Application of Bacillus subtilis BS-N antibacterial culture in the preparation of food antimicrobial agents, antimicrobial foods and their preparation methods

Abstract

This application belongs to the field of microbial technology, specifically relating to the application of antibacterial cultures of Bacillus subtilis BS-N in the preparation of food antibacterial agents, antibacterial foods, and their preparation methods. The antibacterial culture is prepared using the following method: Bacillus subtilis BS-N is inoculated into a culture medium, cultured, and a culture solution is prepared; Bacillus subtilis BS-N is removed from the culture solution, the supernatant is collected, and a liquid antibacterial culture is prepared; or, Bacillus subtilis BS-N is removed from the culture solution, the supernatant is collected, the solvent is removed from the supernatant, and a solid antibacterial culture is prepared; the preservation number of Bacillus subtilis BS-N is GDMCC No: 67746. This application provides a new solution for food antibacterial treatment.

Description

Technical Field

[0001] This application belongs to the field of microbial technology, specifically relating to the application of antibacterial cultures of Bacillus subtilis BS-N in the preparation of food antibacterial agents, antibacterial foods and their preparation methods. Background Technology

[0002] Staphylococcus aureus is one of the main pathogens causing bacterial foodborne illnesses, with contamination problems being particularly prominent in dairy and meat products. Currently, chemical antimicrobial agents (such as sorbates and benzoates) used to control this type of pathogen pose consumer health concerns and risks of bacterial resistance. Among natural antimicrobial substances, nisin is currently the only internationally approved bacteriocin-type food antimicrobial agent; however, its activity is low under neutral or alkaline conditions, its inhibitory effect on some Gram-positive bacteria is limited, and its thermal stability is insufficient (its activity decreases significantly after sterilization at 121°C). Therefore, developing novel natural food antimicrobial agents with better physicochemical stability, a broader antibacterial spectrum, and safety and non-toxicity has significant industrial value. Bacillus subtilis, as a recognized safe probiotic (GRAS strain), exhibits good antibacterial activity in its metabolites, including antimicrobial peptides and lipopeptides. However, most of the existing Bacillus subtilis-derived antimicrobial substances exhibit poor stability in real food matrices, incomplete inhibition of Staphylococcus aureus, and lack systematic safety evaluations. Therefore, screening a Bacillus subtilis strain capable of producing highly effective, stable, and safe antimicrobial substances, and directly applying its sterile supernatant to food antimicrobial treatment, is a pressing technical problem to be solved in this field. Summary of the Invention

[0003] Based on this, one or more embodiments of this application provide the application of Bacillus subtilis BS-N antibacterial culture in the preparation of food antibacterial agents, antibacterial foods, and methods for preparing the same. The technical solutions include the following:

[0004] One or more embodiments of this application provide the application of antibacterial cultures in the preparation of food antimicrobial agents;

[0005] The antibacterial culture was prepared using the following method:

[0006] Bacillus subtilis BS-N was inoculated into the culture medium and cultured to prepare a culture solution;

[0007] Remove Bacillus subtilis BS-N from the culture medium, collect the supernatant, and prepare a liquid antibacterial culture; or,

[0008] Remove Bacillus subtilis BS-N from the culture medium, collect the supernatant, remove the solvent from the supernatant, and prepare a solid antibacterial culture.

[0009] The Bacillus subtilis BS-N has the accession number GDMCC No:67746.

[0010] In some embodiments of this application, the food for which the food antimicrobial agent is applicable includes liquid foods.

[0011] In some embodiments of this application, the liquid food includes dairy products.

[0012] In some embodiments of this application, the food to which the food antimicrobial agent is applicable includes solid foods.

[0013] In some embodiments of this application, the solid food includes meat products.

[0014] In some embodiments of this application, the meat product includes ham.

[0015] In some embodiments of this application, the preparation method satisfies one or more of the following conditions:

[0016] (A1) The culture medium is LB liquid medium or NB medium;

[0017] (A2) The incubation temperature is 25℃~40℃;

[0018] (A3) The incubation time is 12h~36h.

[0019] In some embodiments of this application, the preparation method satisfies one or more of the following conditions:

[0020] (B1) Remove Bacillus subtilis BS-N from the culture medium by centrifugation and collect the supernatant;

[0021] (B2) also includes the step of membrane filtration to remove bacteria from the clear liquid;

[0022] (B3) The solvent in the clear liquid is removed by freeze drying.

[0023] In some embodiments of this application, the preparation method further includes the step of preparing an antibacterial culture from the antibacterial active ingredient with a molecular weight cutoff of ≥10kDa.

[0024] This application provides one or more embodiments of a method for preparing an antibacterial food. The preparation method includes the step of adding an antibacterial component to the food; wherein the antibacterial component includes a food antibacterial agent as defined above.

[0025] In some embodiments of this application, the food includes liquid food.

[0026] In some embodiments of this application, the liquid food includes dairy products.

[0027] In some embodiments of this application, the food includes solid food.

[0028] In some embodiments of this application, the solid food includes meat products.

[0029] In some embodiments of this application, the meat product includes ham.

[0030] This application provides one or more embodiments of an antibacterial food, wherein the antibacterial food is added with a food antibacterial agent as defined above.

[0031] Details of one or more embodiments of this application are set forth in the following description, and other features, objects, and advantages of this application will become apparent from the specification and its claims. Attached Figure Description

[0032] 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.

[0033] Figure 1 The images show the colony morphology and Gram staining results of Bacillus subtilis BS-N; A: Colony morphology of BS-N on LB medium; B: Gram staining characteristics of BS-N (scale bar: 20 μm).

[0034] Figure 2 The results of spore staining of Bacillus subtilis BS-N are shown (at different culture times); A: 2h; B: 4h; C: 6h; D: 8h; E: 10h; F: 12h; G: 14h; H: 16h; I: 18h; J: 20h; K: 22h; L: 24h, scale bar: 20μm.

[0035] Figure 3 The image shows the phylogenetic tree of the 16S rRNA gene of Bacillus subtilis BS-N; ▲ indicates the strain BS-N studied in this study.

[0036] Figure 4 The figures show the growth curves of Bacillus subtilis BS-N in different culture media and temperatures; A: Growth curves in different culture media at 40℃; B: Growth curves in LB medium at different temperatures.

[0037] Figure 5The following figures illustrate the effects of different treatments on BS-N survival rate: A: Effect of different pH treatments on BS-N survival rate; B: Effect of different bile salt concentrations on BS-N survival rate; C: Effect of high temperature treatment on BS-N survival rate; D: Effect of artificial gastrointestinal fluid on BS-N survival rate. Different letters indicate significant differences between different groups (P < 0.05).

