Assessment of Antimicrobial Activity and Selected Safety Attributes of Lactiplantibacillus plantarum and Pediococcus pentosaceus Isolates for Potential Probiotic Use

Assessment of Antimicrobial Activity and Selected Safety Attributes of Lactiplantibacillus plantarum and Pediococcus pentosaceus Isolates for Potential Probiotic Use | 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 Assessment of Antimicrobial Activity and Selected Safety Attributes of Lactiplantibacillus plantarum and Pediococcus pentosaceus Isolates for Potential Probiotic Use Hatice Ahu Kahraman, Melahat Deveci, Elif Naz Gürsel, Aleyna Gacar, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7120721/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 28 You are reading this latest preprint version Abstract The aim of this study is to investigate the some probiotic properties of isolated and identified six LABs (three of Lactiplantibacillus plantarum and three of Pediococcus pentosaceus) , including antibacterial potential against Salmonella Typhimurium (ATCC 14088), Pseudomonas aeruginosa (ATCC 27853), Enterococcus faecalis (ATCC 29212), and Klebsiella pneumoniae (ATCC 700603), their tolerance to low pH, pancreatin and bile salts, cell surface hydrophobicity, autoaggregation as well as determining the antibiotic resistance. While L. plantarum I-2 and P. pentosaceus I-4 showed the highest and lowest resistance in the presence of pepsin and bile salt, respectively, P. pentosaceus I-1 and P. pentosaceus I-4 exhibited the highest and lowest resistance in the presence of pancreatin. Isolates P. pentosaceus I1, L. plantarum I-3, and L. plantarum I-5 formed highest antibacterial activity against P. aeruginosa, S. Typhimurium and E. fecalis. All isolates were susceptible to Amoxicillin–Clavulanic acid and were resistant to Kanamycin. Isolate L. plantarum I-5 (73%) and L. plantarum I-2 (33%) demonstrated the highest hydrophobicity ratio after 24 hours in the presence of chloroform and n-hexane, respectively. The highest and lowest autoaggregation was observed in L. plantarum I-3 (21%) and P. pentosaceus I-4 (2.5%). In conclusion, P. pentosaceus I1, L. plantarum I-2, L. plantarum I-3, and L. plantarum I-5 had desirable in vitro probiotic properties and strong inhibitory activity against the tested pathogens, and they appear to be promising candidates for probiotic bacteria to be used in the food industry. Lactiplantibacillus plantarum Pediococcus pentosaceus Probiotic potential Safety attributes Figures Figure 1 Figure 2 Introduction Lactic acid bacteria (LAB) are generally described as Gram-positive, catalase-negative, facultative anaerobic, non-spore-forming cocci or rods that produce lactic acid as the main end product during carbohydrate fermentation [ 1 ]. Food-associated lactic acid bacteria include species from the genera Carnobacterium, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Oenococcus, Streptococcus, Tetragenococcus, Weissella , and Vagococcus [ 2 , 3 ]. LAB hold a significant place in the food industry due to their well-documented beneficial effects on human health. Probiotics are defined as live microorganisms that confer health benefits to the host when consumed in adequate amounts [ 4 ]. LAB hold an important place among microorganisms used as probiotics. LAB and Bifidobacteria are among the most commonly utilized probiotic bacteria. Today, probiotic bacteria are the subject of extensive research due to their beneficial effects, particularly in the prevention and treatment of common diseases [ 5 ]. Probiotic microorganisms are defined as live microbial cells that confer health benefits to the host when administered in adequate amounts [ 6 ]. Today, probiotic bacteria are widely studied for their beneficial effects on various common diseases. LAB are part of the natural microflora of food, while they can also be used as starters or probiotic cultures for fermented foods [ 7 ]. LAB produce lactic