Screening and Whole-Genome Analysis of a Sheep-Derived Lactic Acid Bacteria with Antibacterial Properties

Screening and Whole-Genome Analysis of a Sheep-Derived Lactic Acid Bacteria with Antibacterial Properties | 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 Screening and Whole-Genome Analysis of a Sheep-Derived Lactic Acid Bacteria with Antibacterial Properties Lingbai Yao, Yao Huang, Linchong Zhang, Yusheng Wang, Jun Jia, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7778181/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Pediococcus pentosaceus is an important lactic acid bacterium widely utilised in fermented foods and animal probiotic preparations. However, strains derived from sheep remain relatively limited in systematic screening and genomic characterisation studies. This research isolated lactic acid bacteria from healthy Chahar sheep and screened strains with application potential through phenotypic evaluation, probiotic property analysis, and whole-genome sequencing. Results indicate that strain SSF2 exhibits rapid acid production capacity, strong bile salt tolerance, survival in simulated gastrointestinal environments, and significant antibacterial activity against major pathogens including Escherichia coli , Salmonella , Staphylococcus aureus , and Vibrio parahaemolyticus . SSF2 exhibits no haemolytic activity and maintains sensitivity to commonly used clinical antibiotics. Genome sequencing revealed SSF2 possesses a 1.85 Mb circular chromosome and two plasmids, encoding functional genes involved in carbohydrate metabolism, amino acid metabolism, stress tolerance, and potential bacteriocin biosynthesis. No virulence factors or antibiotic resistance genes were detected. Overall, both its phenotypic and genomic characteristics indicate that P. pentosaceus SSF2 is a safe and promising probiotic candidate strain with potential applications in ruminant feed additives and food biotechnology. Lactic acid bacteria Pediococcus pentosaceus probiotic functionality whole genome antibacterial activity Figures Figure 1 Figure 2 Figure 3 Figure 4 1 Introduction Probiotics area class of active microorganisms beneficial to the health of the host (humans or animals) [ 1 ] and have been proven to provide health benefits to the host when consumed in sufficient amounts [ 2 ] . They are widely present in the host's oral cavity, skin, gastrointestinal tract and reproductive tract [ 3 – 6 ] . To date, probiotics have become the fastest-growing animal feed additive globally [ 7 ] . Lactic acid bacteria (LAB) are an important component of probiotics [ 8 ] , known for promoting nutrient absorption, antibacterial and anti-infection properties, anti-tumor effects, regulation of gut microbiota, immune modulation, metabolism, and bioremediation [ 9 – 11 ] . LAB has become an ideal choice for commercial development and are widely used in fields such as food, medicine, agriculture, and animal husbandry [ 12 – 14 ] . P. pentosaceus , a Gram-positive bacterium belonging to the family Streptococcaceae and the genus Pediococcus , is a type of lactic acid bacterium [ 15 ] . P. pentosaceus is widely distributed and has been isolated from fermented foods, aquatic products, animal products, and plant products [ 16 ] . Due to its bacteriocins' inhibitory effects on various pathogens and its good thermal stability, P. pentosaceus has attracted significant attention from researchers [ 17 ] . With the increasing awareness of safety among people, the level of animal health farming and welfare farming has been continuously improving, making the safety of veterinary drugs and feed additives a top priority in livestock production. Currently, the global probiotics industry is developing rapidly, but it lacks relevant standards, resulting in inconsistent product quality [ 18 ] . Whole genome sequencing technology provides comprehensive genetic information for the study of LAB, enabling the revelation of metabolic characteristics, potential probiotic functions, and safety of strains [ 19 ] . Through genome analysis, it is possible not only to explore the ecological adaptation mechanisms of LAB in specific environments but also to identify functional genes with application potential, providing a theoretical basis for industrial production and the development of functional foods. Moreover, genomic data can effectively assess the safety of strains, such as screening for antibiotic resistance genes, virulence factors, and the risk of horizontal gene transfer, thereby ensuring their safety in food industry applications [ 20 , 21 ] . As a unique sheep breed specific to Ulanqab City, Chahar sheep have attracted much attention due to their distinctive genomic resources and their significance in traditional nomadic culture. However, the whole genome analysis of LAB derived from Chahar sheep remains quite limited. Existing research mainly focuses on the diversity of microbial communities and preliminary functional exploration, lacking systematic analysis [ 22 ] . In this study, LAB was isolated from the feces of Chahar sheep, and astrain with good acid tolerance, bile salt tolerance, heat resistance, and safety performance was obtained. Whole-genome sequencing and analysis were conducted to explore its probiotic functions, aiming to provide a candidate strain for the development and application of future microecological preparations. 2 Materials and Methods 2.1 Sample collection and screening of acid-producing strains Fresh fecal samples were collected from six healthy adult female Chahar sheep in Hangjin Banner, Ordos City, Inner Mongolia Autonomous Region, China (107°54'21" E, 39°56'56" N). A 5 g sample was weighed, suspended in 45 mL of sterile water, then serially diluted to 10⁻¹ to 10⁻⁹ g/mL. Aliquots of 200 µL of the 10⁻⁵ to 10⁻⁷ dilutions were spread evenly onto MRS agar medium containing bromocresol purple as an indicator (K₂HPO₄ 2.5 g/L, Na₂HPO₄ 2.5 g/L, peptone 2 g/L, yeast extract 0.5 g/L, agar 20 g/L, pH = 7.0). The plates were incubated at 37°C for 24 h under anaerobic conditions. Colonies showing a color change to yellow were identified as acid-producing isolates. To ensure the selected colonies were pure, repeated purification and microscopic examination were performed. The purified colonies were preserved in MRS broth supplemented with 20% glycerol and stored at -80°C (Hope Bio-technology Co., Ltd., Qingdao, China). 2. 2 Acid Production Ability Test of Strains For quantitative determination, the strain was inoculated at 2% (v/v) into 10 mL of MRS liquid medium and cultured under shaking conditions at 37°C and 150 rpm. The pH was measured at specified time points using a calibrated pH meter. All experiments were repeated three times. 2.3 Bile Salt Tolerance of Strains Bile salt tolerance of strains was assessed by inoculating 2% (v/v) overnight culture into MRS liquid medium containing 0.4% (w/v) bile salts (Oxgall). After incubating 10 mL of culture at 37°C for 36 hours, optical density at 600 nm (OD600) was measured using a microplate reader (BioTek Synergy H1). $$\:\text{S}\text{u}\text{r}\text{v}\text{i}\text{v}\text{a}\text{l}\:\text{r}\text{a}\text{t}\text{e}\:\left(\text{\%}\right)\:=\:(\text{O}\text{D}600\_\text{t}\text{r}\text{e}\text{a}\text{t}\text{m}\text{e}\text{n}\text{t}\:\text{g}\text{r}\text{o}\text{u}\text{p}\:/\:\text{O}\text{D}600\_\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}\:\text{g}\text{r}\text{o}\text{u}\text{p})\:\times\:\:100$$ 2.4 Morphological Identification of Strains The candidate strains were subjected to Gram staining and examined under a microscope. Additionally, the morphology of the strains was observed using scanning electron microscopy (SEM). 2.5 Biochemical Identification of Strains The strains were incubated at 37°C for biochemical identification. The results of the LAB physiological and biochemical identification tests were compared with the instructions provided in the LAB biochemical identification kit (Company, SHBG13) to evaluate each indicator. All indicator results were cross-referenced with Bergey’s Manual of Systematic Bacteriology and the Manual for the Systematic Identification of Common Bacteria. The tested indicators included esculin, cellobiose, maltose, mannitol, salicin, sorbitol, sucrose, raffinose, inulin, lactose, and hippuric acid. 2.6 Molecular Biological Identification of Strains Genomic DNA was extracted according to the instructions of the bacterial genome DNA extraction kit (Tiangen Biotech Co., Ltd., Beijing, China). PCR amplification was performed using universal bacterial 16S rDNA primers. The forward primer was 27F (sequence: 5’ - AGAGTTTGATCCTGGCTCA-3’), and the reverse primer was 1492R (sequence: 5’ - GGTTACCTTGTTACGACTT-3’). The PCR amplification conditions were as follows: initial denaturation at 94°C for 5 minutes; followed by 35 cycles of 94°C for 30 seconds (denaturation), 57°C for 45 seconds (annealing), and 72°C for 2 minutes (extension); with a final extension at 72°C for 10 minutes, and storage at 4°C. The PCR products were purified and sent to Beijing Liuhe BGI Technology Co., Ltd. for sequence. The sequencing results were analyzed for homology using the BLAST software available on NCBI, and a phylogenetic tree was constructed using MEGA 6.0 software. 