Fecal bacillus GC-14 strain and application thereof in inhibition of growth of staphylococcus aureus

Abstract

The invention discloses an excrement-derived bacillus GC-14 strain and application thereof in inhibiting growth of staphylococcus aureus, the excrement-derived bacillus GC-14 strain is screened and identified from preserved old vegetables which are naturally stored for the first time, a fermentation product of the strain has a remarkable inhibiting effect on staphylococcus aureus, and experiments prove that the bacillus GC-14 strain has a remarkable inhibiting effect on staphylococcus aureus. The inhibition rate of a fermentation product of the strain on staphylococcus aureus can reach 50% or above. The discovery of the strain not only fills the technical blank of utilizing surface microbial flora participating in traditional food fermentation, but also provides a brand-new microbial resource with development potential for developing a novel, green and efficient staphylococcus aureus inhibitor, and has remarkable application value.

Description

Technical Field

[0001] This invention relates to the field of bacterial fermentation technology, specifically to a fecal Bacillus GC-14 strain and its application in inhibiting the growth of Staphylococcus aureus. Background Technology

[0002] Microorganisms produce a variety of metabolites that not only enhance the flavor and nutritional value of food but may also possess antibacterial activity, thereby extending shelf life and improving food safety. Due to increasing consumer concerns about chemical food additives and the problem of microbial resistance to traditional preservatives and antibiotics, the development of natural and safe food preservatives has become an important trend in the food industry. Therefore, screening strains and their metabolites with antibacterial activity from traditionally fermented foods not only has significant academic research value but also provides a new approach for the development of natural food preservatives and food safety.

[0003] Staphylococcus aureus ( Staphylococcus aureus Staphylococcus aureus (S. aureus) is a common Gram-positive pathogen widely found in air, soil, food, and on human mucous membranes. It can cause food poisoning through food contamination and lead to various infectious diseases such as skin infections, pneumonia, and sepsis, posing a serious threat to food safety and human health. Currently, the control of S. aureus mainly relies on two methods: first, adding chemically synthesized preservatives to food (such as sodium benzoate and potassium sorbate); and second, using antibiotics in clinical and aquaculture fields (such as penicillin and cephalosporins). However, the long-term use of chemical preservatives easily raises consumer concerns about food safety, while the abuse and misuse of antibiotics directly leads to the emergence and spread of multidrug-resistant Staphylococcus aureus, making the effectiveness of traditional control strategies increasingly less effective. Therefore, the development of novel, safe, and drug-resistant antimicrobial agents is urgently needed.

[0004] Screening functional microorganisms with antibacterial activity from the natural environment is an important approach to developing safe and efficient novel biological antibacterial agents. Among numerous microbial resources, Bacillus is considered an ideal resource pool due to its unique advantages: these microorganisms can form spores, exhibiting strong resistance; they are easy to cultivate on a large industrial scale; and they can produce structurally diverse and broadly active antimicrobial metabolites, such as antimicrobial peptides and lipopeptides, which typically have unique mechanisms of action and are less likely to induce drug resistance. Based on this, isolating and screening Bacillus species with inhibitory activity against Staphylococcus aureus from various fermented foods, soil, or water bodies has become a common research and development path in existing technologies. In the Chaoshan region of Guangdong, my country, there is a type of aged pickled radish made through traditional fermentation processes, also known as "Lao Cai Pu" (old pickled radish), which is not only an important part of the local diet but also carries unique cultural value. The raw material is selected from high-quality white radishes harvested in early winter. Using traditional methods, they are pickled with coarse sea salt and stored in old jars, undergoing long-term natural aging under specific conditions. This results in a glossy black color and a unique flavor, possessing special effects such as strengthening the spleen and stomach, clearing stagnation, detoxifying, and relieving hangovers. It can promote appetite and aid digestion, and has good therapeutic effects on bloating, diarrhea, constipation, diabetes, asthma, high cholesterol, and high blood sugar. The longer the pickling period, the more significant the effects. Radishes aged 10-50 years are available on the market, possessing high nutritional and therapeutic value. This unique long-term, dynamic fermentation process inevitably creates an extremely complex microbial ecosystem that may contain special functional microorganisms.

[0005] Studies have shown that more than ten strains of bacteria participate in the fermentation process of "old preserved radish". However, current research focuses on the analysis of flavor substances and the evaluation of nutritional value, while the antibacterial effects of its surface microbial community and its metabolites are not yet fully studied. Summary of the Invention

[0006] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide a fecal-derived Bacillus (FAG) Bacillus stercoris GC-14 strain.