acid as a result of fermentation, and their byproducts contribute to significantly to the discrict flavor, aroma, and overall sensory profile of fermented food products. Moreover, many pathogenic bacteria are sensitive to various antimicrobial compounds produced by LAB, including hydrogen peroxide (H₂O₂), diacetyl, alcohol, carbon dioxide (CO₂), and bacteriocins. Owing to these properties LAB are commonly employed for the biocontrol of foodborne pathogens. These organisms have a wide range of applications, including nutrient production, disease treatment, and the production of macromolecules, enzymes, and metabolites [ 8 ]. Naturally fermented foods without added starter cultures serve as sources of natural probiotics [ 9 ]. Therefore, this study aims to evaluate to some probiotic properties of isolated and identified six LABs (three of Lactobacillus plantarum and three of Pediococcus pentosaceus ), including antibacterial potential against to pathogenic bacteria, their tolerance to low pH, pancreatin and bile salts, cell surface hydrophobicity, autoaggregation as well as determining the antibiotic resistance and to determine the resistance of these isolates to various environmental conditions and to identify the most promising probiotic candidate among them. Material and metod The six lactic acid bacteria (LAB) strains (three Pediococcus pentosaceus and three Lactobacillus plantarum) used in this study were previously isolated from raw milk, white cheese and Turkish sucuk [ 10 ]. Briefly, ten gram of each samples were homogenized with 90 mL peptone water (Merck, Darmstadt, Germany), and serial dilutions were prepared. A 100µL from each sample was spread onto MRS agar (Merck, Darmstadt, Germany), then incubated at 30°C for 48 h anaerobically. Then, colonies were identified colony morphology, gram staining and catalase tests. Gram-positive, and catalase-negative colonies were selected and stored in MRS broth with %15 glycerol at -20°C. Selected potential LAB colonies identified using Matrix Supported Laser Desorption/Ionization Flight Time Mass Spectrometry (MALDI-TOF MS) for species identification with the method previously described by Uysal et al. [ 11 ]. Then, six isolates identified using 16S rDNA gene sequencing following the method by Klindworth et al. [ 12 ] with using the 8F (5' AGAGTTTGATCMTGGCTCAG 3') and 1387R (5' GGGCGGWGTGTACAAGGC 3') primers. Isolates I-1, I-4, and I-6 were exhibited 100% similarity to the reference sequence of Pediococcus pentosaceus and registered to GenBank with the accession numbers PV345696, PV345694 and PV345692, respectively, while isolates I-2, I-3 and I-5 showed 100% similarity to the reference sequence of Lactiplantibacillus plantarum and registered to GenBank with accession numbers PV345698, PV345700 and PV345699, respectively. These identified isolates are given in Table 1 , and these were used in the following analyses for the study. Table 1 Isolates used in the study [ 10 ] Isolate Code Product MALDI-TOF MS Results 16 sRNA Sequencing Results GenBank No I-1 White cheese Pediococcus pentosaceus 091015_01 LBK Pediococcus pentosaceus PV345696 I-2 White cheese Lactobacillus plantarum DSM 20246 DSM Lactiplantibacillus plantarum PV345698 I-3 White cheese Lactobacillus plantarum DSM 2601 DSM Lactiplantibacillus plantarum PV345700 I-4 Raw milk Pediococcus pentosaceus AL908877 BK14924 UKH Pediococcus pentosaceus PV345694 I-5 Sucuk Lactobacillus plantarum DSM 1055 DSM Lactiplantibacillus plantarum PV345699 I-6 Sucuk Pediococcus pentosaceus DSM 20206 DSM Pediococcus pentosaceus PV345692 Determination of Probiotic Properties Hemolytic Activity Hemolytic activity of the isolates was evaluated on Tryptic Soy Agar (TSA; Difco) supplemented with 5% sheep blood, as described by Diguță et al. [ 13 ]. Each bacterial isolate was inoculated onto the surface of the agar using the streaking method, and the plates were incubated at 37°C for 48 hours. Tolerance to Low