2.7 Determination of Strain Growth Performance Following inoculation, the strains were cultured at 37°C. OD600 was measured using a microplate reader at 0, 2, 4, 6, 8, 12, 16, 24, and 38 h to construct the growth curve. 2.8 Tolerance of Strains to Simulated Intestinal Fluids The strains were inoculated into MRS broth at a 2% (v / v) inoculation rate and cultured at 37°C with shaking at 150 r/min for 24 hours. Then, 1 mL of the tested bacterial culture was added to 9 mL of artificial intestinal fluid (0.3% bile salts, 1 g/L trypsin, pH 6.8 adjusted with 4% NaOH, sterilized with a 0.22 µm filter). The cultures were incubated at 37°C with shaking at 150 r/min. The viable cell count was determined by the plate-count method, and the survival rate was calculated using the following formula: $$\:\text{S}\text{u}\text{r}\text{v}\text{i}\text{v}\text{a}\text{l}\:\text{r}\text{a}\text{t}\text{e}\:=\:(\text{c}\text{o}\text{l}\text{o}\text{n}\text{y}\:\text{c}\text{o}\text{u}\text{n}\text{t}\:\text{a}\text{t}\:\text{m}\text{e}\text{a}\text{s}\text{u}\text{r}\text{e}\text{m}\text{e}\text{n}\text{t}\:\text{t}\text{i}\text{m}\text{e}\:/\:\text{c}\text{o}\text{l}\text{o}\text{n}\text{y}\:\text{c}\text{o}\text{u}\text{n}\text{t}\:\text{a}\text{t}\:\text{t}\text{i}\text{m}\text{e}\:0)\:\times\:\:100\text{\%}$$ 2.9 Hemolytic Activity Test By streaking strains onto Columbia blood agar plates (Beikman Bioengineering Co., Ltd., Changde, China) and observing the clear zones surrounding colonies after incubation at 37°C for 24 hours, the haemolytic type (α, β, or γ) was determined. Staphylococcus aureus (CMCC 26003) was used as the control strain. 2.10 Antibiotic Sensitivity A 200 µL aliquot of the activated bacterial culture (cultured for 24 hours) was evenly spread onto MRS solid medium. The antibiotic sensitivity was tested using the Kirby-Bauer disk diffusion method (K-B method). The resistance of the strain to clindamycin (lincomycin), chloramphenicol, furazolidone (nifuroxazide), polymyxin B, vancomycin, ciprofloxacin, ofloxacin, norfloxacin, midecamycin, erythromycin, doxycycline, tetracycline, neomycin, kanamycin, gentamicin, amikacin, cefoperazone, ceftazidime (Fortum), cephaloridine, cefazolin, and cefalexin was determined. 2.11 Antibacterial Activity Test The antibacterial activity of the strain was determined using the Oxford cup agar diffusion method.The strain was inoculated into MRS medium at a 2% (v/v) inoculation rate and cultured at 37°C with shaking at 150 rpm for 24 h. The supernatant was collected and set aside. Fresh bacterial cultures (200 µL each) of Salmonella enteritidis (CMCC 50071), Escherichia coli (ATCC 25922), and Staphylococcus aureus (CMCC 26003) were evenly spread onto MH solid medium. After the bacterial suspension was fully absorbed, three Oxford cups were placed on each plate. Then, 200 µL of the strain supernatant was added to each cup. The plates were incubated at 37°C for 24 h, and the formation of inhibition zones was observed. The diameter of the inhibition zones was measured. 2.12 Genomic DNA extraction Genomic DNA was extracted from the samples using the STE method. The purity and integrity of the DNA were assessed using agarose gel electrophoresis, and the DNA was quantified using a Qubit fluorometer (Life Technologies, USA). Sequencing was performed using the Illumina PE150 system and the PacBio platform at Beijing Novogene Bioinformatics Technology Co., Ltd. 2.13 Library Construction For the Illumina platform, the NEBNext® Ultra™ DNA Library Prep Kit for Illumina (NEB, USA) was used to prepare the second-generation sequencing library. For the PacBio platform, the SMRT Bell library was constructed using the SMRTbell™ Template Kit (version 2.0). The constructed library was quantified using Qubit, and the insert fragment size was assessed using the Agilent 2100 Bioanalyzer. Sequencing was then performed on the PacBio platform. 2.14 Genome Assembly and Analysis After library quality control, genome assembly was performed using the Canu software (version 2.0, https://github.com/marbl/canu/ ) for third generation reads. Error correction of the third-generation sequencing data was conducted using Racon software (version 1.4.13). The genome assembly results were further polished using Pilon software (version 1.22) based on second-generation sequencing data to obtain the final assembly. After assembly, Open Reading Frames (ORFs) were predicted and filtered to identify potential protein-coding regions in the genome. Genome assembly quality was evaluated by observing coverage and GC content distribution. A genome circular map was generated to provide a comprehensive and intuitive visualization of genome characteristics. 2.15 Gene Prediction and Functional Annotation For gene composition prediction, GeneMarkS software (Version 4.17, http://topaz.gatech.edu/GeneMark/ ) was used to predict coding genes. RepeatMasker (Version open- 4.0.5) was used to predict interspersed repeat sequences, and TRF (Tandem Repeats Finder, Version 4.07b) [ 23 ] was used to identify tandem repeat sequences. tRNA prediction was performed using tRNAscan-SE software (Version 1.3.1), and rRNA prediction was conducted using rRNAmmer software (Version 1.2) [ 24 ] . Gene islands were predicted using IslandPath- DIOMB software (Version 0.2) [ 25 ] , and prophages were identified using PhiSpy software (Version 2.3) [ 26 ] . Finally, CRISPR sequences (Clustered Regularly Interspaced Short Palindromic Repeats) were predicted using CRISPRdigger (Version 1.0) [ 27 ] . The coding proteins of the genome were functionally annotated using the NR (Non-Redundant Protein Database), KEGG (Kyoto Encyclopedia of Genes and Genomes), GO (Gene Ontology), COG (Clusters of Orthologous Groups), and CAZy (Carbohydrate-Active Enzymes Database) databases. 2.16 Genomic Safety Assessment Bacterial insertion sequences were predicted using the ISfinder database ( https://www-is.biotoul.fr/ ). Using Blast software, the assembled sequences were compared with the ISfinder database to obtain insertion sequence prediction results. Using Diamond software, the amino acid sequences of the target species, were compared with the VFDB database, and the annotation results were obtained by combining the genes of the target species and their corresponding functional annotation information of virulence factors. Using the RGI (v5.1.0) software, the amino acid sequences of the target species were compared with the CARD database (v3.2.5), and the annotation results were obtained by combining the genes of the target species with their corresponding annotated information on the drug resistance function. 2.17 Statistical Analysis Unless otherwise stated, all experiments were repeated three times. Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism 9 software. Differences between groups were assessed using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. Differences were considered statistically significant at p < 0.05. 3 Results and Discussion 3.1 Selection criteria for sheep-derived LAB and candidate strains From fresh faecal samples of healthy Chahar sheep, 325 colonies capable of forming yellow halos (acid-producing phenotype) were preliminarily selected using MRS–bromocresol purple indicator medium (Figure S1), indicating active lactic acid fermentation capacity [ 28 ] . Subsequently, quantitative assessments of acid production capacity and bile salt tolerance were conducted (Fig. 1 ). Among these isolates, SSF2 exhibited the fastest pH reduction rate, dropping below pH 4.5 within 2 hours, and demonstrated the highest survival rate under 0.4% bile salt conditions. The primary objective of this initial screening was to identify strains capable of surviving in the gastrointestinal environment and rapidly producing acid, characteristics essential for probiotic lactic acid bacteria. Rapid pH decline typically correlates with robust glycolytic activity and lactic acid metabolism, effectively inhibiting pathogen growth during early fermentation stages [ 28 – 30 ] . Bile salt tolerance reflects membrane structural stability and survival capacity through the small intestinal environment [ 31 , 32 ] . This rigorous screening process yielded strains possessing both functional utility for process applications and intestinal survival capability, aligning with established principles for lactic acid bacteria selection. Based on comprehensive evaluation of acid production capacity, bile salt tolerance, and stability, SSF2 was ultimately designated as the candidate strain for subsequent research. 