[0007] A second objective of this invention is to provide a microbial agent, fermentation broth / metabolite.

[0008] A third object of the present invention is to provide the use of the above-mentioned fermentation broth / metabolite in the preparation of products that inhibit Staphylococcus aureus.

[0009] A fourth object of the present invention is to provide a food additive containing the above-mentioned fermentation broth / metabolites.

[0010] The above-mentioned objective of this invention is achieved through the following technical solution: This invention provides a strain of fecal Bacillus (Bacillus) Bacillus stercorisThe GC-14 strain was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on September 26, 2025, with accession number GDMCC No: 67037.

[0011] As a fermented food, "Laocaipu" (preserved radish) may develop unique flavors and potentially bioactive microorganisms during fermentation. This invention first uses a dilution plating method, spreading the "Laocaipu" stock solution on MRS and NB plates at dilution gradients, from which 32 bacterial strains were isolated. Each strain was fermented individually, and the fermentation broth was collected, filtered through a 0.22 μm filter for sterilization, and then freeze-dried to produce a freeze-dried fermentation broth powder. Antibacterial activity testing revealed that strain GC-14 exhibited high specific inhibition against Staphylococcus aureus fermentation broth. Further analysis of the growth characteristics and morphology of this strain showed that it is a Gram-positive rod-shaped bacterium, which can grow singly or in short chains, and enters the logarithmic growth phase after 4 hours of culture following secondary activation. 16S rDNA sequence PCR amplification, sequencing, and phylogenetic tree analysis confirmed that strain GC-14... Bacillus stercoris The strain was classified into the same branch with a confidence level exceeding 90%. Therefore, strain GC-14 was identified as *Bacillus fecalis* (Factoborne Bacillus). Bacillus stercoris The strain was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on September 26, 2025, with accession number GDMCC No: 67037, and classified as follows: Bacillus stercoris GC-14, deposited at No. 100, Xianlie Middle Road, Guangzhou, Guangdong Province.

[0012] Furthermore, the present invention provides a screening method for fecal-derived Bacillus that inhibits the growth of Staphylococcus aureus. The method includes: using "old preserved radish" as the sample source, isolating and purifying single colonies through enrichment culture and dilution plating; using the antibacterial activity of its fermentation products as a key screening indicator for primary and secondary screening; and performing precise molecular biological identification through 16S rDNA sequence analysis to rapidly and accurately obtain the target strain.

[0013] The present invention also provides a bacterial agent containing one or more of the above-mentioned Bacillus foetida GC-14 strain, its culture medium, bacterial suspension, and bacterial cells; the preparation method of the culture medium, bacterial suspension, and bacterial cells is as follows: inoculating and culturing Bacillus foetida GC-14 strain to obtain a culture medium; centrifuging the culture medium and resuspending it to obtain a bacterial suspension; centrifuging the culture medium to obtain bacterial cells.

[0014] The present invention also provides a fermentation broth / metabolite, wherein the fermentation broth / metabolite is obtained by inoculating the above-mentioned Bacillus GC-14 strain of fecal origin or the above-mentioned bacterial agent into a culture medium, fermenting, and sterilizing.

[0015] Furthermore, the culture medium is MRS broth or NB medium.

[0016] Furthermore, the fermentation conditions are 35℃~39℃ for 22h~26h.

[0017] Furthermore, the sterilization condition is filtration using a 0.22 μm filter membrane.

[0018] Furthermore, the sterilization process also includes freeze-drying the fermentation broth / metabolites.

[0019] This invention is the first to screen and identify a strain of fecal-derived Bacillus from naturally stored preserved radish. Bacillus stercoris The GC-14 strain exhibits a significant inhibitory effect on Staphylococcus aureus through its fermentation products. Experiments have confirmed that the fermentation products of this strain achieve an inhibition rate of over 50% against Staphylococcus aureus. This strain demonstrates strong and stable antibacterial activity and shows great application potential. This strain and its metabolites can be used in the development of food preservatives, medical antibacterial agents, or feed additives, providing new microbial resources for the research and development of novel biological antibacterial agents.

[0020] Therefore, the present invention also provides the use of the above-mentioned fermentation broth / metabolite in the preparation of products that inhibit Staphylococcus aureus.

[0021] Furthermore, the product is a food preservative, food additive, medical antibacterial agent, or feed additive.