pH, Pancreatin, and Bile Salts The isolation of LAB was evaluated under low pH and simulated gastrointestinal conditions Low pH, pancreatin, and bile salts were used to simulate these conditions. Initially, overnight (18-hour) incubated cultures were centrifuged (4000 × g, 10 minutes, 4°C), and the pellets were washed twice with 0.9% saline solution. Bacterial cells were suspended in MRS broth and adjusted to pH 3 using 1 N hydrochloric acid. Simultaneously, bacteria were transferred to media containing 0.1% pepsin (pH 3, Sigma-Aldrich), 0.3% bile salts (pH 4.8, Oxgall, Thermo Fisher), and 0.1% pancreatin (pH 8, Sigma-Aldrich). Then, all samples were incubated anaerobically at 37°C for 3 hours. The number of viable bacterial cells (CFU/mL) was determined at the beginning and after 3 hours by inoculation on MRS agar. All tests were performed in triplicate [ 14 ]. Antibacterial Activity of Cell-Free Supernatants The antibacterial activity of LAB isolates was evaluated using cell-free culture supernatants by the agar well diffusion method, as described by Gharib [ 15 ]. The isolates were inoculated into MRS broth and incubated at 37°C for 48 hours. After the incubation, the cultures were centrifuged at 4000 × g for 10 minutes to separate the bacterial cells. The resulting supernatants were filtered through a 0.22 µm pore size filter to obtain fresh, sterilized, cell-free supernatants. Antibacterial activity was tested against four pathogenic bacteria: Salmonella Typhimurium (ATCC 14088), Pseudomonas aeruginosa (ATCC 27853), Enterococcus faecalis (ATCC 29212), and Klebsiella pneumoniae (ATCC 700603). Target bacteria were swabbed onto Mueller-Hinton agar plates, and then 100 µL of cell-free supernatant was added to wells punched into agar plates. The presence of a clear inhibition zone around the wells indicated antibacterial activity, whereas the absence of a zone indicated no inhibitory effect. The diameter of inhibition zones was measured using a digital caliper. Antibiotic Resistance The antibiotic resistance of LAB isolates was determined using the disk diffusion method according to Diguță et al. [ 13 ]. All isolates were adjusted to a turbidity of 0.5 McFarland standard and spread onto MRS agar plates using sterile swab. Antibiotic discs were then placed on the agar surface, and the plates were incubated at 37°C for 48 hours. The isolates were tested for their susceptibility to six antibiotics: Cefoxitin (30 µg), Danofloxacin (5 µg), Lincomycin (15 µg), Kanamycin (5 µg), Amoxicillin–Clavulanic acid (30 µg), and Neomycin (30 µg). After incubation, inhibition zones were measured using a digital caliper. The isolates were categorized based on the diameter of the inhibition zones as follows: Resistant (R): no inhibition zone; Semi-susceptible (SS): inhibition zone between 7–16 mm; Susceptile (S): inhibition zone > 16 mm [ 16 ]. Cell Surface Hydrophobicity The LAB isolates were evaluated for the hydrophobicity according to the method described by Rokana et al. [ 17 ]. Overnight cultures grown in MRS broth were centrifuged at 20,000 rpm for 15 minutes at 4°C. The resulting pellets were washed twice with PBS and resuspended in PBS buffer. The optical density (H₀) of the suspension was adjusted to 0.84–0.89 at 600 nm. Then, 1 mL of n-hexane or chloroform was added to 3 mL of the bacterial suspension, and the mixture was vortexed for 1 minute. After stabilization, the test tubes were incubated at 37°C for 1 hour. The aqueous phase was carefully collected, and its absorbance (H₁) was measured at 600 nm. The percentage of cell surface hydrophobicity was calculated using the following formula: $$\:\%\:Cell\:Surface\:Hydrophobicity\:=\:(1\:-\:H₁\:/\:H₀)\:\times\:\:100$$ Autoaggregation Assay To determine the level of autoaggregation, bacterial suspensions prepared as described above were adjusted to an optical density of 0.80 at 600 nm (OD₆₀₀). A 4 mL aliquot of each cell suspension was transferred into