Functional Microbes from Traditional Fermented Food: Genomics, Lipopeptide Profiling and Antimicrobial Potential of Bacillus velezensis R125

Functional Microbes from Traditional Fermented Food: Genomics, Lipopeptide Profiling and Antimicrobial Potential of Bacillus velezensis R125 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Functional Microbes from Traditional Fermented Food: Genomics, Lipopeptide Profiling and Antimicrobial Potential of Bacillus velezensis R125 FuTian Yu, DengFeng Yang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8646919/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Traditional fermented foods harbor functional microorganisms with antimicrobial potential. This study isolated strain Bacillus velezensis R125 GDMCC 67536 from sour porridge in Guangxi, China, which uniquely exhibited broad-spectrum activity against Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, and Fusarium oxysporum f. sp. cubense race 1 201 among 24 isolates. The strain was identified through phenotypic analysis, 16S rRNA gene sequencing (99.59% identity to the type strain), and whole-genome OrthoANI comparison (97.78% similarity to strain B. amyloliquefaciens subsp. plantarum FZB42 T , and 99.59% identity to the type strain B. velezensis NRRL B-41580 T ). Hybrid sequencing (Illumina HiSeq and PacBio) yielded a complete genome consisting of a circular chromosome (3,970,649 bp, 46.56% GC content) and a circular plasmid (5,981 bp, 52.88% GC content), harboring 3,808 protein-coding genes. Functional annotation using GO, COG, and KEGG databases revealed considerable metabolic versatility, including 51 genes encoding carbohydrate-active enzymes (CAZymes). AntiSMASH analysis predicted 13 biosynthetic gene clusters (BGCs) for secondary metabolites, such as surfactin, fengycin, bacilysin, and macrolactin H. Comparative genomic analysis with strain B. amyloliquefaciens subsp. plantarum FZB42 T , strain B. amyloliquefaciens DSM 7, and strain B. subtilis 168 T indicated that eight BGCs are conserved within the core genome, while five BGCs with low similarity to known clusters appear to be strain-specific, suggesting adaptive divergence. Notably, strain R125 possesses a significantly higher abundance of GH5 family cellulase genes—a genomic feature potentially reflective of adaptation to a plant-derived fermentation substrate. High-performance liquid chromatography-mass spectrometry (HPLC-MS) confirmed the production of three principal lipopeptides: iturin (30.31%), fengycin (22.42%), and surfactin (14.8%). This study elucidates the genomic and metabolomic basis of antimicrobial activity in strain B. velezensis R125 GDMCC 67536, and identifies three strain-specific novel BGCs and a unique GH5 cellulase gene enrichment characteristic associated with fermentation substrate adaptation; the quantitative synergy pattern of its lipopeptides (Iturin > Fengycin > Surfactin) is first reported to mediate broad-spectrum antimicrobial activity, providing experimental basis for the directional utilization of B. velezensis in food preservation and biocontrol. Bacillus velezensis Sour porridge Antimicrobial activity whole-genome sequencing Lipopeptides Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Traditional fermented foods represent a significant repository of microbial resources, harboring a diverse array of functional strains with various biological activities, owing to their unique fermentation environments [ 1 , 2 ]. These microorganisms offer rich raw materials for the development of novel antimicrobial agents, biopesticides, and food preservatives [ 2 – 5 ]. Sour porridge, a traditional fermented food from ethnic minority regions in southern China, hosts a complex microbiota formed through spontaneous fermentation, and this microbiota may contain dominant strains with broad-spectrum antimicrobial potential [ 6 ]. However, systematic screening, identification, and genomic characterization of antimicrobial mechanisms of microbes derived from sour porridge remain limited, constraining the exploitation of these natural microbial resources. Pathogenic microorganisms—including Gram-positive bacteria, Gram-negative bacteria, and phytopathogenic fungi—pose serious global challenges through food spoilage, crop diseases, and clinical infections[ 7 , 8 ]. The overuse of chemical antimicrobials has exacerbated the crisis of antimicrobial resistance, underscoring the urgent need to develop green and efficient natural antibacterial agents. Strain Bacillus velezensis , a widespread probiotic, has attracted considerable attention for its ability to produce diverse antimicrobial secondary metabolites—such as surfactin, fengycin, and bacillomycin—yet the strain-specific nature of its antibacterial activity and the underlying molecular mechanisms require systematic elucidation through whole-genome sequencing and related technologies[ 9 , 10 ]. This study isolates and screens microbial strains from sour porridge collected in Nanning, Guangxi, China, evaluating their inhibitory activity against Escherichia coli ATCC 25922 (Gram-negative), Staphylococcus aureus ATCC 25923 (Gram-positive), and Fusarium oxysporum f. sp. cubense race 1 201 (FoC1, a phytopathogenic fungus) using the agar well diffusion assay. Dominant strains are accurately identified through phenotypic characterization, 16S rRNA gene sequencing, and average nucleotide identity (ANI) analysis. A combined Illumina HiSeq and PacBio sequencing approach is employed for whole-genome sequencing and assembly. Functional gene annotation is performed using GO, COG, and KEGG databases, with particular emphasis on profiling carbohydrate-active enzyme (CAZy) genes and biosynthetic gene clusters (BGCs) for secondary metabolites. This research elucidates the molecular basis of the broad-spectrum antibacterial properties of strain B. velezensis R125, identifies its unique genomic and metabolomic characteristics adapted to fermented food niches, and provides new candidate targets for the mining of novel antimicrobial secondary metabolites and the directional breeding of functional probiotics. Sour porridge is a traditional fermented food of ethnic minorities in South China. Its acidic (typically pH 3.5–4.5), hypertonic, and anaerobic environment creates unique selective pressures on microorganisms. In this study, a total of 24 strains of culturable bacteria were isolated via the gradient dilution plating method from sour porridge samples collected in Nanning, Guangxi. Based on broad-spectrum antimicrobial activity screening (see below), strain B. velezensis R125 was selected for further characterization due to the following reasons: (a) In agar diffusion assays, strain R125 was the only strain that produced clear inhibition zones against Gram-negative bacteria (E. coli ATCC 25922), Gram-positive bacteria ( S. aureus ATCC 25923), and the plant pathogenic fungus (FoC1); (b) The inhibition zones formed by its fermentation supernatant showed sharp edges without diffusion, indicating stable and targeted antimicrobial production; (c) Preliminary identification based on 16S rRNA gene sequencing indicated that the strain belongs to the species B. velezensis , which is known for its significant biocontrol potential. In contrast, among the other 23 isolates, only 4 showed activity against E. coli ATCC 25922, 6 against S. aureus ATCC 25923, and 9 against FoC1, none of which exhibited broad-spectrum antimicrobial properties. Materials and Methods Materials Lysogeny broth (LB) medium, potato dextrose agar (PDA) medium and agar were obtained from Beijing Solarbio Technology Co. Ltd. (Beijing, China). Methanol was of chromatography grade, while hydrochloric acid, ethanol and sodium hydroxide were of analytical grade purchased from Dongguan Sparta Chemical Co., Ltd. (Dongguan, China). Microbial Strains The microbial strains were isolated by our research team from the sour porridge in Nanning, Guangxi, China. Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923 were obtained from American Type Culture Collection. FoC1 201 was stored in a 40% glycerol solution at − 80°C and deposited in the Marine Culture Collection of Guangxi. Strain Bacillus velezensis R125 GDMCC 67536, preserved at the Guangdong Microbial Culture Collection Center in Guangdong Province, China, with the preservation number: GDMCC No. 67536. Determination of the Antimicrobial Activity in Fermentation Supernatants of Strains Isolated from Sour Porridge Antibacterial activity was assessed using a slightly modified agar well diffusion assay [ 11 , 12 ]. The procedure was as follows: Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 were individually cultured to the logarithmic growth phase, and the bacterial suspensions were adjusted to a final concentration of 10⁶ CFU/mL. Each bacterial suspension was then introduced into LB agar medium, which had been cooled to below 50°C, thoroughly mixed, and poured into sterile Petri dishes (90 mm diameter) to create bacteria-seeded plates. After the agar solidified completely, 6 mm diameter wells were aseptically punched into the medium. Each well was filled with 100 µL of fermented supernatant from acidified porridge isolates, while control wells received an equivalent volume of sterile LB medium. The plates were subsequently incubated at 37°C for 16–18 h. The diameters of the inhibition zones surrounding the wells were measured to an accuracy of 0.1 mm using the cross-streak method, thereby quantifying the antibacterial activity of the fermented supernatants. Antifungal activity was evaluated via a mildly adapted agar well diffusion method [ 11 , 13 ]. A 6 mm mycelial plug, obtained from the periphery of a 7-day-old culture of Fusarium oxysporum f. sp. cubense race 1 (FoC1) using a sterile cork borer, was inoculated onto the center of a fresh, sterile PDA plate. Following a 4-day pre-incubation period at 28°C, four 5 mm diameter wells were symmetrically prepared at a distance of 2 cm from the central mycelial plug. Each well received 100 µL of the fermented supernatant, with control wells containing an equal volume of sterile PDA medium. The plates were incubated for an additional 4 days at 28°C, after which the diameters of the inhibition zones were measured to 0.1 mm precision using the cross-streak method, serving as an indicator of antifungal activity. Identification of the R125 Strain The R125 strain was inoculated onto LB solid medium and incubated at 37°C for 24 h. Following incubation, the morphological characteristics of the colonies were examined and documented, and Gram staining was performed for preliminary identification [ 14 ]. Molecular identification was subsequently conducted by amplifying the 16S rRNA gene with the universal primer pair 27F (5′-GGTTACCTTGTTACGACTT-3′) and 1492R (5′-AGAGTTTGATTTGATCCTGGCTAG-3′). The PCR amplification was carried out on a Biometra TC-512 thermocycler (Germany) under the following program: initial denaturation at 94°C for 5 min; 35 cycles of denaturation at 94°C for 30 s, annealing at 64°C for 1 minute, and extension at 72°C for 2 min; followed by a final extension at 72°C for 5 min. The PCR products were sequenced by Sangon Biotech (Shanghai) Co., Ltd [ 15 ]. The obtained 16S rDNA sequence was aligned for homology analysis against known sequences in the GenBank database using BLAST. A phylogenetic tree was constructed with MEGA 7.0 software ( https://www.megasoftware.net/ ) employing the Neighbor-Joining method. Strain R125 was further subjected to genomic typing based on the Average Nucleotide Identity (ANI), computed via the NCBI Prokaryotic Genome Annotation Pipeline (PGAP v2020-07-09.build4716) [ 16 ]. Whole Genome Sequencing and Assembly The R125 strain, preserved at -80°C, was initially revived on LB solid medium and incubated at 37°C for 24 h. Uniform single colonies were then selected and inoculated into LB liquid medium, followed by incubation in a constant-temperature shaking incubator at 37°C and 220 rpm for 12 h. After cultivation, the bacterial culture was centrifuged at 8000 rpm for 15 min to collect the R125 strain cell pellet. The harvested bacterial samples were subsequently sent to Shanghai Majorbio Bio-Pharm Technology Co., Ltd. for genomic DNA extraction and subsequent sequencing. A combined sequencing strategy employing both Illumina HiSeq and PacBio platforms was adopted for genome sequencing. Genomic DNA was fragmented into 8–10 kb segments, purified, and used to construct a SMRT Bell library for PacBio sequencing. Meanwhile, paired-end PE150 sequencing was performed on the Illumina HiSeq X10 platform. Raw sequencing data were subjected to a filtering process to remove reads containing sequencing primers, adapter sequences, and low-quality reads, yielding high-quality Clean Data. A hierarchical genome assembly process (HGAP) and the Canu assembler were employed for genome assembly. After multiple rounds of polishing, a complete genomic sequence was obtained [ 17 , 18 ]. Subsequent genomic bioinformatic analyses were conducted online via the Majorbio Cloud Platform ( https://cloud.majorbio.com ). Annotation and Analysis of Strain B. velezensis R125 Genome The genomic annotation of strain B. velezensis R125 was performed as follows [ 19 ]. Gene prediction was carried out using Glimmer 3.02. Identification of tRNA molecules was conducted with tRNAscan-SE, while rRNA genes were detected using RNAmmer in conjunction with the Rfam database. Functional annotation of predicted genes was achieved through sequence alignment against several reference databases, including the Gene Ontology (GO), Clusters of Orthologous Groups of proteins (COG), and the Kyoto Encyclopedia of Genes and Genomes (KEGG). In addition, carbohydrate-active enzymes (CAZymes) were predicted using the CAZy database, and biosynthetic potential for secondary metabolites was assessed via the antiSMASH software package. Finally, a comprehensive genomic map was generated for visualization using CGView. Lipopeptide Extraction The strain R125 was activated on LB agar plates for 24 h, and a single colony was inoculated into 100 mL of LB liquid medium, followed by incubation at 37°C and 220 r/min for 12 h. The culture was then transferred to a wide-mouth flask at a 4% volume fraction and fermented under the same conditions for 48 h. The fermentation broth was centrifuged at 4°C and 10,000 × g to collect the cell-free supernatant. Strain R125 lipopeptide was extracted using the acid precipitation-methanol extraction method: The supernatant was adjusted to pH 2.0 with 6 mol/L HCl and left to stand at 4°C overnight. It was then centrifuged at 4°C and 10,000 × g for 20 min, and the precipitate was extracted with methanol. The soluble components were collected to obtain the lipopeptide extract [ 20 , 21 ]. Identification of Strain R125 Lipopeptide by High-Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS) The strain R125 lipopeptide was qualitatively identified using high-performance liquid chromatography-mass spectrometry (HPLC-MS) with reference to a literature method after optimization [ 21 , 22 ]. The specific detection conditions were as follows: Chromatographic separation was performed on a Waters Sunfire C18 column (4.6 mm × 100 mm, 3.5 µm). The column temperature was maintained constant at 30°C to ensure separation stability. A UV detection wavelength of 214 nm was selected, providing sensitive detection of peptide bonds in lipopeptides and enabling effective capture of target signals. The mobile phase consisted of a binary gradient system: phase A (1% v/v formic acid in water) and phase B (1% v/v formic acid in acetonitrile). The elution parameters were as follows: flow rate 0.8 mL/min, injection volume 10 µL, total run time 15.00 min. The gradient program was: 0–7 min, phase B increased linearly from 5% to 95%; 7–12 min, phase B maintained at 95%; 12–12.2 min, phase B rapidly decreased from 95% to 5%; 12.2–15 min, phase B maintained at 5% for column equilibration. Mass spectrometry was performed using an electrospray ionization (ESI) source in positive ion scanning mode, which proved highly responsive for the protonated molecular ion peaks of lipopeptides, facilitating structural elucidation. The mass-to-charge ratio (m/z) scanning range was set to 100–2000, covering both the strain R125 lipopeptide and potential degradation products to avoid missing target signals . Statistical Analysis Each assay was performed in triplicate. The experimental data are presented as the mean ± standard deviation. The comparison of the mean values was performed by one-way analysis of variance (ANOVA) and Tukey’s test using SPSS software (IBM Corp., Armonk, NY, USA). A p value of < 0.05 was considered statistically significant. Results Evaluation of Antimicrobial Activity in Fermentation Supernatants of Strains Isolated from Sour Porridge and Screening of Superior Strains The antimicrobial activity of cell-free supernatants (CFSs) from 24 sour porridge isolates was systematically evaluated using the agar well diffusion method. These assays collectively assessed the broad-spectrum antimicrobial potential of the strains, with results presented in Fig. 1 and 2. The results of antibacterial activity assays revealed significant strain-specific variability in the antibacterial activity of CFSs following 48 h of fermentation in LB broth. Against E. coli (Gram-negative), only strains R66, No. 8, R125, and No. 111 displayed distinct antibacterial activity, all forming clear inhibition zones with regular edges and no diffusion or blurring. Among these, strain R125 produced the largest inhibition zone diameter—significantly greater than that of the other three strains—indicating superior anti- E. coli activity. In contrast, against S. aureus (Gram-positive), the number of strains with antibacterial activity increased significantly: CFSs from strains No. 11, No. 12, No. 14, R125, No. 108, and No. 82 effectively inhibited S. aureus growth. Notably, the inhibition zones of strains R125 and No. 11 featured smooth edges and distinct transparent regions, suggesting stronger and more stable inhibitory effects against Gram-positive bacteria. To comprehensively assess the antimicrobial spectrum coverage of the strains, additional antifungal activity screening was performed. Results demonstrated that in FoC1 growth inhibition assays, CFSs from 9 strains (No. 12, No. 72, No. 27, No. 10, No. 41, No. 54, R125, No. 82, and No. 26) exhibited significant antifungal activity, accounting for 37.5% of the total tested strains. Among these, the CFS of strain R125 exhibited more pronounced inhibitory effects on FoC1, with no mycelial spread around the inhibition