3.2 Identification of LAB The SSF2 strain formed round, white colonies with a moist and glossy surface. Microscopically, the cells appeared as oval cocci arranged singly, in pairs, or in short chains ( Figure S2a ). On rich media, the colonies were large and smooth. After Gram staining, the strain was observed under a microscope as purple spherical cells, confirming that it is a Gram-positive coccus ( Figure S2b ). The SEM results further confirmed that strain SSF2 is a coccoid bacterium ( Figure S2c ). The physiological and biochemical identification results of strain SSF2 were shown in Table S1 . The results indicate that strain SSF2 can hydrolyze various carbohydrates, and it is preliminarily identified as Lactococcus [ 29 ] . 16S rRNA gene sequencing results indicate that SSF2 shares 98.7% similarity with P. pentosaceus . Phylogenetic tree analysis further confirms its clustering within the Pediococcus clade. The combined results of morphological, biochemical and phylogenetic analyses confirm SSF2 as belonging to the species P. pentosaceus , a strain widely employed in food fermentation and renowned for its production of pediocin-like bacteriocins. Accurate taxonomic identification is a prerequisite for probiotic evaluation and regulatory approval. This finding aligns with prior research indicating that animal-derived P. pentosaceus strains typically exhibit strong acid tolerance and bacteriocin production capabilities, supporting their potential application as feed additives. (Fig. 2 ). 3.3 Biological characteristics of SSF2 In terms of growth performance, the OD value of P. pentosaceus SSF2 showed little change within the first 2 h, indicating a lag phase. Between 2–16 h, the OD value increased almost linearly, representing the logarithmic growth phase. After 16 h, the strain entered a stationary phase, and no significant decrease in OD value was observed after 16 h, indicating that P. pentosaceus SSF2 has strong stability ( Figure S3a ). This stability is particularly important for industrial applications as it helps ensure the consistency and controllability of fermented product quality. The simulated intestinal fluid experiment further confirms SSF2's adaptability under intestinal conditions, which is a critical indicator for probiotics to maintain viability and exert their functional benefits ( Figure S3b ). Therefore, SSF2 meets the core biological criteria for probiotics: possessing good activity, stability, and resistance to acid and bile salts. 3.4 Evaluation of the Probiotic Effects of SSF2 3.4.1 Safety assessment The haemolysis test results indicated that SSF2 exhibited γ-type haemolysis (no haemolysis observed), confirming its lack of cytotoxic activity (Figure S4). This finding demonstrated that SSF2 complies with the FAO/WHO safety requirements for non-pathogenic lactic acid bacteria used in feed and food [ 33 ] . Antibiotic susceptibility testing results indicate that SSF2 retains sensitivity to most clinically relevant antibiotics (Table S2). Resistance was observed only to vancomycin and polymyxin B. The aforementioned resistance profiles align with the inherent resistance characteristics commonly observed in lactic acid bacteria, with no evidence of abnormal broad-spectrum resistance. This suggests a low risk of acquired resistance [ 34 ] . The high susceptibility facilitates clearance or intervention when clinically necessary. From a regulatory perspective, this is consistent with the European Food Safety Authority's (EFSA) principle that probiotics ‘shall not carry transferable resistance genes and shall present a manageable overall risk of resistance’. Actual regulatory approval typically involves a combined assessment of genomic evaluation and phenotypic results. 3.4.2 Antimicrobial activity of SSF2 P. pentosaceus SSF2 demonstrated significant antibacterial activity against Escherichia coli , Salmonella , Staphylococcus aureus , and Listeria , with the strongest inhibitory effect observed against Vibrio parahaemolyticus , as indicated by the largest inhibition zone diameter (Table 1 ). The second strongest inhibitory effect was against Salmonella . It is noteworthy that SSF2 exhibits particularly potent inhibitory effects against V. parahaemolyticus , given that the genus Vibrio typically possesses strong tolerance and survives in marine and food-borne environments. This indicates that SSF2 generates metabolites capable of overcoming their natural resistance mechanisms. Overall, its antibacterial properties likely result from the synergistic action of multiple mechanisms, including organic acid accumulation, hydrogen peroxide production, and bacteriocin secretion [ 35 ] . SSF2 exhibits potent inhibitory effects against Gram-positive bacteria such as S. aureus and Listeria monocytogenes , consistent with the known spectrum of action for pediocin-like bacteriocins, which typically function by disrupting target cell membrane permeability. The moderate inhibitory effect on Escherichia coli aligns with the protective role of Gram-negative outer membrane structures, yet measurable inhibition zones still occur, indicating that organic acids and other metabolites produced by SSF2 exert some inhibitory influence on these bacteria. Table 1 Statistical table of bacteriostatic experimental results Strain Name Inhibition Zone Diameter Escherichia coli 13.37 ± 0.23d Staphylococcus aureus 18.67 ± 1.04b Salmonella 19.17 ± 0.57b Listeria 17.23 ± 0.23c Vibrio parahaemolyticus 21.87 ± 0.55a Note: The antibacterial ability of the strain corresponds to the size of the inhibition zone. An inhibition zone diameter of 0–10 mm indicates good antibacterial activity, while 10–20 mm indicates excellent antibacterial activity. 3.5 Whole genome analysis of SSF2 The genome of SSF 2 is 1,853,794 bp in size with a GC content of 37.29%. Subsequently, the assembly was corrected through three rounds of error correction using Racon (version 1.4.13) based on third- generation sequencing data, followed by three additional rounds of error correction using Pilon with second-generation sequencing data. The structural integrity and uniformity of sequencing depth validate the high reliability of this assembled genome (Fig. 3 ) . 3.5.1 Genome Composition and Functional Gene Distribution A total of 1,804 coding sequences (CDSs), 54 tRNAs and 6 rRNAs were identified, revealing the compact genomic structure typical of the genus Pediococcus (Figure S5, Table S3). The prediction results of genomic islands and prophages are shown in Table S4 and Figure S6 . A total of 6 genomic islands were predicted, with a total length of 74,287 bp and an average length of 24,762.33 bp. Conforming to the typical scale of GIs in prokaryotes (usually 5-200 kbp), it may have been acquired through horizontal gene transfer (HGT) [ 36 ] .Additionally, 6 prophages were predicted, with a total length of 328,305 bp and an average length of 54,717.5 bp.In the interaction between function and host, the lysogenic conversion phenotype indicates that it may carry beneficial genes (such as bacteriocins, stress proteins), enhancing host adaptability (such as the inhibition of Vibrio parahaemolyticus, Table 1 ) [ 37 ] . The NR annotation results are shown in Figure S7 , where SSF2 has the highest number of matched genes with P. pentosaceus , reaching 1,613, accounting for the vast majority of all matches. This indicates that the genome of SSF2 exhibits a high degree of similarity to the reference genome of P. pentosaceus , further confirming the taxonomic classification of the strain. Based on the KEGG annotation information, the functions of P. pentosaceus SSF 2 were analyzed (Fig. 5 ). The highly specialized metabolic network is prominent, which maybe for rapid energy acquisition and product synthesis [ 38 ] . In carbon source utilization and energy metabolism, the predicted bacteriocin (such as pediocin) synthesis gene cluster (KEGG map01053) of secondary metabolites explains its strong inhibitory activity against pathogenic bacteria, consistent with the results of the bacteriostatic activity test [ 30 ] . In nitrogen and cofactor metabolism,the synthesis of vitamin B group indicates potential probiotic functions (such as intestinal microbial interaction), but the actual synthesis capacity needs to be verified experimentally [ 39 ] . ABC transporters (such as KEGG map02010) may be related to bile salt efflux pump genes (such as bsh), which may contribute to its bile salt tolerance (Fig. 1 b) [ 40 ] . KEGG classification reveals significant enrichment of genes associated with glycolysis, pyruvate metabolism, and phosphotransferase system (PTS)-related transport, indicating this strain possesses efficient carbohydrate-to-lactic acid conversion capabilities. GO and COG functional classification analyses (Figures S8 and 9) further demonstrate that the genome contains numerous genes related to catalytic activity, membrane transport, and stress adaptation. Notably, only one gene was related to antioxidant activity.In the enrichment of core metabolic pathways, the results of COG (136 genes) and KEGG (801 genes) are consistent, supporting its efficient glycolysis and lactic acid fermentation capacity [ 41 ] . Limited synthetic genes (such as branched-chain amino acids) may restrict their growth in low-nitrogen environments, requiring exogenous supplementation [ 42 ] . In terms of environmental adaptability, the enrichment of acid tolerance-related genes (such as F₀F₁-ATPase) is shown, explaining its rapid pH decline ability (Fig. 3A) [ 43 ] . In addition, potential adhesion genes (such as surface proteins) may enhance their colonization ability on the surface of fermentation equipment and also have antibacterial effects. The carbohydrate-active enzyme prediction results (Figure S10) identified multiple CAZy family members, including glycoside hydrolases (GHs) and glycosyltransferases (GTs). These enzymes may confer SSF2's ability to utilise diverse dietary carbohydrates and contribute to its survival within the fibre-rich gastrointestinal environment of ruminants. These genomic-level predictions align with the strain's physiological performance, including rapid acid production, stable growth, and tolerance to bile salts and simulated intestinal fluids [ 44 ] . 