clean glass test tubes and incubated at 37°C for 24 hours under static conditions. After the incubation, 1 mL of the upper phase was carefully collected, and its absorbance (ODₜ) was measured at 600 nm. The percentage of autoaggregation was calculated using the following formula [ 18 ]: $$\:Autoaggregation\:\left(\%\right)\:=\:(1\:-\:ODₜ\:/\:ODᵢ)\:\times\:\:100\:$$ Statistical Analysis All experiments were performed in triplicate, and the results were expressed as mean (M) ± standard deviation (SD). The paired t-test was used to determine differences (p < 0.05). Results Determination of Probiotic Properties The hemolytic activity of the strains was tested on blood agar plates supplemented with 5% sheep blood. None of the isolates exhibited β-hemolysis, indicating that none of the tested LAB strains demonstrated pathogenicity. Tolerance and resistance to low pH, pancreatin, and bile salts are essential for the survival of probiotic LAB in the intestinal tract [ 19 ]. Therefore, tolerance tests are widely used as preliminary screening tools for identifying potential probiotic strains [ 20 ]. In this study, the survival rates of LAB isolates were evaluated under the following conditions: 0.1% pepsin (pH 3), 0.1% pancreatin (pH 8) and 0.3% bile salts (pH 8). The variation in the survival rates of LAB strains under these stress conditions is presented in Table 2 . Table 2 Survival of LAB ısolates under simulated gastrointestinal conditions (log₁₀ CFU/m) 0.1% Pepsin 0.1% Pancreatin 0.3% Bile Salts Isolate Strains Initial 3 h Initial 3 h Initial 3 h I-1 Pediococcus pentosaceus I-1 5.73 ± 0.06 5.91 ± 0.01 ab 6.04 ± 0.06 7.75 ± 0.16 ab 5.52 ± 0.23 5.97 ± 0.03 ab I-2 Lactobacillus plantarum I-2 6.21 ± 0.13 6.91 ± 0.13 a 6.64 ± 0.64 6.55 ± 0.51 a 6.27 ± 0.32 7.60 ± 0.06 a I-3 Lactobacillus plantarum I-3 5.90 ± 0.02 6.06 ± 0.03 b 6.18 ± 0.02 6.27 ± 0.04 ab 6.51 ± 0.42 5.49 ± 0.01 bc I-4 Pediococcus pentosaceus I-4 5.91 ± 0.01 5.87 ± 0.33 ab 5.92 ± 0.09 5.70 ± 0.11 cd 5.89 ± 0.01 5.50 ± 0.03 bc I-5 Lactobacillus plantarum I-5 6.08 ± 0.12 5.90 ± 0.01 ab 6.27 ± 0.11 6.17 ± 0.09 bcd 5.98 ± 0.14 6.62 ± 0.03 b I-6 Pediococcus pentosaceus I-6 5.83 ± 0.01 5.62 ± 0.02 c 6.30 ± 0.49 5.64 ± 0.05 c 5.49 ± 0.15 4.99 ± 0.83 c Results are expressed as mean ± standard deviation (SD). Different superscript letters (a–d) indicate statistically significant differences between groups at the same time point (p < 0.05). Survival of LAB isolates under low pH conditions is critically important for their resistance to gastric acid. The initial bacterial load was adjusted to 10 6 CFU/mL in all samples (Table 2 ). After 3 hours of exposure to 0.1% pepsin (pH 3), the highest survival rate was observed in L. plantarum I-2 (6.91 log₁₀ CFU/mL) and followed by L. plantarum I-3, while the lowest was recorded in isolate P. pentosaceus I-6 (5.62 log₁₀ CFU/mL) (p < 0.05). The highest resistance was determined for isolate P. pentosaceus I-1 for pancreatin (7.75log₁₀ CFU/mL) and followed by I-2 (6.55 log₁₀ CFU/mL). The lowest resistant was determined in P. pentosaceus I-4 (5.70 log₁₀ CFU/mL) and P. pentosaceus I-6 (5.64 log₁₀ CFU/mL). The isolates were tested at 0.3% bile salt concentration to reflects physiological levels in the human gastrointestinal tract. All isolates exhibited survival levels ranging from 4.99 to 7.60 log₁₀ CFU/mL under bile salt exposure. After 3 hours of exposure, the highest viability was observed in isolate L. plantarum I-2 (7.60 log₁₀ CFU/mL), while the lowest was detected in isolate P. pentosaceus I-6 (4.99 log₁₀ CFU/mL). Antimicrobial Activity The diameters of inhibition zones formed by the cell-free supernatants of LAB isolates against Enterococcus faecalis, Pseudomonas aeruginosa, Klebsiella pneumoniae , and Salmonella Typhimurium are presented in Table 3 . Enrofloxacin (40 µg/mL) was used as a positive control for the antimicrobial evaluation. The LAB isolates exhibited varying levels of antimicrobial activity. All