[0022] Furthermore, the food preservatives, medical antibacterial agents, or feed additives also include other acceptable excipients in food, medicine, and feed.

[0023] The present invention also provides a food additive, which includes the above-mentioned fermentation broth / metabolites.

[0024] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a *Bacillus foetida* strain GC-14 and its application in inhibiting the growth of *Staphylococcus aureus*. This invention is the first to screen and identify a *Bacillus foetida* strain GC-14 from naturally stored preserved radish. The fermentation products of this strain have a significant inhibitory effect on *Staphylococcus aureus*. Experiments have confirmed that the fermentation products of this strain can achieve an inhibition rate of over 50% against *Staphylococcus aureus*. The discovery of this strain not only fills the technological gap in utilizing surface microbial communities involved in traditional food fermentation, but also provides a novel and promising microbial resource for developing new, green, and efficient *Staphylococcus aureus* inhibitors, demonstrating significant application value. Attached Figure Description

[0025] Figure 1 For 10 on NB solid-state tablets and MRS solid-state tablets respectively -1 and 10 -2 Colony morphology at different concentrations.

[0026] Figure 2 The results of the antibacterial activity of fermentation broth from 32 strains against Staphylococcus aureus are presented.

[0027] Figure 3 The inhibition rate of the fermentation broth of the strain against Staphylococcus aureus is given.

[0028] Figure 4 The results show the repeated inhibition of Staphylococcus aureus by the fermentation broth of the strains. The results are for strains 6, 7, 14, 17, 28, and 30, in that order.

[0029] Figure 5 The results show the antibacterial effect of MRS broth on Staphylococcus aureus.

[0030] Figure 6 The results of the contents of strain GC-14 inhibiting Staphylococcus aureus.

[0031] Figure 7 The effects of fermentation broth of GC-14 strain obtained by culturing in different culture media on Staphylococcus aureus.

[0032] Figure 8 This is a phylogenetic tree of strains constructed using the collar grafting method based on the 16S rDNA gene sequence. Detailed Implementation

[0033] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0034] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0035] Example 1 Extraction of the strain I. Experimental Methods 1. Extraction of bacterial strains For the isolation of surface bacterial strains, 10 g of preserved radish sample was weighed and placed in a 150 mL sterile Erlenmeyer flask. 30 mL of sterile physiological saline pre-cooled to 4°C was added, and the mixture was homogenized for 3 min to prepare the stock solution. 5 mL of the stock solution was added to an Erlenmeyer flask containing 50 mL of MRS broth liquid medium and incubated at 37°C for 24 h. Then, 1 mL of the mixed bacterial solution was serially diluted with sterile physiological saline and plated on MRS and NB solid plates at 100°C each. -1 and 10 -2For each dilution, three parallel controls were performed, and the samples were incubated at 37°C for two days.

[0036] II. Experimental Results The results are as follows Figure 1 As shown, 10 μL of solid-state plates were applied to NB solid-state plates and MRS solid-state plates, respectively. -1 and 10 -2 Colony morphology at various concentrations. Single colonies exhibiting diverse morphological characteristics were observed on the plates. Thirty-two colonies with distinct morphological differences were initially selected. Each colony was picked up with a 10 μL pipette tip and placed into a 1.5 mL centrifuge tube containing MRS liquid medium, and mixed thoroughly (vortexed for 5 s). 0.5 mL of the bacterial suspension was added to 0.5 mL of 50% glycerol and stored at -80°C for later use.

[0037] Example 2: Inhibitory effect of freeze-dried fermentation broth powder on Staphylococcus aureus I. Experimental Methods 1. Preparation of freeze-dried fermentation broth powder Take the bacterial strain from a 1 mL glycerol tube, dissolve it, and add it to a centrifuge tube containing 40 mL of MRS broth. Incubate at 37°C for 24 h on a shaker for resuscitation and fermentation. After incubation, filter the supernatant through a 0.22 μm filter membrane to remove residual bacteria, ensuring the fermentation broth is sterile, and then stop fermentation. Freeze-dry the sterile fermentation broth to prepare a lyophilized fermentation broth powder. Reconstitute the obtained lyophilized fermentation broth powder into a 10 mg / mL solution using NB medium.