3.5.2 Genomic Safety Assessment This strain did not carry mobile factors known to be associated with clinical risk at the IS element level, supporting its initial biosafety assessment. Comparative analysis based on the VFDB database was determined by similarity threshold (≥ 80%), resulting in no known virulence-related genes with ≥ 80% similarity being detected [ 45 ] . Resistance gene search using CRAD database resulted in no resistance genes detected [ 46 ] . The above negative results support that this strain lacks typical virulence factors with characterised resistance determinants within the range of known databases, suggesting that it has a low potential risk profile. Conclusion In summary, SSF2 possesses potent acid-producing capacity, excellent gastrointestinal tolerance, and broad-spectrum antibacterial activity, rendering it potentially valuable in both food and feed applications. Its inhibition of common intestinal pathogens supports its use as a microbial feed additive, aiding in the maintenance of animal gut health; its significant suppression of V. parahaemolyticus demonstrates its potential for application in aquatic food safety and preservation. Furthermore, SSF2's stable fermentation characteristics and antibacterial capacity render it suitable as a functional strain for food fermentation. Genomic analysis further elucidates its metabolic diversity, stress tolerance, and safety profile, providing a molecular basis for its probiotic properties. Overall, SSF2 represents a promising probiotic strain combining safety with multifunctional characteristics, suitable for applications in food, feed, and related biotechnological fields. Declarations Author Contribution The authors' contributions in this study are as follows:YLB, HY, and ZLC participated in the design of the study; WYS, JJ, Wurentana, and HXR plotted the figures and analyzed data; Swee Sen Teo, YYN, and XZS analyzed the sequencing data; ZYH drafted the manuscript; and CC and SSF criticallyreviewed the manuscript. References Mazziotta C, Tognon M, Martini F, Torreggiani E, Rotondo JC (2023) Probiotics Mechanism of Action on Immune Cells and Beneficial Effects on Human Health. Cells 12(1). 10.3390/cells12010184 Szajewska H, Berni Canani R, Domellöf M et al (2023) Probiotics for the Management of Pediatric Gastrointestinal Disorders: Position Paper of the ESPGHAN Special Interest Group on Gut Microbiota and Modifications. J Pediatr Gastroenterol Nutr 76(2):232–247. 10.1097/mpg.0000000000003633 Xiao J, Fiscella KA, Gill SR (2020) Oral microbiome: possible harbinger for children's health. 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06:41:16","extension":"xml","order_by":33,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":144379,"visible":true,"origin":"","legend":"","description":"","filename":"9323500c1619445b8326669b7c46e01b1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7778181/v1/964d913c837c1673dec135ac.xml"},{"id":98436131,"identity":"73e791d0-fcb7-43a8-9b0f-813732e16d28","added_by":"auto","created_at":"2025-12-17 16:54:58","extension":"html","order_by":34,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":161993,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7778181/v1/2a3c30d3eb112670d2c0da95.html"},{"id":98284844,"identity":"c399a7af-6bf5-4d4e-970f-fdc34fd78f88","added_by":"auto","created_at":"2025-12-16 06:41:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":126399,"visible":true,"origin":"","legend":"\u003cp\u003eScreening of candidate strains. (a) Acid Production Capacity of Bacterial Strains. (b) Bile Salt Tolerance of the Strains.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7778181/v1/130d06097753fb90116010a9.png"},{"id":98284845,"identity":"02cbf086-8a49-47a2-98be-984c191205c4","added_by":"auto","created_at":"2025-12-16 06:41:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":25919,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree of strain SSF2\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7778181/v1/d9c549b1ea634539e8de6923.png"},{"id":98433554,"identity":"2a9ec54f-1e77-405f-a3be-176924c8509e","added_by":"auto","created_at":"2025-12-17 16:50:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":450008,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 4\u003c/strong\u003e Schematic diagram of the circular genome of SSF2. (a) Schematic diagram of the circular genome structure of chromosome (Chr1). The outermost ring depicts coding genes (with sense and antisense strands represented by different colours), the middle ring indicates the positions of tRNAs and rRNAs, and the inner ring shows the distribution of GC content and GC skew. The entire chromosome spans 1,785,410 bp with a GC content of 37.23%. Gene functions are colour-coded according to COG classification. (b) Circular genome diagram of plasmid Plas1. The plasmid measures 10,618 bp with a GC content of 34.95%. (c) Circular genome map of plasmid Plas2. The plasmid length is 57,766 bp with a GC content of 40.98%. Functional genes are coloured according to COG (Clusters of Orthologous Groups) classification, with the legend on the right corresponding to each functional category.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7778181/v1/3b1c3dcbdd32a289c5928a4b.png"},{"id":98434983,"identity":"de8ddc33-6828-41d5-8284-c8db9a25a3f2","added_by":"auto","created_at":"2025-12-17 16:52:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":147813,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 5\u003c/strong\u003e Gene Function Annotation KEGG Metabolic Pathway Classification Map\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7778181/v1/9e75ad20280ea4b89bcacdb5.png"},{"id":109067722,"identity":"48a88eaa-48c0-4d3e-bc3e-77695b886ad1","added_by":"auto","created_at":"2026-05-12 10:00:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":913963,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7778181/v1/e01bca54-fc45-45ee-8d41-d3b9407ff7a1.pdf"},{"id":98284848,"identity":"94fe9a56-5a55-4396-ba26-88862d81eca7","added_by":"auto","created_at":"2025-12-16 06:41:15","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1039037,"visible":true,"origin":"","legend":"","description":"","filename":"Figure.docx","url":"https://assets-eu.researchsquare.com/files/rs-7778181/v1/306ebdcaa71f4bd787b6f62e.docx"},{"id":98434616,"identity":"96b5a435-35f7-4c36-b7be-e34c5e437a44","added_by":"auto","created_at":"2025-12-17 16:52:22","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":21123,"visible":true,"origin":"","legend":"","description":"","filename":"Table.docx","url":"https://assets-eu.researchsquare.com/files/rs-7778181/v1/10ba47c9f1229de170d93f78.