bacillus subtilis bs-q, microbial preparation containing the same, method for preparing bacillus subtilis, bacteriostatic culture and preparation method and application thereof, bacteriostatic agent

Abstract

The application belongs to the technical field of microorganisms, and particularly relates to bacillus subtilis BS-Q, a microbial preparation containing the same, a preparation method of bacillus subtilis, a bacteriostatic culture and a preparation method and application thereof, and a bacteriostatic agent. The bacillus subtilis BS-Q provided in the application has the characteristics of maintaining bacteriostatic activity in a high-salt environment, and can effectively overcome the defects that the metabolic products of traditional bacillus subtilis are prone to conformational change or aggregation inactivation in the high-salt environment of the intestinal tract. Meanwhile, the strain and the bacteriostatic culture thereof exhibit significant inhibitory effects on type A, type C and type G Clostridium perfringens, and have good broad-spectrum bacteriostatic effect.

Description

Technical Field

[0001] This application belongs to the field of microbial technology, specifically relating to Bacillus subtilis BS-Q, microbial preparations containing it, methods for preparing Bacillus subtilis, antibacterial cultures and their preparation methods and applications, and antibacterial agents. Background Technology

[0002] Necrotic enteritis (NE) in poultry is primarily caused by Clostridium perfringens type A or C. This pathogen proliferates rapidly in the intestines and produces toxins, leading to necrosis of the intestinal mucosa, impaired digestion and absorption, and in severe cases, acute death. Globally, NE causes billions of dollars in economic losses to the poultry industry annually, including decreased productivity, increased treatment costs, and higher slaughter waste rates. With the full implementation of antibiotic-free feed policies, the intestinal flora balance previously maintained by sub-therapeutic doses of antibiotics has been disrupted, significantly increasing the colonization and pathogenicity risk of Clostridium perfringens. This has resulted in a sharp rebound in the clinical incidence and recurrence rate of NE, posing a major challenge to healthy poultry farming in the post-antibiotic era.

[0003] Bacillus subtilis, a recognized safe probiotic, contains various antibacterial active substances such as lipopeptides, bacitracin, and enzymes in its metabolites, making it an important candidate to replace antibiotics. However, traditional techniques that directly use Bacillus subtilis fermentation broth or crude extracts have significant shortcomings: firstly, the metabolites react with the high-salt environment of the intestine (such as bile salts, sodium...) + / K + In high-salt environments, these products are prone to conformational changes or aggregation and inactivation, leading to a significant decrease in their antibacterial activity. On the other hand, these products have a weak ability to clear biofilms already formed by Clostridium perfringens, making it difficult to break down the physical barrier of the pathogen. In addition, different toxin types (such as NetB positive and negative) of Clostridium perfringens have different sensitivities to the same metabolite. The metabolites of existing strains are often only effective against some toxin types, resulting in uneven inhibitory effects and making it easy for "escaped strains" to cause secondary outbreaks.

[0004] Although Bacillus subtilis is recognized as a safe probiotic, and its metabolites have been studied for their antibacterial properties, some known Bacillus subtilis metabolites face significant application bottlenecks when used in complex physiological environments such as the animal gut. For example, the high bile salt environment in the gut, leading to hyperosmolar stress, can easily cause conformational changes or aggregation and inactivation of active proteins or lipopeptides in metabolites, thus severely weakening their actual antibacterial efficacy. Furthermore, existing methods lack effective clearance capabilities against established Clostridium perfringens biofilms, and their inhibitory effects on different toxin strains (such as NetB-positive and NetB-negative) are uneven, making it difficult to achieve broad-spectrum and stable control effects.

[0005] Therefore, it is particularly urgent to screen a new strain of Bacillus subtilis with good environmental adaptability. Summary of the Invention

[0006] Based on this, one embodiment of this application provides Bacillus subtilis BS-Q, a microbial preparation containing it, a method for preparing Bacillus subtilis, an antibacterial culture and its preparation method and application, and an antibacterial agent.

[0007] This application provides a strain of Bacillus subtilis BS-Q, taxonomically named Bacillus subtilis BS-Q, which was deposited at the China Center for Type Culture Collection (CCTCC) on January 22, 2026. The accession number of Bacillus subtilis BS-Q is CCTCC NO: M 2026198, and the address is Wuhan University, Wuhan, China. This strain was received and registered by the collection center on January 22, 2026, and was confirmed to be a viable strain by the collection center on January 29, 2026.

