoculated and incubated at 28°C for 7 days. After incubation was complete, starch degradation ability was evaluated by treating the medium with iodine solution and checking for the formation of a clear halo around the cells.

[0112] 2-2. Protein Degradation Ability

[0113] Protein degradation ability was evaluated by the following method.

[0114] Mix A (25 g skim milk, 250 ml distilled water) was prepared and sterilized at 115°C for 13 minutes using an autoclave. Additionally, Mix B (4.55 g R2A agar, 250 ml distilled water) was prepared and sterilized at 121°C for 15 minutes. After cooling Mix A and B to approximately 45°C, Mix B was poured into Mix A and mixed. The mixture of Mix B and Mix A was dispensed into a Petri dish, and the cultured SCB88, SCB64, and SCB92 strains were streak-inoculated. The mixture was then incubated at 28°C for 3 days, and the presence or absence of a clear zone was evaluated.

[0116] 2-3. Evaluation of Growth Medium Range

[0117] To evaluate the growth range of SCB88, SCB64, and SCB92 strains, cultured SCB88 strains were inoculated onto R2A, tryptic soy agar (TSA), nutrient agar (NA), and potato dextrose agar (PDA) media and cultured at 30°C for one week, after which the presence or absence of growth was evaluated.

[0119] 2-4. Evaluation of Growth Temperature Range

[0120] To evaluate the growth temperature range of SCB88, SCB64, and SCB92 strains, cultured SCB88, SCB64, and SCB92 strains were inoculated into R2A medium and cultured at 10–45°C (5°C intervals) for one week, after which the presence or absence of growth was evaluated.

[0122] 2-5. Evaluation of NaCl Growth

[0123] To evaluate the growth range of SCB88, SCB64, and SCB92 strains in a medium containing NaCl, cultured SCB88, SCB64, and SCB92 strains were inoculated into R2A medium containing 1–10% (1% w / v intervals) of NaCl and cultured at 30°C for one week, after which growth in NaCl was evaluated.

[0125] 2-6. Evaluation of Auxin (IAA, Indole-3-acetic acid) Production Capacity

[0126] To evaluate the auxin production ability of SCB88, SCB64, and SCB92 strains, the cultured SCB88, SCB64, and SCB92 strains were harvested by centrifugation and cultured for 24 hours in a medium containing 100 μg / ml of tryptophan in R2A broth. Afterward, the samples were centrifuged at 3000 rpm for 10 minutes, and 1 ml of the supernatant was mixed with 2 ml of Salkowski solution (35% perchloric acid, 1 ml of 0.5 M FeCl3). Subsequently, the auxin production ability was evaluated by measuring the absorbance at 530 nm.

[0128] 2-7. Evaluation of Phosphate Solubilization Capacity

[0129] To evaluate the phosphate solubilization ability of SCB88, SCB64, and SCB92 strains, cultured SCB88, SCB64, and SCB92 strains were cultured in PVK medium (glucose 10 g, Ca3(PO4)25 g, (NH4)2SO4 0.5 g, NaCl 0.2 g, MgSO4·7H2O 0.1 g, KCl 0.2 g, yeast extract 0.5 g, MnSO4·H2O 0.002 g, FeSO4·7H2O 0.002 g, Agar 15.0 g, distilled water 1000 ml, bromo-phenol-blue 0.025 g, pH 7.0) for one week to evaluate whether a ring shape was formed.

[0131] 2-8. Evaluation of ACC deaminase activity

[0132] To evaluate 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity, a drop of suspension of SCB88, SCB64, and SCB92 strains, cultured with 0.5 M ACC (150 µl / plate) plated on DF minimal medium (KH2PO44 g, Na2HPO46 g, MgSO4·7H2O 0.2 g, CuSO4·5H2O 78.22 µg, MoO310 µg, (NH4)2SO42 g, Agar 15 g, distilled water 1000 ml, pH 7.2), was added to evaluate growth.

