Screening and Whole-Genome Analysis of Sheep-Derived Lactic Acid Bacteria

Screening and Whole-Genome Analysis of Sheep-Derived Lactic Acid Bacteria | 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 Sheep-Derived Lactic Acid Bacteria Lingbai Yao, Yao Huang, Linchong Zhang, Yusheng Wang, Jun Jia, and 8 more This is a preprint; it has not been peer reviewed by a journal.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-5985964/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 Probiotics ( lactic acid bacteria ) are widely used as microbial feed additives in livestock production and play an important role in preventing and treating animal diarrhea as well as regulating host immune function. In this study, lactic acid bacteria were isolated from the fresh feces of healthy adult female sheep, and their biological characteristics were analyzed. Based on phylogenetic analysis, strain SSF2 was identified as Pediococcus pentosaceus . SSF2 exhibited tolerance to acid, bile salt concentrations, and simulated artificial gastrointestinal environments. The hemolysis test for SSF2 was negative, it was sensitive to commonly used antibiotics, and it demonstrated significant antibacterial and antioxidant activities, indicating its excellent probiotic potential. Whole-genome sequencing (WGS) was performed using the HiSeq 2500 platform and the PacBio system to explore the genetic characteristics of SSF2. The genome was revealed to consist of a circular chromosome and two plasmids, with sizes of 1,785,410 bp, 10,618 bp, and 57,766 bp, and GC contents of 37.23%, 34.95%, and 40.98%, respectively. The genome was predicted to contain five genomic islands, six prophages, and a potential CRISPR gene editing sequence. Functional annotation through databases such as COG, GO, and KEGG revealed that most genes are related to carbon metabolism, protein and amino acid metabolism, nucleotide metabolism, and membrane transport processes. This study indicates that an in-depth understanding of the functionality and genetic characteristics of Pediococcus pentosaceus SSF2 may enable the potential application of this strain in sheep feed supplements. Lactic acid bacteria Pediococcus pentosaceus probiotic functionality whole genome antibacterial activity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Introduction Probiotics are a class of active microorganisms beneficial to the health of the host (humans or animals) (Mazziotta et al. 2023 ) and have been proven to provide health benefits to the host when consumed in sufficient amounts (Szajewska et al. 2023 ). They are widely present in the host's oral cavity, skin, gastrointestinal tract and reproductive tract (Xiao et al. 2020 , Manzoor et al. 2021 , Tustumi et al. 2023 , Guerra 2024 ). To date, probiotics have become the fastest-growing animal feed additive globally (El-Saadony et al. 2021 ). Lactic acid bacteria (LAB) are an important component of probiotics ,(Roldán-Pérez et al. 2023 ) known for promoting nutrient absorption, antibacterial and anti-infection properties, anti-tumor effects, regulation of gut microbiota, immune modulation, metabolism, and bioremediation (Gomand et al. 2019 , Nagarajan et al. 2022 , Raman et al. 2022 ). LAB have become an ideal choice for commercial development and are widely used in fields such as food, medicine, agriculture, and animal husbandry(Jiang et al. 2021 , Qi et al. 2021 , Ge et al. 2022 ). Pediococcus pentosaceus , a Gram-positive bacterium belonging to the family Streptococcaceae and the genus Pediococcus, is a type of lactic acid bacterium(Kim et al. 2021 ). P. pentosaceus is widely distributed and has been isolated from fermented foods, aquatic products, animal products, and plant products(Nagy et al. 2022 ). Due to its bacteriocins' inhibitory effects on various pathogens and its good thermal stability, Pediococcus pentosaceus has attracted significant attention from researchers(Kahraman-Ilıkkan 2024 ). 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(Phuengjayaem et al. 2021 ). Whole-genome sequencing technology provides comprehensive genetic information for the study of lactic acid bacteria , enabling the revelation of metabolic characteristics, potential probiotic functions, and safety of strains(Rivas et al. 2022 ). Through genome analysis, it is possible not only to explore the ecological adaptation mechanisms of lactic acid bacteria 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(Langille et al. 2013 , Huang et al. 2024 ). 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 lactic acid bacteria derived from Chahar sheep remains quite limited. Existing research mainly focuses on the diversity of microbial communities and preliminary functional exploration, lacking systematic analysis(Behboudi et al. 2023 ). In this study, lactic acid bacteria were isolated from the feces of Chahar sheep, and a strain 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. Materials and Methods Collection and Screening of Strains Fresh fecal samples from six healthy adult female Chahar sheep were collected in Hangjin Banner, Ordos City, Inner Mongolia Autonomous Region, China (longitude 107°54'21" E, latitude 39°56'56" N). A 5 g sample was weighed and added to 45 mL of sterile water, then serially diluted to concentrations ranging from 10⁻¹ to 10⁻⁹ g/mL. Subsequently, 200 µL of the 10⁻⁵ to 10⁻⁷ dilutions were applied to 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, sodium carboxymethyl cellulose 20 g/L, agar 20 g/L, pH = 7.0). The plates were incubated at 37°C for 24 hours, and single colonies that produced acid (indicated by a color change to yellow) were selected. To ensure the selected colonies were pure, repeated purification and microscopic examination were performed. The purified colonies were preserved in MRS broth (Hope Bio-technology Co., Ltd., Qingdao, China) supplemented with 20% glycerol at -80°C. 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). Biochemical Identification of Strains The strains were incubated at 37°C for biochemical identification. The results of the lactic acid bacteria physiological and biochemical identification tests were compared with the instructions provided in the lactic acid bacteria 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. 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 sequencing. 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. Evaluation of Biological Functions of Strains Acid Production Ability Test of Strains Lactic acid bacteria were inoculated and cultured at 37°C with shaking at 150 r/min. The pH value of the bacterial culture was measured at 0, 2, 4, 6, 8, 12, 16, 24, and 36 h. Determination of Strain Growth Performance Lactic acid bacteria were inoculated and cultured at 37°C. The OD600 values were measured at 0, 2, 4, 6, 8, 12, 16, 24, and 38 h using a microplate reader to plot the growth curve of the lactic acid bacteria. Bile Salt Tolerance of Strains Bile salt tolerance test: Lactic acid bacteria were inoculated into MRS broth at a 2% (v/v) inoculation rate and cultured at 37°C with shaking at 150 r/min for 12 hours. The culture was then inoculated into MRS broth containing 0.3% bile salts at a 2% (v/v) inoculation rate. The OD600 values were measured at 0, 2, 4, 6, 8, 12, 16, 24, and 38 hours under 37°C conditions. Data were recorded and analyzed. Tolerance of Strains to Simulated Intestinal Fluids Lactic acid bacteria 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), respectively. The cultures were incubated at 37°C with shaking at 150 r/min. Viable cell counts were determined using the plate spreading method, and survival rates were calculated as follows: Survival rate = (colony count at measurement time / colony count at time 0) × 100% Evaluation of Probiotic Effects of Strains Hemolytic Activity Test Lactic acid bacteria were inoculated into MRS medium at a 2% (v/v) inoculation rate and cultured at 37°C with shaking at 150 r/min for 24 hours. The activated bacterial culture was streaked onto Columbia blood agar plates (Beikman Bioengineering Co., Ltd., Changde, China) and incubated at 37°C for 24 hours in a constant-temperature incubator. The colony morphology and hemolytic activity were observed. Staphylococcus aureus (CMCC 26003) was used as the control strain. 