[0008] This application provides a microbial preparation comprising the aforementioned Bacillus subtilis BS-Q and an acceptable carrier.

[0009] This application also provides a method for preparing Bacillus subtilis, the method comprising the following steps:

[0010] Inoculate the culture medium with the Bacillus subtilis BS-Q or the microbial preparation described above, and culture to prepare a culture solution; the culture solution contains Bacillus subtilis BS-Q.

[0011] In some embodiments, the culture medium is LB liquid medium or NB medium.

[0012] In some of these embodiments, the culture temperature is 25°C-40°C.

[0013] In some of these embodiments, the incubation time is 12-36 hours.

[0014] This application also provides a method for preparing an antibacterial culture, the method comprising the following steps: preparing a culture medium using the preparation method described in 3; removing Bacillus subtilis BS-Q from the culture medium, collecting the supernatant, and directly preparing an antibacterial culture; or removing the solvent from the supernatant to prepare a solid antibacterial culture.

[0015] This application also provides a method for preparing the aforementioned antibacterial culture, resulting in an antibacterial culture.

[0016] This application also provides the use of the aforementioned antibacterial culture in the preparation of antibacterial agents.

[0017] In some embodiments, the antimicrobial agent inhibits one or more of Clostridium perfringens type A, Clostridium perfringens type C, and Clostridium perfringens type G.

[0018] This application also provides the use of the aforementioned antibacterial culture in the preparation of microecological preparations for the prevention and / or treatment of necrotizing enteritis in poultry.

[0019] This application also provides an antibacterial agent, which includes the Bacillus subtilis BS-Q microbial preparation or the antibacterial culture.

[0020] The Bacillus subtilis BS-Q strain provided in this application possesses the characteristic of maintaining antibacterial activity in high-salt environments, effectively overcoming the shortcomings of traditional Bacillus subtilis metabolites, which are prone to conformational changes or aggregation and inactivation in the high-salt intestinal environment. Simultaneously, this strain and its antibacterial culture exhibit significant inhibitory effects against Clostridium perfringens types A, C, and G, demonstrating excellent broad-spectrum antibacterial efficacy. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 Photographs of inhibition zones of different toxin types of Clostridium perfringens provided in an embodiment of this application; Note: Figure 1 In this context, A stands for CP-A; Figure 1 B in the text stands for CP-C; Figure 1C in the text represents CP-G; ①. Penicillin 25 μg / mL; ②. BS-Q fermentation broth; ③. ATCC 6051 fermentation broth; ④. LB negative control;

[0023] Figure 2 This is the result of a localization test of an antibacterial substance provided in an embodiment of this application; Note: Figure 2 In this context, A stands for CP-A; Figure 2 B in the text stands for CP-C; Figure 2 C in the table represents CP-G; ①. Penicillin 25 μg / mL; ②. Sterile supernatant of bacterial cells after washing with PBS, resuspending and sonicating; ③. CFS of sterile supernatant of BS-Q; ④. BS-Q bacterial cells after washing with PBS and resuspending; ⑤. PBS control; ⑥. LB control;

[0024] Figure 3 The results of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) determinations provided in an embodiment of this application are shown below. Note: ①. 268.50 μg / mL; ②. 134.25 μg / mL; ③. 67.13 μg / mL; ④. 33.56 μg / mL; ⑤. 16.78 μg / mL.

[0025] Figure 4 This application provides antibacterial activity after different pH treatments in one embodiment; Note: PC: untreated CFS; NC: 6051-CFS after the same treatment;

[0026] Figure 5 This application provides an embodiment of antibacterial activity after different temperature treatments; Note: PC: untreated CFS; NC: 6051-CFS after the same treatment;

[0027] Figure 6 This application provides antibacterial activities after treatment with different proteases in one embodiment; Note: PC: untreated CFS; NC: 6051-CFS treated in the same way;

[0028] Figure 7 This application provides an embodiment of antibacterial activity after treatment with different salt concentrations; Note: PC: untreated CFS; NC: 6051-CFS after the same treatment;

[0029] Figure 8 The scanning electron microscope provided in one embodiment of this application shows the effect of CFS on bacterial cell morphology; Note: A. CP-A; B. CP-C; C. CP-G; D. CP-A treated with CFS for 0.5 h; E. CP-C treated with CFS for 0.5 h; F. CP-G treated with CFS for 0.5 h;

[0030] Figure 9The effect of CFS on biofilm formation provided in one embodiment of this application; Note: 1 / 16 MBC: C. perfringens treated with 1 / 16 MBC of CFS; 1 / 32 MBC: C. perfringens treated with 1 / 32 MBC of CFS; 1 / 64 MBC: C. perfringens treated with 1 / 64 MBC of CFS; 0: C. perfringens without CFS treatment; Compared with the blank control, , indicating statistical significance (P < 0.05); , indicating that the difference is extremely significant (p < 0.001).