zone—significantly superior to other active strains—indicating potent anti-FoC1 activity. Notably, among all active strains, only strain R125 exhibited potent inhibitory activity against Gram-positive bacteria, Gram-negative bacteria, and phytopathogenic fungi simultaneously. The diameter of inhibition zones produced by its CFS against all three types of indicator microbes was significantly larger than that of the negative control, with excellent activity stability and no observed diffusion or edge blurring of inhibition zones. Identification of the R125 Strain To achieve precise taxonomic identification of strain R125, this study conducted a systematic analysis from two core dimensions—phenotypic characterization and molecular-level verification—to ensure the reliability and comprehensiveness of the identification. In the phenotypic identification phase, cellular and colonial morphological characteristics were first examined. The activated R125 strain was inoculated onto standard LB agar plates (1.5% agar) and incubated statically at 37 °C for 24 h in the dark. Observation using a colony morphology analyzer revealed that the strain formed pale yellow, circular colonies with irregular serrated margins, smooth surfaces, and moist texture, measuring approximately 1.5–2.0 mm in diameter (Fig. 3A). These characteristics are highly consistent with the typical colonial morphology of the genus Bacillus . Subsequently, Gram staining was performed on R125 cells during the logarithmic growth phase. The cells stained purple, exhibited a rod-shaped morphology with blunt ends, and measured approximately 2.0–3.0 μm in length and 0.5–0.8 μm in width (Fig. 3B). According to the principles of Gram staining, the retention of the crystal violet–iodine complex indicates a thick peptidoglycan layer, allowing the preliminary classification of R125 as a Gram-positive bacterium. These phenotypic observations provided a basis for subsequent molecular identification. At the molecular level, 16S rDNA sequence analysis was first performed. Universal bacterial primers were used to amplify the 16S rRNA gene of R125 via PCR. The amplification product was verified by 1.0% agarose gel electrophoresis, which showed a single bright band at approximately 1500 bp (Fig. 3C), indicating high specificity. The purified PCR product was submitted for Sanger sequencing, and the resulting sequence was aligned and corrected using MEGA 11 software. A BLAST homology search was conducted in the GenBank database. Reference strains with coverage ≥98% and similarity ≥95% were selected to construct a phylogenetic tree using the neighbor-joining method with 1000 bootstrap replications. The results indicated that the 16S rDNA sequence of R125 shared 99% similarity with that of Bacillus velezensis strain NRRL B-41580, and the two strains clustered together in the phylogenetic tree with a bootstrap value of 98% (Fig. 3D), suggesting a close evolutionary relationship. To enhance identification accuracy and mitigate potential misclassification due to the high conservation of 16S rDNA sequences, whole-genome sequencing of R125 was conducted. Using the Illumina NovaSeq platform, a PE150 library was constructed with sequencing depth exceeding 50×. A draft genome was obtained after data filtering and assembly. Based on the assembled genome, the Orthologous Average Nucleotide Identity (OrthoANI) between R125 and four reference Bacillus strains — B. velezensis FZB42, B. subtilis 168, B. amyloliquefaciens DSM 7, and B. siamensis KCTC 13613—was calculated using the OAT software with default parameters. Orthologous gene pairs with sequence similarity ≥70% and length ≥100 bp were retained for analysis, and a heatmap of OrthoANI values was generated using TBtools (Figure 3E). The results showed that R125 shared OrthoANI values above 75.04% with all reference strains, with the highest value (97.78%) observed with the model strain B. velezensis FZB42. As OrthoANI analysis is considered the gold standard for evaluating species relatedness at the whole-genome level—based on the average nucleotide identity of orthologous genes—and given that an OrthoANI value ≥95% is widely accepted as the threshold for species delineation in microbial taxonomy (Felczak et al. 2021), the observed value of 97.78% strongly supports the classification of R125 as B. velezensis . Integrating the phenotypic and molecular evidence, R125 exhibits typical characteristics of Bacillus velezensis , including Gram-positive rod-shaped cells and pale-yellow irregular colonies. Its 16S rDNA sequence shows 99% similarity to reference strains of this species, and its OrthoANI value exceeds the species delineation threshold. These multi-faceted and mutually corroborating results allow the confident identification of strain R125 as Bacillus velezensis . In accordance with the International Code of Nomenclature of Prokaryotes, it is formally designated as Bacillus velezensis R125 (abbreviated as B. velezensis R125). Evaluation of Antimicrobial Activity in Fermentation Supernatants of Strains Isolated from Sour Porridge and Screening of Superior Strains The antimicrobial activity of cell-free supernatants (CFSs) from 24 sour porridge isolates was systematically evaluated using the agar well diffusion method. These assays collectively assessed the broad-spectrum antimicrobial potential of the strains, with results presented in Fig. 1 and 2. The results of antibacterial activity assays revealed significant strain-specific variability in the antibacterial activity of CFSs following 48 h of fermentation in LB broth. Against E. coli (Gram-negative), only strains R66, No. 8, R125, and No. 111 displayed distinct antibacterial activity, all forming clear inhibition zones with regular edges and no diffusion or blurring. Among these, strain R125 produced the largest inhibition zone diameter—significantly greater than that of the other three strains—indicating superior anti- E. coli activity. In contrast, against S. aureus (Gram-positive), the number of strains with antibacterial activity increased significantly: CFSs from strains No. 11, No. 12, No. 14, R125, No. 108, and No. 82 effectively inhibited S. aureus growth. Notably, the inhibition zones of strains R125 and No. 11 featured smooth edges and distinct transparent regions, suggesting stronger and more stable inhibitory effects against Gram-positive bacteria. To comprehensively assess the antimicrobial spectrum coverage of the strains, additional antifungal activity screening was performed. Results demonstrated that in FoC1 growth inhibition assays, CFSs from 9 strains (No. 12, No. 72, No. 27, No. 10, No. 41, No. 54, R125, No. 82, and No. 26) exhibited significant antifungal activity, accounting for 37.5% of the total tested strains. Among these, the CFS of strain R125 exhibited more pronounced inhibitory effects on FoC1, with no mycelial spread around the inhibition zone—significantly superior to other active strains—indicating potent anti-FoC1 activity. Notably, among all active strains, only strain R125 exhibited potent inhibitory activity against Gram-positive bacteria, Gram-negative bacteria, and phytopathogenic fungi simultaneously. The diameter of inhibition zones produced by its CFS against all three types of indicator microbes was significantly larger than that of the negative control, with excellent activity stability and no observed diffusion or edge blurring of inhibition zones. Identification of the R125 Strain To achieve precise taxonomic identification of strain R125, this study conducted a systematic analysis from two core dimensions—phenotypic characterization and molecular-level verification—to ensure the reliability and comprehensiveness of the identification. In the phenotypic identification phase, cellular and colonial morphological characteristics were first examined. The activated R125 strain was inoculated onto standard LB agar plates (1.5% agar) and incubated statically at 37 °C for 24 h in the dark. Observation using a colony morphology analyzer revealed that the strain formed pale yellow, circular colonies with irregular serrated margins, smooth surfaces, and moist texture, measuring approximately 1.5–2.0 mm in diameter (Fig. 3A). These characteristics are highly consistent with the typical colonial morphology of the genus Bacillus . Subsequently, Gram staining was performed on R125 cells during the logarithmic growth phase. The cells stained purple, exhibited a rod-shaped morphology with blunt ends, and measured approximately 2.0–3.0 μm in length and 0.5–0.8 μm in width (Fig. 3B). According to the principles of Gram staining, the retention of the crystal violet–iodine complex indicates a thick peptidoglycan layer, allowing the preliminary classification of R125 as a Gram-positive bacterium. These phenotypic observations provided a basis for subsequent molecular identification. At the molecular level, 16S rDNA sequence analysis was first performed. Universal bacterial primers were used to amplify the 16S rRNA gene of R125 via PCR. The amplification product was verified by 1.0% agarose gel electrophoresis, which showed a single bright band at approximately 1500 bp (Fig. 3C), indicating high specificity. The purified PCR product was submitted for Sanger sequencing, and the resulting sequence was aligned and corrected using MEGA 11 software. A BLAST homology search was conducted in the GenBank database. Reference strains with coverage ≥98% and similarity ≥95% were selected to construct a phylogenetic tree using the neighbor-joining method with 1000 bootstrap replications. The results indicated that the 16S rDNA sequence of R125 shared 99% similarity with that of Bacillus velezensis strain NRRL B-41580, and the two strains clustered together in the phylogenetic tree with a bootstrap value of 98% (Fig. 3D), suggesting a close evolutionary relationship. To enhance identification accuracy and mitigate potential misclassification due to the high conservation of 16S rDNA sequences, whole-genome sequencing of R125 was conducted. Using the Illumina NovaSeq platform, a PE150 library was constructed with sequencing depth exceeding 50×. A draft genome was obtained after data filtering and assembly. Based on the assembled genome, the Orthologous Average Nucleotide Identity (OrthoANI) between R125 and four reference Bacillus strains — B. velezensis FZB42, B. subtilis 168, B. amyloliquefaciens DSM 7, and B. siamensis KCTC 13613—was calculated using the OAT software with default parameters. Orthologous gene pairs with sequence similarity ≥70% and length ≥100 bp were retained for analysis, and a heatmap of OrthoANI values was generated using TBtools (Figure 3E). The results showed that R125 shared OrthoANI values above 75.04% with all reference strains, with the highest value (97.78%) observed with the model strain B. velezensis FZB42. As OrthoANI analysis is considered the gold standard for evaluating species relatedness at the whole-genome level—based on the average nucleotide identity of orthologous genes—and given that an OrthoANI value ≥95% is widely accepted as the threshold for species delineation in microbial taxonomy (Felczak et al. 2021), the observed value of 97.78% strongly supports the classification of R125 as B. velezensis . Integrating the phenotypic and molecular evidence, R125 exhibits typical characteristics of Bacillus velezensis , including Gram-positive rod-shaped cells and pale-yellow irregular colonies. Its 16S rDNA sequence shows 99% similarity to reference strains of this species, and its OrthoANI value exceeds the species delineation threshold. These multi-faceted and mutually corroborating results allow the confident identification of strain R125 as Bacillus velezensis . In accordance with the International Code of Nomenclature of Prokaryotes, it is formally designated as Bacillus velezensis R125 (abbreviated as B. velezensis R125). Table 1 Genome statistics of B. velezensis R125. Genomic Feature Chromosome Plasmid Size of the genome assembly (bp) 3,970,649 5981 GC content (%) 46.56 52.88 Protein-coding genes 3808 0 Protein-coding regions (bp) 3506877 0 rRNA genes 24 3 tRNA genes 81 11 tmRNA genes 1 0 CRISPR 2 0 Functional Gene Annotation of B. velezensis R125 Functional annotation of the B. velezensis R125 genome, based on whole-genome sequencing data, is presented in Fig. 5. The Gene Ontology (GO) database provides a standardized classification system for gene functions, systematically categorizing protein roles across three domains: biological process (BP), cellular component (CC), and molecular function (MF). As shown in Fig. 5A, a total of 2,548 genes in the B. velezensis R125 genome were assigned GO terms, corresponding to an annotation rate of approximately 64.9%. Among the three major functional categories, genes associated with biological processes exhibited the highest annotation abundance, indicating functional diversity related to the regulation of cellular activities. This was followed by cellular components, while molecular functions had the lowest representation. Within biological processes, the most abundant functional terms were cellular process, metabolic process, and organic substance metabolic process. The high abundance of metabolic process-related genes suggests active metabolic capacity in this strain. Among cellular components, the top three terms—cell, cell part, and intracellular component—are consistent with the structural characteristics of unicellular bacteria. The enrichment of intracellular component-related genes further reflects the integrity of cellular architecture and function. In the molecular function category, catalytic activity, binding activity, and transferase activity were the most prevalent. The high proportion of catalytic activity genes supports the presence of a diverse repertoire of metabolic enzymes in this strain. The Clusters of Orthologous Groups (COG) database classifies gene functions based on evolutionary relationships among homologous proteins. Our analysis revealed that 3,723 genes in the B. velezensis R125 genome were assigned COG classifications (Fig. 5B). Among the 26 functional categories, the three most represented were: amino acid metabolism and transport (332 genes, 8.9% of annotated genes), transcription (310 genes, 8.3%), and carbohydrate metabolism and transport (244 genes, 6.6%). The transcription category includes genes encoding RNA polymerase subunits and transcription factors, indicative of a complex and complete transcriptional regulatory system. The carbohydrate metabolism and transport category encompasses genes for glycoside hydrolases and transport proteins, underpinning the strain’s ability to efficiently utilize environmental carbohydrates. Additional functional categories, such as signal transduction mechanisms and posttranslational modification, were also identified, collectively supporting a comprehensive physiological regulatory network in the strain. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed using the KEGG database (release 109.0) and the clusterProfiler software, with a significance threshold of p<0.05 based on a hypergeometric test. A total of 3,932 genes in the B. velezensis R125 genome were annotated and assigned to 197 metabolic pathways spanning 12 major categories, including carbohydrate metabolism, amino acid metabolism, and nucleotide metabolism (Fig. 5C). The pathways with the highest gene counts were global and overview maps (1,576 genes), carbohydrate metabolism (427 genes), and amino acid metabolism (309 genes). The global and overview maps category contains genes involved in central metabolic pathways such as glycolysis and the tricarboxylic acid cycle, and its high gene count reflects the completeness of the strain’s core metabolic network. Enrichment of genes related to starch, sucrose, fructose, and mannose metabolism within the carbohydrate metabolism pathway suggests the strain’s potential for utilizing diverse carbon sources. In amino acid metabolism, the presence of genes involved in glutamate and aspartate metabolism supports the strain’s capacity for synthesizing essential amino acids and participating in environmental nitrogen cycling. Together, these findings indicate that B. velezensis R125 possesses substantial metabolic diversity, enabling coordinated multi-pathway involvement in the transformation of sugars, amino acids, and other substances. This metabolic versatility likely underlies its ability to adapt to complex environments and to perform physiological functions such as plant growth promotion and antibacterial activity. Carbohydrate Active Enzyme Annotation Statistics Systematic annotation based on the CAZy (Carbohydrate-Active enZYmes Database) revealed 51 carbohydrate-active enzyme (CAZyme) encoding genes in the genome of B. velezensis R125 (Fig. 5D). These genes span six major CAZyme families: glycoside hydrolases (GH), glycosyl transferases (GT), carbohydrate-binding modules (CBM), carbohydrate esterases (CE), auxiliary activities (AA), and polysaccharide lyases (PL). Among these, GH and GT families were the most abundant, each comprising 22 genes and collectively accounting for 86.28% of all identified CAZymes (43.14% each), thus forming the core of the strain’s CAZyme repertoire. The CBM family included five genes (9.80%), which serve as non-catalytic domains that enhance enzyme binding to carbohydrate substrates. In contrast, CE and AA families were sparsely represented (one gene each), and no PL family genes were detected; together, these minor categories constituted only 3.92% of the total CAZymes. Predictive Analysis of Secondary Metabolite Synthesis Gene Clusters In this study, we employed the AntiSMASH v6.1 online platform (https://antismash.secondarymetabolites.org/) with default parameters to systematically predict and annotate biosynthetic gene clusters (BGCs) in the complete genome sequence of B. velezensis R125 (GenBank accession: PRJNA1358366). The results are summarized in Table 2. A genome-wide screen identified 13 fully annotated BGCs, all located on the bacterial chromosome without notable regional clustering. These include: three NRPS-type clusters, one type III polyketide synthase (T3PKS) cluster, three trans-acyltransferase PKS (transAT-PKS) clusters, two terpene clusters, one thiopeptide cluster, one RRE (Ribosome Binding Site Regulatory Element)-containing cluster, one PKS-like cluster, and one unclassified functional gene cluster. To elucidate the potential secondary metabolite profile of B. velezensis R125, we first conducted targeted homology analysis of the three NRPS-type clusters (Region1, Region8, and Region12) against functionally