Bacillus subtilis GSB-17 Strain Having Food Waste-Degrading Ability and Salt and Pungency Tolerance, Microbial Agent for Food Waste Treatment Comprising the Same, Method for Cultivating Starter Culture for Food Waste Treatment, Method for Producing Microbial Agent for Food Waste Treatment, and Method for Treating Food Waste

Abstract

The present invention relates to a Bacillus subtilis GSB-17 strain (KCTC 16758BP) with excellent food waste decomposition ability and resistance to salt and spiciness, and a microbial preparation for a food waste processor containing the same. The strain was identified as Bacillus subtilis by 16S rRNA gene region analysis and phylogenetic analysis, is capable of growing under conditions of 7 wt% NaCl and 50,000 SHU, and exhibits chitinase, amylase, cellulase, lipase, protease, and gelatinase activities. A pellet-type microbial preparation containing the strain is useful for reducing the volume and weight of food waste, and the decomposed product after treatment can be utilized as a compost composition.

Description

Technology Field

[0001] The present invention relates to a novel microbial strain for the fermentation, decomposition, and reduction of food waste, a microbial preparation containing said strain, a method for manufacturing said microbial preparation, a method for treating food waste, and a compost composition obtained by said treatment. More specifically, the present invention relates to a Bacillus subtilis GSB-17 strain (accession number KCTC 16758BP) which exhibits enzymatic activity for decomposing chitin, starch, cellulose, fat, protein, and gelatin while having resistance to salt and spicy components that are problematic during food waste treatment, and a microbial preparation for a food waste processor using said strain. Background Technology

[0002] Since food waste has high moisture and organic matter content and contains a complex mixture of carbohydrates, fiber, lipids, and proteins, it is difficult to sufficiently suppress odors, leachate, decay, and secondary pollution through simple drying or mechanical grinding alone. Accordingly, there is a high need for technologies that ferment and eliminate or reduce food waste by microorganisms within the source or treatment device.

[0003] Conventionally, microbial preparations for decomposing food waste using strains of the genus Bacillus or combinations of multiple microorganisms, microbial compositions for the fermentation and decomposition of high-concentration organic matter, microbial additives for food processing, and mixed microbial strains have been proposed. These technologies are primarily designed to decompose carbohydrates, cellulose, lipids, and proteins using strains or combinations of strains possessing amylase, cellulase, lipase, and protease activities.

[0004] However, actual food waste, particularly Korean-style food waste, is prone to salt accumulation and often contains spicy components such as chili peppers and seasonings. Since food is fed into the food waste disposer continuously or repeatedly, the salt concentration and spicy components inside the disposer can inhibit the growth of bacterial strains; in this case, reduced decomposition efficiency, odor generation, and spoilage may occur.

[0005] Therefore, there is a need to develop novel strains capable of growing under high-salt and spicy conditions while possessing polyenzymatic activity that can simultaneously decompose the major components of food waste, as well as microbial preparations stably carrierd therein.

[0006] <Prior Art Literature>

[0007] - Published Patent Application No. 10-2016-0089221

[0008] (A novel microbial strain with excellent decomposition ability for food waste)

[0009] - Registered Patent Publication No. 10-2355488

[0010] (Method for manufacturing microbial additives for food processing)

[0011] - Registered Patent Publication No. 10-2599313

[0012] (Mixed microbial strains that decompose food waste) The problem to be solved

[0013] Accordingly, the purpose of the present invention is to provide a novel Bacillus subtilis strain capable of stably proliferating even in the presence of salt accumulation and spicy components within a food waste treatment device, and efficiently decomposing major organic components of food waste, in order to solve the problems of the prior art.

[0014] Another objective of the present invention is to provide a microbial preparation for a food waste processor that is easy to store, transport, and feed by fermenting and pelletizing the strain together with a carrier or nutrient substrate.

[0015] Another objective of the present invention is to provide a treatment method and a compost composition that utilize the microbial agent to increase the reduction rate and reduce the volume of food waste, and enable the decomposition product after treatment to be utilized as compost. means of solving the problem

[0016] In order to achieve the above objectives,

[0017] The present invention provides a Bacillus subtilis GSB-17 strain deposited under accession number KCTC 16758BP.