[0031] Figure 10 The growth inhibition curve of Clostridium perfringens in a co-culture experiment provided in an embodiment of this application; Note: A: Growth trend of CP-A and BS-Q co-cultured for 48 h; B: Growth trend of CP-C and BS-Q co-cultured for 48 h; C: Growth trend of CP-G and BS-Q co-cultured for 48 h; A1: Viable number of CP-A bacteria in the co-culture medium of BS-Q and CP-A; A2: Viable number of CP-A bacteria in the co-culture medium of ATCC 6051 and CP-A; A3: Viable number of CP-A bacteria in the culture medium alone; C1: Viable number of CP-C bacteria in the co-culture medium of BS-Q and CP-C; C2: Viable number of CP-C bacteria in the co-culture medium of ATCC 6051 and CP-C; C3: Viable number of CP-C bacteria in the culture medium alone; G1: Viable number of CP-G bacteria in the co-culture medium of BS-Q and CP-G; G2: ATCC CP-G viable count in co-culture medium of 6051 and CP-G; G3: CP-G viable count in culture medium alone;

[0032] Figure 11 The mucosal immune level (sIgA) provided in one embodiment of this application; Note: A: Compared with the 14d Control group, , indicating statistical significance (p < 0.05); A: This indicates a highly significant difference (p < 0.001). B: Compared with the CP group, , indicating statistical significance (p < 0.05); , indicating that the difference is extremely significant (p < 0.001).

[0033] Figure 12 The effect of Bacillus subtilis BS-Q and its sterile supernatant on the intestinal morphology of chickens challenged with Clostridium perfringens, as provided in an embodiment of this application;

[0034] Figure 13 In one embodiment of this application, total DNA was extracted from cecal contents and subjected to 16S rRNA gene sequencing. PCoA analysis showed that...

[0035] Figure 14 A gate-level composition analysis provided for an embodiment of this application;

[0036] Figure 15 The results of cecal microbiota analysis provided in an embodiment of this application;

[0037] Figure 16 The results of cecal microbiota analysis provided in an embodiment of this application. Detailed Implementation

[0038] The present application will be further described in detail below with reference to the embodiments and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0039] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0040] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0041] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0042] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0043] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0044] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0045] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0046] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0047] In this application, the technical features described in an open-ended manner include both closed technical solutions composed of the listed features and open technical solutions composed of the listed features.

[0048] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0049] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0050] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0051] All references to documents mentioned in this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the inventive purpose and / or technical solution of this application, all cited documents are incorporated herein by reference in their entirety and for all purposes. When citing documents in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. When citing documents in this application, examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0052] This application provides a strain of Bacillus subtilis BS-Q, taxonomically named Bacillus subtilis BS-Q, which was deposited at the China Center for Type Culture Collection (CCTCC) on January 22, 2026. The accession number of Bacillus subtilis BS-Q is CCTCC NO: M 2026198, and the address is Wuhan University, Wuhan, China. This strain was received and registered by the collection center on January 22, 2026, and was confirmed to be a viable strain by the collection center on January 29, 2026.

[0053] This *Bacillus subtilis* strain can maintain and enhance the antibacterial activity of its metabolites in a high-salt environment (8%-10% NaCl concentration), increasing the activity by more than 10% compared to the untreated group. This effectively solves the problem that traditional *Bacillus subtilis* metabolites are prone to conformational changes or aggregation and inactivation in the high-salt intestinal environment. The metabolites of this strain have significant inhibitory effects on *Clostridium perfringens* types A, C, and G, with minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of approximately 30-35 μg / mL (based on total protein content). Furthermore, it exhibits good acid-base stability, thermal stability, and protease sensitivity.