[0134] 2-9. Evaluation of Siderophore Production Capacity

[0135] To evaluate the siderophore production ability of SCB88, SCB64, and SCB92 strains, the CAS diffusion assay was used. Mix A was prepared by mixing a solution of 15.125 mg of chrome azurol S dissolved in 12.5 ml of distilled water, 2.5 ml of iron(III) solution (1 mM FeCl3·6H2O, 10 mM HCl), and 10 ml of an aqueous solution (18.225 mg of HDTMA, 10 ml of distilled water). Subsequently, Mix B (3.38 g of agar, 7.56 g of pipes, 10 mM NaOH, 187.5 ml of distilled water, pH 6.8) was prepared. Mix B and Mix A were slowly mixed, dispensed into a Petri dish, and thoroughly dried. Using a sterile scalpel, the dried medium was divided in half, one half was removed, and R2A was dispensed. Then, the cultured SCB88, SCB64, and SCB92 strains were streak-inoculated onto the R2A surface and cultured at 28°C for one week, and the change from blue to yellow was evaluated.

[0137] Table 1 below shows the results of evaluating microbial activities (starch degradation ability, protein degradation ability, ACC deaminase activity, phosphate solubilization ability, auxin and siderophore production) known to aid in plant survival or growth under various environmental distress conditions and growth characteristics for SCB88, SCB64, and SCB92 strains (+ indicates activity, - indicates inactivity). In addition, the morphological and physiological-biochemical characteristics of SCB88, SCB64, and SCB92 strains are presented.

[0138] The above Bacillus velezensis ( Bacillus velezensis All three strains, SCB64, SCB88, and SCB92, grew in R2A, TSA, NA, and PDA media, and were able to grow in a temperature range of 10–45°C and under 10% NaCl conditions. All three strains exhibited starch and protein degradation capabilities, and as growth-promoting activities, they all demonstrated IAA (Indole-3-acetic acid) production, ACC deaminase production, phosphate solubilization, and siderophore production.

[0139]

[0141] Example 3: Growth-promoting effect of Bacillus velezensis strain on tomatoes

[0142] Tomatoes are Bacillus velezensis ( Bacillus velezensis The group was divided into four groups: SCB64, SCB88, SCB92 strain treatment groups and untreated control groups. Each strain treatment group was drenched under greenhouse conditions, and the growth of tomatoes was compared.

[0143] To this end, tomato seeds (variety: Super Dotarō) were sown in 9 cm plastic pots filled with potting soil and cultivated in a greenhouse for 12 to 16 days. Before the true leaves unfolded, bacterial suspensions of each strain (OD600=0.20, 10 610 ml of CFU / ML was applied as a drench, and after growing tomatoes for 12 to 14 days, plant height, root length, above-ground fresh weight, and below-ground fresh weight were measured.

[0144] As a result, as confirmed in Figure 2, the SCB64, SCB88, and SCB92 strain treatment groups showed increased tomato plant height and fresh weight compared to the untreated control group (Figure 2).

[0146] Example 4: In starfish liquid fertilizer Bacillus velezensis Comparative evaluation of strain growth

[0147] In this embodiment, the self-separated Bacillus velezensis The following experiment was conducted to compare the growth ability of three strains (SCB64, SCB88, SCB92) in starfish liquid fertilizer.

[0149] 4-1. Evaluation of Microbial Growth in Commercially Available Starfish Liquid Fertilizer

[0150] The growth of microorganisms contained in a commercially available starfish liquid fertilizer product (amino acid fermentation byproduct) was evaluated. The main ingredients consist of 50% starfish, 30% molasses, 10% microbial fermentation liquid, 5% organic sulfur, and 5% grain flour. Analysis of the microbial content revealed a very low viable cell count, at approximately 10⁴ CFU / mL.

[0151] In addition, the salinity of the above-mentioned starfish liquid fertilizer product was measured to be in the range of 2.05% to 3.7%, which means that it is difficult for microorganisms that are sensitive to salt or cannot decompose starfish to survive. In fact, it was confirmed that the survival and proliferation of microorganisms present in commercially available starfish liquid fertilizer products are inhibited because they cannot adapt to the starfish components.