[0018] The above strain is identified as belonging to the Bacillus subtilis lineage based on 16S rRNA gene region analysis and phylogenetic analysis. In this specification, the identification and reproduction of the above strain are specified by deposit number KCTC 16758BP, and no specific listing of nucleic acid residues or limitation by sequence identification number is used.

[0019] The above strain has salt tolerance and spiciness resistance capable of growing under 7 wt% NaCl conditions and oleoresin capsicum conditions equivalent to 50,000 SHU.

[0020] The above strain has chitinase, starchase, cellulase, lipase, proteolytic, and gelatinase activities.

[0021] In addition, the present invention provides a microbial preparation for treating food waste comprising one or more of the strain, culture of the strain, culture medium, cell, or spore as an active ingredient.

[0022] In one embodiment, the microbial preparation may be a preparation formed by mixing a Bacillus subtilis GSB-17 starter culture solution with cashew nut meal, rapeseed meal, cypress sawdust, rice bran, and flavor additives, fermenting the mixture, and then forming it into pellets. The starter culture solution may be mixed in an amount of 1 to 20 parts by weight, preferably 3 to 10 parts by weight, per 100 parts by weight of the carrier composition.

[0023] In addition, the present invention provides a method for preparing the above-mentioned starter culture solution, a method for preparing the above-mentioned microbial preparation, a method for treating food waste using the above-mentioned microbial preparation, and a compost composition obtained by the above-mentioned treatment. Effects of the invention

[0024] The Bacillus subtilis GSB-17 strain according to the present invention, configured as described above, exhibits degrading enzyme activity against various organic components, including carbohydrates, cellulose, lipids, and proteinaceous substrates contained in food waste; therefore, it is useful for single-strain-based microbial preparations for food waste treatment.

[0025] Since the strain according to the present invention can proliferate under conditions of 7 wt% NaCl and 50,000 SHU, it is advantageous for maintaining decomposition activity even in the operating environment of a food waste disposer where salt and spicy components may accumulate.

[0026] The microbial preparation according to the present invention can be fermented and pelletized together with a carrier and a nutrient substrate, making storage and input easy, and can be utilized for reducing the volume and volume of food waste in a food waste processor.

[0027] In addition, the decomposed food waste treated with the microbial agent according to the present invention contains organic matter, nitrogen, phosphoric acid, and potassium components, and the levels of harmful substances are below the standard, so it can be utilized as compost. Brief explanation of the drawing

[0028] Figure 1 shows the NCBI BLAST search results for the 16S rRNA gene region of Bacillus subtilis GSB-17. Figure 2 is a phylogenetic tree showing 20 species of the genus Bacillus and a control group. Figure 3 shows photographs of the enzyme activity evaluation of Bacillus subtilis GSB-17 (A: Chitinase, B: Starchase, C: Cellulase, D: Lipase, E: Protease, F: Gelatinase). Figure 4 is a graph comparing the hourly reduction rates of the untreated, Company A, Company B, and GSB-17 microbial agent in a food waste disposer. Figure 5 is a graph comparing the hourly volume reduction rates of the untreated, Company A, Company B, and GSB-17 microbial agents in a food waste disposer. Figure 6 shows comparative photos of tomato growth using food waste composted with microbial agents (T1: untreated, T2: Company A microbial agent, T3: Company B microbial agent, T4: GSB-17 microbial agent). Figure 7 is the deposit certificate for the Bacillus subtilis GSB-17 strain. Specific details for implementing the invention

[0029] The aforementioned objectives, features, and advantages will be described in detail below with reference to the attached drawings, and accordingly, a person skilled in the art to which the present invention pertains will be able to easily implement the technical concept of the present invention.

[0030] In describing the present invention, detailed descriptions of known technologies related to the present invention are omitted if it is determined that such descriptions could unnecessarily obscure the essence of the invention.

[0031] The terms used in this invention have been selected to be as widely used as possible, taking into account their functions in the invention; however, these may vary depending on the intent of those skilled in the art, case law, the emergence of new technologies, etc.

[0032] In addition, in specific cases, there are terms arbitrarily selected by the applicant, and in such cases, their meanings will be described in detail in the description of the relevant invention.

[0033] Therefore, the terms used in this invention should be defined not merely by their names, but based on their meanings and the overall content of the invention.