[0054] This application also provides a microbial preparation comprising the aforementioned Bacillus subtilis BS-Q and an acceptable carrier. The acceptable carrier may be selected from one or more of physiological saline, phosphate buffer, skim milk powder, trehalose, mannitol, glucose, and sucrose. This microbial preparation can be used to prepare feed additives, drinking water additives, or veterinary drug preparations.

[0055] This application also provides a method for preparing Bacillus subtilis, the method comprising the following steps:

[0056] Inoculate the culture medium with the Bacillus subtilis BS-Q or the microbial preparation described above, and culture to prepare a culture solution; the culture solution contains Bacillus subtilis BS-Q.

[0057] In some embodiments, the culture medium is LB liquid medium or NB medium.

[0058] In some embodiments, the culture medium may be selected from any one of LB liquid medium, NB medium, nutrient broth medium, and tryptone soybean broth medium.

[0059] In some embodiments, the culture temperature is 25°C-40°C. For example, the culture temperature can be selected from any one of 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C.

[0060] In some embodiments, the culture time is 12h-36h. For example, the culture time can be selected from any one of 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h, 24h, 25h, 26h, 27h, 28h, 29h, 30h, 31h, 32h, 33h, 34h, 35h, or 36h.

[0061] This application also provides a method for preparing an antibacterial culture, the method comprising the following steps:

[0062] The culture medium is prepared using the method described above; and Bacillus subtilis BS-Q is removed from the culture medium, the clear liquid is collected, and an antibacterial culture is prepared.

[0063] In some embodiments, the process further includes removing Bacillus subtilis BS-Q from the culture medium, collecting the supernatant, removing the solvent from the supernatant, and preparing a solid antibacterial culture.

[0064] This application also provides a method for preparing the aforementioned antibacterial culture, resulting in an antibacterial culture.

[0065] This application also provides the use of the aforementioned antibacterial culture in the preparation of antibacterial agents.

[0066] In some embodiments, the antimicrobial agent inhibits one or more of Clostridium perfringens type A, Clostridium perfringens type C, and Clostridium perfringens type G.

[0067] This application also provides the use of the aforementioned antibacterial culture in the preparation of microecological preparations for the prevention and / or treatment of necrotizing enteritis in poultry.

[0068] This application also provides an antibacterial agent, which includes the Bacillus subtilis BS-Q, the microbial preparation, or the antibacterial culture.

[0069] The Bacillus subtilis BS-Q of this application has enhanced antibacterial activity in high-salt environments. Under conditions of 8%-10% NaCl concentration, the antibacterial activity of its metabolites is increased by more than 10% compared with the untreated group, effectively solving the problem that traditional Bacillus subtilis metabolites are prone to conformational changes or aggregation and inactivation in the high-salt environment of the intestine.

[0070] The antibacterial culture of this application has a significant inhibitory effect on Clostridium perfringens types A, C and G, with a minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of about 30-35 μg / mL, exhibiting good broad-spectrum antibacterial effect.

[0071] The Bacillus subtilis BS-Q and its antibacterial culture of this application have good acid-base stability, thermal stability and biofilm clearance ability, and can maintain stable antibacterial activity in the complex intestinal environment.

[0072] The Bacillus subtilis BS-Q and its antibacterial culture disclosed in this application can effectively prevent and control necrotizing enteritis in poultry through multiple pathways, including improving growth performance, repairing intestinal damage, regulating immunity, and reshaping the microecology, providing an effective antibiotic alternative for healthy poultry farming in the post-antibiotic era.

[0073] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0074] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0075] It should be understood that in the various embodiments of this application, the order of the above-mentioned processes does not imply the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0076] Example 1: Isolation and Identification of Strains

[0077] 1.1 Source of strain

[0078] Bacillus subtilis BS-Q was isolated from fecal samples of wild animals. 1g of fecal sample was added to 9mL of sterile physiological saline, vortexed, and then serially diluted to 10⁻⁶. -4 10 -5 10 -6 Take 100 μL of each culture and spread it onto LB solid medium. Incubate at 37°C for 24 h. Pick single colonies with typical morphology (dry, wrinkled, milky white), streak them three times to obtain pure cultures, and label them BS-Q.

[0079] 1.2 Morphological observation and Gram staining

[0080] Gram staining was performed on pure BS-Q cultures, and bacterial morphology was observed under a microscope (100× oil immersion). Results are as follows: Figure 1 As shown: the bacterial cells are purple (Gram-positive), rod-shaped with blunt ends, and the spores are oval, consistent with the typical morphological characteristics of Bacillus subtilis.