[0038] Figure 6 The results of MIC determination of different concentrations of BS-N CFS against Staphylococcus aureus (96-well plate) are shown; the CFS concentrations in columns 1-10 are 50%, 25%, 12.5%, 6.25%, 3.125%, 1.5625%, 0.781%, 0.391%, 0.195%, and 0.098%, respectively; column 11 is the positive control (only Staphylococcus aureus bacterial suspension was added); column 12 is the negative control (only culture medium was added).

[0039] Figure 7 The results of the MBC determination of BS-N CFS are shown (coated plate photos); A: Coated plate results with 3.125% CFS treatment; B: Coated plate results with 1.5625% CFS treatment.

[0040] Figure 8 The figure shows the antibacterial activity of CFS against Staphylococcus aureus after treatment at different temperatures; different letters indicate significant differences between different groups (P<0.05).

[0041] Figure 9 The figure shows the antibacterial activity of CFS against Staphylococcus aureus after different pH treatments; different letters indicate significant differences between different groups (P<0.05).

[0042] Figure 10 The figure shows the antibacterial activity of CFS after different UV irradiation times; different letters indicate significant differences between different groups (P<0.05).

[0043] Figure 11 The figure shows the antibacterial activity of CFS after treatment with different proteases; different letters indicate significant differences between different groups (P<0.05).

[0044] Figure 12 The antibacterial activity of each component of BS-N CFS ultrafiltration is shown; different letters indicate significant differences between different groups (P<0.05).

[0045] Figure 13The image shows the protein profile differences between BS-N and control strain CFS analyzed by SDS-PAGE; M: molecular weight markers of unstained proteins; 1: 4h BS-N CFS; 2: 4h ATCC 6051 CFS; 3: 12h BS-N CFS; 4: 12h ATCC 6051 CFS.

[0046] Figure 14 The image shows the cytotoxicity of BS-N CFS on IEC-18, MDBK, and HCT-8 cells (MTT assay); left: IEC-18; middle: MDBK; right: HCT-8.

[0047] Figure 15 The figure shows the body weight change curve in mice during an acute oral toxicity test.

[0048] Figure 16A The image shows a pathological section of mouse liver tissue.

[0049] Figure 16B The image shows a histopathological section of a mouse kidney.

[0050] Figure 17 The figure shows the inhibitory effect of BS-N CFS on Staphylococcus aureus in milk under storage conditions of 4℃ and 25℃ (colony count-time curve); A: 4℃; B: 25℃.

[0051] Figure 18 The results show the inhibitory effect of BS-N CFS on Staphylococcus aureus in ham under storage conditions of 4℃ and 25℃; A: 4℃; B: 25℃.

[0052] The Bacillus subtilis BS-N strain provided in this application, with the taxonomic name Bacillus subtilis, was deposited on January 27, 2026, at the Guangdong Microbial Culture Collection Center, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, with accession number GDMCC No:67746. This strain was received and registered by the collection center on January 27, 2026, and was confirmed to be a viable strain by the collection center on the same day. Detailed Implementation

[0053] The present application will be further described in detail below with reference to the accompanying drawings, 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.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for descriptive purposes only and is not intended to be limiting of the application.

[0055] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0056] 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").

[0057] 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.

[0058] 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.

[0059] 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.

[0060] In this document, terms such as “preferred,” “better,” “more suitable,” and “ideal” are merely used to describe implementation methods or examples that achieve better results, and should be understood not to limit the scope of protection of this application.

[0061] In this application, terms such as "further," "even more," and "particularly" are used for descriptive purposes and to indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0062] 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.

[0063] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0064] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0065] 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.

[0066] 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℃.

[0067] 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.

[0068] All references to this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the purpose and / or technical solution of this application, all cited references are incorporated herein by reference in their entirety and for all purposes. When references are cited in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. 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.

[0069] A first aspect of this application provides the application of antibacterial cultures in the preparation of food antibacterial agents;

[0070] The antibacterial culture was prepared using the following method:

[0071] Bacillus subtilis BS-N was inoculated into the culture medium and cultured to prepare a culture solution;

[0072] Remove Bacillus subtilis BS-N from the culture medium, collect the supernatant, and prepare a liquid antibacterial culture; or,

[0073] Remove Bacillus subtilis BS-N from the culture medium, collect the supernatant, remove the solvent from the supernatant, and prepare a solid antibacterial culture.

[0074] The Bacillus subtilis BS-N has the accession number GDMCC No:67746.

[0075] This application does not specifically limit the types of antimicrobial agents used in food; any food can be included, including but not limited to protein foods and pickled foods. Protein foods include, for example, meat, eggs, and milk, including but not limited to dairy products such as milk, yogurt, and protein powder. Pickled foods refer to foods processed using a pickling process, traditionally preserving them by allowing large amounts of salt to penetrate the food's tissues; common examples include pickled vegetables, salted meat, and salted poultry eggs. Based on raw materials, they are mainly divided into pickled vegetable products (such as pickled vegetables in soy sauce and salt) and meat products (such as bacon, ham, and sausage). This application does not specifically limit the physical state of the food, including but not limited to liquid and solid states.

[0076] In some embodiments of this application, the preparation method satisfies one or more of the following conditions:

[0077] (A1) The culture medium is LB liquid medium or NB medium; of course, based on LB liquid medium or NB medium, those skilled in the art can also adjust the formula of the culture medium according to specific needs.

[0078] (A2) The culture temperature is 25℃~40℃, for example 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40℃;

[0079] (A3) The incubation time is 12h~36h, for example 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36h.

[0080] In some examples, LB liquid medium is selected and cultured at 150 rpm to 220 rpm (e.g., 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220 rpm).

[0081] In some embodiments of this application, the preparation method satisfies one or more of the following conditions:

[0082] (B1) Remove Bacillus subtilis BS-N from the culture medium by centrifugation and collect the supernatant. The purpose of centrifugation is mainly to remove bacterial cells, etc. The centrifugation conditions can be: 2℃~6℃ (e.g., 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6℃), 7000rpm~9000rpm (e.g., 7000, 7200, 7400, 7600, 7800, 8000, 8200, 8400, 8600, 8800, 9000rpm) for 10min~20min (e.g., 10, 12, 14, 16, 18, 20min).

[0083] (B2) also includes the step of membrane filtration sterilization of the clear liquid; including but not limited to filtration sterilization using a 0.22μm aqueous filter membrane;

[0084] (B3) The solvent in the clear liquid is removed by freeze drying. This application does not specify the freeze drying conditions. For example, the following conditions are used: pre-freezing at -80°C and then vacuum freeze drying for 48 hours.

[0085] In some embodiments of this application, the preparation method further includes the step of preparing an antibacterial culture from the clear liquid by retaining an antibacterial active ingredient with a molecular weight cutoff of ≥10 kDa. In some examples, the preparation method further includes the step of preparing an antibacterial culture from the clear liquid by retaining an antibacterial active ingredient with a molecular weight cutoff of 15-20 kDa.