[0034] Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings.

[0035] However, the embodiments of the present invention exemplified below may be modified in various other forms, and the scope of the present invention is not limited to the embodiments described below.

[0036] The embodiments of the present invention are provided to more fully explain the invention to those skilled in the art.

[0037] 1. Strain Discovery and Isolation

[0038] Samples were collected from a food waste treatment facility in Hwasun-gun, Jeollanam-do. The collected samples were serially diluted in sterile physiological saline and plated on 0.5% swollen chitin agar medium.

[0039] Bacterial colonies that produced a clear halo in the above medium were selected and purely isolated by plating them 2 to 3 times on a new medium. Among the isolated strains, the strain exhibiting activity suitable for decomposing major components of food waste was named GSB-17.

[0040] 2. Identification and Deposit of Strains

[0041] The 16S rRNA gene region of isolated strain GSB-17 was amplified using 27F and 1492R primers, and the amplification products were analyzed. Specific nucleic acid residues are not listed in this specification, and the identification of the strain is determined by the accession number KCTC 16758BP and the identification results.

[0042] As shown in Figures 1 and 2, the NCBI GenBank BLAST search results showed that the analysis results matched the Bacillus database reference data at a level of 99 to 100%, and the E-value was confirmed to be 0. The strain showing the highest similarity score (Max score) was Bacillus subtilis strain CL2 (MN696247.1).

[0043] Phylogenetic analysis (Maximum likelihood, bootstrap 500) of Bacillus subtilis showed that GSB-17 formed a phylogenetic group with the standard strains Bacillus subtilis MN696247, Bacillus subtilis NR_112116, and Bacillus subtilis NR_102783. The bootstrap support of the upper branch of Bacillus subtilis to which GSB-17 belongs was confirmed to be 99 to 100%.

[0044] In addition, the clade containing GSB-17 was clearly separated from the Bacillus pumilus and Bacillus cereus lineages, and it was confirmed that there is a low possibility of phylogenetic confusion with other closely related species. Therefore, GSB-17 was identified as Bacillus subtilis.

[0045] Bacillus subtilis GSB-17 was deposited with the Korean Collection for Type Cultures (KCTC, KRIBB), an international depository institution under the Treaty of Budapest, as shown in Fig. 7, under deposit number KCTC 16758BP, and according to the deposit certificate, the date of receipt and acceptance is April 23, 2026.

[0046] 3. Evaluation of Salt Tolerance and Spicy Tolerance

[0047] Since food is continuously fed into microbial fermentation food processors, microorganisms may die or their activity may be reduced due to salt and spicy components in the food. Accordingly, the salt and spiciness resistance of GSB-17 was evaluated.

[0048] After inoculating the isolated strains into LB medium (Luria-Bertani agar) containing 7 wt% NaCl and oleoresin capsicum equivalent to 50,000 SHU, respectively, the proliferation was checked.

[0049] As a result, GSB-17 proliferated even under conditions where salt in food accumulated up to about 7%, compared to the average salt content of dried food waste powder being 3 to 4%. In addition, it proliferated even under conditions of 50,000 SHU, which is about 5 times spicier than Cheongyang chili peppers, confirming that it is applicable to food decomposition regardless of whether spicy food is added.

[0050] 4. Evaluation of Degradation Enzyme Activity

[0051] The chitinase, starchase, cellulase, lipase, protease, and gelatinase activities of GSB-17 were evaluated in their respective substrate media (see Fig. 3).

[0052] To evaluate chitinase activity, the strain was inoculated onto an agar medium containing 0.5 wt% swollen chitin and cultured at 37°C for 24 to 48 hours. After culture, the diameter of the clear halo formed around the colony was measured.

[0053] To evaluate starch-degrading enzyme activity, a medium containing 1 wt% soluble starch and 1.5 wt% agar was prepared, and the strain was inoculated and cultured at 37°C for 24 hours. After culture, Lugol solution was applied, and the diameter of the clear halo formed around a single colony was measured.

[0054] To evaluate cellulase activity, the strain was inoculated into an agar medium containing 15 wt% CMC (carboxymethyl cellulose) and cultured at 37°C for 24 hours. After culture, the sample was stained with 0.5 wt% Congo red solution for 15 minutes and decolorized with 1 M NaOH to measure the diameter of the clear zone.