[0038] 2. Antibacterial test against Staphylococcus aureus 2.1 Preparation of Staphylococcus aureus working suspension Staphylococcus aureus was used as the indicator strain. Bacterial samples were taken and thawed at room temperature. Glycerol tubes of Staphylococcus aureus were inoculated into LB broth and cultured with shaking at 37°C for 12–16 h until the late logarithmic growth phase was reached, yielding a bacterial suspension. A portion of the cultured bacterial suspension was transferred to a centrifuge tube and rinsed with physiological saline. An appropriate amount of bacterial suspension and physiological saline were added to the centrifuge tube, mixed, and centrifuged. The supernatant was aspirated using a pipette, and this process was repeated twice. After the final rinse, an equal volume of physiological saline was added to the centrifuge tube to obtain the required working suspension, and the OD was adjusted. 600 The value is adjusted to 0.13, and the resulting solution is the working suspension of Staphylococcus aureus.

[0039] 2.2 Initial screening of bacterial strains like Figure 2As shown, 100 μL of sterile physiological saline was added to each well of a sterile 96-well plate to prevent edge effects. In wells B2-D11 and E2-G7, 50 μL of 5 mg / mL lyophilized bacterial fermentation broth reconstituted with the bacterial culture was added, followed by 50 μL of Staphylococcus aureus working suspension, bringing the total volume of liquid in each well to 100 μL. 50 μL of NB medium and 50 μL of physiological saline were added to wells E8-G9 as a blank control. 50 μL of NB medium and 50 μL of Staphylococcus aureus working suspension were added to wells E10-G11 as a growth control, bringing the final volume of each well to 100 μL. The 96-well plate was then incubated at 37°C for 16-18 h.

[0040] After cultivation, the sample was placed on a microplate reader at a wavelength of 600 nm to measure the absorbance (OD value) of each well. Using the OD value of the blank control group wells as background, the OD value of the growth control group wells should be significantly higher than that of the blank control group, indicating that the experimental system is normal. The OD value was measured using a microplate reader. 600 After setting the value, calculate the antibacterial rate of each well according to the formula:

[0041] 2.3 Secondary screening of antibacterial fermentation broth Based on the calculated inhibition rate, fermentation broths of strains with an inhibition rate of over 40% were selected for secondary screening. For example... Figure 4 As shown, 100 μL of sterile saline was added to each well on the outermost ring of a sterile 96-well plate to prevent edge effects, and 50 μL of sterile saline was added to wells G2-G11. 100 μL of 10 mg / mL lyophilized reconstitution solution of the two strains requiring rescreening was added to wells B2-E2 and B7-E7 of the sterile 96-well plate, respectively. Then, 50 μL of NB medium was added to wells B3-E6 and B8-E11. A dilution process was then performed: 50 μL of solution was pipetted from well B2 into well B3 and mixed, then 50 μL of solution was pipetted into well B4 and mixed, and so on until well B6. After mixing, 50 μL of solution was aspirated and discarded, leaving a liquid volume of 50 μL in each well. The same operation was repeated in wells C2-E6 and B7-E11. Add 50 μL of NB medium to wells F2-G11, using F2-F11 as a growth control and G2-G11 as a blank control. Finally, add 50 μL of Staphylococcus aureus working suspension to wells B2-F11, bringing the final volume of each well in the 96-well plate to 100 μL. Incubate the 96-well plate at 37°C for 16-18 h.

[0042] 3. Effects of MRS broth on Staphylococcus aureus Weigh out the MRS broth lyophilized powder, dissolve it in NB medium to prepare a 10 mg / mL MRS reconstituted solution in NB, and then filter it through a 0.22 μm aqueous microfiltration membrane to ensure its sterility.

[0043] like Figure 5 As shown, 100 μL of sterile physiological saline was added to each well of the outermost ring of a sterile 96-well plate to prevent edge effects. In wells B2-D4 and B9-D11 of the sterile 96-well plate, 50 μL of the reconstituted 10 mg / mL lyophilized powder fermentation broth of the re-screened strain and 50 μL of the reconstituted 10 mg / mL MRS broth were added, respectively. Then, 50 μL of Staphylococcus aureus working suspension was added to each well, bringing the total liquid volume to 100 μL. 50 μL of physiological saline and 50 μL of NB medium were added to wells F2-F11 as blank controls. Finally, 50 μL of NB medium and 50 μL of Staphylococcus aureus working suspension were added to wells E2-E11 as growth controls, bringing the final volume to 100 μL per well. The 96-well plate was incubated at 37°C for 16-18 hours.