[0081] 1.3 Spore staining

[0082] Malachite green staining was used to observe BS-Q spore formation. The specific steps were: smear preparation, flame fixation, addition of malachite green solution, heating until steaming and maintaining for 5 minutes, washing with water, counterstaining with safranin for 45 seconds, and observation under an oil immersion microscope. Results showed that peak spore formation occurred approximately 12 hours after incubation (see [link to relevant documentation]). Figure 2 ).

[0083] 1.4 Identification of 16S rRNA

[0084] Genomic DNA was extracted from BS-Q strains (according to the bacterial DNA extraction kit instructions). PCR amplification was performed using universal 16S rRNA primers 27F (SEQ ID NO.1: 5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (SEQ ID NO.2: 5'-GGTTACCTTGTTACGACTT-3'). PCR reaction conditions: 95℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 55℃ annealing for 90 s, 72℃ extension for 30 s, for a total of 34 cycles; 72℃ extension for 10 min. The amplified product was subjected to 1.5% agarose gel electrophoresis, yielding a specific band of approximately 1500 bp. Sequencing and BLAST alignment showed over 99% homology with the Bacillus subtilis reference strain. Phylogenetic analysis indicated... Figure 3 BS-Q has the smallest genetic distance and the closest kinship with the reference strains of Bacillus subtilis (accession numbers MT240918.1, HQ678655.1, and KM492825.1).

[0085]

[0086] 1.5 Biochemical identification

[0087] Bacterial microbiochemical identification tubes were used for detection. The specific procedure was as follows: a single colony was picked and dissolved in physiological saline to prepare a 0.5% McFarland turbidity bacterial suspension. 100 μL of the suspension was added to each tube (hydrogen sulfide tubes were streaked with an inoculation loop), and the tubes were incubated at 37°C for 24 hours. Results showed: positive fermentation with glucose, mannitol, and sucrose (turning yellow); negative fermentation with lactose and maltose (no change in color); negative fermentation with hydrogen sulfide (no change in color); and a positive VP test. Based on Bergey's Manual of Bacterial Identification, 8th edition, the results are consistent with the characteristics of Bacillus subtilis.

[0088] Example 2: Preparation of sterile supernatant (CFS)

[0089] Bacillus subtilis BS-Q was inoculated at 1% (v / v) in LB liquid medium and cultured at 37°C with shaking at 180 rpm for 12 h. The resulting fermentation broth was centrifuged at 4°C and 8000 rpm for 15 min, and the supernatant was filtered through a 0.22 μm aqueous filter membrane for sterilization to obtain liquid CFS. A portion of the CFS was pre-frozen at -80°C for 24 h, then freeze-dried under vacuum for 48 h, and ground to obtain CFS lyophilized powder. The CFS solids content was determined to be approximately 25 mg / mL, and the total protein concentration was approximately 250-280 μg / mL (determined using the BCA method).

[0090] Example 3: Determination of antibacterial activity (agar diffusion method)

[0091] Clostridium perfringens standard strains of type A (CVCC 52), type C (CVCC 48), and type G (D25) were cultured to the logarithmic growth phase and diluted with PBS to approximately 1×10⁻⁶. 8 CFU / mL. Add 100 μL of indicator bacterial suspension to 15 mL of TSC medium cooled to approximately 40°C, mix well, and pour into a 90 mm Petri dish containing an Oxford cup (8.00 mm outer diameter). After the plates solidify, remove the Oxford cups and add 50 μL of BS-Q fermentation broth, BS-Q CFS, a positive control (penicillin 25 μg / mL), and a negative control (LB medium), respectively. After anaerobic incubation at 37°C for 24 h, measure the diameter of the inhibition zone using calipers. Each group has three replicates. Results are as follows: Figures 1-2 As shown in Table 1.

[0092] Comparison of antibacterial activity with standard strain ATCC 6051: Bacillus subtilis BS-Q and standard strain ATCC 6051 were cultured under the same conditions (LB medium, 37℃, 180 rpm, 12 h) to prepare fermentation broth and CFS. The antibacterial activity against Clostridium perfringens type A was determined using the agar diffusion method. Results showed that neither the ATCC 6051 fermentation broth nor CFS exhibited significant antibacterial activity against Clostridium perfringens type A (inhibition zone diameter was approximately 8.0 mm, comparable to the outer diameter of an Oxford cup). In contrast, the inhibition zone diameter of the BS-Q fermentation broth was 19.67 ± 0.02 mm, and the inhibition zone diameter of the BS-Q CFS was 16.38 ± 0.65 mm (see [link to relevant documentation]). Figure 1 This indicates that the BS-Q strain possesses the ability to produce antimicrobial proteins, which is not present in the ATCC 6051 standard strain.

