WGS analysis and Functional Studies Illustrate Promising Gene- signatures for Probiotic Attributes and Molecular-targeted Therapeutic Prospects of Lactiplantibacillus plantarum LP-ARP2

WGS analysis and Functional Studies Illustrate Promising Gene- signatures for Probiotic Attributes and Molecular-targeted Therapeutic Prospects of Lactiplantibacillus plantarum LP-ARP2 | 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 WGS analysis and Functional Studies Illustrate Promising Gene- signatures for Probiotic Attributes and Molecular-targeted Therapeutic Prospects of Lactiplantibacillus plantarum LP-ARP2 Sinjini Patra, Ritwik Patra, Pradeep Kumar Das, Anshuman Dixit, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7556358/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 Gene-signatures for probiotic attributes expedite screening of novel probiotic strains as well as identify molecular basis of probiotic benefits. Therefore, strain-specific genomic-studies correlating functional-assays are in demand. Here, we present the same for Lactiplantibacillus plantarum LP-ARP2 using genomic-metabolomic-functional approaches. Genomic-features of LP-ARP2 are further compared with clinically relevant L. plantarum strains to elucidate gene-specific commonality. We find robust stress-resilience genetic set-up ( usp/hsl/clp/ ABC-transporters/ ATP-synthase/chaperons dna/gro/grp ) in LP-ARP2 -genome. Our acid/bile tolerance assays also indicate survivability (> 60%) of the strain in harsh conditions. Presence of adhesion-related ( lspA/mapA/eno/srtA/ glycosyltransferases/glycosylhydrolases/lipoproteins) and biofilm-forming genes ( veg/luxS ) further align with its efficacy in autoaggregation (> 60%), adhesion (Caco-2), and biofilm-formation (24 h). CAZyme-genes with significant prebiotic utilization indicate the strain’s ability for gut-microbial-modulation and adaptation. Metabolic-profiling of LP-ARP2 -derived-CFS (HRMS analysis) validates the presence of related genes for SCFAs/vitamins/amino-acids/neurotransmitters GABA/serotonin/acetylcholine etc. Besides, many metabolites are reported antimicrobials. Indeed LP-ARP2 shows significant antibacterial potential against multidrug-resistant bacteria (Gram-positive/Gram-negative), gut-pathogen Salmonella Typhimurium and pathogenic-biofilm (MRSA). Presence of antioxidant-genes in LP-ARP2 -genome (thioredoxin/NADH-dependent- nox/npr/ndh/ glutathione-reductase/glutaredoxin/catalase/peroxidases/methionine sulfoxide reductase) are validated by high radical-scavenging activity of LP-ARP2 (ABTS > 40%, DPPH 25 U/mL, superoxide > 80%, and hydroxyl > 70%). Moreover, in-silico functional-network-analysis reveals LP-ARP2 -derived metabolites target oxidative stress, neuroinflammation, amyloid-beta metabolism, tau-phosphorylation, neurogenesis, and synaptic function, indicating molecular relevance of the therapeutic potential of LP-ARP2. Fascinatingly, genomic-analysis between LP-ARP2 with clinically relevant (depression and intestinal disorders) L. plantarum strains (299v and Lp01) elucidate comparable genetic-features for beneficial probiotics. Thus, study offers potential gene-signatures for probiotic-benefits of L. plantarum and project LP-ARP2 as a promising probiotic with antibacterial, antioxidant and psychobiotic potential. Microbial dysbiosis Probiotics Whole genome sequencing (WGS) analysis Metabolic profiling Human-target based functional-network analysis Gene-signatures Antioxidant and antimicrobial activities Psychobiotic potential Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 1. Introduction Chronic inflammation damages gut-health, causes intestinal disorders. Through gut-organ axes, it further leads to a wide spectrum of systemic diseases like cancer, atherosclerosis, type 2 diabetes, respiratory diseases, and neuropsychiatric disorders (e.g., Alzheimer’s, Parkinson’s, schizophrenia) [ 1 ]. The global burden of diseases related to the gut-organ axis, limited clinical trials, and various side effects of the current therapies highlight the need for safer therapeutic strategies [ 2 , 3 ]. Probiotics could restore gut microbial balance, reduce inflammation, and prevent gut and gut-organ related diseases [ 4 , 5 ]. Therefore, global demand for probiotic supplements is rapidly growing, projected to reach $ 3.5 billion by 2026, reflecting the increasing emphasis on probiotics in health and disease prevention [ 6 ]. Lactic acid bacteria (LAB), especially Lactobacillus species, are the most studied and widely used probiotics, considered GRAS (Generally Recognized as Safe or Qualified Presumption of Safety) by the FDA (Food and Drug Administration) and EFSA (European Food Safety Authority) [ 7 , 8 ]. Among them, Lactiplantibacillus plantarum (formerly Lactobacillus plantarum ) stands out for its versatility and adaptability across diverse environments, from the human body to various fermented products [ 9 ]. Moreover, the broad-spectrum health benefits of L. plantarum range from enhancing gut barrier integrity, immunomodulation, and prevention of infection to lowering cholesterol, indicating widespread applications of the strain in food and pharmaceutical industries [ 9 , 10 ]. However, probiotic effects are highly strain-specific, limiting the use of a single strain for all clinical or industrial purposes [ 11 ]. Therefore, targeted, criteria-based selection and detailed characterization of probiotics are in demand [ 11 , 12 ]. Despite the availability of over 670 annotated L. plantarum genomes in NCBI, only a few strains (299v and Lp01) are commercially used or clinically studied, due to significant genomic variability [ 13 ]. Even detailed genomic mining linking the genomic blueprint to probiotic and functional effects of 299v and Lp01 are also not studied in detail. Whole genome sequencing (WGS) analysis could reveal the probiotic-specific gene signatures to ensure safe and effective applications of the strains. However, a detailed strain-specific WGS study with experimental validation for L. plantarum strains is limited. Moreover, the molecular basis of its therapeutic benefits in disease systems is poorly understood [ 14 ]. Here, we aim to conduct a comprehensive strain-specific genomic, metabolomic and functional study on L. plantarum LP-ARP2 , focusing on the target-based antimicrobial, antioxidant, and psychobiotic properties of the strain. The study presents a thorough WGS analysis of LP-ARP2 , with mining of gene signatures of L. plantarum strains 299v and Lp01 for probiotic-specific functional traits. Interestingly, the genomic properties of LP-ARP2 are comparable to 299v and Lp01 that have been extensively studied in multiple clinical trials and confer beneficial effects in depression, irritable bowel syndrome, and other intestinal disorders [ 30 – 32 ]. Such comparative genomic analysis between the L. plantarum strains ( LP-ARP2 , 299v, and Lp01) could lead to infer species-specific genetic markers for probiotic benefits. Our experimental assays further validate the genomic predictions of the strain LP-ARP2 for the crucial probiotic attributes like acid/bile tolerance, autoaggregation, surface hydrophobicity, adhesion capacity, and biofilm formation. Remarkable prebiotic utilization of the strain indicates potential for gut adaptation and microbiota modulation. In accordance with the predicted gene signatures from WGS analysis, functional assays validate strong antioxidant activity, broad-spectrum antimicrobial effects, and anti-biofilm potential of LP-ARP2- derived cell-free supernatant (CFS). The metabolic profile of CFS by high-resolution mass spectrometry (HRMS) study reveals various bioactive metabolites with health-promoting potential and industrial applications. Target-based functional network analysis with the HRMS-identified metabolites further indicates that LP-ARP2 -derived metabolites could target important neuro-domains through which LP-ARP2 could manifest its psychobiotic properties to alleviate neurodegenerative disorders like Alzheimer’s disease. Such a strategy, using in vitro HRMS data ( LP-ARP2 -derived metabolites) to perform in silico target-based network analysis to find out potential molecular signatures for probiotic function, is novel for any probiotic system. In summary, LP-ARP2 , offers multifunctional therapeutic promise, particularly prominent for antimicrobial, antioxidant, and psychobiotic effects. The study confers promising gene-signatures for probiotic attributes of Lactiplantibacillus plantarum strains and opens up a new avenue to explore the molecular basis of probiotic-derived health-benefits in disease systems ( Fig. 1 ) . 2. Materials and Methods 2.1. Bacterial Culture Pure culture of LP-ARP2 ( L. plantarum MTCC 2621) was obtained from the Microbial Type Culture Collection (MTCC), IMTECH, Chandigarh, India. The strain was routinely cultured with MRS agar and broth (HiMedia) in a standard anaerobic environment. LP-ARP2 and reference strains, L. acidophilus DDS1 and L. rhamnosus GG, were stored at − 80°C in 50% glycerol for long-term use. 2.2. Whole Genome Sequencing and Genomic Characterization of LP-ARP2 2.2.1. Isolation of DNA and preparation of library LP-ARP2 was grown anaerobically in MRS broth at 37°C for 12 h. The genomic DNA was extracted using the QiaAmp DNA mini kit (Cat# 51306), and quantified with the Qubit Fluorometer 3 and the Qubit dsDNA High Sensitivity Assay Kit (Invitrogen, Cat# Q32854). Library preparation was performed using the 5300 Fragment Analyzer (3.1.0.12) and ProSize software 4.0.0.3. Pair-end sequencing (151 bp read length) of the DNA libraries was conducted with the NovaSeq6000 platform (MedGenome, Bangalore, India). Quality-checked sequences were exported as fastq files. Strain identity was confirmed by comparing 16S rRNA gene sequences with reference sequences in NCBI using BLAST. Phylogenetic analysis was conducted using the Neighbor-joining (NJ) method in MEGA12 [ 15 ]. 2.2.2. Determination of the strain identity by whole genome sequencing and genome assembly The complete genome of LP-ARP2 was sequenced [NovaSeq6000 (MedGenome, Bangalore, India)]. The reads were screened for human DNA contamination; ∼10.06–27.65% aligning to the human genome were excluded. The remaining data were quality-checked for sequence quality score distribution, GC content, base quality score, over-represented sequences, average base content per read, and possible PCR or adapter artifacts. Based on these parameters, low-quality reads were trimmed, and adapters were removed from the 3’ ends using fastq mcf (1.04.803). The refined reads were aligned to the Lactiplantibacillus plantarum strain ATCC 8014 reference genome for coverage estimation and reference-guided assembly. Consensus fasta files were developed using Samtools tools (v1.2), while coverage and depth statistics were generated using bedtools (v2.0) and an in-house Perl script. GATK was used to predict variations from the reference-aligned reads, and snpEff to annotate the variants. Genome annotation, including prediction of genes, CDS, and genomic features, was carried out using the Prokaryotic Genome Annotation System (Prokka v1.14.6) [ 16 , 17 ]. The complete genome sequence is available in NCBI under Bioproject PRJNA1248240. 2.2.3. Genomic analysis of LP-ARP2 in comparison to clinically studied L. plantarum strain s (299v and Lp01) Prophage sequences in the LP-ARP2 genome were identified using Rapid Annotations using Subsystems Technology (RAST) server with SEED viewer v2.0. CRISPR, Cas, and truncated Cas sequences were screened using CRISPRCasFinder 1.1.2 [ 18 ]. The Resistance Gene Identifier Tool (RGI 6.0.3) and the Comprehensive Antibiotic Resistance Database (CARD 3.3.0) were used to identify the antibiotic resistance genes (perfect/rigorous hit, high-quality coverage) [ 19 , 20 ]. Acquired antimicrobial resistance genes and/or chromosomal alterations were assessed using ResFinder 4.4.2 (%ID 90.00%, minimum length 60%) [ 21 ]. Virulence and toxin-associated genes were detected by BLAST search against the virulence factor database (VFDB) ( http://www.mgc.ac.cn/cgi-bin/VFs/v5/main.cgi .) [ 22 ]. PathogenFinder 1.1 analyzed potential pathogenicity [ 23 ]. Prokka (1.14.6) and EggNOG-mapper v2.1.12 (Egg-NOG v5.0) were used for functional annotation of LP-ARP2 , L. plantarum 299v, and L. plantarum Lp01 genomes [ 24 ]. Carbohydrate-active enzymes (CAZymes) were identified with dbCAN3 [ 25 ]. KEGG mapper and RAST were employed to annotate pathways linked to probiotic properties [ 26 ]. Biosynthetic gene clusters (BGCs) for bacteriocins were investigated by BAGEL4 [ 27 ]. While primary and secondary metabolites encoding gene clusters were predicted using gutSMASH (Specialised Primary Metabolite Analysis from Anaerobic Bacteria) [ 28 ] and antiSMASH v6.0.1 (strict mode), respectively [ 29 ]. Comparative genomic analysis with clinically studied L. plantarum strains (299v and Lp01), reported to benefit depression, IBS, and other intestinal disorders, provided a framework to infer the potential health-promoting properties of LP-ARP2 [ 30 – 32 ]. 2.3. Validation of Probiotic Attributes of LP-ARP2 2.3.1. Tolerance of LP-ARP2 to acidic pH LP-ARP2 overnight culture was inoculated in MRS broth of pH 4 and pH 3 (pH 6.5, control), incubated at 37°C for 0, 1, 3, and 5 h [ 12 ] At each time point, the culture was serially diluted in PBS (pH 7.4) and plated on MRS agar, followed by anaerobic incubation for 24 h at 37°C. Colony counts were expressed as biomass (Log 10 CFU/mL), and survival rate (%) was calculated as [biomass at pH 4 or 3/biomass at control pH 6.5] x 100. The experiment was performed in triplicate. 2.3.2. Tolerance of LP-ARP2 to bile salt Overnight culture of LP-ARP2 was inoculated in MRS broth with 0.3% and 1% (w/v) bile salts (HiMedia) [ 12 ]. MRS broth without bile salt was the control. Cultures were incubated for 0, 1, 3, and 5 h at 37°C, serially diluted in PBS, and spread on MRS agar. After 24 h anaerobic incubation, biomass (Log 10 CFU/mL) was determined by colony counts. Survival rate (%) was calculated as [biomass at 0.3% or 1% bile salt/biomass in control] x 100. The experiment was carried out in triplicate. 2.3.3. Determination of the self-aggregation property of LP-ARP2 Overnight culture of LP-ARP2 was subcultured in MRS broth until OD 600 = 0.5–0.6, centrifuged (5000 rpm), washed, and resuspended in PBS [ 12 ]. The culture was adjusted to 10 8 CFU/mL (OD 600 0.25 ± 0.1, A 0 ), vortexed (10 sec), and incubated at 37°C for 1–24 h. The absorbance (600 nm) of the upper suspension was measured after each time interval (A time ). The autoaggregation percentage was calculated as [1–(A Time /A 0 ) × 100]. The experiment was carried out in triplicate. 2.3.4. Evaluation of cell surface hydrophobicity of LP-ARP2 In the Bacterial Attachment to Hydrocarbons (BATH) method, LP-ARP2 overnight culture was harvested, washed, and resuspended in 10 mL of phosphate urea magnesium sulphate (PUM) buffer to OD 600 = 0.8–0.9 (A 0 ). [ 30 ]. Adjusted cell suspension (4.8 mL) was mixed with 0.8 mL of n-hexadecane (Sigma)/xylene (Merck), incubated for 10 min, vortexed (2 min), and then incubated at 37°C for 2 h. Absorbance (600 nm) of the lower aqueous phase was measured (A), and hydrophobicity (H%) was calculated as [1–(A/A 0 ) × 100]. The experiment was carried out in triplicate. 2.3.5. Determination of adhesion property of LP-ARP2 using human colon adenocarcinoma cells (Caco-2) Caco-2 cells were cultured in DMEM (Himedia) with 10% heat-inactivated FBS (Gibco) and seeded at 1x10 5 cells/well in six-well plates. Cells were maintained until 80% confluent, followed by 20 days of differentiation to form monolayers [ 31 ]. Before bacterial treatment, the Caco-2 monolayer was washed and co-cultured with LP-ARP2 at 10 8 CFU/mL, followed by incubation for 2 h at 37°C under 5% CO 2 (Galaxy 48R, New Brunswick, Germany). Unattached bacteria were washed, cells were trypsinized, gently aspirated, serially diluted, and plated on MRS agar. After 24 h anaerobic incubation, adhesion rate (%) was expressed (R t /R 0 ) x 100, where R 0 = initial bacterial count (CFU/mL) and R t =count after incubation. The experiment was performed in triplicate. 2.3.6. Evaluation of the biofilm-forming ability of LP-ARP2 An 18 h LP-ARP2 culture in MRS broth was diluted to OD 600 = 0.1 and dispensed into 24-well plates, followed by anaerobic incubation for 24 h at 37°C in a moist chamber [ 32 ]. The media was disposed of after the incubation period, wells were washed and dried at 60°C for 1 h. Biofilms were stained for 45 mins using a 0.1% (w/v) crystal violet (SRL, prepared in 95% ethanol), residual stain was washed, air-dried, and dissolved in 33% acetic acid (Merck). Absorbance was measured at 570 nm. L. rhamnosus GG and L. acidophilus DDS1 served as positive controls. The assay was carried out in triplicate. 2.3.7. Prebiotic utilization of LP-ARP2 Prebiotic utilization of LP-ARP2 was determined in terms of prebiotic score (PS), acidification profile (pH), and prebiotic index (PI) [ 33 – 35 ]. Overnight culture was harvested, washed, and inoculated at 10 6 CFU/mL in MRS-BB (MRS basal broth without any carbon source) supplemented with 1% carbohydrate [glucose or Fructooligosaccharide (FOS), maltodextrin, and inulin], and incubated anaerobically at 37°C for 24 h, 48 h, and 72 h. Dextrose served as the positive control. Growth (OD 600 ) and pH of the culture CFS were measured at each time interval. To assess dose-dependence, prebiotics were tested at 1%, 1.5%, and 2% of maltodextrin. PS was evaluated as (A/B) × 100, where A and B represent the mean OD 600 of LP-ARP2 grown with prebiotic and dextrose, respectively, after 24 h [ 15 ]. For viable counts, cultures were serially diluted in PBS, plated on MRS agar, and incubated anaerobically at 37°C for 24 h. Biomass (Log 10 CFU/mL) was determined, and PI was calculated as PI = [CFU of probiotics in prebiotic carbohydrate/CFU of probiotics in control carbohydrate]. The experiment was performed in triplicate. 2.4. Determination of Safety Attributes of LP-ARP2 : antibiotic susceptibility, haemolytic, and DNase activity For the antibiotic susceptibility, an overnight LP-ARP2 culture was inoculated on MRS agar, and antibiotic discs (HiMedia) were placed [ 12 ]. Plates were incubated anaerobically for 24 h at 37°C, and inhibition zone diameters (mm) were measured. The CLSI standards classified the susceptibility as resistant (R), intermediate susceptible (I), or susceptible (S) [ 36 ]. For hemolysis, culture was spot-inoculated on a sheep blood agar plate (5% w/v, defibrinated, HiMedia), and incubated for 24 h [ 37 ]. Hemolytic activity was categorized as β-hemolysis (clear zones), α-hemolysis (greenish zones), or γ-hemolysis (no zones). Controls included Staphylococcus aureus ATCC 25923 (β-hemolysis), Escherichia coli ATCC 25922 (α-hemolysis), and L. acidophilus DDS1 (γ-hemolysis). For DNase activity, culture was spot-inoculated on DNase agar (HiMedia) and incubated at 37°C for 24 h. After incubation, plates were flooded with 1N HCl to visualize DNase activity as clear zones around colonies [ 12 ]. S. aureus ATCC 25923 served as a positive control, and L. acidophilus DDS1 as a negative control. All the experiments were performed in triplicate. 2.5. Assessment of antimicrobial properties of LP-ARP2 2.5.1. Preparation of the cell-free supernatant (CFS) LP-ARP2 was cultured for 24 h in MRS broth anaerobically at 37°C. The culture was centrifuged for 20 min at 4°C and 4000 rpm. The CFS was filtered using a PVDF filter (0.22 µm, HiMedia) before use in the studies [ 38 ]. 2.5.2. Determination of the antimicrobial activity of LP-ARP2 using Gram-positive, Gram-negative pathogens, and multidrug-resistant hospital isolates For agar well diffusion, the pathogens (0.5 McFarland, OD 600 = 0.5) were swabbed on Mueller-Hinton agar (HiMedia) [ 12 ]. Gram-negative Salmonella Typhimurium ATCC 14028, Gram-positive Staphylococcus aureus ATCC 25923, Methicillin-resistant Staphylococcus aureus ATCC 700699 (MRSA), and multi-drug-resistant clinically isolated Gram-negative strains such as Escherichia coli (ETEC) BCH 04067, Shigella Flexneri BCH 06745, and Vibrio cholerae BCH 09616, were among the pathogens under investigation. 6 mm wells were treated with 100 µL LP-ARP2 CFS (negative control-uninoculated MRS broth) and incubated overnight. The zone of inhibition was measured, and the experiment was carried out in triplicate. 2.5.3. Evaluation of minimum inhibitory percentage (MIP) of LP-ARP2 CFS using the broth microdilution method Three sets of LP-ARP2 CFS were used: Set 1: untreated; Set 2: heat-treated (15 min at 95°C); Set 3: pH-neutralized. Overnight S. Typhimurium culture was centrifuged, washed, and inoculated (100 µL; 10 5 CFU/mL) with varying percentages (100 µL, 1–50% v/v) of the CFS diluted in MRS [ 12 ]. Uninoculated MRS and Mueller-Hinton broth (MHB, HiMedia) were used as blank negative controls. Absorbance (600 nm) was determined after more than 18 h of incubation. The bacteriostatic or bactericidal effects of LP-ARP2 were assessed by spot-inoculating cultures on MHA plates from the same microtiter plate. The experiment was carried out in triplicate. 2.5.4. Determination of anti-biofilm properties of LP-ARP2 CFS To evaluate the antibiofilm property of LP-ARP2 , overnight MRSA strain (OD 600 = 0.1) with glucose (Merck) and NaCl (Merck) was inoculated with 10%-40% v/v LP-ARP2 -CFS (MIP to 4 MIP) in 24-well plates [ 39 , 12 ]. After 24 h incubation, staining was carried out as previously described (section 2.3.6 ). Uninoculated MRS and TSB served as a control. The biofilm formation rate (%) was determined as OD Sample /OD Control x 100. The experiment was carried out in triplicate. 2.6. Evaluation of Antioxidant Properties of LP-ARP2 2.6.1. Preparation of the intact and heat-lysed cells Overnight LP-ARP2 culture was washed and resuspended in distilled water up to an OD 600 of 1.0. Samples were heated to 95°C (water bath) for 30 min to prepare heat-lysed cells [ 40 ]. 2.6.2. Determination of ABTS cation radical scavenging capacity of LP-ARP2 A working solution was prepared by mixing ABTS (Sigma) with potassium persulphate (HiMedia) in 1:1 v/v, followed by dilution up to OD 734 = 0.7 with methanol (HiMedia, HPLC) [ 41 ]. Then, samples (0.6 mL intact or heat-lyzed cells) were combined with ABTS solution (1.2 mL) and incubated (30 min, dark, room temperature). Distilled water was the control. With A C = absorbance of control and A S = absorbance of sample at 734 nm, the ABTS scavenging rate (%) was calculated as (Ac–As)/Ac x 100. The experiment was carried out in triplicate. 2.6.3. Assessment of DPPH free radical scavenging capacity of LP-ARP2 0.2 mM DPPH (CDH) solution (1 mL) in methanol (HiMedia) was mixed with 1 mL intact or heat-lyzed cells, mixed well, and incubated at room temperature (30 min dark) [ 40 ]. Distilled water was the control. DPPH radical scavenging activity (U/mL) = ABS C –ABS S /S x 100, where S = sample volume (mL) and ABS C and ABS S represent the absorbance of the test and control samples, respectively, measured at 517 nm. The experiment was carried out in triplicate. 2.6.4. Estimation of superoxide anion scavenging activity of LP-ARP2 Briefly, 0.2 mL Tris-HCl (Sigma) was mixed with 0.8 mL intact or heat-lyzed cells. 0.1 mL pyrogallol (Sigma) was added, followed by incubation at room temperature (30 min, dark) [ 42 ]. Deionised water was the control. Superoxide anion radical scavenging ability (%) was calculated as [1–(As – A 1 )/A 0 ] x 100, where As = absorbance (320 nm) in the presence of pyrogallol, A 1 = absorbance without pyrogallol, and A 0 = absorbance of the blank solution with pyrogallol. The experiment was carried out in triplicate. 2.6.5. Evaluation of the hydroxyl radical scavenging ability of LP-ARP2 Equal amounts of FeSO 4 (Merck), sodium phosphate buffer (Sigma), and 1,10-phenanthroline (Sigma) were mixed, vortexed, and incubated for 5 to 7 min [ 43 ]. An equal volume of H 2 O 2 (0.12% v/v, CDH) and LP-ARP2 intact or heat-lyzed cells were mixed, and incubated for 60 min at 37°C (water bath). Deionized water was used as the control group in place of sample. Hydroxyl radical scavenging activity (%) was computed as [(As–A 1 )/(A 0 –A 1 )] x 100, where As = absorbance (536 nm) of the sample when H 2 O 2 is present, A 1 = absorbance of the sample when H 2 O 2 is not present, and A 0 is the absorbance of the solution with H 2 O 2 and without a sample. The experiment was carried out three times. 2.7. Determination of metabolite profiling of LP-ARP2 by High-Resolution Mass Spectroscopy (HRMS) Metabolite profiling of LP-ARP2 -derived CFS was performed using an Exactive™ Plus Orbitrap high-resolution mass spectrometer coupled to an Ultimate 3000 HPLC system (Thermo Scientific, USA) [ 11 ]. Equal volumes of CFS and methanol were mixed, filtered, and transferred (0.5 mL) into a DP ID vial (Cat# C4000-1W, Thermo Scientific). Separation was achieved on a Hypersil BDS C18 column (250 mm × 2.1 mm, 5 µm; Thermo Scientific) with 0.1% formic acid in acetonitrile:water (1:1) as the mobile phase. The flow rate was 3 µL/min, column temperature 30°C, and pressure 700 bar. Ionization was performed by electrospray ionization (ESI) at 3 eV, with spectra acquired in both positive and negative ion modes over an m/z range of 50–750 during a 5-min run. Metabolites were annotated using the National Metabolomics Data Repository (NMDR) and cross-referenced against KEGG, LIPID MAPS, ChEBI, HMDB, BMRB, PubChem, and NP Atlas via the Metabolomics Workbench ( https://www.metabolomicsworkbench.org/data/M_form.php#M2 ). Mass tolerance for compound assignment was set at ± 0.2 ppm, and results were exported with compound names and delta PPM values. Data from both ionization modes were integrated, duplicates removed, and identified metabolites validated against published literature, entered into the table. 2.8. Evaluation of Functional Potential of LP-ARP2 -secreted Metabolites 2.8.1. Identification of molecular targets for HRMS-identified metabolites A programmatic multi-database approach was applied to predict potential biological targets of LP-ARP2 -derived bioactive metabolites. Metabolite names were first queried in PubChem via its RESTful API to retrieve chemical descriptors (SMILES, InChIKey, and PubChem CID). These identifiers were then used to extract experimentally validated and computationally predicted protein targets from the PubChem BioAssay database, with additional target information retrieved from BindingDB. All targets were consolidated, deduplicated, and classified into human or microbial categories based on source organism taxonomy. 2.8.2. Construction of compound-target interaction network and functional enrichment analysis Curated compound-target interactions were used to construct a bipartite interaction network in Cytoscape (compounds: source nodes; protein targets: target nodes). Further, the ClueGO plugin in Cytoscape was employed to perform Gene Ontology (GO) with pV ≤ 0.05 and KEGG pathway enrichment analysis to understand the functional roles of the target proteins. The enrichment was conducted using Biological Process, Cellular Component Branch, Molecular Function, and KEGG pathways. To assess disease relevance, the DisGeNET plugin was applied to identify overlaps between predicted human protein targets and Alzheimer’s disease-associated targets, thereby evaluating the potential impact of LP-ARP2 -derived metabolites on Alzheimer’s disease. 2.9. Statistical analysis Statistical analyses were performed using GraphPad Prism v8. Paired t-tests were applied for two-group comparisons, while one-way ANOVA was used for multiple groups with a single variable and two-way ANOVA for comparisons involving multiple groups with two variables. A p-value ≤ 0.05 was considered statistically significant. 3. Results 3.1. Whole Genome Sequencing (WGS) of LP-ARP2 : General features 3.1.1. LP-ARP2 genome and phylogenetic analysis Whole genome sequencing revealed that the LP-ARP2 genome consisted of a single circular chromosome with 3,212,397 bp and 44.55% average GC content ( Fig. 2 a ) . There were 3077 genes identified, and 3006 were the coding DNA sequences (CDSs) in the chromosome (Table S1 ) . An overview of the genome assembly is depicted in Table S2 . Comparative BLAST search against all Lactiplantibacillus genomes placed our strain within the Lactiplantibacillus clade, closely related to Lactiplantibacillus plantarum strain NBRC 15891 ( Fig. 2 b ) . This result was supported by the average nucleotide identity (ANI), where 99.9% of ANI was observed for LP-ARP2 with L. plantarum DOMLa [ 44 ]. 16S rRNA extracted with Barrnapp indicated that Lactiplantibacillus plantarum strain CIP 103151 exhibited the highest similarity with the strain. Therefore, LP-ARP2 was affirmed as Lactiplantibacillus plantarum . 3.1.2. Safety evaluation of LP-ARP2 genome Hidden prophages are common in many Lactobacillus species and can affect the intestinal microbiota upon activation [ 45 ]. Therefore, screening of prophage is crucial for probiotic safety assessment. Functional annotation of the LP-ARP2 genome via RAST-SEED viewer identified genes related to phage replication, tail proteins, capsid proteins, packaging machinery, and phage introns (Table S3) . As L. plantarum is known to be a multihabitat species, prophage elements are more frequent in the genome sequence [ 46 ]. Additionally, to counter phage attacks, bacteria employ the CRISPR/Cas system [ 45 ]. Notably, one CRISPR array and one Cas-associated sequence (CRISPRFinder) were predicted in LP-ARP2 , associated with fewer intact prophages. This suggested the presence of an antiphage defense mechanism in the strain (Table S4) [ 45 ]. Virulence factors (VFs) analysis using core and comprehensive VF datasets showed no significant hits in the LP-ARP2 genome. Using the PathogenFinder tool, LP-ARP2 was identified as a non-human pathogen, showing no alignment with known pathogenic families and exhibiting a very low likelihood (0.2) of being associated with human pathogenicity. Microbes can obtain antibiotic resistance genes through horizontal gene transfer [ 19 ]. Several Lactobacillus strains, including L. plantarum , are intrinsically resistant to vancomycin due to d-Ala-d-lactate in their peptidoglycan [ 47 ]. LP-ARP2 harboured one antibiotic-resistant gene according to the CARD database and RGI analysis (Table S5) , though none were detected by ResFinder. 3.1.3. General functional annotation of the LP-ARP2 genome Functional annotation of the LP-ARP2 genome revealed 1549 CDS (51.5%) classified into 38 functional classes, 204 pathways, and 37 modules (Tables S6). Key pathways included biosynthesis of amino acids, secondary metabolites, and cofactors/vitamins, highlighting strong metabolic versatility. Genes linked to biosynthetic pathways of essential amino acids (phenylalanine, tryptophan, tyrosine, leucine, isoleucine, valine, lysine, methionine, cysteine, serine, threonine, glycine, arginine), pyruvate, and vitamins (riboflavin, thiamine, nicotinate, vitamin B6, nicotinamide) suggested potential health benefits of LP-ARP2 ( Fig. 2 c ) . COG classification (3006 CDS) further highlighted diverse housekeeping and metabolic functions, with genes enabling utilization of glucose (n = 22), mannose and fructose (n = 25), galactose (n = 21), sucrose and starch (n = 26), nucleotide/amino sugars (n = 28), supporting adaptability to varied habitats. Carbohydrate fermentation pathways encoded SCFA production: acetate, lactate, propanoate (n = 10), butanoate (n = 7), pyruvate (n = 22), succinate, a hallmark of probiotic functionality. Additionally, genes for butanoate, polyketide (n = 4), and terpenoid (n = 10) biosynthesis point to applications in food, nutraceutical, and pharmaceutical industries (see discussion). 3.2. WGS analysis: Probiotic-specific genomic features of LP-ARP2 genome 3.2.1. Stress-resilience genetic set-up at LP-ARP2 genome indicated gut-survival potential of the strain To exert health benefits, probiotics must endure in the gastrointestinal tract and food processing stresses ( Fig. 2 d ) . WGS analysis of LP-ARP2 revealed a comprehensive stress-response repertoire, including universal stress proteins (uspA, usp6, yugI) , heat-shock regulators ( ctsR, hrcA ), chaperones ( dnaJ/K, groS/L, grpE, hslU/O ), and proteases ( hslV/U, clpB/C/E/L/P/X ), ensuring protein stability and membrane integrity under heat stress (Table S7) . Cold-shock proteins ( cspA/C ) and proton-translocating systems, including the F₀F₁ ATP synthase operon ( atpA-atpH ), support resilience under cold and acidic conditions [ 48 ]. Additional genes for alkaline shock proteins ( asp2/23 ), acyltransferase ( plsC ), ABC transporters, pyruvate kinase ( pyk ), sodium-proton antiporters ( nhaC ), and DNA repair ( mutL/S2/R/T/Y ) further enhance stress tolerance [ 48 , 49 ]. To withstand bile stress in the small intestine, LP-ARP2 harbours genes for sodium–bile acid symporters, ABC transporters ( glnH–glnPH2 ), oligopeptide transporters ( oppA–F ), glutamine synthetase ( glnA ), as well as pyrophosphatase ( ppaC , preserves membrane integrity) and cyclopropane-fatty-acyl-phospholipid synthase ( cfa , upregulates lipid biosynthesis) [ 48 ]. Further, osmoprotection is ensured by opuCA–D , proV , and proWX , facilitating uptake of glycine betaine, choline, and proline [ 48 ]. Collectively, these genomic features highlight robust adaptation of LP-ARP2 to acidic, alkaline, osmotic, oxidative, and bile stresses, emphasizing its strong probiotic potential [ 50 ]. 3.2.2. Genetic features for adhesion at LP-ARP2 genome indicated the gut- sustainability of the strain Genomic analysis of LP-ARP2 revealed several adhesion-related genes, including lipoprotein signal peptidase II ( lspA ), maltose phosphorylase ( mapA ), enolase ( eno ), a sortase family protein ( srtA ), and abundant glycosyltransferases, along with S-layer (SLPs) and sortase-dependent proteins (SDPs), crucial for colonization potential of probiotics [ 51 , 52 , 11 , 53 ] (Table S7) . Additionally, genes like celA-celE (glycosyl hydrolase family) could enhance gut persistence and antimicrobial activity of LP-ARP2 [ 48 ]. Teichoic acid biosynthetic genes like dltA-dltX may influence host immune responses and adhesion capacity of LP-ARP2 [ 11 , 53 ]. 3.2.3. Genetic markers for biofilm-formation at LP-ARP2 genome indicated potential of the strain for gut- colonization Biofilm formation ability of LP-ARP2 was investigated to assess its potential for enhancing probiotic persistence, gut colonization, and competitive exclusion of pathogens [ 54 , 55 ]. WGS analysis identified veg and luxS genes in the LP-ARP2 genome, where veg stimulates biofilm formation in Gram-positive bacteria, and luxS -mediated quorum sensing via autoinducer-2 (AI-2) regulates attachment and biofilm development [ 56 , 57 ]. Therefore, the presence of biofilm-stimulating genes motivated the experimental exploration of the biofilm-forming ability of LP-ARP2 ( Fig. 2 d ) . 