Complete genome sequence and probiotic characterization of Lactobacillus delbrueckii subsp. indicus DC-3 isolated from traditional indigenous fermented milk

Complete genome sequence and probiotic characterization of Lactobacillus delbrueckii subsp. indicus DC-3 isolated from traditional indigenous fermented milk | 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 Complete genome sequence and probiotic characterization of Lactobacillus delbrueckii subsp. indicus DC-3 isolated from traditional indigenous fermented milk Deepti N. Chaudhari, Jayesh J. Ahire, Anupama N. Devkatte, Amit S. Kulthe This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4487829/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Oct, 2024 Read the published version in Probiotics and Antimicrobial Proteins → Version 1 posted 15 You are reading this latest preprint version Abstract In this study, Lactobacillus delbrueckii subsp. indicus DC-3 was isolated from Indian traditional indigenous fermented milk Dahi and identified using whole genome sequencing. The safety of the strain was evaluated using both genetic and phenotypic analyses, such as the presence of virulence factors, mobile and insertion elements, plasmids, antibiotic resistance, etc . Besides this, the strain was comprehensively investigated for in vitro probiotic traits, biofilm formation, antibacterials, and exopolysaccharide (EPS) production. In results, the strain showed a single circular chromosome (3,145,837 bp) with a GC content of 56.73%, a higher number of accessory and unique genes, an open pan-genome, and the absence of mobile and insertion elements, plasmids, virulence, and transmissible antibiotic resistance genes. The strain was capable of surviving in gastric juice (83% viability at 3 h) and intestinal juice (71% viability at 6 h) and showed 42.5% autoaggregation, adhesion to mucin, 8.7% adhesion to xylene, and 8.3% adhesion to Caco-2 cells. The γ-hemolytic nature, usual antibiotic susceptibility profile, and negative results for mucin and gelatin degradation ensure the safety of the strain. The strain produced 10.5 g/L of D -lactic acid and hydrogen peroxide, capable of inhibiting and co-aggregating Escherichia coli MTCC 1687, Proteus mirabilis MTCC 425, and Candida albicans ATCC 14053. In addition, the strain showed 90 mg/L EPS (48 h) and biofilm formation. In conclusion, this study demonstrates that L. delbrueckii subsp. indicus DC-3 is unique and different than previously reported L. delbrueckii subsp. indicus strains and is a safe potential probiotic candidate. Lactobacillus delbrueckii subsp. indicus DC-3 Probiotic Safety Fermented foods Dahi hydrogen peroxide Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Fermented foods have been one of the integral parts of the human diet for nearly 10,000 years [ 1 ]. They are diverse and serve as important sources of beneficial microbes, microbial-metabolites, vitamins, minerals, proteins, and other nutrients, contributing to 20% of total global food consumption [ 2 ]. However, the exact percentage of consumption is not yet accurately documented. In India, fermented foods were produced by spontaneous fermentation and were rarely produced on a commercial or industrial scale [ 3 ]. Due to huge local and regional variations, India has the largest varieties of traditional fermented foods, which are rich in naturally occurring microbiota [ 4 ]. Studies have shown that Lactic acid bacteria (LAB) such as Lactobacillus , Lactococcus , Leuconostoc , Enterococcus , Oenococcus , Streptococcus , Pediococcus , Alkalibacterium , Carnobacterium , Tetragenococcus , Vagococcus , and Weissella are widely present in many fermented foods and beverages [ 5 ]. Similarly, genera of Bacillus , Kocuria , Micrococcus , Bifidobacterium , and yeasts were also reported based on the type of fermented foods [ 2 , 5 ]. The resident bacteria in the food microbiota interact with the gut microbiome after consumption and have positive impacts on immunity, weight control, cardiovascular and cognitive function, and diabetes management [ 1 ]. A recent review summarized that fermented food consumption increases the alpha diversity in the gut [ 6 ]. Passolli et al. [ 7 ] have shown the possible genetic linking between the LAB of fermented foods and gut microbiome. Thus, considering the wide diversity, health benefits, and genotypic link with gut bacteria, fermented food bacteria could be one of the potential sources for the isolation of beneficial or probiotic bacteria. Probiotics are defined as live microorganisms that, when administered in adequate amounts, confer a health benefit on the host [ 8 ]. According to the overall global safety regulatory structure, the candidate probiotic strain should be identified using whole genome sequencing (WGS), have a history of safe use, or be listed in the Qualified Presumption of Safety (QPS) or generally recognized as safe (GRAS), and be characterized for genotypic and phenotypical properties for pathogenicity and antibiotic resistance [ 9 ]. Besides this, if strains do not have a history of safe use or QPS, then such novel strains should be characterized in detail for genotypic and phenotypical properties, along with animal and human clinical studies [ 10 , 9 ]. Moreover, a candidate probiotic should exhibit resistance to gastric acidity, bile acid, intestinal fluid, adhesion to epithelial cells, and antimicrobial activity [ 10 ]. The genus Lactobacillus includes Gram-positive, facultatively anaerobic, non-spore-forming, fermentative rods [ 11 ]. Lactobacillus delbrueckii is mainly found in fermented products of both plants and animals. In 1983, based on differential phenotypic characteristics, this species was divided into three subspecies namely, delbrueckii , bulgaricus , and lactis [ 11 ]. Lactobacillus delbrueckii subsp. bulgaricus and lactis are mostly present in milk, and the subspecies delbrueckii colonizes vegetable sources [ 12 ]. In 2005, Dellaglio and co-workers reported a novel L. delbrueckii subspecies indicus isolated from Indian dairy products [ 13 ]. Later in 2012 and 2013, two new subspecies such as sunkii , and jakobsenii were proposed [ 14 , 15 ]. To date, apart from the immense potential of L. delbrueckii in dairy fermentations, these bacteria are well-known as a probiotic to impart health benefits in humans and animals [ 16 ]. As probiotic effects are strain-specific, thus it is crucial to isolate newer strains for both fundamental research and the food sector. In this study, strain DC-3 from traditional indigenous fermented milk ( Dahi ) was identified using WGS, genotypically characterized, and comprehensively investigated for in vitro probiotic traits, safety, biofilm formation, antibacterials, and exopolysaccharide production. Materials and Methods Isolation of bacterial strain The traditional fermented food samples viz. fermented milk ( Dahi and Mattha ), and fermented cereal batter ( Anarase and Ragi ) were collected from village Loni-Kalbhor (18.48799103 N 74.01815952 E), Pune, India. The samples ( n = 4) were immediately transferred to the Food Technology Laboratory under cold conditions (4 o C) and serially diluted in MRS (deMan Rogosa Sharpe) broth (HiMedia, India) supplemented with 0.05% (w/v) filter sterilized L-cysteine (Sigma Aldrich, USA). The respective dilutions of each sample were spread plated on MRS agar previously reduced with 0.05% (w/v) sterile L-cysteine. The plates were incubated at 37 o C for 24–48 h. After incubation, the catalase-negative colonies of different morphologies were sub-cultured separately in a fresh 10 mL MRS broth supplemented with L-cysteine. The glycerol stocks were prepared by mixing equal volumes of overnight-grown culture and 40% (w/v) glycerol (Sigma Aldrich, USA) and maintained at – 20 o C. The isolated bacteria were further characterized by Gram staining and morphological features. Carbohydrate (glucose, fructose, lactose, ribose, mannitol, inulin, starch, and glycogen) fermentation ability was evaluated for selected bacteria [ 13 ]. Survival under gastro-intestinal conditions Preparation of bacterial suspension A colony of overnight-grown bacteria was inoculated in 10 mL MRS broth supplemented with 0.05% (w/v) L-cysteine and incubated at 37 o C for 24 h. After incubation, 1 mL culture was further transferred into 100 mL MRS broth containing L-cysteine and incubated at 37 o C for 18 h. The cells were separated by centrifugation at 10000 × g for 10 min under cold conditions (4 o C). The collected cell pellet was washed twice with phosphate buffer saline (PBS, pH 7.3) and re-suspended in the same [ 17 ]. Gastric juice tolerance One milliliter bacterial suspension was mixed with 10 mL filter sterilized (0.2 µm, cellulose acetate, Sartorius, Germany) synthetic gastric juice (lysozyme, 0.1 g; pepsin, 0.0133 gm; proteose peptone, 8.3 g; glucose, 3.5 g; bile, 0.05 g; CaCl 2 , 0.11 g; KCl, 0.37 g; NaCl, 2.05 g; KH 2 PO 4 , 0.6 g; and ultrapure water 1 L, pH 2.5) and incubated at 37 o C for 3 h [ 18 ]. The bacterial survival was determined at the interval of 0, 1, 2, and 3 h on MRS agar plates supplemented with L-cysteine. The viability was expressed as log 10 CFU/mL. Intestinal juice tolerance One milliliter bacterial suspension was mixed with 10 mL filter sterilized intestinal juice (consisting of 1 mg/mL pancreatin (protease 100 U/mg; amylase 100 U/mg; lipase 8 U/ mg), 0.3% (w/v) bile, and 0.85% (w/v) NaCl, pH 8.0) and incubated at 37 o C for 6 h [ 19 ]. The bacterial survival was determined at the interval of 0, 3, and 6 h as described earlier. Adhesion potential Autoaggregation The bacterial strain was cultivated as described earlier, and the cell pellet was dissolved in PBS (pH 7.3) to 0.5 OD units (600 nm). Five milliliters of this homogenous suspension was gently transferred into 14 mL graduated round bottom falcon tube (Corning, USA). The suspension was vortexed (10s) gently and incubated statically at 37°C for 1 h [ 20 ]. After incubation, the upper layer was gently removed and absorbance was recorded at 600 nm. The auto-aggregation percentage was determined as, (OD initial suspension – OD final upper suspension/ OD initial suspension) × 100. Adhesion to mucin The bacterial strain was cultivated and pelleted as described earlier. The cell pellet was dissolved homogeneously in PBS (pH 7.3) containing 0.05% (w/v) Tween 20 to adjust the optical density to 0.5 units (600 nm). A 250 µL aliquots of the above suspension were each gently added to a mucin-coated well [ 17 ] of a 96-well plate (ThermoFisher, USA) and incubated at 4 o C for 24 h. The planktonic cells in the suspension were removed, and wells were gently washed with PBS + Tween 20 (0.05% w/v; pH 7.3) and air-dried. The bacterial adhesion was estimated quantitatively using crystal violet staining [ 21 ]. Adhesion to xylene The bacterial strain was cultivated and pelleted as described earlier. The cell pellet was dissolved homogeneously in 0.1 M KNO 3 (pH 6.2) to 0.5 OD units (600 nm). A 3 mL above suspension was gently added into a 20 mL glass tube (Borosil, India) containing 1 mL xylene. The tubes were incubated statically at 37 o C for 10 min and gently vortexed (5 s). Both aqueous and solvent phases were allowed to separate at 37 o C for 1 h. The aqueous phase was carefully removed and absorbance was recorded at 600 nm [ 22 ]. The percentage of xylene adhesion was determined as, (OD initial suspension – OD aqueous layer / OD initial suspension) × 100. Caco-2 cells adhesion Human colorectal adenocarcinoma (Caco-2) (NCCS, Pune, India) cells (1 × 10 5 cells per mL) were cultivated on glass coverslips placed in 24-well tissue culture plates (ThermoFisher) containing Dulbecco’s modified Eagle’s minimal essential medium (DMEM) for 2 weeks as per the method described by Tuomola et al. [ 23 ]. The DMEM was changed every day for 2 weeks and 1 h before the adhesion assay. A 400 µL bacterial suspension (10 8 cells per mL) prepared in PBS (pH 7.3) was inoculated into a Caco-2 monolayer and incubated at 37 o C for 2 h. After incubation, the Caco-2 monolayers were washed thrice with PBS (pH 7.3), air-dried, and Gram’s stained. The adherent bacteria were counted by selecting 15 random fields per coverslip [ 23 ]. Zeta potential The bacterial strain was cultivated as described earlier, and the cell pellet was dissolved in PBS (pH 7.3) to 0.5 OD units (600 nm). The above suspension was carefully filled in a capillary cell (DTS1070) and subjected to zeta potential measurement using a Nano-ZS Zetasizer (Malvern, UK) [ 24 ]. Safety evaluations Hemolytic activity A 5 µL overnight grown bacterial culture was spotted on a sheep blood agar plate (HiMedia, Mumbai) and incubated at 37°C for 24 − 48 h. Bacillus cereus ATCC 10876 was used as a positive control. Based on the zones surrounding the bacterial growth, the culture was considered as, α-hemolytic (greenish/dark zones), β-hemolytic (clear/light yellow zones), and γ-hemolytic (no zones) [ 19 ]. Gelatinase activity A 5 µL overnight grown bacterial culture was spotted on gelatin-nutrient agar (0.8% gelatin (w/v) + 2.3% nutrient agar) and incubated at 37°C for 24 − 48 h. After incubation, the plates were treated with 5% (w/v) trichloroacetic acid (TCA) and observed for clear zones surrounding the growth. Bacillus cereus ATCC 10876 was used as a positive control. Mucin degradation A 5 µL overnight grown bacterial culture was spotted on mucin (0.3%. w/v) containing minimal agarose medium (pancreatic enzymatic digest of casein, 7.5 g; tryptone, 7.5 g; yeast extract, 3.0 g; meat extract, 5.0 g; cysteine HCl, 0.5 g; K 2 HPO 4 , 3.0 g; KH 2 PO 4 , 0.5 g; NaCl, 5.0 g; MgSO 4 , 0.5 g; agarose, 15 g and ultrapure water 1L; pH 7.2 ± 0.2) plates supplemented with or without 3% (w/v) glucose. The plates were incubated at 37°C for 72 h and stained with 0.1% (w/v) coomassie blue as described by Ahire et al. [ 25 ]. The discolored halo surrounding the bacterial growth indicates mucin lysis. The fecal sample (fecal flora) was used as positive control. Cytotoxicity to HepG-2 and Caco-2 cells Human hepatocarcinoma (HepG-2) and Caco-2 cells were obtained from NCCS, Pune, India. Both HepG2 and Caco-2 were cultivated as per the method described by Tjandrawinata et al. [ 26 ]. A 200 µL bacterial suspension (10 9 cells per mL) prepared in MEM-alpha/MEM was inoculated into 1 × 10 4 cells / well of HepG-2 and or Caco-2 in a 96-well plate (ThermoFisher). The plates were incubated at 37 o C for 24 h. After incubation, the cell viability was determined by using MTT based Cell Proliferation Kit (Merck, USA) as per the manufacturer’s instructions. The percentage of cell viability was determined as, mean absorbance of treated cells / mean absorbance of untreated cells × 100. Antibiotic susceptibility Hundred microliters (~ 10 6 cells per mL) of overnight grown bacterial cells were seeded into the 20 mL molten semi-soft MRS agar (0.7% w/v, agar) and poured into the plates. The antibiotic disks (Table 1 , HiMedia) were placed on surface-dried agar plates. The plates were incubated initially at 4 o C (20 min) for antibiotic diffusion and transferred to 37 o C for 24 h. The zones of growth inhibition were measured in millimeters (mm) and antibiotic susceptibility was evaluated according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Clinical and Laboratory Standards Institute (CLSI) guidelines [ 27 , 28 ]. Table 1 Antibiotic susceptibility of Lactobacillus delbrueckii subsp. indicus DC-3 Class of antibiotic Antibiotic Concentration Zone of inhibition in millimeter (mm) Aminoglycosides Amikacin 30 µg 14 ± 1.0 (R) Neomycin 30 µg 11 ± 1.1 (R) Nystatin 100 Units 0 ± 0.0 (R) Streptomycin 25 µg 10 ± 0.5 (R) β lactam Amoxyclav (Amoxycillin, 20/ clavulanic acid, 10) 30 µg 25 ± 1.2 (S) Penicillin G 10 Units 30 ± 0.9 (S) Cephalosporin Cefazolin 30 µg 21 ± 0.5 (S) Cefdinir 5 µg 17 ± 1.0 (S) Cefixime 5 µg 20 ± 0.5 (S) Cefoperazone 75 µg 23 ± 1.2 (S) Coumarin glycosides Novobiocin 30 µg 0 ± 0.0 (R) Macrolactams Rifampicin 5 µg 14 ± 0.9 (R) Macrolides Azithromycin 15 µg 23 ± 1.1 (S) Clarithromycin 15 µg 27 ± 1.1 (S) Phenylpiperazines Itraconazole 10 µg 0 ± 0.0 (S) Quinolones