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Screening and Whole-Genome Analysis of a Sheep-Derived Lactic Acid Bacteria with Antibacterial Properties","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eProbiotics area class of active microorganisms beneficial to the health of the host (humans or animals)\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e and have been proven to provide health benefits to the host when consumed in sufficient amounts\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. They are widely present in the host's oral cavity, skin, gastrointestinal tract and reproductive tract\u003csup\u003e[\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. To date, probiotics have become the fastest-growing animal feed additive globally\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Lactic acid bacteria (LAB) are an important component of probiotics\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e, known for promoting nutrient absorption, antibacterial and anti-infection properties, anti-tumor effects, regulation of gut microbiota, immune modulation, metabolism, and bioremediation\u003csup\u003e[\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. LAB has become an ideal choice for commercial development and are widely used in fields such as food, medicine, agriculture, and animal husbandry\u003csup\u003e[\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eP. pentosaceus\u003c/em\u003e, a Gram-positive bacterium belonging to the family \u003cem\u003eStreptococcaceae\u003c/em\u003e and the genus \u003cem\u003ePediococcus\u003c/em\u003e, is a type of lactic acid bacterium\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eP. pentosaceus\u003c/em\u003e is widely distributed and has been isolated from fermented foods, aquatic products, animal products, and plant products\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Due to its bacteriocins' inhibitory effects on various pathogens and its good thermal stability, \u003cem\u003eP. pentosaceus\u003c/em\u003e has attracted significant attention from researchers\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. With the increasing awareness of safety among people, the level of animal health farming and welfare farming has been continuously improving, making the safety of veterinary drugs and feed additives a top priority in livestock production. Currently, the global probiotics industry is developing rapidly, but it lacks relevant standards, resulting in inconsistent product quality\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Whole genome sequencing technology provides comprehensive genetic information for the study of LAB, enabling the revelation of metabolic characteristics, potential probiotic functions, and safety of strains\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Through genome analysis, it is possible not only to explore the ecological adaptation mechanisms of LAB in specific environments but also to identify functional genes with application potential, providing a theoretical basis for industrial production and the development of functional foods. Moreover, genomic data can effectively assess the safety of strains, such as screening for antibiotic resistance genes, virulence factors, and the risk of horizontal gene transfer, thereby ensuring their safety in food industry applications\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. As a unique sheep breed specific to Ulanqab City, Chahar sheep have attracted much attention due to their distinctive genomic resources and their significance in traditional nomadic culture. However, the whole genome analysis of LAB derived from Chahar sheep remains quite limited. Existing research mainly focuses on the diversity of microbial communities and preliminary functional exploration, lacking systematic analysis\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. In this study, LAB was isolated from the feces of Chahar sheep, and astrain with good acid tolerance, bile salt tolerance, heat resistance, and safety performance was obtained. Whole-genome sequencing and analysis were conducted to explore its probiotic functions, aiming to provide a candidate strain for the development and application of future microecological preparations.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Sample collection and screening of acid-producing strains\u003c/h2\u003e \u003cp\u003eFresh fecal samples were collected from six healthy adult female Chahar sheep in Hangjin Banner, Ordos City, Inner Mongolia Autonomous Region, China (107\u0026deg;54'21\" E, 39\u0026deg;56'56\" N). A 5 g sample was weighed, suspended in 45 mL of sterile water, then serially diluted to 10⁻\u0026sup1; to 10⁻⁹ g/mL. Aliquots of 200 \u0026micro;L of the 10⁻⁵ to 10⁻⁷ dilutions were spread evenly onto MRS agar medium containing bromocresol purple as an indicator (K₂HPO₄ 2.5 g/L, Na₂HPO₄ 2.5 g/L, peptone 2 g/L, yeast extract 0.5 g/L, agar 20 g/L, pH\u0026thinsp;=\u0026thinsp;7.0). The plates were incubated at 37\u0026deg;C for 24 h under anaerobic conditions. Colonies showing a color change to yellow were identified as acid-producing isolates. To ensure the selected colonies were pure, repeated purification and microscopic examination were performed. The purified colonies were preserved in MRS broth supplemented with 20% glycerol and stored at -80\u0026deg;C (Hope Bio-technology Co., Ltd., Qingdao, China).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e2. 2 Acid Production Ability Test of Strains\u003c/h3\u003e\n\u003cp\u003eFor quantitative determination, the strain was inoculated at 2% (v/v) into 10 mL of MRS liquid medium and cultured under shaking conditions at 37\u0026deg;C and 150 rpm. The pH was measured at specified time points using a calibrated pH meter. All experiments were repeated three times.\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Bile Salt Tolerance of Strains\u003c/h2\u003e \u003cp\u003eBile salt tolerance of strains was assessed by inoculating 2% (v/v) overnight culture into MRS liquid medium containing 0.4% (w/v) bile salts (Oxgall). After incubating 10 mL of culture at 37\u0026deg;C for 36 hours, optical density at 600 nm (OD600) was measured using a microplate reader (BioTek Synergy H1).\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\text{S}\\text{u}\\text{r}\\text{v}\\text{i}\\text{v}\\text{a}\\text{l}\\:\\text{r}\\text{a}\\text{t}\\text{e}\\:\\left(\\text{\\%}\\right)\\:=\\:(\\text{O}\\text{D}600\\_\\text{t}\\text{r}\\text{e}\\text{a}\\text{t}\\text{m}\\text{e}\\text{n}\\text{t}\\:\\text{g}\\text{r}\\text{o}\\text{u}\\text{p}\\:/\\:\\text{O}\\text{D}600\\_\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}\\:\\text{g}\\text{r}\\text{o}\\text{u}\\text{p})\\:\\times\\:\\:100$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Morphological Identification of Strains\u003c/h2\u003e \u003cp\u003eThe candidate strains were subjected to Gram staining and examined under a microscope. Additionally, the morphology of the strains was observed using scanning electron microscopy (SEM).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Biochemical Identification of Strains\u003c/h2\u003e \u003cp\u003eThe strains were incubated at 37\u0026deg;C for biochemical identification. The results of the LAB physiological and biochemical identification tests were compared with the instructions provided in the LAB biochemical identification kit (Company, SHBG13) to evaluate each indicator. All indicator results were cross-referenced with Bergey\u0026rsquo;s Manual of Systematic Bacteriology and the Manual for the Systematic Identification of Common Bacteria. The tested indicators included esculin, cellobiose, maltose, mannitol, salicin, sorbitol, sucrose, raffinose, inulin, lactose, and hippuric acid.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Molecular Biological Identification of Strains\u003c/h2\u003e \u003cp\u003eGenomic DNA was extracted according to the instructions of the bacterial genome DNA extraction kit (Tiangen Biotech Co., Ltd., Beijing, China). PCR amplification was performed using universal bacterial 16S rDNA primers. The forward primer was 27F (sequence: 5\u0026rsquo; - AGAGTTTGATCCTGGCTCA-3\u0026rsquo;), and the reverse primer was 1492R (sequence: 5\u0026rsquo; - GGTTACCTTGTTACGACTT-3\u0026rsquo;). The PCR amplification conditions were as follows: initial denaturation at 94\u0026deg;C for 5 minutes; followed by 35 cycles of 94\u0026deg;C for 30 seconds (denaturation), 57\u0026deg;C for 45 seconds (annealing), and 72\u0026deg;C for 2 minutes (extension); with a final extension at 72\u0026deg;C for 10 minutes, and storage at 4\u0026deg;C. The PCR products were purified and sent to Beijing Liuhe BGI Technology Co., Ltd. for sequence. The sequencing results were analyzed for homology using the BLAST software available on NCBI, and a phylogenetic tree was constructed using MEGA 6.0 software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Determination of Strain Growth Performance\u003c/h2\u003e \u003cp\u003eFollowing inoculation, the strains were cultured at 37\u0026deg;C. OD600 was measured using a microplate reader at 0, 2, 4, 6, 8, 12, 16, 24, and 38 h to construct the growth curve.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Tolerance of Strains to Simulated Intestinal Fluids\u003c/h2\u003e \u003cp\u003eThe strains were inoculated into MRS broth at a 2% (v / v) inoculation rate and cultured at 37\u0026deg;C with shaking at 150 r/min for 24 hours. Then, 1 mL of the tested bacterial culture was added to 9 mL of artificial intestinal fluid (0.3% bile salts, 1 g/L trypsin, pH 6.8 adjusted with 4% NaOH, sterilized with a 0.22 \u0026micro;m filter). The cultures were incubated at 37\u0026deg;C with shaking at 150 r/min. The viable cell count was determined by the plate-count method, and the survival rate was calculated using the following formula:\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:\\text{S}\\text{u}\\text{r}\\text{v}\\text{i}\\text{v}\\text{a}\\text{l}\\:\\text{r}\\text{a}\\text{t}\\text{e}\\:=\\:(\\text{c}\\text{o}\\text{l}\\text{o}\\text{n}\\text{y}\\:\\text{c}\\text{o}\\text{u}\\text{n}\\text{t}\\:\\text{a}\\text{t}\\:\\text{m}\\text{e}\\text{a}\\text{s}\\text{u}\\text{r}\\text{e}\\text{m}\\text{e}\\text{n}\\text{t}\\:\\text{t}\\text{i}\\text{m}\\text{e}\\:/\\:\\text{c}\\text{o}\\text{l}\\text{o}\\text{n}\\text{y}\\:\\text{c}\\text{o}\\text{u}\\text{n}\\text{t}\\:\\text{a}\\text{t}\\:\\text{t}\\text{i}\\text{m}\\text{e}\\:0)\\:\\times\\:\\:100\\text{\\%}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Hemolytic Activity Test\u003c/h2\u003e \u003cp\u003eBy streaking strains onto Columbia blood agar plates (Beikman Bioengineering Co., Ltd., Changde, China) and observing the clear zones surrounding colonies after incubation at 37\u0026deg;C for 24 hours, the haemolytic type (α, β, or γ) was determined. \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (CMCC 26003) was used as the control strain.