characterized NRPS BGCs in the MIBiG v2.0 database. Region1 exhibited 78% amino acid sequence similarity to the surfactin biosynthetic gene cluster (BGC0000433), and its four core NRPS genes (srfA-A, srfA-B, srfA-C, srfA-D) displayed highly conserved modular architectures corresponding to peptide elongation modules in surfactin synthesis. Region8 showed 100% similarity to the fengycin biosynthetic gene cluster (BGC0001095), with identical NRPS module counts and functional domains (condensation, adenylation, and thiolation domains) encoded by fenA, fenB, fenC, and fenD. Region12 displayed 100% similarity to the bacillibactin biosynthetic gene cluster (BGC0000309). Analysis of the three transAT-PKS clusters (Region6, Region7, and Region11) revealed 100% sequence similarity to known BGCs for macrolide H (BGC0000181), bacillaene (BGC0001089), and difficidin (BGC0000176), respectively. The core PKS genes exhibited complete conservation in acyltransferase (AT), ketosynthase (KS), and ketoreductase (KR) domain sequences, suggesting full functional capacity. Furthermore, Region13 showed 100% identity with the bacilysin biosynthetic gene cluster (BGC0001184), which includes key synthesis genes such as bacA, bacB, bacC, and bacD, encoding characteristic enzymes like isochorismatase and aminotransferase essential for bacilysin production. These findings confirm the presence of functional BGCs for surfactin, fengycin, bacillibactin, macrolide H, bacillaene, difficidin, and bacilysin in B. velezensis R125, indicating a high potential for the biosynthesis of these metabolites—all known for their broad-spectrum antimicrobial activities. Surfactin exhibits both surfactant and antibacterial properties, fengycin specifically inhibits fungi, and bacilysin shows significant activity against Gram-positive and Gram-negative bacteria (Cho et al. 2025). According to AntiSMASH annotations, Region2, Region4, Region5, Region9, and Region10 exhibited low sequence similarity (<10%) to known BGCs in the MIBiG database. However, structural integrity assessments confirmed that these clusters contain complete sets of core synthase genes, functionally related genes (e.g., encoding transporters and modifying enzymes), regulatory genes (e.g., transcription factors), and auxiliary genes, with no apparent truncations or deletions. Region2 (~29 kb) showed 4% amino acid similarity to the kijanimicin biosynthetic gene cluster (BGC0000082), an ansamycin-type antibiotic, and encodes characteristic aminotransferase and cyclase genes, suggesting the potential for producing novel ansamycin-like metabolites. Region4 (~41 kb) displayed 7% similarity to the butirosin A/B aminoglycoside biosynthetic gene cluster (BGC0000693), and contains key genes (btrC, btrD) involved in 2-deoxystreptamine synthesis, indicating possible production of novel aminoglycoside derivatives. Regions 5, 9, and 10 (approximately 17 kb, 22 kb, and 41 kb, respectively) showed no significant homology to any known BGCs following comprehensive database searches, suggesting they may represent novel secondary metabolite pathways specific to B. velezensis R125. The structures and biological functions of their encoded metabolites warrant further investigation through heterologous expression and metabolomic profiling. Additionally, analysis based on compound class prediction results revealed that among all predicted compound types, Non-Ribosomal Peptides (NRPs) accounted for the highest proportion. Specifically, among the 13 predicted secondary metabolite gene clusters, five gene clusters were found to encode NRP compounds. Polyketide compounds ranked next, corresponding to four gene clusters, while the remaining compound categories corresponded to a smaller number of gene clusters. Given that NRP-related gene clusters were the most abundant, and considering that lipopeptide compounds predominantly belong to the NRP family, subsequent experiments focused on the extraction and identification of lipopeptide components in the R125 strain, based on these experimental findings. Table 2 Prediction of secondary metabolites of B. velezensis R125 genome. Region Type Most similar known cluster and similarity Category From End Region1 NRPS surfactin (78%) NRP 298,949 363,758 Region2 Thiopeptide Kijanimicin(4%) Polyketide 579,494 608,327 Region3 RRE-containing plantazolicin (91%) RiPP 691,824 715,001 Region4 PKS-like butirosin A / butirosin B(7%) Saccharide 926,732 967,976 Region5 Terpene - - 1,052,869 1,070,201 Region6 TransAT-PKS macrolactin H (100%) Polyketide 1,368,920 1,456,723 Region7 TransAT-PKS bacillaene (100%) Polyketide + NRP 1,676,229 1,776,938 Region8 NRPS fengycin (100%) NRP 1,843,599 1,977,763 Region9 Terpene - - 2,043,517 2,065,400 Region10 T3pks - - 2,138,235 2,179,335 Region11 TransAT-PKS difficidin (100%) Polyketide + NRP 2,349,619 2,443,408 Region12 NRPS bacillibactin (100%) NRP 3,073,123 3,124,915 Region13 Other bacilysin (100%) Other 3,642,167 3,683,585 Identification of Lipopeptide Components in Strain R125 Liquid chromatography-mass spectrometry (LC-MS) was employed for the qualitative and relative quantitative analysis of the extracted R125 lipopeptide fraction, with the identification results presented in Figure 6. Research on lipopeptide compounds has been in-depth, and the component identification system for these compounds is relatively well-established. Based on the accurate molecular weights obtained by mass spectrometry detection and combined with the reported mass spectral characteristic data of lipopeptide compounds in published literature, preliminary qualitative identification of the target fractions can be achieved. As shown in Figure 6A, the R125 lipopeptide fraction mainly comprises three types of typical lipopeptide compounds, namely Iturin (Figures 6C, D), Fengycin (Figures 6E, F), and Surfactin (Figures 6G, H). Further results of the relative quantitative analysis (Figures 6A, B) indicated significant differences in the contents of the three lipopeptide compounds: Iturin exhibited the highest relative content, accounting for 30.31%; Fengycin followed with a relative content of 22.42%; while Surfactin had the lowest relative content at 14.8%. Discussion This study reports the isolation and identification of strain Bacillus velezensis R125 GDMCC 67536 from Guangxi traditional fermented sour porridge, a strain exhibiting broad-spectrum antimicrobial activity. Through whole-genome sequencing and functional annotation, we systematically elucidate the molecular basis underlying its antimicrobial potential. Core findings demonstrate that strain R125 exhibits significant inhibitory activity against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, and Fusarium oxysporum f. sp. cubense race 1 201. Genomic analysis reveals a rich repertoire of biosynthetic gene clusters (BGCs) for secondary metabolites and carbohydrate-active enzymes (CAZymes), which together form a functional network enabling cross-kingdom antimicrobial activity. This discovery not only provides a new paradigm for mining microbial resources from traditional fermented foods, but also offers a candidate strain and genomic foundation for developing green antimicrobial agents and biopesticides. Traditional fermented foods serve as natural microbial “gene pools,” wherein their unique spontaneous fermentation environments—such as the acidic conditions, high osmotic pressure, and complex nutritional matrix of sour porridge—provide inherent advantages for the screening of functional strains [ 25 , 26 ]. Previous research has largely focused on common fermentative microorganisms such as lactic acid bacteria and yeasts, with less attention paid to bacilli, which possess both stress resistance and metabolic diversity [ 27 ]. In this study, strain R125 emerged as the only isolate, out of 24 screened, capable of simultaneously inhibiting both bacteria and fungi. The fermentation supernatant produced distinct zones of inhibition without diffusible artifacts, indicating that the antimicrobial products are stable and target-specific. These results confirm that traditional fermented foods harbor underexplored functional microbial resources and underscore the efficacy of targeted screening from specific fermented substrates—the long-term fermentation process of sour porridge may drive the enrichment and evolution of antimicrobial strains through microbial synergy and competition. Whole-genome analysis provided key insights into the antimicrobial mechanisms of strain R125. First, prediction of secondary metabolite BGCs revealed that strain R125 genome contains 13 complete BGCs, seven of which show 100% sequence homology or high similarity to known clusters for antimicrobial compounds such as surfactin, fengycin, bacilysin, and macrobrevin H. Surfactin, a well-characterized lipopeptide antimicrobial, acts by disrupting bacterial membrane integrity [ 28 , 29 ]; fengycin exhibits high specificity against fungi [ 30 ]; and bacilysin and difficidin further extend the inhibitory spectrum to Gram-positive and Gram-negative bacteria [ 31 ]. The completeness and diversity of these BGCs explain the ability of strain R125 to inhibit diverse pathogens across kingdoms; their synergistic interactions may constitute a multi-target antimicrobial network, thereby reducing the risk of pathogen resistance development. Second, functional annotation of CAZymes uncovered an additional antimicrobial mechanism: strain R125 encodes 51 CAZyme genes spanning six families, including glycoside hydrolases (GHs) and glycosyl transferases (GTs). Among these, GH5, GH9, and CBM6 families are implicated in cellulose degradation, while CBM73 and AA10 target chitin and peptidoglycan—key structural components of fungal and bacterial cell walls, respectively. Degradation of these components directly compromises pathogen structural integrity [ 32 ]. This dual mechanism—combining secondary metabolite inhibition and cell wall degradation—underpins the broad-spectrum antimicrobial activity of strain R125 and distinguishes it from strains relying on a single mode of action, enhancing its stability and efficacy in practical applications. Notably, the GC content of strain R125 plasmid (52.88%) is significantly higher than that of the chromosome (46.56%), suggesting possible acquisition via horizontal gene transfer. The plasmid also carries tRNA and rRNA genes that may participate in regulating the expression of antimicrobial genes, offering a starting point for future studies on adaptive evolution. Lipopeptide Characteristics and Species Consistency: Iturin, Fengycin, and Surfactin are characteristic lipopeptide secondary metabolites of the Bacillus genus, particularly within the Bacillus velezensis species complex. These compounds are synthesized via the nonribosomal peptide synthetase (NRPS) pathway and serve as key chemical markers distinguishing B. velezensis from other Bacillus species (e.g., B. subtilis primarily produces Surfactin, while B. amyloliquefaciens primarily produces Iturin). In this study, HPLC-MS analysis detected that strain R125 produces Iturin (30.31%), Fengycin (22.42%), and Surfactin (14.8%). This finding is highly consistent with the NRPS gene clusters predicted by AntiSMASH (Region 1 surfactin cluster, Region 8 fengycin cluster), confirming the typical metabolic profile of this strain as B. velezensis . Against the backdrop of a growing global antimicrobial resistance crisis, the overuse of chemical antimicrobials has led to serious ecological and public health concerns [ 33 ]. As a naturally sourced probiotic, strain R125 produces fermentation metabolites that are green, efficient, and low in toxicity. It has potential to replace chemical preservatives in food preservation, reducing contamination and safety risks. In agriculture, its strong inhibitory activity against FoC1—evidenced by suppression of mycelial growth in inhibition zones—offers a new biocontrol strategy for soil-borne diseases such as banana Fusarium wilt, aligning with the goals of sustainable agriculture. Moreover, the abundance of CAZymes in the R125 strain genome—such as cellulases and xylanases—suggests potential applications in biomass conversion and environmental degradation of organic waste, thereby expanding the scope of functional microbial utilization. From a scientific standpoint, This study performs whole-genome and metabolomic analysis of B. velezensis R125 isolated from traditional fermented sour porridge, and identifies several unique biological characteristics of this strain adapted to fermented food niches, confirming a molecular mechanism of antimicrobial activity mediated by the synergy between secondary metabolite synthesis and cell wall-degrading enzymes. This enriches our understanding of the regulatory network controlling the antimicrobial spectrum in B. velezensis . Additionally, the identification of three unannotated novel BGCs (Region5, 9, and 10) provides candidate targets for the discovery of novel antimicrobial compounds and genetic resources for microbial metabolic engineering. Genomic prediction identified 13 complete secondary metabolite BGCs in strain R125, and LC-MS analysis directly confirmed the production of three key lipopeptides—Iturin, Fengycin, and Surfactin—consistent with the functional annotation of NRPS-type BGCs. Specifically, the Fengycin BGC (Region8) exhibited 100% sequence similarity to the reference cluster (BGC0001095), and LC-MS detected Fengycin with a relative content of 22.42%; the Surfactin BGC (Region1) showed 78% similarity to BGC0000433, and Surfactin accounted for 14.8% of the detected lipopeptides; Iturin, a well-known antifungal and antibacterial lipopeptide, was the most abundant (30.31%), though its corresponding BGC was not explicitly highlighted in genomic prediction—likely nested within the unclassified or NRPS-type clusters [ 13 ]. This consistency between genomic potential and metabolic output strongly validates the functionality of strain R125’s BGCs, eliminating the uncertainty of “silent clusters” often associated with genomic prediction alone. Each lipopeptide contributes uniquely to the broad-spectrum antimicrobial activity: Iturin disrupts pathogen cell membranes by interacting with phospholipids and sterols, exerting potent activity against both bacteria and fungi [ 13 ]; Fengycin specifically targets fungal cell membranes by binding to ergosterol, a component unique to fungi, which explains strain R125’s strong inhibitory effect on FoC1 [ 30 ]; Surfactin, with its surfactant properties, permeabilizes bacterial membranes and enhances the diffusion of other antimicrobial compounds, synergizing with Iturin and Fengycin to broaden the inhibitory spectrum [ 28 ]. The relative content distribution—Iturin > Fengycin > Surfactin—correlates with strain R125’s comprehensive antimicrobial profile: the high abundance of Iturin provides a foundational broad-spectrum effect, while Fengycin reinforces antifungal specificity, and Surfactin optimizes overall efficacy through synergy. This lipopeptide cocktail effect reduces the risk of pathogen resistance compared to single-component antimicrobial agents. Despite these advances, several limitations remain. First, the identification of secondary metabolites is based solely on genomic prediction; structural validation through techniques such as mass spectrometry and nuclear magnetic resonance, as well as isolation and confirmation of key bioactive compounds via metabolomics and purification experiments, is still required. Second, the in vitro antimicrobial assays do not simulate real-world conditions, such as food matrices or soil microenvironments; the stability and persistence of strain R125’s antimicrobial activity in complex settings need further validation. Third, the synergistic relationship between CAZymes and BGCs has not been experimentally confirmed through gene knockout or heterologous expression studies, and the underlying regulatory pathways warrant further elucidation. Unlike previously reported B. velezensis strains, strain R125 exhibits a unique genomic feature of high abundance of GH5 family cellulase genes, which is a specific adaptive marker for plant-derived fermentation substrates and has not been documented in existing studies. In addition, three novel BGCs (Region5, 9, 10) with no known homologous sequences were identified in strain R125, which are potential targets for the discovery of novel antimicrobial compounds. Furthermore, this study first clarifies the quantitative synergy pattern of lipopeptides in strain R125: the high proportion of Iturin (30.31%) provides a broad-spectrum antibacterial and antifungal foundation, Fengycin (22.42%) specifically enhances the inhibition of phytopathogenic fungi (FoC1), and Surfactin (14.8%) improves the membrane permeability of pathogens and the diffusion of other lipopeptides, forming a unique lipopeptide cocktail effect that reduces the risk of pathogen resistance. In summary, strain Bacillus velezensis R125 GDMCC 67536, isolated from traditional fermented sour porridge, exhibits broad-spectrum antimicrobial activity mediated by the synergy of three key lipopeptides (Iturin, Fengycin, Surfactin) and cell wall-degrading CAZymes. Genomic prediction combined with LC-MS verification confirms the functionality of its secondary metabolite BGCs, while the relative content distribution of lipopeptides explains its comprehensive inhibitory profile against bacteria and fungi. Strain R125 holds great promise as a natural antimicrobial agent, biocontrol agent, and probiotic, with applications spanning food preservation, agriculture, and pharmaceuticals. This study identifies the unique genomic and metabolomic characteristics of strain B. velezensis R125 adapted to fermented food niches, and its specific lipopeptide synergy pattern and novel BGCs provide new experimental data and candidate targets for the research and development of natural antimicrobial agents; the GH5 family cellulase gene enrichment characteristic also provides a new perspective for the study of microbial adaptation to fermentation substrates. By integrating multi-omics technologies, synthetic biology, and applied microbiology, strain R125’s full potential can be harnessed to address global challenges such as antimicrobial resistance, food safety, and sustainable agriculture. Declarations Conflict of interest The authors declare that they have no conflicts of interest. Ethical Approval Not required (This research did not involve humans or animals). Consent for Publication All authors have read and approved this version of the article and due care has been taken to ensure the integrity of the work. Funding This research was funded by financial support from the National Natural Science Foundation of China ( Grant No . 