Nalidixic acid 30 µg 0 ± 0.0 (S) Ofloxacin 5 µg 11 ± 0.5 (R) Steroids / derivatives Fusidic acid 30 µg 0 ± 0.0 (R) Triazole Fluconazole 10 µg 0 ± 0.0 (R) R: resistant (≤ 14 mm); S: sensitive (≥ 14 mm) Minimum inhibitory concentrations (MICs) of selected antibiotics (Table 2 ) were determined as per the agar dilution method described by Andrews [ 29 ]. In brief, the antibiotic stock solutions were prepared and diluted in 20 mL molten MRS agar to achieve concentrations in the range of 0.06 to 128 mg/L per agar plate. A 5 µL bacterial suspension (10 6 cells/mL) was spot inoculated onto the air-dried surface of antibiotic-containing agar plates and incubated at 37 o C for 48 h. MIC was defined as the lowest antibiotic concentration in the agar medium that prevented the growth of tested bacteria [ 25 ]. The MIC values were compared with the MIC cut-off given for obligate homofermentative Lactobacillus by the European Food Safety Authority (EFSA) [ 30 ]. Table 2 Minimal inhibitory concentration (MIC) of selected antibiotics against Lactobacillus delbrueckii subsp. indicus DC-3 Antibiotic MIC (mg/L) DC-3 European Food Safety Authority (EFSA) cut-off Ampicillin 128 - Chloramphenicol 4 4 Clindamycin 0.125 4 Erythromycin 1 1 Gentamicin 64 16 Kanamycin > 128 16 Rifampicin 2 - Tetracycline 8 4 Trimethoprim > 128 - Vancomycin 4 2 -: Not determined Antimicrobial potential Antimicrobial activity and screening for bacteriocin production A 18 h old bacterial cells were spotted (5 µL) on the air-dried surface of MRS agar and incubated at 37 o C for 24 − 48 h. After incubation, 100 µL (10 6 cells per mL) of a 24-hour-old culture of Micrococcus luteus MTCC 106 T , Escherichia coli MTCC 1687, Proteus mirabilis MTCC 425, and Candida albicans ATCC 14053 were each inoculated separately in 20 mL of molten Muller-Hinton (MH) agar and poured on top of bacterial growth. The plates were incubated at 37 o C for 24 h and observed for zone of growth clearance. The results were measured in millimeters (mm) [ 19 ]. Simultaneously, 18 h-old supernatant obtained from the bacteria cultivated as per the method described earlier was neutralized to pH 7.0 and filter sterilized using 0.2 µm cellulose acetate (Sartorius) membrane. A 25 µL of this solution was added into the previously bored wells in Micrococcus luteus MTCC 106 T (10 6 cells per mL) seeded MH agar plates. The plates were incubated at 4 o C for 20 min and later shifted to 30 o C incubator for 48 h. After incubation, the plates were observed for the zone of growth clearance (mm). Lactic acid and hydrogen peroxide (H 2 O 2 ) production A 1% (v/v) overnight-grown bacteria were cultivated in MRS broth (supplemented with 0.05% (w/v) L-cysteine) at 37 o C for 24 h. After incubation, the supernatant was collected and lactic acid ( D - and L - enantiomers) was measured using D -/ L -lactic acid kit (NZY Tech, Lisboa, Portugal), as per the manufacturer’s instructions. H 2 O 2 production was determined as per the method described by Pino et al. [ 31 ] and Ahire et al. [ 25 ]. In brief, an 18 h old bacterial culture was streaked on MRS agar supplemented with 1 mM 3, 3′, 5, 5′-tetramethyl-benzidine (Sigma-Aldrich), and 2 U/mL peroxidase (Sigma-Aldrich). The plates were incubated at 37°C for 48 h. After incubation, the colonies were exposed to air, and the time required to form a blue color in the colonies was recorded. H 2 O 2 production was scored as 0 (no coloration), 1 (low, > 20 min), 2 (medium, 10–20 min), and 3 (high, < 10 min). Co-aggregation with pathogens Both probiotic and pathogenic bacteria ( E. coli MTCC 1687, P. mirabilis MTCC 425, and C. albicans ATCC 14053) were individually cultivated for 18 h in their respective growth media and incubation conditions. After incubation, each bacterial culture was washed twice with PBS (pH 7.3) and re-suspended in PBS to 0.5 OD units. Equal amounts (2:2 mL) of probiotic and pathogen were mixed with gentle vortexing (10 s) and kept undisturbed at 37 o C for 1 h. The upper layer was carefully removed, and the optical density was measured at 600 nm [ 32 ]. A 4 mL suspension of each bacteria was used as a control. The co-aggregation percentage was calculated as, ((OD x + OD y ) / 2) – OD ( x + y ) / OD x + OD y / 2 × 100. where, OD x and OD y : individual aggregation densities of probiotic and pathogen. OD ( x + y ): combined aggregation density [ 25 ]. Exopolysaccharide (EPS) production EPS production ability of the culture was determined by using the ruthenium red skim milk agar plate assay described by Stingele et al. [ 33 ]. In brief, actively growing bacterial cells were spot (5 µL) inoculated on the surface of ruthenium red skim milk agar (ruthenium red, 0.08 g; skim milk powder, 100 g; sucrose, 10 g; yeast extract, 5 g; agar, 15 g, and ultrapure water 1L) plates and incubated at 37°C for 48 h. After incubation, the colony coloration (white: ropy/EPS producer; red: non-ropy/ non-producer) was checked and results were recorded. A 1% (v/v) overnight-grown bacterial culture was inoculated in MRS broth supplemented with 0.05% (w/v) L-cysteine and incubated at 37°C for 48 h. After incubation, the supernatant was separated (8,000 × g for 15 min, 4°C) and mixed with 2 volumes of ice-cold (− 80°C) absolute ethanol. The mixture was kept at 4°C for 48 h to precipitate EPS. The precipitated EPS was collected, dissolved in 8% (w/v) TCA, and incubated overnight at 4°C to precipitate proteins. The protein precipitates were collected by centrifugation at 5000 × g, 10 min, 4°C, and discarded. The supernatant was subjected twice to ice-cold ethanol and TCA precipitation steps to purify the EPS [ 34 ]. The EPS isolated from the above procedure was dialysed at 4°C using a 10 kDa cut-off dialysis membrane (HiMedia) against ultra-pure water. The water during the dialysis was changed 4 times at the interval of 12 h. After dialysis, the EPS concentrate was freeze-dried (Lyo lab, United States), and stored at 4°C until further analysis. The amount of total carbohydrate was determined using Antrone regent and protein by Pierce™ bicinchoninic acid (BCA) kit (Thermo Fisher Scientific, United States), as per the manufacturer’s instructions. Biofilm formation An 18 h old bacterial cells were diluted in MRS broth supplemented with 0.05% (w/v) L-cysteine. Two hundred microliters (3.3 × 10 4 CFU/mL) were dispensed into 96-well plates (ThermoFisher) and incubated at 37 o C for 24 h. Planktonic cells, viable biofilm cells, and total biofilm mass were determined as per the procedure described by Ahire and Dicks [ 21 , 35 ]. Complete genome sequence analysis For the DNA extraction, a single colony of the strain was cultivated overnight in 10 mL of MRS broth supplemented with 0.05% (w/v) L-cysteine. The cells were harvested, and DNA was extracted using MO BIO’s genomic DNA extraction kit (Carlsbad, CA, USA). The quality and quantity of the extracted DNA were checked and measured on a 0.8% agarose gel and the Qubit dsDNA HS assay kit (ThermoFisher Scientific). The DNA fragmentation and library construction were done using the Nextera DNA Flex Library preparation kit (Illumina, San Diego, CA, USA), as per the manufacturer's instructions. After library construction, dual index adapters were ligated at the blunt ends of the DNA fragments, followed by the purification (as described by the manufacturer). The quality and quantity of the fragment library were estimated. The good quality library was normalised, pooled, and subsequently sequenced using 2 × 250 bp chemistry on the Illumina MiSeq platform (Illumina Inc., USA). A 5% PhiX spike in was done during library loading in the sequencing cartage. After sequencing, the quality of paired-end raw reads was checked, and pre-processing sequences (i.e., primers and barcode trimming) was done by the NGS-QC tool to remove low-quality sequences ( http://www.nipgr.res.in/ngsqctoolkit.html ) [ 36 ]. Genome assembly was performed by using SPAdes-3.11.1 assembler (St. Petersburg, Russia). The assembled genome quality was checked using QUAST, and the number of rRNAs was identified and retrieved using RNAmmer 1.2 (USA). The genome annotation of the organism was done using RAST, UniProt/SwissProt, and KEGG database. tRNA and rRNA genes, protein-encoding genes of the genomes, were predicted using tRNA_scan-SE, and RNAmmer, Glimmer version 3.02, respectively. The presence of CRISPR repeats was predicted using the CRISPR Finder tools [ 37 ]. The secondary metabolites were identified using AntiSMASH [ 38 ] and antibiotic resistance genes and virulence factors were determined by CARD [ 39 ] and VFDB [ 40 ]. IslandViewer [ 41 ], CRISPR-Finder [ 37 ] were used to predict the presence of insertion sequences, and bacteriophage associated sequences (Fig. S1 ). The predicted 16S rRNA sequence was used in the EzBioCloud server [ 42 ] to find out the closely related group of organisms. Phylogenetic analysis was constructed using MEGA 7 software [ 43 ]. Based on the constructed phylogenetic tree, a number of genomes of the closely related organisms were downloaded from NCBI website and used for comparative genome analysis by BPGA tool. Strain and whole genome sequence deposition Lactobacillus delbrueckii subsp. indicus DC-3 was deposited at National Centre for Microbial Resource, Pune, with accession number MCC4964. The whole-genome shotgun project has been deposited in GenBank under the following accession number JAOWCA000000000. The BioProject is available at https://www.ncbi.nlm.nih.gov/bioproject/888254 . Statistical analysis All the experiments were performed in triplicates and data are expressed as a mean ± standard deviation (SD). The GraphPad Prism (Version 10, USA) was used for the determination of statistical significance by one-way ANOVA (Tukey) and or t –test. The p < 0.05 was considered as statistically significant. Results Isolation of bacterial strain Fermented milk and cereal batter yielded a number of colonies on MRS agar. Out of that, six colonies showed different morphologies and were designated as isolates DC-1 to 6. The isolates DC-3, 4, and 6 were catalase-negative, Gram’s positive, and non-motile rods. Owing to the better growth and dominant colony type on MRS agar, the fermented milk ( Dahi ) isolate DC-3 was selected and characterized further. On MRS agar, the DC-3 colony appeared whitish, 1–2 mm in size, circular, smooth, and opaque with a convex elevation (Fig. 1 a). The isolate DC-3 was Gram-positive, non-spore-forming, rod-shaped bacteria with rounded ends (Fig. 1bc). The cells were on average 2–6 µm long and 0.5–0.7 µm wide and occurred singly, in pairs, and in short chains (Fig. 1 c). DC-3 fermented glucose, fructose, and lactose without production of carbon dioxide, whereas it failed to ferment ribose, mannitol, inulin, starch, and glycogen. Survival under gastro-intestinal conditions The survival of DC-3 was significantly ( p 0.001) reduced during the incubation in gastric juice. At the end of 1st h, 8.05 ± 0.02 log 10 CFU/mL viable cells were decreased ( p 0.001) to 7.94 ± 0.01 log 10 CFU/mL (Fig. 2 a). Moreover, the viability was further reduced subsequently from 7.94 ± 0.01 log 10 CFU/mL (1 h, p 0.0001) to 6.87 ± 0.01 log 10 CFU/mL (2 h, p 0.0001) and 6.60 ± 0.02 log 10 CFU/mL (3 h, p 0.0001) (Fig. 2 a). In intestinal juice, DC-3 viability was reduced ( p 0.0001) from 4.89 ± 0.01 log 10 CFU/mL to 3.67 ± 0.03 log 10 CFU/mL during the first 3 h of incubation (Fig. 2 b). Later at the end of the 6th h, viability was further reduced ( p 0.001) to 3.51 ± 0.04 log 10 CFU/mL (Fig. 2 b). Adhesion potential DC-3 showed 42.51 ± 1.56% autoaggregation at 1 h of incubation (Fig. 2 c). The adhesion to xylene, mucin and Caco-2 cells was recorded as 8.76 ± 0.75%, 0.66 ± 0.01 OD units (crystal violet equivalence at 550 nm), and 8.3 ± 1.2% respectively (Fig. 2 c, d). The adhesion of DC-3 to Caco-2 cells and mucin is shown Fig. 2 d. The membrane potential measured for DC-3 cells was − 4.85 ± 0.40 mV. Safety evaluations No zone of hemolysis (γ-hemolytic) was observed when DC-3 was cultivated on blood agar for 48 h (Fig. 3 a). On gelatin-nutrient agar plate, DC-3 failed to produce a clear zone of gelatin hydrolysis after the treatment of TCA (Fig. 3 a). In mucin degradation study, DC-3 cultivated on mucin agarose medium with or without glucose failed to produce discolored halo (mucin degradation) after the treatment of coomassie blue (Fig. 3 a). Moreover, in the presence of DC-3, Caco-2 cells exhibited significantly ( p 0.001) higher viability (76 ± 0.16%) as compared to HepG-2 cells (71 ± 1.03%) (Fig. 3 b). DC-3 showed sensitivity to the selected antibiotics of class β lactam, cephalosporin, macrolides, phenylpiperazines and member of quinolones class nalidixic acid (Table 1 ). However, DC-3 was resistant to the selected antibiotics of the class aminoglycosides, coumarin glycosides, macrolactams, steroids / derivatives, triazole, and a member of quinolones class ofloxacin (Table 1 ). In MIC evaluation, the MIC values recorded for gentamicin, kanamycin, tetracycline, and vancomycin were higher than the MIC cut-off values provided by the European Food Safety Authority (EFSA) for obligate homofermentative Lactobacillus (Table 2 ). Antimicrobial potential The 48 h grown DC-3 cells (5–6 mm spot) were inhibited the growth of all tested pathogens (Fig. 3 c). The significantly ( p 0.045) higher zone of growth inhibition was recorded against M. luteus MTCC 106 T (11.33 ± 0.57 mm) as compared to E. coli MTCC 1687 (10 ± 0 mm), whereas growth inhibition differences recorded against E. coli MTCC 1687 (10 ± 0 mm), P. mirabilis MTCC 425 (10.33 ± 0.6 mm), and C. albicans ATCC 14053 (10.33 ± 0.57 mm) remained statistically insignificant ( p > 0.05) (Fig. 3 c). In bacteriocin screening, the neutralized supernatant of DC-3 was failed to inhibit the growth of M. luteus MTCC 106 T . DC-3 produced 10.5 g/L of D -lactic acid and 1.36 g/L of L -lactic acid after 24 h of incubation. Besides this, after exposure to air, DC-3 colonies showed intense blue color in less than 10 min (Fig. 3 d). DC-3 cells co-aggregated significantly ( p 0.013) larger extent with E. coli MTCC 1687 (5.02 ± 0.18%) and then P. mirabilis MTCC 425 (4.48 ± 0.13%). The least co-aggregation was recorded with C. albicans ATCC 14053 (1.57 ± 0.16%). Exopolysaccharide (EPS) production After 48 h incubation on ruthenium red skim milk agar, DC-3 produced white (ropy/EPS producer) growth (Fig. 3 e). Quantitatively, it was determined as 90 mg/L at 48 h incubation in MRS broth containing 2.0% glucose. The 1 mg EPS corresponded to 430.6 ± 20.8 µg carbohydrate and 9.55 ± 3.75 µg protein. Biofilm formation After 24 h of incubation, significantly ( p 0.022) higher optical density readings (0 h: 0.005 ± 0.002; 24 h: 0.058 ± 0.015) were recorded for crystal violet staining of total biofilm (Fig. 3 f). The viable biofilm cells count (Fig. 3 g) was significantly ( p 0.028) increased from 0 h (3.3 × 10 4 ) to 24 h (9.6 × 10 7 ). Simultaneously, the optical densities of planktonic cells (Fig. 3 h) were significantly ( p 0.0001) increased from 0 h (0.048 ± 0.03) to 24 h (1.94 ± 0.07). Complete genome sequence analysis The average quality score for base position in reads was recorded as normal (Fig. S2 ). The genome assembly statistics showed that the strain contained 3,145,837 bp, 3034 scaffolds with maximum scaffold length of 324, N50 length of 17330, and 56.73% G + C. The strain showed 99.19% average nucleotide identity with Lactobacillus delbrueckii subsp. indicus DSM 15996. Similar results were observed with in silico DNA-DNA genome level hybridization analysis (96.64%). After obtaining a genome assembly of sufficient quality, the different types of genome elements were predicted and categorized as pathway genes (972), gene ontology (GO) term genes (378), hypothetical or uncharacterized genes (2223), putative genes (183), non-coding genes (111), tRNA (90), and rRNA (10) genes (Fig. 4 a). The coding sequences were analysed further for their metabolic role and categorized into the 15 abundant functions (Fig. 4 b). The predicted coding sequences (CDS) were utilized to determine the protein clusters of orthologous