[0044] II. Experimental Results An antibacterial experiment was conducted on a 5 mg / mL fermentation broth mixture against Staphylococcus aureus, and the results are as follows: Figure 2 As shown, the fermentation broths of some strains clearly exhibit antibacterial activity against Staphylococcus aureus. The inhibition rates of the fermentation broths of specific antibacterial strains were extracted from parallel experiments, as shown below. Figure 3 As shown.

[0045] Based on the inhibition rate, strains 6, 7, 14, 17, 28, and 30 were used in repeated experiments to replicate the inhibition test against Staphylococcus aureus. The repeated experiments were conducted with concentration gradients of 10, 5, 2.5, 1.25, and 0.625 mg / mL, and four replicates were set up. The results are as follows: Figure 4 The results shown are the inhibition results of strains 6, 7, 14, 17, 28, and 30, respectively. Among them, the fermentation broth of strain GC-14 showed the best inhibitory effect on Staphylococcus aureus. When the concentration of the fermentation broth of strain GC-14 was 10 mg / mL, the inhibitory effect on Staphylococcus aureus reached 53%.

[0046] Meanwhile, a comparative experiment was conducted using lyophilized fermentation broth of GC-14 strain at the same concentration and MRS broth, and the results were as follows: Figure 5 As shown, under the same concentration, MRS broth has a small amount of antibacterial activity against Staphylococcus aureus, while its GC-14 strain fermentation broth has an antibacterial rate of about 50%. That is, the antibacterial effect against Staphylococcus aureus is achieved by the sterile fermentation broth rather than the MRS broth.

[0047] Example 3: The effect of the bacterial cells themselves on Staphylococcus aureus I. Experimental Methods 1. Preparation of cell-free intracellular extract (CFE) The activated bacterial strain was incubated statically in NB medium at 37°C for 18 h, and then centrifuged at 12000 r / min for 15 min at 4°C to collect the bacterial cells. The collected bacterial cells were washed three times with sterile physiological saline, then resuspended in sterile physiological saline, and the bacterial concentration was adjusted to 1×10⁻⁶. 9 CFU / mL was mixed and ultrasonically disrupted. The ultrasonic disruption conditions were: ice bath, 200 W power, pulse disruption for 12 min at 3-5 s intervals (3 s on, 5 s off). The resulting liquid was centrifuged at 12000 rpm for 15 min at 4℃. The supernatant was collected and filtered through a 0.22 μm aqueous microfiltration membrane to obtain CFE.

[0048] 2. Inhibition of Staphylococcus aureus by CFE like Figure 6 As shown, 100 μL of sterile physiological saline was added to each well of the outermost ring of a sterile 96-well plate to prevent edge effects. In wells B2-C5 of the sterile 96-well plate, 50 μL of CFE was added, followed by 50 μL of Staphylococcus aureus working suspension (prepared as in Example 2) and 50 μL of NB medium, bringing the total liquid volume in each well to 150 μL. 50 μL of CFE, 50 μL of physiological saline, and 50 μL of NB medium were added to wells E2-E11 as a blank control. Then, 50 μL of NB medium, 50 μL of Staphylococcus aureus working suspension, and 50 μL of physiological saline were added to wells D2-D11 as a growth control, bringing the final volume of each well to 150 μL. The 96-well plate was then incubated at 37°C for 16-18 hours.

[0049] After cultivation, the sample was placed on a microplate reader at a wavelength of 600 nm to measure the absorbance (OD value) of each well. Using the OD value of the blank control group wells as background, the OD value of the growth control group wells should be significantly higher than that of the blank control group, indicating that the experimental system is normal. The OD value was measured using a microplate reader. 600 After setting the value, calculate the antibacterial rate of each well according to the formula.

[0050] II. Experimental Results The results are as follows Figure 6 As shown, the contents of strain GC-14 (8 replicates) had no inhibitory effect on Staphylococcus aureus.

[0051] Example 4: Effects of different culture media on Staphylococcus aureus I. Experimental Methods 1. Preparation of freeze-dried fermentation broth powder Take 1 mL of the bacterial strain from a glycerol tube, dissolve it, and add it to a centrifuge tube containing 40 mL of NB medium. Incubate at 37°C for 24 h on a shaker for resuscitation and fermentation. After incubation, filter the supernatant through a 0.22 μm filter membrane to remove residual bacteria, ensuring the fermentation broth is sterile, and then stop fermentation. Freeze-dry the sterile fermentation broth to prepare a lyophilized fermentation broth powder. Reconstitute the obtained lyophilized fermentation broth powder with NB medium to prepare a 10 mg / mL reconstituted solution.