isolates demonstrated inhibition zones against E. faecalis , with ranging from 11 to 12 mm, and against P. aeruginosa , with zones of 8 to 10 mm. Isolates L. plantarum I-2, L. plantarum I-3, and L. plantarum I-5 formed 8 mm inhibition zones against S . Typhimurium, whereas the remaining isolates ( P. pentosaceus I-1, P. pentosaceus I-4, and P. pentosaceus I-6) showed no inhibitory effect. Also, none of the isolates showed antimicrobial activity against K. pneumoniae . Table 3 Antimicrobial activity of cell-free supernatants against some pathogenic microorganisms Pseudomonas aeruginosa Klepsiella pneumonia Salmonella Typhimurium Enterococcus faecalis Pediococcus pentosaceus I-1 8 NI NI 11 Lactobacillus plantarum I-2 10 NI 8 12 Lactobacillus plantarum I-3 10 NI 8 12 Pediococcus pentosaceus I-4 9 NI NI 11 Lactobacillus plantarum I-5 9 NI 8 12 Pediococcus pentosaceus I-6 9 NI NI 11 dwell = 6 mm, NI- no inhibition Antibiotic Susceptibility of the Isolates The antibiotic susceptibility of the LAB isolates was evaluated using the disc diffusion method on MRS agar and results are shown in Table 4 . With a few exceptions, the isolates exhibited similar resistance profiles. All isolates were resistant to Kanamycin (5 µg) and susceptible to Amoxicillin–Clavulanic acid (30 µg). Excluding L. plantarum I-3, all isolates were susceptible to Lincomycin (15 µg) and except L. plantarum I-5, the other isolates were semi-susceptible to Cefoxitin (30 µg) and resistant to Danofloxacin (5 µg). Isolates P. pentosaceus I-1, P. pentosaceus I-4, and P. pentosaceus I-6 were resistant to Neomycin (30 µg), while L. plantarum I-2, L. plantarum I-3, and L. plantarum I-5 were found to be semi-susceptible. Table 4 Antibiotic Susceptibility Profiles of LAB Isolates Isolate Cefoxitin (30µg) Danofloxacin (5µg) Lincomycin (15 µg) Kanamycin (5µg) Amoxyclin Clavulonic acid (30µg) Neomycin (30µg) Pediococcus pentosaceus I-1 SR R S R S R Lactobacillus plantarum I-2 SR R S R S SR Lactobacillus plantarum I-3 SR R SR R S SR Pediococcus pentosaceus I-4 SR R S R S R Lactobacillus plantarum I-5 S SR S R S SR Pediococcus pentosaceus I-6 SR R S R S R R – Resistant; SR – Intermediate (zone diameter 7–16 mm); S – Sensitive (zone diameter > 16 mm); disc diameter = 6 mm Hydrophobicity and Autoaggregation of LAB Isolates The surface hydrophobicity of the isolates was assessed using two different solvents: n-hexane and chloroform. The results are presented in Fig. 1 . Isolate L. plantarum I-5 demonstrated the highest hydrophobicity ratio after 24 hours in the presence of chloroform, and followed by L. plantarum I-3 and P. pentosaceus I-6. In the presence of n-hexane, isolate L. plantarum I-2 showed the highest hydrophobicity, while the lowest values were observed in P. pentosaceus I-4 (chloroform) and P. pentosaceus I-1 (n-hexane), respectively. Autoaggregation rates of the isolates are given in Fig. 2 . The highest autoaggregation was observed in L. plantarum I-3, followed by L. plantarum I-5, P. pentosaceus I-1, L. plantarum I-2, and P. pentosaceus I-6. The lowest autoaggregation value was recorded in isolate P. pentosaceus I-4. Discussion Lactic acid bacteria (LAB), widely recognized as probiotics in the food industry, have attracted significant attention due to their health-promoting effects. Accordingly, there is growing interest in isolating and evaluating potential probiotic LAB strains from various food [ 21 ]. In the present study, six LAB isolates which were previously isolated and identified by 16 S rRNA gene sequencing evaluated the probiotic potential by examining their hemolytic activity, tolerance in the gastrointestinal tract, antimicrobial properties, antibiotic resistance, hydrophobicity, and autoaggregation. The ability of probiotic strains to survive and grow under low pH and in the presence of bile salts is considered a desirable characteristic [ 22 ]. Probiotic strains must resist low pH, bile salts, and pancreatic