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. Antibacterial Activity Test The antibacterial activity of the strain was determined using the Oxford cup agar diffusion method. Lactic acid bacteria were inoculated into MRS medium at a 2% (v/v) inoculation rate and cultured at 37°C with shaking at 150 r/min for 24 hours. 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 hours, and the formation of inhibition zones was observed. The diameter of the inhibition zones was measured. Whole Genome Sequencing and Analysis 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. 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. 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. 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)(Patel et al. 2024 ) 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)(Niu et al. 2022 ). Gene islands were predicted using IslandPath-DIOMB software (Version 0.2)(Sun et al. 2020 ), and prophages were identified using PhiSpy software (Version 2.3)(Lee et al. 2021 ). Finally, CRISPR sequences (Clustered Regularly Interspaced Short Palindromic Repeats) were predicted using CRISPRdigger (Version 1.0)(Zhang et al. 2023 ). 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. Results and Discussion Isolation and Identification of Lactic Acid Bacteria Colonies that caused the surrounding medium to change from purple to yellow in the bromocresol purple color reaction were selected, indicating good acid production ability (Figure S1) (Garbacz 2022). The selected strain was designated as SSF2. 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. 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 1) . The physiological and biochemical identification results of strain SSF2 are shown in Table 1 . The results indicate that strain SSF2 can hydrolyze various carbohydrates, and it is preliminarily identified as Lactococcus (Darbandi et al. 2022).Subsequently, homology analysis based on the 16S rRNA sequencing results showed that strain SSF2 had high homology (98.7%) with P. pentosaceus . Combining the morphological characteristics, physiological and biochemical properties, and 16S rDNA sequence analysis, strain SSF2 was ultimately identified as P. pentosaceus and named P. pentosaceus SSF2 (Figure 2) . Evaluation of the Biological Functions of the Strain The acid production performance of P. pentosaceus SSF2 was assessed. The pH of the fermentation broth decreased rapidly during the 0–16 h fermentation period and stabilized after 24 h. The pH dropped most rapidly during the first 0–2 h of fermentation, falling below 5.0 (Figure 3A) , Indicating that the strain has the ability to rapidly initiate lactic acid fermentation. This rapid acid-lowering ability plays an important role in the initial stability of fermented products and the inhibition of contaminant microbial growth(Darbandi et al. 2022, Garbacz 2022, Zhang et al. 2023). 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 3B) . This stability is particularly important for industrial applications as it helps ensure the consistency and controllability of fermented product quality. Additionally, the strain's tolerance to a 0.3% bile salt environment ( Figure 3C ) demonstrates its ability to resist the damaging effects of bile salts on the cell membrane and maintain stability in the intestinal tract (Ren et al. 2018, Liu et al. 2022). The simulated intestinal fluid experiment ( Figure 3D ) further confirms SSF2's adaptability under intestinal conditions, which is a critical indicator for probiotics to maintain viability and exert their functional benefits. Evaluation of the Probiotic Effects of the Strain P. pentosaceus SSF2 does not produce hemolytic zones, indicating that it is a safe strain and can be applied as a potential probiotic in production (Figure 4A) (Pereira et al. 2022). Additionally, the strain exhibited varying sensitivity to 30 antibiotics. It exhibited good sensitivity to clindamycin (lincomycin), chloramphenicol, furazolidone (nifuroxazide), midecamycin, erythromycin, doxycycline, neomycin, gentamicin, cefoperazone, ceftazidime (Fortum), cephaloridine, cefazolin, and cefalexin, indicating a low risk of spreading antibiotic resistance in the food chain or medical environments(Zheng et al. 2020) . It showed moderate sensitivity to tetracycline, kanamycin, and amikacin, while displaying resistance to polymyxin B, vancomycin, ciprofloxacin, ofloxacin, and norfloxacin (Supplementary Table 1) . 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 (21.87 ± 0.55a mm). The second strongest inhibitory effect was against Salmonella , with an inhibition zone diameter of 19.17 ± 0.57b mm . This antibacterial property may be relat0ed to the secretion of its metabolites, such as organic acids, hydrogen peroxide, and bacteriocins(Fugaban et al. 2022) . In particular, the strong inhibitory effect against Vibrio parahaemolyticus indicates that SSF2 has great potential for application in seafood preservation and food safety(Guo et al. 2020) . Whole genome sequencing and analysis Genome Assembly and Prediction The genome of SSF 2 is 1,853,794 bp in size with a GC content of 37.23%. 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 final genome assembly results are detailed in Table 3 . Genomic circular map analysis The assembled genome sequences of the sequencing samples, combined with the predicted results of coding genes, were visualized using the Circos software. If analyses of non-coding RNA and gene functional annotation were also performed, the corresponding results are displayed in the diagram as well. The whole-genome map is shown in Figure 5 , and Plasmids P1/P2 are shown in Figure 6 . Genome Component Analysis Prediction Results of Coding and Non-coding RNA Genes The coding gene prediction for the newly sequenced genome was performed using GeneMarkS software, revealing the following results: the total number of genes is 1,844, with a total gene length of 1,637,667 bp. The average length of coding genes is 888 bp, and the total length of coding regions accounts for 88.34% of the entire genome. The gene lengths are primarily distributed between 200 and 1,500 bp, with a GC content of 37.23% (Figure 7) . The prediction results for non-coding RNA in strain SSF 2 are shown in Table 4, indicating 55 tRNA genes and no rRNA genes. Repetitive Sequence Prediction Repetitive sequences were predicted using RepeatMasker software for interspersed repeats, and TRF (Tandem Repeats Finder) was used to identify tandem repeats in the DNA sequence. The prediction results for interspersed and tandem repeats in strain SSF 2 are as follows: six short interspersed nuclear elements (SINEs) with a total length of 373 bp, 16 long interspersed nuclear elements (LINEs) with a total length of 1,517 bp, and 17 DNA transposons (DNA elements) with an average length of 84 bp. Repetitive sequences account for 43.74% of the genome.The high proportion of DNA transposons and LINEs suggests that the genome may have undergone multiple exogenous DNA integration events, which are common in fermentation environment strains (such as those exposed to phages or plasmids) . The enrichment of repetitive sequences may enhance the strain's rapid evolutionary ability in variable environments (such as pH and salinity fluctuations), promoting local gene rearrangements through homologous recombination . CRISPR Sequence Prediction The prediction results of transposons and CRISPR sequences are shown in Table 5. Prediction of genomic islands and prophages The prediction results of genomic islands and prophages are shown in Table 6 and Figure 8. 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)(Peng et al. 2023) .