[0153] 4-2. Evaluation of the Survival and Proliferative Capacity of Strains in Starfish Liquid Fertilizer

[0154] Separated in the present invention Bacillus velezensis We intended to evaluate whether three strains (SCB64, SCB88, SCB92) could survive and proliferate stably even in a starfish-based liquid fertilizer environment with high salinity (2.05-3.7%).

[0155] First, the three strains mentioned above (SCB64, SCB88, SCB92) were cultured in NB (Nutrient Broth) medium at 30°C for 24 hours, and then the viable cell count (CFU) was measured to 1×10 8 A bacterial suspension was prepared by diluting with sterile physiological saline to a CFU / ml level. 50 μL of the prepared suspension was inoculated into 5 mL of starfish probiotics (liquid fertilizer), so that the initial inoculation concentration in the liquid fertilizer was approximately 1 × 10⁻⁶ 6 The concentration was adjusted to CFU / mL. The inoculated liquid fertilizer was cultured with shaking at 30℃ for 2 days (48 hours).

[0156] After the culture was completed, 100 μL was taken from each sample, serially diluted 10-fold with sterile physiological saline, and plated onto PCA (Plate Count Agar) medium. The plated medium was cultured at 30°C for 24 hours, and the number of colonies formed after culture was counted to calculate the viable cell count (CFU / mL).

[0157] As a result, the SCB88 strain produced 7×10 within 2 days of inoculation. 8 The viable cell count was found to have increased up to CFU / mL, a result demonstrating very high proliferative capacity compared to other strains. On the other hand, SCB92 was 2×10 7 CFU / mL, SCB64 is 4×10 6 It exhibited a relatively low proliferation level at CFU / mL. This confirmed that the SCB88 strain is the most suitable strain for survival and proliferation in a starfish liquid fertilizer environment.

[0158] strain name Initial inoculation concentration (CFU / mL) Live cell count (CFU / mL) after 2 days Proliferation level SCB64 1 × 10 6 4 × 10 6 lowness SCB88 1 × 10 6 7 × 10 8 Very high SCB92 1 × 10 6 2 × 10 7 commonly

[0160] Example 5: Bacillus velezensis Evaluation of Maturity and Changes in Fertilizer Components of Starfish Liquid Fertilizer Following Strain Inoculation

[0161] The separated and identified in Example 1 above Bacillus velezensisWe wanted to determine whether three strains (SCB64, SCB88, SCB92) could promote the maturity of starfish liquid fertilizer with high salinity (2.05-3.7%) and increase the content of fertilizer components (N, P, K).

[0162] First, each strain is 1×10 8 1 mL of a suspension diluted with sterile physiological saline to a CFU / ml level is inoculated into starfish liquid fertilizer (100 mL), so that the initial inoculation concentration in the liquid fertilizer is approximately 1 × 10⁻⁶ 6 The concentration was adjusted to CFU / mL. Using the same method, a total of five treatment groups were formed, including an untreated control (stationary state) and a shaking culture control (150 rpm shaking culture) in addition to the three strain treatment groups. Subsequently, all treatment groups were shaken cultured for 20 days at 30°C under conditions of 150 rpm.

[0163] After cultivation was completed, the maturity status of each treatment group was measured using a liquid fertilizer maturity meter (LMQ 2000) owned by the Sacheon City Agricultural Technology Center, and the total nitrogen (N), phosphorus (P), and potassium (K) content were quantitatively analyzed using a liquid fertilizer component analyzer (SM304-FCM V3).

[0164] As a result of the analysis, the static control group was determined to be in an 'immature' state, with N, P, and K values ​​all appearing as 0. On the other hand, the control group that underwent only shaking culture was determined to be fully mature, but fertilizer components (NPK) were still not detected. In contrast, all experimental groups inoculated with microorganisms were determined to be fully mature, and the group treated with the SCB88 strain recorded the highest fertilizer component values, with nitrogen at 0.250%, phosphorus at 0.031%, and potassium at 0.100%. SCB92 showed nitrogen at 0.152%, phosphorus at 0.023%, and potassium at 0.015%, while SCB64 showed levels of nitrogen at 0.095%, phosphorus at 0.007%, and potassium at 0.040% (Table 3).