[0086] A second aspect of this application provides a method for preparing an antibacterial food. The method includes the step of adding an antibacterial component to the food; wherein the antibacterial component includes a food antibacterial agent as defined in the first aspect above.

[0087] In the second aspect, food is as defined in the first aspect.

[0088] In a third aspect of this application, an antibacterial food is provided, wherein the antibacterial food is added with a food antibacterial agent as defined in the first aspect above.

[0089] 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.

[0090] 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.

[0091] 1. Isolation and Identification of Strains

[0092] 1.1 Source of strains

[0093] Bacillus subtilis BS-N was isolated from natural environmental samples. A pure culture, designated BS-N, was obtained after isolation and purification. The isolation and purification steps were as follows: 1g of camel feces 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-N.

[0094] 1.2 Morphological observation and Gram staining

[0095] BS-N colonies were incubated on LB agar plates at 37°C for 24 hours. The colonies were irregularly round, with a dry, rough surface, typical wrinkles, irregular edges, and a milky white color. Figure 1 Gram staining results showed that the bacteria were purple, short rod-shaped, and blunt at both ends, confirming them as Gram-positive bacteria. Figure 1 .B).

[0096] 1.3 Spore staining

[0097] Malachite green staining was used to observe BS-N spore formation. The specific steps were as follows: 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 spore formation began after approximately 14 hours of culture, and the number of spores increased with prolonged culture time, reaching almost entirely spores in the field of view at approximately 22 hours. Figure 2 ).

[0098] 1.4 Molecular Identification of 16S rRNA

[0099] Genomic DNA of BS-N was extracted and amplified by PCR using universal primers for 16S rRNA. The specific steps are as follows:

[0100] Genomic DNA was extracted from BS-N (refer to the instructions of the bacterial DNA extraction kit) and amplified by PCR using the universal 16S rRNA primers 27F (SEQ ID NO.1: 5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (SEQ ID NO.2: 5'-TACGGTYACCTTGTTGTTACGACTT-3').

[0101] The PCR reaction system is as follows:

[0102] Table 1

[0103]

[0104] 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.

[0105] The amplified product was subjected to 1.5% agarose gel electrophoresis, yielding a specific band of approximately 1400 bp. Sequencing followed by BLAST alignment showed homology with the Bacillus subtilis reference strain ATCC6051 and other Bacillus subtilis strains. Phylogenetic analysis indicated that ( Figure 3 ), BS-N and the standard strain of Bacillus subtilis clustered in the same branch.

[0106] 1.5 Biochemical Identification

[0107] The specific steps are as follows: Micro-biochemical identification tubes were used for testing. The 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 for glucose, mannitol, sucrose, and maltose (turning yellow); negative fermentation for lactose (no change in color); and negative fermentation for hydrogen sulfide (no change in color). Based on Bergey's Manual of Bacterial Identification, this is consistent with the characteristics of Bacillus subtilis.

[0108] 1.6 Comparison of strain characteristics

[0109] The Bacillus subtilis BS-N strain of this application differs significantly from existing Bacillus subtilis strains (including the ATCC 6051 standard strain) in terms of antibacterial properties.

[0110] 1.6.1 Comparison with standard strain ATCC 6051

[0111] CFS (sterile supernatant) was prepared by BS-N and ATCC 6051 under the same conditions (LB medium, 37℃, 12h incubation), and its antibacterial activity was determined using Staphylococcus aureus as the indicator bacterium. The procedure is as follows:

[0112] (1) Activation of strains: BS-N and ATCC 6051 were streaked on LB solid plates and incubated overnight at 37°C.

[0113] (2) Overnight culture: Pick a single colony and inoculate it into 5 mL of LB liquid medium. Incubate at 37°C and 200 rpm for 12 h to obtain the bacterial culture.

[0114] (3) Preparation of CFS: Bacillus subtilis BS-N and ATCC 6051 bacterial cultures were inoculated into LB liquid medium (inoculation amount of about 1%) and cultured at 37℃ and 180 rpm for 12 h. The fermentation broth was centrifuged at 4℃ and 8000 rpm for 15 min, and the supernatant was filtered through a 0.22 μm aqueous filter membrane to remove bacteria, thus obtaining liquid CFS.

[0115] (4) Preparation of indicator bacterial plates: Take overnight culture of Staphylococcus aureus and adjust the turbidity to 0.5 McFarland (approximately 1.5 × 10⁻⁶) with sterile physiological saline. 8 (CFU / mL). Mix 100 μL of bacterial suspension with LB solid medium cooled to 40°C and then pour the mixture onto a plate.

[0116] (5) Place Oxford cups: Place sterile Oxford cups (3 per plate) at equal intervals on the spread plate and press gently to ensure good contact with the culture medium.

[0117] (6) Sample addition: Add 200 μL of the CFS to be tested (BS-N CFS or ATCC 6051 CFS) to an Oxford cup.

[0118] (7) Incubation: Place the plate upright in a 37°C incubator for pre-diffusion for 2 hours, then invert it for 18 hours.

[0119] (8) Measure the inhibition zone: Measure the diameter of the inhibition zone (including the outer diameter of the Oxford cup) using a vernier caliper.

[0120] The outer diameter of the Oxford cup is approximately 8.0 mm. If there is no antibacterial activity, the diameter of the inhibition zone is close to 8.0 mm. Each treatment was performed in triplicate, and results are expressed as mean ± standard deviation.

[0121] The results showed that ATCC 6051 CFS had no significant antibacterial activity against Staphylococcus aureus under the same culture conditions (the inhibition zone diameter was approximately 8.0 mm, comparable to the outer diameter of the Oxford cup), while the inhibition zone diameter of BS-N CFS reached 20.12 ± 0.63 mm, a highly significant difference (P < 0.001). This indicates that the BS-N strain possesses antibacterial ability against Staphylococcus aureus that the ATCC 6051 standard strain does not have.

[0122] 1.6.2 Unique performance advantages of the strain applied for

[0123] Compared to traditional strains such as those described in CN118931800A, CN117511802A, and CN116024114B, the Bacillus subtilis BS-N strain of this application, in addition to exhibiting significant antibacterial activity against Staphylococcus aureus, also possesses the following unique properties:

[0124] (1) Unique protease sensitivity profile: The antibacterial activity of CFS in this application was completely lost after treatment with trypsin and proteinase K, but it still retained about 91% of its antibacterial activity after treatment with pepsin (see Figure 11 This characteristic indicates that its active ingredient possesses a unique spatial structure, enabling it to remain stable in gastric juice, which is beneficial for oral administration. None of the reported Bacillus subtilis-derived antibacterial substances have disclosed data on the sensitivity of this type of protease.