[0093] Furthermore, in the co-culture experiment (Example 8), the viable count of Clostridium perfringens in the BS-Q co-culture group decreased by 1.7-2.3 lg CFU / g at 48 h, while there was no significant difference between the ATCC 6051 co-culture group and the single-culture group (see [link]). Figure 10 The above comparative data further demonstrates the unique antibacterial activity of the BS-Q strain.

[0094] Table 1. Antibacterial activity of CFS against three types of Clostridium perfringens.

[0095]

[0096] Example 4: Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)

[0097] The micro-dilution method was used. In 96-well plates, CFS was serially diluted with LB medium to volume concentrations of 50%, 25%, 12.5%, 6.25%, 3.125%, 1.5625%, 0.781%, 0.391%, 0.195%, and 0.098%. 100 μL of Staphylococcus aureus culture (approximately 1 × 10⁻⁶) was added to each well. 6 (CFU / mL) , incubated at 37℃ for 12 h. Wells without bacterial culture served as negative controls, and wells with bacterial culture only served as positive controls. The turbidity of the culture medium in the wells was observed, and the lowest CFS concentration that inhibited bacterial growth was defined as the MIC. 100 μL samples were taken from the MIC and the wells above the previous concentration and spread onto TSC plates. After anaerobic incubation at 37℃ for 24 h, the bacteria were counted, and the lowest concentration that killed 99.9% of the bacteria was defined as the MBC. Results are as follows. Figure 3 As shown, the MIC and MBC of CFS against the three Clostridium perfringens species were both approximately 30-35 μg / mL.

[0098] Example 5: Stability Evaluation

[0099] Acid-base stability: The pH of CFS was adjusted to approximately 3.0, 5.0, 9.0, and 11.0, respectively, and then adjusted back to the original pH after appropriate treatment time. The antibacterial activity was then measured. The results showed that CFS maintained significant antibacterial activity even after extreme pH treatment (e.g., ...). Figure 4 (As shown).

[0100] Thermal stability: CFS was treated at approximately 45℃, 65℃, 85℃, and 100℃ for 30 min, and its antibacterial activity was measured after cooling. The results showed that CFS retained its antibacterial activity after treatment at 100℃ (e.g., Figure 5 (As shown).

[0101] Protease stability: Trypsin, pepsin, and proteinase K (final concentration approximately 1 mg / mL) were added to CFS, and the proteases were inactivated after appropriate treatment time. Antibacterial activity was then measured. Results showed that the antibacterial activity of all three proteases decreased significantly after treatment (e.g., ...). Figure 6 (As shown).

[0102] Salt concentration stability: Solutions with final NaCl concentrations of 2%, 4%, 6%, 8%, and 10% were prepared using CFS, with CFS without NaCl serving as a control. The antibacterial activity of each sample against Staphylococcus aureus was determined using the Oxford cup method. The results showed that when the NaCl concentration reached 8%-10%, the antibacterial activity of CFS was significantly improved compared to the untreated group, with an increase of 12%-24%. p <0.01), showing a dose-dependent enhancement (e.g. Figure 7 (As shown in the figures). Other stability (pH, temperature, protease) procedures are similar and have been described in detail in the figures.

[0103] Example 6: Effect on bacterial cell morphology (scanning electron microscopy)

[0104] CFS (approximately 1 / 2 MIC, i.e., approximately 15-17 μg / mL) was mixed with Clostridium perfringens (CP-A, CP-C, CP-G) bacterial suspensions and anaerobically cultured at 37°C for 30 min. The bacterial cells were collected by centrifugation, washed twice with PBS, fixed with 2.5% glutaraldehyde, and incubated overnight at 4°C. Subsequently, the cells were washed three times with 0.1M phosphate buffer, fixed with 1% osmium tetroxide for 1 h, dehydrated with a gradient of ethanol (30%, 50%, 70%, 80%, 90%, 95%, 100%, 15 min each), and replaced with isoamyl acetate for 15 min. The cells were dried using a critical point dryer, sputter-coated with gold (approximately 30 s), and observed under a scanning electron microscope (HITACHI SU8100) at an accelerating voltage of 5 kV and a magnification of 20000×.