32360244) and the Science and Technology Major Project of Guangxi ( Guike AA24206044). Author Contribution FuTian Yu designed, performed the experiment and wrote the original draft. DengFeng Yang was involved in conceptualization. All authors reviewed and edited the manuscript. References An F, Sun H, Wu J, Zhao C, Li T, Huang H, Fang Q, Mu E, Wu R (2021) Investigating the core microbiota and its influencing factors in traditional Chinese pickles. Food Res Int. 147: 110543 Bhattacharjee MJ, Bala A, Khan MR, Mukherjee AK (2025) Functional impact of bioactive peptides derived from fermented foods on diverse human populations. Food Chemistry. 492(Pt 1): 145416 Bielen A, Babic I, Vuk Surjan M, Kazazic S, Simatovic A, Lajtner J, Udikovic-Kolic N, Mesic Z, Hudina S (2024) Comparison of MALDI-TOF mass spectrometry and 16S rDNA sequencing for identification of environmental bacteria: a case study of cave mussel-associated culturable microorganisms. Environ Sci Pollut Res Int. 31(14): 21752-21764 Chen HJ, Liu Y, Zhong YS, Li MZ, Lai JJ, Luo YY, Huang SL, Liu SQ, Yu GH, Sun YH, Shao MW (2025) Modulating Surfactin Biosynthesis in Bacillus subtilis R31 Enhances Behavioural Traits and Biocontrol Efficacy Against Banana Fusarium Wilt . Microb Biotechnol. 18(11): e70261 Cho YT, Ting HM, Wang BW, Tsai YC, Wang HY, Yang YL, Ariyawansa HA (2025) Integrating genomics and targeted metabolite profiling to elucidate disease-suppression mechanisms of Bacillus velezensis GFB08. Curr Res Microb Sci. 9: 100503 Choi H, Son S (2025) Emerging nano-vaccine technologies to overcome anti-microbial resistance. J Control Release. 386: 114076 Felczak MM, Bowers RM, Woyke T, Teravest MA (2021) Zymomonas diversity and potential for biofuel production. Biotechnol Biofuels. 14(1): 112 Gilliard G, Demortier T, Boubsi F, Jijakli MH, Ongena M, De Clerck C, Deleu M (2024) Deciphering the distinct biocontrol activities of lipopeptides fengycin and surfactin through their differential impact on lipid membranes. Colloids Surf B Biointerfaces. 239: 113933 Illikoud N, Do Carmo FLR, Daniel N, Jan G, Gagnaire V (2023) Development of innovative fermented products by exploiting the diversity of immunomodulatory properties and fermentative activity of lactic and propionic acid bacteria. Food Res Int. 166: 112557 Isil C, Koydemir HC, Eryilmaz M, De Haan K, Pillar N, Mentesoglu K, Unal AF, Rivenson Y, Chandrasekaran S, Garner OB, Ozcan A (2025) Virtual Gram staining of label-free bacteria using dark-field microscopy and deep learning. Sci Adv. 11(2): eads2757 Jumpathong J, Uawisetwathana U, Intha N, Phakdee N, Pathaichindachote W, Rod-In W, Sirijan M (2026) Antagonistic activity and application of lipopeptide-producing Bacillus velezensis as biocontrol agent against rice bacterial leaf blight in vivo. Physiological and Molecular Plant Pathology. 142: Kayiran SD, Bolgen UMG, Akgul E, Kadiroglu P, Cengiz N, Cevikelli T, Onan D, Ozkan EE, Bozdogan G, Boga M, Ozogul Y, Esatbeyoglu T, Ozogul F (2025) Chemical composition, characterization, and antimicrobial properties of Thymbra spicata essential oil-based nanoemulsions and its application on curd cheese. Front Nutr. 12: 1615832 Lee YR, Lee HW, Park KM, Lee NK, Paik HD (2025) Anti-inflammatory effects of Lactiplantibacillus plantarum strains through MAPK, NF-kappaB, and AP-1 signaling pathways and its application in soy milk. Food Res Int. 208: 116216 Li Z, Jiang J, Sun K, Ye S (2025) Unveiling the Potential of Endophytic Bacillus amyloliquefaciens LJ1 from Nanguo Pear: A Genomic and Functional Study for Biocontrol of Post-Harvest Rot. Foods. 14(17): 3020 Liang SM, Li MX, Gong X, Luo Z, Yang JM (2026) Determination and pharmacokinetic study of thyroid hormone receptor-β agonist resmetirom in beagle dogs using HPLC-MS/MS method. Journal of Pharmaceutical and Biomedical Analysis. 269: Liu Y, Teng K, Huang F, Xia T, Zhang J, Wang T, Zhong J (2022) High-throughput discovery of novel lanthipeptides and producers by metagenomic mining of isolates population (MMIP) from Chinese spicy cabbage. Food Res Int. 154: 110991 Nguyen HD, Vu NH, Do HDK, Vu MT (2025) Comparative chloroplast genomic analysis of Pithecellobium dulce (Roxb.) Benth 1844 and related species within Caesalpinioideae. Genetica. 153(1): 19 Oufensou S, Ul-Hassan Z, Badr K, Balmas V, Jaoua S, Migheli Q (2025) Guns 'N Roses: Fungal Volatile Warfare in Postharvest Disease Control. Annu Rev Phytopathol. 63(1): 477-499 Qiao Y, Zhang K, Zhang Z, Zhang C, Sun Y, Feng Z (2022) Fermented soybean foods: A review of their functional components, mechanism of action and factors influencing their health benefits. Food Res Int. 158: 111575 Salamaga B, Kong L, Pasquina-Lemonche L, Lafage L, Von Und Zur Muhlen M, Gibson JF, Grybchuk D, Tooke AK, Panchal V, Culp EJ, Tatham E, O'kane ME, Catley TE, Renshaw SA, Wright GD, Plevka P, Bullough PA, Han A, Hobbs JK, Foster SJ (2021) Demonstration of the role of cell wall homeostasis in Staphylococcus aureus growth and the action of bactericidal antibiotics. Proc Natl Acad Sci U S A. 118(44): e2106022118 Snyder AB, Martin N, Wiedmann M (2024) Microbial food spoilage: impact, causative agents and control strategies. Nat Rev Microbiol. 22(9): 528-542 Villa-Rodriguez E, Moreno-Ulloa A, Castro-Longoria E, Parra-Cota FI, De Los Santos-Villalobos S (2021) Integrated omics approaches for deciphering antifungal metabolites produced by a novel Bacillus species , B. cabrialesii TE3(T), against the spot blotch disease of wheat ( Triticum turgidum L. subsp. durum ). Microbiological Research. 251: 126826 Wang R, Wu J, Jiang N, Lin H, An F, Wu C, Yue X, Shi H, Wu R (2023) Recent developments in horizontal gene transfer with the adaptive innovation of fermented foods. Crit Rev Food Sci Nutr. 63(5): 569-584 Wang Z, Xu T, Wang J, Sun G, Chen Q, Sun H, Shi B (2025) Probiotic potential of Bacillus velezensis MZ-09: Assessing characteristics and safety through in vitro and in vivo analyses. Microbiological Research. 301: 128321 Wu Y, Li A, Cheng L, Chen Q, Li J, Xu Y, Huo D (2022) Deep Shotgun metagenomic and 16S rRNA analysis revealed the microbial diversity of lactic acid bacteria in traditional fermented foods of eastern Hainan, China. Food Funct. 13(24): 12938-12952 Ye C, Zhao W, Liu D, Yang R, Cui Z, Zou D, Li D, Wei X, Xiong H, Niu C (2024) Screening, identification, engineering, and characterization of Bacillus -derived alpha-amylase for effective tobacco starch degradation. Int J Biol Macromol. 282(Pt 6): 137364 Yu F, Shen Y, Qin Y, Pang Y, Fan H, Peng J, Pei X, Liu X (2022) Isolation and purification of antibacterial lipopeptides from Bacillus velezensis YA215 isolated from sea mangroves. Frontiers in Nutrition. 9: 1064764 Yu FT, Shen YY, Chen SL, Fan HL, Pang YY, Liu MY, Peng JJ, Pei XD, Liu XL (2024) Analysis of the Genomic Sequences and Metabolites of Bacillus velezensis YA215. Biochemical Genetics. 62(6): 5073-5091 Yu FT, Shen YY, Pang YY, Fan HL, Liu MY, Liu XL (2024) Effects of branched-chain amino acids on surfactin structure and antibacterial activity in Bacillus velezensis YA215. World Journal of Microbiology & Biotechnology. 40(9): 281 Zhang P, Hu M, Meng W, Wang J, Yuan Z, Wang F, Li S (2025) Effect of Lactiplantibacillus plantarum MX14 and Pichia membranifaciens PY33 co-fermentation on quality characteristics of red sour soup. Food Chemistry. 492(Pt 2): 145473 Zhang X, Li S, Liu H, Wongta A, Xu Z, Zhou K, Hongsibsong S (2025) Isolation and Characterizations of Histamine- and Tyramine-Producing Strains Isolated from Fermented Soybean Food: Soy Sauce and Soybean Paste. Foods. 14(14): 2407 Zhao J, Pachiadaki M, Conrad RE, Hatt JK, Bristow LA, Rodriguez RL, Rossello-Mora R, Stewart FJ, Konstantinidis KT (2025) Promiscuous and genome-wide recombination underlies the sequence-discrete species of the SAR11 lineage in the deep ocean. Isme j. 19(1): wraf072 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 22 Jan, 2026 Editor assigned by journal 20 Jan, 2026 Submission checks completed at journal 20 Jan, 2026 First submitted to journal 20 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8646919","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":578736816,"identity":"5ab59a1d-69e2-4ce8-88db-e500cc29ea0c","order_by":0,"name":"FuTian Yu","email":"","orcid":"","institution":"Guangxi Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"FuTian","middleName":"","lastName":"Yu","suffix":""},{"id":578736817,"identity":"db3c1902-9ee2-4383-8668-7d73328f21ea","order_by":1,"name":"DengFeng Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8UlEQVRIiWNgGAWjYDCCA0BcAeN8MLCxI07LGSibcUZBWjJpWph5PhxibCCkg+947+EXByru2G04fvbwaxuDA8wM7IePbsCnRfLMuTSLA2eeJW84k5dmnWNwh4+BJy3tBj4tBjdyzIw/th1ONjgAZOQYPGNmkOAxw6/l/hszg4P/gFrOvzEztjA4zNhAUMsNHuMHBxsO2wGtM37MQIwWyTM5ZgwHjh1OkLzxxoyxxyAtmY2QX/iOnzH+cKDmsD3f+RzjDz/+2Njxsx8+hlcLELBJAInEBQcgDAY2AspBgPkDkLCXb4AwRsEoGAWjYBRgAABN0FlFskV5OwAAAABJRU5ErkJggg==","orcid":"","institution":"Guangxi Academy of Sciences","correspondingAuthor":true,"prefix":"","firstName":"DengFeng","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2026-01-20 08:50:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8646919/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8646919/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105808805,"identity":"b14383fe-7cfa-45e0-a28b-ea4a15cbcfee","added_by":"auto","created_at":"2026-03-31 10:57:59","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":108735,"visible":true,"origin":"","legend":"\u003cp\u003eResults of Antibacterial Activity in Fermentation Supernatants of Strains Isolated from Sour Porridge.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8646919/v1/245c00d134df2d6873904762.jpg"},{"id":105808803,"identity":"a0f8bf9d-550f-4813-b9ff-a3f3a4b2c6d5","added_by":"auto","created_at":"2026-03-31 10:57:59","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":76733,"visible":true,"origin":"","legend":"\u003cp\u003eResults of Antifungal Activity in Fermentation Supernatants of Strains Isolated from Sour Porridge.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8646919/v1/b1641bd6f7e4a75b7e91bd4c.jpg"},{"id":105808804,"identity":"ae7c2f63-194f-419d-a631-179bec58a5f3","added_by":"auto","created_at":"2026-03-31 10:57:59","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":161478,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of the R125 strain. (A) Colonial morphology of the R125 strain. (B) Gram staining of the R125 strain. (C) Agarose gel electrophoresis image of the R125 strain. (D) Neighbor-joining phylogenetic tree of the R125 strain. (E) OrthoANI values between strain R125 and selected \u003cem\u003eBacillus spp\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8646919/v1/5669154e60f8b211f544ca58.jpg"},{"id":105904713,"identity":"ed7016f3-ec69-4e58-a74d-abefd7ca0772","added_by":"auto","created_at":"2026-04-01 10:10:22","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":132084,"visible":true,"origin":"","legend":"\u003cp\u003eMap of the chromosome and plasmid of the \u003cem\u003eB. velezensis\u003c/em\u003e R125 genome. (A) Chromosome circle map of \u003cem\u003eB. velezensis\u003c/em\u003e R125 genome; (B) Gene circle map of \u003cem\u003eB. velezensis\u003c/em\u003e R125 plasmid. From inside to outside, the first circle represents the scale; the second circle represents the GC skew; the third circle represents the GC content; the fourth and seventh circles represent the COG to which each CDS belongs; and the fifth and sixth circles represent the positions of the CDS, tRNA, and rRNA on the genome.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8646919/v1/a6669bedeb482d3a33885dc8.jpg"},{"id":105808828,"identity":"36c29e4c-a256-448e-9202-a92a26a0cc1f","added_by":"auto","created_at":"2026-03-31 10:58:03","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":198023,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional gene analysis of \u003cem\u003eB. velezensis \u003c/em\u003eR125. (A) Clusters of GO annotations; (B) Clusters of COG annotations; (C) Clusters of KEGG annotations; (D) Gene count distributions of carbohydrate-active enzyme families.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8646919/v1/03b681fb790207f802c6db33.jpg"},{"id":105808808,"identity":"4eb55127-191e-44f8-a712-307a95c313f8","added_by":"auto","created_at":"2026-03-31 10:57:59","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":210908,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of lipopeptide components in strain R125. (A) Liquid chromatography results of lipopeptides from strain R125. (B) Peak area results from liquid chromatography. (C-D) Mass spectrometry results of iturin. (E-F) Mass spectrometry results of fengycin. (G-H) Mass spectrometry results of surfactin.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8646919/v1/45fd12e2e469a55ce0015aa1.jpg"},{"id":105908450,"identity":"72bc668b-c3c2-46d3-8842-6a8ee123e534","added_by":"auto","created_at":"2026-04-01 10:37:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1864220,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8646919/v1/46c0192f-cc70-42d9-86f6-aa3ea3fd06ac.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Functional Microbes from Traditional Fermented Food: Genomics, Lipopeptide Profiling and Antimicrobial Potential of Bacillus velezensis R125","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTraditional fermented foods represent a significant repository of microbial resources, harboring a diverse array of functional strains with various biological activities, owing to their unique fermentation environments [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. These microorganisms offer rich raw materials for the development of novel antimicrobial agents, biopesticides, and food preservatives [\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Sour porridge, a traditional fermented food from ethnic minority regions in southern China, hosts a complex microbiota formed through spontaneous fermentation, and this microbiota may contain dominant strains with broad-spectrum antimicrobial potential [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, systematic screening, identification, and genomic characterization of antimicrobial mechanisms of microbes derived from sour porridge remain limited, constraining the exploitation of these natural microbial resources.\u003c/p\u003e \u003cp\u003ePathogenic microorganisms\u0026mdash;including Gram-positive bacteria, Gram-negative bacteria, and phytopathogenic fungi\u0026mdash;pose serious global challenges through food spoilage, crop diseases, and clinical infections[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The overuse of chemical antimicrobials has exacerbated the crisis of antimicrobial resistance, underscoring the urgent need to develop green and efficient natural antibacterial agents. Strain \u003cem\u003eBacillus velezensis\u003c/em\u003e, a widespread probiotic, has attracted considerable attention for its ability to produce diverse antimicrobial secondary metabolites\u0026mdash;such as surfactin, fengycin, and bacillomycin\u0026mdash;yet the strain-specific nature of its antibacterial activity and the underlying molecular mechanisms require systematic elucidation through whole-genome sequencing and related technologies[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study isolates and screens microbial strains from sour porridge collected in Nanning, Guangxi, China, evaluating their inhibitory activity against \u003cem\u003eEscherichia coli\u003c/em\u003e ATCC 25922 (Gram-negative), \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC 25923 (Gram-positive), and \u003cem\u003eFusarium oxysporum\u003c/em\u003e f. sp. cubense race 1 201 (FoC1, a phytopathogenic fungus) using the agar well diffusion assay. Dominant strains are accurately identified through phenotypic characterization, 16S rRNA gene sequencing, and average nucleotide identity (ANI) analysis. A combined Illumina HiSeq and PacBio sequencing approach is employed for whole-genome sequencing and assembly. Functional gene annotation is performed using GO, COG, and KEGG databases, with particular emphasis on profiling carbohydrate-active enzyme (CAZy) genes and biosynthetic gene clusters (BGCs) for secondary metabolites. This research elucidates the molecular basis of the broad-spectrum antibacterial properties of strain \u003cem\u003eB. velezensis\u003c/em\u003e R125, identifies its unique genomic and metabolomic characteristics adapted to fermented food niches, and provides new candidate targets for the mining of novel antimicrobial secondary metabolites and the directional breeding of functional probiotics.\u003c/p\u003e \u003cp\u003eSour porridge is a traditional fermented food of ethnic minorities in South China. Its acidic (typically pH 3.5\u0026ndash;4.5), hypertonic, and anaerobic environment creates unique selective pressures on microorganisms. In this study, a total of 24 strains of culturable bacteria were isolated via the gradient dilution plating method from sour porridge samples collected in Nanning, Guangxi. Based on broad-spectrum antimicrobial activity screening (see below), strain \u003cem\u003eB. velezensis\u003c/em\u003e R125 was selected for further characterization due to the following reasons: (a) In agar diffusion assays, strain R125 was the only strain that produced clear inhibition zones against Gram-negative bacteria \u003cem\u003e(E. coli\u003c/em\u003e ATCC 25922), Gram-positive bacteria (\u003cem\u003eS. aureus\u003c/em\u003e ATCC 25923), and the plant pathogenic fungus (FoC1); (b) The inhibition zones formed by its fermentation supernatant showed sharp edges without diffusion, indicating stable and targeted antimicrobial production; (c) Preliminary identification based on 16S rRNA gene sequencing indicated that the strain belongs to the species \u003cem\u003eB. velezensis\u003c/em\u003e, which is known for its significant biocontrol potential. In contrast, among the other 23 isolates, only 4 showed activity against \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922, 6 against \u003cem\u003eS. aureus\u003c/em\u003e ATCC 25923, and 9 against FoC1, none of which exhibited broad-spectrum antimicrobial properties.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eLysogeny broth (LB) medium, potato dextrose agar (PDA) medium and agar were obtained from Beijing Solarbio Technology Co. Ltd. (Beijing, China). Methanol was of chromatography grade, while hydrochloric acid, ethanol and sodium hydroxide were of analytical grade purchased from Dongguan Sparta Chemical Co., Ltd. (Dongguan, China).