3.2.4. Carbohydrate-active enzymes (CAZymes) at LP-ARP2 -genome indicated potential of the strain for gut-adaptation and modulation CAZymes are central to gut microbial adaptation by enabling the degradation of complex polysaccharides [ 58 ]. KEGG and COG analyses of LP-ARP2 revealed a total of 85 CAZyme genes, categorized into glycoside hydrolases (GHs, n = 37, 14 families), glycosyltransferases (GTs, n = 45, 8 families), and carbohydrate-binding modules (CBMs, n = 4, 2 families) ( Table 1 ) . GHs (47%) predominantly included GH13, GH31, and GH65, which target mono-, oligo-, and polysaccharides such as glucose, fructose, galactose, α-/β-glucans, arabinoxylans, and cellulose (GH9, GH26) (Fig. S1 ) [ 59 , 60 ]. GTs (48%) catalyze sugar transfer and influence host-microbe immune interactions [ 48 ]. While CBMs (4%; CBM48, CBM50) mediate polysaccharide hydrolysis (Fig. S1 ) . This broad CAZyme repertoire indicated that LP-ARP2 could metabolize diverse types of carbohydrates, enabling the ecological adaptation of other gut-bacteria with beneficial functions like immune modulation and pathogenic defence. In addition, LP-ARP2 harbored an enriched set of sugar phosphotransferase (PTS) genes (e.g., pts4ABC, pts10A–C, pts23A–C, pts30ABC, pts36A–C, ulaA–B, celA–D, chbA/C ), emphasizing efficient carbon transport. Functional diversity was observed across mannose/sorbose/fructose ( manA/L/Y/N, dhaA–T, agaB/C ), sorbitol/glucitol ( srlA/B/E/M ), and cellobiose/lactose ( mtlA/D/F/R, mngA/B ) PTS systems. Genes encoding alcohol dehydrogenase, L-lactate dehydrogenases, phosphate acetyltransferase, phosphoketolase, and pyruvate formate lyase suggested metabolic flexibility through both homo- and hetero-fermentative pathways. Together, these genetic-features (Table 1 ) implied that LP-ARP2 could be a lucrative candidate for probiotic-formulations [ 48 ] (See discussion). Table 1 Carbohydrate-active enzymes (CAZymes) detected in the LP-ARP2 genome Preferred name Cazyme Function glgB CBM48, GH13, GH31 formation of alpha-1,6-glucosidic bond in glycogen glgD GT5 Nucleotidyl transferase glgA GT5 Synthesizes alpha-1,4-glucan chains using ADP-glucose glgP GT35 allosteric enzyme in carbohydrate metabolism malQ CBM48, GH13, GH31, GH77 Belongs to the glycosyl hydrolase 13 family pgmB GH37, GH65 beta-phosphoglucomutase trePP GH37, GH65 Glycosyl hydrolase family 65 pgmB GH37, GH65 beta-phosphoglucomutase malL GH13, GH31 Alpha amylase, catalytic domain malS GH13 Glycogen debranching enzyme mapA GH65 hydrolase, family 65, central catalytic scrB GH32 invertase treC GH13 Alpha amylase, catalytic domain protein celA GT1 Belongs to the glycosyl hydrolase 1 family csbB GT2 Glycosyltransferase like family 2 cps4F GT4, GT5 Glycosyl transferases group 1 ica2 GT2 Glycosyl transferase family group 2 mltD CBM50 NlpC P60 family protein murG GT28 Cell wall formation tagE GT4 Glycosyl transferases group 1 pgaC GT2 Glycosyl transferase nplT GH13 Belongs to the glycosyl hydrolase 13 family celA GT1 Belongs to the glycosyl hydrolase 1 family tagE5 GT4 Poly (Glycerol-phosphate) alpha-glucosyltransferase tagE6 GT4 Glycosyl transferases group 1 pbg6, celA GT1 Belongs to the glycosyl hydrolase 1 family malA GH13, GH31 Alpha amylase, catalytic domain protein mngB GH38 Glycosyl hydrolases family 38 N-terminal domain mltD CBM50 PFAM NLP P60 protein celE GH5, GH9 GDSL-like Lipase/Acylhydrolase family lacM GH101, GH29 beta-galactosidase bglH, pbg10 GT1 Belongs to the glycosyl hydrolase 1 family treP GH65 hydrolase, family 65, central catalytic malZ GH31 Belongs to the glycosyl hydrolase 31 family aglB GH4, GT4 Family 4 glycosyl hydrolase C-terminal domain malL GH13 Alpha amylase, catalytic domain protein bgl GT1 Belongs to the glycosyl hydrolase 1 family mngB GH38 Glycosyl hydrolases family 38 N-terminal domain dexB GH13 Alpha amylase, catalytic domain protein tagA GT26 Takes part in the de novo synthesis of teichoic acid sidC GT2, GT4 DNA recombination arbB GT1 Belongs to the glycosyl hydrolase 1 family nagH GH20, GH26, GH5, GH9 MucBP domain sacB GT2, GT4 Stealth protein CR2, conserved region 2 cps1B GT2, GT4 Glycosyl transferases group 1 recX, mgs, cpoA, tagE1, tagE2, tagE3 GT4 Regulatory protein RecX pbp2A GT51 penicillin-binding protein ykoT, pgaC GT2 Glycosyl transferase family 2 mapA GH65 hydrolase, family 65, central catalytic ponA GT51 penicillin-binding protein 1A 3.2.5. Genes for amino acid biosynthesis at LP-ARP2 genome indicated probiotic efficacy Functional annotation revealed that the LP-ARP2 genome contained genes for the biosynthesis of essential amino acids. This includes a) aromatic amino acids ( aroA/C/D/E/K ) like tryptophan ( trpA-G ), tyrosine ( tyrA/S ), phenylalanine ( pheS/T ); b) branched-chain amino acids (BCAAs) like isoleucine, leucine, valine ( ilvE, leuS ), as well as c) other essential amino acids like threonine (hom, hom1, asd, thrB/C/S/E) , histidine (hisA-I/K/Z/S) , and lysine (lysC/A) [ 61 ]. 3.2.6. Gene clusters at the LP-ARP2 genome indicated the metabolic versatility of the strain Functional annotation of the LP-ARP2 genome revealed the presence of genes associated with the short-chain fatty acids (SCFA) synthesis ( ackA , nagA , pta , acyP , adhE , pdh , fabI-H , patB , ldhA , accA-D , poxB , gabD , dapA-E , malY , eda , and scfaA , Fig. 2 d). Genes related to vitamin biosynthesis pathways were also identified ( adk , rsgA , nudF , pdxK/B , birA , fabI/Z/H/F , nadD , ycsE , yitU , and iscS , riboflavin ( ribBA/D/E/H/T/F ), thiamine ( ThiM/N/D/E/I ), vitamine K2 ( menG/A ), folate ( folA/P/E/B/C ), coenzyme A ( coaA-E)) [ 62 , 63 , 61 ] (Table S7) . LP-ARP2 harbored gene signatures associated with exopolysaccharide (EPS) biosynthesis and regulation ( cpsY/4J/4I/4G/4F/4D/1B/2I, epsB-V ). EPS facilitates gut-colonization, biofilm formation, and host interactions [ 49 ]. Next, metabolic gene cluster (MGC) analysis using gutSMASH predicted gene clusters in LP-ARP2- genome, responsible for synthesis of primary and secondary metabolites involved in host metabolism and immune responses [ 28 ]. Two MGC regions were detected: a) pyruvate to acetate-formate (region 10.1), involved in SCFA (acetate, butyrate, and propionate) production ( Fig. 3 a ) , and b) nitrate reductase (region 23.1), involved in nitrate/nitrite reduction to ammonia and nitric oxide (NO). These are important for immune regulation and host defence ( Fig. 3 b ) . Further, KEGG and COG analyses revealed a diverse gene repertoire for secondary metabolite biosynthesis and transport, including terpenoids and polyketides. antiSMASH identified three major clusters: a) T3PKS cluster (region 2.1; Chal_sti_synt_N domain) for polyketide synthesis ( Fig. 3 c ) , b) terpene cluster (region 8.1), and c) cyclic-lactone-autoinducer cluster (region 10.1) ( Fig. 4 a–b ) . PKS and terpene clusters are implicated in producing bioactive metabolites with applications in the food industry [ 64 ]. While the cyclic-lactone-autoinducer cluster suggests quorum-sensing functions in defence. COG analysis supported these findings, with ~ 2.88% of genes linked to defence mechanisms (Table S8) . 3.2.7. Prediction of bacteriocin in the LP-ARP2 genome: Antimicrobial efficacy Bacteriocins are antimicrobial peptides secreted by many probiotic bacteria. They confer beneficial effects in food preservation and pathogen inhibition. Interestingly, BAGEL4 analysis detected potential bacteriocin-encoding gene-clusters in LP-ARP2 genome, including Plantaricin F (bit score = 105.14) and Plantaricin E (bit score = 112.46) as major hits. Besides, LP-ARP2 genome also contained ORFs encoding a) bacteriocin transport and sensor proteins: orf00020, b) potential bacteriocin immunity protein; orf00025, and c) others like histidine kinase related to bacteriocin production; LanT, bacteriocin ABC-transporter, ATP-binding, and permease protein PlnG; hlyD gene, encoding an accessory factor for the ABC transporter PlnH ( Fig. 4 c ). 3.2.8. Genetic features at LP-ARP2 genome showed antioxidant activities Probiotics harboring oxidative stress tolerance genes could function as natural antioxidants to alleviate gut inflammation. Genome annotation revealed that LP-ARP2 harboured three major antioxidant mechanisms: glutathione biosynthesis, glutathione redox cycle, glutaredoxin systems, and gamma-glutamyl cycle. Genome mining identified entire thioredoxin ( tpx, trxb/a ) and NADH-dependent ( nox, npr , ndh ) antioxidant gene signatures, along with key redox-regulators such as gshR1 (glutathione reductase), nrdH (glutaredoxin), katA (catalase), and peroxidases ( Fig. 2 d ) . Additionally, genes encoding msrA and msrB , components of the methionine sulfoxide reductase system, indicated protection against ROS-mediated protein oxidation (Table S7) . Therefore, these genetic features indicated that LP-ARP2 could possess strong antioxidant potential. 3.3. Probiotic Attributes of LP-ARP2 : Experimental Validation of WGS Analysis 3.3.1. Stress-adaptation potential of LP-ARP2 : tolerance to acid and bile WGS analysis revealed stress-response genes, indicating the adaptation potential of LP-ARP2 to a stressful environment. Acid tolerance assay showed 80% and 60% survival of LP-ARP2 at pH 3 after 3 and 5 h, respectively, with stable biomass up to 1 h at both pH 3 and 4 (p > 0.05); viability declined significantly only after 5 h at pH 3 (****p < 0.0001), indicating strong acid stress resilience ( Fig. 5 a ) . Under bile stress, LP-ARP2 maintained high survival (97% at 0.3% and 96% at 1% bile) after 3–5 h ( Fig. 5 b ) , with biomass increasing significantly after 3 h (***p < 0.001) and 5 h (****p < 0.0001). These genomic and in vitro results together confirm robust acid-bile tolerance, highlighting the adaptability of the strain to harsh gastrointestinal conditions. 3.3.2. Gut-sustainability of LP-ARP2 : Autoaggregation, cell surface hydrophobicity, adhesion to Caco-2 cells, and biofilm formation assays Probiotic surface proteins mediate host interaction and adhesion, and LP-ARP2 contained putative adhesion-related genes, indicating strong colonization potential. Autoaggregation assay of LP-ARP2 showed a significant increase (****p < 0.0001) in aggregation capacity from 5.9–44.74% within 1–12 h, exhibiting the highest aggregation (66.44%) at 24 h ( Fig. 5 c ) . LP-ARP2 showed significant surface hydrophobicity with n-hexadecane (58.93%) and xylene (58.27%) ( Fig. 5 d ) . This finding implied the promising adhesion ability of LP-ARP2 with the complex hydrophobic surface of gut epithelial cells. The adhesion efficacy of LP-ARP2 to Caco-2 was significant, with an adhesion rate of 7.1% after 2 h, corroborating genomic predictions ( Fig. 5 e ) . Notably, LP-ARP2 formed robust biofilms within 24 h without additional supplements, surpassing L. acidophilus DDS1 (**p 0.05) ( Fig. 5 f ). Collectively, genomic and in vitro evidence confirmed strong adhesion, biofilm formation, and persistence, strengthening the ability of LP-ARP2 to colonize the gut mucosa and enhance therapeutic efficacy. 3.3.3. Gut-modulation and adaptability of LP-ARP2 : Prebiotic utilization assay Genomic analysis revealed a diverse array of CAZymes in LP-ARP2 , indicating metabolic flexibility in degrading complex carbohydrates. Prebiotic utilization assays with FOS, maltodextrin, and inulin (dextrose control) showed highest efficiency with maltodextrin (pH 5.13, after 12 h), followed by inulin (pH 5.81) and FOS (pH 6.28), with corresponding organic acid production (Fig. S2) . Dose-dependent assays with maltodextrin (1%, 1.5%, and 2% w/v), revealed significant drop in pH (****p 0.05) ( Fig. 5 h and 5 i ) . Biomass (Log 10 CFU/mL) also increased with maltodextrin, consistent with PS, and remained comparable to the control ( ns p>0.05) ( Fig. 5 j ) . A PI > 1 signifies enhanced probiotic growth over the control [ 34 ]. Hence, PI confirmed enhanced growth with 1.5% FOS and 1% maltodextrin, showing PI > 1 compared to dextrose ( Fig. 5 k ) . Thus, both genomic and experimental analyses established dose-dependent prebiotic utilization capacity of LP-ARP2 , particularly for maltodextrin, consistent with its CAZyme repertoire. 3.3.4. Safety Attributes of LP-ARP2 : In vitro Validation of WGS Analysis 3.3.4.1. Antibiotic susceptibility of LP-ARP2 Based on the clinical risk of the transfer of antibiotic-resistant genes, antibiotic susceptibility is essential for probiotic evaluation [ 65 ]. LP-ARP2 was sensitive to most conventional antibiotics (ampicillin, amoxicillin/clavulanic acid, clindamycin, chloramphenicol, erythromycin, penicillin, and tetracycline) but resistant to vancomycin and gentamicin, consistent with profiles of other probiotics (Table S9) . 3.3.4.2. Non-haemolytic and DNase-negative attributes of LP-ARP2 LP-ARP2 showed no hemolysis on the blood agar plate (non-haemolytic) and no DNase activity on the DNase agar plate (Table S10) . Therefore, in silico studies and in vitro assays assured the non-pathogenic nature of LP-ARP2 , reinforcing its suitability as a safe and functional probiotic candidate. 3.3.5. Promising Antimicrobial Properties of LP-ARP2 3.3.5.1. Robust antimicrobial potential of LP-ARP2 across the diverse pathogens Effective probiotics should exhibit broad-spectrum antimicrobial activity. Indeed, LP-ARP2 showed significant inhibition for Gram-positive and Gram-negative enteric bacteria, with the most pronounced effects observed for S. aureus , V. cholerae , and S. flexneri ( Fig. 6 a ) . Notably, V. cholerae and S. flexneri strains used in this study were multidrug-resistant clinical isolates. 3.3.5.2. Anti- Salmonella effect of LP-ARP2 : Co‑culture assay S. Typhimurium (ST), a major foodborne pathogen, was employed to strengthen the antimicrobial effects of LP-ARP2 . In the agar well diffusion assay, the heat-treated CFS of LP-ARP2 retained inhibition (12.66 mm ± 0.5) as the normal CFS (12.66 mm ± 0.5), however the activity was abolished (0 mm) upon pH-neutralization, confirming acid-driven antibacterial effects ( Fig. 6 b ) . Broth microdilution showed significant reduction of ST growth at 1% (*p < 0.05) and 5% v/v (****p < 0.0001) LP-ARP2 CFS, with complete inhibition (MIP) of ST at 10% (v/v) CFS ( Fig. 6 c-e ) . The agar spot assay revealed bacteriostatic activity at 10% and bactericidal activity at 20% CFS ( Fig. 6 f ) . 3.3.5.3. Anti-biofilm activity of LP-ARP2 against MRSA Pathogenic biofilms cause major risks to food safety and clinical industries, with MRSA being a strong biofilm-former, contributing to its virulence [ 66 ]. LP-ARP2 CFS showed potent anti-biofilm activity, with 10% (v/v) CFS identified as the MIP for MRSA ( Fig. 6 g; Fig. S3) , significantly reducing biofilm formation at 10% (**p < 0.005) and more strongly inhibiting biofilm formation at 20–40% (****p < 0.0001) of LP-ARP2 CFS ( Fig. 6 h ) . 3.3.6. Antioxidant potential of LP-ARP2 3.3.6.1. Ability of LP-ARP2 to scavenge cations by ABTS assay In the ABTS assay, LP-ARP2 heat-lysed cells showed significantly higher (**p < 0.05) scavenging rate (43.17%) than the intact cells (36.43%) ( Fig. 7 a ) . This result suggested that the intracellular components of LP-ARP2 contributed more to its strain-specific antioxidant potential [ 67 ]. 3.3.6.2. Free radical scavenging capacity of LP-ARP2 by DPPH assay DPPH assay measures antioxidant efficacy by the reduction in absorbance at 517 nm as radicals convert to non-radical forms [ 42 ]. The DPPH assay showed significantly higher (***p < 0.05) free radical scavenging activity (25 U/mL) of LP-ARP2 heat-lysed cells than whole cells (12.7 U/mL) ( Fig. 7 b ) , indicating stronger antioxidant potential in the intracellular components of the strain. 3.3.6.3. Ability of LP-ARP2 to scavenge superoxide radical Superoxide anions trigger lipid oxidation by generating singlet oxygen. LP-ARP2 heat-lysed cells showed significantly higher (***p < 0.05) superoxide anion scavenging activity (83.95%) than intact cells (67.21%), indicating strong antioxidant potential of the intracellular components of the strain ( Fig. 7 c ) . 3.3.6.4. Capacity of LP-ARP2 to scavenge hydroxyl radical Hydrogen peroxide generates harmful hydroxyl radicals that damage lipids, proteins, and tissues [ 42 ]. LP-ARP2 intact cells showed significantly higher (**p < 0.05) scavenging activity (72.82%) than heat-lysed cells (60.62%) (**p < 0.05) ( Fig. 7 d ) , suggesting greater antioxidant potential in the whole cells than in intracellular components, highlighting a distinct, strain-specific probiotic trait. 3.4. Metabolomics of LP-ARP2 : Identification of Bioactive Metabolites Using WGS and HRMS Analyses 3.4.1. Detection of antimicrobial metabolites in LP-ARP2 CFS WGS analysis of LP-ARP2 revealed multiple genes associated with antimicrobial metabolite biosynthetic pathways, including SCFA and EPS biosynthesis and regulation. These signatures suggested enhanced potential for production of antimicrobial compounds, crucial for defence and pathogen exclusion. Consistent with genomic predictions, HRMS profiling of LP-ARP2 -derived CFS detected a wide array of antimicrobial metabolites ( Table 2 ) . SCFAs, including acetic, isobutyric, butyric, lactic, and hydroxypropionic acids, were abundant, corroborating gutSMASH results ( Fig. 3 a ) . These bioactive compounds are recognized as primary agents in antimicrobial activity and modulation of the immune system [ 11 ]. Butyrate, a major colonocyte energy source and immunomodulator, was notably present [ 12 ]. Organic acids (valeric, oxaloacetate, oxoglutarate) and antimicrobial compounds such as homoserine and homoserine lactone were also identified. Thus, WGS and HRMS validated the potential of LP-ARP2 to produce antimicrobial metabolites, reinforcing its functional and clinical relevance. Table 2 Metabolite Profiling of the Cell-Free Supernatant (CFS) of LP-ARP2 Name Input Mass Matched Mass Significance Butyric acid 89.02 89.0597 Energy source for colon cells, important for gut-health, reduction of inflammation Alanine 90.05 90.0549 Amino acid Glycine 74.06 74.0248 Amino acid Phenylalanine 166.1 166.0862 Essential amino acid Lactamide 90.05 90.0553 Has applications in pharmaceutical industries Dimethylethanolamine 90.05 90.0913 Has anti-inflammatory, antioxidant activities, used as a firming and anti-aging product Oxalic acid 91.00 91.0026 Used in cleaning or bleaching D-glyceraldehyde 89.02 89.0244 Cellular metabolite Hydroxypropionic acid 89.02 89.0244 Cellular metabolite Acetoacetic acid 101 101.0244 Cellular metabolite Valeric acid 101.1 101.0608 Cellular metabolite 2.3-butanediol 89.02 89.0608 Has applications in chemical, cosmetics, agriculture, and pharmaceutical industries Acetate 60.04 60.0206 Fermentation and acidification, cholesterol synthesis Betaine 118.1 118.0862 Lactic acid fermentation, prevention of liver injury Proline 116.1 116.0706 Amino acid Threonine 118.1 118.0510 Protein synthesis Gamma-Aminobutyric acid 102 102.0561 Neurotransmitter Fructose 179.1 179.0561 Cellular metabolite Glucose 179.1 179.0561 Cellular metabolite Galactose 179.1 179.0561 Cellular metabolite Citric acid 191 191.0197 Mineral absorption, source of flavonoids, antioxidants, and vitamin C Dihydroxyacetone 89.02 89.0244 Used in the cosmetic industry, intermediate in lipid biosynthesis, and glycolysis Lactic acid 89.02 89.0244 Antimicrobial, antiviral and immunomodulatory properties Dimethylglycine 102 102.0561 Important for boosting energy and immunity Acetoacetamide 102 102.0549 Has applications in textile industry Hydroxypyruvic acid 105 105.0182 Cellular metabolite Malonic acid 105 105.0182 Has application as flavoring agents, fragrances, and pharmaceuticals Dioxosuccinic acid 147.1 146.9924 Used in different chemical reactions Arabinonic acid 167 167.0550 Takes part in an organism's growth, development, or reproduction Leucine 132.1 132.1019 Essential amino acid Isoleucine 132.1 132.1019 Essential amino acid Pyruvic acid 89.02 89.0233 Potential anti-inflammatory and antioxidant agent Acetoin 89.02 89.0597 Flavour additive in food, cosmetics, synthesis of optically active pharmaceuticals Valine 118.1 118.0862 Essential amino acid Sarcosine 90.05 90.0549 Has application in schizophrenia treatment, cosmetic and pharmaceutical industries Oxaloacetate ion 132.1 132.0053 Cellular metabolite. Can alleviate liver injury Aminocaproic acid 132.1 132.1019 It is used as an antifibrinolytic agent to treat bleeding Isobutyric acid 89.02 89.0597 Energy source for colonocytes and has antimutagenic activity Methyl propionate 89.02 89.0597 Antibacterial and antifungal activity 3-Hydroxypropionate 89.02 89.0233 Useful as a platform for synthesis of biodegradable plastic Acetylglycine 118.1 118.0499 Has a role as a metabolite in peptide and amino acid modification Aminocaproic acid 132.1 132.1019 Useful in the treatment of reducing bleeding and cardiac surgery Oxoglutaric acid 145 145.0143 Cellular metabolite Methylglutaric acid 145 145.0506 Organic acid can reduce cholesterol synthesis Glutamine 145 145.0619 Protein synthesis Lysine 145 145.0983 Can reduce anxiety, and cold sores, improve calcium absorption, protein synthesis Arginine 175.1 175.1189 Helps in protein synthesis Acetylcholine 145 145.1108 Major neurotransmitter Tartaric acid 149 149.0092 Has applications in food industries Arabinose 149 149.0456 Sugar metabolite Homoserine Lactone 102 102.0549 Quorum-sensing Ascorbic acid (vitamin C) 175.1 175.0248 Helps in wound healing, mineral absorption, immune system Serotonin 175.1 175.0877 Neurotransmitter Aspartic acid 132.1 132.0302 Protein synthesis Homoserine 118.1 118.0510 Antibacterial and anticancer activity Pyridoxal (vitamin B 6 ) 166.1 166.0510 Metabolism of lipids, carbohydrates, coenzymes, and hormones. Brain function Malonic semialdehyde 89.02 89.0233 Important for cosmetic and pharmaceutical industries Adipic acid 145 145.0506 Has applications in food and beverages, pharmaceuticals 5-keto-D-fructose 179 179.0550 Natural diketone, present in honey Gamma-amino-beta-hydroxybutyric acid 118.1 118.0510 A derivative of the neurotransmitter gamma-aminobutyric acid Ethanolamine 60.04 60.0455 Diverse application in pharmaceutical and chemical industry 3-Aminobutanoic acid 102 102.0561 Secondary metabolite, can act as a defense or signaling molecule 3.4.2. Detection of LP-ARP2- metabolites for health-promoting functions and industrial significance Genome mining of LP-ARP2 revealed extensive genomic potential for biosynthesis of health-promoting metabolites like vitamins, essential amino acids, and secondary metabolites, highlighting its metabolic versatility. HRMS profiling of LP-ARP2 -derived CFS validated these predictions by identifying essential amino acids (isoleucine, leucine, valine, threonine, phenylalanine, lysine) and vitamins, such as pyridoxal (B6) and ascorbic acid (C), aligning with genome annotations ( Table 2 ) . In addition, HRMS detected metabolites of industrial importance, including lactamide, oxalic acid, dihydroxyacetone, malonic acid, ethanolamine, and adipic acid, with applications in pharmaceutical, food, and cosmetic sectors. Notably, the quorum-sensing molecule homoserine lactone was identified, confirming antiSMASH predictions ( Fig. 4 b ) . Metabolites with physiological significance, like glyceraldehyde, acetoacetic acid, hydroxypyruvic acid, oxaloacetate, valeric acid, and oxoglutaric acid, were also detected, nourishing host energy metabolism. Therefore, WGS and HRMS demonstrated LP-ARP2 as a potential source of health-promoting and industrially valuable metabolites. 3.4.3. Detection of LP-ARP2 -derived metabolites with anti-oxidant and psychobiotic potential WGS analysis of LP-ARP2 revealed multiple antioxidant systems, including glutathione biosynthesis and redox cycling, glutaredoxin, γ-glutamyl pathways, thioredoxin, and NADH-dependent systems. HRMS of LP-ARP2 -derived CFS corroborated this by detecting redox-active metabolites, such as dimethylethanolamine, citric acid, lactic acid, and pyruvic acid, along with abundant SCFAs and organic acids (acetic, lactic, isobutyric, butyric, and hydroxypropionic acids), known for antioxidant and host-protective effects ( Table 2 ) . Antioxidant attributes alleviate oxidative stress-driven neuroinflammation, reinforcing gut-brain axis balance and psychobiotic potential of probiotics. WGS analysis highlighted gene signatures for biosynthesis of neuromodulatory metabolites, including glutamate decarboxylase ( gadB ) and GABA transaminase ( gabT ), leading to GABA biosynthesis and metabolism. HRMS confirmed GABA in LP-ARP2 -CFS, along with acetylcholine and serotonin, indicating psychobiotic potential ( Table 2 ) . Branched-chain amino acids (BCAAs), predicted by genome mining and detected in CFS (isoleucine, leucine, valine), further support neurotransmitter balance, while osmolyte betaine provides neuroprotection. The presence of the complete trp operon ( trpA–G ) in the genome with serotonin in CFS of LP-ARP2 demonstrated an active tryptophan–serotonin pathway, which could contribute to restoring neurotransmission disrupted in neurodegenerative conditions. Together, WGS analysis and HRMS study illustrated strong concordance between genomic capacity and metabolite expression, establishing LP-ARP2 as a promising producer of antioxidant and psychobiotic compounds with potential roles in mitigation of oxidative stress and modulation of inflammation across gut-brain axis. 3.5. Human-target based functional-network analysis with LP-ARP2 -derived metabolites: Molecular basis of therapeutic potential Genome annotation and HRMS analysis corroborated the presence of metabolites in LP-ARP2- CFS with antibacterial and antioxidant properties. Our in vitro experiments further validated robust antimicrobial and antioxidant activities of LP-ARP2 , strengthening the promise of the strain to reduce gut inflammation and related diseases. To elucidate the molecular basis underlying these effects, we further performed a human-target-based functional-network analysis with LP-ARP2 -derived metabolites, aiming to uncover the pathways and mechanistic insights into their roles in disease prevention and modulation. Network analysis using Cytoscape identified 4442 nodes and 4957 edges, reflecting broad metabolite-host associations ( Fig. 8 a ) . The Cytoscape plug-in CytoHubba identified hub nodes based on degree, maximum clique centrality (MCC), and bottleneck algorithms. Interestingly, all neurotransmitters, serotonin (87), glycine (59), acetylcholine (54), and Gamma-Aminobutyric acid (36), were detected holding a high degree and MCC scores, suggesting a central role of LP-ARP2 -metabolites in modulating the gut-brain axis by influencing neurotransmitter-related pathways. Functional enrichment analysis of the metabolites-target network based on molecular function ( Fig. 8 b ) , biological process, and KEGG pathways (Fig. S4-S5) corroborated this, revealing significant enrichment of neurotransmitter receptor activity, serotonin and GABA signaling, and GABAergic synapse pathways. Disease-gene association analysis showed strong links with neurological and cognitive disorders (Fig. S6) . Further, as a case study, Alzheimer’s disease (AD) was examined using the DisGeNET plug-in. The analysis revealed overlaps between the targets for LP-ARP2- derived metabolite and AD-associated genes (Fig. S7) . The curated network demonstrated direct interactions of LP-ARP2 metabolites with critical AD targets ( Fig. 9 a ) . Enrichment of this AD-specific network highlighted modulation of pathways central to AD pathophysiology, including amyloid-beta metabolism, synaptic transmission, tau hyperphosphorylation, neuroinflammation, and oxidative stress ( Fig. 9 b, S8–S10 ) . Notably, LP-ARP2 -secreted metabolites such as pyruvic acid, glycine, α-ketoglutarate, citric acid, betaine, and serotonin were predicted to influence these processes (Table S11) , suggesting that LP-ARP2 may exert neuroprotective effects by stabilizing neurotransmission and mitigating AD-related pathologies ( Fig. 10 ) . Together, these results highlight the potential of LP-ARP2 as a psychobiotic strain capable of reinforcing the gut-brain axis and providing therapeutic benefits in neurodegenerative and inflammation-associated disorders. 3.6. Promising Gene-Signatures for probiotic L. plantarum : Comparative Genomic Signatures between LP-ARP2 with clinically relevant strains LP 299v, and Lp01 Comparative genomic analysis of LP-ARP2 with clinically studied strains LP 299v and Lp01 revealed strong conservation of core probiotic attributes. Stress tolerance determinants, including ABC transporters and acid- and bile-responsive genes ( atp, nha and asp, gln, opp, opu , details Table 3 ), indicated that LP-ARP2 has adaptation capacities similar to the clinically relevant strains. Conserved adhesion ( lsp, map, srt , eno, cel , details Table 3 ) and immunomodulation genes ( dlt , details Table 3 ) across these strains further reinforced their gut-sustainability potentials. Indeed LP 299v and Lp01 are able to survive, persist and therefore show clinical benefits in human patients. Also, for LP-ARP2 , our in vitro experimental validation (acid/bile/aggregation/adhesion/biofilm formation assays) highlighted the similar potential of LP-ARP2 for gastrointestinal survival and persistence. Notably, the CAZyme repertoire and diverse sugar transport systems of LP-ARP2 were significantly prominent and comparable to those of LP 299v and Lp01, reflecting enhanced ecological adaptability and versatile carbohydrate utilization ( Fig. 11 , Table 3 ) . Table 3 Comparative genomic analysis of LP-ARP2 with L. plantarum (LP) 299v and L. plantarum (LP) Lp01 showing promising gene signatures for probiotic attributes, beneficial properties and therapeutic potential Promising gene signatures Potential probiotic/functional attribute Comment LP-ARP2 LP 299v Lp01 uspA, usp6, yugI uspA, usp6, yugI uspA, usp6, yugI Universal stress tolerance Prevent the aggregation of cellular proteins and maintain membrane stabilization under heat stress [ 63 ] ctsR, hrcA ctsR, hrcA ctsR, hrcA Heat-shock tolerance dnaJ, dnaK, groS, groL, grpE, hslU, hslO dnaJ, dnaK, groS, groL, grpE, hslU, hslO dnaJ, dnaK, groS, groL, grpE, hslU, hslO Molecular chaperones hslV, hslU, clpB, clpC, clpE, clpL, clpP, clpX hslV, hslU, clpB, clpC, clpE, clpL, clpP, clpX hslV, hslU, clpB, clpC, clpE, clpL, clpP, clpX Proteases cspA, cspC cspA, cspC cspA, cspC Cold-shock tolerance Help to sustain cold-shock related deleterious effects [ 63 ] atpA–atpH atpA–atpH atpA–atpH Acid stress tolerance Help in proton translocation across membranes to maintain cytoplasmic pH under acidic stress [ 63 ] plsC, asp2, asp23, nhaC, pyk plsC, asp, asp2, nhaC, pyk plsC, asp, asp2, aspB, aspA, aspS, nhaC, pyk Acid and alkaline stress tolerance ppaC, cfa, oppA-F, glnA, glnH, glnP, glnR, glnQ, glnPH2, opuCA, opuCB, opuCC, opuCD, proV, proWX ppaC, cfa, oppA-F, glnA, glnH, glnP, glnR, glnQ, glnPH2, opuCA, opuCB, opuCC, opuCD, proV, proWX ppaC, cfa, oppA-F, glnA, glnH, glnP, glnR, glnQ, glnPH2, opuCA, opuCB, opuCC, opuCD, proV, proWX Bile stress tolerance Assist to preserve membrane integrity under bile stress by upregulation of lipid biosynthesis, uptake of osmoprotectants [ 63 ] mutL, mutS2, mutR, mutT, mutY mutL, mutS2, mutT, mutY mutL, mutS2, mutT, mutY DNA repair Aid in mismatch repair [ 64 ] lspA, mapA, eno, srtA, celA-celE , lspA, mapA, eno, srtA, celA-celE lspA, mapA, eno, srtA, celA, celB, celE Adhesion to gut epithelial cells Encode cell surface proteins of bacteria for colonization to host cells [96,66–68] dltA-D, dltX dltA-D, dltX dltA-D, dltX Adhesion and immunomodulation Influence interaction with host epithelial cells and immune components [ 68 ] veg, luxS veg, luxS veg, luxS Biofilm formation Encode autoinducer-2 (AI-2) which influences both the attachment and biofilm development of Lactobacillus sp. [ 71 , 72 ] tpx, trxb , trxa, nox, npr , ndh, gshR1, nrdH, katA, msrA, msrB tpx, trxb , trxa, nox, npr , ndh, gshF, gshR, gshR1, nrdH, katA, msrA, msrB tpx, trxb , trxa, nox, npr , ndh, gshF, gshR, gshR1, nrdH, katA, msrA, msrB Antioxidant activity Assist in scavenging of ROS, peroxidase detoxification, redox-regulation in the host protecting ROS-mediated protein oxidation [ 63 ] ackA , nagA , pta , acyP , adhE , pdh , fabI-H , patB , ldhA , accA-D , poxB , gabD , dapA-E , malY , eda , scfaA ackA , nagA , pta , acyP , adhE , pdh , fabI-H , patB , ldhA , accA-D , poxB , gabD , dapA-F , malY ackA , nagA , pta , acyP , adhE , pdh , fabI-H , patB , ldhA , accA-D , poxB , gabD , dapA-F , malY SCFA biosynthesis Encode different enzymes involved in fermentation pathways [ 73 ] adk , ribD/E/BA/H/T/F , ThiM/N/D/E/I , rsgA , nudF , pdxK/B , folA-C/E/P , birA , fabI/Z/H/F , nadD , ycsE , yitU , iscS, coaA-D/E/G, menA/G adk , ribD/E/BA/H/T/F , ThiM/N/D/E/I , rsgA , nudF , pdxK/B , folA-E/K/P/T , birA , fabI/Z/H/F , nadD , ycsE , yitU , iscS, coaA-D/E, menA/G adk , ribD/E/BA/H/T/F , ThiM/N/D/E/I , rsgA , nudF , pdxK/B , folA/B/C/E/K , birA , fabI/Z/H/F , nadD , ycsE , yitU , iscS, coaA-D/E, menA/G Vitamin (riboflavin, folate, coenzyme A, thiamine, vitamin K2) biosynthesis Encode different proteins involved in vitamin biosynthesis pathways [ 73 – 75 ] CBM, GHs, GTs, pts, man, mtl, dha, srl CBM, GHs, GTs, AAs, pts, man, mtl, dha, srl CBM, GHs, GTs, AAs, pts, man, mtl, dha, srl Complex carbohydrate hydrolysis and sugar transport Assist in hydrolysis of glycosidic bonds in complex carbohydrates and transfer of sugar molecules [ 63 ] gadB, gabT gabT, gabR, gabD, gadB gabT, gabR, gabD, gadB GABA biosynthesis and metabolism Participate in the synthesis of GABA [97] trpA/B/C/D/E/F/G trpA/B/D/E/F/G/S trpA/B/D/E/F/G/S tryptophan-serotonin metabolism Involved in the synthesis of tryptophan [98] aroA/C/D/E/K, hisA-I/K/S/Z, tyrA/S, pheS/T, ilvE, leuS, hom, hom1, asd, thrB/C/E/S, lysA/C, proA-C/S/V aroA-F/K, hisA-I/K/S/Z, tyrA/S, pheS/T, ilvE, leuS, hom, hom1, asd, thrB/C/E/S, lysA/C/P/M/S, proA-C/S/V aroA-F/K, hisA-I/K/S/Z, tyrA/S, pheS/T, ilvE, leuS, hom, hom1, asd, thrB/C/E/S, lysA/C/P/M/S, proA-C/S/V/WX Essential amino acids (phenylalanine, tyrosine, histidine, isoleucine, valine, leucine, threonine, proline) biosynthesis Associated with the biosynthesis of essential amino acids [ 75 ] cpsY/4D/4J/4I/4G/4F, cps1B/2I, epsB/V cpsY/4D/4J/4I/4G/4F, cps1B/2I, epsB/V cpsY/4D/4J/4I/4G/4F, cps2I, epsB/V Exopolysaccharide biosynthesis and secretion Involved in the synthesis and secretion of exopolysaccharides [ 64 ] Conserved pathways for the biosynthesis of essential amino acids ( aro, trp, his, tyr, phe, tyr, lys , etc.), vitamins ( rib, thi, fol, coa etc. ), and SCFAs ( ack, adh, pdh, scfa etc. ) were consistently observed across LP-ARP2 , LP 299v, and Lp01 strains ( Fig. 11 , Table 3 ) . Indeed, several essential amino acids, vitamins, and SCFAs were validated in LP-ARP2 CFS (HRMS study). Since Lp01 has been shown to be effective in synbiotic formulations [ 68 ], the presence of similar gene signatures for these beneficial metabolites in the LP-ARP2 genome, with HRMS-detection, enhanced its promise as a lucrative candidate for synbiotic applications as a functional food. Genomic signatures of probiotics LP 299v and Lp01 also correlated with their reported functional and clinical outcomes. Antioxidant gene repertoires ( tpx, nox, ndh, gsh, kat , details Table 3 ) were similar across all three strains. Recall, LP 299v and Lp01 were previously shown to mitigate gut inflammation in IBS and other gastrointestinal disorders ( Fig. 11 , Table 3 ) . The presence of similar antioxidant-linked genes and robust free radical scavenging activity of LP-ARP2 strongly suggests a comparable potential to alleviate oxidative stress-driven gastrointestinal diseases. Concurrently, comparable pathways ( trp, gabT, gadB , details Table 3 ) for neuroactive metabolites, including GABA, SCFAs, branched-chain amino acids (BCAAs), and tryptophan, were identified in all three strains. LP 299v has been clinically shown to improve depressive symptoms, while Lp01 has shown immunomodulatory roles in allergic conditions. Consistently, our network-based analysis of LP-ARP2 -derived metabolites revealed neurotransmitter- and neuroinflammation-associated human targets (GABA receptor, amyloid-beta binding, serotonin receptor signaling pathways, etc), indicating comparable psychobiotic potential. Therefore, above analysis highlights the commonality of validated genetic signatures among three strains of L. plantarum for probiotic properties with potential clinical significance for mitigating gut inflammation–related diseases, particularly neuroinflammation-associated disorders. 