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 parahaemolyticu s, Figure 3D)(Bertelli et al. 2022) . Gene Function Annotation NR Annotation Results The NR annotation results are shown in Figure 9 , 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. KEGG annotation Results Based on the KEGG annotation information, the functions of P . pentosaceus SSF 2 were analyzed (Figure 10). A total of six classification levels (KEGG A classes) were identified, including 44 genes annotated as related to Cellular Processes, 105 genes related to Environmental Information Processing, 156 genes related to Genetic Information Processing, 54 genes related to Human Diseases, 801 genes related to Metabolism, and 22 genes related to Organismal Systems.The highly specialized metabolic network is prominent, which may be for rapid energy acquisition and product synthesis(Santoriello and Bassler 2024) . 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 (Figure 3D)(Zhang et al. 2023) . 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(Vermeulen et al. 2021) . 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 (Figure 3C)(Szczyrek et al. 2021) . GO Annotation Results Through analysis using the GO database, a total of 6,292 annotated gene sequences were identified, categorized into three main categories and 46 subcategories (Figure 11). Among them, 3,190 sequences were classified under biological processes, 1,325 under cellular components, and 1,777 under molecular functions. 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(Lewis et al. 2024) . Limited synthetic genes (such as branched-chain amino acids) may restrict its growth in low-nitrogen environments, requiring exogenous supplementation(Wang et al. 2024) . In terms of environmental adaptability, the enrichment of acid tolerance-related genes (such as F₀F₁-ATPase) is shown, explaining its rapid pH decline ability (Figure 3A)(Li et al. 2023) . 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. COG Annotation Results The COG database can be divided into 26 functional categories, and the statistical results are shown in Figure 12 . Among them, there are 185 genes related to translation, ribosomal structure, and biogenesis; 94 genes related to replication, recombination, and repair; 136 genes related to carbohydrate transport and metabolism; and 109 genes related to amino acid transport and metabolism. CAZy Annotation Results The CAZy annotation results for P. pentosaceus SSF2 are shown in Figure 13 , indicating a total of 57 carbohydrate-related enzymes classified into four categories. These include 19 glycosyltransferases (Glycosyl Transferases), 26 glycoside hydrolases (Glycoside Hydrolases), 9 carbohydrate-binding modules (Carbohydrate-Binding Modules), and 3 carbohydrate esterases (Carbohydrate Esterases).The GH28 present therein can explain the ability to degrade plant cell walls(Carli et al. 2019) . Conclusion In this study, a whole-genome analysis of P. pentosaceus SSF2 was conducted, revealing its exceptional biological characteristics and potential application value. The genome size is 1,853,794 bp with a GC content of 37.23%, containing 1,844 coding genes. Functional annotation of the genes indicates its rich biological functions in carbohydrate metabolism, amino acid metabolism, and other pathways. SSF2 exhibits excellent bile salt resistance, and survival ability in simulated gastrointestinal fluids, along with significant antibacterial activity against various pathogenic bacteria. The strain shows no hemolytic activity, indicating it is a safe strain suitable for potential probiotic applications in food production. Overall, P. pentosaceus SSF2 demonstrates broad application prospects in the fields of food fermentation and health. Declarations Submission statement This study has been approved by the Ethics Review Committee of Jining Normal University (approval number: [2024-ER-0456]). All participants signed informed consent prior to participating in the study. All authors have read and agreed to the final version of the manuscript and agreed to contribute it to the [Annals of Microbiology]. All data and materials generated in this study were obtained from the corresponding authors upon reasonable request. The authors declare that this study is free of any conflicts of interest that might affect the fairness of the findings. 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 critically reviewed the manuscript. This study was supported by Key technologies in Ulanqab Project assignment (2021GJ105), Jining Normal University PhD Innovation Fund (jsbsjj2306)，Central Guidance for Local Science and Technology Development Fund Project (2024ZY0098), Subproject of the National Key Research and Development Program (2024YFD2000505-03), National Natural Science Foundation of China (32460830), Ordos Key Research and Development Program Project (YF20240039). We thank them for their support. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We sincerely thank the editorial department of Annals of Microbiology and the reviewers for their review and valuable comments on this paper. Consent for publication Not applicable Data Availability Statement The datasets [GENERATED/ANALYZED] for this study can be found in the [NAME OF REPOSITORY] [LINK]. Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Funding Key technologies in Ulanqab Project assignment (2021GJ105), Jining Normal University PhD Innovation Fund (jsbsjj2306)，Central Guidance for Local Science and Technology Development Fund Project (2024ZY0098), Subproject of the National Key Research and Development Program (2024YFD2000505-03), National Natural Science Foundation of China (32460830), Ordos Key Research and Development Program Project (YF20240039). Author Contributions Conceptualization, data analysis, and drafting the manuscript, Lingbai Yao and Yao Huang; sampling, and determination, Linchong Zhang, Yusheng Wang, Jun Jia, and Zhaoshui Xing; data analysis: Xuran Hai, Wurentana and Swee Sen Teo; supervision, review, and editing, Shaofeng Su, Chao Cheng and Haiqing Wu. 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Nutrients 13(7) Tustumi F, Arienzo VP, Sunye IR, Lucas PFS, Colonno BB, Quintas JG, Lisboa EN, Szor DJ (2023) Esophageal Dysbiosis in Achalasia and Cancer Development: A Critical Review. Genes (Basel) 14(8) Vermeulen R, Deane S, Dicks L, Rohwer J, van Staden ADP (2021) Manganese Privation-Induced Transcriptional Upregulation of the Class IIa Bacteriocin Plantaricin 423 in Lactobacillus plantarum Strain 423. Appl Environ Microbiol 87(21):e0097621 Wang J, Xu X, Liu Y, Wang W, Ren C, Guo Y, Wang J, Wang N, He L, Zhao F (2024) Unknown bacterial species lead to soil CO(2) emission reduction by promoting lactic fermentation in alpine meadow on the Qinghai-Tibetan Plateau. Sci Total Environ 906:167610 Xiao J, Fiscella KA, Gill SR (2020) Oral microbiome: possible harbinger for children's health. Int J Oral Sci 12(1):12 Zhang J, Ren L, Zhang L, Gong Y, Xu T, Wang X, Guo C, Zhai L, Yu X, Li Y, Zhu P, Chen R, Jing X, Jing G, Zhou S, Xu M, Wang C, Niu C, Ge Y, Ma B, Shang G, Cui Y, Yao S, Xu J (2023) Single-cell rapid identification, in situ viability and vitality profiling, and genome-based source-tracking for probiotics products. Imeta 2(3): e117 Zhang J, Xiao Y, Wang H, Zhang H, Chen W, Lu W (2023) Lactic acid bacteria-derived exopolysaccharide: Formation, immunomodulatory ability, health effects, and structure-function relationship. Microbiol Res 274:127432 Zheng J, Wittouck S, Salvetti E, Franz C, Harris HMB, Mattarelli P, O'Toole PW, Pot B, Vandamme P, Walter J, Watanabe K, Wuyts S, Felis GE, Gänzle MG, Lebeer S (2020) A taxonomic note on the genus Lactobacillus: Description of 23 novel genera, emended description of the genus Lactobacillus Beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae. Int J Syst Evol Microbiol 70(4):2782–2858 Tables Tables 1 to 6 are available in the Supplementary Files section. Supplementary Files table.xlsx floatimage14.jpeg Figure S1 Acid Production Reaction of the Strain with Bromocresol Purple. Cite Share Download PDF Status: Posted Version 1 posted 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-5985964","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":452193682,"identity":"54bff688-610b-43b7-9e27-eefb5d383b83","order_by":0,"name":"Lingbai 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07:36:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5985964/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5985964/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82395141,"identity":"77f17913-d31e-48ab-8517-47bd4ef85121","added_by":"auto","created_at":"2025-05-09 20:06:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":196212,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological identification of strain SSF2. (A) Colony appearance (B) Gram staining of the strain (C) Electron micrograph of the strain.