[0125] (2) Excellent physicochemical stability: The antibacterial activity of CFS in this application did not decrease significantly after treatment for 30 min in a wide temperature range from -20℃ to 100℃. Figure 8 It maintained its antibacterial activity after being treated for 2 hours in the pH range of 2.0 to 10.0. Figure 9 The activity is stable within 60 minutes of ultraviolet irradiation. Figure 10 This multidimensional stability enables it to adapt to various environmental conditions during food processing and storage.

[0126] (3) Complete eradication effect in real food matrices: This application provides, for the first time, systematic data on the inhibitory effect of BS-N CFS on Staphylococcus aureus in two representative food matrices, milk and ham, at two storage temperatures of 4℃ and 25℃. At 4℃, the target bacteria were completely eradicated in the milk treatment group on day 5, and in the ham treatment group on day 3. Figure 17-18 This "complete elimination" effect is extremely rare in existing reports of the food application of Bacillus subtilis-derived antimicrobial substances.

[0127] (4) Complete in vitro and in vivo safety data: The safety evaluation of BS-N CFS was completed in this application system. The hemolysis rate was 2.78% ± 0.24%, which is lower than the 5% safety threshold (Table 3); the survival rates of IEC-18, MDBK and HCT-8 cells at a concentration of 5 mg / mL were 73%, 98% and 121%, respectively, all of which are higher than the 70% safety standard ( Figure 14 In an acute oral toxicity test in mice, a dose of 5000 mg / kg was administered via gavage. No deaths or abnormal signs were observed, liver and kidney function indicators were normal, and histopathological examination was normal. Figure 15 , Figure 16A , Figure 16B According to the acute toxicity classification standard, it belongs to the practically non-toxic category. This safety data provides comprehensive support for the commercial application of CFS as a food antimicrobial agent.

[0128] (5) First discovery of specific antimicrobial protein: SDS-PAGE analysis showed that BS-N showed a specific protein band in the 15~20kDa range after 12h of culture, while the control strain ATCC6051 and BS-N 4h samples did not show this band. Figure 13The amino acid sequence of the protein (SEQ ID NO.4) was obtained by mass spectrometry. Sequence alignment and functional search revealed no reported antibacterial activity of this protein in existing literature and patents. This application discloses for the first time the antibacterial function of this protein against Staphylococcus aureus.

[0129] In summary, even though other Bacillus subtilis strains' CFS exhibit some antibacterial activity against Staphylococcus aureus, the advantages of the BS-N CFS in this application in terms of the aforementioned multiple properties (especially the unique pepsin resistance, complete removal effect in real food matrices, and the discovery of specific new proteins) are unpredictable by existing technologies.

[0130] 1.6.3 Application Validation and Technical Challenges in Food Matrices

[0131] Most reported applications of Bacillus subtilis are concentrated in feed additives or plant disease control, with scarce data on its application in real food matrices (such as dairy and meat products). This is not accidental, as the complexity of food matrices (especially milk and ham) poses multiple challenges to the activity of antibacterial substances:

[0132] (1) Protein interference: Milk contains about 3% protein (mainly casein and whey protein). These proteins can bind to antimicrobial peptides or protein-based antimicrobial substances through hydrophobic interactions, electrostatic adsorption, etc., leading to a decrease in their free concentration and activity. Several studies have reported that milk proteins can significantly reduce the inhibitory effect of bacteriocins such as Nisin on Staphylococcus aureus.

[0133] (2) Interference from fat and salt: Fat in the matrix of high-fat and high-salt foods such as ham can encapsulate antimicrobial substances, while salt can alter the conformation of antimicrobial peptides, affecting their interaction with bacterial cell membranes. Especially under refrigeration conditions at 4°C, fat coagulation may further reduce the diffusion efficiency of antimicrobial substances.

[0134] (3) pH and temperature fluctuations: pH changes (such as pH decrease due to lactic acid fermentation) and temperature fluctuations (such as cold chain interruption) during food processing and storage can affect the stability of antimicrobial substances.

[0135] This application provides, for the first time, systematic data on the inhibitory effect of BS-N CFS on Staphylococcus aureus in two representative food matrices, milk and ham, at two storage temperatures of 4°C and 25°C, and verifies that the target bacteria can be completely eliminated at 4°C (day 5 in milk and day 3 in ham). This "complete elimination" effect is extremely rare in existing reports on the food application of Bacillus subtilis-derived antimicrobial substances, demonstrating the unique effect of the BS-N CFS strain in this application.

[0136] In addition, this application has systematically completed the in vitro and in vivo safety evaluation of BS-N CFS (hemolysis rate, cytotoxicity, acute oral toxicity), providing complete safety data support for its commercial application as a food antimicrobial agent, which is not available in most existing patents.

[0137] 2. Growth characteristics and tolerance of the strain

[0138] 2.1 Growth Curve

[0139] The specific steps are as follows:

[0140] (1) Activation of strain: BS-N was streaked on LB solid plates and incubated overnight at 37°C.

[0141] (2) Overnight culture: Pick a single colony and inoculate it into 5 mL of LB liquid medium. Incubate at 37°C and 200 rpm for 12 h to obtain the bacterial culture.

[0142] (3) The above activated bacterial solution was inoculated into LB and NB medium at an inoculation rate of 1%, and cultured at 40℃ and 180 rpm. Samples were taken every 2 hours to measure OD. 600 .

[0143] (4) The activated bacterial solution was inoculated into LB medium at a rate of 1% and cultured under the same conditions at 25℃, 37℃ and 40℃ respectively.

[0144] The results showed that, at 40℃, BS-N grew better in LB medium than in NB medium, with a maximum OD value of [missing value]. 600 The value is approximately 2.7. In LB medium, the growth rate and plateau OD value at 37°C are both higher than at 25°C and 40°C. The optimal culture conditions are determined to be: LB medium, 37°C (…). Figure 4 At 37°C in LB medium, the logarithmic growth phase lasts approximately 2–12 hours, and the stationary phase lasts 12–20 hours.

[0145] 2.2 Acid resistance

[0146] The survival rate of BS-N was determined after treatment at different pH conditions for 2 hours. The specific steps are as follows:

[0147] (1) Activation of strain: BS-N was streaked on LB solid plates and incubated overnight at 37°C.

[0148] (2) Overnight culture: Pick a single colony and inoculate it into 5 mL of LB liquid medium. Incubate at 37°C and 200 rpm for 12 h to obtain the bacterial culture.

[0149] (3) Resuspend the BS-N bacterial culture in PBS to approximately 1×10⁻⁶. 9CFU / mL, inoculated at a 1% inoculum into LB medium at pH 2.0, 3.0, 4.0, 5.0, and 6.0 (LB medium inoculated to pH 7.1 served as the control group), treated at 37°C for 2 hours, and then plated for counting. Survival rate = (number of viable bacteria in the treatment group / number of viable bacteria in the control group) × 100%.