4. Discussion The present work emerges as a thorough investigation of WGS analysis of Lactiplantibacillus plantarum LP-ARP2 with experimental validation for probiotic traits, HRMS-based metabolite profiling, and functional analysis to uncover the promising genomic marker for the health-promoting properties of the strain ( Table 3 ) . Strikingly, when we compared genomic features of LP-ARP2 with clinically relevant L. plantarum strains, L. plantarum 299v and L. plantarum Lp01, the analyses confer comparable genetic signatures for therapeutic benefits ( Fig. 11 , Table 3 ) . Such strain-specific marker studies could further help to build up a database, which could speed up initial screening of novel probiotic strains using the genome sequence only. Next, these genetic markers also identify the molecular basis of probiotic benefits. To elucidate this further, we used LP-ARP2- derived metabolites to generate a target-based functional network to find out the molecular logic of the LP-ARP2- driven therapeutic benefits. To our knowledge, such a strategy has never been implemented in any probiotic system. The consequences are diverse since the strategy provides a novel road map for further mechanistic studies on any probiotic interventions, focusing on any particular disease model. Robust antioxidant properties of LP-ARP2 suggest that it could reduce oxidative stress and restore redox homeostasis in the gut. The antioxidant gene-signatures of LP-ARP2 are similar to those of LP 299v and Lp01. Recall that LP 299v and Lp01 have been shown to be effective in clinical trials to benefit patients with intestinal disorders. Fascinatingly, the antioxidant activity of LP-ARP2 is also comparable to commercially available and clinically tested (mouse model) L. plantarum strain (MWFLp-182). Together, these indicate the potential roles of the strain in antioxidant defence mechanisms and translational promise as functional foods and nutraceuticals targeting redox imbalance [ 69 , 70 ]. Oxidative stress can also trigger neuroinflammation, which leads to neuronal loss and neurodegeneration. The remarkable antioxidative effects of LP-ARP2 could reduce oxidative stress and neuroinflammation, two major drivers for neurodegeneration ( Fig. 7 ) . LP-ARP2 -CFS secretes key neurotransmitters such as acetylcholine, GABA, and serotonin, which are linked to appetite regulation, energy metabolism, and behavioral modulation, highlighting the promising role of the strain in gut-brain communication (see below). Genome annotation identified gadB and gabT genes, enabling GABA biosynthesis and metabolism, respectively, supporting HRMS findings of GABA in the LP-ARP2 -derived CFS [ 71 ]. GABA plays a vital neuroprotective role by modulating neuronal excitability and reducing oxidative damage and inflammation. Therefore, GABA could control neurological disorders, including epilepsy, Parkinson’s, and Alzheimer’s disease [ 13 ]. Additionally, branched-chain amino acids (BCAAs), important for neurotransmitter balance, are detected in the CFS of the strain. Neuroprotective osmolyte betaine is also detected in LP-ARP2 -CFS. The presence of the comprehensive trp operon ( trpA-G genes) in the genome and serotonin in the CFS supports the involvement in tryptophan-serotonin pathways, which could potentially restore dysregulated neurotransmission observed in AD [ 72 ]. Interestingly, similar gene signatures for GABA, tryptophan, and branched-chain amino acids (BCAAs) were identified in L. plantarum 299v, a strain reported to induce metabolic alterations in patients with major depression [ 73 ]. Next, pathway and network analyses indicate that LP-ARP2 -derived metabolites could target key pathways involved in Alzheimer’s disease, suggesting the potential of the strain to mitigate AD through metabolite-driven mechanisms ( Fig. 10 ; Table S11) . LP-ARP2 -derived metabolites such as butyrate, acetate, citric acid, and dihydroxyacetone exhibit antioxidant properties and therefore could reduce oxidative stress and neuroinflammation. Glycine, alpha-ketoglutarate, betaine, acetate, pyruvate, and serotonin could help restore synaptic transmission and correct neurotransmitter imbalances. LP-ARP2 -secreted metabolites like dihydroxyacetone, glycine, alanine, alpha-ketoglutarate, pyruvate, citric acid, acetate, and aspartic acid are linked to neurogenesis and may counteract neurodegeneration. Alpha-ketoglutarate, alanine, oxalic acid, citric acid, and betaine are associated with regulating neuronal apoptosis, where alpha-ketoglutarate could increase brain-derived neurotrophic factor (BDNF) levels and cognitive function. Pyruvic acid is predicted to target amyloid-beta metabolism. Citric acid could reduce tau protein accumulation, a hallmark of AD, and SCFAs like butyrate and acetate could inhibit GSK3β, reducing tau hyperphosphorylation [ 74 ]. Therefore, these findings suggest that LP-ARP2 -derived metabolites may exert neuroprotective effects by modulating multiple AD-related pathways and strengthening the integrity of gut-brain axis in the host. LP-ARP2 -derived metabolites include essential amino acids, vitamins (C, B 3 ), and energy metabolism-related compounds (e.g., pyruvic acid, oxaloacetate, acetoacetic acid), elucidating the significance of the strain as a nutritional supplement. The genome of LP-ARP2 also harbors numerous genes for essential amino acid biosynthesis, comparable to LP 299v and Lp01, with documented health benefits. This repertoire suggests a capacity to augment host amino acid pools, supporting gut barrier function, neurotransmitter synthesis, and metabolic health. LP-ARP2 shows a broad spectrum of antimicrobial activity with comparable efficacy to commercial L. plantarum strains. Rich repertoire of CAZymes (especially GHs) in LP-ARP2 genome, together with the prebiotic-utilizing ability of LP-ARP2 highlights the therapeutic promise of the strain for effective synbiotic formulations to improve gut health. Interestingly, the therapeutic potential of synbiotic formulations has been evaluated earlier for Lp01 in allergic conditions [ 68 ]. These reinforce the potential application of the strain for microbial control and health-promoting formulations [ 75 ]. The genomic analysis predicts T3PKS and terpene regions in the LP-ARP2 genome ( Fig. 3 c and 4 a ) , which specifically encode for lipid components and bioactive secondary metabolites with lucrative industrial applications. Indeed, LP-ARP2 -derived metabolites (e.g., citric acid, tartaric acid, and acetoin) have diverse applications in food, cosmetic, and poultry industries [ 11 ]. While adipic acid can be used across food processing sectors (food additive), other metabolites like lactamide, malonic semialdehyde, and 2,3-butanediol are widely used in pharmaceutical, cosmetic, and agricultural industries [ 76 , 12 ]. 5. Conclusion This study presents an integrated genomic, metabolomic, and functional profiling of LP-ARP2 , unveiling genetic signatures of the strain for probiotic attributes and therapeutic potential with antimicrobial (anti-biofilm efficacy as well), anti-oxidant, and psychobiotic functions. The comparable genetic features of LP-ARP2 with clinically significant L. plantarum strains for intestinal disorders and depression further indicate the potential application of LP-ARP2 for similar therapeutic benefits. Target-based in silico investigation indeed indicates LP-ARP2 -derived metabolites could modulate multiple neurodegenerative disease-related pathways, suggesting their roles in neuroprotection through metabolite-driven mechanisms. Additionally, the detection of numerous industrially relevant secondary metabolites highlights the promising application of LP-ARP2 in the food, cosmetic, and poultry sectors. Abbreviations WHO, World Health Organization; WGS, Whole Genome Sequencing; ATCC, American Type Culture Collection; MRS, De Man Rogosa Sharpe agar; DMEM, Dulbecco's Modified Eagle Medium; FBS, Fetal Bovine Serum; MTCC, Microbial Type Culture Collection; PBS, Phosphate Buffered Saline; DPPH, 1,1-diphenyl-2-picrylhydrazyl (DPPH)-2,2-diphenyl-1-picrylhydrazyl; ABTS, 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid; PCR, polymerase chain reaction; CFU, colony forming unit; ANI, Average Nucleotide Identity, CRISPR, Clustered Regularly Interspaced Short Palindromic Repeats; PKs, Polyketide Synthases Declarations Authors’ Contributions SP performed all experiments, including analysis, and drafted the manuscript. SP, RP, and PKD performed the bioinformatics experiments and analysis of Figures 8 and 9. AD supervised and analysed Figures 8 and 9. ARC conceived the study, designed the approach, evaluated the data, and was involved in manuscript preparation. Finally, all authors reviewed and edited the manuscript. Ethical statements The author(s) state that there is no conflict of interest. Declaration of Competing Interest The authors report no declaration of interest. Acknowledgments This study was supported by the intramural funding provided by IIT Bhubaneswar. SP receives an Institutional fellowship from IIT Bhubaneswar. RP and PKD acknowledge the MOES DOM-Project and CSIR-JRF for their fellowships, respectively. We are thankful to Dr. Nrisingha Dey, ILS Bhubaneswar, for the Gram-positive pathogens [Methicillin-resistant Staphylococcus aureus ATCC 700699 (MRSA) and Staphylococcus aureus ATCC 25923], Dr. Asish Kumar Mukhopadhyay, NICED Kolkata for Gram-negative clinical isolates [ Escherichia coli (ETEC) BCH 04067 , Vibrio cholerae BCH 09616 , Shigella Flexneri BCH 06745] and Dr. Biswaranjan Pradhan, SKBET, IIT Bhubaneswar, for Lactobacillus acidophilus DDS1. We thank the ILS-High Performance Computing Facility for the computational resources and support. References Hou K, Wu ZX, Chen XY, Wang JQ, Zhang D, Xiao C, Zhu D, Koya JB, Wei L, Li J, Chen ZS (2022) Microbiota in health and diseases. Signal Transduct Target Ther 7:135. https://10.1038/s41392-022-00974-4 Yeshi K, Ruscher R, Hunter L, Daly NL, Loukas A, Wangchuk P (2020) Revisiting Inflammatory Bowel Disease: Pathology, Treatments, Challenges and Emerging Therapeutics Including Drug Leads from Natural Products. J Clin Med 9. https://doi.org/10.3390/jcm9051273 Zhang J, Zhang Y, Wang J, Xia Y, Zhang J, Chen L (2024) Recent advances in Alzheimer's disease: Mechanisms, clinical trials and new drug development strategies. Signal Transduct Target Ther 9:211. https://doi.org/10.1038/s41392-024-01911-3 Patra S, Sahu N, Saxena S, Pradhan B, Nayak SK, Roychowdhury A (2022) Effects of Probiotics at the Interface of Metabolism and Immunity to Prevent Colorectal Cancer-Associated Gut Inflammation: A Systematic Network and Meta-Analysis With Molecular Docking Studies. Front Microbiol 13:878297. https://10.3389/fmicb.2022.878297 Patra S, Saxena S, Sahu N, Pradhan B, Roychowdhury A (2021) Systematic Network and Meta-analysis on the Antiviral Mechanisms of Probiotics: A Preventive and Treatment Strategy to Mitigate SARS-CoV-2 Infection. Probiotics Antimicrob Proteins 13:1138-1156. https://doi.org/10.1007/s12602-021-09748-w Joint F (2002) WHO working group report on drafting guidelines for the evaluation of probiotics in food. London, Ontario, Canada 30:16-22 Ayed L, M'Hir S, Nuzzolese D, Di Cagno R, Filannino P (2024) Harnessing the Health and Techno-Functional Potential of Lactic Acid Bacteria: A Comprehensive Review. Foods 13. https://doi.org/10.3390/foods13101538 Behera SS, Ray RC, Zdolec N (2018) Lactobacillus plantarum with Functional Properties: An Approach to Increase Safety and Shelf-Life of Fermented Foods. Biomed Res Int 2018:9361614. https://10.1155/2018/9361614 Gizachew S, Engidawork E (2024) Genomic Characterization of Lactiplantibacillus plantarum Strains: Potential Probiotics from Ethiopian Traditional Fermented Cottage Cheese. Genes (Basel) 15. https://doi.org/10.3390/genes15111389 Garcia-Gonzalez N, Battista N, Prete R, Corsetti A (2021) Health-Promoting Role of Lactiplantibacillus plantarum Isolated from Fermented Foods. Microorganisms 9. https://10.3390/microorganisms9020349 Dash J, Sethi M, Deb S, Parida D, Kar S, Mahapatra S, Minz AP, Pradhan B, Prasad P, Senapati S (2023) Biochemical, functional and genomic characterization of a new probiotic Ligilactobacillus salivarius F14 from the gut of tribes of Odisha. World J Microbiol Biotechnol 39:171. https://doi.org/10.1007/s11274-023-03626-z Patra S, Pradhan B, Roychowdhury A (2025) Complete genome sequence, metabolic profiling and functional studies reveal Ligilactobacillus salivarius LS-ARS2 is a promising biofilm-forming probiotic with significant antioxidant, antibacterial, and antibiofilm potential. Front Microbiol 16:1535388. https://10.3389/fmicb.2025.1535388 Surve S, Shinde DB, Kulkarni R (2022) Isolation, characterization and comparative genomics of potentially probiotic Lactiplantibacillus plantarum strains from Indian foods. Sci Rep 12:1940. https://10.1038/s41598-022-05850-3 Hu F, Gao Q, Zheng C, Zhang W, Yang Z, Wang S, Zhang Y, Lu T (2024) Encapsulated lactiplantibacillus plantarum improves Alzheimer's symptoms in APP/PS1 mice. J Nanobiotechnology 22:582. https://doi.org/10.1186/s12951-024-02862-1 Kumar S, Stecher G, Suleski M, Sanderford M, Sharma S, Tamura K (2024) MEGA12: Molecular Evolutionary Genetic Analysis Version 12 for Adaptive and Green Computing. Mol Biol Evol 41. https://doi.org/10.1093/molbev/msae263 Seemann T (2014) Prokka: rapid prokaryotic genome annotation. Bioinformatics 30:2068-2069. https://doi.org/10.1093/bioinformatics/btu153 Li H, Durbin R (2009) Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25:1754-1760. https://doi.org/10.1093/bioinformatics/btp324 Grissa I, Vergnaud G, Pourcel C (2007) CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats. Nucleic Acids Res 35:W52-57. https://doi.org/10.1093/nar/gkm360 Jia B, Raphenya AR, Alcock B, Waglechner N, Guo P, Tsang KK, Lago BA, Dave BM, Pereira S, Sharma AN, Doshi S, Courtot M, Lo R, Williams LE, Frye JG, Elsayegh T, Sardar D, Westman EL, Pawlowski AC, Johnson TA, Brinkman FS, Wright GD, McArthur AG (2017) CARD 2017: expansion and model-centric curation of the comprehensive antibiotic resistance database. Nucleic Acids Res 45:D566-D573. https://doi.org/10.1093/nar/gkw1004 Zankari E, Hasman H, Cosentino S, Vestergaard M, Rasmussen S, Lund O, Aarestrup FM, Larsen MV (2012) Identification of acquired antimicrobial resistance genes. J Antimicrob Chemother 67:2640-2644. https://doi.org/10.1093/jac/dks261 Bortolaia V, Kaas RS, Ruppe E, Roberts MC, Schwarz S, Cattoir V, Philippon A, Allesoe RL, Rebelo AR, Florensa AF, Fagelhauer L, Chakraborty T, Neumann B, Werner G, Bender JK, Stingl K, Nguyen M, Coppens J, Xavier BB, Malhotra-Kumar S, Westh H, Pinholt M, Anjum MF, Duggett NA, Kempf I, Nykasenoja S, Olkkola S, Wieczorek K, Amaro A, Clemente L, Mossong J, Losch S, Ragimbeau C, Lund O, Aarestrup FM (2020) ResFinder 4.0 for predictions of phenotypes from genotypes. J Antimicrob Chemother 75:3491-3500. https://doi.org/10.1093/jac/dkaa345 Liu B, Zheng D, Jin Q, Chen L, Yang J (2019) VFDB 2019: a comparative pathogenomic platform with an interactive web interface. Nucleic Acids Res 47:D687-D692. https://10.1093/nar/gky1080 Cosentino S, Voldby Larsen M, Moller Aarestrup F, Lund O (2013) PathogenFinder--distinguishing friend from foe using bacterial whole genome sequence data. PLoS One 8:e77302. https://10.1371/journal.pone.0077302 Dietzsch S, Braesigk A, Seidel C, Remmele J, Kitzing R, Schlender T, Mynarek M, Geismar D, Jablonska K, Schwarz R, Pazos M, Weber DC, Frick S, Gurtner K, Matuschek C, Harrabi SB, Gluck A, Lewitzki V, Dieckmann K, Benesch M, Gerber NU, Obrecht D, Rutkowski S, Timmermann B, Kortmann RD (2022) Types of deviation and review criteria in pretreatment central quality control of tumor bed boost in medulloblastoma-an analysis of the German Radiotherapy Quality Control Panel in the SIOP PNET5 MB trial. Strahlenther Onkol 198:282-290. https://doi.org/10.1007/s00066-021-01822-0 Zheng J, Ge Q, Yan Y, Zhang X, Huang L, Yin Y (2023) dbCAN3: automated carbohydrate-active enzyme and substrate annotation. Nucleic Acids Res 51:W115-W121. https://doi.org/10.1093/nar/gkad328 Kanehisa M, Sato Y, Morishima K (2016) BlastKOALA and GhostKOALA: KEGG Tools for Functional Characterization of Genome and Metagenome Sequences. J Mol Biol 428:726-731. https://10.1016/j.jmb.2015.11.006 de Jong A, van Hijum SA, Bijlsma JJ, Kok J, Kuipers OP (2006) BAGEL: a web-based bacteriocin genome mining tool. Nucleic Acids Res 34:W273-279. https://doi.org/10.1093/nar/gkl237 Pascal Andreu V, Roel-Touris J, Dodd D, Fischbach MA, Medema MH (2021) The gutSMASH web server: automated identification of primary metabolic gene clusters from the gut microbiota. Nucleic Acids Res 49:W263-W270. https://doi.org/10.1093/nar/gkab353 Blin K, Shaw S, Kloosterman AM, Charlop-Powers Z, van Wezel GP, Medema MH, Weber T (2021) antiSMASH 6.0: improving cluster detection and comparison capabilities. Nucleic Acids Res 49:W29-W35. https://doi.org/10.1093/nar/gkab335 Rosenberg M, Gutnick D, Rosenberg E (1980) Adherence of bacteria to hydrocarbons: a simple method for measuring cell-surface hydrophobicity. FEMS Microbiol Lett 9:29-33 Breyer GM, Arechavaleta NN, Siqueira FM, de Souza da Motta A (2021) Characterization of Lactic Acid Bacteria in Raw Buffalo Milk: a Screening for Novel Probiotic Candidates and Their Transcriptional Response to Acid Stress. Probiotics Antimicrob Proteins 13:468-483. https://doi.org/10.1007/s12602-020-09700-4 Rocchetti MT, Russo P, De Simone N, Capozzi V, Spano G, Fiocco D (2024) Immunomodulatory activity on human macrophages by cell-free supernatants to explore the probiotic and postbiotic potential of Lactiplantibacillus plantarum strains of plant origin. Probiotics Antimicrob Proteins 16:911-926. https://doi.org/10.1007/s12602-023-10084-4 Anand S, Mandal S, Tomar SK (2019) Effect of Lactobacillus rhamnosus NCDC 298 with FOS in Combination on Viability and Toxin Production of Enterotoxigenic Escherichia coli. Probiotics Antimicrob Proteins 11:23-29. https://10.1007/s12602-017-9327-1 Palframan R, Gibson GR, Rastall RA (2003) Development of a quantitative tool for the comparison of the prebiotic effect of dietary oligosaccharides. Lett Appl Microbiol 37:281-284. https://10.1046/j.1472-765x.2003.01398.x Pabari K, Pithva S, Kothari C, Purama RK, Kondepudi KK, Vyas BRM, Kothari R, Ambalam P (2020) Evaluation of Probiotic Properties and Prebiotic Utilization Potential of Weissella paramesenteroides Isolated From Fruits. Probiotics Antimicrob Proteins 12:1126-1138. https://10.1007/s12602-019-09630-w Wayne P (2011) Clinical and laboratory standards institute. Performance standards for antimicrobial susceptibility testing. Fonseca HC, de Sousa Melo D, Ramos CL, Dias DR, Schwan RF (2021) Probiotic properties of lactobacilli and their ability to inhibit the adhesion of enteropathogenic bacteria to Caco-2 and HT-29 cells. Probiotics Antimicrob Proteins 13:102-112. https://doi.org/10.1007/s12602-020-09659-2 Chen CC, Lai CC, Huang HL, Huang WY, Toh HS, Weng TC, Chuang YC, Lu YC, Tang HJ (2019) Antimicrobial Activity of Lactobacillus Species Against Carbapenem-Resistant Enterobacteriaceae. Front Microbiol 10:789. https://doi.org/10.3389/fmicb.2019.00789 Lee J-E, Lee N-K, Paik H-D (2021) Antimicrobial and anti-biofilm effects of probiotic Lactobacillus plantarum KU200656 isolated from kimchi. Food Sci Biotechnol 30:97-106. https://doi.org/10.1007/s10068-020-00837-0 Ramalho JB, Soares MB, Spiazzi CC, Bicca DF, Soares VM, Pereira JG, Da Silva WP, Sehn CP, Cibin FW (2019) In vitro probiotic and antioxidant potential of Lactococcus lactis subsp. cremoris LL95 and its effect in mice behaviour. Nutrients 11:901. https://doi.org/10.3390/nu11040901 Kim S, Lee JY, Jeong Y, Kang C-H (2022) Antioxidant activity and probiotic properties of lactic acid bacteria. Fermentation 8:29. https://doi.org/10.3390/fermentation8010029 Gao D, Gao Z, Zhu G (2013) Antioxidant effects of Lactobacillus plantarum via activation of transcription factor Nrf2. Food Funct 4:982-989. https://doi.org/10.1039/c3fo30316k Xiong L, Ni X, Niu L, Zhou Y, Wang Q, Khalique A, Liu Q, Zeng Y, Shu G, Pan K, Jing B, Zeng D (2019) Isolation and Preliminary Screening of a Weissella confusa Strain from Giant Panda (Ailuropoda melanoleuca). Probiotics Antimicrob Proteins 11:535-544. https://doi.org/10.1007/s12602-018-9402-2 Tenea GN, Ortega C (2021) Genome Characterization of Lactiplantibacillus plantarum Strain UTNGt2 Originated from Theobroma grandiflorum (White Cacao) of Ecuadorian Amazon: Antimicrobial Peptides from Safety to Potential Applications. Antibiotics (Basel) 10. https://doi.org/10.3390/antibiotics10040383 Pei Z, Sadiq FA, Han X, Zhao J, Zhang H, Ross RP, Lu W, Chen W (2021) Comprehensive Scanning of Prophages in Lactobacillus: Distribution, Diversity, Antibiotic Resistance Genes, and Linkages with CRISPR-Cas Systems. mSystems 6:e0121120. https://doi.org/10.1128/mSystems.01211-20 Jarocki P, Komon-Janczara E, Mlodzinska A, Sadurski J, Kolodzinska K, Laczmanski L, Panek J, Frac M (2023) Occurrence and genetic diversity of prophage sequences identified in the genomes of L. casei group bacteria. Sci Rep 13:8603. https://10.1038/s41598-023-35823-z Deghorain M, Goffin P, Fontaine L, Mainardi JL, Daniel R, Errington J, Hallet B, Hols P (2007) Selectivity for D-lactate incorporation into the peptidoglycan precursors of Lactobacillus plantarum: role of Aad, a VanX-like D-alanyl-D-alanine dipeptidase. J Bacteriol 189:4332-4337. https://doi.org/10.1128/JB.01829-06 Kandasamy S, Lee KH, Yoo J, Yun J, Kang HB, Kim JE, Oh MH, Ham JS (2024) Whole genome sequencing of Lacticaseibacillus casei KACC92338 strain with strong antioxidant activity, reveals genes and gene clusters of probiotic and antimicrobial potential. Front Microbiol 15:1458221. https://doi.org/10.3389/fmicb.2024.1458221 Sun J, Li W, Li R, Liu W, Wang J, Sun Z, Menghe B (2025) Genomic analysis of probiotics from breast milk Limosilactobacillus fermentum UP-1 and application in fermented milk. J Dairy Sci. https://10.3168/jds.2025-26535 Heunis T, Deane S, Smit S, Dicks LM (2014) Proteomic profiling of the acid stress response in Lactobacillus plantarum 423. J Proteome Res 13:4028-4039. https://doi.org/10.1021/pr500353x Monteagudo-Mera A, Rastall RA, Gibson GR, Charalampopoulos D, Chatzifragkou A (2019) Adhesion mechanisms mediated by probiotics and prebiotics and their potential impact on human health. Appl Microbiol Biotechnol 103:6463-6472. https://10.1007/s00253-019-09978-7 Brockhausen I (2014) Crossroads between Bacterial and Mammalian Glycosyltransferases. Front Immunol 5:492. https://10.3389/fimmu.2014.00492 Lebeer S, Vanderleyden J, De Keersmaecker SC (2008) Genes and molecules of lactobacilli supporting probiotic action. Microbiol Mol Biol Rev 72:728-764, Table of Contents. https://doi.org/10.1128/MMBR.00017-08 Salas-Jara MJ, Ilabaca A, Vega M, Garcia A (2016) Biofilm Forming Lactobacillus: New Challenges for the Development of Probiotics. Microorganisms 4. https://doi.org/10.3390/microorganisms4030035 Gao J, Sadiq FA, Zheng Y, Zhao J, He G, Sang Y (2022) Biofilm-based delivery approaches and specific enrichment strategies of probiotics in the human gut. Gut Microbes 14:2126274. https://doi.org/10.1080/19490976.2022.2126274 Lei Y, Oshima T, Ogasawara N, Ishikawa S (2013) Functional analysis of the protein Veg, which stimulates biofilm formation in Bacillus subtilis. J Bacteriol 195:1697-1705. https://doi.org/10.1128/JB.02201-12 Mgomi FC, Yang YR, Cheng G, Yang ZQ (2023) Lactic acid bacteria biofilms and their antimicrobial potential against pathogenic microorganisms. Biofilm 5:100118. https://10.1016/j.bioflm.2023.100118 Liang X, Dai N, Yang F, Zhu H, Zhang G, Wang Y (2024) Molecular identification and safety assessment of the potential probiotic strain Bacillus paralicheniformis HMPM220325 isolated from artisanal fruit dairy products. Food Funct 15:747-765. https://doi.org/10.1039/d3fo04625g Kavanova K, Kostovova I, Moravkova M, Kubasova T, Babak V, Crhanova M (2024) Comparative Genome Analysis and Characterization of the Probiotic Properties of Lactic Acid Bacteria Isolated from the Gastrointestinal Tract of Wild Boars in the Czech Republic. Probiotics Antimicrob Proteins. https://doi.org/10.1007/s12602-024-10259-7 Shrestha P, Karmacharya J, Han SR, Lee JH, Oh TJ (2024) Elucidation of bacterial trehalose-degrading trehalase and trehalose phosphorylase: physiological significance and its potential applications. Glycobiology 34. https://doi.org/10.1093/glycob/cwad084 Jakaria Al-Mujahidy SM, Kryukov K, Ikeo K, Saito K, Uddin ME, Ibn Sina AA (2024) Functional genomic analysis of the isolated potential probiotic Lactobacillus delbrueckii subsp. indicus TY-11 and its comparison with other Lactobacillus delbrueckii strains. Microbiol Spectr 12:e0347023. https://10.1128/spectrum.03470-23 Sethi M, Ok A, Dash J, Parida D, Kar S, Mishra S, Minz AP, Padhi A, Das KR, Pradhan B, Prakash T, Senapati S (2025) Whole Genome Mining and Characterization of a New Probiotic Strain Levilactobacillus brevis ILSH3 from Handia: An Ethnic Fermented Beverage of Odisha, India. Probiotics Antimicrob Proteins 17:1596-1614. https://doi.org/10.1007/s12602-024-10217-3 Abriouel H, Manetsberger J, Caballero Gomez N, Benomar N (2022) In silico genomic analysis of the potential probiotic Lactiplantibacillus pentosus CF2-10N reveals promising beneficial effects with health promoting properties. Front Microbiol 13:989824. https://10.3389/fmicb.2022.989824 Naughton LM, Romano S, O'Gara F, Dobson ADW (2017) Identification of Secondary Metabolite Gene Clusters in the Pseudovibrio Genus Reveals Encouraging Biosynthetic Potential toward the Production of Novel Bioactive Compounds. Front Microbiol 8:1494. https://doi.org/10.3389/fmicb.2017.01494 Wong A, Ngu DY, Dan LA, Ooi A, Lim RL (2015) Detection of antibiotic resistance in probiotics of dietary supplements. Nutr J 14:95. https://doi.org/10.1186/s12937-015-0084-2 Kaushik A, Kest H, Sood M, Steussy BW, Thieman C, Gupta S (2024) Biofilm Producing Methicillin-Resistant Staphylococcus aureus (MRSA) Infections in Humans: Clinical Implications and Management. Pathogens 13. https://doi.org/10.3390/pathogens13010076 Pique N, Berlanga M, Minana-Galbis D (2019) Health Benefits of Heat-Killed (Tyndallized) Probiotics: An Overview. Int J Mol Sci 20. https://doi.org/10.3390/ijms20102534 Fassio F, Guagnini F (2018) House dust mite-related respiratory allergies and probiotics: a narrative review. Clin Mol Allergy 16:15. https://10.1186/s12948-018-0092-9 Gawlik-Dziki U, Baraniak B, Sikora M, Jakubczyk A, Kapusta I, Swieca M (2021) Potentially Bioaccessible Phenolic and Antioxidant Potential of Fresh and Stored Lentil Sprouts-Effect of Lactobacillus plantarum 299v Enrichment. Molecules 26. https://doi.org/10.3390/molecules26082109 Nie H, Wang X, Luo Y, Kong F, Mu G, Wu X (2024) Mechanism Explanation on Improved Cognitive Ability of D-Gal Inducing Aged Mice Model by Lactiplantibacillus plantarum MWFLp-182 via the Microbiota-Gut-Brain Axis. J Agric Food Chem 72:9795-9806. https://doi.org/10.1021/acs.jafc.3c09675 Diez-Gutiérrez L, San Vicente L, Barrón LJR, del Carmen Villarán M, Chávarri M (2020) Gamma-aminobutyric acid and probiotics: Multiple health benefits and their future in the global functional food and nutraceuticals market. J Funct Foods 64:103669. https://doi.org/10.1016/j.jff.2019.103669 Liang X, He J, Zhang N, Muhammad A, Lu X, Shao Y (2022) Probiotic potentials of the silkworm gut symbiont Enterococcus casseliflavus ECB140, a promising L‐tryptophan producer living inside the host. J Appl Microbiol 133:1620-1635. https://doi.org/10.1111/jam.15675 Godzien J, Kalaska B, Rudzki L, Barbas-Bernardos C, Swieton J, Lopez-Gonzalvez A, Ostrowska L, Szulc A, Waszkiewicz N, Ciborowski M, Garcia A, Kretowski A, Barbas C, Pawlak D (2025) Probiotic Lactobacillus plantarum 299v supplementation in patients with major depression in a double-blind, randomized, placebo-controlled trial: A metabolomics study. J Affect Disord 368:180-190. https://10.1016/j.jad.2024.09.058 Qian XH, Xie RY, Liu XL, Chen SD, Tang HD (2022) Mechanisms of Short-Chain Fatty Acids Derived from Gut Microbiota in Alzheimer's Disease. Aging Dis 13:1252-1266. https://10.14336/AD.2021.1215 Wullt M, Johansson Hagslatt ML, Odenholt I, Berggren A (2007) Lactobacillus plantarum 299v enhances the concentrations of fecal short-chain fatty acids in patients with recurrent clostridium difficile-associated diarrhea. Dig Dis Sci 52:2082-2086. https://10.1007/s10620-006-9123-3 Horn H, Holland EG, Hazleton L (1957) Food additives, safety of adipic acid as compared with citric and tartaric acid. J Agric Food Chem 5:759-762. https://doi.org/10.1021/jf60080a007 Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.doc 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-7556358","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":514703933,"identity":"f21d5349-b557-4646-a909-4180810e0ff1","order_by":0,"name":"Sinjini Patra","email":"","orcid":"","institution":"Indian Institute of Technology Bhubaneswar","correspondingAuthor":false,"prefix":"","firstName":"Sinjini","middleName":"","lastName":"Patra","suffix":""},{"id":514703935,"identity":"0c76fc98-6930-41ce-9efd-850e6d8abed7","order_by":1,"name":"Ritwik Patra","email":"","orcid":"","institution":"Institute of Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Ritwik","middleName":"","lastName":"Patra","suffix":""},{"id":514703936,"identity":"1a618938-52eb-41ea-aa78-6fbb86287e16","order_by":2,"name":"Pradeep Kumar Das","email":"","orcid":"","institution":"Institute of Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Pradeep","middleName":"Kumar","lastName":"Das","suffix":""},{"id":514703937,"identity":"e94da062-352c-494f-ac8c-7c3be064f03e","order_by":3,"name":"Anshuman Dixit","email":"","orcid":"","institution":"Institute of Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Anshuman","middleName":"","lastName":"Dixit","suffix":""},{"id":514703939,"identity":"33389b25-2c7b-457b-b944-ef3f32544fcb","order_by":4,"name":"Anasuya Roychowdhury","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAElEQVRIiWNgGAWjYLACxgYgcQPEqpDgYQAiMGAjTssZCR4e0rQwtjEwwLXgAvyzDx+T+LnDJrHvdo/ZZ955FjL20r2HPzDU2DHwSTdg1SJxLi1NsvdMWuLMO2eMZ/NuAzpM5lyaBMOxZAY2mQPYrTnDYybN2HY4d8ONHGNmsBaJHDOgRw4wsEkkYNUhj6plDliL8QeGf7i1GKBqaQBrMZBgbMOtxfAMW7Jlb1ta/cwbacWMc44BtdwB+iWxL5kHlxa5M8wHb/xsszHmu5G8meFNTZ09+2xgiH34ZicnPwO7FiBgkYCxmOAxAlSML3aYP8BYjD/wKBsFo2AUjIKRCwDEOFX0wuHCewAAAABJRU5ErkJggg==","orcid":"","institution":"Indian Institute of Technology Bhubaneswar","correspondingAuthor":true,"prefix":"","firstName":"Anasuya","middleName":"","lastName":"Roychowdhury","suffix":""}],"badges":[],"createdAt":"2025-09-07 12:53:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7556358/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7556358/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91503979,"identity":"9cf1f604-9313-4850-a988-13dd069ed61b","added_by":"auto","created_at":"2025-09-17 08:03:54","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1723975,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical abstract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig.1.tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7556358/v1/221e1325548bb4ab20d1a9c8.jpg"},{"id":91504814,"identity":"0722c594-6d04-47c0-9a56-cfae1549857e","added_by":"auto","created_at":"2025-09-17 08:11:54","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2481723,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea Circular genome diagram of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLP-ARP2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003eFrom the inner to the outer: the first ring represented the genome size (3,212,397 bp); the second depicted the GC skew (G + C/G – C); the third and fourth showed forward and reverse CDS (coding sequences) annotated with Prokka (the sites of CDSs/rRNA/tRNA/tmRNA on the genome are marked). \u003cstrong\u003eb Phylogenetic analysis based on NCBI blast and MEGA 12 reaffirmed that \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLP-ARP2 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ebelongs to \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLactiplantibacillus plantarum. \u003c/strong\u003e\u003c/em\u003ePhylogenetic tree indicates that the closest clade for \u003cem\u003eLP-ARP2\u003c/em\u003eis \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e strain NBRC 15891. \u003cstrong\u003ec\u003c/strong\u003e \u003cstrong\u003eGenome mining of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLP-ARP2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003egenome and prediction of major pathways. The KEGG enriched pathways.\u003c/strong\u003e Enriched KEGG pathways were analysed from the genome of \u003cem\u003eLP-ARP2\u003c/em\u003e. Carbohydrate metabolic pathways, biosynthesis of essential amino acids, and biosynthesis of secondary metabolites were major pathways. \u003cstrong\u003ed Genome mining of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLP-ARP2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e genome and prediction of probiotic gene signatures. \u003c/strong\u003eWGS analysis identified major gene signatures associated with probiotic and functional properties in the \u003cem\u003eLP-ARP2\u003c/em\u003e genome\u003c/p\u003e","description":"","filename":"Fig.2.tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7556358/v1/04771b7033f53d562718aec6.jpg"},{"id":91505648,"identity":"55a74177-2d90-401b-87b1-c00ac7e22f8e","added_by":"auto","created_at":"2025-09-17 08:19:54","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2428663,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e \u003cstrong\u003ePrediction of genes for primary metabolites in Metabolic Gene Clusters (MGCs) of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLP-ARP2 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003egenome. \u003c/strong\u003eGutSMASH run of the \u003cem\u003eLP-ARP2\u003c/em\u003egenome predicted type pyruvate to acetate-formate MGC region, which is responsible for the production of SCFAs. \u003cstrong\u003eComparative analysis\u003c/strong\u003e between different bacterial reference genes (pre-computed in gutSMASH) for the most similar MGCs based on a gutSMASH run. Genes with the same colour indicated putative homologs based on significant Blast hits between \u003cem\u003eLP-ARP2\u003c/em\u003e and the reference bacterial genes. \u003cstrong\u003eThe KnownClusterBlast analysis\u003c/strong\u003e showed the gene similarity of the predicted region (Pyruvate2acetate-formate) with MGCs associated with known functions of other \u003cem\u003eLactobacillus\u003c/em\u003e reference genomes. \u003cstrong\u003eThe ClusterBlast analysis \u003c/strong\u003eshowed that the predicted MGC does not have homology with other \u003cem\u003eLactobacillus\u003c/em\u003e. White genes have no relationship. \u003cstrong\u003eb GutSMASH run of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLP-ARP2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e genome predicted the type nitrate reductase MGC region\u003c/strong\u003e, which is responsible for the production of nitric oxide. \u003cstrong\u003ec\u003c/strong\u003e \u003cstrong\u003ePrediction of genes for secondary metabolites in the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLP-ARP2 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003egenome. antiSMASH run of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLP-ARP2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e genome\u003c/strong\u003e predicted the type T3PKS region, which is responsible for the production of metabolites with diverse applications in the food industry\u003c/p\u003e","description":"","filename":"Fig.3.tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7556358/v1/b4395d4e3b2277f48eda4ceb.jpg"},{"id":91504816,"identity":"c15e86dd-9cc4-468e-8d10-262f074b4ae9","added_by":"auto","created_at":"2025-09-17 08:11:54","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2386966,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea Prediction of genes for secondary metabolites in the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLP-ARP2 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003egenome. antiSMASH run of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLP-ARP2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e genome\u003c/strong\u003e predicted a type of Terpene region, responsible for the production of secondary metabolites with applications in the pharmaceutical industry. \u003cstrong\u003eb\u003c/strong\u003e \u003cstrong\u003eantiSMASH run of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLP-ARP2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003egenome\u003c/strong\u003e predicted a type cyclic-lactone-autoinducer region, which is responsible for the production of cyclic peptides, important for quorum sensing\u003cstrong\u003e c\u003c/strong\u003e \u003cstrong\u003ePrediction of genes for bacteriocin and accessory proteins in Biosynthetic Gene Clusters (BCGs) organization in the genome of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLP-ARP2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e(BAGEL4 analysis)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig.4.tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7556358/v1/4871eeba975477aa03994c79.jpg"},{"id":91503988,"identity":"545e7a6c-2b93-4fb4-aec4-406b28cbacae","added_by":"auto","created_at":"2025-09-17 08:03:54","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1802708,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProbiotic attributes of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLP-ARP2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. a Acid tolerance. \u003c/strong\u003eSurvival of \u003cem\u003eLP-ARP2\u003c/em\u003e at pH 3 and pH 4 at different time intervals indicated the viability of the strain at acidic pH. Data was presented as mean ± SD from three independent biological replicates. Statistical analysis was done using a two-way ANOVA with a Bonferroni post hoc test; *p\u0026lt;0.05; ****p\u0026lt;0.0001. \u003cstrong\u003eb Bile tolerance of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLP-ARP2.