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMicrobial Strains\u003c/h3\u003e\n\u003cp\u003eThe microbial strains were isolated by our research team from the sour porridge in Nanning, Guangxi, China. \u003cem\u003eEscherichia coli\u003c/em\u003e ATCC 25922, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC 25923 were obtained from American Type Culture Collection. FoC1 201 was stored in a 40% glycerol solution at \u0026minus;\u0026thinsp;80\u0026deg;C and deposited in the Marine Culture Collection of Guangxi. Strain \u003cem\u003eBacillus velezensis\u003c/em\u003e R125 GDMCC 67536, preserved at the Guangdong Microbial Culture Collection Center in Guangdong Province, China, with the preservation number: GDMCC No. 67536.\u003c/p\u003e\n\u003ch3\u003eDetermination of the Antimicrobial Activity in Fermentation Supernatants of Strains Isolated from Sour Porridge\u003c/h3\u003e\n\u003cp\u003eAntibacterial activity was assessed using a slightly modified agar well diffusion assay [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The procedure was as follows: \u003cem\u003eEscherichia coli\u003c/em\u003e ATCC 25922 and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC 25923 were individually cultured to the logarithmic growth phase, and the bacterial suspensions were adjusted to a final concentration of 10⁶ CFU/mL. Each bacterial suspension was then introduced into LB agar medium, which had been cooled to below 50\u0026deg;C, thoroughly mixed, and poured into sterile Petri dishes (90 mm diameter) to create bacteria-seeded plates. After the agar solidified completely, 6 mm diameter wells were aseptically punched into the medium. Each well was filled with 100 \u0026micro;L of fermented supernatant from acidified porridge isolates, while control wells received an equivalent volume of sterile LB medium. The plates were subsequently incubated at 37\u0026deg;C for 16\u0026ndash;18 h. The diameters of the inhibition zones surrounding the wells were measured to an accuracy of 0.1 mm using the cross-streak method, thereby quantifying the antibacterial activity of the fermented supernatants.\u003c/p\u003e \u003cp\u003eAntifungal activity was evaluated via a mildly adapted agar well diffusion method [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. A 6 mm mycelial plug, obtained from the periphery of a 7-day-old culture of Fusarium oxysporum f. sp. cubense race 1 (FoC1) using a sterile cork borer, was inoculated onto the center of a fresh, sterile PDA plate. Following a 4-day pre-incubation period at 28\u0026deg;C, four 5 mm diameter wells were symmetrically prepared at a distance of 2 cm from the central mycelial plug. Each well received 100 \u0026micro;L of the fermented supernatant, with control wells containing an equal volume of sterile PDA medium. The plates were incubated for an additional 4 days at 28\u0026deg;C, after which the diameters of the inhibition zones were measured to 0.1 mm precision using the cross-streak method, serving as an indicator of antifungal activity.\u003c/p\u003e\n\u003ch3\u003eIdentification of the R125 Strain\u003c/h3\u003e\n\u003cp\u003eThe R125 strain was inoculated onto LB solid medium and incubated at 37\u0026deg;C for 24 h. Following incubation, the morphological characteristics of the colonies were examined and documented, and Gram staining was performed for preliminary identification [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Molecular identification was subsequently conducted by amplifying the 16S rRNA gene with the universal primer pair 27F (5\u0026prime;-GGTTACCTTGTTACGACTT-3\u0026prime;) and 1492R (5\u0026prime;-AGAGTTTGATTTGATCCTGGCTAG-3\u0026prime;). The PCR amplification was carried out on a Biometra TC-512 thermocycler (Germany) under the following program: initial denaturation at 94\u0026deg;C for 5 min; 35 cycles of denaturation at 94\u0026deg;C for 30 s, annealing at 64\u0026deg;C for 1 minute, and extension at 72\u0026deg;C for 2 min; followed by a final extension at 72\u0026deg;C for 5 min. The PCR products were sequenced by Sangon Biotech (Shanghai) Co., Ltd [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe obtained 16S rDNA sequence was aligned for homology analysis against known sequences in the GenBank database using BLAST. A phylogenetic tree was constructed with MEGA 7.0 software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.megasoftware.net/\u003c/span\u003e\u003cspan address=\"https://www.megasoftware.net/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) employing the Neighbor-Joining method. Strain R125 was further subjected to genomic typing based on the Average Nucleotide Identity (ANI), computed via the NCBI Prokaryotic Genome Annotation Pipeline (PGAP v2020-07-09.build4716) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eWhole Genome Sequencing and Assembly\u003c/h3\u003e\n\u003cp\u003eThe R125 strain, preserved at -80\u0026deg;C, was initially revived on LB solid medium and incubated at 37\u0026deg;C for 24 h. Uniform single colonies were then selected and inoculated into LB liquid medium, followed by incubation in a constant-temperature shaking incubator at 37\u0026deg;C and 220 rpm for 12 h. After cultivation, the bacterial culture was centrifuged at 8000 rpm for 15 min to collect the R125 strain cell pellet. The harvested bacterial samples were subsequently sent to Shanghai Majorbio Bio-Pharm Technology Co., Ltd. for genomic DNA extraction and subsequent sequencing.\u003c/p\u003e \u003cp\u003eA combined sequencing strategy employing both Illumina HiSeq and PacBio platforms was adopted for genome sequencing. Genomic DNA was fragmented into 8\u0026ndash;10 kb segments, purified, and used to construct a SMRT Bell library for PacBio sequencing. Meanwhile, paired-end PE150 sequencing was performed on the Illumina HiSeq X10 platform. Raw sequencing data were subjected to a filtering process to remove reads containing sequencing primers, adapter sequences, and low-quality reads, yielding high-quality Clean Data. A hierarchical genome assembly process (HGAP) and the Canu assembler were employed for genome assembly. After multiple rounds of polishing, a complete genomic sequence was obtained [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Subsequent genomic bioinformatic analyses were conducted online via the Majorbio Cloud Platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cloud.majorbio.com\u003c/span\u003e\u003cspan address=\"https://cloud.majorbio.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnnotation and Analysis of Strain\u003c/b\u003e \u003cb\u003eB. velezensis\u003c/b\u003e \u003cb\u003eR125 Genome\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe genomic annotation of strain \u003cem\u003eB. velezensis\u003c/em\u003e R125 was performed as follows [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Gene prediction was carried out using Glimmer 3.02. Identification of tRNA molecules was conducted with tRNAscan-SE, while rRNA genes were detected using RNAmmer in conjunction with the Rfam database. Functional annotation of predicted genes was achieved through sequence alignment against several reference databases, including the Gene Ontology (GO), Clusters of Orthologous Groups of proteins (COG), and the Kyoto Encyclopedia of Genes and Genomes (KEGG). In addition, carbohydrate-active enzymes (CAZymes) were predicted using the CAZy database, and biosynthetic potential for secondary metabolites was assessed via the antiSMASH software package. Finally, a comprehensive genomic map was generated for visualization using CGView.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eLipopeptide Extraction\u003c/h2\u003e \u003cp\u003eThe strain R125 was activated on LB agar plates for 24 h, and a single colony was inoculated into 100 mL of LB liquid medium, followed by incubation at 37\u0026deg;C and 220 r/min for 12 h. The culture was then transferred to a wide-mouth flask at a 4% volume fraction and fermented under the same conditions for 48 h. The fermentation broth was centrifuged at 4\u0026deg;C and 10,000 \u0026times; g to collect the cell-free supernatant.\u003c/p\u003e \u003cp\u003eStrain R125 lipopeptide was extracted using the acid precipitation-methanol extraction method: The supernatant was adjusted to pH 2.0 with 6 mol/L HCl and left to stand at 4\u0026deg;C overnight. It was then centrifuged at 4\u0026deg;C and 10,000 \u0026times; g for 20 min, and the precipitate was extracted with methanol. The soluble components were collected to obtain the lipopeptide extract [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003e\u003cb\u003eIdentification of Strain R125 Lipopeptide by High-Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS)\u003c/b\u003e\u003c/div\u003e \u003cp\u003eThe strain R125 lipopeptide was qualitatively identified using high-performance liquid chromatography-mass spectrometry (HPLC-MS) with reference to a literature method after optimization [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The specific detection conditions were as follows: Chromatographic separation was performed on a Waters Sunfire C18 column (4.6 mm \u0026times; 100 mm, 3.5 \u0026micro;m). The column temperature was maintained constant at 30\u0026deg;C to ensure separation stability. A UV detection wavelength of 214 nm was selected, providing sensitive detection of peptide bonds in lipopeptides and enabling effective capture of target signals. The mobile phase consisted of a binary gradient system: phase A (1% v/v formic acid in water) and phase B (1% v/v formic acid in acetonitrile). The elution parameters were as follows: flow rate 0.8 mL/min, injection volume 10 \u0026micro;L, total run time 15.00 min. The gradient program was: 0\u0026ndash;7 min, phase B increased linearly from 5% to 95%; 7\u0026ndash;12 min, phase B maintained at 95%; 12\u0026ndash;12.2 min, phase B rapidly decreased from 95% to 5%; 12.2\u0026ndash;15 min, phase B maintained at 5% for column equilibration. Mass spectrometry was performed using an electrospray ionization (ESI) source in positive ion scanning mode, which proved highly responsive for the protonated molecular ion peaks of lipopeptides, facilitating structural elucidation. The mass-to-charge ratio (m/z) scanning range was set to 100\u0026ndash;2000, covering both the strain R125 lipopeptide and potential degradation products to avoid missing target signals .\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eEach assay was performed in triplicate. The experimental data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. The comparison of the mean values was performed by one-way analysis of variance (ANOVA) and Tukey\u0026rsquo;s test using SPSS software (IBM Corp., Armonk, NY, USA). A p value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eEvaluation of Antimicrobial Activity in Fermentation Supernatants of Strains Isolated from Sour Porridge and Screening of Superior Strains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe antimicrobial activity of cell-free supernatants (CFSs) from 24 sour porridge isolates was systematically evaluated using the agar well diffusion method. These assays collectively assessed the broad-spectrum antimicrobial potential of the strains, with results presented in Fig. 1 and 2. The results of antibacterial activity assays revealed significant strain-specific variability in the antibacterial activity of CFSs following 48 h of fermentation in LB broth. Against \u003cem\u003eE. coli\u003c/em\u003e (Gram-negative), only strains R66, No. 8, R125, and No. 111 displayed distinct antibacterial activity, all forming clear inhibition zones with regular edges and no diffusion or blurring. Among these, strain R125 produced the largest inhibition zone diameter\u0026mdash;significantly greater than that of the other three strains\u0026mdash;indicating superior anti-\u003cem\u003eE. coli\u003c/em\u003e activity. In contrast, against \u003cem\u003eS. aureus\u003c/em\u003e (Gram-positive), the number of strains with antibacterial activity increased significantly: CFSs from strains No. 11, No. 12, No. 14, R125, No. 108, and No. 82 effectively inhibited S. aureus growth. Notably, the inhibition zones of strains R125 and No. 11 featured smooth edges and distinct transparent regions, suggesting stronger and more stable inhibitory effects against Gram-positive bacteria.\u003c/p\u003e\n\u003cp\u003eTo comprehensively assess the antimicrobial spectrum coverage of the strains, additional antifungal activity screening was performed. Results demonstrated that in FoC1 growth inhibition assays, CFSs from 9 strains (No. 12, No. 72, No. 27, No. 10, No. 41, No. 54, R125, No. 82, and No. 26) exhibited significant antifungal activity, accounting for 37.5% of the total tested strains. Among these, the CFS of strain R125 exhibited more pronounced inhibitory effects on FoC1, with no mycelial spread around the inhibition zone\u0026mdash;significantly superior to other active strains\u0026mdash;indicating potent anti-FoC1 activity. Notably, among all active strains, only strain R125 exhibited potent inhibitory activity against Gram-positive bacteria, Gram-negative bacteria, and phytopathogenic fungi simultaneously. The diameter of inhibition zones produced by its CFS against all three types of indicator microbes was significantly larger than that of the negative control, with excellent activity stability and no observed diffusion or edge blurring of inhibition zones.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIdentification of the R125 Strain\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo achieve precise taxonomic identification of strain R125, this study conducted a systematic analysis from two core dimensions\u0026mdash;phenotypic characterization and molecular-level verification\u0026mdash;to ensure the reliability and comprehensiveness of the identification. In the phenotypic identification phase, cellular and colonial morphological characteristics were first examined. The activated R125 strain was inoculated onto standard LB agar plates (1.5% agar) and incubated statically at 37 \u0026deg;C for 24 h in the dark. Observation using a colony morphology analyzer revealed that the strain formed pale yellow, circular colonies with irregular serrated margins, smooth surfaces, and moist texture, measuring approximately 1.5\u0026ndash;2.0 mm in diameter (Fig. 3A). These characteristics are highly consistent with the typical colonial morphology of the genus \u003cem\u003eBacillus\u003c/em\u003e. Subsequently, Gram staining was performed on R125 cells during the logarithmic growth phase. The cells stained purple, exhibited a rod-shaped morphology with blunt ends, and measured approximately 2.0\u0026ndash;3.0 \u0026mu;m in length and 0.5\u0026ndash;0.8 \u0026mu;m in width (Fig. 3B). According to the principles of Gram staining, the retention of the crystal violet\u0026ndash;iodine complex indicates a thick peptidoglycan layer, allowing the preliminary classification of R125 as a Gram-positive bacterium. These phenotypic observations provided a basis for subsequent molecular identification.\u003c/p\u003e\n\u003cp\u003eAt the molecular level, 16S rDNA sequence analysis was first performed. Universal bacterial primers were used to amplify the 16S rRNA gene of R125 via PCR. The amplification product was verified by 1.0% agarose gel electrophoresis, which showed a single bright band at approximately 1500 bp (Fig. 3C), indicating high specificity. The purified PCR product was submitted for Sanger sequencing, and the resulting sequence was aligned and corrected using MEGA 11 software. A BLAST homology search was conducted in the GenBank database. Reference strains with coverage \u0026ge;98% and similarity \u0026ge;95% were selected to construct a phylogenetic tree using the neighbor-joining method with 1000 bootstrap replications. The results indicated that the 16S rDNA sequence of R125 shared 99% similarity with that of \u003cem\u003eBacillus velezensis\u003c/em\u003e strain NRRL B-41580, and the two strains clustered together in the phylogenetic tree with a bootstrap value of 98% (Fig. 3D), suggesting a close evolutionary relationship.\u003c/p\u003e\n\u003cp\u003eTo enhance identification accuracy and mitigate potential misclassification due to the high conservation of 16S rDNA sequences, whole-genome sequencing of R125 was conducted. Using the Illumina NovaSeq platform, a PE150 library was constructed with sequencing depth exceeding 50\u0026times;. A draft genome was obtained after data filtering and assembly. Based on the assembled genome, the Orthologous Average Nucleotide Identity (OrthoANI) between R125 and four reference \u003cem\u003eBacillus strains\u003c/em\u003e\u0026mdash;\u003cem\u003eB. velezensis\u003c/em\u003e FZB42, \u003cem\u003eB. subtilis\u003c/em\u003e 168, \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e DSM 7, and \u003cem\u003eB. siamensis\u003c/em\u003e KCTC 13613\u0026mdash;was calculated using the OAT software with default parameters. Orthologous gene pairs with sequence similarity \u0026ge;70% and length \u0026ge;100 bp were retained for analysis, and a heatmap of OrthoANI values was generated using TBtools (Figure 3E). The results showed that R125 shared OrthoANI values above 75.04% with all reference strains, with the highest value (97.78%) observed with the model strain \u003cem\u003eB. velezensis\u003c/em\u003e FZB42. As OrthoANI analysis is considered the gold standard for evaluating species relatedness at the whole-genome level\u0026mdash;based on the average nucleotide identity of orthologous genes\u0026mdash;and given that an OrthoANI value \u0026ge;95% is widely accepted as the threshold for species delineation in microbial taxonomy (Felczak et al. 2021), the observed value of 97.78% strongly supports the classification of R125 as \u003cem\u003eB. velezensis\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eIntegrating the phenotypic and molecular evidence, R125 exhibits typical characteristics of \u003cem\u003eBacillus velezensis\u003c/em\u003e, including Gram-positive rod-shaped cells and pale-yellow irregular colonies. Its 16S rDNA sequence shows 99% similarity to reference strains of this species, and its OrthoANI value exceeds the species delineation threshold. These multi-faceted and mutually corroborating results allow the confident identification of strain R125 as \u003cem\u003eBacillus velezensis\u003c/em\u003e. In accordance with the International Code of Nomenclature of Prokaryotes, it is formally designated as \u003cem\u003eBacillus velezensis\u003c/em\u003e R125 (abbreviated as \u003cem\u003eB. velezensis\u003c/em\u003e R125).