[0105] The results are as follows Figure 8As shown: the control group (A, B, C) cells are rod-shaped with a smooth and plump surface; the CFS-treated group (D, E, F) cells have a rough, wrinkled, or even ruptured surface, indicating that CFS can destroy the cellular structural integrity of Clostridium perfringens.

[0106] Example 7: Determination of anti-biofilm activity (crystal violet staining method)

[0107] Adjust the *Clostridium perfringens* population to approximately 2 × 10⁻⁶. 8 CFU / mL was mixed with 100 μL each of CFS at different dilutions (1 / 16 MBC, 1 / 32 MBC, 1 / 64 MBC) in 96-well plates and anaerobically incubated at 37°C for 48 h. The supernatant was discarded, the plates were washed three times with PBS, fixed with methanol for 15 min, stained with 0.1% crystal violet for 15 min, rinsed with running water, dried, and dissolved in 33% glacial acetic acid. OD was then measured. 595 Values. Each group has 3 replicates. Results are as follows: Figure 9 As shown.

[0108] Example 8: Co-culture experiment

[0109] BS-Q cells were collected by centrifugation, washed three times with PBS, and resuspended in FTG medium to a final volume of 2.5 × 10⁻⁶. 5 CFU / mL. Mixed with Clostridium perfringens types A, C, and G (final concentration 2.5 × 10⁻⁶). 4 The mixture (CFU / mL) was incubated anaerobically at 37℃ for 48 h. Samples were taken at 0 h, 12 h, 24 h, 36 h, and 48 h, serially diluted, and plated on TSC plates for counting. ATCC 6051 co-culture group and Clostridium perfringens culture group served as controls. Results are as follows: Figure 10 As shown.

[0110] Example 9: Animal Experiment Design

[0111] One-day-old healthy broiler chickens (medium-growth type, purchased from Guangdong Zhiwei Agricultural Technology Co., Ltd.) were selected and randomly divided into several groups, with three replicates per group. The experimental groups included: blank control group, challenge control group, BS-Q high / low dose prevention group, BS-Q high / low dose treatment group, CFS high / low dose prevention group, CFS high / low dose treatment group, and positive drug control group.

[0112] Bacterial intervention: BS-Q bacterial suspension was administered via drinking water, with a high dose of approximately 10. 8 CFU / animal / day, low dose approximately 10 6CFU / animal / day. CFS intervention: CFS was administered via drinking water, with a high dose of approximately 10 mL / animal / day and a low dose of approximately 1 mL / animal / day. The prevention group received administration before challenge, while the treatment group received administration after challenge. Challenge protocol: Starting from a specific age, except for the blank control group, all groups received approximately 10 mL of fresh Clostridium perfringens bacterial suspension orally daily. 8 CFU / animal), continuously challenged with the virus for several days.

[0113] Example 10: Growth Performance Measurement

[0114] Chickens were weighed on an empty stomach before and at the end of the experiment to calculate the average daily weight gain. The results are shown in Table 2. Compared with the challenge control group, the average daily weight gain of the high-dose BS-Q bacterial prophylaxis group, low-dose treatment group, and high-dose treatment group was significantly increased (p<0.05-0.001), with the high-dose treatment group showing the most significant effect. There were no statistically significant differences between the CFS intervention groups and the positive drug control group and the challenge control group.

[0115] Table 2. Average daily weight gain of each experimental group (12-19 days old)

[0116]

[0117] Note: Compared with the virus challenge group, p<0.05; p<0.001.

[0118] Example 11: Intestinal lesion scoring

[0119] After the challenge, the animals were euthanized, and duodenal lesions were observed and scored according to a 0-4 scale. The results are shown in Table 3. The average lesion score of the challenge control group was approximately 1.8, significantly higher than that of the blank control group (approximately 0.9, p<0.001). Compared with the challenge control group, except for a few groups, the lesion scores of all intervention groups decreased significantly (p<0.05-0.001), with the low-dose bacterial prophylaxis group, high-dose treatment group, and all CFS intervention groups showing the most significant effects.

[0120] Table 3 Duodenal Lesion Scoring

[0121]

[0122] Note: p<0.001 compared with the virus challenge group.