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Antibiotic Sensitivity\u003c/h2\u003e \u003cp\u003eA 200 \u0026micro;L aliquot of the activated bacterial culture (cultured for 24 hours) was evenly spread onto MRS solid medium. The antibiotic sensitivity was tested using the Kirby-Bauer disk diffusion method (K-B method). The resistance of the strain to clindamycin (lincomycin), chloramphenicol, furazolidone (nifuroxazide), polymyxin B, vancomycin, ciprofloxacin, ofloxacin, norfloxacin, midecamycin, erythromycin, doxycycline, tetracycline, neomycin, kanamycin, gentamicin, amikacin, cefoperazone, ceftazidime (Fortum), cephaloridine, cefazolin, and cefalexin was determined.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Antibacterial Activity Test\u003c/h2\u003e \u003cp\u003eThe antibacterial activity of the strain was determined using the Oxford cup agar diffusion method.The strain was inoculated into MRS medium at a 2% (v/v) inoculation rate and cultured at 37\u0026deg;C with shaking at 150 rpm for 24 h. The supernatant was collected and set aside. Fresh bacterial cultures (200 \u0026micro;L each) of Salmonella enteritidis (CMCC 50071), Escherichia coli (ATCC 25922), and Staphylococcus aureus (CMCC 26003) were evenly spread onto MH solid medium. After the bacterial suspension was fully absorbed, three Oxford cups were placed on each plate. Then, 200 \u0026micro;L of the strain supernatant was added to each cup. The plates were incubated at 37\u0026deg;C for 24 h, and the formation of inhibition zones was observed. The diameter of the inhibition zones was measured.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Genomic DNA extraction\u003c/h2\u003e \u003cp\u003eGenomic DNA was extracted from the samples using the STE method. The purity and integrity of the DNA were assessed using agarose gel electrophoresis, and the DNA was quantified using a Qubit fluorometer (Life Technologies, USA). Sequencing was performed using the Illumina PE150 system and the PacBio platform at Beijing Novogene Bioinformatics Technology Co., Ltd.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13 Library Construction\u003c/h2\u003e \u003cp\u003eFor the Illumina platform, the NEBNext\u0026reg; Ultra\u0026trade; DNA Library Prep Kit for Illumina (NEB, USA) was used to prepare the second-generation sequencing library. For the PacBio platform, the SMRT Bell library was constructed using the SMRTbell\u0026trade; Template Kit (version 2.0). The constructed library was quantified using Qubit, and the insert fragment size was assessed using the Agilent 2100 Bioanalyzer. Sequencing was then performed on the PacBio platform.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14 Genome Assembly and Analysis\u003c/h2\u003e \u003cp\u003eAfter library quality control, genome assembly was performed using the Canu software (version 2.0, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/marbl/canu/\u003c/span\u003e\u003cspan address=\"https://github.com/marbl/canu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for third generation reads. Error correction of the third-generation sequencing data was conducted using Racon software (version 1.4.13). The genome assembly results were further polished using Pilon software (version 1.22) based on second-generation sequencing data to obtain the final assembly. After assembly, Open Reading Frames (ORFs) were predicted and filtered to identify potential protein-coding regions in the genome. Genome assembly quality was evaluated by observing coverage and GC content distribution. A genome circular map was generated to provide a comprehensive and intuitive visualization of genome characteristics.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.15 Gene Prediction and Functional Annotation\u003c/h2\u003e \u003cp\u003eFor gene composition prediction, GeneMarkS software (Version 4.17, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://topaz.gatech.edu/GeneMark/\u003c/span\u003e\u003cspan address=\"http://topaz.gatech.edu/GeneMark/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to predict coding genes. RepeatMasker (Version open- 4.0.5) was used to predict interspersed repeat sequences, and TRF (Tandem Repeats Finder, Version 4.07b)\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003ewas used to identify tandem repeat sequences. tRNA prediction was performed using tRNAscan-SE software (Version 1.3.1), and rRNA prediction was conducted using rRNAmmer software (Version 1.2)\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Gene islands were predicted using IslandPath- DIOMB software (Version 0.2)\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e, and prophages were identified using PhiSpy software (Version 2.3)\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Finally, CRISPR sequences (Clustered Regularly Interspaced Short Palindromic Repeats) were predicted using CRISPRdigger (Version 1.0)\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. The coding proteins of the genome were functionally annotated using the NR (Non-Redundant Protein Database), KEGG (Kyoto Encyclopedia of Genes and Genomes), GO (Gene Ontology), COG (Clusters of Orthologous Groups), and CAZy (Carbohydrate-Active Enzymes Database) databases.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.16 Genomic Safety Assessment\u003c/h2\u003e \u003cp\u003eBacterial insertion sequences were predicted using the ISfinder database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www-is.biotoul.fr/\u003c/span\u003e\u003cspan address=\"https://www-is.biotoul.fr/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Using Blast software, the assembled sequences were compared with the ISfinder database to obtain insertion sequence prediction results. Using Diamond software, the amino acid sequences of the target species, were compared with the VFDB database, and the annotation results were obtained by combining the genes of the target species and their corresponding functional annotation information of virulence factors.\u0026emsp;Using the RGI (v5.1.0) software, the amino acid sequences of the target species were compared with the CARD database (v3.2.5), and the annotation results were obtained by combining the genes of the target species with their corresponding annotated information on the drug resistance function.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2.17 Statistical Analysis\u003c/h2\u003e \u003cp\u003eUnless otherwise stated, all experiments were repeated three times. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical analysis was performed using GraphPad Prism 9 software. Differences between groups were assessed using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. Differences were considered statistically significant at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results and Discussion","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Selection criteria for sheep-derived LAB and candidate strains\u003c/h2\u003e \u003cp\u003eFrom fresh faecal samples of healthy Chahar sheep, 325 colonies capable of forming yellow halos (acid-producing phenotype) were preliminarily selected using MRS\u0026ndash;bromocresol purple indicator medium (Figure S1), indicating active lactic acid fermentation capacity \u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. Subsequently, quantitative assessments of acid production capacity and bile salt tolerance were conducted (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Among these isolates, SSF2 exhibited the fastest pH reduction rate, dropping below pH 4.5 within 2 hours, and demonstrated the highest survival rate under 0.4% bile salt conditions.\u003c/p\u003e \u003cp\u003eThe primary objective of this initial screening was to identify strains capable of surviving in the gastrointestinal environment and rapidly producing acid, characteristics essential for probiotic lactic acid bacteria. Rapid pH decline typically correlates with robust glycolytic activity and lactic acid metabolism, effectively inhibiting pathogen growth during early fermentation stages \u003csup\u003e[\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Bile salt tolerance reflects membrane structural stability and survival capacity through the small intestinal environment \u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. This rigorous screening process yielded strains possessing both functional utility for process applications and intestinal survival capability, aligning with established principles for lactic acid bacteria selection.