enzymes to survive passage through the upper gastrointestinal tract [ 23 ]. In this study, all isolates remained viable after 3 hours of incubation under simulated gastrointestinal conditions, (low pH, pancreatin, and bile salts) indicating strong tolerance to gastric and intestinal environments. L. plantarum I-2 has the highest survival rates of the pepsin, pancreatin and bile salts condition. The resistance of Lactobacillus species to bile salts is largely attributed to the presence of the bsh-1 and bsh-2 genes, which encode bile salt hydrolase enzymes that hydrolyze bile salts [ 24 ]. Similar results have been seen with other L. plantarum strains, known for their robust resistance to tough gastrointestinal conditions [ 19 ]. This aligns with previous studies highlighting the strong acid resistance of L. plantarum strains [ 25 ]. Similar findings regarding the probiotic properties of LAB have been reported in other studies, consistent with the results of the present study [ 24 , 26 ]. The resistance to pancreatin, which simulates intestinal protease activity, varied among the isolates. P. pentosaceus I-1 showed the highest survival rate under pancreatin condition. In contrast, strains P. pentosaceus I-4 and P. pentosaceus I-6 had reduced survival, likely due to sensitivity to enzymes or differences in cell wall composition. When exposed to 0.3% bile salts, which represent a normal concentration of bile in the intestine, all isolates showed varying levels of tolerance. L. plantarum I-2 demonstrated the highest tolerance, suggesting better adaptability to intestinal conditions, On the other hand, P. pentosaceus I-6 showed the lowest resistance, which may indicate a susceptibility of the membrane to bile-induced destabilization [ 27 ]. LAB are capable of inhibiting pathogen colonization and growth in the gastrointestinal tract. This inhibitory effect may be due to the production of antimicrobial agents or by preventing pathogen adhesion to the intestinal mucosa [ 19 , 28 ]. In the present study, LAB isolates were tested against major foodborne pathogens, including Enterococcus faecalis, Pseudomonas aeruginosa , Salmonella Typhimurium , and Klebsiella pneumonia . The isolates exhibited varying degrees of inhibition zones against E. faecalis and P. aeruginosa while only L. plantarum strains (I-2, I-3, I-5) inhibited Salmonella Typhimurium and none of the isolates showed inhibitory effect on K. pneumoniae . The lack of antibacterial activity against K. pneumoniae suggests the antimicrobial effects are specific to certain pathogens and might relate to differences in cell wall structure or sensitivity to LAB-derived substances [ 29 ]. Similarly, Fossi and Ndjouenkeu identified thermotolerant LAB strains ( L. plantarum and L. acidophilus ). These selected strains showed strong antimicrobial activity against major foodborne pathogens such as Salmonella Enteritidis , Salmonella Typhimurium, Escherichia coli, Staphylococcus aureus , and Listeria monocytogenes , indicating their potential for use in industrial probiotic production. Previous studies on the antibacterial effects of LAB strains are consistent with our findings, confirming that the isolated strains exhibit strong antagonistic activity against the selected pathogens [ 30 , 31 , 32 , 33 ]. Examining the antibiotic resistance profile of potential probiotic strains is a crucial step, as the a risk of transferring resistance genes to the gut microbiota [ 34 ]. All isolates were found resistant to kanamycin, which is common for LAB due to their intrinsic resistance due to low membrane permeability and the presence of aminoglycoside-modifying enzymes [ 35 , 36 ]. Many Lactobacillus species are naturally resistant to aminoglycosides (gentamicin, kanamycin, streptomycin, and neomycin), ciprofloxacin, and trimethoprim, while they are generally sensitive to penicillin and β-lactam antibiotics, as well as chloramphenicol, tetracycline, erythromycin, linezolid, and