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEvaluation of Antimicrobial Activity in Fermentation Supernatants of Strains Isolated from Sour Porridge and Screening of Superior Strains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe antimicrobial activity of cell-free supernatants (CFSs) from 24 sour porridge isolates was systematically evaluated using the agar well diffusion method. These assays collectively assessed the broad-spectrum antimicrobial potential of the strains, with results presented in Fig. 1 and 2. The results of antibacterial activity assays revealed significant strain-specific variability in the antibacterial activity of CFSs following 48 h of fermentation in LB broth. Against \u003cem\u003eE. coli\u003c/em\u003e (Gram-negative), only strains R66, No. 8, R125, and No. 111 displayed distinct antibacterial activity, all forming clear inhibition zones with regular edges and no diffusion or blurring. Among these, strain R125 produced the largest inhibition zone diameter\u0026mdash;significantly greater than that of the other three strains\u0026mdash;indicating superior anti-\u003cem\u003eE. coli\u003c/em\u003e activity. In contrast, against \u003cem\u003eS. aureus\u003c/em\u003e (Gram-positive), the number of strains with antibacterial activity increased significantly: CFSs from strains No. 11, No. 12, No. 14, R125, No. 108, and No. 82 effectively inhibited S. aureus growth. Notably, the inhibition zones of strains R125 and No. 11 featured smooth edges and distinct transparent regions, suggesting stronger and more stable inhibitory effects against Gram-positive bacteria.\u003c/p\u003e\n\u003cp\u003eTo comprehensively assess the antimicrobial spectrum coverage of the strains, additional antifungal activity screening was performed. Results demonstrated that in FoC1 growth inhibition assays, CFSs from 9 strains (No. 12, No. 72, No. 27, No. 10, No. 41, No. 54, R125, No. 82, and No. 26) exhibited significant antifungal activity, accounting for 37.5% of the total tested strains. Among these, the CFS of strain R125 exhibited more pronounced inhibitory effects on FoC1, with no mycelial spread around the inhibition zone\u0026mdash;significantly superior to other active strains\u0026mdash;indicating potent anti-FoC1 activity. Notably, among all active strains, only strain R125 exhibited potent inhibitory activity against Gram-positive bacteria, Gram-negative bacteria, and phytopathogenic fungi simultaneously. The diameter of inhibition zones produced by its CFS against all three types of indicator microbes was significantly larger than that of the negative control, with excellent activity stability and no observed diffusion or edge blurring of inhibition zones.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIdentification of the R125 Strain\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo achieve precise taxonomic identification of strain R125, this study conducted a systematic analysis from two core dimensions\u0026mdash;phenotypic characterization and molecular-level verification\u0026mdash;to ensure the reliability and comprehensiveness of the identification. In the phenotypic identification phase, cellular and colonial morphological characteristics were first examined. The activated R125 strain was inoculated onto standard LB agar plates (1.5% agar) and incubated statically at 37 \u0026deg;C for 24 h in the dark. Observation using a colony morphology analyzer revealed that the strain formed pale yellow, circular colonies with irregular serrated margins, smooth surfaces, and moist texture, measuring approximately 1.5\u0026ndash;2.0 mm in diameter (Fig. 3A). These characteristics are highly consistent with the typical colonial morphology of the genus \u003cem\u003eBacillus\u003c/em\u003e. Subsequently, Gram staining was performed on R125 cells during the logarithmic growth phase. The cells stained purple, exhibited a rod-shaped morphology with blunt ends, and measured approximately 2.0\u0026ndash;3.0 \u0026mu;m in length and 0.5\u0026ndash;0.8 \u0026mu;m in width (Fig. 3B). According to the principles of Gram staining, the retention of the crystal violet\u0026ndash;iodine complex indicates a thick peptidoglycan layer, allowing the preliminary classification of R125 as a Gram-positive bacterium. These phenotypic observations provided a basis for subsequent molecular identification.\u003c/p\u003e\n\u003cp\u003eAt the molecular level, 16S rDNA sequence analysis was first performed. Universal bacterial primers were used to amplify the 16S rRNA gene of R125 via PCR. The amplification product was verified by 1.0% agarose gel electrophoresis, which showed a single bright band at approximately 1500 bp (Fig. 3C), indicating high specificity. The purified PCR product was submitted for Sanger sequencing, and the resulting sequence was aligned and corrected using MEGA 11 software. A BLAST homology search was conducted in the GenBank database. Reference strains with coverage \u0026ge;98% and similarity \u0026ge;95% were selected to construct a phylogenetic tree using the neighbor-joining method with 1000 bootstrap replications. The results indicated that the 16S rDNA sequence of R125 shared 99% similarity with that of \u003cem\u003eBacillus velezensis\u003c/em\u003e strain NRRL B-41580, and the two strains clustered together in the phylogenetic tree with a bootstrap value of 98% (Fig. 3D), suggesting a close evolutionary relationship.\u003c/p\u003e\n\u003cp\u003eTo enhance identification accuracy and mitigate potential misclassification due to the high conservation of 16S rDNA sequences, whole-genome sequencing of R125 was conducted. Using the Illumina NovaSeq platform, a PE150 library was constructed with sequencing depth exceeding 50\u0026times;. A draft genome was obtained after data filtering and assembly. Based on the assembled genome, the Orthologous Average Nucleotide Identity (OrthoANI) between R125 and four reference \u003cem\u003eBacillus strains\u003c/em\u003e\u0026mdash;\u003cem\u003eB. velezensis\u003c/em\u003e FZB42, \u003cem\u003eB. subtilis\u003c/em\u003e 168, \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e DSM 7, and \u003cem\u003eB. siamensis\u003c/em\u003e KCTC 13613\u0026mdash;was calculated using the OAT software with default parameters. Orthologous gene pairs with sequence similarity \u0026ge;70% and length \u0026ge;100 bp were retained for analysis, and a heatmap of OrthoANI values was generated using TBtools (Figure 3E). The results showed that R125 shared OrthoANI values above 75.04% with all reference strains, with the highest value (97.78%) observed with the model strain \u003cem\u003eB. velezensis\u003c/em\u003e FZB42. As OrthoANI analysis is considered the gold standard for evaluating species relatedness at the whole-genome level\u0026mdash;based on the average nucleotide identity of orthologous genes\u0026mdash;and given that an OrthoANI value \u0026ge;95% is widely accepted as the threshold for species delineation in microbial taxonomy (Felczak et al. 2021), the observed value of 97.78% strongly supports the classification of R125 as \u003cem\u003eB. velezensis\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eIntegrating the phenotypic and molecular evidence, R125 exhibits typical characteristics of \u003cem\u003eBacillus velezensis\u003c/em\u003e, including Gram-positive rod-shaped cells and pale-yellow irregular colonies. Its 16S rDNA sequence shows 99% similarity to reference strains of this species, and its OrthoANI value exceeds the species delineation threshold. These multi-faceted and mutually corroborating results allow the confident identification of strain R125 as \u003cem\u003eBacillus velezensis\u003c/em\u003e. In accordance with the International Code of Nomenclature of Prokaryotes, it is formally designated as \u003cem\u003eBacillus velezensis\u003c/em\u003e R125 (abbreviated as \u003cem\u003eB. velezensis\u003c/em\u003e R125).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e Genome statistics of \u003cem\u003eB. velezensis\u003c/em\u003e R125.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"449\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eGenomic Feature\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eChromosome\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ePlasmid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eSize of the genome assembly (bp)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3,970,649\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5981\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eGC content (%)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e46.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e52.88\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eProtein-coding genes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3808\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eProtein-coding regions (bp)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3506877\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003erRNA genes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003etRNA genes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003etmRNA genes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eCRISPR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eFunctional Gene Annotation of \u003cem\u003eB. velezensis\u0026nbsp;\u003c/em\u003eR125\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunctional annotation of the \u003cem\u003eB. velezensis\u003c/em\u003e R125 genome, based on whole-genome sequencing data, is presented in Fig. 5. The Gene Ontology (GO) database provides a standardized classification system for gene functions, systematically categorizing protein roles across three domains: biological process (BP), cellular component (CC), and molecular function (MF). As shown in Fig. 5A, a total of 2,548 genes in the \u003cem\u003eB. velezensis\u0026nbsp;\u003c/em\u003eR125 genome were assigned GO terms, corresponding to an annotation rate of approximately 64.9%. Among the three major functional categories, genes associated with biological processes exhibited the highest annotation abundance, indicating functional diversity related to the regulation of cellular activities. This was followed by cellular components, while molecular functions had the lowest representation. Within biological processes, the most abundant functional terms were cellular process, metabolic process, and organic substance metabolic process. The high abundance of metabolic process-related genes suggests active metabolic capacity in this strain. Among cellular components, the top three terms\u0026mdash;cell, cell part, and intracellular component\u0026mdash;are consistent with the structural characteristics of unicellular bacteria. The enrichment of intracellular component-related genes further reflects the integrity of cellular architecture and function. In the molecular function category, catalytic activity, binding activity, and transferase activity were the most prevalent. The high proportion of catalytic activity genes supports the presence of a diverse repertoire of metabolic enzymes in this strain.\u003c/p\u003e\n\u003cp\u003eThe Clusters of Orthologous Groups (COG) database classifies gene functions based on evolutionary relationships among homologous proteins. Our analysis revealed that 3,723 genes in the \u003cem\u003eB. velezensis\u0026nbsp;\u003c/em\u003eR125 genome were assigned COG classifications (Fig. 5B). Among the 26 functional categories, the three most represented were: amino acid metabolism and transport (332 genes, 8.9% of annotated genes), transcription (310 genes, 8.3%), and carbohydrate metabolism and transport (244 genes, 6.6%). The transcription category includes genes encoding RNA polymerase subunits and transcription factors, indicative of a complex and complete transcriptional regulatory system. The carbohydrate metabolism and transport category encompasses genes for glycoside hydrolases and transport proteins, underpinning the strain\u0026rsquo;s ability to efficiently utilize environmental carbohydrates. Additional functional categories, such as signal transduction mechanisms and posttranslational modification, were also identified, collectively supporting a comprehensive physiological regulatory network in the strain.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eKyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed using the KEGG database (release 109.0) and the clusterProfiler software, with a significance threshold of p\u0026lt;0.05 based on a hypergeometric test. A total of 3,932 genes in the \u003cem\u003eB. velezensis\u0026nbsp;\u003c/em\u003eR125 genome were annotated and assigned to 197 metabolic pathways spanning 12 major categories, including carbohydrate metabolism, amino acid metabolism, and nucleotide metabolism (Fig. 5C). The pathways with the highest gene counts were global and overview maps (1,576 genes), carbohydrate metabolism (427 genes), and amino acid metabolism (309 genes). The global and overview maps category contains genes involved in central metabolic pathways such as glycolysis and the tricarboxylic acid cycle, and its high gene count reflects the completeness of the strain\u0026rsquo;s core metabolic network. Enrichment of genes related to starch, sucrose, fructose, and mannose metabolism within the carbohydrate metabolism pathway suggests the strain\u0026rsquo;s potential for utilizing diverse carbon sources. In amino acid metabolism, the presence of genes involved in glutamate and aspartate metabolism supports the strain\u0026rsquo;s capacity for synthesizing essential amino acids and participating in environmental nitrogen cycling. Together, these findings indicate that \u003cem\u003eB. velezensis\u0026nbsp;\u003c/em\u003eR125 possesses substantial metabolic diversity, enabling coordinated multi-pathway involvement in the transformation of sugars, amino acids, and other substances. This metabolic versatility likely underlies its ability to adapt to complex environments and to perform physiological functions such as plant growth promotion and antibacterial activity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCarbohydrate Active Enzyme Annotation Statistics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSystematic annotation based on the CAZy (Carbohydrate-Active enZYmes Database) revealed 51 carbohydrate-active enzyme (CAZyme) encoding genes in the genome of \u003cem\u003eB. velezensis\u003c/em\u003e R125 (Fig. 5D). These genes span six major CAZyme families: glycoside hydrolases (GH), glycosyl transferases (GT), carbohydrate-binding modules (CBM), carbohydrate esterases (CE), auxiliary activities (AA), and polysaccharide lyases (PL). Among these, GH and GT families were the most abundant, each comprising 22 genes and collectively accounting for 86.28% of all identified CAZymes (43.14% each), thus forming the core of the strain\u0026rsquo;s CAZyme repertoire. The CBM family included five genes (9.80%), which serve as non-catalytic domains that enhance enzyme binding to carbohydrate substrates. In contrast, CE and AA families were sparsely represented (one gene each), and no PL family genes were detected; together, these minor categories constituted only 3.92% of the total CAZymes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePredictive Analysis of Secondary Metabolite Synthesis Gene Clusters\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, we employed the AntiSMASH v6.1 online platform (https://antismash.secondarymetabolites.org/) with default parameters to systematically predict and annotate biosynthetic gene clusters (BGCs) in the complete genome sequence of \u003cem\u003eB. velezensis\u003c/em\u003e R125 (GenBank accession: PRJNA1358366). The results are summarized in Table 2. A genome-wide screen identified 13 fully annotated BGCs, all located on the bacterial chromosome without notable regional clustering. These include: three NRPS-type clusters, one type III polyketide synthase (T3PKS) cluster, three trans-acyltransferase PKS (transAT-PKS) clusters, two terpene clusters, one thiopeptide cluster, one RRE (Ribosome Binding Site Regulatory Element)-containing cluster, one PKS-like cluster, and one unclassified functional gene cluster.\u003c/p\u003e\n\u003cp\u003eTo elucidate the potential secondary metabolite profile of \u003cem\u003eB. velezensis\u0026nbsp;\u003c/em\u003eR125, we first conducted targeted homology analysis of the three NRPS-type clusters (Region1, Region8, and Region12) against functionally characterized NRPS BGCs in the MIBiG v2.0 database. Region1 exhibited 78% amino acid sequence similarity to the surfactin biosynthetic gene cluster (BGC0000433), and its four core NRPS genes (srfA-A, srfA-B, srfA-C, srfA-D) displayed highly conserved modular architectures corresponding to peptide elongation modules in surfactin synthesis. Region8 showed 100% similarity to the fengycin biosynthetic gene cluster (BGC0001095), with identical NRPS module counts and functional domains (condensation, adenylation, and thiolation domains) encoded by fenA, fenB, fenC, and fenD. Region12 displayed 100% similarity to the bacillibactin biosynthetic gene cluster (BGC0000309).\u003c/p\u003e\n\u003cp\u003eAnalysis of the three transAT-PKS clusters (Region6, Region7, and Region11) revealed 100% sequence similarity to known BGCs for macrolide H (BGC0000181), bacillaene (BGC0001089), and difficidin (BGC0000176), respectively. The core PKS genes exhibited complete conservation in acyltransferase (AT), ketosynthase (KS), and ketoreductase (KR) domain sequences, suggesting full functional capacity. Furthermore, Region13 showed 100% identity with the bacilysin biosynthetic gene cluster (BGC0001184), which includes key synthesis genes such as bacA, bacB, bacC, and bacD, encoding characteristic enzymes like isochorismatase and aminotransferase essential for bacilysin production. These findings confirm the presence of functional BGCs for surfactin, fengycin, bacillibactin, macrolide H, bacillaene, difficidin, and bacilysin in \u003cem\u003eB. velezensis\u0026nbsp;\u003c/em\u003eR125, indicating a high potential for the biosynthesis of these metabolites\u0026mdash;all known for their broad-spectrum antimicrobial activities. Surfactin exhibits both surfactant and antibacterial properties, fengycin specifically inhibits fungi, and bacilysin shows significant activity against Gram-positive and Gram-negative bacteria (Cho et al. 2025).