[0055] To evaluate lipase activity, an agar medium containing 1 wt% TBN (tributyrin) was emulsified by sonication for 1 hour, then the strain was inoculated and cultured at 37°C for 24 hours.

[0056] To evaluate proteolytic enzyme activity, the strain was inoculated into an agar medium containing 2 wt% skim milk and cultured at 37°C for 24 hours. To evaluate gelatinase activity, an agar medium containing 3 wt% gelatin was prepared, and after inoculating the strain and culturing at 37°C for 24 hours, 30 wt% trichloroacetic acid was added to measure the diameter of the clear zone.

[0057] As shown in Table 1, GSB-17 formed clear zones of chitinase 1.2 cm, amylase 1.5 cm, cellulase 1.7 cm, lipase 0.7 cm, protease 1.9 cm, and gelatinase 2.2 cm. Therefore, GSB-17 possesses complex enzyme activity capable of degrading major macromolecular components contained in food waste.

[0058] No. Strain No. Chitinase Amylase Cellulase Lipase Protease Gelatinase 1 GSB-03 0.8 1.1 1.4 0 1.2 1.1 2 GSB-08 0.2 0.8 1.5 0.1 2.0 2.2 3 GSB-10 0.2 0.7 1.2 0.5 1.9 1.8 4 GSB-14 0.4 0.9 0.8 0.9 2.0 2.0 5 GSB-15 0.5 0.7 1.7 0.4 2.1 2.0 6 GSB-16 0.1 0.4 0.9 0.3 1.5 1.3 7 GSB-21 0.2 0.1 0.5 1.0 1.3 1.1 8 GSB-23 0.8 0.1 0.6 0 1.4 1.5 9 GSB-34 0.1 0.2 0.8 0 0.7 0.9 10 GSB-37 0.7 0.8 0.5 0 1.6 1.5 11 GSB-39 0.7 0.1 1.2 0 1.5 1.3 12 GSB-45 0.9 1.1 1.1 0.2 1.5 1.1 13 GSB-49 0.1 0.7 1.2 0.3 1.1 1.0 14 GSB-54 0.5 1.7 1.0 0 1.0 1.2 15 GSB-55 1.0 1.1 0.8 0.1 1.4 1.3 16 GSB-58 0.5 1.1 1.0 0.1 1.2 1.1 17 GSB-67 0.4 0.1 0.9 0.4 1.2 1.0 18 GSB-69 1.3 0.5 1.4 0 1.1 0.9 19 GSB-78 0.9 0.7 0.7 0.5 1.0 1.2 20 GSB-79 0.4 0.4 1.3 0.1 1.6 1.3 21 GSB-86 1.1 0.9 1.1 0.7 1.7 1.4 22 GSB-88 0.7 0.8 1.1 0.7 1.1 1.0 23 GSB-91 1.0 0.8 1.4 0 1.0 0.8 24 GSB-93 1.0 0.7 0.8 0.8 0.9 1.1 25 GSB-94 1.1 1.0 0.5 0.3 0.8 0.5 26 GSB-97 0.7 0.5 0.3 0.5 1.4 1.1 GSB-17 1.2 1.5 1.7 0.7 1.9 2.2

[0059] 5. Culture of starter cultures for food waste disposers

[0060] A total of 1 L of culture medium was prepared and sterilized to contain 0.2 wt% Na2HPO4, 0.1 wt% K2HPO4, 0.05 wt% NaCl, 0.1 wt% NH4Cl, 0.05 wt% MgSO4·7H2O, 0.05 wt% CaCl2·2H2O, and 1 wt% dried food powder.

[0061] Bacillus subtilis GSB-17 (KCTC 16758BP) was inoculated into a sterile medium and cultured at 37°C for 2 days to prepare a decomposition microbial inoculum culture solution for a food waste disposer.

[0062] Since the above medium contains dried powder derived from food waste, it is advantageous for allowing the strain to proliferate in a state adapted to the complex carbon source contained in actual food waste.