[0150] The results showed that the survival rate was approximately 4%–5% at pH 2.0 and 3.0, approximately 9% at pH 4.0, approximately 50% at pH 5.0, and approximately 69% at pH 6.0. The survival rate at pH 7.1 (control group) was set at 100%. Figure 5 A).

[0151] 2.3 Tolerance to bile salts

[0152] The specific steps are as follows:

[0153] (1) Activation of strain: BS-N was streaked on LB solid plates and incubated overnight at 37°C.

[0154] (2) Overnight culture: Pick a single colony and inoculate it into 5 mL of LB liquid medium. Incubate at 37°C and 200 rpm for 12 h to obtain the bacterial culture.

[0155] (3) Resuspend the BS-N bacterial culture in PBS to approximately 1×10⁻⁶. 9 CFU / mL, porcine bile salts were inoculated at a 1% inoculum into LB medium at final concentrations of 0.1wt%, 0.2wt%, and 0.3wt%, respectively. A control group without porcine bile salts was used. After treatment at 37℃ for 2 hours, the culture was spread and counted. Survival rate = (number of viable bacteria in the treatment group / number of viable bacteria in the control group) × 100%.

[0156] The results showed that after treatment with bile salt concentrations of 0.1 wt%, 0.2 wt%, and 0.3 wt% for 2 hours, the survival rates of BS-N were 9.34% ± 1.28%, 3.21% ± 0.67%, and 1.08% ± 0.43%, respectively, exhibiting a concentration-dependent decrease. Figure 5 .B).

[0157] 2.4 High temperature resistance

[0158] The specific steps are as follows:

[0159] (1) Activation of strain: BS-N was streaked on LB solid plates and incubated overnight at 37°C.

[0160] (2) Overnight culture: Pick a single colony and inoculate it into 5 mL of LB liquid medium. Incubate at 37°C and 200 rpm for 12 h to obtain the bacterial culture.

[0161] (3) Take 1 mL of bacterial suspension and treat it in an 80℃ water bath for 10 min, then immediately place it on an ice bath and spread it for counting. The control group was not treated with an 80℃ water bath. Survival rate = number of viable bacteria in the treatment group / number of viable bacteria in the control group × 100%.

[0162] The results showed that after treatment in an 80℃ water bath for 10 minutes, the survival rate of BS-N was approximately 45%. Figure 5 .C).

[0163] 2.5 Simulated gastrointestinal fluid tolerance

[0164] The specific steps are as follows:

[0165] (1) Activation of strain: BS-N was streaked on LB solid plates and incubated overnight at 37°C.

[0166] (2) Overnight culture: Pick a single colony and inoculate it into 5 mL of LB liquid medium. Incubate at 37°C and 200 rpm for 12 h to obtain the bacterial culture.

[0167] (3) The bacterial suspension was inoculated into artificial gastric fluid (pH 1.5) and artificial intestinal fluid (pH 6.8) at an inoculation rate of 10% (v / v), and treated at 37°C for 2 h. An equal volume of PBS was used as a control, and the bacteria were counted by spreading. Survival rate = number of viable bacteria in the treatment group / number of viable bacteria in the control group × 100%.

[0168] The results showed that the survival rate was approximately 13% after 2 hours of treatment in artificial gastric fluid and approximately 56% after 2 hours of treatment in artificial intestinal fluid. Figure 5 .D).

[0169] 2.6 Determination of Antibacterial Spectrum

[0170] The inhibitory effect of BS-N culture medium and sterile supernatant (CFS) on indicator bacteria was determined using the Oxford cup method. The specific steps are as follows:

[0171] (1) Activation of strain: BS-N was streaked on LB solid plates and incubated overnight at 37°C.

[0172] (2) Overnight culture: Pick a single colony and inoculate it into 5 mL of LB liquid medium. Incubate at 37°C and 200 rpm for 12 h to obtain the bacterial culture.

[0173] (3) Preparation of BS-N culture medium and BS-N CFS: Bacillus subtilis BS-N bacterial suspension was inoculated into LB liquid medium (inoculation amount of about 1%) and cultured at 37℃ and 180rpm for 12h to prepare BS-N culture medium; the culture medium was centrifuged at 4℃ and 8000rpm for 15min, and the supernatant was filtered through a 0.22μm aqueous filter membrane to remove bacteria, thus obtaining liquid BS-N CFS.

[0174] (4) Adjust the indicator bacteria (Staphylococcus aureus, Clostridium perfringens type A and G, Escherichia coli, Salmonella) to approximately 1×10⁻⁶. 8 CFU / mL. Take 100 μL of indicator bacterial suspension and add it to 15 mL of LB agar medium cooled to about 40°C. Mix well and pour into a 90 mm Petri dish containing an Oxford cup.

[0175] (5) Place Oxford cups and add BS-N culture medium, BS-N CFS (filtered through a 0.22μm filter membrane), positive control (ampicillin 25μg / mL) and negative control (LB medium) respectively. After incubation at 37℃ for 12h, measure the diameter of the inhibition zone.

[0176] The results are shown in Table 2. BS-N culture medium and CFS showed significant inhibitory effects on Staphylococcus aureus and Clostridium perfringens (types A and G), weak inhibition on Escherichia coli (culture medium only), and no inhibition on Salmonella.

[0177] Table 2. Antibacterial effects of BS-N culture medium and CFS on different indicator bacteria

[0178]

[0179] Unless otherwise specified, all indicator bacteria mentioned in this application are those listed in Table 1.

[0180] The Bacillus subtilis BS-N described in this application was deposited on January 27, 2026, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No: 67746), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province.

[0181] SEQ ID NO.3: 16S rRNA sequence of BS-N strain (approximately 1400 bp).

[0182]

[0183] According to items 1 and 2, the strain of this application possesses the following biological characteristics: In LB medium at 37℃, it enters the logarithmic growth phase after approximately 2–12 hours of cultivation, and the stationary phase after 12–20 hours; spore formation begins after approximately 14 hours of cultivation, and sporulation is essentially completed after 22 hours; it exhibits good survival ability in weakly acidic to neutral environments (pH 5.0–7.0), but is sensitive to strongly acidic environments (pH ≤ 4.0); it has a certain survival ability under bile salt concentrations ≤ 0.1%, but the survival rate significantly decreases at higher bile salt concentrations (≥ 0.2%); it has a high survival rate in simulated intestinal fluid, but a low survival rate in simulated gastric fluid. Its culture medium and sterile supernatant have significant inhibitory effects on Staphylococcus aureus and Clostridium perfringens, with inhibition zone diameters exceeding 15 mm. This application also provides microbial preparations containing the aforementioned Bacillus subtilis BS-N, and the application of this strain in the preparation of feed additives, microecological preparations, or products against foodborne pathogens.