[0123] Example 12: Measurement of mucosal immune level (sIgA)

[0124] Duodenal lavage fluid was collected, and the sIgA content was determined using an ELISA kit. Figure 11As shown in the results, the sIgA level in the control group was significantly higher than that in the blank control group after challenge (p<0.01). Compared with the challenge control group, the sIgA levels in the BS-Q high / low dose prevention group, high dose treatment group, and CFS high dose treatment group were significantly lower (8%-11%, p<0.05-0.001).

[0125] Example 13: Observation of intestinal villus morphology

[0126] Prepare paraffin sections of the duodenum, measure villus height and crypt depth, and calculate the villus-crypt ratio. Figure 12 As shown, the results indicated that the villus height was reduced, the crypt depth was increased, and the villus-crypt ratio was decreased in the challenge control group. All intervention groups significantly improved the above morphological parameters, with the villus-crypt ratio showing the most significant improvement in the low-dose bacterial prevention group and the high-dose CFS treatment group (p<0.001).

[0127] Example 14: Analysis of the cecal microbiota

[0128] Total DNA was extracted from the cecal contents, and 16S rRNA gene sequencing was performed. PCoA analysis showed (e.g.) Figure 13 As shown in AF), the samples from the challenge control group and each intervention group were clearly separated, and some intervention group samples were adjacent to the blank control group, indicating that both bacterial cell and CFS interventions can reverse the bacterial flora disorder caused by challenge. Phylogenetic composition analysis (such as...) Figure 14 As shown in the figure, challenge led to an increase in the relative abundance of Bacillota and a decrease in the relative abundance of Bacteroidota, and the intervention group was able to restore the ratio of the two.

[0129] LEfSE analysis (e.g.) Figures 15-16 As shown in the figure, the bacterial intervention group was enriched with beneficial bacteria such as Lactobacillus and Enterococcus, the CFS intervention group was enriched with groups such as Erysipelotrichales and Subdoligranulum, and the positive drug control group was enriched with other specific groups.

[0130] Conclusion: This application confirms that Bacillus subtilis BS-Q cells and their sterile supernatant can prevent and control necrotizing enteritis in poultry through multiple pathways, including improving growth performance, repairing intestinal damage, regulating immunity, and reshaping the microecology. Furthermore, the intervention effect of the bacterial cells is superior to that of CFS. Both can be used alone or in combination to prepare microecological preparations, replacing antibiotics in broiler farming.

[0131] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A strain of Bacillus subtilis BS-Q, characterized in that, It was deposited at the China Center for Type Culture Collection on January 22, 2026, with the accession number CCTCC NO:M 2026198, address: Wuhan University, Wuhan, China.

2. A microbial preparation, characterized in that, The microbial preparation comprises Bacillus subtilis BS-Q as described in claim 1 and an acceptable carrier.

3. A method for preparing Bacillus subtilis, characterized in that, The preparation method includes the following steps: Inoculate the culture medium with Bacillus subtilis BS-Q as described in claim 1 or the microbial preparation as described in claim 2, culture, and prepare a culture medium; the culture medium contains Bacillus subtilis BS-Q.

4. The method for preparing Bacillus subtilis according to claim 3, characterized in that, The preparation method satisfies one or more of the following conditions: (1) The culture medium is LB liquid medium or NB medium; (2) The incubation temperature is 25℃-40℃; (3) The incubation time is 12h-36h.

5. A method for preparing an antibacterial culture, characterized in that, The preparation method includes the following steps: The culture medium was prepared using the preparation method described in claim 3; and, Remove Bacillus subtilis BS-Q from the culture medium, collect the supernatant, and directly prepare an antibacterial culture; or remove the solvent from the supernatant to prepare a solid antibacterial culture.

6. The antibacterial culture prepared by the method of claim 5.

7. The use of the antibacterial culture according to claim 6 in the preparation of antibacterial agents.

8. The application according to claim 7, characterized in that, The antibacterial agent inhibits one or more of the following: Clostridium perfringens type A, Clostridium perfringens type C, and Clostridium perfringens type G.

9. The use of the antibacterial culture according to claim 6 in the preparation of a microecological preparation for the prevention and / or treatment of necrotizing enteritis in poultry.

10. An antibacterial agent, characterized in that, The antibacterial agent includes Bacillus subtilis BS-Q as described in claim 1, the microbial preparation as described in claim 2, or the antibacterial culture as described in claim 6.

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