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5985964/v1/3a8f01d24d12ca90944bc802.png"},{"id":82396247,"identity":"c8ffa956-e7b4-417e-84a5-4b160ab35223","added_by":"auto","created_at":"2025-05-09 20:30:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":12051,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree of strain SSF2\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5985964/v1/9f0d28216b0605d206cec35f.png"},{"id":82395774,"identity":"466dda11-acf9-4b77-8acc-967770a34024","added_by":"auto","created_at":"2025-05-09 20:22:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":47664,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of the biological functions of strain SSF2. (A) Acid production performance; (B) Growth performance; (C) Bile salt tolerance; (D) Tolerance to simulated intestinal fluid.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5985964/v1/2c07912342abf27178b4c9c2.png"},{"id":82395777,"identity":"701e14de-fea4-4030-a4c0-10bef392d250","added_by":"auto","created_at":"2025-05-09 20:22:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":383297,"visible":true,"origin":"","legend":"\u003cp\u003eHemolytic activity assay of the strains: (A) Results for SSF2, (B) Results for \u003cem\u003eStaphylococcus aureus\u003c/em\u003e.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.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5985964/v1/492e3c836adbfd859d307268.png"},{"id":82395775,"identity":"309328e3-fb9c-4815-b551-95ba549e458d","added_by":"auto","created_at":"2025-05-09 20:22:38","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3047384,"visible":true,"origin":"","legend":"\u003cp\u003eGenome circle map of SSF 2 Note: The outermost circle represents the genomic sequence position coordinates. From the outside to the inside, the circles display the following information: gene functional annotation results (including COG/KOG annotation results based on the specific project), ncRNA, and genomic GC content. The GC content is calculated using a window size of (chromosome length/1000) bp and a step size of (chromosome length/1000) bp. The inward blue regions indicate areas where the GC content is lower than the average GC content of the whole genome, while the outward red regions indicate the opposite, with higher peaks representing greater deviations from the average GC content. The innermost circle shows the genomic GC skew values, calculated using a window size of (chromosome length/1000) bp and a step size of (chromosome length/1000) bp, with the formula (G-C)/(G+C). The inward green regions indicate areas where the G content is lower than the C content,\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5985964/v1/feb53f08a4d098d928954d50.png"},{"id":82395145,"identity":"f5e1285a-7daf-413b-89e5-f52ec0850b76","added_by":"auto","created_at":"2025-05-09 20:06:38","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":345483,"visible":true,"origin":"","legend":"\u003cp\u003eSSF 2 plasmid map. (A) Plasmid 1; (B) Plasmid 2. From the outside to the inside, the circles represent the following: COG functional annotation classified genes (arrows pointing clockwise indicate coding on the positive strand), genomic sequence position coordinates, genomic GC content (calculated with a window size of 500 bp and a step size of 20 bp). The inward blue regions indicate areas where the GC content is lower than the average GC content of the whole genome, while the outward red regions indicate the opposite, with higher peaks representing greater deviations from the average GC content. The innermost circle shows the genomic GC skew values (calculated with a window size of 500 bp and a step size of 20 bp), using the formula (G-C)/(G+C). The inward green regions indicate areas where the G content is lower than the C content, while the outward orange regions indicate the opposite.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5985964/v1/60099c2e6fca384e429ebd58.jpeg"},{"id":82395675,"identity":"fcf8b759-a102-495b-b8d2-f4c125fd266b","added_by":"auto","created_at":"2025-05-09 20:14:38","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":58884,"visible":true,"origin":"","legend":"\u003cp\u003eLength distribution of coding gene sequence\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-5985964/v1/52b675c9b1d077b57f68b62a.png"},{"id":82395149,"identity":"4b358fbf-b126-4ca1-8136-89f6e6c57249","added_by":"auto","created_at":"2025-05-09 20:06:38","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":65768,"visible":true,"origin":"","legend":"\u003cp\u003eStatistical map of gene distribution in gene island\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-5985964/v1/d66efd779f32b92bfbf9a178.png"},{"id":82396249,"identity":"b4af70fe-4b20-45e8-8ff5-f1823facce5a","added_by":"auto","created_at":"2025-05-09 20:30:38","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":34687,"visible":true,"origin":"","legend":"\u003cp\u003eNr Database Annotated Species Statistics Chart Note: The horizontal axis represents species IDs, and the vertical axis represents the number of annotated genes.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-5985964/v1/ce80f99390a820081cb70c10.png"},{"id":82395157,"identity":"08c14a93-aa77-4720-8b97-f01aac12b4d2","added_by":"auto","created_at":"2025-05-09 20:06:38","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":147813,"visible":true,"origin":"","legend":"\u003cp\u003eGene Function Annotation KEGG Metabolic Pathway Classification Map\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-5985964/v1/9374a61786378dbef11c8ddb.png"},{"id":82395676,"identity":"24481f48-a63c-441a-9511-93179dc85d05","added_by":"auto","created_at":"2025-05-09 20:14:38","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":151284,"visible":true,"origin":"","legend":"\u003cp\u003eGene Function Annotation GO Functional Classification Map Note: The x-axis represents the next level of GO terms under the three main GO categories, and the y-axis represents the number of genes annotated under each term (including its sub-terms). The three different classifications represent the three basic GO categories (from left to right: biological process, cellular component, and molecular function).\u003c/p\u003e","description":"","filename":"floatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-5985964/v1/18bcb6bb236072d82688affa.png"},{"id":82396546,"identity":"5ccce4db-aad3-492a-a290-fb4032044262","added_by":"auto","created_at":"2025-05-09 20:38:38","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":51662,"visible":true,"origin":"","legend":"\u003cp\u003eGene Function Annotation COG Functional Classification Map\u003c/p\u003e","description":"","filename":"floatimage12.png","url":"https://assets-eu.researchsquare.com/files/rs-5985964/v1/af780707466afb717de35a74.png"},{"id":82395684,"identity":"ef980d08-83cb-4b1a-9d9a-4dd145b2e3cb","added_by":"auto","created_at":"2025-05-09 20:14:38","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":18651,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional Classification Of CAZy And Statistical Map Of Corresponding Gene Guantity\u003c/p\u003e","description":"","filename":"floatimage13.png","url":"https://assets-eu.researchsquare.com/files/rs-5985964/v1/72ee28c13a319ad951b9d2af.png"},{"id":84641683,"identity":"2d6c3709-b047-4ede-9db6-433dfb226ef2","added_by":"auto","created_at":"2025-06-15 15:03:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2974369,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5985964/v1/3a22aaf2-8584-40fa-abed-7db5742bf920.pdf"},{"id":82395138,"identity":"4f93d478-78ec-46ce-a71d-fbab70cd7254","added_by":"auto","created_at":"2025-05-09 20:06:38","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14649,"visible":true,"origin":"","legend":"","description":"","filename":"table.