groups (COGs) using the EggNOG web service (Fig. 4 c). No antibiotic resistance genes (ARs) were detected using a strict criterion. However, using loose cut-off values, a total of 312 genes were predicted. Out of which, only more than 60% of identity records were considered (Table S1 ). Besides this, no virulence factor genes, mobile and insertion elements, and plasmids were detected or identified in strain DC-3. The AntiSMASH analysis identified the genes to produce lanthipeptide class III. Phylogenetic analysis showed the close similarity of the strain DC-3 with Lactobacillus delbrueckii subsp. indicus DSM 15996/JCM15610 (Fig. S3). In comparative genome analysis, the closest neighbours such as L. delbrueckii subsp. indicus , L. equicursoris and L. porci were selected based on the phylogenetic analysis as the priority and pruned down using genome availability status. The number of accessory genes and unique genes was higher in strain DC-3 as compared with selected organisms (Fig. 5 a). The estimated pan-genome size of strain DC-3 was 4,777.86, and the parameter ‘b’ was calculated to be 0.558297 (Fig. 5 b). The phylogeny of the core genome showed that strain DC-3 shares the close relatedness with L. delbrueckii subsp. indicus (Fig. S4). Furthermore, the details of CDS, tRNA, rRNA, tmRNA, repeat region, and CARD were given in circular genome map of strain DC-3 (Fig. 6 ). Discussion The traditional diversity in the preparation of fermented foods and substrates is crucial for their microbiota and unique compositions [ 6 ]. Most of the members of these microbial communities were capable of surviving gastrointestinal transit and helping to strengthen the gut microbiome [ 1 ]. Several studies have indicated that fermented foods are a good source of probiotic bacteria [ 44 ]. However, isolation of probiotic bacteria from local and traditional fermented foods remained under-reported. In this study, we investigated probiotic attributes, adhesion ability, safety, antimicrobial potential, exopolysaccharide production, biofilm formation, and whole genome sequence analysis of fermented milk ( Dahi ) isolate L. delbrueckii subsp. indicus DC-3. The survival under gastro-intestinal conditions is one of the prerequisite properties for probiotic bacteria. In order survive under these conditions lactic acid bacteria stimulate the activity of F 0 F 1 ATP proton pumps to maintain cytoplasmic pH, produce alkaline compounds in the cell cytoplasm, modify the cell membrane integrity and fluidity, and upregulate the genes for amino acid decarboxylation and repair or protection of macromolecules [ 45 ]. In this study, strain DC-3 showed 83% viability for up to 3 hours in gastric juice and 71% viability for 6 hours in intestinal juice, which falls between a reasonable survival rate of 70 − 80% for in vitro static experiments [ 46 , 47 ]. Studies suggest that the bacterial survival rate for gastro-intestinal conditions is largely dependent on exposure time and strain [ 32 , 48 ]. Moreover, the gastric and intestinal juice viability of strain DC-3 was comparatively higher (> 70%) than the gastric and intestinal juice viability (< 70%) reported for L. delbrueckii subsp. indicus CRL1447 [ 47 ]. Adhesion of probiotic candidates to the intestinal epithelial cells for colonization is crucial for inhibition of pathogens, nutrient absorption, and immunity [ 49 , 50 ]. It has been suggested that probiotic bacteria interact with epithelial cells through electrostatic and hydrophobic interactions mediated through teichoic (TA) and lipoteichoic acid (LTA), environmental DNA (eDNA), polysaccharides, and complex polymers, or by producing bioactive metabolites [ 51 ]. In this study, the higher percentage of DC-3 autoaggregation suggested the ability of the strain to interact with or recognize surface proteins, TA, LTA, organelles, eDNA, or exopolysaccharides (EPS) for colonization. Besides this, the DC-3 adhesion to the polar solvent xylene demonstrated hydrophobic cell surfaces. The negatively charged membrane potential indicated the presence of TA and LTA, a phosphate-rich cell surface glycolpolymer essential for the attachment of bacterial cells to epithelial cells [ 52 ]. Moreover, adhesion to mucin and CaCo-2 cells further confirmed the adhesion potential of the L. delbrueckii subsp. indicus DC-3, which is well coordinated with previous findings [ 47 ]. In order to establish safety, candidate probiotics must have data on genotypic and phenotypic identity, whole genome sequencing (WGS), studies on virulence, toxin production, and antibiotic resistance [ 10 ]. Besides this, when a candidate probiotic doesn’t have a history of safe use and is not listed in the qualified presumption of safety (QPS), extensive safety testing is required, including animal and human studies [ 9 ]. The strain DC-3 reported in this study was isolated from traditional fermented milk ( Dahi ), which has a long history of safe use. It doesn’t produce hemolysin to lyse red blood cells, gelatinase to break down extracellular matrix, and is incapable of degrading mucin, a core structural element of the mucosal surfaces of the digestive tract. The cytotoxic effect of DC-3 observed against hepato- and colon-carcinoma cells could be due to their anti-carcinogenic abilities. The MICs of ampicillin, chloramphenicol, clindamycin, and erythromycin observed against DC-3 were as per the cut-off values provided by EFSA [ 30 ]. However, higher MICs of gentamycin, kanamycin, tetracycline, and vancomycin could not pose the risk of transmissible antibiotic resistance genes from food and feed, as it is an outcome of the intrinsic resistance of the Lactobacillus [ 53 ]. Similarly, resistance to selected antibiotics in the classes aminoglycosides, coumarin glycosides, macrolactams, steroids / derivatives, and ofloxacin, a quinolone, could be intrinsic, i.e., on the chromosome. These findings are well aligned with antibiotic resistance in probiotic microorganisms [ 54 ]. Moreover, the antibiotic- and antifungal (triazole)-resistant trait of the strain could be useful for its application during or along with antibiotic or antifungal agents. Overall, based on in vitro safety and antibiotic susceptibility profiles, strain DC-3 might be regarded a safe strain for probiotic use. The antimicrobial activity of the probiotics is essential to eliminating pathogens, which are the main competitors for nutrients and colonization in the gut [ 55 ]. Studies indicated that probiotic bacteria are capable of producing lactic acid, acetic acid, diacetyl, bacteriocins and bacteriocin-like inhibitory substances (BLIS), hydrogen peroxide, and surface active molecules to inhibit the pathogens [ 56 ]. In this investigation, strain DC-3 inhibited the growth of both Gram-positive and Gram-negative bacteria as well as yeast. Based on the zones of growth inhibition and the strains' inability to inhibit bacterial growth following neutralization of cell-free supernatant, it was determined that the action was attributable to lactic acid and/or hydrogen peroxide rather than bacteriocins. The production of a higher amount of lactic acid and hydrogen peroxide further confirmed the findings. Karnaouri et al. [ 57 ] showed that, as starter cultures in dairy fermentation, L. delbrueckii subsp. are efficient lactic acid producers. Similarly, the hydrogen peroxide production ability of L. delbrueckii subsp. bulgaricus was previously reported by Kot et al. [ 58 ]. Besides antimicrobial production, co-aggregation of probiotic bacteria with pathogens is one of the important properties of manipulating the aberrant intestinal microbiota [ 59 ]. This property increases the colonization efficiency of probiotic bacteria. In this study, strain DC-3 showed co-aggregations with E. coli , P. mirabilis , and C. albicans , indicating its ability to lower both bacteria and yeast colonization in the gut. The co-aggregation percentages were mostly dependent on strains and the time of the assay, and the results obtained in this study are well coordinated with previous findings [ 59 , 25 ]. The exopolysaccharide of lactic acid bacteria (LAB) is known to enhance their tolerance to harsh gastro-intestinal conditions and colonization in the gut [ 60 ]. It is also reported for numerous other health benefits like immunomodulation, antiviral, anti-cancer, anti-inflammatory, anti-yeast, and putative antimicrobial activities [ 61 ]. In this study, strain DC-3 showed its ability to produce EPS, which is important for its survival and colonization in the gut. The quantity of EPS reported in this study was comparatively lower than that of L. delbrueckii ssp. indicus WDS-7 [ 62 ]. In addition to EPS production, strain DC-3 exhibited biofilm formation, which is considered an advantageous trait for efficient colonization. Studies have shown that LAB-biofilms enhance their antimicrobial capacity and could act as a biocontrol agent for pathogens and pathogenic biofilms [ 63 ]. The strain DC-3 biofilm could be an ideal combination of a good probiotic strain and its antimicrobials (lactic acid and hydrogen peroxide) to inhibit invading pathogens. To date, regulatory standards for probiotic safety have been well established in Europe and some Asian countries [ 9 ]. According to those standards, whole genome sequencing (WGS) is one of the requirements for establishing the safety of probiotic bacteria [ 9 , 10 ]. In this study, WGS of strain DC-3 isolated from traditional fermented milk ( Dahi ) showed a single circular chromosome (3,145,837 bp) with a GC content of 56.73% and was identified as L. delbrueckii subsp. indicus . The higher genomic GC content of the DC-3 than previously reported L. delbrueckii subsp. indicus JCM 15610 T (49.4%) indicated high thermal stability [ 64 ]. Recently, Teng et al. [ 65 ] showed that genomic GC has a considerable impact on the average amino acid characteristics of proteomes, including the N/C ratio and hydrophobicity, which may determine bacterial fitness. In comparative genome analysis with the closest neighbours ( L. delbrueckii subsp. indicus , L. equicursoris , and L. porci ), strain DC-3 showed a higher number of accessory and unique genes, which might be useful to enhance the strains' ability to adapt to their hosts or the surrounding environment [ 66 ]. Moreover, the open pan-genome of the DC-3 could indicate a probability of finding novel function genes. These results corroborate well with previous findings that the pan-genomes of most species of the Lactobacillaceae family are moderately open compared to other bacterial species [ 67 ]. In coding sequence analysis, each of the 15% genes was found to contribute to amino acid and carbohydrate metabolism, 10% to protein metabolism, 9% to cofactors and vitamins, 5% to membrane transport, 3% to stress response, 2% to regulation of cell signaling, 1% to chemotaxis, etc. No virulence factor genes, mobile and insertion elements, or plasmids were detected or identified in DC-3, which indicated the strain’s safety. The antibiotic resistance gene profiling results were well coordinated with the in vitro findings of the strain and further confirmed the strain's safety. Furthermore, the lanthipeptide class III identified in the WGS analysis was not detected in vitro as, most of the class III lanthipeptides often lack antibacterial activities [ 68 ]. Lactobacillus delbrueckii often possessed Lanthipeptide III and their conserved residues, i.e., lyase, kinase, and cyclase enzymes [ 69 ]. Conclusion Lactobacillus delbrueckii subsp. indicus DC-3 isolated from traditional Indian fermented milk ( Dahi ) showed good survival capabilities under in vitro gastrointestinal stress conditions, adhesion, lactic acid and hydrogen peroxide-mediated antimicrobial activity, exopolysaccharide production, and biofilm formation. The absence of genes for transmissible antibiotic resistance, virulence factor, mobile and insertion elements, plasmids, in vitro hemolysin and gelatinase production, and mucin degrading abilities confirmed the strain’s genotypic and phenotypic safety. In genotypic analysis, high GC content, the presence of a higher number of accessory and unique genes, and an open pan-genome exhibited thermal stability and strains' ability to adapt to their hosts or the surrounding environment. Overall, strain DC-3 isolated from traditional fermented milk ( Dahi ), which has a long history of safe use, is a safe probiotic candidate for further commercial applications. Declarations Funding The authors did not receive support from any organization for the submitted work. Competing Interests Ahire JJ was employed by Dr. Reddy’s Laboratories Limited. Dr. Reddy’s Laboratories had no direct and indirect role in the design/analysis/writing/publication of this research article. Other authors have no conflict of interest to declare. Data Availability Statement All data is included in the text, however, the raw data of this article will be made available by the authors, without undue reservation, to any qualified researcher. Author Contributions Conceptualization: Chaudhari DN, Ahire JJ, Devkatte AN; Methodology: Chaudhari DN, Ahire JJ; Formal analysis and investigation: Chaudhari DN, Ahire JJ; Writing - original draft preparation: Chaudhari DN, Ahire JJ; Writing – Chaudhari DN, Ahire JJ; Resources: Kulthe AS, Devkatte AN; Supervision: Ahire JJ, Devkatte AN. 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Microorganisms 9(11):2336. https://doi.org/10.3390/microorganisms9112336 Campedelli I, Mathur H, Salvetti E, Clarke S, Rea MC, Torriani S, Ross RP, Hill C, O'Toole PW (2018) Genus-wide assessment of antibiotic resistance in Lactobacillus spp. Appl Environ Microbiol 85(1):e01738–e01718. https://doi.org/10.1128/AEM.01738-18 Li T, Teng D, Mao R, Hao Y, Wang X, Wang J (2020) A critical review of antibiotic resistance in probiotic bacteria. Food Res Int 19:109571. https://doi.org/10.1016/j.foodr es.2020.109571 Petrariu O-A, Barbu IC, Niculescu A-G, Constantin M, Grigore GA, Cristian R-E, Mihaescu G, Vrancianu CO (2024) Role of probiotics in managing various human diseases, from oral pathology to cancer and gastrointestinal diseases. Front Microbiol 14:1296447. https://doi.org/10.3389/fmicb.2023.1296447 Amaning Danquah C, Minkah PAB, Osei Duah Junior I, Amankwah KB, Somuah SO (2022) Antimicrobial compounds from microorganisms. Antibiotics 11(3):285. https://doi.org/10.3390/antibiotics11030285 Karnaouri A, Georgia A, Kalogiannis KG, Lappas A, Topakas E (2020) Efficient d-lactic acid production by Lactobacillus delbrueckii subsp. bulgaricus through conversion of organosolv pretreated lignocellulosic biomass. Biomass Bioenergy 140:105672. https://doi.org/10.1016/j.biombioe.2020.105672 Kot E, Furmanov S, Bezkorovainy A (1996) Hydrogen peroxide production and oxidation of ferrous iron by Lactobacillus delbrueckii ssp. bulgaricus . J Dairy Sci 79(5):758–766. https://doi.org/10.3168/jds.S0022-0302(96)76423-8 Collado MC, Meriluoto J, Salminen S (2008) Adhesion and aggregation properties of probiotic and pathogen strains. Eur Food Res Technol 226:1065–1073. https://doi.org/10.1007/s00217-007-0632-x Lu Y, Han S, Zhang S, Wang K, Lv L, McClements DJ, Xiao H, Berglund B, Yao M, Li L (2022) The role of probiotic exopolysaccharides in adhesion to mucin in different gastrointestinal conditions. Curr Res Food Sci 5:581–589. https://doi.org/10.1016/j.crfs.2022.02.015 Korcz E, Varga L (2021) Exopolysaccharides from lactic acid bacteria: Techno-functional application in the food industry. Trends Food Sci Technol 110:375–384. https://doi.org/10.1016/j.tifs.2021.02.014 Wu C, Dai C, Tong L, Lv H, Zhou X (2022) Evaluation of the probiotic potential of Lactobacillus delbrueckii ssp. indicus WDS-7 isolated from chinese traditional fermented buffalo milk in vitro . Pol J Microbiol 71(1):91–105. https://doi.org/10.33073/pjm-2022-012 Mgomi FC, Yang YR, Cheng G, Yang ZQ (2023) Lactic acid bacteria biofilms and their antimicrobial potential against pathogenic microorganisms. Biofilm 5:100118. https://doi.org/10.1016/j.bioflm.2023.100118 Baek MG, Kim KW, Yi H (2023) Subspecies-level genome comparison of Lactobacillus delbrueckii . Sci Rep 13(1):3171. https://doi.org/10.1038/s41598-023-29404-3 Teng W, Liao B, Chen M, Shu W (2023) Genomic legacies of ancient adaptation illuminate GC-content evolution in bacteria. Microbiol Spectr 11(1):e0214522. https://doi.org/10.1128/spectrum.02145-22 Zhang W, Wang J, Zhang D, Liu H, Wang S, Wang Y, Ji H (2019) Complete genome sequencing and comparative genome characterization of Lactobacillus johnsonii ZLJ010, a potential probiotic with health-promoting properties. Front Genet 10:812. https://doi.org/10.3389/fgene.2019.00812 Rajput A, Chauhan SM, Mohite OS, Hyun JC, Ardalani O, Jahn LJ, Sommer MO, Palsson BO (2023) Pangenome analysis reveals the genetic basis for taxonomic classification of the Lactobacillaceae family. Food Microbiol 115:104334. https://doi.org/10.1016/j.fm.2023.104334 Hegemann JD, Sussmuth RD (2020) Matters of class: coming of age of class III and IV lanthipeptides. RSC Chem Biol 1:110–127. https://doi.org/10.1039/D0CB00073F Lang H, Liu Y, Duan H, Zhang W, Hu X, Zheng H (2023) Identification of peptides from honeybee gut symbionts as potential antimicrobial agents against Melissococcus plutonius . Nat Commun 14(1):7650. https://doi.org/10.1038/s41467-023-43352-6 Additional Declarations Competing interest reported. Ahire JJ was employed by Dr. Reddy’s Laboratories Limited. Dr. Reddy’s Laboratories had no direct and indirect role in the design/analysis/writing/publication of this research article. Other authors have no conflict of interest to declare. Supplementary Files SupplementaryFigures.docx TableS1.docx Cite Share Download PDF Status: Published Journal Publication published 17 Oct, 2024 Read the published version in Probiotics and Antimicrobial Proteins → Version 1 posted Editorial decision: Revision requested 01 Jul, 2024 Reviews received at journal 24 Jun, 2024 Reviews received at journal 20 Jun, 2024 Reviews received at journal 19 Jun, 2024 Reviewers agreed at journal 15 Jun, 2024 Reviews received at journal 15 Jun, 2024 Reviewers agreed at journal 10 Jun, 2024 Reviewers agreed at journal 10 Jun, 2024 Reviewers agreed at journal 10 Jun, 2024 Reviewers agreed at journal 10 Jun, 2024 Reviewers agreed at journal 09 Jun, 2024 Reviewers invited by journal 09 Jun, 2024 Submission checks completed at journal 29 May, 2024 Editor assigned by journal 29 May, 2024 First submitted to journal 27 May, 2024 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-4487829","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":312561379,"identity":"8c7361f0-cc12-4d20-a999-168bbc425fa0","order_by":0,"name":"Deepti N. Chaudhari","email":"","orcid":"","institution":"MIT School of Food Technology, MIT-ADT University","correspondingAuthor":false,"prefix":"","firstName":"Deepti","middleName":"N.","lastName":"Chaudhari","suffix":""},{"id":312561380,"identity":"5f4660fe-8262-41ab-801a-7bad314d5828","order_by":1,"name":"Jayesh J. Ahire","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwklEQVRIiWNgGAWjYBACxoYDbAwJDAxyIM6BB6RoMQZrSSDSIjYQkdgAIonSwtx4+NmDh3ts0ueHHX4ItMVOTreBoMOOmRskPEvL3Xg7zQCoJdnY7ABBLQfMJBIOHM7dODsBpOVA4jbCWo5/A2r5n244O/0DsVrOgGw5kCAvnUO0LWfKgFqSDTdI5xQcSDAgwi+GM45vk/xxwE5efnb65g8fKuzkiNACVWEApg0IKAcBef4GKKOBCNWjYBSMglEwMgEAXfBM1TwZzzoAAAAASUVORK5CYII=","orcid":"","institution":"Dr. Reddy’s Laboratories Limited","correspondingAuthor":true,"prefix":"","firstName":"Jayesh","middleName":"J.","lastName":"Ahire","suffix":""},{"id":312561381,"identity":"5f1b5664-fc72-4763-95f3-e5d91739829b","order_by":2,"name":"Anupama N. Devkatte","email":"","orcid":"","institution":"MIT School of Food Technology, MIT-ADT University","correspondingAuthor":false,"prefix":"","firstName":"Anupama","middleName":"N.","lastName":"Devkatte","suffix":""},{"id":312561382,"identity":"8a470bd6-dda7-41c6-88d1-8a3d65ad4858","order_by":3,"name":"Amit S. Kulthe","email":"","orcid":"","institution":"MIT School of Food Technology, MIT-ADT University","correspondingAuthor":false,"prefix":"","firstName":"Amit","middleName":"S.","lastName":"Kulthe","suffix":""}],"badges":[],"createdAt":"2024-05-28 03:30:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4487829/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4487829/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12602-024-10385-2","type":"published","date":"2024-10-17T15:56:57+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":58111203,"identity":"b82c58b4-35d1-4214-aee7-8d89cfa2e571","added_by":"auto","created_at":"2024-06-11 09:29:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":566948,"visible":true,"origin":"","legend":"\u003cp\u003eColony morphology on MRS agar (a), Gram’s character (b), and scanning electron micrograph (SEM) (c) of \u003cem\u003eL. delbrueckii \u003c/em\u003esubsp.\u003cem\u003e indicus \u003c/em\u003eDC-3. The SEM images were obtained as per Ahire et al. [24] using EVO 18 SEM (Carl Zeiss, Germany). Cell size was measured using ImageJ software (USA). Single head arrow indicates colonies. Two head arrow indicates size.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-4487829/v1/3c3b0d284bbd94e8da4f1b71.png"},{"id":58111651,"identity":"d51deef9-942d-4aae-aa62-8d7b308057c9","added_by":"auto","created_at":"2024-06-11 09:37:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":267918,"visible":true,"origin":"","legend":"\u003cp\u003eGastric juice tolerance (a), intestinal juice tolerance (b), autoaggregation and adhesion to xylene (c), and adhesion to mucin and Caco-2 cell (inset shows the adhesion of DC-3 to mucin and Caco-2 cells) (d) of \u003cem\u003eL. delbrueckii \u003c/em\u003esubsp.\u003cem\u003e indicus \u003c/em\u003eDC-3. Data are represented as mean ± SD. ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, and **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-4487829/v1/e5ed02aff73659835e41ec17.png"},{"id":58111206,"identity":"554ae1ea-c632-47f4-90d2-8932ad6a34f0","added_by":"auto","created_at":"2024-06-11 09:29:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1121445,"visible":true,"origin":"","legend":"\u003cp\u003eHemolytic, gelatinase and mucin degradation activities (a), cytotoxicity (b), antimicrobial activity (c), hydrogen peroxide producing blue colonies (d) EPS producing white/ropy growth (e), total biofilm formation (f) (inset shows total biofilm after CV straining), viable cells in biofilm (g), and planktonic cells (h) of \u003cem\u003eL. delbrueckii \u003c/em\u003esubsp.\u003cem\u003e indicus \u003c/em\u003eDC-3. Data are represented as mean ± SD. ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.05, and ns \u003cem\u003ep\u003c/em\u003e\u0026gt; 0.05. Arrow indicates a positive test. Note: The mucin degradation image is a representative of mucin degradations with or without glucose supplementation.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-4487829/v1/d0edf5fcf1ec732488c8aedd.png"},{"id":58111205,"identity":"71284579-6731-4345-8c33-bb40e697fd81","added_by":"auto","created_at":"2024-06-11 09:29:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":206147,"visible":true,"origin":"","legend":"\u003cp\u003eDifferent type of genomic elements predicted from the assembly genome (a), major metabolic pathways in the genome (b), and cluster of orthologous groups (COG) distribution of the genes (c) of\u003cem\u003e L. delbrueckii \u003c/em\u003esubsp.\u003cem\u003e indicus \u003c/em\u003eDC-3. GO: Gene Ontology. Capital alphabets indicate COG category.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-4487829/v1/83e07ccff0693aa5d890ebf1.png"},{"id":58111960,"identity":"4a56e6b6-0e1f-4b96-8209-376742d9b6de","added_by":"auto","created_at":"2024-06-11 09:45:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":719314,"visible":true,"origin":"","legend":"\u003cp\u003ePan-genome analysis. (a) distribution of genes, (b) core-pan genome plot of \u003cem\u003eL. delbrueckii \u003c/em\u003esubsp.\u003cem\u003e indicus \u003c/em\u003eDC-3 and closely related organisms.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-4487829/v1/2e68c74aac852e6aed803005.png"},{"id":58111652,"identity":"59dceab1-f546-4300-9e32-54677b40affd","added_by":"auto","created_at":"2024-06-11 09:37:45","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":564823,"visible":true,"origin":"","legend":"\u003cp\u003eCircular genome map of \u003cem\u003eL. delbrueckii \u003c/em\u003esubsp.\u003cem\u003e indicus \u003c/em\u003eDC-3.\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-4487829/v1/816fc369a27ca17fdb0caa86.png"},{"id":67148821,"identity":"2a0f6dfa-bfeb-4dab-adcd-01668415aed7","added_by":"auto","created_at":"2024-10-21 16:08:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4679975,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4487829/v1/20839c81-e41a-420a-96e8-8e98da30886f.pdf"},{"id":58111210,"identity":"1f0514a2-4b87-4a58-b9e8-80e90214199b","added_by":"auto","created_at":"2024-06-11 09:29:45","extension":"docx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":661252,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-4487829/v1/4a86956ef2f134eb6ad9c315.docx"},{"id":58111208,"identity":"f5f6657a-9910-448e-88b1-8ed0133af5d6","added_by":"auto","created_at":"2024-06-11 09:29:45","extension":"docx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":13461,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4487829/v1/6791e067bd17958f276b22f0.docx"}],"financialInterests":"Competing interest reported. Ahire JJ was employed by Dr. Reddy’s Laboratories Limited. Dr. Reddy’s Laboratories had no direct and indirect role in the design/analysis/writing/publication of this research article. Other authors have no conflict of interest to declare.","formattedTitle":"Complete genome sequence and probiotic characterization of Lactobacillus delbrueckii subsp. indicus DC-3 isolated from traditional indigenous fermented milk","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFermented foods have been one of the integral parts of the human diet for nearly 10,000 years [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. They are diverse and serve as important sources of beneficial microbes, microbial-metabolites, vitamins, minerals, proteins, and other nutrients, contributing to 20% of total global food consumption [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, the exact percentage of consumption is not yet accurately documented. In India, fermented foods were produced by spontaneous fermentation and were rarely produced on a commercial or industrial scale [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Due to huge local and regional variations, India has the largest varieties of traditional fermented foods, which are rich in naturally occurring microbiota [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Studies have shown that Lactic acid bacteria (LAB) such as \u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003eLactococcus\u003c/em\u003e, \u003cem\u003eLeuconostoc\u003c/em\u003e, \u003cem\u003eEnterococcus\u003c/em\u003e, \u003cem\u003eOenococcus\u003c/em\u003e, \u003cem\u003eStreptococcus\u003c/em\u003e, \u003cem\u003ePediococcus\u003c/em\u003e, \u003cem\u003eAlkalibacterium\u003c/em\u003e, \u003cem\u003eCarnobacterium\u003c/em\u003e, \u003cem\u003eTetragenococcus\u003c/em\u003e, \u003cem\u003eVagococcus\u003c/em\u003e, and \u003cem\u003eWeissella\u003c/em\u003e are widely present in many fermented foods and beverages [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Similarly, genera of \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003eKocuria\u003c/em\u003e, \u003cem\u003eMicrococcus\u003c/em\u003e, \u003cem\u003eBifidobacterium\u003c/em\u003e, and yeasts were also reported based on the type of fermented foods [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe resident bacteria in the food microbiota interact with the gut microbiome after consumption and have positive impacts on immunity, weight control, cardiovascular and cognitive function, and diabetes management [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. A recent review summarized that fermented food consumption increases the alpha diversity in the gut [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Passolli et al. [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] have shown the possible genetic linking between the LAB of fermented foods and gut microbiome. Thus, considering the wide diversity, health benefits, and genotypic link with gut bacteria, fermented food bacteria could be one of the potential sources for the isolation of beneficial or probiotic bacteria.\u003c/p\u003e \u003cp\u003eProbiotics are defined as live microorganisms that, when administered in adequate amounts, confer a health benefit on the host [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. According to the overall global safety regulatory structure, the candidate probiotic strain should be identified using whole genome sequencing (WGS), have a history of safe use, or be listed in the Qualified Presumption of Safety (QPS) or generally recognized as safe (GRAS), and be characterized for genotypic and phenotypical properties for pathogenicity and antibiotic resistance [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Besides this, if strains do not have a history of safe use or QPS, then such novel strains should be characterized in detail for genotypic and phenotypical properties, along with animal and human clinical studies [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Moreover, a candidate probiotic should exhibit resistance to gastric acidity, bile acid, intestinal fluid, adhesion to epithelial cells, and antimicrobial activity [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe genus \u003cem\u003eLactobacillus\u003c/em\u003e includes Gram-positive, facultatively anaerobic, non-spore-forming, fermentative rods [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. \u003cem\u003eLactobacillus delbrueckii\u003c/em\u003e is mainly found in fermented products of both plants and animals. In 1983, based on differential phenotypic characteristics, this species was divided into three subspecies namely, \u003cem\u003edelbrueckii\u003c/em\u003e, \u003cem\u003ebulgaricus\u003c/em\u003e, and \u003cem\u003elactis\u003c/em\u003e [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. \u003cem\u003eLactobacillus delbrueckii\u003c/em\u003e subsp. \u003cem\u003ebulgaricus\u003c/em\u003e and \u003cem\u003elactis\u003c/em\u003e are mostly present in milk, and the subspecies \u003cem\u003edelbrueckii\u003c/em\u003e colonizes vegetable sources [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In 2005, Dellaglio and co-workers reported a novel \u003cem\u003eL. delbrueckii\u003c/em\u003e subspecies \u003cem\u003eindicus\u003c/em\u003e isolated from Indian dairy products [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Later in 2012 and 2013, two new subspecies such as \u003cem\u003esunkii\u003c/em\u003e, and \u003cem\u003ejakobsenii\u003c/em\u003e were proposed [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. To date, apart from the immense potential of \u003cem\u003eL. delbrueckii\u003c/em\u003e in dairy fermentations, these bacteria are well-known as a probiotic to impart health benefits in humans and animals [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. As probiotic effects are strain-specific, thus it is crucial to isolate newer strains for both fundamental research and the food sector.\u003c/p\u003e \u003cp\u003eIn this study, strain DC-3 from traditional indigenous fermented milk (\u003cem\u003eDahi\u003c/em\u003e) was identified using WGS, genotypically characterized, and comprehensively investigated for \u003cem\u003ein vitro\u003c/em\u003e probiotic traits, safety, biofilm formation, antibacterials, and exopolysaccharide production.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eIsolation of bacterial strain\u003c/h2\u003e \u003cp\u003eThe traditional fermented food samples \u003cem\u003eviz.