quinupristin-dalfopristin [ 37 ]. In a recent study, Duche et al. [ 38 ] evaluated the antibiotic resistance profiles of LAB strains. The resistance rates were approximately 100% for ceftazidime, cefoxitin, kanamycin, nalidixic acid, vancomycin, teicoplanin, methicillin, and norfloxacin. Resistance to cefmetazole, polymyxin B, tobramycin, and moxifloxacin ranged from 91.2–97.2%. For ciprofloxacin, gentamicin, fusidic acid, and gatifloxacin, resistance rates were 76.5%, 76.5%, 79.4%, and 82.4%, respectively. Intermediate resistance was observed against penicillin G (52.9%) and clindamycin (58.5%). In contrast, high sensitivity (70.6–100%) was recorded for amoxicillin-clavulanate (amoxiclav), tetracycline, tigecycline, meropenem, imipenem, trimethoprim, co-trimoxazole, and azithromycin. However, the susceptibility to amoxicillin-clavulanic acid and lincomycin (except L. plantarum I-3), indicating a low risk of contributing to the spread of antibiotic resistance and suggesting positive safety profiles. The differing reactions to neomycin and danofloxacin reveal strain-specific variations in resistance patterns. Intermediate resistance to cefoxitin and neomycin in particular Pediococcus strains points to the need for further genetic testing to confirm the absence of transferable resistance genes [ 39 ]. Cell surface hydrophobicity is an important trait that facilitates the adhesion of probiotic bacteria to epithelial cells and promotes colonization [ 26 ]. In the present study, the hydrophobicity of the isolates was evaluated in the presence of two different solvents. Among all tested isolates, the highest hydrophobicity was observed in L. plantarum I-5 in chloroform, while I-2 showed a strong affinity for n-hexane. This probably suggests potential differences in cell wall composition, especially between protein and lipid structures [ 18 ]. Similarly, studies by Coulibaly et al. [ 40 ] and Rokana et al. [ 17 ] also reported the highest hydrophobicity when chloroform was used. Autoaggregation is another key factor contributing to adhesion and colonization on epithelial surfaces and is often used to assess the probiotic potential of strains [ 41 ]. According to Kang et al. [ 42 ] and Kim et al. [ 43 ], autoaggregation levels can be classified as low (16–35%), moderate (35–50%), or high (≥ 50%). After 24 hours of incubation, the highest autoaggregation level was observed in L. plantarum I3, with a value of 22%, which falls within the low aggregation category. High autoaggregation in L. plantarum I-3 and L. plantarum I-5 may support their colonization and survival in the gastrointestinal mucosa, by the way of the competitive exclusion of pathogens [ 44 ]. In contrast, P. pentosaceus I-4's low autoaggregation could reduce its probiotic potential [ 34 ]. In contrast, P. pentosaceus I-4 has minimal hydrophobicity and autoaggregation, which may limit its ability to adhere to mucosal surfaces. Conclusion Based on the results obtained, P. pentosaceus I-1, L. plantarum I-2, L. plantarum I-3, and L. plantarum I-5, demonstrated the most promising probiotic traits, including strong gastrointestinal tolerance, antimicrobial activity, and favorable cell surface characteristics. The variability seen between strains highlights the importance of thorough phenotypic and genotypic evaluation in probiotic screening. However, further studies, including in vivo trials, are required to confirm their health-promoting effects and to investigate their potential application in food systems. Declarations Competing Interests and Funding The authors declare no potential conflicts of interest. This research was supported by TUBİTAK 2209A Student Scientific Research Grant (grant no. 1919B012220737). Authorship Contributions HAK designed the experiments. HAK, MD, ENG, AG and NÖ carried out the analyses. HAK contributed to interpreting the results and took the lead in writing the manuscript. Both authors provided