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5985964/v1/34695c88a480eb94d5bb1d8f.xlsx"},{"id":82395672,"identity":"7dbfb0da-d985-46f9-99a6-67110209bbad","added_by":"auto","created_at":"2025-05-09 20:14:38","extension":"jpeg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":30487,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S1 Acid Production Reaction of the Strain with Bromocresol Purple.\u003c/p\u003e","description":"","filename":"floatimage14.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5985964/v1/676374abb9caf294d7929dc8.jpeg"}],"financialInterests":"","formattedTitle":"Screening and Whole-Genome Analysis of Sheep-Derived Lactic Acid Bacteria","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eProbiotics\u003c/em\u003e are a class of active microorganisms beneficial to the health of the host (humans or animals) (Mazziotta et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and have been proven to provide health benefits to the host when consumed in sufficient amounts (Szajewska et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). They are widely present in the host's oral cavity, skin, gastrointestinal tract and reproductive tract (Xiao et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Manzoor et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Tustumi et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2023\u003c/span\u003e, Guerra \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). To date, \u003cem\u003eprobiotics\u003c/em\u003e have become the fastest-growing animal feed additive globally (El-Saadony et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). \u003cem\u003eLactic acid bacteria\u003c/em\u003e (LAB) are an important component of \u003cem\u003eprobiotics\u003c/em\u003e ,(Rold\u0026aacute;n-P\u0026eacute;rez et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) known for promoting nutrient absorption, antibacterial and anti-infection properties, anti-tumor effects, regulation of gut microbiota, immune modulation, metabolism, and bioremediation (Gomand et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Nagarajan et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Raman et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). LAB have become an ideal choice for commercial development and are widely used in fields such as food, medicine, agriculture, and animal husbandry(Jiang et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Qi et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Ge et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). \u003cem\u003ePediococcus pentosaceus\u003c/em\u003e, a Gram-positive bacterium belonging to the family Streptococcaceae and the genus Pediococcus, is a type of lactic acid bacterium(Kim et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). \u003cem\u003eP. pentosaceus\u003c/em\u003e is widely distributed and has been isolated from fermented foods, aquatic products, animal products, and plant products(Nagy et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Due to its bacteriocins' inhibitory effects on various pathogens and its good thermal stability, \u003cem\u003ePediococcus pentosaceus\u003c/em\u003e has attracted significant attention from researchers(Kahraman-Ilıkkan \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\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 \u003cem\u003eprobiotics\u003c/em\u003e industry is developing rapidly, but it lacks relevant standards, resulting in inconsistent product quality(Phuengjayaem et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Whole-genome sequencing technology provides comprehensive genetic information for the study of \u003cem\u003elactic acid bacteria\u003c/em\u003e, enabling the revelation of metabolic characteristics, potential probiotic functions, and safety of strains(Rivas et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Through genome analysis, it is possible not only to explore the ecological adaptation mechanisms of \u003cem\u003elactic acid bacteria\u003c/em\u003e 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(Langille et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, Huang et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\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 \u003cem\u003elactic acid bacteria\u003c/em\u003e derived from Chahar sheep remains quite limited. Existing research mainly focuses on the diversity of microbial communities and preliminary functional exploration, lacking systematic analysis(Behboudi et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In this study, \u003cem\u003elactic acid bacteria\u003c/em\u003e were isolated from the feces of Chahar sheep, and a strain 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":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCollection and Screening of Strains\u003c/h2\u003e \u003cp\u003eFresh fecal samples from six healthy adult female Chahar sheep were collected in Hangjin Banner, Ordos City, Inner Mongolia Autonomous Region, China (longitude 107\u0026deg;54'21\" E, latitude 39\u0026deg;56'56\" N). A 5 g sample was weighed and added to 45 mL of sterile water, then serially diluted to concentrations ranging from 10⁻\u0026sup1; to 10⁻⁹ g/mL. Subsequently, 200 \u0026micro;L of the 10⁻⁵ to 10⁻⁷ dilutions were applied to 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, sodium carboxymethyl cellulose 20 g/L, agar 20 g/L, pH\u0026thinsp;=\u0026thinsp;7.0). The plates were incubated at 37\u0026deg;C for 24 hours, and single colonies that produced acid (indicated by a color change to yellow) were selected. To ensure the selected colonies were pure, repeated purification and microscopic examination were performed. The purified colonies were preserved in MRS broth (Hope Bio-technology Co., Ltd., Qingdao, China) supplemented with 20% glycerol at -80\u0026deg;C.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMorphological Identification of Strains\u003c/h3\u003e\n\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\n\u003ch3\u003eBiochemical Identification of Strains\u003c/h3\u003e\n\u003cp\u003eThe strains were incubated at 37\u0026deg;C for biochemical identification. The results of the \u003cem\u003elactic acid bacteria\u003c/em\u003e physiological and biochemical identification tests were compared with the instructions provided in the \u003cem\u003elactic acid bacteria\u003c/em\u003e biochemical identification kit (Company, SHBG13) to evaluate each indicator. All indicator results were cross-referenced with \u003cem\u003eBergey\u0026rsquo;s Manual of Systematic Bacteriology\u003c/em\u003e and the \u003cem\u003eManual for the Systematic Identification of Common Bacteria\u003c/em\u003e. The tested indicators included esculin, cellobiose, maltose, mannitol, salicin, sorbitol, sucrose, raffinose, inulin, lactose, and hippuric acid.\u003c/p\u003e\n\u003ch3\u003eMolecular Biological Identification of Strains\u003c/h3\u003e\n\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 sequencing. 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\n\u003ch3\u003eEvaluation of Biological Functions of Strains\u003c/h3\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAcid Production Ability Test of Strains\u003c/h2\u003e \u003cp\u003e \u003cem\u003eLactic acid bacteria\u003c/em\u003e were inoculated and cultured at 37\u0026deg;C with shaking at 150 r/min. The pH value of the bacterial culture was measured at 0, 2, 4, 6, 8, 12, 16, 24, and 36 h.