\u003c/em\u003e fermented milk (\u003cem\u003eDahi\u003c/em\u003e and \u003cem\u003eMattha\u003c/em\u003e), and fermented cereal batter (\u003cem\u003eAnarase\u003c/em\u003e and \u003cem\u003eRagi\u003c/em\u003e) were collected from village Loni-Kalbhor (18.48799103 N 74.01815952 E), Pune, India. The samples (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4) were immediately transferred to the Food Technology Laboratory under cold conditions (4 \u003csup\u003eo\u003c/sup\u003eC) and serially diluted in MRS (deMan Rogosa Sharpe) broth (HiMedia, India) supplemented with 0.05% (w/v) filter sterilized L-cysteine (Sigma Aldrich, USA). The respective dilutions of each sample were spread plated on MRS agar previously reduced with 0.05% (w/v) sterile L-cysteine. The plates were incubated at 37 \u003csup\u003eo\u003c/sup\u003eC for 24\u0026ndash;48 h. After incubation, the catalase-negative colonies of different morphologies were sub-cultured separately in a fresh 10 mL MRS broth supplemented with L-cysteine. The glycerol stocks were prepared by mixing equal volumes of overnight-grown culture and 40% (w/v) glycerol (Sigma Aldrich, USA) and maintained at \u0026ndash; 20 \u003csup\u003eo\u003c/sup\u003eC. The isolated bacteria were further characterized by Gram staining and morphological features. Carbohydrate (glucose, fructose, lactose, ribose, mannitol, inulin, starch, and glycogen) fermentation ability was evaluated for selected bacteria [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eSurvival under gastro-intestinal conditions\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003ePreparation of bacterial suspension\u003c/h2\u003e \u003cp\u003eA colony of overnight-grown bacteria was inoculated in 10 mL MRS broth supplemented with 0.05% (w/v) L-cysteine and incubated at 37 \u003csup\u003eo\u003c/sup\u003eC for 24 h. After incubation, 1 mL culture was further transferred into 100 mL MRS broth containing L-cysteine and incubated at 37 \u003csup\u003eo\u003c/sup\u003eC for 18 h. The cells were separated by centrifugation at 10000 \u0026times;\u003cem\u003eg\u003c/em\u003e for 10 min under cold conditions (4 \u003csup\u003eo\u003c/sup\u003eC). The collected cell pellet was washed twice with phosphate buffer saline (PBS, pH 7.3) and re-suspended in the same [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eGastric juice tolerance\u003c/h2\u003e \u003cp\u003eOne milliliter bacterial suspension was mixed with 10 mL filter sterilized (0.2 \u0026micro;m, cellulose acetate, Sartorius, Germany) synthetic gastric juice (lysozyme, 0.1 g; pepsin, 0.0133 gm; proteose peptone, 8.3 g; glucose, 3.5 g; bile, 0.05 g; CaCl\u003csub\u003e2\u003c/sub\u003e, 0.11 g; KCl, 0.37 g; NaCl, 2.05 g; KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 0.6 g; and ultrapure water 1 L, pH 2.5) and incubated at 37 \u003csup\u003eo\u003c/sup\u003eC for 3 h [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The bacterial survival was determined at the interval of 0, 1, 2, and 3 h on MRS agar plates supplemented with L-cysteine. The viability was expressed as log\u003csub\u003e10\u003c/sub\u003e CFU/mL.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eIntestinal juice tolerance\u003c/h2\u003e \u003cp\u003eOne milliliter bacterial suspension was mixed with 10 mL filter sterilized intestinal juice (consisting of 1 mg/mL pancreatin (protease 100 U/mg; amylase 100 U/mg; lipase 8 U/ mg), 0.3% (w/v) bile, and 0.85% (w/v) NaCl, pH 8.0) and incubated at 37 \u003csup\u003eo\u003c/sup\u003eC for 6 h [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The bacterial survival was determined at the interval of 0, 3, and 6 h as described earlier.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAdhesion potential\u003c/h2\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eAutoaggregation\u003c/h2\u003e \u003cp\u003eThe bacterial strain was cultivated as described earlier, and the cell pellet was dissolved in PBS (pH 7.3) to 0.5 OD units (600 nm). Five milliliters of this homogenous suspension was gently transferred into 14 mL graduated round bottom falcon tube (Corning, USA). The suspension was vortexed (10s) gently and incubated statically at 37\u0026deg;C for 1 h [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. After incubation, the upper layer was gently removed and absorbance was recorded at 600 nm. The auto-aggregation percentage was determined as, (OD initial suspension \u0026ndash; OD final upper suspension/ OD initial suspension) \u0026times; 100.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eAdhesion to mucin\u003c/h2\u003e \u003cp\u003eThe bacterial strain was cultivated and pelleted as described earlier. The cell pellet was dissolved homogeneously in PBS (pH 7.3) containing 0.05% (w/v) Tween 20 to adjust the optical density to 0.5 units (600 nm). A 250 \u0026micro;L aliquots of the above suspension were each gently added to a mucin-coated well [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] of a 96-well plate (ThermoFisher, USA) and incubated at 4 \u003csup\u003eo\u003c/sup\u003eC for 24 h. The planktonic cells in the suspension were removed, and wells were gently washed with PBS\u0026thinsp;+\u0026thinsp;Tween 20 (0.05% w/v; pH 7.3) and air-dried. The bacterial adhesion was estimated quantitatively using crystal violet staining [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAdhesion to xylene\u003c/h2\u003e \u003cp\u003eThe bacterial strain was cultivated and pelleted as described earlier. The cell pellet was dissolved homogeneously in 0.1 M KNO\u003csub\u003e3\u003c/sub\u003e (pH 6.2) to 0.5 OD units (600 nm). A 3 mL above suspension was gently added into a 20 mL glass tube (Borosil, India) containing 1 mL xylene. The tubes were incubated statically at 37 \u003csup\u003eo\u003c/sup\u003eC for 10 min and gently vortexed (5 s). Both aqueous and solvent phases were allowed to separate at 37 \u003csup\u003eo\u003c/sup\u003eC for 1 h. The aqueous phase was carefully removed and absorbance was recorded at 600 nm [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The percentage of xylene adhesion was determined as, (OD initial suspension \u0026ndash; OD aqueous layer / OD initial suspension) \u0026times; 100.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCaco-2 cells adhesion\u003c/h2\u003e \u003cp\u003eHuman colorectal adenocarcinoma (Caco-2) (NCCS, Pune, India) cells (1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per mL) were cultivated on glass coverslips placed in 24-well tissue culture plates (ThermoFisher) containing Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s minimal essential medium (DMEM) for 2 weeks as per the method described by Tuomola et al. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The DMEM was changed every day for 2 weeks and 1 h before the adhesion assay. A 400 \u0026micro;L bacterial suspension (10\u003csup\u003e8\u003c/sup\u003e cells per mL) prepared in PBS (pH 7.3) was inoculated into a Caco-2 monolayer and incubated at 37 \u003csup\u003eo\u003c/sup\u003eC for 2 h. After incubation, the Caco-2 monolayers were washed thrice with PBS (pH 7.3), air-dried, and Gram\u0026rsquo;s stained. The adherent bacteria were counted by selecting 15 random fields per coverslip [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eZeta potential\u003c/h2\u003e \u003cp\u003eThe bacterial strain was cultivated as described earlier, and the cell pellet was dissolved in PBS (pH 7.3) to 0.5 OD units (600 nm). The above suspension was carefully filled in a capillary cell (DTS1070) and subjected to zeta potential measurement using a Nano-ZS Zetasizer (Malvern, UK) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSafety evaluations\u003c/h2\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003eHemolytic activity\u003c/h2\u003e \u003cp\u003eA 5 \u0026micro;L overnight grown bacterial culture was spotted on a sheep blood agar plate (HiMedia, Mumbai) and incubated at 37\u0026deg;C for 24\u0026thinsp;\u0026minus;\u0026thinsp;48 h. \u003cem\u003eBacillus cereus\u003c/em\u003e ATCC 10876 was used as a positive control. Based on the zones surrounding the bacterial growth, the culture was considered as, α-hemolytic (greenish/dark zones), β-hemolytic (clear/light yellow zones), and γ-hemolytic (no zones) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eGelatinase activity\u003c/h2\u003e \u003cp\u003eA 5 \u0026micro;L overnight grown bacterial culture was spotted on gelatin-nutrient agar (0.8% gelatin (w/v)\u0026thinsp;+\u0026thinsp;2.3% nutrient agar) and incubated at 37\u0026deg;C for 24\u0026thinsp;\u0026minus;\u0026thinsp;48 h. After incubation, the plates were treated with 5% (w/v) trichloroacetic acid (TCA) and observed for clear zones surrounding the growth. \u003cem\u003eBacillus cereus\u003c/em\u003e ATCC 10876 was used as a positive control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eMucin degradation\u003c/h2\u003e \u003cp\u003eA 5 \u0026micro;L overnight grown bacterial culture was spotted on mucin (0.3%. w/v) containing minimal agarose medium (pancreatic enzymatic digest of casein, 7.5 g; tryptone, 7.5 g; yeast extract, 3.0 g; meat extract, 5.0 g; cysteine HCl, 0.5 g; K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 3.0 g; KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 0.5 g; NaCl, 5.0 g; MgSO\u003csub\u003e4\u003c/sub\u003e, 0.5 g; agarose, 15 g and ultrapure water 1L; pH 7.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2) plates supplemented with or without 3% (w/v) glucose. The plates were incubated at 37\u0026deg;C for 72 h and stained with 0.1% (w/v) coomassie blue as described by Ahire et al. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The discolored halo surrounding the bacterial growth indicates mucin lysis. The fecal sample (fecal flora) was used as positive control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eCytotoxicity to HepG-2 and Caco-2 cells\u003c/h2\u003e \u003cp\u003eHuman hepatocarcinoma (HepG-2) and Caco-2 cells were obtained from NCCS, Pune, India. Both HepG2 and Caco-2 were cultivated as per the method described by Tjandrawinata et al. [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. A 200 \u0026micro;L bacterial suspension (10\u003csup\u003e9\u003c/sup\u003e cells per mL) prepared in MEM-alpha/MEM was inoculated into 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells / well of HepG-2 and or Caco-2 in a 96-well plate (ThermoFisher). The plates were incubated at 37 \u003csup\u003eo\u003c/sup\u003eC for 24 h. After incubation, the cell viability was determined by using MTT based Cell Proliferation Kit (Merck, USA) as per the manufacturer\u0026rsquo;s instructions. The percentage of cell viability was determined as, mean absorbance of treated cells / mean absorbance of untreated cells \u0026times; 100.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eAntibiotic susceptibility\u003c/h2\u003e \u003cp\u003eHundred microliters (~\u0026thinsp;10\u003csup\u003e6\u003c/sup\u003e cells per mL) of overnight grown bacterial cells were seeded into the 20 mL molten semi-soft MRS agar (0.7% w/v, agar) and poured into the plates. The antibiotic disks (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, HiMedia) were placed on surface-dried agar plates. The plates were incubated initially at 4 \u003csup\u003eo\u003c/sup\u003eC (20 min) for antibiotic diffusion and transferred to 37 \u003csup\u003eo\u003c/sup\u003eC for 24 h. The zones of growth inhibition were measured in millimeters (mm) and antibiotic susceptibility was evaluated according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Clinical and Laboratory Standards Institute (CLSI) guidelines [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\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\u003eAntibiotic susceptibility of \u003cem\u003eLactobacillus delbrueckii\u003c/em\u003e subsp. \u003cem\u003eindicus\u003c/em\u003e DC-3\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=\"left\" 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\u003eClass of antibiotic\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAntibiotic\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eConcentration\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eZone of inhibition in millimeter (mm)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eAminoglycosides\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAmikacin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30 \u0026micro;g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 (R)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNeomycin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30 \u0026micro;g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1 (R)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNystatin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100 Units\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0 (R)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStreptomycin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25 \u0026micro;g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 (R)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eβ lactam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAmoxyclav (Amoxycillin, 20/\u003c/p\u003e \u003cp\u003eclavulanic acid, 10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30 \u0026micro;g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 (S)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePenicillin G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10 Units\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 (S)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eCephalosporin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCefazolin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30 \u0026micro;g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 (S)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCefdinir\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5 \u0026micro;g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 (S)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCefixime\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5 \u0026micro;g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 (S)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCefoperazone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e75 \u0026micro;g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 (S)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCoumarin glycosides\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNovobiocin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30 \u0026micro;g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0 (R)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMacrolactams\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRifampicin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5 \u0026micro;g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 (R)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMacrolides\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAzithromycin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15 \u0026micro;g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1 (S)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eClarithromycin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15 \u0026micro;g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e27\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1 (S)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhenylpiperazines\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eItraconazole\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10 \u0026micro;g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0 (S)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eQuinolones\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNalidixic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30 \u0026micro;g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0 (S)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOfloxacin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5 \u0026micro;g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 (R)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSteroids / derivatives\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFusidic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30 \u0026micro;g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0 (R)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTriazole\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFluconazole\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10 \u0026micro;g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0 (R)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eR: resistant (\u0026le;\u0026thinsp;14 mm); S: sensitive (\u0026ge;\u0026thinsp;14 mm)\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eMinimum inhibitory concentrations (MICs) of selected antibiotics (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) were determined as per the agar dilution method described by Andrews [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In brief, the antibiotic stock solutions were prepared and diluted in 20 mL molten MRS agar to achieve concentrations in the range of 0.06 to 128 mg/L per agar plate. A 5 \u0026micro;L bacterial suspension (10\u003csup\u003e6\u003c/sup\u003e cells/mL) was spot inoculated onto the air-dried surface of antibiotic-containing agar plates and incubated at 37 \u003csup\u003eo\u003c/sup\u003eC for 48 h. MIC was defined as the lowest antibiotic concentration in the agar medium that prevented the growth of tested bacteria [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The MIC values were compared with the MIC cut-off given for obligate homofermentative \u003cem\u003eLactobacillus\u003c/em\u003e by the European Food Safety Authority (EFSA) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\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\u003eMinimal inhibitory concentration (MIC) of selected antibiotics against \u003cem\u003eLactobacillus delbrueckii\u003c/em\u003e subsp. \u003cem\u003eindicus\u003c/em\u003e DC-3\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\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAntibiotic\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eMIC (mg/L)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDC-3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eEuropean Food Safety Authority (EFSA) cut-off\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmpicillin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCiprofloxacin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChloramphenicol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClindamycin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.125\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eErythromycin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGentamicin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKanamycin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRifampicin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTetracycline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTrimethoprim\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVancomycin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003e-: Not determined\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eAntimicrobial potential\u003c/h2\u003e \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e \u003ch2\u003eAntimicrobial activity and screening for bacteriocin production\u003c/h2\u003e \u003cp\u003eA 18 h old bacterial cells were spotted (5 \u0026micro;L) on the air-dried surface of MRS agar and incubated at 37 \u003csup\u003eo\u003c/sup\u003eC for 24 \u0026minus;\u0026thinsp;48 h. After incubation, 100 \u0026micro;L (10\u003csup\u003e6\u003c/sup\u003e cells per mL) of a 24-hour-old culture of \u003cem\u003eMicrococcus luteus\u003c/em\u003e MTCC 106\u003csup\u003eT\u003c/sup\u003e, \u003cem\u003eEscherichia coli\u003c/em\u003e MTCC 1687, \u003cem\u003eProteus mirabilis\u003c/em\u003e MTCC 425, and \u003cem\u003eCandida albicans\u003c/em\u003e ATCC 14053 were each inoculated separately in 20 mL of molten Muller-Hinton (MH) agar and poured on top of bacterial growth. The plates were incubated at 37 \u003csup\u003eo\u003c/sup\u003eC for 24 h and observed for zone of growth clearance. The results were measured in millimeters (mm) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSimultaneously, 18 h-old supernatant obtained from the bacteria cultivated as per the method described earlier was neutralized to pH 7.0 and filter sterilized using 0.2 \u0026micro;m cellulose acetate (Sartorius) membrane. A 25 \u0026micro;L of this solution was added into the previously bored wells in \u003cem\u003eMicrococcus luteus\u003c/em\u003e MTCC 106\u003csup\u003eT\u003c/sup\u003e (10\u003csup\u003e6\u003c/sup\u003e cells per mL) seeded MH agar plates. The plates were incubated at 4 \u003csup\u003eo\u003c/sup\u003eC for 20 min and later shifted to 30 \u003csup\u003eo\u003c/sup\u003eC incubator for 48 h. After incubation, the plates were observed for the zone of growth clearance (mm).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eLactic acid and hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) production\u003c/h2\u003e \u003cp\u003eA 1% (v/v) overnight-grown bacteria were cultivated in MRS broth (supplemented with 0.05% (w/v) L-cysteine) at 37 \u003csup\u003eo\u003c/sup\u003eC for 24 h. After incubation, the supernatant was collected and lactic acid (\u003csub\u003eD\u003c/sub\u003e- and \u003csub\u003eL\u003c/sub\u003e- enantiomers) was measured using \u003csub\u003eD\u003c/sub\u003e-/\u003csub\u003eL\u003c/sub\u003e-lactic acid kit (NZY Tech, Lisboa, Portugal), as per the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e production was determined as per the method described by Pino et al. [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] and Ahire et al. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In brief, an 18 h old bacterial culture was streaked on MRS agar supplemented with 1 mM 3, 3\u0026prime;, 5, 5\u0026prime;-tetramethyl-benzidine (Sigma-Aldrich), and 2 U/mL peroxidase (Sigma-Aldrich). The plates were incubated at 37\u0026deg;C for 48 h. After incubation, the colonies were exposed to air, and the time required to form a blue color in the colonies was recorded. H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e production was scored as 0 (no coloration), 1 (low, \u0026gt; 20 min), 2 (medium, 10\u0026ndash;20 min), and 3 (high, \u0026lt; 10 min).\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eCo-aggregation with pathogens\u003c/h2\u003e \u003cp\u003eBoth probiotic and pathogenic bacteria (\u003cem\u003eE. coli\u003c/em\u003e MTCC 1687, \u003cem\u003eP. mirabilis\u003c/em\u003e MTCC 425, and \u003cem\u003eC. albicans\u003c/em\u003e ATCC 14053) were individually cultivated for 18 h in their respective growth media and incubation conditions. After incubation, each bacterial culture was washed twice with PBS (pH 7.3) and re-suspended in PBS to 0.5 OD units. Equal amounts (2:2 mL) of probiotic and pathogen were mixed with gentle vortexing (10 s) and kept undisturbed at 37 \u003csup\u003eo\u003c/sup\u003eC for 1 h. The upper layer was carefully removed, and the optical density was measured at 600 nm [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. A 4 mL suspension of each bacteria was used as a control. The co-aggregation percentage was calculated as, ((OD\u003cem\u003ex\u003c/em\u003e\u0026thinsp;+\u0026thinsp;OD\u003cem\u003ey\u003c/em\u003e) / 2) \u0026ndash; OD (\u003cem\u003ex\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003ey\u003c/em\u003e) / OD\u003cem\u003ex\u003c/em\u003e\u0026thinsp;+\u0026thinsp;OD\u003cem\u003ey\u003c/em\u003e / 2 \u0026times; 100. where, OD\u003cem\u003ex\u003c/em\u003e and OD\u003cem\u003ey\u003c/em\u003e: individual aggregation densities of probiotic and pathogen. OD (\u003cem\u003ex\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003ey\u003c/em\u003e): combined aggregation density [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eExopolysaccharide (EPS) production\u003c/h2\u003e \u003cp\u003eEPS production ability of the culture was determined by using the ruthenium red skim milk agar plate assay described by Stingele et al. [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In brief, actively growing bacterial cells were spot (5 \u0026micro;L) inoculated on the surface of ruthenium red skim milk agar (ruthenium red, 0.08 g; skim milk powder, 100 g; sucrose, 10 g; yeast extract, 5 g; agar, 15 g, and ultrapure water 1L) plates and incubated at 37\u0026deg;C for 48 h. After incubation, the colony coloration (white: ropy/EPS producer; red: non-ropy/ non-producer) was checked and results were recorded.\u003c/p\u003e \u003cp\u003eA 1% (v/v) overnight-grown bacterial culture was inoculated in MRS broth supplemented with 0.05% (w/v) L-cysteine and incubated at 37\u0026deg;C for 48 h. After incubation, the supernatant was separated (8,000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 15 min, 4\u0026deg;C) and mixed with 2 volumes of ice-cold (\u0026minus;\u0026thinsp;80\u0026deg;C) absolute ethanol. The mixture was kept at 4\u0026deg;C for 48 h to precipitate EPS. The precipitated EPS was collected, dissolved in 8% (w/v) TCA, and incubated overnight at 4\u0026deg;C to precipitate proteins. The protein precipitates were collected by centrifugation at 5000 \u0026times; g, 10 min, 4\u0026deg;C, and discarded. The supernatant was subjected twice to ice-cold ethanol and TCA precipitation steps to purify the EPS [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The EPS isolated from the above procedure was dialysed at 4\u0026deg;C using a 10 kDa cut-off dialysis membrane (HiMedia) against ultra-pure water. The water during the dialysis was changed 4 times at the interval of 12 h. After dialysis, the EPS concentrate was freeze-dried (Lyo lab, United States), and stored at 4\u0026deg;C until further analysis. The amount of total carbohydrate was determined using Antrone regent and protein by Pierce\u0026trade; bicinchoninic acid (BCA) kit (Thermo Fisher Scientific, United States), as per the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eBiofilm formation\u003c/h2\u003e \u003cp\u003eAn 18 h old bacterial cells were diluted in MRS broth supplemented with 0.05% (w/v) L-cysteine. Two hundred microliters (3.3 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e CFU/mL) were dispensed into 96-well plates (ThermoFisher) and incubated at 37 \u003csup\u003eo\u003c/sup\u003eC for 24 h. Planktonic cells, viable biofilm cells, and total biofilm mass were determined as per the procedure described by Ahire and Dicks [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eComplete genome sequence analysis\u003c/h2\u003e \u003cp\u003eFor the DNA extraction, a single colony of the strain was cultivated overnight in 10 mL of MRS broth supplemented with 0.05% (w/v) L-cysteine. The cells were harvested, and DNA was extracted using MO BIO\u0026rsquo;s genomic DNA extraction kit (Carlsbad, CA, USA). The quality and quantity of the extracted DNA were checked and measured on a 0.8% agarose gel and the Qubit dsDNA HS assay kit (ThermoFisher Scientific).\u003c/p\u003e \u003cp\u003eThe DNA fragmentation and library construction were done using the Nextera DNA Flex Library preparation kit (Illumina, San Diego, CA, USA), as per the manufacturer's instructions. After library construction, dual index adapters were ligated at the blunt ends of the DNA fragments, followed by the purification (as described by the manufacturer). The quality and quantity of the fragment library were estimated. The good quality library was normalised, pooled, and subsequently sequenced using 2 \u0026times; 250 bp chemistry on the Illumina MiSeq platform (Illumina Inc., USA). A 5% PhiX spike in was done during library loading in the sequencing cartage. After sequencing, the quality of paired-end raw reads was checked, and pre-processing sequences (i.e., primers and barcode trimming) was done by the NGS-QC tool to remove low-quality sequences (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.nipgr.res.in/ngsqctoolkit.html\u003c/span\u003e\u003cspan address=\"http://www.nipgr.res.in/ngsqctoolkit.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGenome assembly was performed by using SPAdes-3.11.1 assembler (St. Petersburg, Russia). The assembled genome quality was checked using QUAST, and the number of rRNAs was identified and retrieved using RNAmmer 1.2 (USA). The genome annotation of the organism was done using RAST, UniProt/SwissProt, and KEGG database. tRNA and rRNA genes, protein-encoding genes of the genomes, were predicted using tRNA_scan-SE, and RNAmmer, Glimmer version 3.02, respectively. The presence of CRISPR repeats was predicted using the CRISPR Finder tools [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The secondary metabolites were identified using AntiSMASH [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] and antibiotic resistance genes and virulence factors were determined by CARD [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] and VFDB [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. IslandViewer [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], CRISPR-Finder [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] were used to predict the presence of insertion sequences, and bacteriophage associated sequences (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The predicted 16S rRNA sequence was used in the EzBioCloud server [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] to find out the closely related group of organisms. Phylogenetic analysis was constructed using MEGA 7 software [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Based on the constructed phylogenetic tree, a number of genomes of the closely related organisms were downloaded from NCBI website and used for comparative genome analysis by BPGA tool.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eStrain and whole genome sequence deposition\u003c/h2\u003e \u003cp\u003e \u003cem\u003eLactobacillus delbrueckii\u003c/em\u003e subsp. \u003cem\u003eindicus\u003c/em\u003e DC-3 was deposited at National Centre for Microbial Resource, Pune, with accession number MCC4964. The whole-genome shotgun project has been deposited in GenBank under the following accession number JAOWCA000000000. The BioProject is available at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/bioproject/888254\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/bioproject/888254\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll the experiments were performed in triplicates and data are expressed as a mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). The GraphPad Prism (Version 10, USA) was used for the determination of statistical significance by one-way ANOVA (Tukey) and or \u003cem\u003et\u003c/em\u003e \u0026ndash;test. The \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered as statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003eIsolation of bacterial strain\u003c/h2\u003e \u003cp\u003eFermented milk and cereal batter yielded a number of colonies on MRS agar. Out of that, six colonies showed different morphologies and were designated as isolates DC-1 to 6. The isolates DC-3, 4, and 6 were catalase-negative, Gram\u0026rsquo;s positive, and non-motile rods. Owing to the better growth and dominant colony type on MRS agar, the fermented milk (\u003cem\u003eDahi\u003c/em\u003e) isolate DC-3 was selected and characterized further. On MRS agar, the DC-3 colony appeared whitish, 1\u0026ndash;2 mm in size, circular, smooth, and opaque with a convex elevation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). The isolate DC-3 was Gram-positive, non-spore-forming, rod-shaped bacteria with rounded ends (Fig.\u0026nbsp;1bc). The cells were on average 2\u0026ndash;6 \u0026micro;m long and 0.5\u0026ndash;0.7 \u0026micro;m wide and occurred singly, in pairs, and in short chains (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). DC-3 fermented glucose, fructose, and lactose without production of carbon dioxide, whereas it failed to ferment ribose, mannitol, inulin, starch, and glycogen.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003eSurvival under gastro-intestinal conditions\u003c/h2\u003e \u003cp\u003eThe survival of DC-3 was significantly (\u003cem\u003ep\u003c/em\u003e 0.001) reduced during the incubation in gastric juice. At the end of 1st h, 8.