\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eViability of \u003cem\u003eLP-ARP2 \u003c/em\u003ein the presence of 0.3% and 1% bile salt for 3 and 5 h of incubation time indicated tolerance of the strain at high bile concentrations. Data were represented as mean ± SD based on three independent biological replicates. Statistical analysis was done using a two-way ANOVA with a Bonferroni post hoc test; *p\u0026lt;0.05; ****p\u0026lt;0.0001. \u003cstrong\u003ec\u003c/strong\u003e \u003cstrong\u003eAutoaggregation attributes of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLP-ARP2.\u003c/strong\u003e\u003c/em\u003e The aggregation property of \u003cem\u003eLP-ARP2\u003c/em\u003e was assessed at different time points up to 24 h. The results were depicted as mean ± SD based on three independent biological replicates. Statistical analysis was done using two-way ANOVA with Bonferroni post hoc test; p\u0026gt;0.05. \u003cstrong\u003ed\u003c/strong\u003e \u003cstrong\u003eCell surface hydrophobicity property\u003c/strong\u003e \u003cstrong\u003eof \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLP-ARP2.\u003c/strong\u003e\u003c/em\u003e The hydrophobicity percentage of \u003cem\u003eLP-ARP2 was \u003c/em\u003eevaluated with n-hexadecane and xylene (2 h of incubation). All the results were shown as mean ± SD based on three independent biological replicates. Statistical analysis was done using one-way ANOVA with Bonferroni post hoc test; *p\u0026lt;0.05; ****p\u0026lt;0.0001. \u003cstrong\u003ee\u003c/strong\u003e \u003cstrong\u003eAdhesion efficacy\u003c/strong\u003e \u003cstrong\u003eof \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLP-ARP2\u003c/strong\u003e\u003c/em\u003e. Attachment efficiency of \u003cem\u003eLP-ARP2\u003c/em\u003e was shown with intestinal epithelial Caco-2 cells. \u003cstrong\u003ef Biofilm-forming ability of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLP-ARP2\u003c/strong\u003e\u003c/em\u003e. The assay was performed compared with two standard probiotic strains, \u003cem\u003eL. acidophilus\u003c/em\u003e DDS1 and \u003cem\u003eL. rhamnosus\u003c/em\u003e GG (\u003cem\u003eLR-GG\u003c/em\u003e). Results were represented as mean ± SD based on three independent biological replicates. Statistical analysis was done using one-way ANOVA with Bonferroni post hoc test; ****p\u0026lt;0.0001. \u003cstrong\u003eg Effect of prebiotics on the growth of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLP-ARP2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003eAcidification profile of \u003cem\u003eLP-ARP2 \u003c/em\u003ein MRS basal media supplemented with 1%, 1.5%, and 2% maltodextrin compared with the same doses of dextrose. Statistical analysis was done using one-way ANOVA with Bonferroni post hoc test; ****p\u0026lt;0.0001. \u003cstrong\u003eh\u003c/strong\u003e \u003cstrong\u003eGrowth of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLP-ARP2 \u003c/strong\u003e\u003c/em\u003ein MRS basal media supplemented with different doses of dextrose and maltodextrin (1%, 1.5%, and 2%). Statistical analysis was done using one-way ANOVA with Bonferroni post hoc test; \u003csup\u003ens\u003c/sup\u003ep\u0026gt;0.05.\u003cstrong\u003e i Prebiotic score of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLP-ARP2 \u003c/strong\u003e\u003c/em\u003ein different doses of maltodextrin (1%, 1.5%, and 2%) as compared to glucose. \u003cstrong\u003ej\u003c/strong\u003e \u003cstrong\u003eBiomass (Log\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e10\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e CFU/mL) of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLP-ARP2 \u003c/strong\u003e\u003c/em\u003ein MRS basal media supplemented with different doses of dextrose and maltodextrin (1%, 1.5%, and 2%). Statistical analysis was done using one-way ANOVA with Bonferroni post hoc test; \u003csup\u003ens\u003c/sup\u003ep\u0026gt;0.05. \u003cstrong\u003ek Prebiotic index of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLP-ARP2 \u003c/strong\u003e\u003c/em\u003ein different doses of maltodextrin (1%, 1.5%, and 2%) as compared to dextrose\u003c/p\u003e","description":"","filename":"Fig.5.tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7556358/v1/cd1676477d31d9c47c40c322.jpg"},{"id":91503991,"identity":"bdbc276f-c395-4552-8467-186ea1f29077","added_by":"auto","created_at":"2025-09-17 08:03:54","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1427348,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntimicrobial properties of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLP-ARP2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. a Agar well diffusion assay.\u003c/strong\u003e The result showed diverse inhibition zones, signifying the antimicrobial activity of \u003cem\u003eLP-ARP2\u003c/em\u003e against multiple Gram-positive and Gram-negative enteric pathogens. The data were represented as mean ± SD based on three independent biological replicates. \u003cstrong\u003eb Agar well diffusion assay for \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSalmonella Typhimurium\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003eThe results showed the effect of normal, pH-neutralized, and heat-denatured cell-free supernatant (CFS) of \u003cem\u003eLP-ARP2\u003c/em\u003e on the growth of \u003cem\u003eS. Typhimurium\u003c/em\u003e (ST). The results were shown as mean ± SD based on three independent biological replicates. \u003cstrong\u003ec, d, e\u003c/strong\u003e \u003cstrong\u003eDetermination of\u003c/strong\u003e \u003cstrong\u003ethe\u003c/strong\u003e \u003cstrong\u003eminimum inhibitory percentage (MIP) of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLP-ARP2 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eCFS for ST. \u003c/strong\u003eST was incubated in various percentages of \u003cstrong\u003ec\u003c/strong\u003enormal, \u003cstrong\u003ed\u003c/strong\u003e pH-neutralized, and \u003cstrong\u003ee\u003c/strong\u003e heat-treated CFS of \u003cem\u003eLP-ARP2\u003c/em\u003e to estimate the MIP of the CFS against the pathogen. The data presented from three biological replicates; *p\u0026lt;0.05; ****p\u0026lt;0.0001 using one-way ANOVA with Bonferroni post hoc test. \u003cstrong\u003ef Estimation of the viability of ST in the presence of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLP-ARP2 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eCFS. \u003c/strong\u003eFrom every well of ST grown in \u003cem\u003eLP-ARP2\u003c/em\u003e CFS, the culture was spotted to find out the bactericidal or bacteriostatic effect. All the data are represented as the mean ± SD from three independent biological replicates. \u003cstrong\u003eDetermination of minimum inhibitory percentage (MIP) of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLP-ARP2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-derived CFS for Methicillin-resistant \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eStaphylococcus aureus \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e(MRSA). g\u003c/strong\u003e\u003cem\u003e \u003c/em\u003e\u003cstrong\u003eTo determine the MIP, \u003c/strong\u003eMRSA was grown in different percentages of \u003cem\u003eLP-ARP2\u003c/em\u003e CFS.\u003cstrong\u003e \u003c/strong\u003eAll the data were represented as mean ± SD of three replicates.\u003cstrong\u003e h Anti-biofilm Assay. \u003c/strong\u003eCell-free supernatant (CFS) of \u003cem\u003eLP-ARP2\u003c/em\u003e showed a significant reduction in the biofilm formation of Methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC 700699. The results were represented as the mean ± SD based on three independent biological replicates. Statistical analysis was done using one-way ANOVA with a Bonferroni post hoc test; ****p\u0026lt;0.0001\u003c/p\u003e","description":"","filename":"Fig.6.tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7556358/v1/67add5dbba056662287854fb.jpg"},{"id":91503987,"identity":"12d9e51f-e913-4e71-a3b6-52d2024521bb","added_by":"auto","created_at":"2025-09-17 08:03:54","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1445377,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnti-oxidant properties of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLP-ARP2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. a ABTS cation radical scavenging assay.\u003c/strong\u003e The result depicted a significant cation radical scavenging rate of intact and heat-lysed cells of \u003cem\u003eLP-ARP2\u003c/em\u003e. \u003cstrong\u003eb DPPH free radical scavenging assay.\u003c/strong\u003e The result demonstrated the free radical scavenging activity of \u003cem\u003eLP-ARP2\u003c/em\u003e intact and heat-lysed cells. \u003cstrong\u003ec Superoxide free radical scavenging assay.\u003c/strong\u003e The data showed the superoxide anion scavenging capacity of \u003cem\u003eLP-ARP2\u003c/em\u003e intact and heat-lysed cells. \u003cstrong\u003ed Hydroxyl free radical scavenging assay.\u003c/strong\u003e The result indicated the hydroxyl radical scavenging ability of \u003cem\u003eLP-ARP2\u003c/em\u003e intact and heat-lysed cells. The results were shown as mean ± SD from three independent biological replicates. Statistical analysis was done using a paired t-test. *p\u0026lt;0.05; ****p\u0026lt;0.0001. The antioxidant activity of \u003cem\u003eLP-ARP2\u003c/em\u003e could potentially alleviate oxidative stress, leading to the mitigation of gut-inflammation and neuroinflammation, promoting gut/brain health\u003c/p\u003e","description":"","filename":"Fig.7.tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7556358/v1/2e4bdf64f831257841d92772.jpg"},{"id":91504817,"identity":"d743dbdc-6ff1-4cf4-aa51-03c0041bcf3e","added_by":"auto","created_at":"2025-09-17 08:11:54","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1341788,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMolecular basis of therapeutic potential of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLP-ARP2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-derived metabolites. a Cytoscape-based \u003c/strong\u003enetwork of \u003cem\u003eLP-ARP2\u003c/em\u003e-derived metabolites and metabolite-associated human targets.\u003cstrong\u003e b Functional enrichment analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLP-ARP2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-derived metabolites and the human target network. Molecular function (gene ontology) enrichment analysis \u003c/strong\u003ereveals the enrichment of neurotransmitters (GABA, serotonin) receptor activity\u003cstrong\u003e, \u003c/strong\u003epostsynaptic neurotransmitter receptor activity\u003cstrong\u003e \u003c/strong\u003eassociated with the \u003cem\u003eLP-ARP2\u003c/em\u003e-secreted metabolites, and their human targets\u003c/p\u003e","description":"","filename":"Fig.8.tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7556358/v1/691e36f426d92940f102f0b7.jpg"},{"id":91503994,"identity":"0cf54ddd-c4ce-45ea-9521-81276b419b57","added_by":"auto","created_at":"2025-09-17 08:03:54","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1429188,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNetwork analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLP-ARP2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-derived metabolites and Alzheimer's Disease-associated targets.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e \u003cstrong\u003eCytoscape-based network\u003c/strong\u003e shows \u003cem\u003eLP-ARP2\u003c/em\u003e-secreted metabolites and their human targets, mainly involved in Alzheimer's Disease pathogenesis. Blue nodes represent \u003cem\u003eLP-ARP2\u003c/em\u003e-derived metabolites, and orange nodes represent metabolite-associated Alzheimer's Disease\u003cstrong\u003e \u003c/strong\u003etargets.\u003cstrong\u003e b\u003c/strong\u003e \u003cstrong\u003eFunctional enrichment analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLP-ARP2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-derived metabolites and Alzheimer's Disease-associated human targets network. Network analysis of metabolites and Alzheimer's Disease-associated targets.\u003c/strong\u003e Molecular function (gene ontology), enrichment analysis of metabolites, and Alzheimer's Disease-associated human targets network show important molecular functions that could be targeted by \u003cem\u003eLP-ARP2\u003c/em\u003e-secreted metabolites to alleviate the disease conditions\u003c/p\u003e","description":"","filename":"Fig.9.tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7556358/v1/e8e620d722a14dc675548653.jpg"},{"id":91503989,"identity":"eb7ce202-f6d1-472e-8f13-266ce3a9dcbf","added_by":"auto","created_at":"2025-09-17 08:03:54","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":1650254,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe mechanistic model on the alleviation of Alzheimer's disease pathogenesis by \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLP-ARP2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-secreted bioactive metabolites\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig.10.tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7556358/v1/8835ad9310340cc4544bde97.jpg"},{"id":91504823,"identity":"a1cd6d85-1202-4188-8362-ef7b8e1f2f8b","added_by":"auto","created_at":"2025-09-17 08:11:54","extension":"jpg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":1073029,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparative genomic analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLP-ARP2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e with \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eL. plantarum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e (LP) 299v and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eL. plantarum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e(LP) Lp01. \u003c/strong\u003eWGS analysis identified major gene signatures associated with probiotic and functional properties in the genomes of \u003cem\u003eLP-ARP2,\u003c/em\u003e LP 299v, and Lp01\u003c/p\u003e","description":"","filename":"Fig.11.tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7556358/v1/a185a76bead90359a09c3ff1.jpg"},{"id":93737454,"identity":"01982a76-e24c-4f93-a4bf-4affac6d378e","added_by":"auto","created_at":"2025-10-17 04:01:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":24105938,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7556358/v1/8b350da8-4586-4695-9146-7fb1177777d3.pdf"},{"id":91504819,"identity":"d3f6b794-d129-4d9e-9590-46570b4e31b7","added_by":"auto","created_at":"2025-09-17 08:11:54","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6449152,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.doc","url":"https://assets-eu.researchsquare.com/files/rs-7556358/v1/16a58928130b01669a9e9263.doc"}],"financialInterests":"No competing interests reported.","formattedTitle":"WGS analysis and Functional Studies Illustrate Promising Gene- signatures for Probiotic Attributes and Molecular-targeted Therapeutic Prospects of Lactiplantibacillus plantarum LP-ARP2","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eChronic inflammation damages gut-health, causes intestinal disorders. Through gut-organ axes, it further leads to a wide spectrum of systemic diseases like cancer, atherosclerosis, type 2 diabetes, respiratory diseases, and neuropsychiatric disorders (e.g., Alzheimer\u0026rsquo;s, Parkinson\u0026rsquo;s, schizophrenia) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The global burden of diseases related to the gut-organ axis, limited clinical trials, and various side effects of the current therapies highlight the need for safer therapeutic strategies [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Probiotics could restore gut microbial balance, reduce inflammation, and prevent gut and gut-organ related diseases [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Therefore, global demand for probiotic supplements is rapidly growing, projected to reach \u003cspan\u003e$\u003c/span\u003e3.5\u0026nbsp;billion by 2026, reflecting the increasing emphasis on probiotics in health and disease prevention [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eLactic acid bacteria (LAB), especially \u003cem\u003eLactobacillus\u003c/em\u003e species, are the most studied and widely used probiotics, considered GRAS (Generally Recognized as Safe or Qualified Presumption of Safety) by the FDA (Food and Drug Administration) and EFSA (European Food Safety Authority) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Among them, \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e (formerly \u003cem\u003eLactobacillus plantarum\u003c/em\u003e) stands out for its versatility and adaptability across diverse environments, from the human body to various fermented products [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Moreover, the broad-spectrum health benefits of \u003cem\u003eL. plantarum\u003c/em\u003e range from enhancing gut barrier integrity, immunomodulation, and prevention of infection to lowering cholesterol, indicating widespread applications of the strain in food and pharmaceutical industries [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, probiotic effects are highly strain-specific, limiting the use of a single strain for all clinical or industrial purposes [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Therefore, targeted, criteria-based selection and detailed characterization of probiotics are in demand [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Despite the availability of over 670 annotated \u003cem\u003eL. plantarum\u003c/em\u003e genomes in NCBI, only a few strains (299v and Lp01) are commercially used or clinically studied, due to significant genomic variability [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Even detailed genomic mining linking the genomic blueprint to probiotic and functional effects of 299v and Lp01 are also not studied in detail. Whole genome sequencing (WGS) analysis could reveal the probiotic-specific gene signatures to ensure safe and effective applications of the strains. However, a detailed strain-specific WGS study with experimental validation for \u003cem\u003eL. plantarum\u003c/em\u003e strains is limited. Moreover, the molecular basis of its therapeutic benefits in disease systems is poorly understood [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHere, we aim to conduct a comprehensive strain-specific genomic, metabolomic and functional study on \u003cem\u003eL. plantarum LP-ARP2\u003c/em\u003e, focusing on the target-based antimicrobial, antioxidant, and psychobiotic properties of the strain. The study presents a thorough WGS analysis of \u003cem\u003eLP-ARP2\u003c/em\u003e, with mining of gene signatures of \u003cem\u003eL. plantarum\u003c/em\u003e strains 299v and Lp01 for probiotic-specific functional traits. Interestingly, the genomic properties of \u003cem\u003eLP-ARP2\u003c/em\u003e are comparable to 299v and Lp01 that have been extensively studied in multiple clinical trials and confer beneficial effects in depression, irritable bowel syndrome, and other intestinal disorders [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Such comparative genomic analysis between the \u003cem\u003eL. plantarum\u003c/em\u003e strains (\u003cem\u003eLP-ARP2\u003c/em\u003e, 299v, and Lp01) could lead to infer species-specific genetic markers for probiotic benefits. Our experimental assays further validate the genomic predictions of the strain \u003cem\u003eLP-ARP2\u003c/em\u003e for the crucial probiotic attributes like acid/bile tolerance, autoaggregation, surface hydrophobicity, adhesion capacity, and biofilm formation. Remarkable prebiotic utilization of the strain indicates potential for gut adaptation and microbiota modulation. In accordance with the predicted gene signatures from WGS analysis, functional assays validate strong antioxidant activity, broad-spectrum antimicrobial effects, and anti-biofilm potential of \u003cem\u003eLP-ARP2-\u003c/em\u003ederived cell-free supernatant (CFS). The metabolic profile of CFS by high-resolution mass spectrometry (HRMS) study reveals various bioactive metabolites with health-promoting potential and industrial applications. Target-based functional network analysis with the HRMS-identified metabolites further indicates that \u003cem\u003eLP-ARP2\u003c/em\u003e-derived metabolites could target important neuro-domains through which \u003cem\u003eLP-ARP2\u003c/em\u003e could manifest its psychobiotic properties to alleviate neurodegenerative disorders like Alzheimer\u0026rsquo;s disease. Such a strategy, using \u003cem\u003ein vitro\u003c/em\u003e HRMS data (\u003cem\u003eLP-ARP2\u003c/em\u003e-derived metabolites) to perform \u003cem\u003ein silico\u003c/em\u003e target-based network analysis to find out potential molecular signatures for probiotic function, is novel for any probiotic system.\u003c/p\u003e\u003cp\u003eIn summary, \u003cem\u003eLP-ARP2\u003c/em\u003e, offers multifunctional therapeutic promise, particularly prominent for antimicrobial, antioxidant, and psychobiotic effects. The study confers promising gene-signatures for probiotic attributes of \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e strains and opens up a new avenue to explore the molecular basis of probiotic-derived health-benefits in disease systems \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Bacterial Culture\u003c/h2\u003e\u003cp\u003ePure culture of \u003cem\u003eLP-ARP2\u003c/em\u003e (\u003cem\u003eL. plantarum\u003c/em\u003e MTCC 2621) was obtained from the Microbial Type Culture Collection (MTCC), IMTECH, Chandigarh, India. The strain was routinely cultured with MRS agar and broth (HiMedia) in a standard anaerobic environment. \u003cem\u003eLP-ARP2\u003c/em\u003e and reference strains, \u003cem\u003eL. acidophilus\u003c/em\u003e DDS1 and \u003cem\u003eL. rhamnosus\u003c/em\u003e GG, were stored at \u0026minus;\u0026thinsp;80\u0026deg;C in 50% glycerol for long-term use.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Whole Genome Sequencing and Genomic Characterization of \u003cem\u003eLP-ARP2\u003c/em\u003e\u003c/h2\u003e\u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\u003ch2\u003e2.2.1. Isolation of DNA and preparation of library\u003c/h2\u003e\u003cp\u003e\u003cem\u003eLP-ARP2\u003c/em\u003e was grown anaerobically in MRS broth at 37\u0026deg;C for 12 h. The genomic DNA was extracted using the QiaAmp DNA mini kit (Cat# 51306), and quantified with the Qubit Fluorometer 3 and the Qubit dsDNA High Sensitivity Assay Kit (Invitrogen, Cat# Q32854). Library preparation was performed using the 5300 Fragment Analyzer (3.1.0.12) and ProSize software 4.0.0.3. Pair-end sequencing (151 bp read length) of the DNA libraries was conducted with the NovaSeq6000 platform (MedGenome, Bangalore, India). Quality-checked sequences were exported as fastq files. Strain identity was confirmed by comparing 16S rRNA gene sequences with reference sequences in NCBI using BLAST. Phylogenetic analysis was conducted using the Neighbor-joining (NJ) method in MEGA12 [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e2.2.2. Determination of the strain identity by whole genome sequencing and genome assembly\u003c/h2\u003e\u003cp\u003eThe complete genome of \u003cem\u003eLP-ARP2\u003c/em\u003e was sequenced [NovaSeq6000 (MedGenome, Bangalore, India)]. The reads were screened for human DNA contamination; \u0026sim;10.06\u0026ndash;27.65% aligning to the human genome were excluded. The remaining data were quality-checked for sequence quality score distribution, GC content, base quality score, over-represented sequences, average base content per read, and possible PCR or adapter artifacts. Based on these parameters, low-quality reads were trimmed, and adapters were removed from the 3\u0026rsquo; ends using fastq mcf (1.04.803). The refined reads were aligned to the \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e strain ATCC 8014 reference genome for coverage estimation and reference-guided assembly. Consensus fasta files were developed using Samtools tools (v1.2), while coverage and depth statistics were generated using bedtools (v2.0) and an in-house Perl script. GATK was used to predict variations from the reference-aligned reads, and snpEff to annotate the variants. Genome annotation, including prediction of genes, CDS, and genomic features, was carried out using the Prokaryotic Genome Annotation System (Prokka v1.14.6) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The complete genome sequence is available in NCBI under Bioproject PRJNA1248240.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.2.3. Genomic analysis of \u003cem\u003eLP-ARP2\u003c/em\u003e in comparison to clinically studied \u003cem\u003eL. plantarum strain\u003c/em\u003es (299v and Lp01)\u003c/h2\u003e\u003cp\u003eProphage sequences in the \u003cem\u003eLP-ARP2\u003c/em\u003e genome were identified using Rapid Annotations using Subsystems Technology (RAST) server with SEED viewer v2.0. CRISPR, Cas, and truncated Cas sequences were screened using CRISPRCasFinder 1.1.2 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The Resistance Gene Identifier Tool (RGI 6.0.3) and the Comprehensive Antibiotic Resistance Database (CARD 3.3.0) were used to identify the antibiotic resistance genes (perfect/rigorous hit, high-quality coverage) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Acquired antimicrobial resistance genes and/or chromosomal alterations were assessed using ResFinder 4.4.2 (%ID 90.00%, minimum length 60%) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Virulence and toxin-associated genes were detected by BLAST search against the virulence factor database (VFDB) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.mgc.ac.cn/cgi-bin/VFs/v5/main.cgi\u003c/span\u003e\u003cspan address=\"http://www.mgc.ac.cn/cgi-bin/VFs/v5/main.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. PathogenFinder 1.1 analyzed potential pathogenicity [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eProkka (1.14.6) and EggNOG-mapper v2.1.12 (Egg-NOG v5.0) were used for functional annotation of \u003cem\u003eLP-ARP2\u003c/em\u003e, \u003cem\u003eL. plantarum\u003c/em\u003e 299v, and \u003cem\u003eL. plantarum\u003c/em\u003e Lp01 genomes [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Carbohydrate-active enzymes (CAZymes) were identified with dbCAN3 [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. KEGG mapper and RAST were employed to annotate pathways linked to probiotic properties [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Biosynthetic gene clusters (BGCs) for bacteriocins were investigated by BAGEL4 [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. While primary and secondary metabolites encoding gene clusters were predicted using gutSMASH (Specialised Primary Metabolite Analysis from Anaerobic Bacteria) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] and antiSMASH v6.0.1 (strict mode), respectively [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Comparative genomic analysis with clinically studied \u003cem\u003eL. plantarum\u003c/em\u003e strains (299v and Lp01), reported to benefit depression, IBS, and other intestinal disorders, provided a framework to infer the potential health-promoting properties of \u003cem\u003eLP-ARP2\u003c/em\u003e [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Validation of Probiotic Attributes of \u003cem\u003eLP-ARP2\u003c/em\u003e\u003c/h2\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.3.1. Tolerance of \u003cem\u003eLP-ARP2\u003c/em\u003e to acidic pH\u003c/h2\u003e\u003cp\u003e\u003cem\u003eLP-ARP2\u003c/em\u003e overnight culture was inoculated in MRS broth of pH 4 and pH 3 (pH 6.5, control), incubated at 37\u0026deg;C for 0, 1, 3, and 5 h [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] At each time point, the culture was serially diluted in PBS (pH 7.4) and plated on MRS agar, followed by anaerobic incubation for 24 h at 37\u0026deg;C. Colony counts were expressed as biomass (Log\u003csub\u003e10\u003c/sub\u003e CFU/mL), and survival rate (%) was calculated as [biomass at pH 4 or 3/biomass at control pH 6.5] x 100. The experiment was performed in triplicate.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003ch2\u003e2.3.2. Tolerance of \u003cem\u003eLP-ARP2\u003c/em\u003e to bile salt\u003c/h2\u003e\u003cp\u003eOvernight culture of \u003cem\u003eLP-ARP2\u003c/em\u003e was inoculated in MRS broth with 0.3% and 1% (w/v) bile salts (HiMedia) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. MRS broth without bile salt was the control. Cultures were incubated for 0, 1, 3, and 5 h at 37\u0026deg;C, serially diluted in PBS, and spread on MRS agar. After 24 h anaerobic incubation, biomass (Log\u003csub\u003e10\u003c/sub\u003e CFU/mL) was determined by colony counts. Survival rate (%) was calculated as [biomass at 0.3% or 1% bile salt/biomass in control] x 100. The experiment was carried out in triplicate.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003ch2\u003e2.3.3. Determination of the self-aggregation property of \u003cem\u003eLP-ARP2\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eOvernight culture of \u003cem\u003eLP-ARP2\u003c/em\u003e was subcultured in MRS broth until OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.5\u0026ndash;0.6, centrifuged (5000 rpm), washed, and resuspended in PBS [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The culture was adjusted to 10\u003csup\u003e8\u003c/sup\u003e CFU/mL (OD\u003csub\u003e600\u003c/sub\u003e 0.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1, A\u003csub\u003e0\u003c/sub\u003e), vortexed (10 sec), and incubated at 37\u0026deg;C for 1\u0026ndash;24 h. The absorbance (600 nm) of the upper suspension was measured after each time interval (A\u003csub\u003etime\u003c/sub\u003e). The autoaggregation percentage was calculated as [1\u0026ndash;(A\u003csub\u003eTime\u003c/sub\u003e/A\u003csub\u003e0\u003c/sub\u003e) \u0026times; 100]. The experiment was carried out in triplicate.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003e2.3.4. Evaluation of cell surface hydrophobicity of \u003cem\u003eLP-ARP2\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eIn the Bacterial Attachment to Hydrocarbons (BATH) method, \u003cem\u003eLP-ARP2\u003c/em\u003e overnight culture was harvested, washed, and resuspended in 10 mL of phosphate urea magnesium sulphate (PUM) buffer to OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.8\u0026ndash;0.9 (A\u003csub\u003e0\u003c/sub\u003e). [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Adjusted cell suspension (4.8 mL) was mixed with 0.8 mL of n-hexadecane (Sigma)/xylene (Merck), incubated for 10 min, vortexed (2 min), and then incubated at 37\u0026deg;C for 2 h. Absorbance (600 nm) of the lower aqueous phase was measured (A), and hydrophobicity (H%) was calculated as [1\u0026ndash;(A/A\u003csub\u003e0\u003c/sub\u003e) \u0026times; 100]. The experiment was carried out in triplicate.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003ch2\u003e2.3.5. Determination of adhesion property of \u003cem\u003eLP-ARP2\u003c/em\u003e using human colon adenocarcinoma cells (Caco-2)\u003c/h2\u003e\u003cp\u003eCaco-2 cells were cultured in DMEM (Himedia) with 10% heat-inactivated FBS (Gibco) and seeded at 1x10\u003csup\u003e5\u003c/sup\u003e cells/well in six-well plates. Cells were maintained until 80% confluent, followed by 20 days of differentiation to form monolayers [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Before bacterial treatment, the Caco-2 monolayer was washed and co-cultured with \u003cem\u003eLP-ARP2\u003c/em\u003e at 10\u003csup\u003e8\u003c/sup\u003e CFU/mL, followed by incubation for 2 h at 37\u0026deg;C under 5% CO\u003csub\u003e2\u003c/sub\u003e (Galaxy 48R, New Brunswick, Germany). Unattached bacteria were washed, cells were trypsinized, gently aspirated, serially diluted, and plated on MRS agar. After 24 h anaerobic incubation, adhesion rate (%) was expressed (R\u003csub\u003et\u003c/sub\u003e/R\u003csub\u003e0\u003c/sub\u003e) x 100, where R\u003csub\u003e0\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;initial bacterial count (CFU/mL) and R\u003csub\u003et\u003c/sub\u003e=count after incubation. The experiment was performed in triplicate.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003ch2\u003e2.3.6. Evaluation of the biofilm-forming ability of \u003cem\u003eLP-ARP2\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eAn 18 h \u003cem\u003eLP-ARP2\u003c/em\u003e culture in MRS broth was diluted to OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.1 and dispensed into 24-well plates, followed by anaerobic incubation for 24 h at 37\u0026deg;C in a moist chamber [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The media was disposed of after the incubation period, wells were washed and dried at 60\u0026deg;C for 1 h. Biofilms were stained for 45 mins using a 0.1% (w/v) crystal violet (SRL, prepared in 95% ethanol), residual stain was washed, air-dried, and dissolved in 33% acetic acid (Merck). Absorbance was measured at 570 nm. \u003cem\u003eL. rhamnosus GG\u003c/em\u003e and \u003cem\u003eL. acidophilus\u003c/em\u003e DDS1 served as positive controls. The assay was carried out in triplicate.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\u003ch2\u003e2.3.7. Prebiotic utilization of \u003cem\u003eLP-ARP2\u003c/em\u003e\u003c/h2\u003e\u003cp\u003ePrebiotic utilization of \u003cem\u003eLP-ARP2\u003c/em\u003e was determined in terms of prebiotic score (PS), acidification profile (pH), and prebiotic index (PI) [\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Overnight culture was harvested, washed, and inoculated at 10\u003csup\u003e6\u003c/sup\u003e CFU/mL in MRS-BB (MRS basal broth without any carbon source) supplemented with 1% carbohydrate [glucose or Fructooligosaccharide (FOS), maltodextrin, and inulin], and incubated anaerobically at 37\u0026deg;C for 24 h, 48 h, and 72 h. Dextrose served as the positive control. Growth (OD\u003csub\u003e600\u003c/sub\u003e) and pH of the culture CFS were measured at each time interval. To assess dose-dependence, prebiotics were tested at 1%, 1.5%, and 2% of maltodextrin. PS was evaluated as (A/B) \u0026times; 100, where A and B represent the mean OD\u003csub\u003e600\u003c/sub\u003e of \u003cem\u003eLP-ARP2\u003c/em\u003e grown with prebiotic and dextrose, respectively, after 24 h [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. For viable counts, cultures were serially diluted in PBS, plated on MRS agar, and incubated anaerobically at 37\u0026deg;C for 24 h. Biomass (Log\u003csub\u003e10\u003c/sub\u003e CFU/mL) was determined, and PI was calculated as PI = [CFU of probiotics in prebiotic carbohydrate/CFU of probiotics in control carbohydrate]. The experiment was performed in triplicate.