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDetermination of Strain Growth Performance\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003eLactic acid bacteria\u003c/em\u003e were inoculated and cultured at 37\u0026deg;C. The OD600 values were measured at 0, 2, 4, 6, 8, 12, 16, 24, and 38 h using a microplate reader to plot the growth curve of the \u003cem\u003elactic acid bacteria.\u003c/em\u003e\u003c/p\u003e\n\u003ch3\u003eBile Salt Tolerance of Strains\u003c/h3\u003e\n\u003cp\u003eBile salt tolerance test: \u003cem\u003eLactic acid bacteria\u003c/em\u003e 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 12 hours. The culture was then inoculated into MRS broth containing 0.3% bile salts at a 2% (v/v) inoculation rate. The OD600 values were measured at 0, 2, 4, 6, 8, 12, 16, 24, and 38 hours under 37\u0026deg;C conditions. Data were recorded and analyzed.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTolerance of Strains to Simulated Intestinal Fluids\u003c/h2\u003e \u003cp\u003e \u003cem\u003eLactic acid bacteria\u003c/em\u003e 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), respectively. The cultures were incubated at 37\u0026deg;C with shaking at 150 r/min. Viable cell counts were determined using the plate spreading method, and survival rates were calculated as follows:\u003c/p\u003e \u003cp\u003eSurvival rate = (colony count at measurement time / colony count at time 0) \u0026times; 100%\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of Probiotic Effects of Strains\u003c/h2\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003eHemolytic Activity Test\u003c/h2\u003e \u003cp\u003e \u003cem\u003eLactic acid bacteria\u003c/em\u003e were inoculated into MRS medium at a 2% (v/v) inoculation rate and cultured at 37\u0026deg;C with shaking at 150 r/min for 24 hours. The activated bacterial culture was streaked onto Columbia blood agar plates (Beikman Bioengineering Co., Ltd., Changde, China) and incubated at 37\u0026deg;C for 24 hours in a constant-temperature incubator. The colony morphology and hemolytic activity were observed. \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (CMCC 26003) was used as the control strain.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAntibiotic 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=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eAntibacterial Activity Test\u003c/h2\u003e \u003cp\u003eThe antibacterial activity of the strain was determined using the Oxford cup agar diffusion method. \u003cem\u003eLactic acid bacteria\u003c/em\u003e were inoculated into MRS medium at a 2% (v/v) inoculation rate and cultured at 37\u0026deg;C with shaking at 150 r/min for 24 hours. The supernatant was collected and set aside. Fresh bacterial cultures (200 \u0026micro;L each) of \u003cem\u003eSalmonella enteritidis\u003c/em\u003e (CMCC 50071 ), \u003cem\u003eEscherichia coli\u003c/em\u003e (ATCC 25922 ), and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (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 hours, and the formation of inhibition zones was observed. The diameter of the inhibition zones was measured.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eWhole Genome Sequencing and Analysis\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=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eLibrary 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=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eGenome 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=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eGene 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)(Patel et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) 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)(Niu et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Gene islands were predicted using IslandPath-DIOMB software (Version 0.2)(Sun et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and prophages were identified using PhiSpy software (Version 2.3)(Lee et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Finally, CRISPR sequences (Clustered Regularly Interspaced Short Palindromic Repeats) were predicted using CRISPRdigger (Version 1.0)(Zhang et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2023\u003c/span\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"},{"header":"Results and Discussion","content":"\u003cp\u003e\u003cstrong\u003eIsolation and Identification of Lactic Acid Bacteria\u003c/strong\u003e\u003c/p\u003e\n\u003cp id=\"_Toc106277044\"\u003eColonies that caused the surrounding medium to change from purple to yellow in the bromocresol purple color reaction were selected, indicating good acid production ability \u003cstrong\u003e(Figure S1)\u003c/strong\u003e(Garbacz 2022). The selected strain was designated as SSF2. 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. 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 \u003cstrong\u003e(Figure 1)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eThe physiological and biochemical identification results of strain SSF2 are shown in \u003cstrong\u003eTable\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e1\u003c/strong\u003e. The results indicate that strain SSF2 can hydrolyze various carbohydrates, and it is preliminarily identified as\u003cem\u003e\u0026nbsp;Lactococcus\u003c/em\u003e(Darbandi et al. 2022).Subsequently, homology analysis based on the 16S rRNA sequencing results showed that strain SSF2 had high homology (98.7%) with \u003cem\u003eP. pentosaceus\u003c/em\u003e. Combining the morphological characteristics, physiological and biochemical properties, and 16S rDNA sequence analysis, strain SSF2 was ultimately identified as P. \u003cem\u003epentosaceus\u0026nbsp;\u003c/em\u003eand named P. \u003cem\u003epentosaceus\u0026nbsp;\u003c/em\u003eSSF2 \u003cstrong\u003e(Figure 2)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEvaluation of the Biological Functions of the Strain\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe acid production performance of P. \u003cem\u003epentosaceus\u0026nbsp;\u003c/em\u003eSSF2 was assessed. The pH of the fermentation broth decreased rapidly during the 0\u0026ndash;16 h fermentation period and stabilized after 24 h. The pH dropped most rapidly during the first 0\u0026ndash;2 h of fermentation, falling below 5.0 \u003cstrong\u003e(Figure 3A)\u003c/strong\u003e, Indicating that the strain has the ability to rapidly initiate lactic acid fermentation. This rapid acid-lowering ability plays an important role in the initial stability of fermented products and the inhibition of contaminant microbial growth(Darbandi et al. 2022, Garbacz 2022, Zhang et al. 2023). In 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 (\u003cstrong\u003eFigure 3B)\u003c/strong\u003e. This stability is particularly important for industrial applications as it helps ensure the consistency and controllability of fermented product quality.\u003c/p\u003e\n\u003cp\u003eAdditionally, the strain\u0026apos;s tolerance to a 0.3% bile salt environment (\u003cstrong\u003eFigure 3C\u003c/strong\u003e) demonstrates its ability to resist the damaging effects of bile salts on the cell membrane and maintain stability in the intestinal tract (Ren et al. 2018, Liu et al. 2022). The simulated intestinal fluid experiment (\u003cstrong\u003eFigure 3D\u003c/strong\u003e) further confirms SSF2\u0026apos;s adaptability under intestinal conditions, which is a critical indicator for \u003cem\u003eprobiotics\u003c/em\u003e to maintain viability and exert their functional benefits.