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 log\u003csub\u003e10\u003c/sub\u003e CFU/mL viable cells were decreased (\u003cem\u003ep\u003c/em\u003e 0.001) to 7.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 log\u003csub\u003e10\u003c/sub\u003e CFU/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Moreover, the viability was further reduced subsequently from 7.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 log\u003csub\u003e10\u003c/sub\u003e CFU/mL (1 h, \u003cem\u003ep\u003c/em\u003e 0.0001) to 6.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 log\u003csub\u003e10\u003c/sub\u003e CFU/mL (2 h, \u003cem\u003ep\u003c/em\u003e 0.0001) and 6.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 log\u003csub\u003e10\u003c/sub\u003e CFU/mL (3 h, \u003cem\u003ep\u003c/em\u003e 0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn intestinal juice, DC-3 viability was reduced (\u003cem\u003ep\u003c/em\u003e 0.0001) from 4.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 log\u003csub\u003e10\u003c/sub\u003e CFU/mL to 3.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 log\u003csub\u003e10\u003c/sub\u003e CFU/mL during the first 3 h of incubation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Later at the end of the 6th h, viability was further reduced (\u003cem\u003ep\u003c/em\u003e 0.001) to 3.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 log\u003csub\u003e10\u003c/sub\u003e CFU/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003eAdhesion potential\u003c/h2\u003e \u003cp\u003eDC-3 showed 42.51\u0026thinsp;\u0026plusmn;\u0026thinsp;1.56% autoaggregation at 1 h of incubation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). The adhesion to xylene, mucin and Caco-2 cells was recorded as 8.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75%, 0.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 OD units (crystal violet equivalence at 550 nm), and 8.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2% respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, d). The adhesion of DC-3 to Caco-2 cells and mucin is shown Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed. The membrane potential measured for DC-3 cells was \u0026minus;\u0026thinsp;4.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40 mV.\u003c/p\u003e \u003cdiv id=\"Sec33\" class=\"Section3\"\u003e \u003ch2\u003eSafety evaluations\u003c/h2\u003e \u003cp\u003eNo zone of hemolysis (γ-hemolytic) was observed when DC-3 was cultivated on blood agar for 48 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). On gelatin-nutrient agar plate, DC-3 failed to produce a clear zone of gelatin hydrolysis after the treatment of TCA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). In mucin degradation study, DC-3 cultivated on mucin agarose medium with or without glucose failed to produce discolored halo (mucin degradation) after the treatment of coomassie blue (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Moreover, in the presence of DC-3, Caco-2 cells exhibited significantly (\u003cem\u003ep\u003c/em\u003e 0.001) higher viability (76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16%) as compared to HepG-2 cells (71\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDC-3 showed sensitivity to the selected antibiotics of class β lactam, cephalosporin, macrolides, phenylpiperazines and member of quinolones class nalidixic acid (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). However, DC-3 was resistant to the selected antibiotics of the class aminoglycosides, coumarin glycosides, macrolactams, steroids / derivatives, triazole, and a member of quinolones class ofloxacin (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In MIC evaluation, the MIC values recorded for gentamicin, kanamycin, tetracycline, and vancomycin were higher than the MIC cut-off values provided by the European Food Safety Authority (EFSA) for obligate homofermentative \u003cem\u003eLactobacillus\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec34\" class=\"Section3\"\u003e \u003ch2\u003eAntimicrobial potential\u003c/h2\u003e \u003cp\u003eThe 48 h grown DC-3 cells (5\u0026ndash;6 mm spot) were inhibited the growth of all tested pathogens (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). The significantly (\u003cem\u003ep\u003c/em\u003e 0.045) higher zone of growth inhibition was recorded against \u003cem\u003eM. luteus\u003c/em\u003e MTCC 106\u003csup\u003eT\u003c/sup\u003e (11.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57 mm) as compared to \u003cem\u003eE. coli\u003c/em\u003e MTCC 1687 (10\u0026thinsp;\u0026plusmn;\u0026thinsp;0 mm), whereas growth inhibition differences recorded against \u003cem\u003eE. coli\u003c/em\u003e MTCC 1687 (10\u0026thinsp;\u0026plusmn;\u0026thinsp;0 mm), \u003cem\u003eP. mirabilis\u003c/em\u003e MTCC 425 (10.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 mm), and \u003cem\u003eC. albicans\u003c/em\u003e ATCC 14053 (10.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57 mm) remained statistically insignificant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). In bacteriocin screening, the neutralized supernatant of DC-3 was failed to inhibit the growth of \u003cem\u003eM. luteus\u003c/em\u003e MTCC 106\u003csup\u003eT\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDC-3 produced 10.5 g/L of \u003csub\u003eD\u003c/sub\u003e-lactic acid and 1.36 g/L of \u003csub\u003eL\u003c/sub\u003e-lactic acid after 24 h of incubation. Besides this, after exposure to air, DC-3 colonies showed intense blue color in less than 10 min (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eDC-3 cells co-aggregated significantly (\u003cem\u003ep\u003c/em\u003e 0.013) larger extent with \u003cem\u003eE. coli\u003c/em\u003e MTCC 1687 (5.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18%) and then \u003cem\u003eP. mirabilis\u003c/em\u003e MTCC 425 (4.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13%). The least co-aggregation was recorded with \u003cem\u003eC. albicans\u003c/em\u003e ATCC 14053 (1.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16%).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eExopolysaccharide (EPS) production\u003c/h3\u003e\n\u003cp\u003eAfter 48 h incubation on ruthenium red skim milk agar, DC-3 produced white (ropy/EPS producer) growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). Quantitatively, it was determined as 90 mg/L at 48 h incubation in MRS broth containing 2.0% glucose. The 1 mg EPS corresponded to 430.6\u0026thinsp;\u0026plusmn;\u0026thinsp;20.8 \u0026micro;g carbohydrate and 9.55\u0026thinsp;\u0026plusmn;\u0026thinsp;3.75 \u0026micro;g protein.\u003c/p\u003e\n\u003ch3\u003eBiofilm formation\u003c/h3\u003e\n\u003cp\u003eAfter 24 h of incubation, significantly (\u003cem\u003ep\u003c/em\u003e 0.022) higher optical density readings (0 h: 0.005\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002; 24 h: 0.058\u0026thinsp;\u0026plusmn;\u0026thinsp;0.015) were recorded for crystal violet staining of total biofilm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). The viable biofilm cells count (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg) was significantly (\u003cem\u003ep\u003c/em\u003e 0.028) increased from 0 h (3.3 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e) to 24 h (9.6 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e). Simultaneously, the optical densities of planktonic cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh) were significantly (\u003cem\u003ep\u003c/em\u003e 0.0001) increased from 0 h (0.048\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03) to 24 h (1.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07).\u003c/p\u003e \u003cdiv id=\"Sec37\" class=\"Section2\"\u003e \u003ch2\u003eComplete genome sequence analysis\u003c/h2\u003e \u003cp\u003eThe average quality score for base position in reads was recorded as normal (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The genome assembly statistics showed that the strain contained 3,145,837 bp, 3034 scaffolds with maximum scaffold length of 324, N50 length of 17330, and 56.73% G\u0026thinsp;+\u0026thinsp;C. The strain showed 99.19% average nucleotide identity with \u003cem\u003eLactobacillus delbrueckii\u003c/em\u003e subsp. \u003cem\u003eindicus\u003c/em\u003e DSM 15996. Similar results were observed with \u003cem\u003ein silico\u003c/em\u003e DNA-DNA genome level hybridization analysis (96.64%).\u003c/p\u003e \u003cp\u003eAfter obtaining a genome assembly of sufficient quality, the different types of genome elements were predicted and categorized as pathway genes (972), gene ontology (GO) term genes (378), hypothetical or uncharacterized genes (2223), putative genes (183), non-coding genes (111), tRNA (90), and rRNA (10) genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). The coding sequences were analysed further for their metabolic role and categorized into the 15 abundant functions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). The predicted coding sequences (CDS) were utilized to determine the protein clusters of orthologous groups (COGs) using the EggNOG web service (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNo antibiotic resistance genes (ARs) were detected using a strict criterion. However, using loose cut-off values, a total of 312 genes were predicted. Out of which, only more than 60% of identity records were considered (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Besides this, no virulence factor genes, mobile and insertion elements, and plasmids were detected or identified in strain DC-3. The AntiSMASH analysis identified the genes to produce lanthipeptide class III.\u003c/p\u003e \u003cp\u003ePhylogenetic analysis showed the close similarity of the strain DC-3 with \u003cem\u003eLactobacillus delbrueckii\u003c/em\u003e subsp. \u003cem\u003eindicus\u003c/em\u003e DSM 15996/JCM15610 (Fig. S3). In comparative genome analysis, the closest neighbours such as \u003cem\u003eL. delbrueckii\u003c/em\u003e subsp. \u003cem\u003eindicus\u003c/em\u003e, \u003cem\u003eL. equicursoris\u003c/em\u003e and \u003cem\u003eL. porci\u003c/em\u003e were selected based on the phylogenetic analysis as the priority and pruned down using genome availability status. The number of accessory genes and unique genes was higher in strain DC-3 as compared with selected organisms (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). The estimated pan-genome size of strain DC-3 was 4,777.86, and the parameter \u0026lsquo;b\u0026rsquo; was calculated to be 0.558297 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). The phylogeny of the core genome showed that strain DC-3 shares the close relatedness with \u003cem\u003eL. delbrueckii\u003c/em\u003e subsp. \u003cem\u003eindicus\u003c/em\u003e (Fig. S4).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, the details of CDS, tRNA, rRNA, tmRNA, repeat region, and CARD were given in circular genome map of strain DC-3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe traditional diversity in the preparation of fermented foods and substrates is crucial for their microbiota and unique compositions [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Most of the members of these microbial communities were capable of surviving gastrointestinal transit and helping to strengthen the gut microbiome [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Several studies have indicated that fermented foods are a good source of probiotic bacteria [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. However, isolation of probiotic bacteria from local and traditional fermented foods remained under-reported. In this study, we investigated probiotic attributes, adhesion ability, safety, antimicrobial potential, exopolysaccharide production, biofilm formation, and whole genome sequence analysis of fermented milk (\u003cem\u003eDahi\u003c/em\u003e) isolate \u003cem\u003eL. delbrueckii\u003c/em\u003e subsp. \u003cem\u003eindicus\u003c/em\u003e DC-3.\u003c/p\u003e \u003cp\u003eThe survival under gastro-intestinal conditions is one of the prerequisite properties for probiotic bacteria. In order survive under these conditions lactic acid bacteria stimulate the activity of F\u003csub\u003e0\u003c/sub\u003eF\u003csub\u003e1\u003c/sub\u003e ATP proton pumps to maintain cytoplasmic pH, produce alkaline compounds in the cell cytoplasm, modify the cell membrane integrity and fluidity, and upregulate the genes for amino acid decarboxylation and repair or protection of macromolecules [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In this study, strain DC-3 showed 83% viability for up to 3 hours in gastric juice and 71% viability for 6 hours in intestinal juice, which falls between a reasonable survival rate of 70\u0026thinsp;\u0026minus;\u0026thinsp;80% for \u003cem\u003ein vitro\u003c/em\u003e static experiments [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Studies suggest that the bacterial survival rate for gastro-intestinal conditions is largely dependent on exposure time and strain [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Moreover, the gastric and intestinal juice viability of strain DC-3 was comparatively higher (\u0026gt;\u0026thinsp;70%) than the gastric and intestinal juice viability (\u0026lt;\u0026thinsp;70%) reported for \u003cem\u003eL. delbrueckii\u003c/em\u003e subsp. \u003cem\u003eindicus\u003c/em\u003e CRL1447 [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAdhesion of probiotic candidates to the intestinal epithelial cells for colonization is crucial for inhibition of pathogens, nutrient absorption, and immunity [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. It has been suggested that probiotic bacteria interact with epithelial cells through electrostatic and hydrophobic interactions mediated through teichoic (TA) and lipoteichoic acid (LTA), environmental DNA (eDNA), polysaccharides, and complex polymers, or by producing bioactive metabolites [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. In this study, the higher percentage of DC-3 autoaggregation suggested the ability of the strain to interact with or recognize surface proteins, TA, LTA, organelles, eDNA, or exopolysaccharides (EPS) for colonization. Besides this, the DC-3 adhesion to the polar solvent xylene demonstrated hydrophobic cell surfaces. The negatively charged membrane potential indicated the presence of TA and LTA, a phosphate-rich cell surface glycolpolymer essential for the attachment of bacterial cells to epithelial cells [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Moreover, adhesion to mucin and CaCo-2 cells further confirmed the adhesion potential of the \u003cem\u003eL. delbrueckii\u003c/em\u003e subsp. \u003cem\u003eindicus\u003c/em\u003e DC-3, which is well coordinated with previous findings [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn order to establish safety, candidate probiotics must have data on genotypic and phenotypic identity, whole genome sequencing (WGS), studies on virulence, toxin production, and antibiotic resistance [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Besides this, when a candidate probiotic doesn\u0026rsquo;t have a history of safe use and is not listed in the qualified presumption of safety (QPS), extensive safety testing is required, including animal and human studies [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The strain DC-3 reported in this study was isolated from traditional fermented milk (\u003cem\u003eDahi\u003c/em\u003e), which has a long history of safe use. It doesn\u0026rsquo;t produce hemolysin to lyse red blood cells, gelatinase to break down extracellular matrix, and is incapable of degrading mucin, a core structural element of the mucosal surfaces of the digestive tract. The cytotoxic effect of DC-3 observed against hepato- and colon-carcinoma cells could be due to their anti-carcinogenic abilities. The MICs of ampicillin, chloramphenicol, clindamycin, and erythromycin observed against DC-3 were as per the cut-off values provided by EFSA [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. However, higher MICs of gentamycin, kanamycin, tetracycline, and vancomycin could not pose the risk of transmissible antibiotic resistance genes from food and feed, as it is an outcome of the intrinsic resistance of the \u003cem\u003eLactobacillus\u003c/em\u003e [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Similarly, resistance to selected antibiotics in the classes aminoglycosides, coumarin glycosides, macrolactams, steroids / derivatives, and ofloxacin, a quinolone, could be intrinsic, i.e., on the chromosome. These findings are well aligned with antibiotic resistance in probiotic microorganisms [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Moreover, the antibiotic- and antifungal (triazole)-resistant trait of the strain could be useful for its application during or along with antibiotic or antifungal agents. Overall, based on \u003cem\u003ein vitro\u003c/em\u003e safety and antibiotic susceptibility profiles, strain DC-3 might be regarded a safe strain for probiotic use.