\u003c/p\u003e\n\u003cp\u003eAccording to AntiSMASH annotations, Region2, Region4, Region5, Region9, and Region10 exhibited low sequence similarity (\u0026lt;10%) to known BGCs in the MIBiG database. However, structural integrity assessments confirmed that these clusters contain complete sets of core synthase genes, functionally related genes (e.g., encoding transporters and modifying enzymes), regulatory genes (e.g., transcription factors), and auxiliary genes, with no apparent truncations or deletions. Region2 (~29 kb) showed 4% amino acid similarity to the kijanimicin biosynthetic gene cluster (BGC0000082), an ansamycin-type antibiotic, and encodes characteristic aminotransferase and cyclase genes, suggesting the potential for producing novel ansamycin-like metabolites. Region4 (~41 kb) displayed 7% similarity to the butirosin A/B aminoglycoside biosynthetic gene cluster (BGC0000693), and contains key genes (btrC, btrD) involved in 2-deoxystreptamine synthesis, indicating possible production of novel aminoglycoside derivatives.\u003c/p\u003e\n\u003cp\u003eRegions 5, 9, and 10 (approximately 17 kb, 22 kb, and 41 kb, respectively) showed no significant homology to any known BGCs following comprehensive database searches, suggesting they may represent novel secondary metabolite pathways specific to \u003cem\u003eB. velezensis\u0026nbsp;\u003c/em\u003eR125. The structures and biological functions of their encoded metabolites warrant further investigation through heterologous expression and metabolomic profiling.\u003c/p\u003e\n\u003cp\u003eAdditionally, analysis based on compound class prediction results revealed that among all predicted compound types, Non-Ribosomal Peptides (NRPs) accounted for the highest proportion. Specifically, among the 13 predicted secondary metabolite gene clusters, five gene clusters were found to encode NRP compounds. Polyketide compounds ranked next, corresponding to four gene clusters, while the remaining compound categories corresponded to a smaller number of gene clusters. Given that NRP-related gene clusters were the most abundant, and considering that lipopeptide compounds predominantly belong to the NRP family, subsequent experiments focused on the extraction and identification of lipopeptide components in the R125 strain, based on these experimental findings.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e Prediction of secondary metabolites of \u003cem\u003eB. velezensis\u0026nbsp;\u003c/em\u003eR125 genome.\u003c/p\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"593\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eRegion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eType\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eMost similar known cluster and similarity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eCategory\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eFrom\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eEnd\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eRegion1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eNRPS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003esurfactin (78%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eNRP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e298,949\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e363,758\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eRegion2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eThiopeptide\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eKijanimicin(4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ePolyketide\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e579,494\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e608,327\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eRegion3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eRRE-containing\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eplantazolicin (91%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eRiPP\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e691,824\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e715,001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eRegion4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ePKS-like\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ebutirosin A / butirosin B(7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSaccharide\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e926,732\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e967,976\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eRegion5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eTerpene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1,052,869\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1,070,201\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eRegion6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eTransAT-PKS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003emacrolactin H (100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ePolyketide\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1,368,920\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1,456,723\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eRegion7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eTransAT-PKS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ebacillaene (100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ePolyketide + NRP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1,676,229\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1,776,938\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eRegion8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eNRPS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003efengycin (100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eNRP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1,843,599\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1,977,763\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eRegion9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eTerpene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2,043,517\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2,065,400\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eRegion10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eT3pks\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2,138,235\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2,179,335\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eRegion11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eTransAT-PKS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003edifficidin (100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ePolyketide + NRP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2,349,619\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2,443,408\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eRegion12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eNRPS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ebacillibactin (100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eNRP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3,073,123\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3,124,915\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eRegion13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eOther\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ebacilysin (100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eOther\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3,642,167\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3,683,585\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eIdentification of Lipopeptide Components in Strain R125\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLiquid chromatography-mass spectrometry (LC-MS) was employed for the qualitative and relative quantitative analysis of the extracted R125 lipopeptide fraction, with the identification results presented in Figure 6. Research on lipopeptide compounds has been in-depth, and the component identification system for these compounds is relatively well-established. Based on the accurate molecular weights obtained by mass spectrometry detection and combined with the reported mass spectral characteristic data of lipopeptide compounds in published literature, preliminary qualitative identification of the target fractions can be achieved.\u003c/p\u003e\n\u003cp\u003eAs shown in Figure 6A, the R125 lipopeptide fraction mainly comprises three types of typical lipopeptide compounds, namely Iturin (Figures 6C, D), Fengycin (Figures 6E, F), and Surfactin (Figures 6G, H). Further results of the relative quantitative analysis (Figures 6A, B) indicated significant differences in the contents of the three lipopeptide compounds: Iturin exhibited the highest relative content, accounting for 30.31%; Fengycin followed with a relative content of 22.42%; while Surfactin had the lowest relative content at 14.8%.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study reports the isolation and identification of strain \u003cem\u003eBacillus velezensis\u003c/em\u003e R125 GDMCC 67536 from Guangxi traditional fermented sour porridge, a strain exhibiting broad-spectrum antimicrobial activity. Through whole-genome sequencing and functional annotation, we systematically elucidate the molecular basis underlying its antimicrobial potential. Core findings demonstrate that strain R125 exhibits significant inhibitory activity against \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC 25923, \u003cem\u003eEscherichia coli\u003c/em\u003e ATCC 25922, and \u003cem\u003eFusarium oxysporum\u003c/em\u003e f. sp. cubense race 1 201. Genomic analysis reveals a rich repertoire of biosynthetic gene clusters (BGCs) for secondary metabolites and carbohydrate-active enzymes (CAZymes), which together form a functional network enabling cross-kingdom antimicrobial activity. This discovery not only provides a new paradigm for mining microbial resources from traditional fermented foods, but also offers a candidate strain and genomic foundation for developing green antimicrobial agents and biopesticides.\u003c/p\u003e \u003cp\u003eTraditional fermented foods serve as natural microbial \u0026ldquo;gene pools,\u0026rdquo; wherein their unique spontaneous fermentation environments\u0026mdash;such as the acidic conditions, high osmotic pressure, and complex nutritional matrix of sour porridge\u0026mdash;provide inherent advantages for the screening of functional strains [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Previous research has largely focused on common fermentative microorganisms such as lactic acid bacteria and yeasts, with less attention paid to bacilli, which possess both stress resistance and metabolic diversity [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In this study, strain R125 emerged as the only isolate, out of 24 screened, capable of simultaneously inhibiting both bacteria and fungi. The fermentation supernatant produced distinct zones of inhibition without diffusible artifacts, indicating that the antimicrobial products are stable and target-specific. These results confirm that traditional fermented foods harbor underexplored functional microbial resources and underscore the efficacy of targeted screening from specific fermented substrates\u0026mdash;the long-term fermentation process of sour porridge may drive the enrichment and evolution of antimicrobial strains through microbial synergy and competition.\u003c/p\u003e \u003cp\u003eWhole-genome analysis provided key insights into the antimicrobial mechanisms of strain R125. First, prediction of secondary metabolite BGCs revealed that strain R125 genome contains 13 complete BGCs, seven of which show 100% sequence homology or high similarity to known clusters for antimicrobial compounds such as surfactin, fengycin, bacilysin, and macrobrevin H. Surfactin, a well-characterized lipopeptide antimicrobial, acts by disrupting bacterial membrane integrity [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]; fengycin exhibits high specificity against fungi [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]; and bacilysin and difficidin further extend the inhibitory spectrum to Gram-positive and Gram-negative bacteria [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The completeness and diversity of these BGCs explain the ability of strain R125 to inhibit diverse pathogens across kingdoms; their synergistic interactions may constitute a multi-target antimicrobial network, thereby reducing the risk of pathogen resistance development. Second, functional annotation of CAZymes uncovered an additional antimicrobial mechanism: strain R125 encodes 51 CAZyme genes spanning six families, including glycoside hydrolases (GHs) and glycosyl transferases (GTs). Among these, GH5, GH9, and CBM6 families are implicated in cellulose degradation, while CBM73 and AA10 target chitin and peptidoglycan\u0026mdash;key structural components of fungal and bacterial cell walls, respectively. Degradation of these components directly compromises pathogen structural integrity [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. This dual mechanism\u0026mdash;combining secondary metabolite inhibition and cell wall degradation\u0026mdash;underpins the broad-spectrum antimicrobial activity of strain R125 and distinguishes it from strains relying on a single mode of action, enhancing its stability and efficacy in practical applications. Notably, the GC content of strain R125 plasmid (52.88%) is significantly higher than that of the chromosome (46.56%), suggesting possible acquisition via horizontal gene transfer. The plasmid also carries tRNA and rRNA genes that may participate in regulating the expression of antimicrobial genes, offering a starting point for future studies on adaptive evolution.\u003c/p\u003e \u003cp\u003eLipopeptide Characteristics and Species Consistency: Iturin, Fengycin, and Surfactin are characteristic lipopeptide secondary metabolites of the \u003cem\u003eBacillus\u003c/em\u003e genus, particularly within the \u003cem\u003eBacillus velezensis\u003c/em\u003e species complex. These compounds are synthesized via the nonribosomal peptide synthetase (NRPS) pathway and serve as key chemical markers distinguishing \u003cem\u003eB. velezensis\u003c/em\u003e from other \u003cem\u003eBacillus\u003c/em\u003e species (e.g., \u003cem\u003eB. subtilis\u003c/em\u003e primarily produces Surfactin, while \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e primarily produces Iturin). In this study, HPLC-MS analysis detected that strain R125 produces Iturin (30.31%), Fengycin (22.42%), and Surfactin (14.8%). This finding is highly consistent with the NRPS gene clusters predicted by AntiSMASH (Region 1 surfactin cluster, Region 8 fengycin cluster), confirming the typical metabolic profile of this strain as \u003cem\u003eB. velezensis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eAgainst the backdrop of a growing global antimicrobial resistance crisis, the overuse of chemical antimicrobials has led to serious ecological and public health concerns [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. As a naturally sourced probiotic, strain R125 produces fermentation metabolites that are green, efficient, and low in toxicity. It has potential to replace chemical preservatives in food preservation, reducing contamination and safety risks. In agriculture, its strong inhibitory activity against FoC1\u0026mdash;evidenced by suppression of mycelial growth in inhibition zones\u0026mdash;offers a new biocontrol strategy for soil-borne diseases such as banana Fusarium wilt, aligning with the goals of sustainable agriculture. Moreover, the abundance of CAZymes in the R125 strain genome\u0026mdash;such as cellulases and xylanases\u0026mdash;suggests potential applications in biomass conversion and environmental degradation of organic waste, thereby expanding the scope of functional microbial utilization.\u003c/p\u003e \u003cp\u003eFrom a scientific standpoint, This study performs whole-genome and metabolomic analysis of \u003cem\u003eB. velezensis\u003c/em\u003e R125 isolated from traditional fermented sour porridge, and identifies several unique biological characteristics of this strain adapted to fermented food niches, confirming a molecular mechanism of antimicrobial activity mediated by the synergy between secondary metabolite synthesis and cell wall-degrading enzymes. This enriches our understanding of the regulatory network controlling the antimicrobial spectrum in \u003cem\u003eB. velezensis\u003c/em\u003e. Additionally, the identification of three unannotated novel BGCs (Region5, 9, and 10) provides candidate targets for the discovery of novel antimicrobial compounds and genetic resources for microbial metabolic engineering.\u003c/p\u003e \u003cp\u003eGenomic prediction identified 13 complete secondary metabolite BGCs in strain R125, and LC-MS analysis directly confirmed the production of three key lipopeptides\u0026mdash;Iturin, Fengycin, and Surfactin\u0026mdash;consistent with the functional annotation of NRPS-type BGCs. Specifically, the Fengycin BGC (Region8) exhibited 100% sequence similarity to the reference cluster (BGC0001095), and LC-MS detected Fengycin with a relative content of 22.42%; the Surfactin BGC (Region1) showed 78% similarity to BGC0000433, and Surfactin accounted for 14.8% of the detected lipopeptides; Iturin, a well-known antifungal and antibacterial lipopeptide, was the most abundant (30.31%), though its corresponding BGC was not explicitly highlighted in genomic prediction\u0026mdash;likely nested within the unclassified or NRPS-type clusters [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. This consistency between genomic potential and metabolic output strongly validates the functionality of strain R125\u0026rsquo;s BGCs, eliminating the uncertainty of \u0026ldquo;silent clusters\u0026rdquo; often associated with genomic prediction alone.\u003c/p\u003e \u003cp\u003eEach lipopeptide contributes uniquely to the broad-spectrum antimicrobial activity: Iturin disrupts pathogen cell membranes by interacting with phospholipids and sterols, exerting potent activity against both bacteria and fungi [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]; Fengycin specifically targets fungal cell membranes by binding to ergosterol, a component unique to fungi, which explains strain R125\u0026rsquo;s strong inhibitory effect on FoC1 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]; Surfactin, with its surfactant properties, permeabilizes bacterial membranes and enhances the diffusion of other antimicrobial compounds, synergizing with Iturin and Fengycin to broaden the inhibitory spectrum [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The relative content distribution\u0026mdash;Iturin\u0026thinsp;\u0026gt;\u0026thinsp;Fengycin\u0026thinsp;\u0026gt;\u0026thinsp;Surfactin\u0026mdash;correlates with strain R125\u0026rsquo;s comprehensive antimicrobial profile: the high abundance of Iturin provides a foundational broad-spectrum effect, while Fengycin reinforces antifungal specificity, and Surfactin optimizes overall efficacy through synergy. This lipopeptide cocktail effect reduces the risk of pathogen resistance compared to single-component antimicrobial agents.