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Determination of Safety Attributes of \u003cem\u003eLP-ARP2\u003c/em\u003e: antibiotic susceptibility, haemolytic, and DNase activity\u003c/h2\u003e\u003cp\u003eFor the antibiotic susceptibility, an overnight \u003cem\u003eLP-ARP2\u003c/em\u003e culture was inoculated on MRS agar, and antibiotic discs (HiMedia) were placed [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Plates were incubated anaerobically for 24 h at 37\u0026deg;C, and inhibition zone diameters (mm) were measured. The CLSI standards classified the susceptibility as resistant (R), intermediate susceptible (I), or susceptible (S) [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. For hemolysis, culture was spot-inoculated on a sheep blood agar plate (5% w/v, defibrinated, HiMedia), and incubated for 24 h [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Hemolytic activity was categorized as β-hemolysis (clear zones), α-hemolysis (greenish zones), or γ-hemolysis (no zones). Controls included \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC 25923 (β-hemolysis), \u003cem\u003eEscherichia coli\u003c/em\u003e ATCC 25922 (α-hemolysis), and \u003cem\u003eL. acidophilus\u003c/em\u003e DDS1 (γ-hemolysis). For DNase activity, culture was spot-inoculated on DNase agar (HiMedia) and incubated at 37\u0026deg;C for 24 h. After incubation, plates were flooded with 1N HCl to visualize DNase activity as clear zones around colonies [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. \u003cem\u003eS. aureus\u003c/em\u003e ATCC 25923 served as a positive control, and \u003cem\u003eL. acidophilus\u003c/em\u003e DDS1 as a negative control. All the experiments were performed in triplicate.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Assessment of antimicrobial properties of \u003cem\u003eLP-ARP2\u003c/em\u003e\u003c/h2\u003e\u003cdiv id=\"Sec18\" class=\"Section3\"\u003e\u003ch2\u003e2.5.1. Preparation of the cell-free supernatant (CFS)\u003c/h2\u003e\u003cp\u003e\u003cem\u003eLP-ARP2\u003c/em\u003e was cultured for 24 h in MRS broth anaerobically at 37\u0026deg;C. The culture was centrifuged for 20 min at 4\u0026deg;C and 4000 rpm. The CFS was filtered using a PVDF filter (0.22 \u0026micro;m, HiMedia) before use in the studies [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section3\"\u003e\u003ch2\u003e2.5.2. Determination of the antimicrobial activity of \u003cem\u003eLP-ARP2\u003c/em\u003e using Gram-positive, Gram-negative pathogens, and multidrug-resistant hospital isolates\u003c/h2\u003e\u003cp\u003eFor agar well diffusion, the pathogens (0.5 McFarland, OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.5) were swabbed on Mueller-Hinton agar (HiMedia) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Gram-negative \u003cem\u003eSalmonella Typhimurium\u003c/em\u003e ATCC 14028, Gram-positive \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC 25923, Methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC 700699 (MRSA), and multi-drug-resistant clinically isolated Gram-negative strains such as \u003cem\u003eEscherichia coli\u003c/em\u003e (ETEC) BCH 04067, \u003cem\u003eShigella Flexneri\u003c/em\u003e BCH 06745, and \u003cem\u003eVibrio cholerae\u003c/em\u003e BCH 09616, were among the pathogens under investigation. 6 mm wells were treated with 100 \u0026micro;L \u003cem\u003eLP-ARP2\u003c/em\u003e CFS (negative control-uninoculated MRS broth) and incubated overnight. The zone of inhibition was measured, and the experiment was carried out in triplicate.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section3\"\u003e\u003ch2\u003e2.5.3. Evaluation of minimum inhibitory percentage (MIP) of \u003cem\u003eLP-ARP2\u003c/em\u003e CFS using the broth microdilution method\u003c/h2\u003e\u003cp\u003eThree sets of \u003cem\u003eLP-ARP2\u003c/em\u003e CFS were used: Set 1: untreated; Set 2: heat-treated (15 min at 95\u0026deg;C); Set 3: pH-neutralized. Overnight \u003cem\u003eS. Typhimurium\u003c/em\u003e culture was centrifuged, washed, and inoculated (100 \u0026micro;L; 10\u003csup\u003e5\u003c/sup\u003e CFU/mL) with varying percentages (100 \u0026micro;L, 1\u0026ndash;50% v/v) of the CFS diluted in MRS [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Uninoculated MRS and Mueller-Hinton broth (MHB, HiMedia) were used as blank negative controls. Absorbance (600 nm) was determined after more than 18 h of incubation. The bacteriostatic or bactericidal effects of \u003cem\u003eLP-ARP2\u003c/em\u003e were assessed by spot-inoculating cultures on MHA plates from the same microtiter plate. The experiment was carried out in triplicate.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section3\"\u003e\u003ch2\u003e2.5.4. Determination of anti-biofilm properties of \u003cem\u003eLP-ARP2\u003c/em\u003e CFS\u003c/h2\u003e\u003cp\u003eTo evaluate the antibiofilm property of \u003cem\u003eLP-ARP2\u003c/em\u003e, overnight MRSA strain (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.1) with glucose (Merck) and NaCl (Merck) was inoculated with 10%-40% v/v \u003cem\u003eLP-ARP2\u003c/em\u003e-CFS (MIP to 4 MIP) in 24-well plates [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. After 24 h incubation, staining was carried out as previously described (section \u003cspan refid=\"Sec14\" class=\"InternalRef\"\u003e2.3.6\u003c/span\u003e). Uninoculated MRS and TSB served as a control. The biofilm formation rate (%) was determined as OD\u003csub\u003eSample\u003c/sub\u003e/OD\u003csub\u003eControl\u003c/sub\u003e x 100. The experiment was carried out in triplicate.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Evaluation of Antioxidant Properties of \u003cem\u003eLP-ARP2\u003c/em\u003e\u003c/h2\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003e2.6.1. Preparation of the intact and heat-lysed cells\u003c/h2\u003e\u003cp\u003eOvernight \u003cem\u003eLP-ARP2\u003c/em\u003e culture was washed and resuspended in distilled water up to an OD\u003csub\u003e600\u003c/sub\u003e of 1.0. Samples were heated to 95\u0026deg;C (water bath) for 30 min to prepare heat-lysed cells [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section3\"\u003e\u003ch2\u003e2.6.2. Determination of ABTS cation radical scavenging capacity of \u003cem\u003eLP-ARP2\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eA working solution was prepared by mixing ABTS (Sigma) with potassium persulphate (HiMedia) in 1:1 v/v, followed by dilution up to OD\u003csub\u003e734\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.7 with methanol (HiMedia, HPLC) [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Then, samples (0.6 mL intact or heat-lyzed cells) were combined with ABTS solution (1.2 mL) and incubated (30 min, dark, room temperature). Distilled water was the control. With A\u003csub\u003eC\u003c/sub\u003e = absorbance of control and A\u003csub\u003eS\u003c/sub\u003e = absorbance of sample at 734 nm, the ABTS scavenging rate (%) was calculated as (Ac\u0026ndash;As)/Ac x 100. The experiment was carried out in triplicate.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003e2.6.3. Assessment of DPPH free radical scavenging capacity of \u003cem\u003eLP-ARP2\u003c/em\u003e\u003c/h2\u003e\u003cp\u003e0.2 mM DPPH (CDH) solution (1 mL) in methanol (HiMedia) was mixed with 1 mL intact or heat-lyzed cells, mixed well, and incubated at room temperature (30 min dark) [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Distilled water was the control. DPPH radical scavenging activity (U/mL)\u0026thinsp;=\u0026thinsp;ABS\u003csub\u003eC\u003c/sub\u003e\u0026ndash;ABS\u003csub\u003eS\u003c/sub\u003e/S x 100, where S\u0026thinsp;=\u0026thinsp;sample volume (mL) and ABS\u003csub\u003eC\u003c/sub\u003e and ABS\u003csub\u003eS\u003c/sub\u003e represent the absorbance of the test and control samples, respectively, measured at 517 nm. The experiment was carried out in triplicate.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003e2.6.4. Estimation of superoxide anion scavenging activity of \u003cem\u003eLP-ARP2\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eBriefly, 0.2 mL Tris-HCl (Sigma) was mixed with 0.8 mL intact or heat-lyzed cells. 0.1 mL pyrogallol (Sigma) was added, followed by incubation at room temperature (30 min, dark) [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Deionised water was the control. Superoxide anion radical scavenging ability (%) was calculated as [1\u0026ndash;(As \u0026ndash; A\u003csub\u003e1\u003c/sub\u003e)/A\u003csub\u003e0\u003c/sub\u003e] x 100, where As =\u0026thinsp;absorbance (320 nm) in the presence of pyrogallol, A\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;absorbance without pyrogallol, and A\u003csub\u003e0\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;absorbance of the blank solution with pyrogallol. The experiment was carried out in triplicate.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\u003ch2\u003e2.6.5. Evaluation of the hydroxyl radical scavenging ability of \u003cem\u003eLP-ARP2\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eEqual amounts of FeSO\u003csub\u003e4\u003c/sub\u003e (Merck), sodium phosphate buffer (Sigma), and 1,10-phenanthroline (Sigma) were mixed, vortexed, and incubated for 5 to 7 min [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. An equal volume of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (0.12% v/v, CDH) and \u003cem\u003eLP-ARP2\u003c/em\u003e intact or heat-lyzed cells were mixed, and incubated for 60 min at 37\u0026deg;C (water bath). Deionized water was used as the control group in place of sample. Hydroxyl radical scavenging activity (%) was computed as [(As\u0026ndash;A\u003csub\u003e1\u003c/sub\u003e)/(A\u003csub\u003e0\u003c/sub\u003e\u0026ndash;A\u003csub\u003e1\u003c/sub\u003e)] x 100, where As =\u0026thinsp;absorbance (536 nm) of the sample when H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e is present, A\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;absorbance of the sample when H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e is not present, and A\u003csub\u003e0\u003c/sub\u003e is the absorbance of the solution with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and without a sample. The experiment was carried out three times.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Determination of metabolite profiling of \u003cem\u003eLP-ARP2\u003c/em\u003e by High-Resolution Mass Spectroscopy (HRMS)\u003c/h2\u003e\u003cp\u003eMetabolite profiling of \u003cem\u003eLP-ARP2\u003c/em\u003e-derived CFS was performed using an Exactive\u0026trade; Plus Orbitrap high-resolution mass spectrometer coupled to an Ultimate 3000 HPLC system (Thermo Scientific, USA) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Equal volumes of CFS and methanol were mixed, filtered, and transferred (0.5 mL) into a DP ID vial (Cat# C4000-1W, Thermo Scientific). Separation was achieved on a Hypersil BDS C18 column (250 mm \u0026times; 2.1 mm, 5 \u0026micro;m; Thermo Scientific) with 0.1% formic acid in acetonitrile:water (1:1) as the mobile phase. The flow rate was 3 \u0026micro;L/min, column temperature 30\u0026deg;C, and pressure 700 bar. Ionization was performed by electrospray ionization (ESI) at 3 eV, with spectra acquired in both positive and negative ion modes over an m/z range of 50\u0026ndash;750 during a 5-min run. Metabolites were annotated using the National Metabolomics Data Repository (NMDR) and cross-referenced against KEGG, LIPID MAPS, ChEBI, HMDB, BMRB, PubChem, and NP Atlas via the Metabolomics Workbench (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.metabolomicsworkbench.org/data/M_form.php#M2\u003c/span\u003e\u003cspan address=\"https://www.metabolomicsworkbench.org/data/M_form.php#M2\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Mass tolerance for compound assignment was set at \u0026plusmn;\u0026thinsp;0.2 ppm, and results were exported with compound names and delta PPM values. Data from both ionization modes were integrated, duplicates removed, and identified metabolites validated against published literature, entered into the table.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Evaluation of Functional Potential of \u003cem\u003eLP-ARP2\u003c/em\u003e-secreted Metabolites\u003c/h2\u003e\u003cdiv id=\"Sec30\" class=\"Section3\"\u003e\u003ch2\u003e2.8.1. Identification of molecular targets for HRMS-identified metabolites\u003c/h2\u003e\u003cp\u003eA programmatic multi-database approach was applied to predict potential biological targets of \u003cem\u003eLP-ARP2\u003c/em\u003e-derived bioactive metabolites. Metabolite names were first queried in PubChem via its RESTful API to retrieve chemical descriptors (SMILES, InChIKey, and PubChem CID). These identifiers were then used to extract experimentally validated and computationally predicted protein targets from the PubChem BioAssay database, with additional target information retrieved from BindingDB. All targets were consolidated, deduplicated, and classified into human or microbial categories based on source organism taxonomy.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec31\" class=\"Section3\"\u003e\u003ch2\u003e2.8.2. Construction of compound-target interaction network and functional enrichment analysis\u003c/h2\u003e\u003cp\u003eCurated compound-target interactions were used to construct a bipartite interaction network in Cytoscape (compounds: source nodes; protein targets: target nodes). Further, the ClueGO plugin in Cytoscape was employed to perform Gene Ontology (GO) with pV \u0026le; 0.05 and KEGG pathway enrichment analysis to understand the functional roles of the target proteins. The enrichment was conducted using Biological Process, Cellular Component Branch, Molecular Function, and KEGG pathways. To assess disease relevance, the DisGeNET plugin was applied to identify overlaps between predicted human protein targets and Alzheimer\u0026rsquo;s disease-associated targets, thereby evaluating the potential impact of \u003cem\u003eLP-ARP2\u003c/em\u003e-derived metabolites on Alzheimer\u0026rsquo;s disease.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec32\" class=\"Section2\"\u003e\u003ch2\u003e2.9. Statistical analysis\u003c/h2\u003e\u003cp\u003eStatistical analyses were performed using GraphPad Prism v8. Paired t-tests were applied for two-group comparisons, while one-way ANOVA was used for multiple groups with a single variable and two-way ANOVA for comparisons involving multiple groups with two variables. A p-value\u0026thinsp;\u0026le;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec34\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Whole Genome Sequencing (WGS) of \u003cem\u003eLP-ARP2\u003c/em\u003e: General features\u003c/h2\u003e\u003cdiv id=\"Sec35\" class=\"Section3\"\u003e\u003ch2\u003e3.1.1. \u003cem\u003eLP-ARP2\u003c/em\u003e genome and phylogenetic analysis\u003c/h2\u003e\u003cp\u003eWhole genome sequencing revealed that the \u003cem\u003eLP-ARP2\u003c/em\u003e genome consisted of a single circular chromosome with 3,212,397 bp and 44.55% average GC content \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. There were 3077 genes identified, and 3006 were the coding DNA sequences (CDSs) in the chromosome \u003cb\u003e(Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e)\u003c/b\u003e. An overview of the genome assembly is depicted in \u003cb\u003eTable S2\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eComparative BLAST search against all \u003cem\u003eLactiplantibacillus\u003c/em\u003e genomes placed our strain within the \u003cem\u003eLactiplantibacillus\u003c/em\u003e clade, closely related to \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e strain NBRC 15891 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. This result was supported by the average nucleotide identity (ANI), where 99.9% of ANI was observed for \u003cem\u003eLP-ARP2\u003c/em\u003e with \u003cem\u003eL. plantarum\u003c/em\u003e DOMLa [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. 16S rRNA extracted with Barrnapp indicated that \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e strain CIP 103151 exhibited the highest similarity with the strain. Therefore, \u003cem\u003eLP-ARP2\u003c/em\u003e was affirmed as \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec36\" class=\"Section3\"\u003e\u003ch2\u003e3.1.2. Safety evaluation of \u003cem\u003eLP-ARP2\u003c/em\u003e genome\u003c/h2\u003e\u003cp\u003eHidden prophages are common in many \u003cem\u003eLactobacillus\u003c/em\u003e species and can affect the intestinal microbiota upon activation [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Therefore, screening of prophage is crucial for probiotic safety assessment. Functional annotation of the \u003cem\u003eLP-ARP2\u003c/em\u003e genome via RAST-SEED viewer identified genes related to phage replication, tail proteins, capsid proteins, packaging machinery, and phage introns \u003cb\u003e(Table S3)\u003c/b\u003e. As \u003cem\u003eL. plantarum\u003c/em\u003e is known to be a multihabitat species, prophage elements are more frequent in the genome sequence [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Additionally, to counter phage attacks, bacteria employ the CRISPR/Cas system [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Notably, one CRISPR array and one Cas-associated sequence (CRISPRFinder) were predicted in \u003cem\u003eLP-ARP2\u003c/em\u003e, associated with fewer intact prophages. This suggested the presence of an antiphage defense mechanism in the strain \u003cb\u003e(Table S4)\u003c/b\u003e [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eVirulence factors (VFs) analysis using core and comprehensive VF datasets showed no significant hits in the \u003cem\u003eLP-ARP2\u003c/em\u003e genome. Using the PathogenFinder tool, \u003cem\u003eLP-ARP2\u003c/em\u003e was identified as a non-human pathogen, showing no alignment with known pathogenic families and exhibiting a very low likelihood (0.2) of being associated with human pathogenicity.\u003c/p\u003e\u003cp\u003eMicrobes can obtain antibiotic resistance genes through horizontal gene transfer [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Several \u003cem\u003eLactobacillus\u003c/em\u003e strains, including \u003cem\u003eL. plantarum\u003c/em\u003e, are intrinsically resistant to vancomycin due to d-Ala-d-lactate in their peptidoglycan [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. \u003cem\u003eLP-ARP2\u003c/em\u003e harboured one antibiotic-resistant gene according to the CARD database and RGI analysis \u003cb\u003e(Table S5)\u003c/b\u003e, though none were detected by ResFinder.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec37\" class=\"Section3\"\u003e\u003ch2\u003e3.1.3. General functional annotation of the \u003cem\u003eLP-ARP2\u003c/em\u003e genome\u003c/h2\u003e\u003cp\u003eFunctional annotation of the \u003cem\u003eLP-ARP2\u003c/em\u003e genome revealed 1549 CDS (51.5%) classified into 38 functional classes, 204 pathways, and 37 modules \u003cb\u003e(Tables S6).\u003c/b\u003e Key pathways included biosynthesis of amino acids, secondary metabolites, and cofactors/vitamins, highlighting strong metabolic versatility. Genes linked to biosynthetic pathways of essential amino acids (phenylalanine, tryptophan, tyrosine, leucine, isoleucine, valine, lysine, methionine, cysteine, serine, threonine, glycine, arginine), pyruvate, and vitamins (riboflavin, thiamine, nicotinate, vitamin B6, nicotinamide) suggested potential health benefits of \u003cem\u003eLP-ARP2\u003c/em\u003e \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e. COG classification (3006 CDS) further highlighted diverse housekeeping and metabolic functions, with genes enabling utilization of glucose (n\u0026thinsp;=\u0026thinsp;22), mannose and fructose (n\u0026thinsp;=\u0026thinsp;25), galactose (n\u0026thinsp;=\u0026thinsp;21), sucrose and starch (n\u0026thinsp;=\u0026thinsp;26), nucleotide/amino sugars (n\u0026thinsp;=\u0026thinsp;28), supporting adaptability to varied habitats. Carbohydrate fermentation pathways encoded SCFA production: acetate, lactate, propanoate (n\u0026thinsp;=\u0026thinsp;10), butanoate (n\u0026thinsp;=\u0026thinsp;7), pyruvate (n\u0026thinsp;=\u0026thinsp;22), succinate, a hallmark of probiotic functionality. Additionally, genes for butanoate, polyketide (n\u0026thinsp;=\u0026thinsp;4), and terpenoid (n\u0026thinsp;=\u0026thinsp;10) biosynthesis point to applications in food, nutraceutical, and pharmaceutical industries (see discussion).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec38\" class=\"Section2\"\u003e\u003ch2\u003e3.2. WGS analysis: Probiotic-specific genomic features of \u003cem\u003eLP-ARP2\u003c/em\u003e genome\u003c/h2\u003e\u003cdiv id=\"Sec39\" class=\"Section3\"\u003e\u003ch2\u003e3.2.1. Stress-resilience genetic set-up at \u003cem\u003eLP-ARP2\u003c/em\u003e genome indicated gut-survival potential of the strain\u003c/h2\u003e\u003cp\u003eTo exert health benefits, probiotics must endure in the gastrointestinal tract and food processing stresses \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e. WGS analysis of \u003cem\u003eLP-ARP2\u003c/em\u003e revealed a comprehensive stress-response repertoire, including universal stress proteins \u003cem\u003e(uspA, usp6, yugI)\u003c/em\u003e, heat-shock regulators (\u003cem\u003ectsR, hrcA\u003c/em\u003e), chaperones (\u003cem\u003ednaJ/K, groS/L, grpE, hslU/O\u003c/em\u003e), and proteases (\u003cem\u003ehslV/U, clpB/C/E/L/P/X\u003c/em\u003e), ensuring protein stability and membrane integrity under heat stress \u003cb\u003e(Table S7)\u003c/b\u003e. Cold-shock proteins (\u003cem\u003ecspA/C\u003c/em\u003e) and proton-translocating systems, including the F₀F₁ ATP synthase operon (\u003cem\u003eatpA-atpH\u003c/em\u003e), support resilience under cold and acidic conditions [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Additional genes for alkaline shock proteins (\u003cem\u003easp2/23\u003c/em\u003e), acyltransferase (\u003cem\u003eplsC\u003c/em\u003e), ABC transporters, pyruvate kinase (\u003cem\u003epyk\u003c/em\u003e), sodium-proton antiporters (\u003cem\u003enhaC\u003c/em\u003e), and DNA repair (\u003cem\u003emutL/S2/R/T/Y\u003c/em\u003e) further enhance stress tolerance [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTo withstand bile stress in the small intestine, \u003cem\u003eLP-ARP2\u003c/em\u003e harbours genes for sodium\u0026ndash;bile acid symporters, ABC transporters (\u003cem\u003eglnH\u0026ndash;glnPH2\u003c/em\u003e), oligopeptide transporters (\u003cem\u003eoppA\u0026ndash;F\u003c/em\u003e), glutamine synthetase (\u003cem\u003eglnA\u003c/em\u003e), as well as pyrophosphatase (\u003cem\u003eppaC\u003c/em\u003e, preserves membrane integrity) and cyclopropane-fatty-acyl-phospholipid synthase (\u003cem\u003ecfa\u003c/em\u003e, upregulates lipid biosynthesis) [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Further, osmoprotection is ensured by \u003cem\u003eopuCA\u0026ndash;D\u003c/em\u003e, \u003cem\u003eproV\u003c/em\u003e, and \u003cem\u003eproWX\u003c/em\u003e, facilitating uptake of glycine betaine, choline, and proline [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Collectively, these genomic features highlight robust adaptation of \u003cem\u003eLP-ARP2\u003c/em\u003e to acidic, alkaline, osmotic, oxidative, and bile stresses, emphasizing its strong probiotic potential [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec40\" class=\"Section3\"\u003e\u003ch2\u003e3.2.2. Genetic features for adhesion at \u003cem\u003eLP-ARP2\u003c/em\u003e genome indicated the gut- sustainability of the strain\u003c/h2\u003e\u003cp\u003eGenomic analysis of \u003cem\u003eLP-ARP2\u003c/em\u003e revealed several adhesion-related genes, including lipoprotein signal peptidase II (\u003cem\u003elspA\u003c/em\u003e), maltose phosphorylase (\u003cem\u003emapA\u003c/em\u003e), enolase (\u003cem\u003eeno\u003c/em\u003e), a sortase family protein (\u003cem\u003esrtA\u003c/em\u003e), and abundant glycosyltransferases, along with S-layer (SLPs) and sortase-dependent proteins (SDPs), crucial for colonization potential of probiotics [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e] \u003cb\u003e(Table S7)\u003c/b\u003e. Additionally, genes like \u003cem\u003ecelA-celE\u003c/em\u003e (glycosyl hydrolase family) could enhance gut persistence and antimicrobial activity of \u003cem\u003eLP-ARP2\u003c/em\u003e [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Teichoic acid biosynthetic genes like \u003cem\u003edltA-dltX\u003c/em\u003e may influence host immune responses and adhesion capacity of \u003cem\u003eLP-ARP2\u003c/em\u003e [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec41\" class=\"Section3\"\u003e\u003ch2\u003e3.2.3. Genetic markers for biofilm-formation at \u003cem\u003eLP-ARP2\u003c/em\u003e genome indicated potential of the strain for gut- colonization\u003c/h2\u003e\u003cp\u003eBiofilm formation ability of \u003cem\u003eLP-ARP2\u003c/em\u003e was investigated to assess its potential for enhancing probiotic persistence, gut colonization, and competitive exclusion of pathogens [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. WGS analysis identified \u003cem\u003eveg\u003c/em\u003e and \u003cem\u003eluxS\u003c/em\u003e genes in the \u003cem\u003eLP-ARP2\u003c/em\u003e genome, where \u003cem\u003eveg\u003c/em\u003e stimulates biofilm formation in Gram-positive bacteria, and \u003cem\u003eluxS\u003c/em\u003e-mediated quorum sensing via autoinducer-2 (AI-2) regulates attachment and biofilm development [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Therefore, the presence of biofilm-stimulating genes motivated the experimental exploration of the biofilm-forming ability of \u003cem\u003eLP-ARP2\u003c/em\u003e \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec42\" class=\"Section3\"\u003e\u003ch2\u003e3.2.4. Carbohydrate-active enzymes (CAZymes) at \u003cem\u003eLP-ARP2\u003c/em\u003e-genome indicated potential of the strain for gut-adaptation and modulation\u003c/h2\u003e\u003cp\u003eCAZymes are central to gut microbial adaptation by enabling the degradation of complex polysaccharides [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. KEGG and COG analyses of \u003cem\u003eLP-ARP2\u003c/em\u003e revealed a total of 85 CAZyme genes, categorized into glycoside hydrolases (GHs, n\u0026thinsp;=\u0026thinsp;37, 14 families), glycosyltransferases (GTs, n\u0026thinsp;=\u0026thinsp;45, 8 families), and carbohydrate-binding modules (CBMs, n\u0026thinsp;=\u0026thinsp;4, 2 families) \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. GHs (47%) predominantly included GH13, GH31, and GH65, which target mono-, oligo-, and polysaccharides such as glucose, fructose, galactose, α-/β-glucans, arabinoxylans, and cellulose (GH9, GH26) \u003cb\u003e(Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e)\u003c/b\u003e [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. GTs (48%) catalyze sugar transfer and influence host-microbe immune interactions [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. While CBMs (4%; CBM48, CBM50) mediate polysaccharide hydrolysis \u003cb\u003e(Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e)\u003c/b\u003e. This broad CAZyme repertoire indicated that \u003cem\u003eLP-ARP2\u003c/em\u003e could metabolize diverse types of carbohydrates, enabling the ecological adaptation of other gut-bacteria with beneficial functions like immune modulation and pathogenic defence. In addition, \u003cem\u003eLP-ARP2\u003c/em\u003e harbored an enriched set of sugar phosphotransferase (PTS) genes (e.g., \u003cem\u003epts4ABC, pts10A\u0026ndash;C, pts23A\u0026ndash;C, pts30ABC, pts36A\u0026ndash;C, ulaA\u0026ndash;B, celA\u0026ndash;D, chbA/C\u003c/em\u003e), emphasizing efficient carbon transport. Functional diversity was observed across mannose/sorbose/fructose (\u003cem\u003emanA/L/Y/N, dhaA\u0026ndash;T, agaB/C\u003c/em\u003e), sorbitol/glucitol (\u003cem\u003esrlA/B/E/M\u003c/em\u003e), and cellobiose/lactose (\u003cem\u003emtlA/D/F/R, mngA/B\u003c/em\u003e) PTS systems. Genes encoding alcohol dehydrogenase, L-lactate dehydrogenases, phosphate acetyltransferase, phosphoketolase, and pyruvate formate lyase suggested metabolic flexibility through both homo- and hetero-fermentative pathways. Together, these genetic-features (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e implied that \u003cem\u003eLP-ARP2\u003c/em\u003e could be a lucrative candidate for probiotic-formulations [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] (See discussion).\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\u003eCarbohydrate-active enzymes (CAZymes) detected in the \u003cem\u003eLP-ARP2\u003c/em\u003e genome\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\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\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePreferred name\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCazyme\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFunction\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eglgB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCBM48, GH13, GH31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eformation of alpha-1,6-glucosidic bond in glycogen\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eglgD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGT5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNucleotidyl transferase\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eglgA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGT5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSynthesizes alpha-1,4-glucan chains using ADP-glucose\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eglgP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGT35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eallosteric enzyme in carbohydrate metabolism\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003emalQ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCBM48, GH13, GH31, GH77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBelongs to the glycosyl hydrolase 13 family\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003epgmB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGH37, GH65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ebeta-phosphoglucomutase\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003etrePP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGH37, GH65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGlycosyl hydrolase family 65\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003epgmB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGH37, GH65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ebeta-phosphoglucomutase\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003emalL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGH13, GH31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAlpha amylase, catalytic domain\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003emalS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGH13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGlycogen debranching enzyme\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003emapA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGH65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ehydrolase, family 65, central catalytic\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003escrB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGH32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003einvertase\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003etreC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGH13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAlpha amylase, catalytic domain protein\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ecelA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGT1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBelongs to the glycosyl hydrolase 1 family\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ecsbB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGT2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGlycosyltransferase like family 2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ecps4F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGT4, GT5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGlycosyl transferases group 1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eica2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGT2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGlycosyl transferase family group 2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003emltD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCBM50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNlpC P60 family protein\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003emurG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGT28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCell wall formation\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003etagE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGT4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGlycosyl transferases group 1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003epgaC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGT2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGlycosyl transferase\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003enplT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGH13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBelongs to the glycosyl hydrolase 13 family\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ecelA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGT1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBelongs to the glycosyl hydrolase 1 family\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003etagE5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGT4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePoly (Glycerol-phosphate) alpha-glucosyltransferase\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003etagE6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGT4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGlycosyl transferases group 1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003epbg6, celA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGT1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBelongs to the glycosyl hydrolase 1 family\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003emalA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGH13, GH31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAlpha amylase, catalytic domain protein\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003emngB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGH38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGlycosyl hydrolases family 38 N-terminal domain\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003emltD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCBM50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePFAM NLP P60 protein\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ecelE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGH5, GH9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGDSL-like Lipase/Acylhydrolase