\u003c/p\u003e \u003cp\u003eThe antimicrobial activity of the probiotics is essential to eliminating pathogens, which are the main competitors for nutrients and colonization in the gut [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Studies indicated that probiotic bacteria are capable of producing lactic acid, acetic acid, diacetyl, bacteriocins and bacteriocin-like inhibitory substances (BLIS), hydrogen peroxide, and surface active molecules to inhibit the pathogens [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. In this investigation, strain DC-3 inhibited the growth of both Gram-positive and Gram-negative bacteria as well as yeast. Based on the zones of growth inhibition and the strains' inability to inhibit bacterial growth following neutralization of cell-free supernatant, it was determined that the action was attributable to lactic acid and/or hydrogen peroxide rather than bacteriocins. The production of a higher amount of lactic acid and hydrogen peroxide further confirmed the findings. Karnaouri et al. [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e] showed that, as starter cultures in dairy fermentation, \u003cem\u003eL. delbrueckii\u003c/em\u003e subsp. are efficient lactic acid producers. Similarly, the hydrogen peroxide production ability of \u003cem\u003eL. delbrueckii\u003c/em\u003e subsp. \u003cem\u003ebulgaricus\u003c/em\u003e was previously reported by Kot et al. [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBesides antimicrobial production, co-aggregation of probiotic bacteria with pathogens is one of the important properties of manipulating the aberrant intestinal microbiota [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. This property increases the colonization efficiency of probiotic bacteria. In this study, strain DC-3 showed co-aggregations with \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eP. mirabilis\u003c/em\u003e, and \u003cem\u003eC. albicans\u003c/em\u003e, indicating its ability to lower both bacteria and yeast colonization in the gut. The co-aggregation percentages were mostly dependent on strains and the time of the assay, and the results obtained in this study are well coordinated with previous findings [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe exopolysaccharide of lactic acid bacteria (LAB) is known to enhance their tolerance to harsh gastro-intestinal conditions and colonization in the gut [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. It is also reported for numerous other health benefits like immunomodulation, antiviral, anti-cancer, anti-inflammatory, anti-yeast, and putative antimicrobial activities [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. In this study, strain DC-3 showed its ability to produce EPS, which is important for its survival and colonization in the gut. The quantity of EPS reported in this study was comparatively lower than that of \u003cem\u003eL. delbrueckii\u003c/em\u003e ssp. \u003cem\u003eindicus\u003c/em\u003e WDS-7 [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. In addition to EPS production, strain DC-3 exhibited biofilm formation, which is considered an advantageous trait for efficient colonization. Studies have shown that LAB-biofilms enhance their antimicrobial capacity and could act as a biocontrol agent for pathogens and pathogenic biofilms [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. The strain DC-3 biofilm could be an ideal combination of a good probiotic strain and its antimicrobials (lactic acid and hydrogen peroxide) to inhibit invading pathogens.\u003c/p\u003e \u003cp\u003eTo date, regulatory standards for probiotic safety have been well established in Europe and some Asian countries [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. According to those standards, whole genome sequencing (WGS) is one of the requirements for establishing the safety of probiotic bacteria [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In this study, WGS of strain DC-3 isolated from traditional fermented milk (\u003cem\u003eDahi\u003c/em\u003e) showed a single circular chromosome (3,145,837 bp) with a GC content of 56.73% and was identified as \u003cem\u003eL. delbrueckii\u003c/em\u003e subsp. \u003cem\u003eindicus\u003c/em\u003e. The higher genomic GC content of the DC-3 than previously reported \u003cem\u003eL. delbrueckii\u003c/em\u003e subsp. \u003cem\u003eindicus\u003c/em\u003e JCM 15610\u003csup\u003eT\u003c/sup\u003e (49.4%) indicated high thermal stability [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Recently, Teng et al. [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e] showed that genomic GC has a considerable impact on the average amino acid characteristics of proteomes, including the N/C ratio and hydrophobicity, which may determine bacterial fitness. In comparative genome analysis with the closest neighbours (\u003cem\u003eL. delbrueckii\u003c/em\u003e subsp. \u003cem\u003eindicus\u003c/em\u003e, \u003cem\u003eL. equicursoris\u003c/em\u003e, and \u003cem\u003eL. porci\u003c/em\u003e), strain DC-3 showed a higher number of accessory and unique genes, which might be useful to enhance the strains' ability to adapt to their hosts or the surrounding environment [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Moreover, the open pan-genome of the DC-3 could indicate a probability of finding novel function genes. These results corroborate well with previous findings that the pan-genomes of most species of the Lactobacillaceae family are moderately open compared to other bacterial species [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. In coding sequence analysis, each of the 15% genes was found to contribute to amino acid and carbohydrate metabolism, 10% to protein metabolism, 9% to cofactors and vitamins, 5% to membrane transport, 3% to stress response, 2% to regulation of cell signaling, 1% to chemotaxis, etc. No virulence factor genes, mobile and insertion elements, or plasmids were detected or identified in DC-3, which indicated the strain\u0026rsquo;s safety. The antibiotic resistance gene profiling results were well coordinated with the \u003cem\u003ein vitro\u003c/em\u003e findings of the strain and further confirmed the strain's safety. Furthermore, the lanthipeptide class III identified in the WGS analysis was not detected \u003cem\u003ein vitro\u003c/em\u003e as, most of the class III lanthipeptides often lack antibacterial activities [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. \u003cem\u003eLactobacillus delbrueckii\u003c/em\u003e often possessed Lanthipeptide III and their conserved residues, i.e., lyase, kinase, and cyclase enzymes [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003e \u003cem\u003eLactobacillus delbrueckii\u003c/em\u003e subsp. \u003cem\u003eindicus\u003c/em\u003e DC-3 isolated from traditional Indian fermented milk (\u003cem\u003eDahi\u003c/em\u003e) showed good survival capabilities under \u003cem\u003ein vitro\u003c/em\u003e gastrointestinal stress conditions, adhesion, lactic acid and hydrogen peroxide-mediated antimicrobial activity, exopolysaccharide production, and biofilm formation. The absence of genes for transmissible antibiotic resistance, virulence factor, mobile and insertion elements, plasmids, \u003cem\u003ein vitro\u003c/em\u003e hemolysin and gelatinase production, and mucin degrading abilities confirmed the strain\u0026rsquo;s genotypic and phenotypic safety. In genotypic analysis, high GC content, the presence of a higher number of accessory and unique genes, and an open pan-genome exhibited thermal stability and strains' ability to adapt to their hosts or the surrounding environment. Overall, strain DC-3 isolated from traditional fermented milk (\u003cem\u003eDahi\u003c/em\u003e), which has a long history of safe use, is a safe probiotic candidate for further commercial applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors did not receive support from any organization for the submitted work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAhire JJ was employed by Dr. Reddy\u0026rsquo;s Laboratories Limited. Dr. Reddy\u0026rsquo;s Laboratories had no direct and indirect role in the design/analysis/writing/publication of this research article. Other authors have no conflict of interest to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data is included in the text, however, the raw data of this article will be made available by the authors, without undue reservation, to any qualified researcher.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: Chaudhari DN, Ahire JJ, Devkatte AN; Methodology: Chaudhari DN, Ahire JJ; Formal analysis and investigation: Chaudhari DN, Ahire JJ; Writing - original draft preparation: Chaudhari DN, Ahire JJ; Writing \u0026ndash; Chaudhari DN, Ahire JJ; Resources: Kulthe AS, Devkatte AN; Supervision: Ahire JJ, Devkatte AN.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study does not contain any work related with participation of humans and/or animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed consent\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLeeuwendaal NK, Stanton C, O\u0026rsquo;Toole PW, Beresford TP (2022) Fermented foods, health and the gut microbiome. 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Nat Commun 14(1):7650. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41467-023-43352-6\u003c/span\u003e\u003cspan address=\"10.1038/s41467-023-43352-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"probiotics-and-antimicrobial-proteins","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"paap","sideBox":"Learn more about [Probiotics and Antimicrobial Proteins](http://link.springer.com/journal/12601)","snPcode":"12602","submissionUrl":"https://submission.nature.com/new-submission/12602/3","title":"Probiotics and Antimicrobial Proteins","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Lactobacillus delbrueckii subsp. indicus DC-3, Probiotic, Safety, Fermented foods, Dahi, hydrogen peroxide","lastPublishedDoi":"10.21203/rs.3.rs-4487829/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4487829/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn this study, \u003cem\u003eLactobacillus delbrueckii\u003c/em\u003e subsp. \u003cem\u003eindicus\u003c/em\u003e DC-3 was isolated from Indian traditional indigenous fermented milk \u003cem\u003eDahi\u003c/em\u003e and identified using whole genome sequencing. The safety of the strain was evaluated using both genetic and phenotypic analyses, such as the presence of virulence factors, mobile and insertion elements, plasmids, antibiotic resistance, \u003cem\u003eetc\u003c/em\u003e. Besides this, the strain was comprehensively investigated for \u003cem\u003ein vitro\u003c/em\u003e probiotic traits, biofilm formation, antibacterials, and exopolysaccharide (EPS) production. In results, the strain showed a single circular chromosome (3,145,837 bp) with a GC content of 56.73%, a higher number of accessory and unique genes, an open pan-genome, and the absence of mobile and insertion elements, plasmids, virulence, and transmissible antibiotic resistance genes. The strain was capable of surviving in gastric juice (83% viability at 3 h) and intestinal juice (71% viability at 6 h) and showed 42.5% autoaggregation, adhesion to mucin, 8.7% adhesion to xylene, and 8.3% adhesion to Caco-2 cells. The γ-hemolytic nature, usual antibiotic susceptibility profile, and negative results for mucin and gelatin degradation ensure the safety of the strain. The strain produced 10.5 g/L of \u003csub\u003eD\u003c/sub\u003e-lactic acid and hydrogen peroxide, capable of inhibiting and co-aggregating \u003cem\u003eEscherichia coli\u003c/em\u003e MTCC 1687, \u003cem\u003eProteus mirabilis\u003c/em\u003e MTCC 425, and \u003cem\u003eCandida albicans\u003c/em\u003e ATCC 14053. In addition, the strain showed 90 mg/L EPS (48 h) and biofilm formation. In conclusion, this study demonstrates that \u003cem\u003eL. delbrueckii\u003c/em\u003e subsp. \u003cem\u003eindicus\u003c/em\u003e DC-3 is unique and different than previously reported \u003cem\u003eL. delbrueckii\u003c/em\u003e subsp. \u003cem\u003eindicus\u003c/em\u003e strains and is a safe potential probiotic candidate.\u003c/p\u003e","manuscriptTitle":"Complete genome sequence and probiotic characterization of Lactobacillus delbrueckii subsp. indicus DC-3 isolated from traditional indigenous fermented milk","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-11 09:29:40","doi":"10.21203/rs.3.rs-4487829/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-02T00:01:59+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-24T17:08:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-20T14:55:40+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-19T18:59:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"80701342752669435033336533079638932969","date":"2024-06-15T17:13:00+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-15T04:34:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"96613824619844961071739756492899148047","date":"2024-06-10T09:30:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"261652995303995215087936532470065540327","date":"2024-06-10T09:21:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"50300277839884958716548702220396102653","date":"2024-06-10T05:55:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"59417592175715887785401548414387446668","date":"2024-06-10T04:59:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"272218061229077257435612663208644526799","date":"2024-06-10T03:46:42+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-10T03:44:58+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-29T07:40:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-29T07:40:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Probiotics and Antimicrobial Proteins","date":"2024-05-28T03:29:04+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"probiotics-and-antimicrobial-proteins","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"paap","sideBox":"Learn more about [Probiotics and Antimicrobial Proteins](http://link.springer.com/journal/12601)","snPcode":"12602","submissionUrl":"https://submission.nature.com/new-submission/12602/3","title":"Probiotics and Antimicrobial Proteins","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"23033a38-15fd-43ca-8d23-a745d20beeca","owner":[],"postedDate":"June 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-10-21T15:59:08+00:00","versionOfRecord":{"articleIdentity":"rs-4487829","link":"https://doi.org/10.1007/s12602-024-10385-2","journal":{"identity":"probiotics-and-antimicrobial-proteins","isVorOnly":false,"title":"Probiotics and Antimicrobial Proteins"},"publishedOn":"2024-10-17 15:56:57","publishedOnDateReadable":"October 17th, 2024"},"versionCreatedAt":"2024-06-11 09:29:40","video":"","vorDoi":"10.1007/s12602-024-10385-2","vorDoiUrl":"https://doi.org/10.1007/s12602-024-10385-2","workflowStages":[]},"version":"v1","identity":"rs-4487829","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4487829","identity":"rs-4487829","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}