[0184] 3. Preparation of CFS

[0185] (1) Activation of strain: BS-N was streaked on LB solid plates and incubated overnight at 37°C.

[0186] (2) Overnight culture: Pick a single colony and inoculate it into 5 mL of LB liquid medium. Incubate at 37°C and 200 rpm for 12 h to obtain the bacterial culture.

[0187] (3) Bacillus subtilis BS-N bacterial culture was inoculated into LB liquid medium (inoculation amount approximately 1%, v / v) and cultured at 37°C and 180 rpm for 12 h. The 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 CFS was pre-frozen at -80°C for at least 4 h (or overnight), and then freeze-dried at a cold trap temperature of -55°C to -60°C and a vacuum degree ≤0.1 mbar for 48 h. The resulting CFS lyophilized powder had a solids content of approximately 25 mg / mL. The liquid CFS and CFS lyophilized powder prepared under this procedure were used for subsequent tests.

[0188] 4. MIC and MBC measurements

[0189] The micro-dilution method was used. In a 96-well plate, 100 μL of LB medium was pre-added to each well. 100 μL of liquid CFS was added to well 1, mixed well, and 100 μL was transferred to well 2. This serial dilution was repeated until well 10, discarding the first 10 μL. The final CFS concentrations (v / v) in wells 1 through 10 were 50%, 25%, 12.5%, 6.25%, 3.125%, 1.5625%, 0.781%, 0.391%, 0.195%, and 0.098%, respectively. Subsequently, 100 μL of Staphylococcus aureus culture (approximately 1 × 10⁻⁶) was added to each well (wells 1 through 11). 6 The CFS concentration was further diluted by half (CFU / mL). Well 11 was the positive control (only bacterial culture was added, without CFS), and well 12 was the negative control (only culture medium was added, without bacterial culture or CFS). Results were observed after incubation at 37°C for 12 hours.

[0190] result( Figure 6 When the final concentration of liquid CFS was ≥1.5625%, the wells became clear, and the MIC was determined to be 1.5625% (v / v). 100 μL samples were taken from the wells containing the MIC (1.5625%) and the previous concentration (3.125%), spread onto LB plates, and incubated at 37°C for 24 h. No colonies grew in the 3.125% group. Figure 7 The MBC was determined to be 3.125% (v / v).

[0191] 5. Stability Evaluation

[0192] (1) Temperature stability

[0193] Liquid CFS was treated at -20℃, 4℃, 25℃, 37℃, 60℃, 80℃, and 100℃ for 30 min, and its antibacterial activity was determined. The specific steps are as follows: Liquid CFS was dispensed into sealed centrifuge tubes and treated at -20℃, 4℃, 25℃, 37℃, 60℃, 80℃, and 100℃ for 30 min, respectively. Untreated CFS was used as the control group. The antibacterial activity against Staphylococcus aureus was determined using the Oxford cup method (as described in section 2.6).

[0194] The results showed that the diameter of the inhibition zone in each temperature treatment group was not significantly different from that in the control group. Figure 8 ).

[0195] (2) pH stability

[0196] The pH of liquid CFS was adjusted to 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and 10.0, respectively, and then returned to the original pH (approximately 8.4) after 2 hours of treatment. The antibacterial activity was then measured. The specific steps are as follows: The pH of liquid CFS was adjusted to 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and 10.0 respectively using 1M HCl solution or 1M NaOH solution. After 2 hours of treatment, the pH was returned to the original pH. Simultaneously, liquid CFS without pH adjustment was used as a control group. The antibacterial activity was measured, and the method for measuring antibacterial activity is described in section 2.6.

[0197] The results showed that CFS maintained its antibacterial activity within the pH range of 2.0 to 10.0. Figure 9 ) Flocculent precipitate appears under acidic conditions (pH≤4), but the supernatant is deactivated after the precipitate is removed, indicating that the active substance exists in an insoluble form under acidic conditions.

[0198] (3) UV stability

[0199] Liquid CFS was irradiated under ultraviolet light for 10–60 min, and its antibacterial activity was measured. The specific steps are as follows: Liquid CFS was placed in a sterile petri dish and irradiated under an ultraviolet lamp (30 cm away from the lamp) in a clean bench for 10, 20, 30, 40, 50, and 60 min respectively. Liquid CFS not irradiated under ultraviolet light was used as a control group. The antibacterial activity was measured, and the method for measuring the antibacterial activity is described in section 2.6.

[0200] The results showed that the antibacterial activity did not decrease significantly within 60 minutes. Figure 10 ).

[0201] (4) Protease stability

[0202] Pepsin (porcine gastric mucosa, 1:30000, Beijing Solarbio Science & Technology Co., Ltd., catalog number P8160), trypsin (1:250, bovine pancreas, Beijing Solarbio Science & Technology Co., Ltd., catalog number T8150), and proteinase K (1:10000, derived from Candida albicans, Beijing Solarbio Science & Technology Co., Ltd., catalog number P9460) (final concentration approximately 1 mg / mL) were added to liquid CFS. After treatment under suitable conditions for 2 hours, the proteases were inactivated, and the antibacterial activity was measured. The specific steps are as follows: Pepsin, trypsin, and proteinase K (final concentration 1 mg / mL for each) were added to liquid CFS, and the mixture was treated in a water bath at 37℃ (pepsin, trypsin) or 55℃ (proteinase K) for 2 hours, respectively. Then, the proteases were inactivated by heating at 100℃ for 10 minutes. After cooling, the antibacterial activity was measured. The method for measuring the antibacterial activity is described in section 2.6. Liquid CFS without added proteases served as the control group.

[0203] The results showed that the antibacterial activity of trypsin and proteinase K was significantly lost after treatment, while pepsin retained about 91% of its activity after treatment. Figure 11 ).

[0204] 6. Preliminary identification of antibacterial substances

[0205] (1) Ultrafiltration retention

[0206] Liquid CFS was sequentially passed through 30kDa and 10kDa ultrafiltration membranes, and the antibacterial activity of each component was measured. The specific steps are as follows: Take 20mL of liquid CFS and centrifuge it sequentially using 30kDa and 10kDa ultrafiltration tubes (4℃, 5000rpm). Collect the three components >30kDa, 10~30kDa, and <10kDa. Make up the volume to the original volume with LB medium. Use liquid CFS without ultrafiltration treatment as a control group. Measure the antibacterial activity of each component. The method for measuring antibacterial activity is described in section 2.6.

[0207] The results showed that the >30kDa and 10~30kDa fractions had antibacterial activity, while the <10kDa fraction had no activity. Figure 12 ).