\u003c/p\u003e \u003cp\u003eDespite these advances, several limitations remain. First, the identification of secondary metabolites is based solely on genomic prediction; structural validation through techniques such as mass spectrometry and nuclear magnetic resonance, as well as isolation and confirmation of key bioactive compounds via metabolomics and purification experiments, is still required. Second, the in vitro antimicrobial assays do not simulate real-world conditions, such as food matrices or soil microenvironments; the stability and persistence of strain R125\u0026rsquo;s antimicrobial activity in complex settings need further validation. Third, the synergistic relationship between CAZymes and BGCs has not been experimentally confirmed through gene knockout or heterologous expression studies, and the underlying regulatory pathways warrant further elucidation.\u003c/p\u003e \u003cp\u003eUnlike previously reported \u003cem\u003eB. velezensis\u003c/em\u003e strains, strain R125 exhibits a unique genomic feature of high abundance of GH5 family cellulase genes, which is a specific adaptive marker for plant-derived fermentation substrates and has not been documented in existing studies. In addition, three novel BGCs (Region5, 9, 10) with no known homologous sequences were identified in strain R125, which are potential targets for the discovery of novel antimicrobial compounds. Furthermore, this study first clarifies the quantitative synergy pattern of lipopeptides in strain R125: the high proportion of Iturin (30.31%) provides a broad-spectrum antibacterial and antifungal foundation, Fengycin (22.42%) specifically enhances the inhibition of phytopathogenic fungi (FoC1), and Surfactin (14.8%) improves the membrane permeability of pathogens and the diffusion of other lipopeptides, forming a unique lipopeptide cocktail effect that reduces the risk of pathogen resistance.\u003c/p\u003e \u003cp\u003eIn summary, strain \u003cem\u003eBacillus velezensis\u003c/em\u003e R125 GDMCC 67536, isolated from traditional fermented sour porridge, exhibits broad-spectrum antimicrobial activity mediated by the synergy of three key lipopeptides (Iturin, Fengycin, Surfactin) and cell wall-degrading CAZymes. Genomic prediction combined with LC-MS verification confirms the functionality of its secondary metabolite BGCs, while the relative content distribution of lipopeptides explains its comprehensive inhibitory profile against bacteria and fungi. Strain R125 holds great promise as a natural antimicrobial agent, biocontrol agent, and probiotic, with applications spanning food preservation, agriculture, and pharmaceuticals. This study identifies the unique genomic and metabolomic characteristics of strain \u003cem\u003eB. velezensis\u003c/em\u003e R125 adapted to fermented food niches, and its specific lipopeptide synergy pattern and novel BGCs provide new experimental data and candidate targets for the research and development of natural antimicrobial agents; the GH5 family cellulase gene enrichment characteristic also provides a new perspective for the study of microbial adaptation to fermentation substrates. By integrating multi-omics technologies, synthetic biology, and applied microbiology, strain R125\u0026rsquo;s full potential can be harnessed to address global challenges such as antimicrobial resistance, food safety, and sustainable agriculture.\u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eConflict of interest\u003c/strong\u003e \u003cp\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eEthical Approval\u003c/strong\u003e \u003cp\u003eNot required (This research did not involve humans or animals).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for Publication\u003c/strong\u003e \u003cp\u003eAll authors have read and approved this version of the article and due care has been taken to ensure the integrity of the work.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was funded by financial support from the National Natural Science Foundation of China (\u003cb\u003eGrant No\u003c/b\u003e. 32360244) and the Science and Technology Major Project of Guangxi (\u003cb\u003eGuike\u003c/b\u003e AA24206044).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eFuTian Yu designed, performed the experiment and wrote the original draft. DengFeng Yang was involved in conceptualization. All authors reviewed and edited the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAn F, Sun H, Wu J, Zhao C, Li T, Huang H, Fang Q, Mu E, Wu R (2021) Investigating the core microbiota and its influencing factors in traditional Chinese pickles. Food Res Int. 147: 110543\u003c/li\u003e\n\u003cli\u003eBhattacharjee MJ, Bala A, Khan MR, Mukherjee AK (2025) Functional impact of bioactive peptides derived from fermented foods on diverse human populations. Food Chemistry. 492(Pt 1): 145416\u003c/li\u003e\n\u003cli\u003eBielen A, Babic I, Vuk Surjan M, Kazazic S, Simatovic A, Lajtner J, Udikovic-Kolic N, Mesic Z, Hudina S (2024) Comparison of MALDI-TOF mass spectrometry and 16S rDNA sequencing for identification of environmental bacteria: a case study of cave mussel-associated culturable microorganisms. Environ Sci Pollut Res Int. 31(14): 21752-21764\u003c/li\u003e\n\u003cli\u003eChen HJ, Liu Y, Zhong YS, Li MZ, Lai JJ, Luo YY, Huang SL, Liu SQ, Yu GH, Sun YH, Shao MW (2025) Modulating Surfactin Biosynthesis in \u003cem\u003eBacillus subtilis\u003c/em\u003e R31 Enhances Behavioural Traits and Biocontrol Efficacy Against Banana \u003cem\u003eFusarium Wilt\u003c/em\u003e. Microb Biotechnol. 18(11): e70261\u003c/li\u003e\n\u003cli\u003eCho YT, Ting HM, Wang BW, Tsai YC, Wang HY, Yang YL, Ariyawansa HA (2025) Integrating genomics and targeted metabolite profiling to elucidate disease-suppression mechanisms of \u003cem\u003eBacillus velezensis\u003c/em\u003e GFB08. Curr Res Microb Sci. 9: 100503\u003c/li\u003e\n\u003cli\u003eChoi H, Son S (2025) Emerging nano-vaccine technologies to overcome anti-microbial resistance. J Control Release. 386: 114076\u003c/li\u003e\n\u003cli\u003eFelczak MM, Bowers RM, Woyke T, Teravest MA (2021) Zymomonas diversity and potential for biofuel production. Biotechnol Biofuels. 14(1): 112\u003c/li\u003e\n\u003cli\u003eGilliard G, Demortier T, Boubsi F, Jijakli MH, Ongena M, De Clerck C, Deleu M (2024) Deciphering the distinct biocontrol activities of lipopeptides fengycin and surfactin through their differential impact on lipid membranes. Colloids Surf B Biointerfaces. 239: 113933\u003c/li\u003e\n\u003cli\u003eIllikoud N, Do Carmo FLR, Daniel N, Jan G, Gagnaire V (2023) Development of innovative fermented products by exploiting the diversity of immunomodulatory properties and fermentative activity of lactic and propionic acid bacteria. Food Res Int. 166: 112557\u003c/li\u003e\n\u003cli\u003eIsil C, Koydemir HC, Eryilmaz M, De Haan K, Pillar N, Mentesoglu K, Unal AF, Rivenson Y, Chandrasekaran S, Garner OB, Ozcan A (2025) Virtual Gram staining of label-free bacteria using dark-field microscopy and deep learning. Sci Adv. 11(2): eads2757\u003c/li\u003e\n\u003cli\u003eJumpathong J, Uawisetwathana U, Intha N, Phakdee N, Pathaichindachote W, Rod-In W, Sirijan M (2026) Antagonistic activity and application of lipopeptide-producing Bacillus velezensis as biocontrol agent against rice bacterial leaf blight in vivo. Physiological and Molecular Plant Pathology. 142: \u003c/li\u003e\n\u003cli\u003eKayiran SD, Bolgen UMG, Akgul E, Kadiroglu P, Cengiz N, Cevikelli T, Onan D, Ozkan EE, Bozdogan G, Boga M, Ozogul Y, Esatbeyoglu T, Ozogul F (2025) Chemical composition, characterization, and antimicrobial properties of Thymbra spicata essential oil-based nanoemulsions and its application on curd cheese. Front Nutr. 12: 1615832\u003c/li\u003e\n\u003cli\u003eLee YR, Lee HW, Park KM, Lee NK, Paik HD (2025) Anti-inflammatory effects of\u003cem\u003e Lactiplantibacillus plantarum\u003c/em\u003e strains through MAPK, NF-kappaB, and AP-1 signaling pathways and its application in soy milk. Food Res Int. 208: 116216\u003c/li\u003e\n\u003cli\u003eLi Z, Jiang J, Sun K, Ye S (2025) Unveiling the Potential of Endophytic\u003cem\u003e Bacillus amyloliquefaciens\u003c/em\u003e LJ1 from Nanguo Pear: A Genomic and Functional Study for Biocontrol of Post-Harvest Rot. Foods. 14(17): 3020\u003c/li\u003e\n\u003cli\u003eLiang SM, Li MX, Gong X, Luo Z, Yang JM (2026) Determination and pharmacokinetic study of thyroid hormone receptor-\u0026beta; agonist resmetirom in beagle dogs using HPLC-MS/MS method. Journal of Pharmaceutical and Biomedical Analysis. 269: \u003c/li\u003e\n\u003cli\u003eLiu Y, Teng K, Huang F, Xia T, Zhang J, Wang T, Zhong J (2022) High-throughput discovery of novel lanthipeptides and producers by metagenomic mining of isolates population (MMIP) from Chinese spicy cabbage. Food Res Int. 154: 110991\u003c/li\u003e\n\u003cli\u003eNguyen HD, Vu NH, Do HDK, Vu MT (2025) Comparative chloroplast genomic analysis of\u003cem\u003e Pithecellobium dulce\u003c/em\u003e (Roxb.) Benth 1844 and related species within Caesalpinioideae. Genetica. 153(1): 19\u003c/li\u003e\n\u003cli\u003eOufensou S, Ul-Hassan Z, Badr K, Balmas V, Jaoua S, Migheli Q (2025) Guns \u0026apos;N Roses: Fungal Volatile Warfare in Postharvest Disease Control. Annu Rev Phytopathol. 63(1): 477-499\u003c/li\u003e\n\u003cli\u003eQiao Y, Zhang K, Zhang Z, Zhang C, Sun Y, Feng Z (2022) Fermented soybean foods: A review of their functional components, mechanism of action and factors influencing their health benefits. Food Res Int. 158: 111575\u003c/li\u003e\n\u003cli\u003eSalamaga B, Kong L, Pasquina-Lemonche L, Lafage L, Von Und Zur Muhlen M, Gibson JF, Grybchuk D, Tooke AK, Panchal V, Culp EJ, Tatham E, O\u0026apos;kane ME, Catley TE, Renshaw SA, Wright GD, Plevka P, Bullough PA, Han A, Hobbs JK, Foster SJ (2021) Demonstration of the role of cell wall homeostasis in \u003cem\u003eStaphylococcus aureus\u003c/em\u003e growth and the action of bactericidal antibiotics. Proc Natl Acad Sci U S A. 118(44): e2106022118\u003c/li\u003e\n\u003cli\u003eSnyder AB, Martin N, Wiedmann M (2024) Microbial food spoilage: impact, causative agents and control strategies. Nat Rev Microbiol. 22(9): 528-542\u003c/li\u003e\n\u003cli\u003eVilla-Rodriguez E, Moreno-Ulloa A, Castro-Longoria E, Parra-Cota FI, De Los Santos-Villalobos S (2021) Integrated omics approaches for deciphering antifungal metabolites produced by a novel \u003cem\u003eBacillus species\u003c/em\u003e, \u003cem\u003eB. cabrialesii\u003c/em\u003e TE3(T), against the spot blotch disease of wheat (\u003cem\u003eTriticum turgidum L. subsp. durum\u003c/em\u003e). Microbiological Research. 251: 126826\u003c/li\u003e\n\u003cli\u003eWang R, Wu J, Jiang N, Lin H, An F, Wu C, Yue X, Shi H, Wu R (2023) Recent developments in horizontal gene transfer with the adaptive innovation of fermented foods. Crit Rev Food Sci Nutr. 63(5): 569-584\u003c/li\u003e\n\u003cli\u003eWang Z, Xu T, Wang J, Sun G, Chen Q, Sun H, Shi B (2025) Probiotic potential of \u003cem\u003eBacillus velezensis\u003c/em\u003e MZ-09: Assessing characteristics and safety through in vitro and in vivo analyses. Microbiological Research. 301: 128321\u003c/li\u003e\n\u003cli\u003eWu Y, Li A, Cheng L, Chen Q, Li J, Xu Y, Huo D (2022) Deep Shotgun metagenomic and 16S rRNA analysis revealed the microbial diversity of lactic acid bacteria in traditional fermented foods of eastern Hainan, China. Food Funct. 13(24): 12938-12952\u003c/li\u003e\n\u003cli\u003eYe C, Zhao W, Liu D, Yang R, Cui Z, Zou D, Li D, Wei X, Xiong H, Niu C (2024) Screening, identification, engineering, and characterization of \u003cem\u003eBacillus\u003c/em\u003e-derived alpha-amylase for effective tobacco starch degradation. Int J Biol Macromol. 282(Pt 6): 137364\u003c/li\u003e\n\u003cli\u003eYu F, Shen Y, Qin Y, Pang Y, Fan H, Peng J, Pei X, Liu X (2022) Isolation and purification of antibacterial lipopeptides from \u003cem\u003eBacillus velezensis\u003c/em\u003e YA215 isolated from sea mangroves. Frontiers in Nutrition. 9: 1064764\u003c/li\u003e\n\u003cli\u003eYu FT, Shen YY, Chen SL, Fan HL, Pang YY, Liu MY, Peng JJ, Pei XD, Liu XL (2024) Analysis of the Genomic Sequences and Metabolites of \u003cem\u003eBacillus velezensis\u003c/em\u003e YA215. Biochemical Genetics. 62(6): 5073-5091\u003c/li\u003e\n\u003cli\u003eYu FT, Shen YY, Pang YY, Fan HL, Liu MY, Liu XL (2024) Effects of branched-chain amino acids on surfactin structure and antibacterial activity in \u003cem\u003eBacillus velezensis\u003c/em\u003e YA215. World Journal of Microbiology \u0026amp; Biotechnology. 40(9): 281\u003c/li\u003e\n\u003cli\u003eZhang P, Hu M, Meng W, Wang J, Yuan Z, Wang F, Li S (2025) Effect of \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e MX14 and \u003cem\u003ePichia membranifaciens\u003c/em\u003e PY33 co-fermentation on quality characteristics of red sour soup. Food Chemistry. 492(Pt 2): 145473\u003c/li\u003e\n\u003cli\u003eZhang X, Li S, Liu H, Wongta A, Xu Z, Zhou K, Hongsibsong S (2025) Isolation and Characterizations of Histamine- and Tyramine-Producing Strains Isolated from Fermented Soybean Food: Soy Sauce and Soybean Paste. Foods. 14(14): 2407\u003c/li\u003e\n\u003cli\u003eZhao J, Pachiadaki M, Conrad RE, Hatt JK, Bristow LA, Rodriguez RL, Rossello-Mora R, Stewart FJ, Konstantinidis KT (2025) Promiscuous and genome-wide recombination underlies the sequence-discrete species of the SAR11 lineage in the deep ocean. Isme j. 19(1): wraf072\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":false,"email":"","identity":"current-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Current Microbiology","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":false,"inReviewRevisionsEnabled":false},"keywords":"Bacillus velezensis, Sour porridge, Antimicrobial activity, whole-genome sequencing, Lipopeptides","lastPublishedDoi":"10.21203/rs.3.rs-8646919/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8646919/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTraditional fermented foods harbor functional microorganisms with antimicrobial potential. This study isolated strain \u003cem\u003eBacillus velezensis\u003c/em\u003e R125 GDMCC 67536 from sour porridge in Guangxi, China, which uniquely exhibited broad-spectrum activity against \u003cem\u003eEscherichia coli\u003c/em\u003e ATCC 25922, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC 25923, and \u003cem\u003eFusarium oxysporum\u003c/em\u003e f. sp. cubense race 1 201 among 24 isolates. The strain was identified through phenotypic analysis, 16S rRNA gene sequencing (99.59% identity to the type strain), and whole-genome OrthoANI comparison (97.78% similarity to strain \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e subsp. plantarum FZB42\u003csup\u003eT\u003c/sup\u003e, and 99.59% identity to the type strain \u003cem\u003eB. velezensis\u003c/em\u003e NRRL B-41580\u003csup\u003eT\u003c/sup\u003e). Hybrid sequencing (Illumina HiSeq and PacBio) yielded a complete genome consisting of a circular chromosome (3,970,649 bp, 46.56% GC content) and a circular plasmid (5,981 bp, 52.88% GC content), harboring 3,808 protein-coding genes. Functional annotation using GO, COG, and KEGG databases revealed considerable metabolic versatility, including 51 genes encoding carbohydrate-active enzymes (CAZymes). AntiSMASH analysis predicted 13 biosynthetic gene clusters (BGCs) for secondary metabolites, such as surfactin, fengycin, bacilysin, and macrolactin H. Comparative genomic analysis with strain \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e subsp. plantarum FZB42\u003csup\u003eT\u003c/sup\u003e, strain \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e DSM 7, and strain \u003cem\u003eB. subtilis\u003c/em\u003e 168\u003csup\u003eT\u003c/sup\u003e indicated that eight BGCs are conserved within the core genome, while five BGCs with low similarity to known clusters appear to be strain-specific, suggesting adaptive divergence. Notably, strain R125 possesses a significantly higher abundance of GH5 family cellulase genes\u0026mdash;a genomic feature potentially reflective of adaptation to a plant-derived fermentation substrate. High-performance liquid chromatography-mass spectrometry (HPLC-MS) confirmed the production of three principal lipopeptides: iturin (30.31%), fengycin (22.42%), and surfactin (14.8%). This study elucidates the genomic and metabolomic basis of antimicrobial activity in strain \u003cem\u003eB. velezensis\u003c/em\u003e R125 GDMCC 67536, and identifies three strain-specific novel BGCs and a unique GH5 cellulase gene enrichment characteristic associated with fermentation substrate adaptation; the quantitative synergy pattern of its lipopeptides (Iturin\u0026thinsp;\u0026gt;\u0026thinsp;Fengycin\u0026thinsp;\u0026gt;\u0026thinsp;Surfactin) is first reported to mediate broad-spectrum antimicrobial activity, providing experimental basis for the directional utilization of \u003cem\u003eB. velezensis\u003c/em\u003e in food preservation and biocontrol.\u003c/p\u003e","manuscriptTitle":"Functional Microbes from Traditional Fermented Food: Genomics, Lipopeptide Profiling and Antimicrobial Potential of Bacillus velezensis R125","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-31 10:57:14","doi":"10.21203/rs.3.rs-8646919/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-22T12:03:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-20T20:26:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-20T15:35:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"Current Microbiology","date":"2026-01-20T08:26:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":false,"email":"","identity":"current-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Current Microbiology","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":false,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f4f0678a-21c7-4b7e-ae14-b1820e45cdf8","owner":[],"postedDate":"March 31st, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-31T10:57:14+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-31 10:57:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8646919","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8646919","identity":"rs-8646919","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}