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEvaluation of the Probiotic Effects of the Strain\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eP. pentosaceus\u0026nbsp;\u003c/em\u003eSSF2 does not produce hemolytic zones, indicating that it is a safe strain and can be applied as a potential probiotic in production\u003cstrong\u003e\u0026nbsp;(Figure 4A)\u003c/strong\u003e(Pereira et al. 2022). Additionally, the strain exhibited varying sensitivity to 30 antibiotics. It exhibited good sensitivity to clindamycin (lincomycin), chloramphenicol, furazolidone (nifuroxazide), midecamycin, erythromycin, doxycycline, neomycin, gentamicin, cefoperazone, ceftazidime (Fortum), cephaloridine, cefazolin, and cefalexin, indicating a low risk of spreading antibiotic resistance in the food chain or medical environments(Zheng et al. 2020) . It showed moderate sensitivity to tetracycline, kanamycin, and amikacin, while displaying resistance to polymyxin B, vancomycin, ciprofloxacin, ofloxacin, and norfloxacin \u003cstrong\u003e(Supplementary Table 1)\u003c/strong\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 (21.87 \u0026plusmn; 0.55a mm). The second strongest inhibitory effect was against\u003cem\u003e\u0026nbsp;Salmonella\u003c/em\u003e, with an inhibition zone diameter of 19.17 \u0026plusmn; 0.57b mm . This antibacterial property may be relat0ed to the secretion of its metabolites, such as organic acids, hydrogen peroxide, and bacteriocins(Fugaban et al. 2022) . In particular, the strong inhibitory effect against \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e indicates that SSF2 has great potential for application in seafood preservation and food safety(Guo et al. 2020) .\u003c/p\u003e\n\u003ch2\u003eWhole genome sequencing and analysis\u003c/h2\u003e\n\u003ch2\u003eGenome Assembly and Prediction\u003c/h2\u003e\n\u003cp\u003eThe genome of SSF 2 is 1,853,794 bp in size with a GC content of 37.23%. 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 final genome assembly results are detailed in \u003cstrong\u003eTable\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e3\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Genomic circular map analysis\u003c/p\u003e\n\u003cp\u003eThe assembled genome sequences of the sequencing samples, combined with the predicted results of coding genes, were visualized using the Circos software. If analyses of non-coding RNA and gene functional annotation were also performed, the corresponding results are displayed in the diagram as well. The whole-genome map is shown in \u003cstrong\u003eFigure\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e5\u003c/strong\u003e, and Plasmids P1/P2 are shown in \u003cstrong\u003eFigure 6\u003c/strong\u003e.\u003c/p\u003e\n\u003ch2\u003eGenome Component Analysis\u003c/h2\u003e\n\u003ch2 id=\"_Toc122175764\"\u003e\u0026nbsp;Prediction Results of Coding and Non-coding RNA Genes\u003c/h2\u003e\n\u003cp\u003eThe coding gene prediction for the newly sequenced genome was performed using GeneMarkS software, revealing the following results: the total number of genes is 1,844, with a total gene length of 1,637,667 bp. The average length of coding genes is 888 bp, and the total length of coding regions accounts for 88.34% of the entire genome. The gene lengths are primarily distributed between 200 and 1,500 bp, with a GC content of 37.23% \u003cstrong\u003e(Figure 7)\u003c/strong\u003e. The prediction results for non-coding RNA in strain SSF 2 are shown in Table 4, indicating 55 tRNA genes and no rRNA genes.\u003c/p\u003e\n\u003ch2\u003eRepetitive Sequence Prediction\u003c/h2\u003e\n\u003cp\u003eRepetitive sequences were predicted using RepeatMasker software for interspersed repeats, and TRF (Tandem Repeats Finder) was used to identify tandem repeats in the DNA sequence. The prediction results for interspersed and tandem repeats in strain SSF 2 are as follows: six short interspersed nuclear elements (SINEs) with a total length of 373 bp, 16 long interspersed nuclear elements (LINEs) with a total length of 1,517 bp, and 17 DNA transposons (DNA elements) with an average length of 84 bp. Repetitive sequences account for 43.74% of the genome.The high proportion of DNA transposons and LINEs suggests that the genome may have undergone multiple exogenous DNA integration events, which are common in fermentation environment strains (such as those exposed to phages or plasmids) . The enrichment of repetitive sequences may enhance the strain\u0026apos;s rapid evolutionary ability in variable environments (such as pH and salinity fluctuations), promoting local gene rearrangements through homologous recombination .\u003c/p\u003e\n\u003ch2\u003eCRISPR \u0026nbsp; Sequence Prediction\u003c/h2\u003e\n\u003cp\u003eThe prediction results of transposons and CRISPR sequences are shown in Table 5.\u003c/p\u003e\n\u003ch2\u003ePrediction of genomic islands and prophages\u003c/h2\u003e\n\u003cp\u003eThe prediction results of genomic islands and prophages are shown in Table 6 and Figure 8. 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)(Peng et al. 2023) .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 \u003cem\u003eVibrio parahaemolyticu\u003c/em\u003es, Figure 3D)(Bertelli et al. 2022) .\u003c/p\u003e\n\u003ch2\u003eGene Function Annotation\u003c/h2\u003e\n\u003ch2\u003eNR Annotation Results\u003c/h2\u003e\n\u003cp\u003eThe NR annotation results are shown in \u003cstrong\u003eFigure\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e9\u003c/strong\u003e, where SSF2 has the highest number of matched genes with \u003cem\u003eP\u003c/em\u003e\u003cem\u003e.\u003c/em\u003e\u003cem\u003e\u0026nbsp;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\u003c/em\u003e\u003cem\u003e.\u003c/em\u003e\u003cem\u003e\u0026nbsp;pentosaceus\u003c/em\u003e, further confirming the taxonomic classification of the strain.\u003c/p\u003e\n\u003ch3\u003eKEGG annotation Results\u003c/h3\u003e\n\u003cp\u003eBased on the KEGG annotation information, the functions of \u003cem\u003eP\u003c/em\u003e\u003cem\u003e.\u003c/em\u003e\u003cem\u003e\u0026nbsp;pentosaceus\u003c/em\u003e SSF 2 were analyzed (Figure 10). A total of six classification levels (KEGG A classes) were identified, including 44 genes annotated as related to Cellular Processes, 105 genes related to Environmental Information Processing, 156 genes related to Genetic Information Processing, 54 genes related to Human Diseases, 801 genes related to Metabolism, and 22 genes related to Organismal Systems.The highly specialized metabolic network is prominent, which may be for rapid energy acquisition and product synthesis(Santoriello and Bassler 2024) . 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 (Figure 3D)(Zhang et al. 2023) . 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(Vermeulen et al. 2021) . 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 (Figure 3C)(Szczyrek et al. 2021) .\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eGO Annotation Results\u003c/h3\u003e\n\u003cp\u003eThrough analysis using the GO database, a total of 6,292 annotated gene sequences were identified, categorized into three main categories and 46 subcategories (Figure 11). Among them, 3,190 sequences were classified under biological processes, 1,325 under cellular components, and 1,777 under molecular functions. 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(Lewis et al. 2024) . Limited synthetic genes (such as branched-chain amino acids) may restrict its growth in low-nitrogen environments, requiring exogenous supplementation(Wang et al. 2024) . In terms of environmental adaptability, the enrichment of acid tolerance-related genes (such as F₀F₁-ATPase) is shown, explaining its rapid pH decline ability (Figure 3A)(Li et al. 2023) . 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.\u003c/p\u003e\n\u003ch3\u003eCOG Annotation Results\u003c/h3\u003e\n\u003cp\u003eThe COG database can be divided into 26 functional categories, and the statistical results are shown in\u003cstrong\u003e\u0026nbsp;Figure 12\u003c/strong\u003e. Among them, there are 185 genes related to translation, ribosomal structure, and biogenesis; 94 genes related to replication, recombination, and repair; 136 genes related to carbohydrate transport and metabolism; and 109 genes related to amino acid transport and metabolism.