\u003c/p\u003e \u003cp\u003eBased on comprehensive evaluation of acid production capacity, bile salt tolerance, and stability, SSF2 was ultimately designated as the candidate strain for subsequent research.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Identification of LAB\u003c/h2\u003e \u003cp\u003eThe SSF2 strain formed round, white colonies with a moist and glossy surface. Microscopically, the cells appeared as oval cocci arranged singly, in pairs, or in short chains (\u003cb\u003eFigure S2a\u003c/b\u003e). On rich media, the colonies were large and smooth. After Gram staining, the strain was observed under a microscope as purple spherical cells, confirming that it is a Gram-positive coccus (\u003cb\u003eFigure S2b\u003c/b\u003e). The SEM results further confirmed that strain SSF2 is a coccoid bacterium (\u003cb\u003eFigure S2c\u003c/b\u003e). The physiological and biochemical identification results of strain SSF2 were shown in \u003cb\u003eTable S1\u003c/b\u003e. The results indicate that strain SSF2 can hydrolyze various carbohydrates, and it is preliminarily identified as Lactococcus\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. 16S rRNA gene sequencing results indicate that SSF2 shares 98.7% similarity with \u003cem\u003eP. pentosaceus\u003c/em\u003e. Phylogenetic tree analysis further confirms its clustering within the \u003cem\u003ePediococcus\u003c/em\u003e clade. The combined results of morphological, biochemical and phylogenetic analyses confirm SSF2 as belonging to the species \u003cem\u003eP. pentosaceus\u003c/em\u003e, a strain widely employed in food fermentation and renowned for its production of pediocin-like bacteriocins. Accurate taxonomic identification is a prerequisite for probiotic evaluation and regulatory approval. This finding aligns with prior research indicating that animal-derived \u003cem\u003eP. pentosaceus\u003c/em\u003e strains typically exhibit strong acid tolerance and bacteriocin production capabilities, supporting their potential application as feed additives. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Biological characteristics of SSF2\u003c/h2\u003e \u003cp\u003eIn terms of growth performance, the OD value of \u003cem\u003eP. pentosaceus\u003c/em\u003e SSF2 showed little change within the first 2 h, indicating a lag phase. Between 2\u0026ndash;16 h, the OD value increased almost linearly, representing the logarithmic growth phase. After 16 h, the strain entered a stationary phase, and no significant decrease in OD value was observed after 16 h, indicating that \u003cem\u003eP. pentosaceus\u003c/em\u003e SSF2 has strong stability (\u003cb\u003eFigure S3a\u003c/b\u003e). This stability is particularly important for industrial applications as it helps ensure the consistency and controllability of fermented product quality. The simulated intestinal fluid experiment further confirms SSF2's adaptability under intestinal conditions, which is a critical indicator for probiotics to maintain viability and exert their functional benefits (\u003cb\u003eFigure S3b\u003c/b\u003e). Therefore, SSF2 meets the core biological criteria for probiotics: possessing good activity, stability, and resistance to acid and bile salts.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Evaluation of the Probiotic Effects of SSF2\u003c/h2\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003e3.4.1 Safety assessment\u003c/h2\u003e \u003cp\u003eThe haemolysis test results indicated that SSF2 exhibited γ-type haemolysis (no haemolysis observed), confirming its lack of cytotoxic activity (Figure S4). This finding demonstrated that SSF2 complies with the FAO/WHO safety requirements for non-pathogenic lactic acid bacteria used in feed and food \u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAntibiotic susceptibility testing results indicate that SSF2 retains sensitivity to most clinically relevant antibiotics (Table S2). Resistance was observed only to vancomycin and polymyxin B. The aforementioned resistance profiles align with the inherent resistance characteristics commonly observed in lactic acid bacteria, with no evidence of abnormal broad-spectrum resistance. This suggests a low risk of acquired resistance\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. The high susceptibility facilitates clearance or intervention when clinically necessary. From a regulatory perspective, this is consistent with the European Food Safety Authority's (EFSA) principle that probiotics \u0026lsquo;shall not carry transferable resistance genes and shall present a manageable overall risk of resistance\u0026rsquo;. Actual regulatory approval typically involves a combined assessment of genomic evaluation and phenotypic results.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003e3.4.2 Antimicrobial activity of SSF2\u003c/h2\u003e \u003cp\u003e \u003cem\u003eP. pentosaceus\u003c/em\u003e SSF2 demonstrated significant antibacterial activity against \u003cem\u003eEscherichia coli\u003c/em\u003e, \u003cem\u003eSalmonella\u003c/em\u003e, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, and \u003cem\u003eListeria\u003c/em\u003e, with the strongest inhibitory effect observed against \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e, as indicated by the largest inhibition zone diameter (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The second strongest inhibitory effect was against \u003cem\u003eSalmonella\u003c/em\u003e. It is noteworthy that SSF2 exhibits particularly potent inhibitory effects against \u003cem\u003eV. parahaemolyticus\u003c/em\u003e, given that the genus \u003cem\u003eVibrio\u003c/em\u003e typically possesses strong tolerance and survives in marine and food-borne environments. This indicates that SSF2 generates metabolites capable of overcoming their natural resistance mechanisms. Overall, its antibacterial properties likely result from the synergistic action of multiple mechanisms, including organic acid accumulation, hydrogen peroxide production, and bacteriocin secretion \u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. SSF2 exhibits potent inhibitory effects against Gram-positive bacteria such as \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eListeria monocytogenes\u003c/em\u003e, consistent with the known spectrum of action for pediocin-like bacteriocins, which typically function by disrupting target cell membrane permeability. The moderate inhibitory effect on Escherichia coli aligns with the protective role of Gram-negative outer membrane structures, yet measurable inhibition zones still occur, indicating that organic acids and other metabolites produced by SSF2 exert some inhibitory influence on these bacteria.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStatistical table of bacteriostatic experimental results\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrain Name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInhibition Zone Diameter\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23d\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSalmonella\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eListeria\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e17.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23c\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"2\"\u003eNote: The antibacterial ability of the strain corresponds to the size of the inhibition zone. An inhibition zone diameter of 0\u0026ndash;10 mm indicates good antibacterial activity, while 10\u0026ndash;20 mm indicates excellent antibacterial activity.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Whole genome analysis of SSF2\u003c/h2\u003e \u003cp\u003eThe genome of SSF 2 is 1,853,794 bp in size with a GC content of 37.29%. Subsequently, the assembly was corrected through three rounds of error correction using Racon (version 1.4.13) based on third- generation sequencing data, followed by three additional rounds of error correction using Pilon with second-generation sequencing data. The structural integrity and uniformity of sequencing depth validate the high reliability of this assembled genome (Fig.\u0026nbsp;3\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec28\" class=\"Section3\"\u003e \u003ch2\u003e3.5.1 Genome Composition and Functional Gene Distribution\u003c/h2\u003e \u003cp\u003eA total of 1,804 coding sequences (CDSs), 54 tRNAs and 6 rRNAs were identified, revealing the compact genomic structure typical of the genus \u003cem\u003ePediococcus\u003c/em\u003e (Figure S5, Table S3).