[0208] (2) SDS-PAGE

[0209] CFS concentrations of BS-N and control strain (ATCC6051) after culturing for 4 h and 12 h were concentrated by ultrafiltration and then subjected to electrophoresis. The specific steps are as follows:

[0210] A. Activation of strains: BS-N and ATCC 6051 were streaked onto LB agar plates and incubated overnight at 37°C.

[0211] B. Overnight culture: Pick a single colony and inoculate it into 5 mL of LB liquid medium, and culture at 37°C and 200 rpm for 12 h to obtain the bacterial culture.

[0212] C. Preparation of CFS: Bacillus subtilis BS-N and ATCC 6051 bacterial cultures were inoculated into LB liquid medium (approximately 1% inoculum) and cultured at 37°C and 180 rpm. During the culture, fermentation broths of each strain were collected after 4 h and 12 h of fermentation and centrifuged at 4°C and 8000 rpm for 15 min. The supernatant was filtered through a 0.22 μm aqueous filter membrane for sterilization, yielding four liquid CFS: 4h BS-NCFS; 4h ATCC 6051 CFS; 12h BS-N CFS; and 12h ATCC 6051 CFS.

[0213] D. After concentration with a 10kDa ultrafiltration tube, mix with 5× loading buffer, boil to denature, and perform SDS-PAGE (15% separating gel, 5% stacking gel), followed by Coomassie brilliant blue staining.

[0214] The results showed that BS-N CFS exhibited a specific band in the 15-20 kDa range at 12 h, while the control strain and the 4 h BS-N CFS sample did not show this band. Figure 13 Correspondingly, the inhibition zone diameter of 4h BS-N CFS against Staphylococcus aureus was approximately 8.0 mm (comparable to the outer diameter of the Oxford cup), while the inhibition zone diameter of 12h BS-N CFS reached 20.12±0.63 mm, indicating that the appearance of this specific band is closely related to the generation of antibacterial activity.

[0215] To further identify the protein in this specific band, LC-MS / MS analysis was performed on BS-N 12h CFS. Mass spectrometry analysis revealed that the amino acid sequence of the protein, with a molecular weight of approximately 15–20 kDa, is shown in SEQ ID NO. 4. No antibacterial activity of this protein in Bacillus subtilis has been reported in published literature and patents. Combined with the results of protease sensitivity testing (complete inactivation by trypsin and proteinase K treatment, and retention of approximately 91% activity by pepsin treatment), it can be determined that the main antibacterial substance in BS-N CFS is a protein, and this protein has not previously been reported to have antibacterial function against Staphylococcus aureus.

[0216] The protein sequence of the specific band is: MLSQIYPQAQHPYSFELNKDMHISAAHFIPRESAGACSRVHGHTYTVNITVAGDELDDSGFLVNFSVLKKLVHGNYDHTLLNDHEDFSQDDRYSLPTTEVVAKTIYDNVQAYLDTLENKPTCVQVFVRETPTSYCVYRPKKGGLNG (SEQ ID NO.4).

[0217] 7. Safety Evaluation

[0218] (1) Hemolysis rate determination

[0219] Fresh blood was collected from mice, anticoagulated with EDTA, and a 2% erythrocyte suspension was prepared. In a 96-well plate, 100 μL of different concentrations of BS-N CFS (100%, 50%, 25%, 12.5%, 6.25% v / v) and 100 μL of erythrocyte suspension were added to each well. The plate was incubated at 37°C for 1 h, and the supernatant was collected after centrifugation to determine the OD (oxidative stress). 540 The negative control was PBS, and the positive control was distilled water. Hemolysis rate = (Sample A - Negative A) / (Positive A - Negative A) × 100%.

[0220] The results showed that the hemolysis rate of direct use of CFS (i.e., the 100% group) was 2.78% ± 0.24%, which was lower than the 5% safety threshold (Table 3).

[0221] Table 3. Results of hemolysis rate determination

[0222]

[0223] Note: Different letters indicate significant differences between different groups (P < 0.05).

[0224] (2) Cytotoxicity test

[0225] The effects of BS-N CFS lyophilized powder on IEC-18, MDBK, and HCT-8 cells were determined using the MTT assay. The specific steps were as follows: IEC-18 (rat ileal epithelial cells), MDBK (bovine kidney epithelial cells), and HCT-8 (human colon cancer cells) were seeded into 96-well plates (1 × 10⁶ cells per well). 4 ~5×10 4 Cells were cultured to 80% confluence. BS-N CFS lyophilized powder was added and dissolved in the corresponding culture medium to final concentrations of 5, 2.5, 1.25, 0.625, 0.3125, and 0.15625 mg / mL, with three replicates for each concentration. The cells were cultured for another 24 hours. MTT reagent (final concentration 0.5 mg / mL) was added, and the cells were incubated for 4 hours. The soluble solution was then added, and the OD was measured. 570 Cell viability = (OD of experimental group - OD of blank group) / (OD of control group - OD of blank group) × 100%.

[0226] The results showed that at the highest final concentration of 5 mg / mL, the cell viability rates of IEC-18, MDBK, and HCT-8 were 73%, 98%, and 121%, respectively, all exceeding the safety standard of 70%. Figure 14 ).

[0227] (3) Acute oral toxicity test in mice

[0228] Twenty SPF-grade Kunming mice (half male and half female), weighing 20-25g, were selected and acclimatized for 3 days. Using a dose-limiting method, the experimental group (n=10) was administered a single dose of 5000mg / kg (BS-N CFS lyophilized powder dissolved in sterile water) via gavage, while the control group (n=10) was administered an equal volume of sterile water via gavage. Mice were fasted for 5 hours before and 2 hours after gavage. Observations were conducted for 14 days, and body weight, symptoms of poisoning, and mortality were recorded. After the experiment, blood was collected from the orbital sinus of the mice. Whole blood was allowed to stand at 4℃ for 2 hours, and then centrifuged at 3000rpm for 10 minutes to separate serum. The alanine aminotransferase (ALT / GPT) assay kit (catalog number C009-2-1) and aspartate aminotransferase (AST / GOT) assay kit (catalog number C010-2-1) from Nanjing Jiancheng Biotechnology Institute were used according to the instructions. The absorbance was measured at 510nm using a microplate reader, and serum ALT and AST activities were calculated. Mice were euthanized, and liver and kidney tissues were collected, fixed with 4% paraformaldehyde, dehydrated, embedded in paraffin, sectioned (approximately 5 μm thick), stained with hematoxylin and eosin (HE), and then histopathological changes were observed under an optical microscope.