family\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003elacM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGH101, GH29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ebeta-galactosidase\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ebglH, pbg10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGT1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBelongs to the glycosyl hydrolase 1 family\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003etreP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGH65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ehydrolase, family 65, central catalytic\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003emalZ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGH31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBelongs to the glycosyl hydrolase 31 family\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eaglB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGH4, GT4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFamily 4 glycosyl hydrolase C-terminal domain\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003emalL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGH13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAlpha amylase, catalytic domain protein\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ebgl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGT1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBelongs to the glycosyl hydrolase 1 family\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003emngB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGH38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGlycosyl hydrolases family 38 N-terminal domain\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003edexB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGH13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAlpha amylase, catalytic domain protein\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003etagA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGT26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTakes part in the de novo synthesis of teichoic acid\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003esidC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGT2, GT4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDNA recombination\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003earbB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGT1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBelongs to the glycosyl hydrolase 1 family\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003enagH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGH20, GH26, GH5, GH9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMucBP domain\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003esacB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGT2, GT4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eStealth protein CR2, conserved region 2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ecps1B\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGT2, GT4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGlycosyl transferases group 1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003erecX, mgs, cpoA, tagE1, tagE2, tagE3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGT4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRegulatory protein RecX\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003epbp2A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGT51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003epenicillin-binding protein\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eykoT, pgaC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGT2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGlycosyl transferase family 2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003emapA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGH65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ehydrolase, family 65, central catalytic\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eponA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGT51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003epenicillin-binding protein 1A\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec43\" class=\"Section3\"\u003e\u003ch2\u003e3.2.5. Genes for amino acid biosynthesis at \u003cem\u003eLP-ARP2\u003c/em\u003e genome indicated probiotic efficacy\u003c/h2\u003e\u003cp\u003eFunctional annotation revealed that the \u003cem\u003eLP-ARP2\u003c/em\u003e genome contained genes for the biosynthesis of essential amino acids. This includes a) aromatic amino acids (\u003cem\u003earoA/C/D/E/K\u003c/em\u003e) like tryptophan (\u003cem\u003etrpA-G\u003c/em\u003e), tyrosine (\u003cem\u003etyrA/S\u003c/em\u003e), phenylalanine (\u003cem\u003epheS/T\u003c/em\u003e); b) branched-chain amino acids (BCAAs) like isoleucine, leucine, valine (\u003cem\u003eilvE, leuS\u003c/em\u003e), as well as c) other essential amino acids like threonine \u003cem\u003e(hom, hom1, asd, thrB/C/S/E)\u003c/em\u003e, histidine \u003cem\u003e(hisA-I/K/Z/S)\u003c/em\u003e, and lysine \u003cem\u003e(lysC/A)\u003c/em\u003e [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec44\" class=\"Section3\"\u003e\u003ch2\u003e3.2.6. Gene clusters at the \u003cem\u003eLP-ARP2\u003c/em\u003e genome indicated the metabolic versatility of the strain\u003c/h2\u003e\u003cp\u003eFunctional annotation of the \u003cem\u003eLP-ARP2\u003c/em\u003e genome revealed the presence of genes associated with the short-chain fatty acids (SCFA) synthesis (\u003cem\u003eackA\u003c/em\u003e, \u003cem\u003enagA\u003c/em\u003e, \u003cem\u003epta\u003c/em\u003e, \u003cem\u003eacyP\u003c/em\u003e, \u003cem\u003eadhE\u003c/em\u003e, \u003cem\u003epdh\u003c/em\u003e, \u003cem\u003efabI-H\u003c/em\u003e, \u003cem\u003epatB\u003c/em\u003e, \u003cem\u003eldhA\u003c/em\u003e, \u003cem\u003eaccA-D\u003c/em\u003e, \u003cem\u003epoxB\u003c/em\u003e, \u003cem\u003egabD\u003c/em\u003e, \u003cem\u003edapA-E\u003c/em\u003e, \u003cem\u003emalY\u003c/em\u003e, \u003cem\u003eeda\u003c/em\u003e, and \u003cem\u003escfaA\u003c/em\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Genes related to vitamin biosynthesis pathways were also identified (\u003cem\u003eadk\u003c/em\u003e, \u003cem\u003ersgA\u003c/em\u003e, \u003cem\u003enudF\u003c/em\u003e, \u003cem\u003epdxK/B\u003c/em\u003e, \u003cem\u003ebirA\u003c/em\u003e, \u003cem\u003efabI/Z/H/F\u003c/em\u003e, \u003cem\u003enadD\u003c/em\u003e, \u003cem\u003eycsE\u003c/em\u003e, \u003cem\u003eyitU\u003c/em\u003e, and \u003cem\u003eiscS\u003c/em\u003e, riboflavin (\u003cem\u003eribBA/D/E/H/T/F\u003c/em\u003e), thiamine (\u003cem\u003eThiM/N/D/E/I\u003c/em\u003e), vitamine K2 (\u003cem\u003emenG/A\u003c/em\u003e), folate (\u003cem\u003efolA/P/E/B/C\u003c/em\u003e), coenzyme A (\u003cem\u003ecoaA-E))\u003c/em\u003e [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e] \u003cb\u003e(Table S7)\u003c/b\u003e. \u003cem\u003eLP-ARP2\u003c/em\u003e harbored gene signatures associated with exopolysaccharide (EPS) biosynthesis and regulation (\u003cem\u003ecpsY/4J/4I/4G/4F/4D/1B/2I, epsB-V\u003c/em\u003e). EPS facilitates gut-colonization, biofilm formation, and host interactions [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eNext, metabolic gene cluster (MGC) analysis using gutSMASH predicted gene clusters in \u003cem\u003eLP-ARP2-\u003c/em\u003egenome, responsible for synthesis of primary and secondary metabolites involved in host metabolism and immune responses [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Two MGC regions were detected: a) pyruvate to acetate-formate (region 10.1), involved in SCFA (acetate, butyrate, and propionate) production \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e, and b) nitrate reductase (region 23.1), involved in nitrate/nitrite reduction to ammonia and nitric oxide (NO). These are important for immune regulation and host defence \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFurther, KEGG and COG analyses revealed a diverse gene repertoire for secondary metabolite biosynthesis and transport, including terpenoids and polyketides. antiSMASH identified three major clusters: a) T3PKS cluster (region 2.1; Chal_sti_synt_N domain) for polyketide synthesis \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e, b) terpene cluster (region 8.1), and c) cyclic-lactone-autoinducer cluster (region 10.1) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea\u0026ndash;b\u003cb\u003e)\u003c/b\u003e. PKS and terpene clusters are implicated in producing bioactive metabolites with applications in the food industry [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. While the cyclic-lactone-autoinducer cluster suggests quorum-sensing functions in defence. COG analysis supported these findings, with ~\u0026thinsp;2.88% of genes linked to defence mechanisms \u003cb\u003e(Table S8)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec45\" class=\"Section3\"\u003e\u003ch2\u003e3.2.7. Prediction of bacteriocin in the \u003cem\u003eLP-ARP2\u003c/em\u003e genome: Antimicrobial efficacy\u003c/h2\u003e\u003cp\u003eBacteriocins are antimicrobial peptides secreted by many probiotic bacteria. They confer beneficial effects in food preservation and pathogen inhibition. Interestingly, BAGEL4 analysis detected potential bacteriocin-encoding gene-clusters in \u003cem\u003eLP-ARP2\u003c/em\u003e genome, including Plantaricin F (bit score\u0026thinsp;=\u0026thinsp;105.14) and Plantaricin E (bit score\u0026thinsp;=\u0026thinsp;112.46) as major hits. Besides, \u003cem\u003eLP-ARP2\u003c/em\u003e genome also contained ORFs encoding a) bacteriocin transport and sensor proteins: orf00020, b) potential bacteriocin immunity protein; orf00025, and c) others like histidine kinase related to bacteriocin production; LanT, bacteriocin ABC-transporter, ATP-binding, and permease protein PlnG; \u003cem\u003ehlyD\u003c/em\u003e gene, encoding an accessory factor for the ABC transporter PlnH \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec46\" class=\"Section3\"\u003e\u003ch2\u003e3.2.8. Genetic features at \u003cem\u003eLP-ARP2\u003c/em\u003e genome showed antioxidant activities\u003c/h2\u003e\u003cp\u003eProbiotics harboring oxidative stress tolerance genes could function as natural antioxidants to alleviate gut inflammation. Genome annotation revealed that \u003cem\u003eLP-ARP2\u003c/em\u003e harboured three major antioxidant mechanisms: glutathione biosynthesis, glutathione redox cycle, glutaredoxin systems, and gamma-glutamyl cycle. Genome mining identified entire thioredoxin (\u003cem\u003etpx, trxb/a\u003c/em\u003e) and NADH-dependent (\u003cem\u003enox, npr\u003c/em\u003e, \u003cem\u003endh\u003c/em\u003e) antioxidant gene signatures, along with key redox-regulators such as \u003cem\u003egshR1\u003c/em\u003e (glutathione reductase), \u003cem\u003enrdH\u003c/em\u003e (glutaredoxin), \u003cem\u003ekatA\u003c/em\u003e (catalase), and peroxidases \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e. Additionally, genes encoding \u003cem\u003emsrA\u003c/em\u003e and \u003cem\u003emsrB\u003c/em\u003e, components of the methionine sulfoxide reductase system, indicated protection against ROS-mediated protein oxidation \u003cb\u003e(Table S7)\u003c/b\u003e. Therefore, these genetic features indicated that \u003cem\u003eLP-ARP2\u003c/em\u003e could possess strong antioxidant potential.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec47\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Probiotic Attributes of \u003cem\u003eLP-ARP2\u003c/em\u003e: Experimental Validation of WGS Analysis\u003c/h2\u003e\u003cdiv id=\"Sec48\" class=\"Section3\"\u003e\u003ch2\u003e3.3.1. Stress-adaptation potential of \u003cem\u003eLP-ARP2\u003c/em\u003e: tolerance to acid and bile\u003c/h2\u003e\u003cp\u003eWGS analysis revealed stress-response genes, indicating the adaptation potential of \u003cem\u003eLP-ARP2\u003c/em\u003e to a stressful environment. Acid tolerance assay showed 80% and 60% survival of \u003cem\u003eLP-ARP2\u003c/em\u003e at pH 3 after 3 and 5 h, respectively, with stable biomass up to 1 h at both pH 3 and 4 (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05); viability declined significantly only after 5 h at pH 3 (****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), indicating strong acid stress resilience \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. Under bile stress, \u003cem\u003eLP-ARP2\u003c/em\u003e maintained high survival (97% at 0.3% and 96% at 1% bile) after 3\u0026ndash;5 h \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e, with biomass increasing significantly after 3 h (***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and 5 h (****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). These genomic and \u003cem\u003ein vitro\u003c/em\u003e results together confirm robust acid-bile tolerance, highlighting the adaptability of the strain to harsh gastrointestinal conditions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec49\" class=\"Section3\"\u003e\u003ch2\u003e3.3.2. Gut-sustainability of \u003cem\u003eLP-ARP2\u003c/em\u003e: Autoaggregation, cell surface hydrophobicity, adhesion to Caco-2 cells, and biofilm formation assays\u003c/h2\u003e\u003cp\u003eProbiotic surface proteins mediate host interaction and adhesion, and \u003cem\u003eLP-ARP2\u003c/em\u003e contained putative adhesion-related genes, indicating strong colonization potential. Autoaggregation assay of \u003cem\u003eLP-ARP2\u003c/em\u003e showed a significant increase (****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) in aggregation capacity from 5.9\u0026ndash;44.74% within 1\u0026ndash;12 h, exhibiting the highest aggregation (66.44%) at 24 h \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e. \u003cem\u003eLP-ARP2\u003c/em\u003e showed significant surface hydrophobicity with n-hexadecane (58.93%) and xylene (58.27%) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e. This finding implied the promising adhesion ability of \u003cem\u003eLP-ARP2\u003c/em\u003e with the complex hydrophobic surface of gut epithelial cells. The adhesion efficacy of \u003cem\u003eLP-ARP2\u003c/em\u003e to Caco-2 was significant, with an adhesion rate of 7.1% after 2 h, corroborating genomic predictions \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee\u003cb\u003e)\u003c/b\u003e. Notably, \u003cem\u003eLP-ARP2\u003c/em\u003e formed robust biofilms within 24 h without additional supplements, surpassing \u003cem\u003eL. acidophilus\u003c/em\u003e DDS1 (**p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and comparable to \u003cem\u003eL. rhamnosus\u003c/em\u003e GG (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef\u003cb\u003e).\u003c/b\u003e Collectively, genomic and \u003cem\u003ein vitro\u003c/em\u003e evidence confirmed strong adhesion, biofilm formation, and persistence, strengthening the ability of \u003cem\u003eLP-ARP2\u003c/em\u003e to colonize the gut mucosa and enhance therapeutic efficacy.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec50\" class=\"Section3\"\u003e\u003ch2\u003e3.3.3. Gut-modulation and adaptability of \u003cem\u003eLP-ARP2\u003c/em\u003e: Prebiotic utilization assay\u003c/h2\u003e\u003cp\u003eGenomic analysis revealed a diverse array of CAZymes in \u003cem\u003eLP-ARP2\u003c/em\u003e, indicating metabolic flexibility in degrading complex carbohydrates. Prebiotic utilization assays with FOS, maltodextrin, and inulin (dextrose control) showed highest efficiency with maltodextrin (pH 5.13, after 12 h), followed by inulin (pH 5.81) and FOS (pH 6.28), with corresponding organic acid production \u003cb\u003e(Fig. S2)\u003c/b\u003e. Dose-dependent assays with maltodextrin (1%, 1.5%, and 2% w/v), revealed significant drop in pH (****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; 1%, pH 4.75; 1.5%, pH 4.46; 2%, pH 4.23) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg\u003cb\u003e)\u003c/b\u003e, increased growth (OD\u003csub\u003e600\u003c/sub\u003e), and higher PS (91.3% at 2%, 88.16% at 1.5%, 83.7% at 1%), comparable to dextrose (\u003csup\u003ens\u003c/sup\u003ep\u0026gt;0.05) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ei\u003cb\u003e)\u003c/b\u003e. Biomass (Log\u003csub\u003e10\u003c/sub\u003e CFU/mL) also increased with maltodextrin, consistent with PS, and remained comparable to the control (\u003csup\u003ens\u003c/sup\u003ep\u0026gt;0.05) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ej\u003cb\u003e)\u003c/b\u003e. A PI\u0026thinsp;\u0026gt;\u0026thinsp;1 signifies enhanced probiotic growth over the control [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Hence, PI confirmed enhanced growth with 1.5% FOS and 1% maltodextrin, showing PI\u0026thinsp;\u0026gt;\u0026thinsp;1 compared to dextrose \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ek\u003cb\u003e)\u003c/b\u003e. Thus, both genomic and experimental analyses established dose-dependent prebiotic utilization capacity of \u003cem\u003eLP-ARP2\u003c/em\u003e, particularly for maltodextrin, consistent with its CAZyme repertoire.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec51\" class=\"Section3\"\u003e\u003ch2\u003e3.3.4. Safety Attributes of \u003cem\u003eLP-ARP2\u003c/em\u003e: \u003cem\u003eIn vitro\u003c/em\u003e Validation of WGS Analysis\u003c/h2\u003e\u003cdiv id=\"Sec52\" class=\"Section4\"\u003e\u003ch2\u003e3.3.4.1. Antibiotic susceptibility of \u003cem\u003eLP-ARP2\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eBased on the clinical risk of the transfer of antibiotic-resistant genes, antibiotic susceptibility is essential for probiotic evaluation [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. \u003cem\u003eLP-ARP2\u003c/em\u003e was sensitive to most conventional antibiotics (ampicillin, amoxicillin/clavulanic acid, clindamycin, chloramphenicol, erythromycin, penicillin, and tetracycline) but resistant to vancomycin and gentamicin, consistent with profiles of other probiotics \u003cb\u003e(Table S9)\u003c/b\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec53\" class=\"Section4\"\u003e\u003ch2\u003e3.3.4.2. Non-haemolytic and DNase-negative attributes of \u003cem\u003eLP-ARP2\u003c/em\u003e\u003c/h2\u003e\u003cp\u003e\u003cem\u003eLP-ARP2\u003c/em\u003e showed no hemolysis on the blood agar plate (non-haemolytic) and no DNase activity on the DNase agar plate \u003cb\u003e(Table S10)\u003c/b\u003e. Therefore, \u003cem\u003ein silico\u003c/em\u003e studies and \u003cem\u003ein vitro\u003c/em\u003e assays assured the non-pathogenic nature of \u003cem\u003eLP-ARP2\u003c/em\u003e, reinforcing its suitability as a safe and functional probiotic candidate.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec54\" class=\"Section3\"\u003e\u003ch2\u003e3.3.5. Promising Antimicrobial Properties of \u003cem\u003eLP-ARP2\u003c/em\u003e\u003c/h2\u003e\u003cdiv id=\"Sec55\" class=\"Section4\"\u003e\u003ch2\u003e3.3.5.1. Robust antimicrobial potential of \u003cem\u003eLP-ARP2\u003c/em\u003e across the diverse pathogens\u003c/h2\u003e\u003cp\u003eEffective probiotics should exhibit broad-spectrum antimicrobial activity. Indeed, \u003cem\u003eLP-ARP2\u003c/em\u003e showed significant inhibition for Gram-positive and Gram-negative enteric bacteria, with the most pronounced effects observed for \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eV. cholerae\u003c/em\u003e, and \u003cem\u003eS. flexneri\u003c/em\u003e \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. Notably, \u003cem\u003eV. cholerae\u003c/em\u003e and \u003cem\u003eS. flexneri\u003c/em\u003e strains used in this study were multidrug-resistant clinical isolates.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec56\" class=\"Section4\"\u003e\u003ch2\u003e3.3.5.2. Anti-\u003cem\u003eSalmonella\u003c/em\u003e effect of \u003cem\u003eLP-ARP2\u003c/em\u003e: Co‑culture assay\u003c/h2\u003e\u003cp\u003e\u003cem\u003eS. Typhimurium\u003c/em\u003e (ST), a major foodborne pathogen, was employed to strengthen the antimicrobial effects of \u003cem\u003eLP-ARP2\u003c/em\u003e. In the agar well diffusion assay, the heat-treated CFS of \u003cem\u003eLP-ARP2\u003c/em\u003e retained inhibition (12.66 mm\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5) as the normal CFS (12.66 mm\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5), however the activity was abolished (0 mm) upon pH-neutralization, confirming acid-driven antibacterial effects \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. Broth microdilution showed significant reduction of ST growth at 1% (*p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and 5% v/v (****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) \u003cem\u003eLP-ARP2\u003c/em\u003e CFS, with complete inhibition (MIP) of ST at 10% (v/v) CFS \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec-e\u003cb\u003e)\u003c/b\u003e. The agar spot assay revealed bacteriostatic activity at 10% and bactericidal activity at 20% CFS \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec57\" class=\"Section4\"\u003e\u003ch2\u003e3.3.5.3. Anti-biofilm activity of \u003cem\u003eLP-ARP2\u003c/em\u003e against MRSA\u003c/h2\u003e\u003cp\u003ePathogenic biofilms cause major risks to food safety and clinical industries, with MRSA being a strong biofilm-former, contributing to its virulence [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. \u003cem\u003eLP-ARP2\u003c/em\u003e CFS showed potent anti-biofilm activity, with 10% (v/v) CFS identified as the MIP for MRSA \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg; \u003cb\u003eFig. S3)\u003c/b\u003e, significantly reducing biofilm formation at 10% (**p\u0026thinsp;\u0026lt;\u0026thinsp;0.005) and more strongly inhibiting biofilm formation at 20\u0026ndash;40% (****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) of \u003cem\u003eLP-ARP2\u003c/em\u003e CFS \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eh\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec58\" class=\"Section3\"\u003e\u003ch2\u003e3.3.6. Antioxidant potential of \u003cem\u003eLP-ARP2\u003c/em\u003e\u003c/h2\u003e\u003cdiv id=\"Sec59\" class=\"Section4\"\u003e\u003ch2\u003e3.3.6.1. Ability of \u003cem\u003eLP-ARP2\u003c/em\u003e to scavenge cations by ABTS assay\u003c/h2\u003e\u003cp\u003eIn the ABTS assay, \u003cem\u003eLP-ARP2\u003c/em\u003e heat-lysed cells showed significantly higher (**p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) scavenging rate (43.17%) than the intact cells (36.43%) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. This result suggested that the intracellular components of \u003cem\u003eLP-ARP2\u003c/em\u003e contributed more to its strain-specific antioxidant potential [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec60\" class=\"Section4\"\u003e\u003ch2\u003e3.3.6.2. Free radical scavenging capacity of \u003cem\u003eLP-ARP2\u003c/em\u003e by DPPH assay\u003c/h2\u003e\u003cp\u003eDPPH assay measures antioxidant efficacy by the reduction in absorbance at 517 nm as radicals convert to non-radical forms [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The DPPH assay showed significantly higher (***p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) free radical scavenging activity (25 U/mL) of \u003cem\u003eLP-ARP2\u003c/em\u003e heat-lysed cells than whole cells (12.7 U/mL) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e, indicating stronger antioxidant potential in the intracellular components of the strain.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec61\" class=\"Section4\"\u003e\u003ch2\u003e3.3.6.3. Ability of \u003cem\u003eLP-ARP2\u003c/em\u003e to scavenge superoxide radical\u003c/h2\u003e\u003cp\u003eSuperoxide anions trigger lipid oxidation by generating singlet oxygen. \u003cem\u003eLP-ARP2\u003c/em\u003e heat-lysed cells showed significantly higher (***p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) superoxide anion scavenging activity (83.95%) than intact cells (67.21%), indicating strong antioxidant potential of the intracellular components of the strain \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec62\" class=\"Section4\"\u003e\u003ch2\u003e3.3.6.4. Capacity of \u003cem\u003eLP-ARP2\u003c/em\u003e to scavenge hydroxyl radical\u003c/h2\u003e\u003cp\u003eHydrogen peroxide generates harmful hydroxyl radicals that damage lipids, proteins, and tissues [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. \u003cem\u003eLP-ARP2\u003c/em\u003e intact cells showed significantly higher (**p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) scavenging activity (72.82%) than heat-lysed cells (60.62%) (**p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e, suggesting greater antioxidant potential in the whole cells than in intracellular components, highlighting a distinct, strain-specific probiotic trait.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec63\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Metabolomics of \u003cem\u003eLP-ARP2\u003c/em\u003e: Identification of Bioactive Metabolites Using WGS and HRMS Analyses\u003c/h2\u003e\u003cdiv id=\"Sec64\" class=\"Section3\"\u003e\u003ch2\u003e3.4.1. Detection of antimicrobial metabolites in \u003cem\u003eLP-ARP2\u003c/em\u003e CFS\u003c/h2\u003e\u003cp\u003eWGS analysis of \u003cem\u003eLP-ARP2\u003c/em\u003e revealed multiple genes associated with antimicrobial metabolite biosynthetic pathways, including SCFA and EPS biosynthesis and regulation. These signatures suggested enhanced potential for production of antimicrobial compounds, crucial for defence and pathogen exclusion.\u003c/p\u003e\u003cp\u003eConsistent with genomic predictions, HRMS profiling of \u003cem\u003eLP-ARP2\u003c/em\u003e-derived CFS detected a wide array of antimicrobial metabolites \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. SCFAs, including acetic, isobutyric, butyric, lactic, and hydroxypropionic acids, were abundant, corroborating gutSMASH results \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. These bioactive compounds are recognized as primary agents in antimicrobial activity and modulation of the immune system [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Butyrate, a major colonocyte energy source and immunomodulator, was notably present [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Organic acids (valeric, oxaloacetate, oxoglutarate) and antimicrobial compounds such as homoserine and homoserine lactone were also identified. Thus, WGS and HRMS validated the potential of \u003cem\u003eLP-ARP2\u003c/em\u003e to produce antimicrobial metabolites, reinforcing its functional and clinical relevance.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMetabolite Profiling of the Cell-Free Supernatant (CFS) of \u003cem\u003eLP-ARP2\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\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\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eName\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eInput Mass\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMatched Mass\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSignificance\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eButyric acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e89.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e89.0597\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eEnergy source for colon cells, important for gut-health, reduction of inflammation\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAlanine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e90.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e90.0549\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAmino acid\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlycine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e74.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e74.0248\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAmino acid\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePhenylalanine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e166.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e166.0862\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eEssential amino acid\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLactamide\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e90.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e90.0553\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHas applications in pharmaceutical industries\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDimethylethanolamine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e90.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e90.0913\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHas anti-inflammatory, antioxidant activities, used as a firming and anti-aging product\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOxalic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e91.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e91.0026\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUsed in cleaning or bleaching\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eD-glyceraldehyde\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e89.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e89.0244\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCellular metabolite\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHydroxypropionic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e89.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e89.0244\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCellular metabolite\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAcetoacetic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e101\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e101.0244\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCellular metabolite\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eValeric acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e101.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e101.0608\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCellular metabolite\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2.3-butanediol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e89.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e89.0608\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHas applications in chemical, cosmetics, agriculture, and pharmaceutical industries\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAcetate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e60.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e60.0206\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFermentation and acidification, cholesterol synthesis\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBetaine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e118.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e118.0862\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLactic acid fermentation, prevention of liver injury\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eProline\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e116.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e116.0706\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAmino acid\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eThreonine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e118.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e118.0510\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eProtein synthesis\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGamma-Aminobutyric acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e102\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e102.0561\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNeurotransmitter\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFructose\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e179.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e179.0561\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCellular metabolite\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlucose\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e179.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e179.0561\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCellular metabolite\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGalactose\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e179.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e179.0561\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCellular metabolite\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCitric acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e191\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e191.0197\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMineral absorption, source of flavonoids, antioxidants, and vitamin C\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDihydroxyacetone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e89.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e89.0244\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUsed in the cosmetic industry, intermediate in lipid biosynthesis, and glycolysis\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLactic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e89.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e89.0244\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAntimicrobial, antiviral and immunomodulatory properties\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDimethylglycine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e102\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e102.0561\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eImportant for boosting energy and immunity\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAcetoacetamide\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e102\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e102.0549\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHas applications in textile industry\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHydroxypyruvic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e105\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e105.0182\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCellular metabolite\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMalonic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e105\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e105.0182\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHas application as flavoring agents, fragrances, and pharmaceuticals\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDioxosuccinic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e147.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e146.9924\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUsed in different chemical reactions\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eArabinonic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e167\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e167.0550\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTakes part in an organism's growth, development, or reproduction\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLeucine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e132.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e132.1019\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eEssential amino acid\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIsoleucine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e132.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e132.1019\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eEssential amino acid\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePyruvic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e89.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e89.0233\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePotential anti-inflammatory and antioxidant agent\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAcetoin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e89.