\u003c/p\u003e\n\u003ch3\u003eCAZy Annotation Results\u003c/h3\u003e\n\u003cp\u003eThe CAZy annotation results for \u003cem\u003eP. pentosaceus\u003c/em\u003e SSF2 are shown in \u003cstrong\u003eFigure 13\u003c/strong\u003e, indicating a total of 57 carbohydrate-related enzymes classified into four categories. These include 19 glycosyltransferases (Glycosyl Transferases), 26 glycoside hydrolases (Glycoside Hydrolases), 9 carbohydrate-binding modules (Carbohydrate-Binding Modules), and 3 carbohydrate esterases (Carbohydrate Esterases).The GH28 present therein can explain the ability to degrade plant cell walls(Carli et al. 2019) .\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, a whole-genome analysis of \u003cem\u003eP. pentosaceus\u003c/em\u003e SSF2 was conducted, revealing its exceptional biological characteristics and potential application value. The genome size is 1,853,794 bp with a GC content of 37.23%, containing 1,844 coding genes. Functional annotation of the genes indicates its rich biological functions in carbohydrate metabolism, amino acid metabolism, and other pathways. SSF2 exhibits excellent bile salt resistance, and survival ability in simulated gastrointestinal fluids, along with significant antibacterial activity against various pathogenic bacteria. The strain shows no hemolytic activity, indicating it is a safe strain suitable for potential probiotic applications in food production. Overall, \u003cem\u003eP. pentosaceus\u003c/em\u003e SSF2 demonstrates broad application prospects in the fields of food fermentation and health.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSubmission statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study has been approved by the Ethics Review Committee of Jining Normal University (approval number: [2024-ER-0456]). All participants signed informed consent prior to participating in the study. All authors have read and agreed to the final version of the manuscript and agreed to contribute it to the [Annals of Microbiology]. All data and materials generated in this study were obtained from the corresponding authors upon reasonable request. The authors declare that this study is free of any conflicts of interest that might affect the fairness of the findings.\u003c/p\u003e\n\u003cp\u003eThe authors\u0026apos; 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 critically reviewed the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study was supported by Key technologies in Ulanqab Project assignment (2021GJ105), Jining Normal University PhD Innovation Fund (jsbsjj2306)，Central Guidance for Local Science and Technology Development Fund Project (2024ZY0098), Subproject of the National Key Research and Development Program (2024YFD2000505-03), National Natural Science Foundation of China (32460830), Ordos Key Research and Development Program Project (YF20240039). We thank them for their support. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\u003c/p\u003e\n\u003cp\u003eWe sincerely thank the editorial department of Annals of Microbiology and the reviewers for their review and valuable comments on this paper.\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets [GENERATED/ANALYZED] for this study can be found in the [NAME OF REPOSITORY] [LINK].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Conflict of Interest\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eKey technologies in Ulanqab Project assignment (2021GJ105), Jining Normal University PhD Innovation Fund (jsbsjj2306)，Central Guidance for Local Science and Technology Development Fund Project (2024ZY0098), Subproject of the National Key Research and Development Program (2024YFD2000505-03),\u0026nbsp;National Natural Science Foundation of China\u0026nbsp;(32460830),\u0026nbsp;Ordos Key Research and Development Program Project (YF20240039).\u003c/p\u003e\n\u003cp\u003eAuthor Contributions\u003c/p\u003e\n\u003cp\u003eConceptualization, data analysis, and drafting the manuscript, Lingbai Yao and Yao Huang; sampling, and determination, Linchong Zhang, Yusheng Wang, Jun Jia, and Zhaoshui Xing; data analysis: Xuran Hai, \u0026nbsp; Wurentana and Swee Sen Teo; supervision, review, and editing, Shaofeng Su, Chao Cheng and Haiqing Wu. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003eAcknowledgments\u003c/p\u003e\n\u003cp\u003eWe would like to thank Dr. Zhao Junli for his contribution during the manuscript preparation process.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBehboudi R, Nouri-Baygi M, Naghibzadeh M (2023) RPTRF: A rapid perfect tandem repeat finder tool for DNA sequences. BioSystems 226:104869\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBertelli C, Gray KL, Woods N, Lim AC, Tilley KE, Winsor GL, Hoad GR, Roudgar A, Spencer A, Peltier J, Warren D, Raphenya AR, McArthur AG, Brinkman FSL (2022) Enabling genomic island prediction and comparison in multiple genomes to investigate bacterial evolution and outbreaks. 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Int J Syst Evol Microbiol 70(4):2782\u0026ndash;2858\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 6 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"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},"keywords":"Lactic acid bacteria, Pediococcus pentosaceus, probiotic functionality, whole genome, antibacterial activity","lastPublishedDoi":"10.21203/rs.3.rs-5985964/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5985964/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eProbiotics\u003c/em\u003e (\u003cem\u003elactic acid bacteria\u003c/em\u003e) are widely used as microbial feed additives in livestock production and play an important role in preventing and treating animal diarrhea as well as regulating host immune function. In this study, \u003cem\u003elactic acid bacteria\u003c/em\u003e were isolated from the fresh feces of healthy adult female sheep, and their biological characteristics were analyzed. Based on phylogenetic analysis, strain SSF2 was identified as \u003cem\u003ePediococcus pentosaceus\u003c/em\u003e. SSF2 exhibited tolerance to acid, bile salt concentrations, and simulated artificial gastrointestinal environments. The hemolysis test for SSF2 was negative, it was sensitive to commonly used antibiotics, and it demonstrated significant antibacterial and antioxidant activities, indicating its excellent probiotic potential. Whole-genome sequencing (WGS) was performed using the HiSeq 2500 platform and the PacBio system to explore the genetic characteristics of SSF2. The genome was revealed to consist of a circular chromosome and two plasmids, with sizes of 1,785,410 bp, 10,618 bp, and 57,766 bp, and GC contents of 37.23%, 34.95%, and 40.98%, respectively. The genome was predicted to contain five genomic islands, six prophages, and a potential CRISPR gene editing sequence. Functional annotation through databases such as COG, GO, and KEGG revealed that most genes are related to carbon metabolism, protein and amino acid metabolism, nucleotide metabolism, and membrane transport processes. This study indicates that an in-depth understanding of the functionality and genetic characteristics of \u003cem\u003ePediococcus pentosaceus\u003c/em\u003e SSF2 may enable the potential application of this strain in sheep feed supplements.\u003c/p\u003e","manuscriptTitle":"Screening and Whole-Genome Analysis of Sheep-Derived Lactic Acid Bacteria","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-09 20:06:33","doi":"10.21203/rs.3.rs-5985964/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":"2d72699b-378a-4318-a319-53a95dd5c064","owner":[],"postedDate":"May 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-15T14:55:41+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-09 20:06:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5985964","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5985964","identity":"rs-5985964","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}