[0229] The results showed that no animal deaths or abnormal signs were observed, and there was no significant difference in weight gain between the experimental group and the control group. Figure 15 Serum ALT and AST levels in the experimental group were not significantly different from those in the control group, and histopathological examinations of the liver and kidneys showed no abnormalities. Figure 16A , Figure 16B According to the acute toxicity classification criteria, BS-N CFS belongs to the practically non-toxic category.

[0230] As described in items 3 through 7, this application provides a sterile supernatant (CFS) of Bacillus subtilis BS-N (accession number: GDMCC No: 67746). This CFS exhibits significant inhibitory activity against Staphylococcus aureus, with a minimum inhibitory concentration (MIC) of approximately 1.5%–3% and a minimum bactericidal concentration (MBC) of approximately 3%. It remains stable within a temperature range of -20°C to 100°C, pH 2–10, and after 60 minutes of UV irradiation. Its antibacterial activity is sensitive to trypsin and proteinase K, but resistant to pepsin. Ultrafiltration and electrophoretic analysis show that the antibacterial substance is a protein with a molecular weight of approximately 15–20 kDa. The hemolysis rate is less than 5%, and it exhibits no cytotoxicity. Acute oral toxicity tests in mice have confirmed it to be practically non-toxic. This CFS can be used to prepare food antimicrobial agents or disinfectant products against Staphylococcus aureus.

[0231] 8. Antibacterial effect of milk

[0232] Commercially available pasteurized milk was dispensed into 10mL tubes and artificially contaminated with Staphylococcus aureus (final concentration approximately 10). 3Add BS-N CFS (approximately 2% by volume) and store at 4°C and 25°C for 5 days, respectively. Use an equal volume of PBS as a control group. Samples were taken every other day, diluted, and plated onto Baird-Parker plates, incubated at 37°C, and counted.

[0233] The results are as follows Figure 17 As shown:

[0234] 4℃: The number of Staphylococcus aureus in the control group increased slowly, reaching approximately 3.3 log CFU / mL on day 5; the number of colonies in the treatment group continued to decrease, dropping to approximately 1.5 log CFU / mL on day 3, and was completely eliminated (not detected) on day 5.

[0235] 25℃: Staphylococcus aureus in the control group showed exponential growth, rising to 7.5 log CFU / mL on day 1 and 8.4 log CFU / mL on day 2; growth in the treatment group was significantly inhibited, at approximately 2.9 log CFU / mL on day 1 and approximately 3.5 log CFU / mL on day 2, and remained 2-4 log units lower than the control group during storage.

[0236] 9. Antibacterial effects in ham

[0237] Commercially available Jinhua ham was peeled in a clean bench and cut into small pieces of approximately 0.5 cm³. Each piece was inoculated with 50 μL of Staphylococcus aureus bacterial suspension (final concentration approximately 1 × 10⁻⁶). 4 (CFU / g) Pre-incubate at 25℃ for 60 min. Then, completely immerse the ham samples in BS-N liquid CFS (approximately 200 μL per sample) at room temperature for 15 min, gently shaking 2-3 times during this period to ensure uniform surface contact. Remove the ham samples and air dry on sterile filter paper (approximately 10-15 min, until no obvious droplets remain on the surface), then seal in sterile Erlenmeyer flasks. The control group uses an equal volume of PBS buffer instead of BS-N liquid CFS, similarly immersing for 15 min, air drying, and sealing. Store at 4℃ and 25℃ for 5 days, respectively. Samples are taken every 24 h, homogenized with PBS, serially diluted, and plated on Baird-Parker plates for counting; three replicates are performed for each group.

[0238] The results are as follows Figure 18 As shown:

[0239] 4℃: In the control group, the level of Staphylococcus aureus increased from about 2.9 log CFU / g to about 5.2 log CFU / g on day 5; in the treatment group, the level decreased to about 2.0 log CFU / g on day 1, decreased to 1.5 log CFU / g on day 2, and was undetectable from day 3 onwards.

[0240] 25℃: The control group increased to 5.4 log CFU / g on day 1 and reached 8.0 log CFU / g on day 2; the growth of the treatment group was significantly slowed down, and the difference with the control group was still about 1.4 log CFU / g on day 5.

[0241] As indicated in items 8 and 9, BS-N CFS exhibits significant inhibitory effects against Staphylococcus aureus in both food matrices and at both storage temperatures, with even better results under refrigeration and more thorough effects in liquid foods. This characteristic makes it suitable as a natural food antimicrobial agent, particularly for antimicrobial preservation of cold chain foods, while also providing additional safety for foods stored at room temperature.

[0242] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0243] 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 and drawings can be used to interpret the content of the claims.

Claims

1. Application of antibacterial cultures in the preparation of food antibacterial agents; The antibacterial culture was prepared using the following method: Bacillus subtilis BS-N was inoculated into the culture medium and cultured to prepare a culture solution; Remove Bacillus subtilis BS-N from the culture medium, collect the supernatant, and prepare a liquid antibacterial culture; or, Remove Bacillus subtilis BS-N from the culture medium, collect the supernatant, remove the solvent from the supernatant, and prepare a solid antibacterial culture. The Bacillus subtilis BS-N has the accession number GDMCC No:67746.

2. The application according to claim 1, characterized in that, The food antimicrobial agent is applicable to liquid foods; Optionally, the liquid food includes dairy products.

3. The application according to claim 1, characterized in that, The food antimicrobial agent is applicable to solid foods; Optionally, the solid food includes meat products; Optionally, the meat product includes ham.

4. The application according to any one of claims 1 to 3, characterized in that, The preparation method satisfies one or more of the following conditions: (A1) The culture medium is LB liquid medium or NB medium; (A2) The incubation temperature is 25℃~40℃; (A3) The incubation time is 12h~36h.

5. The application according to any one of claims 1 to 3, characterized in that, The preparation method satisfies one or more of the following conditions: (B1) Remove Bacillus subtilis BS-N from the culture medium by centrifugation and collect the supernatant; (B2) also includes the step of membrane filtration to remove bacteria from the clear liquid; (B3) The solvent in the clear liquid is removed by freeze drying.

6. The application according to any one of claims 1 to 3, characterized in that, The preparation method further includes the step of preparing an antibacterial culture from the antibacterial active ingredient with a molecular weight cutoff of ≥10kDa.

7. A method for preparing an antibacterial food, characterized in that, The preparation method includes the step of adding an antimicrobial component to the food; wherein the antimicrobial component includes a food antimicrobial agent as defined in any one of claims 1 to 6.

8. The method for preparing antibacterial food according to claim 7, characterized in that, The food includes liquid food; Optionally, the liquid food includes dairy products.

9. The method for preparing antibacterial food according to claim 7, characterized in that, The food includes solid food; Optionally, the solid food includes meat products; Optionally, the meat product includes ham.

10. An antibacterial food, characterized in that, The antibacterial food contains a food antibacterial agent as defined in any one of claims 1 to 6.

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