[0165] Through these results, it was confirmed that the SCB88 strain is the most effective strain not only for survival and proliferation within starfish liquid fertilizer but also for promoting the composting of the liquid fertilizer and increasing the fertilizer component content.

[0166] Treatment area Assessment of maturity Nitrogen (N, %) Phosphorus (P, %) Potassium (K, %) Untreated control group (stationary state) Unripe 0 0 0 Shaking culture control (150 rpm) Fermentation complete 0 0 0 SCB64 vaccination group Fermentation complete 0.095 0.007 0.04 SCB88 vaccination group Fermentation complete 0.25 0.031 0.1 SCB92 vaccination group Fermentation complete 0.152 0.023 0.015

[0168] Statistical analysis

[0169] Statistical analysis of plant shoot height, root length, and fresh weight was performed using R packages, including analysis of variance (ANOVA) and multiple variance testing of biomass between treatments. The least significant difference (LSD) test was used for multiple variance testing at P < 0.05, and all experiments were conducted at least three times.

[0171] Name of Depositing Institution: National Institute of Agricultural Sciences Accession Number: KACC92641P Date of Deposit: 2025-03-24

Claims

Claim 1 Bacillus velezensis deposited as KACC 92641P, which has the ability to colonize and proliferate in starfish liquid fertilizer and enhances the content of one or more fertilizer components selected from the group consisting of nitrogen (N), potassium (K), and phosphorus (P) in starfish liquid fertilizer ( Bacillus velezensis ) SCB88 strain. Claim 2 In paragraph 1, the strain is Bacillus velezensis, which promotes tomato growth ( Bacillus velezensis ) SCB88 strain. Claim 3 In paragraph 1, the strain is Bacillus velezensis, which promotes the maturation of starfish liquid fertilizer ( Bacillus velezensis ) SCB88 strain. Claim 4 delete Claim 5 In claim 1, the strain is Bacillus velezensis having a nucleotide sequence 16S rRNA represented by SEQ ID NO. 3 ( Bacillus velezensis ) SCB88 strain. Claim 6 delete Claim 7 Bacillus velezensis of Article 1 ( Bacillus velezensis A composition for promoting tomato growth comprising the SCB88 strain, a culture thereof, or a culture extract as an active ingredient. Claim 8 A method for promoting tomato growth comprising the step of spraying or applying the tomato growth-promoting composition of claim 7 to a tomato plant. Claim 9 Bacillus velezensis of Article 1 ( Bacillus velezensis A method for preparing a composition for promoting tomato growth, comprising the step of culturing the SCB88 strain. Claim 10 Bacillus velezensis of Article 1 ( Bacillus velezensis A starfish liquid fertilizer composition for promoting tomato growth, comprising the SCB88 strain, a culture thereof, or a culture extract as an active ingredient. Claim 11 Bacillus velezensis of Article 1 ( Bacillus velezensis A probiotic composition for promoting tomato growth, comprising the SCB88 strain, a culture thereof, or a culture extract as an active ingredient. Claim 12 Bacillus velezensis of Article 1 ( Bacillus velezensis Bacillus velezensis in starfish liquid fertilizer containing the SCB88 strain, its culture, or culture extract as an active ingredient ( Bacillus velezensis Composition for promoting colonization of the SCB88 strain. Claim 13 Bacillus velezensis of Article 1 ( Bacillus velezensis A composition for promoting the maturation of starfish liquid fertilizer, comprising the SCB88 strain, a culture thereof, or a culture extract thereof as an active ingredient. Claim 14 Bacillus velezensis of Article 1 ( Bacillus velezensis A composition for enhancing the fertilizer component content of one or more starfish liquid fertilizers selected from the group consisting of nitrogen (N), potassium (K), and phosphorus (P), comprising the SCB88 strain, a culture thereof, or a culture extract thereof as an active ingredient.

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