[0063] 6. Manufacture of microbial preparations for food waste disposers

[0064] A Bacillus subtilis GSB-17 (KCTC 16758BP) spawn culture solution was mixed with a carrier composition comprising 30% by weight of cashew nut meal, 25% by weight of rapeseed meal, 19% by weight of cypress sawdust, 25% by weight of rice bran, and 1% by weight of a flavor additive. At this time, the spawn culture solution was mixed in such a way that it was 5% by weight per 100% by weight of the carrier composition.

[0065] The above mixture was fermented for 2 days to produce a fermented base material, and the fermented base material was fed into a pelletizer and molded into a pellet shape.

[0066] In another embodiment, the carrier composition may comprise 20 to 40 weight% cashew nut meal, 15 to 35 weight% rapeseed meal, 10 to 30 weight% cypress sawdust, 15 to 35 weight% rice bran, and 0.1 to 5 weight% flavor additive, and within the above range, it may be adjusted considering fermentability, moldability, storageability, and feedability into a processor.

[0067] In another embodiment, the inoculum culture solution may be mixed in an amount of 1 to 20 parts by weight per 100 parts by weight of the carrier composition, preferably in an amount of 3 to 10 parts by weight. The above range is suitable for maintaining the moldability and drying properties of the carrier while ensuring the initial colonization of the strain.

[0068] The above formulation may be used as a liquid culture medium, or may be manufactured in the form of a carrier adsorbent, powder, granules, tablets, or pellets.

[0069] 7. Evaluation of the food waste decomposition ability of microbial agents for food waste disposers

[0070] To evaluate the food waste treatment efficacy of a microbial agent prepared with Bacillus subtilis GSB-17 (KCTC 16758BP), an experiment was conducted using the DTR-ADV004 model food waste processor from Datoro Biotech Co., Ltd.

[0071] The experimental groups were divided into an untreated group containing only food waste, a group treated with a microbial agent for Company A's food waste disposer, a group treated with a microbial agent for Company B's food waste disposer, and a group treated with the GSB-17 microbial agent.

[0072] Each experimental group was evaluated under identical treatment equipment, food waste input conditions, stirring conditions, and operating time conditions. The weight loss rate was calculated as the ratio of the remaining weight after operation to the initial input weight, and the volume reduction rate was calculated based on the degree of decrease in volume-related sensory characteristics or indicators compared to the untreated group.

[0073] As shown in Figures 4 and 5, the GSB-17 microbial preparation treatment group showed overall higher weight loss and volume reduction rates compared to the untreated group, the A company treatment group, and the B company treatment group during the observation period of 12 to 60 hours.

[0074] 8. Evaluation of composting of food waste

[0075] The suitability of food waste decomposed with the GSB-17 microbial agent for use as compost was evaluated. Conditions for organic matter to be used as compost include organic matter content, levels below the standard for hazardous components, and the degree of maturity.

[0076] Food waste decomposed by the GSB-17 microbial agent was found to be suitable for use as compost and was confirmed to contain fertilizer components such as nitrogen, phosphorus, and potassium.

[0077] Analysis Items (Unit) Standard specifications Analysis results GSB-17 microbial preparation decomposed food standard Organic matter (%) 30 or more 65.10 harmful ingredients Arsenic (mg / kg) [dry weight] 45 or less Non-detectable Cadmium (mg / kg) [Dry Weight] 5 or less Non-detectable Mercury (mg / kg) [dry weight] 2 or less Non-detectable Lead (mg / kg) [Dry Weight] 130 or less 1.98 Chromium (mg / kg) [Dry Weight] 200 or less 6.90 Copper (mg / kg) [building weight] 360 or less 14.03 Nickel (mg / kg) [Dry Weight] 45 or less 2.29 Zinc (mg / kg) [Dry Weight] 900 or less 76.68 E. coli O157:H7 Non-detectable Non-detectable Salmonella Non-detectable Non-detectable Other specifications Organic matter to nitrogen ratio 45 or less 44.28 Salt (%) [Dry Weight] 2.0 or lower 1.36 moisture(%) 55 or less 11.55 Maturity [Comeback Method] Completion of maturation (70%) or less Fully ripened Hydrochloric acid insoluble matter (%) 25 or less 3.05 Nutrients Total Nitrogen (%) No standards 1.47 Total Phosphoric Acid (%) No standards 1.19 Total potassium (%) No standards 0.87

[0078] 9. Evaluation of Plant Growth Promotion by Composted Food Waste

[0079] As shown in Fig. 6, the effect on tomato growth was evaluated when food waste decomposed by the untreated (T1), Company A microbial agent (T2), Company B microbial agent (T3), and GSB-17 microbial agent (T4) was used as compost.