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e89.0597\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFlavour additive in food, cosmetics, synthesis of optically active pharmaceuticals\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eValine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e118.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e118.0862\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eEssential amino acid\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSarcosine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e90.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e90.0549\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHas application in schizophrenia treatment, cosmetic and pharmaceutical industries\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOxaloacetate ion\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e132.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e132.0053\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCellular metabolite. Can alleviate liver injury\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAminocaproic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e132.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e132.1019\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eIt is used as an antifibrinolytic agent to treat bleeding\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIsobutyric acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e89.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e89.0597\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eEnergy source for colonocytes and has antimutagenic activity\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMethyl propionate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e89.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e89.0597\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAntibacterial and antifungal activity\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3-Hydroxypropionate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e89.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e89.0233\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUseful as a platform for synthesis of biodegradable plastic\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAcetylglycine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e118.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e118.0499\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHas a role as a metabolite in peptide and amino acid modification\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAminocaproic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e132.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e132.1019\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUseful in the treatment of reducing bleeding and cardiac surgery\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOxoglutaric acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e145\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e145.0143\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCellular metabolite\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMethylglutaric acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e145\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e145.0506\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eOrganic acid can reduce cholesterol synthesis\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlutamine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e145\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e145.0619\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eProtein synthesis\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLysine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e145\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e145.0983\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCan reduce anxiety, and cold sores, improve calcium absorption, protein synthesis\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eArginine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e175.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e175.1189\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHelps in protein synthesis\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAcetylcholine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e145\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e145.1108\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMajor neurotransmitter\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTartaric acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e149\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e149.0092\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHas applications in food industries\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eArabinose\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e149\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e149.0456\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSugar metabolite\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHomoserine Lactone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e102\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e102.0549\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eQuorum-sensing\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAscorbic acid (vitamin C)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e175.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e175.0248\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHelps in wound healing, mineral absorption, immune system\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSerotonin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e175.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e175.0877\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNeurotransmitter\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAspartic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e132.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e132.0302\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eProtein synthesis\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHomoserine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e118.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e118.0510\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAntibacterial and anticancer activity\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePyridoxal (vitamin B\u003csub\u003e6\u003c/sub\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e166.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e166.0510\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMetabolism of lipids, carbohydrates, coenzymes, and hormones. Brain function\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMalonic semialdehyde\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e89.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e89.0233\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eImportant for cosmetic and pharmaceutical industries\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAdipic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e145\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e145.0506\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHas applications in food and beverages, pharmaceuticals\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5-keto-D-fructose\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e179\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e179.0550\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNatural diketone, present in honey\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGamma-amino-beta-hydroxybutyric acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e118.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e118.0510\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eA derivative of the neurotransmitter gamma-aminobutyric acid\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEthanolamine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e60.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e60.0455\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eDiverse application in pharmaceutical and chemical industry\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3-Aminobutanoic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e102\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e102.0561\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSecondary metabolite, can act as a defense or signaling molecule\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec65\" class=\"Section3\"\u003e\u003ch2\u003e3.4.2. Detection of \u003cem\u003eLP-ARP2-\u003c/em\u003emetabolites for health-promoting functions and industrial significance\u003c/h2\u003e\u003cp\u003eGenome mining of \u003cem\u003eLP-ARP2\u003c/em\u003e revealed extensive genomic potential for biosynthesis of health-promoting metabolites like vitamins, essential amino acids, and secondary metabolites, highlighting its metabolic versatility.\u003c/p\u003e\u003cp\u003eHRMS profiling of \u003cem\u003eLP-ARP2\u003c/em\u003e-derived CFS validated these predictions by identifying essential amino acids (isoleucine, leucine, valine, threonine, phenylalanine, lysine) and vitamins, such as pyridoxal (B6) and ascorbic acid (C), aligning with genome annotations \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. In addition, HRMS detected metabolites of industrial importance, including lactamide, oxalic acid, dihydroxyacetone, malonic acid, ethanolamine, and adipic acid, with applications in pharmaceutical, food, and cosmetic sectors. Notably, the quorum-sensing molecule homoserine lactone was identified, confirming antiSMASH predictions \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. Metabolites with physiological significance, like glyceraldehyde, acetoacetic acid, hydroxypyruvic acid, oxaloacetate, valeric acid, and oxoglutaric acid, were also detected, nourishing host energy metabolism. Therefore, WGS and HRMS demonstrated \u003cem\u003eLP-ARP2\u003c/em\u003e as a potential source of health-promoting and industrially valuable metabolites.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec66\" class=\"Section3\"\u003e\u003ch2\u003e3.4.3. Detection of \u003cem\u003eLP-ARP2\u003c/em\u003e-derived metabolites with anti-oxidant and psychobiotic potential\u003c/h2\u003e\u003cp\u003eWGS analysis of \u003cem\u003eLP-ARP2\u003c/em\u003e revealed multiple antioxidant systems, including glutathione biosynthesis and redox cycling, glutaredoxin, γ-glutamyl pathways, thioredoxin, and NADH-dependent systems. HRMS of \u003cem\u003eLP-ARP2\u003c/em\u003e-derived CFS corroborated this by detecting redox-active metabolites, such as dimethylethanolamine, citric acid, lactic acid, and pyruvic acid, along with abundant SCFAs and organic acids (acetic, lactic, isobutyric, butyric, and hydroxypropionic acids), known for antioxidant and host-protective effects \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eAntioxidant attributes alleviate oxidative stress-driven neuroinflammation, reinforcing gut-brain axis balance and psychobiotic potential of probiotics. WGS analysis highlighted gene signatures for biosynthesis of neuromodulatory metabolites, including glutamate decarboxylase (\u003cem\u003egadB\u003c/em\u003e) and GABA transaminase (\u003cem\u003egabT\u003c/em\u003e), leading to GABA biosynthesis and metabolism. HRMS confirmed GABA in \u003cem\u003eLP-ARP2\u003c/em\u003e-CFS, along with acetylcholine and serotonin, indicating psychobiotic potential \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Branched-chain amino acids (BCAAs), predicted by genome mining and detected in CFS (isoleucine, leucine, valine), further support neurotransmitter balance, while osmolyte betaine provides neuroprotection. The presence of the complete \u003cem\u003etrp\u003c/em\u003e operon (\u003cem\u003etrpA\u0026ndash;G\u003c/em\u003e) in the genome with serotonin in CFS of \u003cem\u003eLP-ARP2\u003c/em\u003e demonstrated an active tryptophan\u0026ndash;serotonin pathway, which could contribute to restoring neurotransmission disrupted in neurodegenerative conditions.\u003c/p\u003e\u003cp\u003eTogether, WGS analysis and HRMS study illustrated strong concordance between genomic capacity and metabolite expression, establishing \u003cem\u003eLP-ARP2\u003c/em\u003e as a promising producer of antioxidant and psychobiotic compounds with potential roles in mitigation of oxidative stress and modulation of inflammation across gut-brain axis.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec67\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e3.5. Human-target based functional-network analysis with\u003c/b\u003e \u003cb\u003eLP-ARP2\u003c/b\u003e\u003cb\u003e-derived metabolites: Molecular basis of therapeutic potential\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eGenome annotation and HRMS analysis corroborated the presence of metabolites in \u003cem\u003eLP-ARP2-\u003c/em\u003eCFS with antibacterial and antioxidant properties. Our \u003cem\u003ein vitro\u003c/em\u003e experiments further validated robust antimicrobial and antioxidant activities of \u003cem\u003eLP-ARP2\u003c/em\u003e, strengthening the promise of the strain to reduce gut inflammation and related diseases. To elucidate the molecular basis underlying these effects, we further performed a human-target-based functional-network analysis with \u003cem\u003eLP-ARP2\u003c/em\u003e-derived metabolites, aiming to uncover the pathways and mechanistic insights into their roles in disease prevention and modulation.\u003c/p\u003e\u003cp\u003eNetwork analysis using Cytoscape identified 4442 nodes and 4957 edges, reflecting broad metabolite-host associations \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. The Cytoscape plug-in CytoHubba identified hub nodes based on degree, maximum clique centrality (MCC), and bottleneck algorithms. Interestingly, all neurotransmitters, serotonin (87), glycine (59), acetylcholine (54), and Gamma-Aminobutyric acid (36), were detected holding a high degree and MCC scores, suggesting a central role of \u003cem\u003eLP-ARP2\u003c/em\u003e-metabolites in modulating the gut-brain axis by influencing neurotransmitter-related pathways. Functional enrichment analysis of the metabolites-target network based on molecular function \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e, biological process, and KEGG pathways \u003cb\u003e(Fig. S4-S5)\u003c/b\u003e corroborated this, revealing significant enrichment of neurotransmitter receptor activity, serotonin and GABA signaling, and GABAergic synapse pathways. Disease-gene association analysis showed strong links with neurological and cognitive disorders \u003cb\u003e(Fig. S6)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFurther, as a case study, Alzheimer\u0026rsquo;s disease (AD) was examined using the DisGeNET plug-in. The analysis revealed overlaps between the targets for \u003cem\u003eLP-ARP2-\u003c/em\u003ederived metabolite and AD-associated genes \u003cb\u003e(Fig. S7)\u003c/b\u003e. The curated network demonstrated direct interactions of \u003cem\u003eLP-ARP2\u003c/em\u003e metabolites with critical AD targets \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. Enrichment of this AD-specific network highlighted modulation of pathways central to AD pathophysiology, including amyloid-beta metabolism, synaptic transmission, tau hyperphosphorylation, neuroinflammation, and oxidative stress \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eb, S8\u0026ndash;S10\u003cb\u003e)\u003c/b\u003e. Notably, \u003cem\u003eLP-ARP2\u003c/em\u003e-secreted metabolites such as pyruvic acid, glycine, α-ketoglutarate, citric acid, betaine, and serotonin were predicted to influence these processes \u003cb\u003e(Table S11)\u003c/b\u003e, suggesting that \u003cem\u003eLP-ARP2\u003c/em\u003e may exert neuroprotective effects by stabilizing neurotransmission and mitigating AD-related pathologies \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Together, these results highlight the potential of \u003cem\u003eLP-ARP2\u003c/em\u003e as a psychobiotic strain capable of reinforcing the gut-brain axis and providing therapeutic benefits in neurodegenerative and inflammation-associated disorders.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.6. Promising Gene-Signatures for probiotic\u003c/b\u003e \u003cb\u003eL. plantarum\u003c/b\u003e: \u003cb\u003eComparative Genomic Signatures between\u003c/b\u003e \u003cb\u003eLP-ARP2\u003c/b\u003e \u003cb\u003ewith clinically relevant strains LP 299v, and Lp01\u003c/b\u003e\u003c/p\u003e\u003cp\u003eComparative genomic analysis of \u003cem\u003eLP-ARP2\u003c/em\u003e with clinically studied strains LP 299v and Lp01 revealed strong conservation of core probiotic attributes. Stress tolerance determinants, including ABC transporters and acid- and bile-responsive genes (\u003cem\u003eatp, nha\u003c/em\u003e and \u003cem\u003easp, gln, opp, opu\u003c/em\u003e, details Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), indicated that \u003cem\u003eLP-ARP2\u003c/em\u003e has adaptation capacities similar to the clinically relevant strains. Conserved adhesion (\u003cem\u003elsp, map, srt\u003c/em\u003e, \u003cem\u003eeno, cel\u003c/em\u003e, details Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) and immunomodulation genes (\u003cem\u003edlt\u003c/em\u003e, details Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) across these strains further reinforced their gut-sustainability potentials. Indeed LP 299v and Lp01 are able to survive, persist and therefore show clinical benefits in human patients. Also, for \u003cem\u003eLP-ARP2\u003c/em\u003e, our \u003cem\u003ein vitro\u003c/em\u003e experimental validation (acid/bile/aggregation/adhesion/biofilm formation assays) highlighted the similar potential of \u003cem\u003eLP-ARP2\u003c/em\u003e for gastrointestinal survival and persistence. Notably, the CAZyme repertoire and diverse sugar transport systems of \u003cem\u003eLP-ARP2\u003c/em\u003e were significantly prominent and comparable to those of LP 299v and Lp01, reflecting enhanced ecological adaptability and versatile carbohydrate utilization \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eComparative genomic analysis of LP-ARP2 with L. plantarum (LP) 299v and L. plantarum (LP) Lp01 showing promising gene signatures for probiotic attributes, beneficial properties and therapeutic potential\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\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\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e\u003cp\u003ePromising gene signatures\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePotential probiotic/functional attribute\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eComment\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eLP-ARP2\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLP 299v\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLp01\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003euspA, usp6, yugI\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003euspA, usp6, yugI\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003euspA, usp6, yugI\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUniversal stress tolerance\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003ePrevent the aggregation of cellular proteins and maintain membrane stabilization under heat stress [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ectsR, hrcA\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003ectsR, hrcA\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003ectsR, hrcA\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHeat-shock tolerance\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ednaJ, dnaK, groS, groL, grpE, hslU, hslO\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003ednaJ, dnaK, groS, groL, grpE, hslU, hslO\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003ednaJ, dnaK, groS, groL, grpE, hslU, hslO\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMolecular chaperones\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ehslV, hslU, clpB, clpC, clpE, clpL, clpP, clpX\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003ehslV, hslU, clpB, clpC, clpE, clpL, clpP, clpX\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003ehslV, hslU, clpB, clpC, clpE, clpL, clpP, clpX\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eProteases\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ecspA, cspC\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003ecspA, cspC\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003ecspA, cspC\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCold-shock tolerance\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eHelp to sustain cold-shock related deleterious effects [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eatpA\u0026ndash;atpH\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eatpA\u0026ndash;atpH\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eatpA\u0026ndash;atpH\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAcid stress tolerance\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eHelp in proton translocation across membranes to maintain cytoplasmic pH under acidic stress [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eplsC, asp2, asp23, nhaC, pyk\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eplsC, asp, asp2, nhaC, pyk\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eplsC, asp, asp2, aspB, aspA, aspS, nhaC, pyk\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAcid and alkaline stress tolerance\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eppaC, cfa, oppA-F, glnA, glnH, glnP, glnR, glnQ, glnPH2, opuCA, opuCB, opuCC, opuCD, proV, proWX\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eppaC, cfa, oppA-F, glnA, glnH, glnP, glnR, glnQ, glnPH2, opuCA, opuCB, opuCC, opuCD, proV, proWX\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eppaC, cfa, oppA-F, glnA, glnH, glnP, glnR, glnQ, glnPH2, opuCA, opuCB, opuCC, opuCD, proV, proWX\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eBile stress tolerance\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAssist to preserve membrane integrity under bile stress by upregulation of lipid biosynthesis, uptake of osmoprotectants [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003emutL, mutS2, mutR, mutT, mutY\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003emutL, mutS2, mutT, mutY\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003emutL, mutS2, mutT, mutY\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eDNA repair\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAid in mismatch repair [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003elspA, mapA, eno, srtA, celA-celE\u003c/em\u003e,\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003elspA, mapA, eno, srtA, celA-celE\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003elspA, mapA, eno, srtA, celA, celB, celE\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAdhesion to gut epithelial cells\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eEncode cell surface proteins of bacteria for colonization to host cells [96,66\u0026ndash;68]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003edltA-D, dltX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003edltA-D, dltX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003edltA-D, dltX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAdhesion and immunomodulation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eInfluence interaction with host epithelial cells and immune components [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eveg, luxS\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eveg, luxS\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eveg, luxS\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eBiofilm formation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eEncode autoinducer-2 (AI-2) which influences both the attachment and biofilm development of \u003cem\u003eLactobacillus\u003c/em\u003e sp. [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003etpx, trxb\u003c/em\u003e, \u003cem\u003etrxa, nox, npr\u003c/em\u003e, \u003cem\u003endh, gshR1, nrdH, katA, msrA, msrB\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003etpx, trxb\u003c/em\u003e, \u003cem\u003etrxa, nox, npr\u003c/em\u003e, \u003cem\u003endh, gshF, gshR, gshR1, nrdH, katA, msrA, msrB\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003etpx, trxb\u003c/em\u003e, \u003cem\u003etrxa, nox, npr\u003c/em\u003e, \u003cem\u003endh, gshF, gshR, gshR1, nrdH, katA, msrA, msrB\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAntioxidant activity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAssist in scavenging of ROS, peroxidase detoxification, redox-regulation in the host protecting ROS-mediated protein oxidation [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eackA\u003c/em\u003e, \u003cem\u003enagA\u003c/em\u003e, \u003cem\u003epta\u003c/em\u003e, \u003cem\u003eacyP\u003c/em\u003e, \u003cem\u003eadhE\u003c/em\u003e, \u003cem\u003epdh\u003c/em\u003e, \u003cem\u003efabI-H\u003c/em\u003e, \u003cem\u003epatB\u003c/em\u003e, \u003cem\u003eldhA\u003c/em\u003e, \u003cem\u003eaccA-D\u003c/em\u003e, \u003cem\u003epoxB\u003c/em\u003e, \u003cem\u003egabD\u003c/em\u003e, \u003cem\u003edapA-E\u003c/em\u003e, \u003cem\u003emalY\u003c/em\u003e, \u003cem\u003eeda\u003c/em\u003e, \u003cem\u003escfaA\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eackA\u003c/em\u003e, \u003cem\u003enagA\u003c/em\u003e, \u003cem\u003epta\u003c/em\u003e, \u003cem\u003eacyP\u003c/em\u003e, \u003cem\u003eadhE\u003c/em\u003e, \u003cem\u003epdh\u003c/em\u003e, \u003cem\u003efabI-H\u003c/em\u003e, \u003cem\u003epatB\u003c/em\u003e, \u003cem\u003eldhA\u003c/em\u003e, \u003cem\u003eaccA-D\u003c/em\u003e, \u003cem\u003epoxB\u003c/em\u003e, \u003cem\u003egabD\u003c/em\u003e, \u003cem\u003edapA-F\u003c/em\u003e, \u003cem\u003emalY\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eackA\u003c/em\u003e, \u003cem\u003enagA\u003c/em\u003e, \u003cem\u003epta\u003c/em\u003e, \u003cem\u003eacyP\u003c/em\u003e, \u003cem\u003eadhE\u003c/em\u003e, \u003cem\u003epdh\u003c/em\u003e, \u003cem\u003efabI-H\u003c/em\u003e, \u003cem\u003epatB\u003c/em\u003e, \u003cem\u003eldhA\u003c/em\u003e, \u003cem\u003eaccA-D\u003c/em\u003e, \u003cem\u003epoxB\u003c/em\u003e, \u003cem\u003egabD\u003c/em\u003e, \u003cem\u003edapA-F\u003c/em\u003e, \u003cem\u003emalY\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSCFA biosynthesis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eEncode different enzymes involved in fermentation pathways [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eadk\u003c/em\u003e, \u003cem\u003eribD/E/BA/H/T/F\u003c/em\u003e, \u003cem\u003eThiM/N/D/E/I\u003c/em\u003e, \u003cem\u003ersgA\u003c/em\u003e, \u003cem\u003enudF\u003c/em\u003e, \u003cem\u003epdxK/B\u003c/em\u003e, \u003cem\u003efolA-C/E/P\u003c/em\u003e, \u003cem\u003ebirA\u003c/em\u003e, \u003cem\u003efabI/Z/H/F\u003c/em\u003e, \u003cem\u003enadD\u003c/em\u003e, \u003cem\u003eycsE\u003c/em\u003e, \u003cem\u003eyitU\u003c/em\u003e, \u003cem\u003eiscS, coaA-D/E/G, menA/G\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eadk\u003c/em\u003e, \u003cem\u003eribD/E/BA/H/T/F\u003c/em\u003e, \u003cem\u003eThiM/N/D/E/I\u003c/em\u003e, \u003cem\u003ersgA\u003c/em\u003e, \u003cem\u003enudF\u003c/em\u003e, \u003cem\u003epdxK/B\u003c/em\u003e, \u003cem\u003efolA-E/K/P/T\u003c/em\u003e, \u003cem\u003ebirA\u003c/em\u003e, \u003cem\u003efabI/Z/H/F\u003c/em\u003e, \u003cem\u003enadD\u003c/em\u003e, \u003cem\u003eycsE\u003c/em\u003e, \u003cem\u003eyitU\u003c/em\u003e, \u003cem\u003eiscS, coaA-D/E, menA/G\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eadk\u003c/em\u003e, \u003cem\u003eribD/E/BA/H/T/F\u003c/em\u003e, \u003cem\u003eThiM/N/D/E/I\u003c/em\u003e, \u003cem\u003ersgA\u003c/em\u003e, \u003cem\u003enudF\u003c/em\u003e, \u003cem\u003epdxK/B\u003c/em\u003e, \u003cem\u003efolA/B/C/E/K\u003c/em\u003e, \u003cem\u003ebirA\u003c/em\u003e, \u003cem\u003efabI/Z/H/F\u003c/em\u003e, \u003cem\u003enadD\u003c/em\u003e, \u003cem\u003eycsE\u003c/em\u003e, \u003cem\u003eyitU\u003c/em\u003e, \u003cem\u003eiscS, coaA-D/E, menA/G\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eVitamin (riboflavin, folate, coenzyme A, thiamine, vitamin K2) biosynthesis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eEncode different proteins involved in vitamin biosynthesis pathways [\u003cspan additionalcitationids=\"CR74\" citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCBM, GHs, GTs, \u003cem\u003epts, man, mtl, dha, srl\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCBM, GHs, GTs, AAs, \u003cem\u003epts, man, mtl, dha, srl\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCBM, GHs, GTs, AAs, \u003cem\u003epts, man, mtl, dha, srl\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eComplex carbohydrate hydrolysis and sugar transport\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAssist in hydrolysis of glycosidic bonds in complex carbohydrates and transfer of sugar molecules [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003egadB, gabT\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003egabT, gabR, gabD, gadB\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003egabT, gabR, gabD, gadB\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eGABA biosynthesis and metabolism\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eParticipate in the synthesis of GABA [97]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003etrpA/B/C/D/E/F/G\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003etrpA/B/D/E/F/G/S\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003etrpA/B/D/E/F/G/S\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003etryptophan-serotonin metabolism\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eInvolved in the synthesis of tryptophan [98]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003earoA/C/D/E/K, hisA-I/K/S/Z, tyrA/S, pheS/T, ilvE, leuS, hom, hom1, asd, thrB/C/E/S, lysA/C, proA-C/S/V\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003earoA-F/K, hisA-I/K/S/Z, tyrA/S, pheS/T, ilvE, leuS, hom, hom1, asd, thrB/C/E/S, lysA/C/P/M/S, proA-C/S/V\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003earoA-F/K, hisA-I/K/S/Z, tyrA/S, pheS/T, ilvE, leuS, hom, hom1, asd, thrB/C/E/S, lysA/C/P/M/S, proA-C/S/V/WX\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eEssential amino acids (phenylalanine, tyrosine, histidine, isoleucine, valine, leucine, threonine, proline) biosynthesis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAssociated with the biosynthesis of essential amino acids [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ecpsY/4D/4J/4I/4G/4F, cps1B/2I, epsB/V\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003ecpsY/4D/4J/4I/4G/4F, cps1B/2I, epsB/V\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003ecpsY/4D/4J/4I/4G/4F, cps2I, epsB/V\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eExopolysaccharide biosynthesis and secretion\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eInvolved in the synthesis and secretion of exopolysaccharides [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eConserved pathways for the biosynthesis of essential amino acids (\u003cem\u003earo, trp, his, tyr, phe, tyr, lys\u003c/em\u003e, etc.), vitamins (\u003cem\u003erib, thi, fol, coa etc.\u003c/em\u003e), and SCFAs (\u003cem\u003eack, adh, pdh, scfa etc.\u003c/em\u003e) were consistently observed across \u003cem\u003eLP-ARP2\u003c/em\u003e, LP 299v, and Lp01 strains \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Indeed, several essential amino acids, vitamins, and SCFAs were validated in \u003cem\u003eLP-ARP2\u003c/em\u003e CFS (HRMS study). Since Lp01 has been shown to be effective in synbiotic formulations [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e], the presence of similar gene signatures for these beneficial metabolites in the \u003cem\u003eLP-ARP2\u003c/em\u003e genome, with HRMS-detection, enhanced its promise as a lucrative candidate for synbiotic applications as a functional food.\u003c/p\u003e\u003cp\u003eGenomic signatures of probiotics LP 299v and Lp01 also correlated with their reported functional and clinical outcomes. Antioxidant gene repertoires (\u003cem\u003etpx, nox, ndh, gsh, kat\u003c/em\u003e, details Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) were similar across all three strains. Recall, LP 299v and Lp01 were previously shown to mitigate gut inflammation in IBS and other gastrointestinal disorders \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. The presence of similar antioxidant-linked genes and robust free radical scavenging activity of \u003cem\u003eLP-ARP2\u003c/em\u003e strongly suggests a comparable potential to alleviate oxidative stress-driven gastrointestinal diseases. Concurrently, comparable pathways (\u003cem\u003etrp, gabT, gadB\u003c/em\u003e, details Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) for neuroactive metabolites, including GABA, SCFAs, branched-chain amino acids (BCAAs), and tryptophan, were identified in all three strains. LP 299v has been clinically shown to improve depressive symptoms, while Lp01 has shown immunomodulatory roles in allergic conditions. Consistently, our network-based analysis of \u003cem\u003eLP-ARP2\u003c/em\u003e-derived metabolites revealed neurotransmitter- and neuroinflammation-associated human targets (GABA receptor, amyloid-beta binding, serotonin receptor signaling pathways, etc), indicating comparable psychobiotic potential.\u003c/p\u003e\u003cp\u003eTherefore, above analysis highlights the commonality of validated genetic signatures among three strains of \u003cem\u003eL. plantarum\u003c/em\u003e for probiotic properties with potential clinical significance for mitigating gut inflammation\u0026ndash;related diseases, particularly neuroinflammation-associated disorders.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe present work emerges as a thorough investigation of WGS analysis of \u003cem\u003eLactiplantibacillus plantarum LP-ARP2\u003c/em\u003e with experimental validation for probiotic traits, HRMS-based metabolite profiling, and functional analysis to uncover the promising genomic marker for the health-promoting properties of the strain \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Strikingly, when we compared genomic features of \u003cem\u003eLP-ARP2\u003c/em\u003e with clinically relevant \u003cem\u003eL. plantarum\u003c/em\u003e strains, \u003cem\u003eL. plantarum\u003c/em\u003e 299v and \u003cem\u003eL. plantarum\u003c/em\u003e Lp01, the analyses confer comparable genetic signatures for therapeutic benefits \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Such strain-specific marker studies could further help to build up a database, which could speed up initial screening of novel probiotic strains using the genome sequence only. Next, these genetic markers also identify the molecular basis of probiotic benefits. To elucidate this further, we used \u003cem\u003eLP-ARP2-\u003c/em\u003ederived metabolites to generate a target-based functional network to find out the molecular logic of the \u003cem\u003eLP-ARP2-\u003c/em\u003edriven therapeutic benefits. To our knowledge, such a strategy has never been implemented in any probiotic system. The consequences are diverse since the strategy provides a novel road map for further mechanistic studies on any probiotic interventions, focusing on any particular disease model.