[0052] 2. Antibacterial test against Staphylococcus aureus To prevent edge effects, add 100 μL of sterile physiological saline to each well on the outermost ring of a sterile 96-well plate. Add 50 μL of 10 mg / mL lyophilized powder reconstitution solution of the re-screened MRS and NB fermentation broths to wells B2-D4 and B9-D11, respectively, followed by 50 μL of Staphylococcus aureus working suspension to each well, bringing the total liquid volume to 100 μL. Add MRS and NB fermentation broths to wells F2-F4 and F5-F8, respectively, followed by 50 μL of physiological saline to each well, ensuring a total liquid volume of 100 μL, as blank controls. Add 50 μL of NB medium and 50 μL of Staphylococcus aureus working suspension to wells E2-E11 as growth controls, bringing the final volume to 100 μL per well. Incubate the 96-well plate at 37°C for 16-18 hours.

[0053] II. Experimental Results The results are as follows Figure 7 As shown, the sterile fermentation broth of GC-14 strain fermented in NB medium also has a certain inhibitory effect on Staphylococcus aureus, but the effect is not as good as that of the sterile fermentation broth of GC-14 strain fermented in MRS broth.

[0054] Example 5 Identification of the strain I. Experimental Methods A small amount of bacterial culture was taken and Gram staining was used to observe the morphology of GC-14 cells. DNA was extracted from the purified GC-14 strain using SDS-PAGE, and conserved sequences were amplified using universal primers. The amplified products were then sent to a sequencing company for sequencing, and the resulting sequences were assembled and compared with the NCBI database.

[0055] II. Experimental Results After staining with Gram stain, the growth characteristics and morphological analysis of the strain showed that it is a Gram-positive rod-shaped bacterium that can grow singly or in short chains. After secondary activation, it entered the logarithmic growth phase after 4 hours of culture.

[0056] The 16S rRNA gene sequence of this strain, totaling 1450 bases, is shown in SEQ ID No. 1. After comparison with the NCBI database, phylogenetic trees were constructed using MEGA 5.0 software for 16S rRNA gene sequences with high similarity. The results are as follows: Figure 8 As shown, strain GC-14 belongs to the same branch as Bacillus, with a similarity of 99.58% (Table 1). Based on morphological and cultural characteristics, strain GC-14 was identified as a fecal Bacillus. Bacillus stercoris The strain was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on September 26, 2025, with accession number GDMCC No: 67037, and classified as follows: Bacillus stercoris GC-14, deposited at No. 100, Xianlie Middle Road, Guangzhou, Guangdong Province.

[0057] Table 1. Homology comparison information for GC-14

Claims

1. A strain of fecal-derived Bacillus ( Bacillus stercoris GC-14 strain, characterized in that, The strain was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on September 26, 2025, with accession number GDMCC No: 67037.

2. A microbial agent, characterized in that, The bacterial agent contains one or more of the following: the *Bacillus foetida* GC-14 strain as described in claim 1, its culture medium, bacterial suspension, and bacterial cells; the culture medium, bacterial suspension, and bacterial cells are prepared by inoculating and culturing the *Bacillus foetida* GC-14 strain to obtain a culture medium; centrifuging the culture medium and resuspending it to obtain a bacterial suspension; and centrifuging the culture medium to obtain bacterial cells.

3. A fermentation broth / metabolite, characterized in that, The fermentation broth / metabolite is obtained by inoculating the fecal Bacillus GC-14 strain of claim 1 or the bacterial agent of claim 2 into a culture medium, fermenting, and sterilizing.

4. The fermentation broth / metabolite according to claim 3, characterized in that, The culture medium is MRS broth or NB medium.

5. The fermentation broth / metabolite according to claim 3, characterized in that, The fermentation conditions are 35℃~39℃ for 22h~26h.

6. The fermentation broth / metabolite according to claim 3, characterized in that, The sterilization condition is filtration using a 0.22 μm filter membrane.

7. The fermentation broth / metabolite according to claim 3, characterized in that, The sterilization process also includes freeze-drying the fermentation broth / metabolites.

8. The use of the fermentation broth / metabolite of claim 3 in the preparation of a product that inhibits Staphylococcus aureus.

9. The application according to claim 8, characterized in that, The product is a food preservative, food additive, medical antibacterial agent, or feed additive.

10. A food additive, characterized in that, The food additive includes the fermentation broth / metabolite described in claim 3.

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