[0080] As a result of plant experiments, tomato growth tended to be relatively good when food decomposed by the GSB-17 microbial agent (T4) was used as compost.

[0081] Accordingly, the Bacillus subtilis GSB-17 strain of the present invention and the microbial preparation containing it can be introduced into food waste processors for household, commercial, multi-unit housing, and treatment facilities to be used for the fermentation and decomposition, reduction, and volume reduction of food waste.

[0082] In addition, since the decomposed food waste treated with the microbial agent of the present invention can be utilized as a compost composition, it becomes applicable to the field of resource recovery and circular utilization of food waste.

[0083] <Deposit Information>

[0084] Depository Name: Korea Research Institute of Biotechnology and Bioengineering Biological Resource Center (KCTC)

[0085] Trustee Number: KCTC 16758BP

[0086] Date of Trust: April 23, 2026

[0087] As specific parts of the present invention have been described in detail above, it will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the present invention.

[0088] Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims

Claim 1 Bacillus subtilis GSB-17 strain deposited under accession number KCTC 16758BP, which has the function of decomposing food waste. Claim 2 Bacillus subtilis GSB-17 strain according to claim 1, characterized in that the strain is capable of growing in LB medium containing 7% by weight NaCl and LB medium containing oleoresin capsicum equivalent to 50,000 SHU. Claim 3 A Bacillus subtilis GSB-17 strain according to claim 1, characterized in that the strain has chitinase, amylase, cellulase, lipase, protease, and gelatinase activities and forms a clear halo around a single colony in an agar medium containing each substrate. Claim 4 A microbial preparation for treating food waste comprising, as an active ingredient, one or more of the following: a Bacillus subtilis GSB-17 strain deposited under accession number KCTC 16758BP having the function of decomposing food waste, and a culture, culture medium, cell, or spore of said strain. Claim 5 A microbial preparation for treating food waste according to claim 4, characterized in that the microbial preparation is a pellet-type or granular preparation obtained by inoculating a culture solution of the strain onto a carrier or nutrient substrate comprising cashew nut meal, rapeseed meal, cypress sawdust, rice bran, and flavor additives and fermenting it. Claim 6 A microbial preparation for treating food waste according to claim 5, wherein the carrier or nutrient material comprises 20 to 40 weight% cashew nut meal, 15 to 35 weight% rapeseed meal, 10 to 30 weight% cypress sawdust, 15 to 35 weight% rice bran, and 0.1 to 5 weight% flavor additives based on 100 weight% of the total carrier composition. Claim 7 A method for culturing starter culture for treating food waste, comprising the step of inoculating Bacillus subtilis GSB-17 (KCTC 16758BP) into a medium comprising 0.1 to 0.3 wt% Na2HPO4, 0.05 to 0.2 wt% K2HPO4, 0.01 to 0.1 wt% NaCl, 0.05 to 0.2 wt% NH4Cl, 0.01 to 0.1 wt% MgSO4·7H2O, 0.01 to 0.1 wt% CaCl2·2H2O, and 0.5 to 2.0 wt% dried food powder, and culturing at 30 to 40°C for 1 to 3 days. Claim 8 A method for manufacturing a microbial agent for treating food waste, comprising the step of mixing a starter culture obtained by the method of claim 7 with a carrier composition comprising 20 to 40 weight% cashew nut meal, 15 to 35 weight% rapeseed meal, 10 to 30 weight% cypress sawdust, 15 to 35 weight% rice bran, and 0.1 to 5 weight% flavor additives based on 100 weight% of the total carrier composition, fermenting the mixture for 1 to 4 days, and forming the fermented composition into pellets, wherein the starter culture is mixed in an amount of 1 to 20 weight parts per 100 weight parts of the carrier composition. Claim 9 A method for treating food waste comprising the steps of: introducing the microbial agent of claim 4 into a food waste processor; introducing food waste into the food waste processor; and fermenting and decomposing the food waste inside the food waste processor under aerobic and stirring conditions. Claim 10 delete

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