\u003c/p\u003e \u003cp\u003eThe prediction results of genomic islands and prophages are shown in \u003cb\u003eTable S4\u003c/b\u003e and \u003cb\u003eFigure S6\u003c/b\u003e. A total of 6 genomic islands were predicted, with a total length of 74,287 bp and an average length of 24,762.33 bp. Conforming to the typical scale of GIs in prokaryotes (usually 5-200 kbp), it may have been acquired through horizontal gene transfer (HGT)\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e.Additionally, 6 prophages were predicted, with a total length of 328,305 bp and an average length of 54,717.5 bp.In the interaction between function and host, the lysogenic conversion phenotype indicates that it may carry beneficial genes (such as bacteriocins, stress proteins), enhancing host adaptability (such as the inhibition of Vibrio parahaemolyticus, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e)\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe NR annotation results are shown in \u003cb\u003eFigure S7\u003c/b\u003e, where SSF2 has the highest number of matched genes with \u003cem\u003eP. pentosaceus\u003c/em\u003e, reaching 1,613, accounting for the vast majority of all matches. This indicates that the genome of SSF2 exhibits a high degree of similarity to the reference genome of \u003cem\u003eP. pentosaceus\u003c/em\u003e, further confirming the taxonomic classification of the strain.\u003c/p\u003e \u003cp\u003eBased on the KEGG annotation information, the functions of \u003cem\u003eP. pentosaceus\u003c/em\u003e SSF 2 were analyzed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The highly specialized metabolic network is prominent, which maybe for rapid energy acquisition and product synthesis\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. In carbon source utilization and energy metabolism, the predicted bacteriocin (such as pediocin) synthesis gene cluster (KEGG map01053) of secondary metabolites explains its strong inhibitory activity against pathogenic bacteria, consistent with the results of the bacteriostatic activity test\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. In nitrogen and cofactor metabolism,the synthesis of vitamin B group indicates potential probiotic functions (such as intestinal microbial interaction), but the actual synthesis capacity needs to be verified experimentally\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. ABC transporters (such as KEGG map02010) may be related to bile salt efflux pump genes (such as bsh), which may contribute to its bile salt tolerance (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb)\u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eKEGG classification reveals significant enrichment of genes associated with glycolysis, pyruvate metabolism, and phosphotransferase system (PTS)-related transport, indicating this strain possesses efficient carbohydrate-to-lactic acid conversion capabilities. GO and COG functional classification analyses (Figures S8 and 9) further demonstrate that the genome contains numerous genes related to catalytic activity, membrane transport, and stress adaptation. Notably, only one gene was related to antioxidant activity.In the enrichment of core metabolic pathways, the results of COG (136 genes) and KEGG (801 genes) are consistent, supporting its efficient glycolysis and lactic acid fermentation capacity \u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. Limited synthetic genes (such as branched-chain amino acids) may restrict their growth in low-nitrogen environments, requiring exogenous supplementation\u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e. In terms of environmental adaptability, the enrichment of acid tolerance-related genes (such as F₀F₁-ATPase) is shown, explaining its rapid pH decline ability (Fig.\u0026nbsp;3A)\u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. In addition, potential adhesion genes (such as surface proteins) may enhance their colonization ability on the surface of fermentation equipment and also have antibacterial effects. The carbohydrate-active enzyme prediction results (Figure S10) identified multiple CAZy family members, including glycoside hydrolases (GHs) and glycosyltransferases (GTs). These enzymes may confer SSF2's ability to utilise diverse dietary carbohydrates and contribute to its survival within the fibre-rich gastrointestinal environment of ruminants. These genomic-level predictions align with the strain's physiological performance, including rapid acid production, stable growth, and tolerance to bile salts and simulated intestinal fluids\u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section3\"\u003e \u003ch2\u003e3.5.2 Genomic Safety Assessment\u003c/h2\u003e \u003cp\u003eThis strain did not carry mobile factors known to be associated with clinical risk at the IS element level, supporting its initial biosafety assessment. Comparative analysis based on the VFDB database was determined by similarity threshold (\u0026ge;\u0026thinsp;80%), resulting in no known virulence-related genes with \u0026ge;\u0026thinsp;80% similarity being detected \u003csup\u003e[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e. Resistance gene search using CRAD database resulted in no resistance genes detected\u003csup\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e. The above negative results support that this strain lacks typical virulence factors with characterised resistance determinants within the range of known databases, suggesting that it has a low potential risk profile.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, SSF2 possesses potent acid-producing capacity, excellent gastrointestinal tolerance, and broad-spectrum antibacterial activity, rendering it potentially valuable in both food and feed applications. Its inhibition of common intestinal pathogens supports its use as a microbial feed additive, aiding in the maintenance of animal gut health; its significant suppression of \u003cem\u003eV. parahaemolyticus\u003c/em\u003e demonstrates its potential for application in aquatic food safety and preservation. Furthermore, SSF2's stable fermentation characteristics and antibacterial capacity render it suitable as a functional strain for food fermentation. Genomic analysis further elucidates its metabolic diversity, stress tolerance, and safety profile, providing a molecular basis for its probiotic properties. Overall, SSF2 represents a promising probiotic strain combining safety with multifunctional characteristics, suitable for applications in food, feed, and related biotechnological fields.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eThe authors' contributions in this study are as follows:YLB, HY, and ZLC participated in the design of the study; WYS, JJ, Wurentana, and HXR plotted the figures and analyzed data; Swee Sen Teo, YYN, and XZS analyzed the sequencing data; ZYH drafted the manuscript; and CC and SSF criticallyreviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMazziotta C, Tognon M, Martini F, Torreggiani E, Rotondo JC (2023) Probiotics Mechanism of Action on Immune Cells and Beneficial Effects on Human Health. 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However, strains derived from sheep remain relatively limited in systematic screening and genomic characterisation studies. This research isolated lactic acid bacteria from healthy Chahar sheep and screened strains with application potential through phenotypic evaluation, probiotic property analysis, and whole-genome sequencing. Results indicate that strain SSF2 exhibits rapid acid production capacity, strong bile salt tolerance, survival in simulated gastrointestinal environments, and significant antibacterial activity against major pathogens including \u003cem\u003eEscherichia coli\u003c/em\u003e, \u003cem\u003eSalmonella\u003c/em\u003e, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, and \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e. SSF2 exhibits no haemolytic activity and maintains sensitivity to commonly used clinical antibiotics. Genome sequencing revealed SSF2 possesses a 1.85 Mb circular chromosome and two plasmids, encoding functional genes involved in carbohydrate metabolism, amino acid metabolism, stress tolerance, and potential bacteriocin biosynthesis. No virulence factors or antibiotic resistance genes were detected. Overall, both its phenotypic and genomic characteristics indicate that \u003cem\u003eP. pentosaceus\u003c/em\u003e SSF2 is a safe and promising probiotic candidate strain with potential applications in ruminant feed additives and food biotechnology.\u003c/p\u003e","manuscriptTitle":"Screening and Whole-Genome Analysis of a Sheep-Derived Lactic Acid Bacteria with Antibacterial Properties","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-16 06:41:10","doi":"10.21203/rs.3.rs-7778181/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"56fd5f92-120e-444e-bf11-a8e4f03359ed","owner":[],"postedDate":"December 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-12T07:01:36+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-16 06:41:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7778181","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7778181","identity":"rs-7778181","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}