\u003c/p\u003e\u003cp\u003eRobust antioxidant properties of \u003cem\u003eLP-ARP2\u003c/em\u003e suggest that it could reduce oxidative stress and restore redox homeostasis in the gut. The antioxidant gene-signatures of \u003cem\u003eLP-ARP2\u003c/em\u003e are similar to those of LP 299v and Lp01. Recall that LP 299v and Lp01 have been shown to be effective in clinical trials to benefit patients with intestinal disorders. Fascinatingly, the antioxidant activity of \u003cem\u003eLP-ARP2\u003c/em\u003e is also comparable to commercially available and clinically tested (mouse model) \u003cem\u003eL. plantarum\u003c/em\u003e strain (MWFLp-182). Together, these indicate the potential roles of the strain in antioxidant defence mechanisms and translational promise as functional foods and nutraceuticals targeting redox imbalance [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOxidative stress can also trigger neuroinflammation, which leads to neuronal loss and neurodegeneration. The remarkable antioxidative effects of \u003cem\u003eLP-ARP2\u003c/em\u003e could reduce oxidative stress and neuroinflammation, two major drivers for neurodegeneration \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. \u003cem\u003eLP-ARP2\u003c/em\u003e-CFS secretes key neurotransmitters such as acetylcholine, GABA, and serotonin, which are linked to appetite regulation, energy metabolism, and behavioral modulation, highlighting the promising role of the strain in gut-brain communication (see below). Genome annotation identified \u003cem\u003egadB\u003c/em\u003e and \u003cem\u003egabT\u003c/em\u003e genes, enabling GABA biosynthesis and metabolism, respectively, supporting HRMS findings of GABA in the \u003cem\u003eLP-ARP2\u003c/em\u003e-derived CFS [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. GABA plays a vital neuroprotective role by modulating neuronal excitability and reducing oxidative damage and inflammation. Therefore, GABA could control neurological disorders, including epilepsy, Parkinson\u0026rsquo;s, and Alzheimer\u0026rsquo;s disease [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Additionally, branched-chain amino acids (BCAAs), important for neurotransmitter balance, are detected in the CFS of the strain. Neuroprotective osmolyte betaine is also detected in \u003cem\u003eLP-ARP2\u003c/em\u003e-CFS. The presence of the comprehensive \u003cem\u003etrp\u003c/em\u003e operon (\u003cem\u003etrpA-G\u003c/em\u003e genes) in the genome and serotonin in the CFS supports the involvement in tryptophan-serotonin pathways, which could potentially restore dysregulated neurotransmission observed in AD [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. Interestingly, similar gene signatures for GABA, tryptophan, and branched-chain amino acids (BCAAs) were identified in \u003cem\u003eL. plantarum\u003c/em\u003e 299v, a strain reported to induce metabolic alterations in patients with major depression [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eNext, pathway and network analyses indicate that \u003cem\u003eLP-ARP2\u003c/em\u003e-derived metabolites could target key pathways involved in Alzheimer\u0026rsquo;s disease, suggesting the potential of the strain to mitigate AD through metabolite-driven mechanisms \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e; \u003cb\u003eTable S11)\u003c/b\u003e. \u003cem\u003eLP-ARP2\u003c/em\u003e-derived metabolites such as butyrate, acetate, citric acid, and dihydroxyacetone exhibit antioxidant properties and therefore could reduce oxidative stress and neuroinflammation. Glycine, alpha-ketoglutarate, betaine, acetate, pyruvate, and serotonin could help restore synaptic transmission and correct neurotransmitter imbalances. \u003cem\u003eLP-ARP2\u003c/em\u003e-secreted metabolites like dihydroxyacetone, glycine, alanine, alpha-ketoglutarate, pyruvate, citric acid, acetate, and aspartic acid are linked to neurogenesis and may counteract neurodegeneration. Alpha-ketoglutarate, alanine, oxalic acid, citric acid, and betaine are associated with regulating neuronal apoptosis, where alpha-ketoglutarate could increase brain-derived neurotrophic factor (BDNF) levels and cognitive function. Pyruvic acid is predicted to target amyloid-beta metabolism. Citric acid could reduce tau protein accumulation, a hallmark of AD, and SCFAs like butyrate and acetate could inhibit GSK3β, reducing tau hyperphosphorylation [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. Therefore, these findings suggest that \u003cem\u003eLP-ARP2\u003c/em\u003e-derived metabolites may exert neuroprotective effects by modulating multiple AD-related pathways and strengthening the integrity of gut-brain axis in the host.\u003c/p\u003e\u003cp\u003e\u003cem\u003eLP-ARP2\u003c/em\u003e-derived metabolites include essential amino acids, vitamins (C, B\u003csub\u003e3\u003c/sub\u003e), and energy metabolism-related compounds (e.g., pyruvic acid, oxaloacetate, acetoacetic acid), elucidating the significance of the strain as a nutritional supplement. The genome of \u003cem\u003eLP-ARP2\u003c/em\u003e also harbors numerous genes for essential amino acid biosynthesis, comparable to LP 299v and Lp01, with documented health benefits. This repertoire suggests a capacity to augment host amino acid pools, supporting gut barrier function, neurotransmitter synthesis, and metabolic health.\u003c/p\u003e\u003cp\u003e\u003cem\u003eLP-ARP2\u003c/em\u003e shows a broad spectrum of antimicrobial activity with comparable efficacy to commercial \u003cem\u003eL. plantarum\u003c/em\u003e strains. Rich repertoire of CAZymes (especially GHs) in \u003cem\u003eLP-ARP2\u003c/em\u003e genome, together with the prebiotic-utilizing ability of \u003cem\u003eLP-ARP2\u003c/em\u003e highlights the therapeutic promise of the strain for effective synbiotic formulations to improve gut health. Interestingly, the therapeutic potential of synbiotic formulations has been evaluated earlier for Lp01 in allergic conditions [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. These reinforce the potential application of the strain for microbial control and health-promoting formulations [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe genomic analysis predicts T3PKS and terpene regions in the \u003cem\u003eLP-ARP2\u003c/em\u003e genome \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e, which specifically encode for lipid components and bioactive secondary metabolites with lucrative industrial applications. Indeed, \u003cem\u003eLP-ARP2\u003c/em\u003e-derived metabolites (e.g., citric acid, tartaric acid, and acetoin) have diverse applications in food, cosmetic, and poultry industries [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. While adipic acid can be used across food processing sectors (food additive), other metabolites like lactamide, malonic semialdehyde, and 2,3-butanediol are widely used in pharmaceutical, cosmetic, and agricultural industries [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study presents an integrated genomic, metabolomic, and functional profiling of \u003cem\u003eLP-ARP2\u003c/em\u003e, unveiling genetic signatures of the strain for probiotic attributes and therapeutic potential with antimicrobial (anti-biofilm efficacy as well), anti-oxidant, and psychobiotic functions. The comparable genetic features of \u003cem\u003eLP-ARP2\u003c/em\u003e with clinically significant \u003cem\u003eL.\u003c/em\u003e plantarum strains for intestinal disorders and depression further indicate the potential application of \u003cem\u003eLP-ARP2\u003c/em\u003e for similar therapeutic benefits. Target-based \u003cem\u003ein silico\u003c/em\u003e investigation indeed indicates \u003cem\u003eLP-ARP2\u003c/em\u003e-derived metabolites could modulate multiple neurodegenerative disease-related pathways, suggesting their roles in neuroprotection through metabolite-driven mechanisms. Additionally, the detection of numerous industrially relevant secondary metabolites highlights the promising application of \u003cem\u003eLP-ARP2\u003c/em\u003e in the food, cosmetic, and poultry sectors.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eWHO, World Health Organization; WGS, Whole Genome Sequencing; ATCC, American Type Culture Collection; MRS, De Man Rogosa Sharpe agar; DMEM,\u0026nbsp;\u003cem\u003eDulbecco\u0026apos;s Modified Eagle Medium; FBS, Fetal Bovine Serum;\u0026nbsp;\u003c/em\u003eMTCC, Microbial Type Culture Collection; PBS, Phosphate Buffered Saline; DPPH, \u003cem\u003e1,1-diphenyl-2-picrylhydrazyl (DPPH)-2,2-diphenyl-1-picrylhydrazyl;\u0026nbsp;\u003c/em\u003eABTS, 2,2\u0026apos;-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid; PCR, polymerase chain reaction; CFU, colony forming unit; ANI, \u003cem\u003eAverage Nucleotide Identity,\u0026nbsp;\u003c/em\u003eCRISPR, Clustered Regularly Interspaced Short Palindromic Repeats; PKs, Polyketide Synthases\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSP performed all experiments, including analysis, and drafted the manuscript. SP, RP, and PKD performed the bioinformatics experiments and analysis of Figures 8 and 9. AD supervised and analysed Figures 8 and 9. ARC conceived the study, designed the approach, evaluated the data, and was involved in manuscript preparation. Finally, all authors reviewed and edited the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical statements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author(s) state that there is no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors report no declaration of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the intramural funding provided by IIT Bhubaneswar. SP receives an Institutional fellowship from IIT Bhubaneswar. RP and PKD acknowledge the MOES DOM-Project and CSIR-JRF for their fellowships, respectively. \u0026nbsp;We are thankful to Dr. Nrisingha Dey, ILS Bhubaneswar, for the Gram-positive pathogens [Methicillin-resistant \u003cem\u003eStaphylococcus aureus\u0026nbsp;\u003c/em\u003eATCC 700699 (MRSA) and\u003cem\u003e\u0026nbsp;Staphylococcus aureus\u0026nbsp;\u003c/em\u003eATCC 25923], Dr. Asish Kumar Mukhopadhyay, NICED Kolkata for Gram-negative clinical isolates [\u003cem\u003eEscherichia coli\u0026nbsp;\u003c/em\u003e(ETEC) BCH 04067\u003cem\u003e, Vibrio cholerae\u0026nbsp;\u003c/em\u003eBCH 09616\u003cem\u003e, Shigella Flexneri\u0026nbsp;\u003c/em\u003eBCH 06745]\u0026nbsp;and Dr. Biswaranjan Pradhan, SKBET, IIT Bhubaneswar, for \u003cem\u003eLactobacillus acidophilus\u003c/em\u003e DDS1. We thank the ILS-High Performance Computing Facility for the computational resources and support.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHou K, Wu ZX, Chen XY, Wang JQ, Zhang D, Xiao C, Zhu D, Koya JB, Wei L, Li J, Chen ZS (2022) Microbiota in health and diseases. Signal Transduct Target Ther 7:135. https://10.1038/s41392-022-00974-4\u003c/li\u003e\n\u003cli\u003eYeshi K, Ruscher R, Hunter L, Daly NL, Loukas A, Wangchuk P (2020) Revisiting Inflammatory Bowel Disease: Pathology, Treatments, Challenges and Emerging Therapeutics Including Drug Leads from Natural Products. J Clin Med 9. https://doi.org/10.3390/jcm9051273\u003c/li\u003e\n\u003cli\u003eZhang J, Zhang Y, Wang J, Xia Y, Zhang J, Chen L (2024) Recent advances in Alzheimer\u0026apos;s disease: Mechanisms, clinical trials and new drug development strategies. Signal Transduct Target Ther 9:211. https://doi.org/10.1038/s41392-024-01911-3\u003c/li\u003e\n\u003cli\u003ePatra S, Sahu N, Saxena S, Pradhan B, Nayak SK, Roychowdhury A (2022) Effects of Probiotics at the Interface of Metabolism and Immunity to Prevent Colorectal Cancer-Associated Gut Inflammation: A Systematic Network and Meta-Analysis With Molecular Docking Studies. Front Microbiol 13:878297. https://10.3389/fmicb.2022.878297\u003c/li\u003e\n\u003cli\u003ePatra S, Saxena S, Sahu N, Pradhan B, Roychowdhury A (2021) Systematic Network and Meta-analysis on the Antiviral Mechanisms of Probiotics: A Preventive and Treatment Strategy to Mitigate SARS-CoV-2 Infection. Probiotics Antimicrob Proteins 13:1138-1156. https://doi.org/10.1007/s12602-021-09748-w\u003c/li\u003e\n\u003cli\u003eJoint F (2002) WHO working group report on drafting guidelines for the evaluation of probiotics in food. London, Ontario, Canada 30:16-22\u003c/li\u003e\n\u003cli\u003eAyed L, M\u0026apos;Hir S, Nuzzolese D, Di Cagno R, Filannino P (2024) Harnessing the Health and Techno-Functional Potential of Lactic Acid Bacteria: A Comprehensive Review. Foods 13. https://doi.org/10.3390/foods13101538\u003c/li\u003e\n\u003cli\u003eBehera SS, Ray RC, Zdolec N (2018) Lactobacillus plantarum with Functional Properties: An Approach to Increase Safety and Shelf-Life of Fermented Foods. Biomed Res Int 2018:9361614. https://10.1155/2018/9361614\u003c/li\u003e\n\u003cli\u003eGizachew S, Engidawork E (2024) Genomic Characterization of Lactiplantibacillus plantarum Strains: Potential Probiotics from Ethiopian Traditional Fermented Cottage Cheese. Genes (Basel) 15. https://doi.org/10.3390/genes15111389\u003c/li\u003e\n\u003cli\u003eGarcia-Gonzalez N, Battista N, Prete R, Corsetti A (2021) Health-Promoting Role of Lactiplantibacillus plantarum Isolated from Fermented Foods. Microorganisms 9. https://10.3390/microorganisms9020349\u003c/li\u003e\n\u003cli\u003eDash J, Sethi M, Deb S, Parida D, Kar S, Mahapatra S, Minz AP, Pradhan B, Prasad P, Senapati S (2023) Biochemical, functional and genomic characterization of a new probiotic Ligilactobacillus salivarius F14 from the gut of tribes of Odisha. World J Microbiol Biotechnol 39:171. https://doi.org/10.1007/s11274-023-03626-z\u003c/li\u003e\n\u003cli\u003ePatra S, Pradhan B, Roychowdhury A (2025) Complete genome sequence, metabolic profiling and functional studies reveal Ligilactobacillus salivarius LS-ARS2 is a promising biofilm-forming probiotic with significant antioxidant, antibacterial, and antibiofilm potential. Front Microbiol 16:1535388. https://10.3389/fmicb.2025.1535388\u003c/li\u003e\n\u003cli\u003eSurve S, Shinde DB, Kulkarni R (2022) Isolation, characterization and comparative genomics of potentially probiotic Lactiplantibacillus plantarum strains from Indian foods. Sci Rep 12:1940. https://10.1038/s41598-022-05850-3\u003c/li\u003e\n\u003cli\u003eHu F, Gao Q, Zheng C, Zhang W, Yang Z, Wang S, Zhang Y, Lu T (2024) Encapsulated lactiplantibacillus plantarum improves Alzheimer\u0026apos;s symptoms in APP/PS1 mice. J Nanobiotechnology 22:582. https://doi.org/10.1186/s12951-024-02862-1\u003c/li\u003e\n\u003cli\u003eKumar S, Stecher G, Suleski M, Sanderford M, Sharma S, Tamura K (2024) MEGA12: Molecular Evolutionary Genetic Analysis Version 12 for Adaptive and Green Computing. Mol Biol Evol 41. https://doi.org/10.1093/molbev/msae263\u003c/li\u003e\n\u003cli\u003eSeemann T (2014) Prokka: rapid prokaryotic genome annotation. Bioinformatics 30:2068-2069. https://doi.org/10.1093/bioinformatics/btu153\u003c/li\u003e\n\u003cli\u003eLi H, Durbin R (2009) Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25:1754-1760. https://doi.org/10.1093/bioinformatics/btp324\u003c/li\u003e\n\u003cli\u003eGrissa I, Vergnaud G, Pourcel C (2007) CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats. Nucleic Acids Res 35:W52-57. https://doi.org/10.1093/nar/gkm360\u003c/li\u003e\n\u003cli\u003eJia B, Raphenya AR, Alcock B, Waglechner N, Guo P, Tsang KK, Lago BA, Dave BM, Pereira S, Sharma AN, Doshi S, Courtot M, Lo R, Williams LE, Frye JG, Elsayegh T, Sardar D, Westman EL, Pawlowski AC, Johnson TA, Brinkman FS, Wright GD, McArthur AG (2017) CARD 2017: expansion and model-centric curation of the comprehensive antibiotic resistance database. Nucleic Acids Res 45:D566-D573. https://doi.org/10.1093/nar/gkw1004\u003c/li\u003e\n\u003cli\u003eZankari E, Hasman H, Cosentino S, Vestergaard M, Rasmussen S, Lund O, Aarestrup FM, Larsen MV (2012) Identification of acquired antimicrobial resistance genes. J Antimicrob Chemother 67:2640-2644. https://doi.org/10.1093/jac/dks261\u003c/li\u003e\n\u003cli\u003eBortolaia V, Kaas RS, Ruppe E, Roberts MC, Schwarz S, Cattoir V, Philippon A, Allesoe RL, Rebelo AR, Florensa AF, Fagelhauer L, Chakraborty T, Neumann B, Werner G, Bender JK, Stingl K, Nguyen M, Coppens J, Xavier BB, Malhotra-Kumar S, Westh H, Pinholt M, Anjum MF, Duggett NA, Kempf I, Nykasenoja S, Olkkola S, Wieczorek K, Amaro A, Clemente L, Mossong J, Losch S, Ragimbeau C, Lund O, Aarestrup FM (2020) ResFinder 4.0 for predictions of phenotypes from genotypes. J Antimicrob Chemother 75:3491-3500. https://doi.org/10.1093/jac/dkaa345\u003c/li\u003e\n\u003cli\u003eLiu B, Zheng D, Jin Q, Chen L, Yang J (2019) VFDB 2019: a comparative pathogenomic platform with an interactive web interface. Nucleic Acids Res 47:D687-D692. https://10.1093/nar/gky1080\u003c/li\u003e\n\u003cli\u003eCosentino S, Voldby Larsen M, Moller Aarestrup F, Lund O (2013) PathogenFinder--distinguishing friend from foe using bacterial whole genome sequence data. PLoS One 8:e77302. https://10.1371/journal.pone.0077302\u003c/li\u003e\n\u003cli\u003eDietzsch S, Braesigk A, Seidel C, Remmele J, Kitzing R, Schlender T, Mynarek M, Geismar D, Jablonska K, Schwarz R, Pazos M, Weber DC, Frick S, Gurtner K, Matuschek C, Harrabi SB, Gluck A, Lewitzki V, Dieckmann K, Benesch M, Gerber NU, Obrecht D, Rutkowski S, Timmermann B, Kortmann RD (2022) Types of deviation and review criteria in pretreatment central quality control of tumor bed boost in medulloblastoma-an analysis of the German Radiotherapy Quality Control Panel in the SIOP PNET5 MB trial. Strahlenther Onkol 198:282-290. https://doi.org/10.1007/s00066-021-01822-0\u003c/li\u003e\n\u003cli\u003eZheng J, Ge Q, Yan Y, Zhang X, Huang L, Yin Y (2023) dbCAN3: automated carbohydrate-active enzyme and substrate annotation. Nucleic Acids Res 51:W115-W121. https://doi.org/10.1093/nar/gkad328\u003c/li\u003e\n\u003cli\u003eKanehisa M, Sato Y, Morishima K (2016) BlastKOALA and GhostKOALA: KEGG Tools for Functional Characterization of Genome and Metagenome Sequences. J Mol Biol 428:726-731. https://10.1016/j.jmb.2015.11.006\u003c/li\u003e\n\u003cli\u003ede Jong A, van Hijum SA, Bijlsma JJ, Kok J, Kuipers OP (2006) BAGEL: a web-based bacteriocin genome mining tool. Nucleic Acids Res 34:W273-279. https://doi.org/10.1093/nar/gkl237\u003c/li\u003e\n\u003cli\u003ePascal Andreu V, Roel-Touris J, Dodd D, Fischbach MA, Medema MH (2021) The gutSMASH web server: automated identification of primary metabolic gene clusters from the gut microbiota. Nucleic Acids Res 49:W263-W270. https://doi.org/10.1093/nar/gkab353\u003c/li\u003e\n\u003cli\u003eBlin K, Shaw S, Kloosterman AM, Charlop-Powers Z, van Wezel GP, Medema MH, Weber T (2021) antiSMASH 6.0: improving cluster detection and comparison capabilities. Nucleic Acids Res 49:W29-W35. https://doi.org/10.1093/nar/gkab335\u003c/li\u003e\n\u003cli\u003eRosenberg M, Gutnick D, Rosenberg E (1980) Adherence of bacteria to hydrocarbons: a simple method for measuring cell-surface hydrophobicity. FEMS Microbiol Lett 9:29-33\u003c/li\u003e\n\u003cli\u003eBreyer GM, Arechavaleta NN, Siqueira FM, de Souza da Motta A (2021) Characterization of Lactic Acid Bacteria in Raw Buffalo Milk: a Screening for Novel Probiotic Candidates and Their Transcriptional Response to Acid Stress. Probiotics Antimicrob Proteins 13:468-483. https://doi.org/10.1007/s12602-020-09700-4\u003c/li\u003e\n\u003cli\u003eRocchetti MT, Russo P, De Simone N, Capozzi V, Spano G, Fiocco D (2024) Immunomodulatory activity on human macrophages by cell-free supernatants to explore the probiotic and postbiotic potential of Lactiplantibacillus plantarum strains of plant origin. Probiotics Antimicrob Proteins 16:911-926. https://doi.org/10.1007/s12602-023-10084-4\u003c/li\u003e\n\u003cli\u003eAnand S, Mandal S, Tomar SK (2019) Effect of Lactobacillus rhamnosus NCDC 298 with FOS in Combination on Viability and Toxin Production of Enterotoxigenic Escherichia coli. Probiotics Antimicrob Proteins 11:23-29. https://10.1007/s12602-017-9327-1\u003c/li\u003e\n\u003cli\u003ePalframan R, Gibson GR, Rastall RA (2003) Development of a quantitative tool for the comparison of the prebiotic effect of dietary oligosaccharides. Lett Appl Microbiol 37:281-284. https://10.1046/j.1472-765x.2003.01398.x\u003c/li\u003e\n\u003cli\u003ePabari K, Pithva S, Kothari C, Purama RK, Kondepudi KK, Vyas BRM, Kothari R, Ambalam P (2020) Evaluation of Probiotic Properties and Prebiotic Utilization Potential of Weissella paramesenteroides Isolated From Fruits. Probiotics Antimicrob Proteins 12:1126-1138. https://10.1007/s12602-019-09630-w\u003c/li\u003e\n\u003cli\u003eWayne P (2011) Clinical and laboratory standards institute. Performance standards for antimicrobial susceptibility testing. \u003c/li\u003e\n\u003cli\u003eFonseca HC, de Sousa Melo D, Ramos CL, Dias DR, Schwan RF (2021) Probiotic properties of lactobacilli and their ability to inhibit the adhesion of enteropathogenic bacteria to Caco-2 and HT-29 cells. Probiotics Antimicrob Proteins 13:102-112. https://doi.org/10.1007/s12602-020-09659-2\u003c/li\u003e\n\u003cli\u003eChen CC, Lai CC, Huang HL, Huang WY, Toh HS, Weng TC, Chuang YC, Lu YC, Tang HJ (2019) Antimicrobial Activity of Lactobacillus Species Against Carbapenem-Resistant Enterobacteriaceae. Front Microbiol 10:789. https://doi.org/10.3389/fmicb.2019.00789\u003c/li\u003e\n\u003cli\u003eLee J-E, Lee N-K, Paik H-D (2021) Antimicrobial and anti-biofilm effects of probiotic Lactobacillus plantarum KU200656 isolated from kimchi. Food Sci Biotechnol 30:97-106. https://doi.org/10.1007/s10068-020-00837-0\u003c/li\u003e\n\u003cli\u003eRamalho JB, Soares MB, Spiazzi CC, Bicca DF, Soares VM, Pereira JG, Da Silva WP, Sehn CP, Cibin FW (2019) In vitro probiotic and antioxidant potential of Lactococcus lactis subsp. cremoris LL95 and its effect in mice behaviour. Nutrients 11:901. https://doi.org/10.3390/nu11040901\u003c/li\u003e\n\u003cli\u003eKim S, Lee JY, Jeong Y, Kang C-H (2022) Antioxidant activity and probiotic properties of lactic acid bacteria. Fermentation 8:29. https://doi.org/10.3390/fermentation8010029\u003c/li\u003e\n\u003cli\u003eGao D, Gao Z, Zhu G (2013) Antioxidant effects of Lactobacillus plantarum via activation of transcription factor Nrf2. Food Funct 4:982-989. https://doi.org/10.1039/c3fo30316k\u003c/li\u003e\n\u003cli\u003eXiong L, Ni X, Niu L, Zhou Y, Wang Q, Khalique A, Liu Q, Zeng Y, Shu G, Pan K, Jing B, Zeng D (2019) Isolation and Preliminary Screening of a Weissella confusa Strain from Giant Panda (Ailuropoda melanoleuca). Probiotics Antimicrob Proteins 11:535-544. https://doi.org/10.1007/s12602-018-9402-2\u003c/li\u003e\n\u003cli\u003eTenea GN, Ortega C (2021) Genome Characterization of Lactiplantibacillus plantarum Strain UTNGt2 Originated from Theobroma grandiflorum (White Cacao) of Ecuadorian Amazon: Antimicrobial Peptides from Safety to Potential Applications. Antibiotics (Basel) 10. https://doi.org/10.3390/antibiotics10040383\u003c/li\u003e\n\u003cli\u003ePei Z, Sadiq FA, Han X, Zhao J, Zhang H, Ross RP, Lu W, Chen W (2021) Comprehensive Scanning of Prophages in Lactobacillus: Distribution, Diversity, Antibiotic Resistance Genes, and Linkages with CRISPR-Cas Systems. mSystems 6:e0121120. https://doi.org/10.1128/mSystems.01211-20\u003c/li\u003e\n\u003cli\u003eJarocki P, Komon-Janczara E, Mlodzinska A, Sadurski J, Kolodzinska K, Laczmanski L, Panek J, Frac M (2023) Occurrence and genetic diversity of prophage sequences identified in the genomes of L. casei group bacteria. Sci Rep 13:8603. https://10.1038/s41598-023-35823-z\u003c/li\u003e\n\u003cli\u003eDeghorain M, Goffin P, Fontaine L, Mainardi JL, Daniel R, Errington J, Hallet B, Hols P (2007) Selectivity for D-lactate incorporation into the peptidoglycan precursors of Lactobacillus plantarum: role of Aad, a VanX-like D-alanyl-D-alanine dipeptidase. J Bacteriol 189:4332-4337. https://doi.org/10.1128/JB.01829-06\u003c/li\u003e\n\u003cli\u003eKandasamy S, Lee KH, Yoo J, Yun J, Kang HB, Kim JE, Oh MH, Ham JS (2024) Whole genome sequencing of Lacticaseibacillus casei KACC92338 strain with strong antioxidant activity, reveals genes and gene clusters of probiotic and antimicrobial potential. Front Microbiol 15:1458221. https://doi.org/10.3389/fmicb.2024.1458221\u003c/li\u003e\n\u003cli\u003eSun J, Li W, Li R, Liu W, Wang J, Sun Z, Menghe B (2025) Genomic analysis of probiotics from breast milk Limosilactobacillus fermentum UP-1 and application in fermented milk. J Dairy Sci. https://10.3168/jds.2025-26535\u003c/li\u003e\n\u003cli\u003eHeunis T, Deane S, Smit S, Dicks LM (2014) Proteomic profiling of the acid stress response in Lactobacillus plantarum 423. J Proteome Res 13:4028-4039. https://doi.org/10.1021/pr500353x\u003c/li\u003e\n\u003cli\u003eMonteagudo-Mera A, Rastall RA, Gibson GR, Charalampopoulos D, Chatzifragkou A (2019) Adhesion mechanisms mediated by probiotics and prebiotics and their potential impact on human health. Appl Microbiol Biotechnol 103:6463-6472. https://10.1007/s00253-019-09978-7\u003c/li\u003e\n\u003cli\u003eBrockhausen I (2014) Crossroads between Bacterial and Mammalian Glycosyltransferases. Front Immunol 5:492. https://10.3389/fimmu.2014.00492\u003c/li\u003e\n\u003cli\u003eLebeer S, Vanderleyden J, De Keersmaecker SC (2008) Genes and molecules of lactobacilli supporting probiotic action. Microbiol Mol Biol Rev 72:728-764, Table of Contents. https://doi.org/10.1128/MMBR.00017-08\u003c/li\u003e\n\u003cli\u003eSalas-Jara MJ, Ilabaca A, Vega M, Garcia A (2016) Biofilm Forming Lactobacillus: New Challenges for the Development of Probiotics. Microorganisms 4. https://doi.org/10.3390/microorganisms4030035\u003c/li\u003e\n\u003cli\u003eGao J, Sadiq FA, Zheng Y, Zhao J, He G, Sang Y (2022) Biofilm-based delivery approaches and specific enrichment strategies of probiotics in the human gut. Gut Microbes 14:2126274. https://doi.org/10.1080/19490976.2022.2126274\u003c/li\u003e\n\u003cli\u003eLei Y, Oshima T, Ogasawara N, Ishikawa S (2013) Functional analysis of the protein Veg, which stimulates biofilm formation in Bacillus subtilis. J Bacteriol 195:1697-1705. https://doi.org/10.1128/JB.02201-12\u003c/li\u003e\n\u003cli\u003eMgomi FC, Yang YR, Cheng G, Yang ZQ (2023) Lactic acid bacteria biofilms and their antimicrobial potential against pathogenic microorganisms. Biofilm 5:100118. https://10.1016/j.bioflm.2023.100118\u003c/li\u003e\n\u003cli\u003eLiang X, Dai N, Yang F, Zhu H, Zhang G, Wang Y (2024) Molecular identification and safety assessment of the potential probiotic strain Bacillus paralicheniformis HMPM220325 isolated from artisanal fruit dairy products. Food Funct 15:747-765. https://doi.org/10.1039/d3fo04625g\u003c/li\u003e\n\u003cli\u003eKavanova K, Kostovova I, Moravkova M, Kubasova T, Babak V, Crhanova M (2024) Comparative Genome Analysis and Characterization of the Probiotic Properties of Lactic Acid Bacteria Isolated from the Gastrointestinal Tract of Wild Boars in the Czech Republic. Probiotics Antimicrob Proteins. https://doi.org/10.1007/s12602-024-10259-7\u003c/li\u003e\n\u003cli\u003eShrestha P, Karmacharya J, Han SR, Lee JH, Oh TJ (2024) Elucidation of bacterial trehalose-degrading trehalase and trehalose phosphorylase: physiological significance and its potential applications. Glycobiology 34. https://doi.org/10.1093/glycob/cwad084\u003c/li\u003e\n\u003cli\u003eJakaria Al-Mujahidy SM, Kryukov K, Ikeo K, Saito K, Uddin ME, Ibn Sina AA (2024) Functional genomic analysis of the isolated potential probiotic Lactobacillus delbrueckii subsp. indicus TY-11 and its comparison with other Lactobacillus delbrueckii strains. Microbiol Spectr 12:e0347023. https://10.1128/spectrum.03470-23\u003c/li\u003e\n\u003cli\u003eSethi M, Ok A, Dash J, Parida D, Kar S, Mishra S, Minz AP, Padhi A, Das KR, Pradhan B, Prakash T, Senapati S (2025) Whole Genome Mining and Characterization of a New Probiotic Strain Levilactobacillus brevis ILSH3 from Handia: An Ethnic Fermented Beverage of Odisha, India. Probiotics Antimicrob Proteins 17:1596-1614. https://doi.org/10.1007/s12602-024-10217-3\u003c/li\u003e\n\u003cli\u003eAbriouel H, Manetsberger J, Caballero Gomez N, Benomar N (2022) In silico genomic analysis of the potential probiotic Lactiplantibacillus pentosus CF2-10N reveals promising beneficial effects with health promoting properties. Front Microbiol 13:989824. https://10.3389/fmicb.2022.989824\u003c/li\u003e\n\u003cli\u003eNaughton LM, Romano S, O\u0026apos;Gara F, Dobson ADW (2017) Identification of Secondary Metabolite Gene Clusters in the Pseudovibrio Genus Reveals Encouraging Biosynthetic Potential toward the Production of Novel Bioactive Compounds. Front Microbiol 8:1494. https://doi.org/10.3389/fmicb.2017.01494\u003c/li\u003e\n\u003cli\u003eWong A, Ngu DY, Dan LA, Ooi A, Lim RL (2015) Detection of antibiotic resistance in probiotics of dietary supplements. Nutr J 14:95. https://doi.org/10.1186/s12937-015-0084-2\u003c/li\u003e\n\u003cli\u003eKaushik A, Kest H, Sood M, Steussy BW, Thieman C, Gupta S (2024) Biofilm Producing Methicillin-Resistant Staphylococcus aureus (MRSA) Infections in Humans: Clinical Implications and Management. Pathogens 13. https://doi.org/10.3390/pathogens13010076\u003c/li\u003e\n\u003cli\u003ePique N, Berlanga M, Minana-Galbis D (2019) Health Benefits of Heat-Killed (Tyndallized) Probiotics: An Overview. Int J Mol Sci 20. https://doi.org/10.3390/ijms20102534\u003c/li\u003e\n\u003cli\u003eFassio F, Guagnini F (2018) House dust mite-related respiratory allergies and probiotics: a narrative review. Clin Mol Allergy 16:15. https://10.1186/s12948-018-0092-9\u003c/li\u003e\n\u003cli\u003eGawlik-Dziki U, Baraniak B, Sikora M, Jakubczyk A, Kapusta I, Swieca M (2021) Potentially Bioaccessible Phenolic and Antioxidant Potential of Fresh and Stored Lentil Sprouts-Effect of Lactobacillus plantarum 299v Enrichment. Molecules 26. https://doi.org/10.3390/molecules26082109\u003c/li\u003e\n\u003cli\u003eNie H, Wang X, Luo Y, Kong F, Mu G, Wu X (2024) Mechanism Explanation on Improved Cognitive Ability of D-Gal Inducing Aged Mice Model by Lactiplantibacillus plantarum MWFLp-182 via the Microbiota-Gut-Brain Axis. J Agric Food Chem 72:9795-9806. https://doi.org/10.1021/acs.jafc.3c09675\u003c/li\u003e\n\u003cli\u003eDiez-Guti\u0026eacute;rrez L, San Vicente L, Barr\u0026oacute;n LJR, del Carmen Villar\u0026aacute;n M, Ch\u0026aacute;varri M (2020) Gamma-aminobutyric acid and probiotics: Multiple health benefits and their future in the global functional food and nutraceuticals market. J Funct Foods 64:103669. https://doi.org/10.1016/j.jff.2019.103669\u003c/li\u003e\n\u003cli\u003eLiang X, He J, Zhang N, Muhammad A, Lu X, Shao Y (2022) Probiotic potentials of the silkworm gut symbiont Enterococcus casseliflavus ECB140, a promising L‐tryptophan producer living inside the host. J Appl Microbiol 133:1620-1635. https://doi.org/10.1111/jam.15675\u003c/li\u003e\n\u003cli\u003eGodzien J, Kalaska B, Rudzki L, Barbas-Bernardos C, Swieton J, Lopez-Gonzalvez A, Ostrowska L, Szulc A, Waszkiewicz N, Ciborowski M, Garcia A, Kretowski A, Barbas C, Pawlak D (2025) Probiotic Lactobacillus plantarum 299v supplementation in patients with major depression in a double-blind, randomized, placebo-controlled trial: A metabolomics study. J Affect Disord 368:180-190. https://10.1016/j.jad.2024.09.058\u003c/li\u003e\n\u003cli\u003eQian XH, Xie RY, Liu XL, Chen SD, Tang HD (2022) Mechanisms of Short-Chain Fatty Acids Derived from Gut Microbiota in Alzheimer\u0026apos;s Disease. Aging Dis 13:1252-1266. https://10.14336/AD.2021.1215\u003c/li\u003e\n\u003cli\u003eWullt M, Johansson Hagslatt ML, Odenholt I, Berggren A (2007) Lactobacillus plantarum 299v enhances the concentrations of fecal short-chain fatty acids in patients with recurrent clostridium difficile-associated diarrhea. Dig Dis Sci 52:2082-2086. https://10.1007/s10620-006-9123-3\u003c/li\u003e\n\u003cli\u003eHorn H, Holland EG, Hazleton L (1957) Food additives, safety of adipic acid as compared with citric and tartaric acid. J Agric Food Chem 5:759-762. https://doi.org/10.1021/jf60080a007\u003c/li\u003e\n\u003c/ol\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":"Microbial dysbiosis, Probiotics, Whole genome sequencing (WGS) analysis, Metabolic profiling, Human-target based functional-network analysis, Gene-signatures, Antioxidant and antimicrobial activities, Psychobiotic potential","lastPublishedDoi":"10.21203/rs.3.rs-7556358/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7556358/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGene-signatures for probiotic attributes expedite screening of novel probiotic strains as well as identify molecular basis of probiotic benefits. Therefore, strain-specific genomic-studies correlating functional-assays are in demand. Here, we present the same for \u003cem\u003eLactiplantibacillus plantarum LP-ARP2\u003c/em\u003e using genomic-metabolomic-functional approaches. Genomic-features of \u003cem\u003eLP-ARP2\u003c/em\u003e are further compared with clinically relevant \u003cem\u003eL. plantarum\u003c/em\u003e strains to elucidate gene-specific commonality.\u003c/p\u003e\u003cp\u003eWe find robust stress-resilience genetic set-up (\u003cem\u003eusp/hsl/clp/\u003c/em\u003eABC-transporters/ ATP-synthase/chaperons \u003cem\u003edna/gro/grp\u003c/em\u003e) in \u003cem\u003eLP-ARP2\u003c/em\u003e-genome. Our acid/bile tolerance assays also indicate survivability (\u0026gt;\u0026thinsp;60%) of the strain in harsh conditions. Presence of adhesion-related (\u003cem\u003elspA/mapA/eno/srtA/\u003c/em\u003eglycosyltransferases/glycosylhydrolases/lipoproteins) and biofilm-forming genes (\u003cem\u003eveg/luxS\u003c/em\u003e) further align with its efficacy in autoaggregation (\u0026gt;\u0026thinsp;60%), adhesion (Caco-2), and biofilm-formation (24 h). CAZyme-genes with significant prebiotic utilization indicate the strain\u0026rsquo;s ability for gut-microbial-modulation and adaptation. Metabolic-profiling of \u003cem\u003eLP-ARP2\u003c/em\u003e-derived-CFS (HRMS analysis) validates the presence of related genes for SCFAs/vitamins/amino-acids/neurotransmitters GABA/serotonin/acetylcholine etc. Besides, many metabolites are reported antimicrobials. Indeed \u003cem\u003eLP-ARP2\u003c/em\u003e shows significant antibacterial potential against multidrug-resistant bacteria (Gram-positive/Gram-negative), gut-pathogen \u003cem\u003eSalmonella Typhimurium\u003c/em\u003e and pathogenic-biofilm (MRSA). Presence of antioxidant-genes in \u003cem\u003eLP-ARP2\u003c/em\u003e-genome (thioredoxin/NADH-dependent-\u003cem\u003enox/npr/ndh/\u003c/em\u003eglutathione-reductase/glutaredoxin/catalase/peroxidases/methionine sulfoxide reductase) are validated by high radical-scavenging activity of \u003cem\u003eLP-ARP2\u003c/em\u003e (ABTS\u0026thinsp;\u0026gt;\u0026thinsp;40%, DPPH 25 U/mL, superoxide\u0026thinsp;\u0026gt;\u0026thinsp;80%, and hydroxyl\u0026thinsp;\u0026gt;\u0026thinsp;70%). Moreover, \u003cem\u003ein-silico\u003c/em\u003e functional-network-analysis reveals \u003cem\u003eLP-ARP2\u003c/em\u003e-derived metabolites target oxidative stress, neuroinflammation, amyloid-beta metabolism, tau-phosphorylation, neurogenesis, and synaptic function, indicating molecular relevance of the therapeutic potential of \u003cem\u003eLP-ARP2.\u003c/em\u003e Fascinatingly, genomic-analysis between \u003cem\u003eLP-ARP2\u003c/em\u003e with clinically relevant (depression and intestinal disorders) \u003cem\u003eL. plantarum\u003c/em\u003e strains (299v and Lp01) elucidate comparable genetic-features for beneficial probiotics.\u003c/p\u003e\u003cp\u003eThus, study offers potential gene-signatures for probiotic-benefits of \u003cem\u003eL. plantarum\u003c/em\u003e and project \u003cem\u003eLP-ARP2\u003c/em\u003e as a promising probiotic with antibacterial, antioxidant and psychobiotic potential.\u003c/p\u003e","manuscriptTitle":"WGS analysis and Functional Studies Illustrate Promising Gene- signatures for Probiotic Attributes and Molecular-targeted Therapeutic Prospects of Lactiplantibacillus plantarum LP-ARP2","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-17 08:03:49","doi":"10.21203/rs.3.rs-7556358/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":"bfee0ab2-00e1-4c7e-9da9-53975b5db93f","owner":[],"postedDate":"September 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-11T09:08:58+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-17 08:03:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7556358","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7556358","identity":"rs-7556358","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}