critical feedback and helped shape the research, analysis, and manuscript. Funding This research was supported by TUBİTAK 2209A Student Scientific Research Grant (grant no. 1919B012220737). Data Availability The data supporting this study's findings are available from the corresponding author upon reasonable request. Conflicts of Interest The authors declare no potential conflicts of interest. Ethical Approval This study did not involve animal or human subjects. References Axelsson L (2004) In: Salminen S, Von Wright A (eds) Lactic acid bacteria: microbiological and functional aspects, 3rd edn. CRC, Boca Raton Vandamme P, Pot B, Gillis M, De Vos P, Kersters K, Swings J (1996) Polyphasic taxonomy, a consensus approach to bacterial systematics. 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Probiotics Antimicrob proteins 2:162–174. http://doi.org/10.1007/s12602-010-9050-7 Adetoye A, Pinloche E, Adeniyi BA, Ayeni FA (2018) Characterization and anti-salmonella activities of lactic acid bacteria isolated from cattle faeces. BMC Microbiol 18:1–11. https://doi.org/10.1186/s12866-018-1248-y Zhao H, Liu L, Peng S, Yuan L, Li H, Wang H (2019) Heterologous expression of argininosuccinate synthase from Oenococcus oeni enhances the acid resistance of Lactobacillus plantarum. Front Microbiol 10:1393. https://doi.org/10.3389/fmicb.2019.01393 Krausova G, Hyrslova I, Hynstova I (2019) In vitro evaluation of adhesion capacity, hydrophobicity, and auto-aggregation of newly isolated potential probiotic strains. Fermentation 5(4):100. https://doi.org/10.3390/fermentation5040100 Begley M, Gahan CG, Hill C (2005) The interaction between bacteria and bile. 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Probiotics Antimicrob Proteins 13(4):957–969. https://doi.org/10.1007/s12602-021-09754-y Pavli F, Argyri A, Papadopoulou O, Nychas G, Chorianopoulos N, Tassou C (2016) Probiotic potential of lactic acid bacteria from traditional fermented dairy and meat products: Assessment by in vitro tests and molecular characterization. J Prob Health 4(157):10–4172 Silva BC, Jung LRC, Sandes SHC, Alvim LB, Bomfim MRQ, Nicoli JR, Nunes AC (2013) In vitro assessment of functional properties of lactic acid bacteria isolated from faecal microbiota of healthy dogs for potential use as probiotics. Beneficial Microbes 4(3):267–276. https://doi.org/10.3920/BM2012.0048 Zommiti M, Bouffartigues E, Maillot O, Barreau M, Szunerits S, Sebei K, Ferchichi M (2018) In vitro assessment of the probiotic properties and bacteriocinogenic potential of Pediococcus pentosaceus MZF16 isolated from artisanal Tunisian meat Dried Ossban. Front Microbiol 9:2607. https://doi.org/10.3389/fmicb.2018.02607 Mathur S, Singh R (2005) Antibiotic resistance in food lactic acid bacteria–a review. Int J Food Microbiol 105(3):281–295. https://doi.org/10.1016/j.ijfoodmicro.2005.03.008 Cao Z, Pan H, Li S, Shi C, Wang S, Wang F, Zhao Z (2019) In vitro evaluation of probiotic potential of lactic acid bacteria isolated from Yunnan De’ang pickled tea. Probiotics Antimicrob proteins 11:103–112. https://doi.org/10.1007/s12602-018-9395-x Abriouel H, Casado Muñoz MDC, Lavilla Lerma L, Pérez Montoro B, Bockelmann W, Pichner R, Kabisch J, Cho G-S, Franz CMAP, Gálvez A, Benomar N (2015) New insights in antibiotic resistance of Lactobacillus species from fermented foods. Food Res Int 78:465–481. https://doi.org/10.1016/j.foodres.2015.09.016 Duche RT, Singh A, Wandhare AG, Sangwan V, Sihag MK, Nwagu TN, Ezeogu LI (2023) Antibiotic resistance in potential probiotic lactic acid bacteria of fermented foods and human origin from Nigeria. BMC Microbiol 23(1):142. https://doi.org/10.1186/s12866-023-02883-0 EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) (2012) Guidance on the assessment of bacterial susceptibility to antimicrobials of human and veterinary importance. EFSA J 10(6):2740. https://doi.org/10.2903/j.efsa.2012.2740 Coulibaly WH, Camara F, Diguta C, Matei F (2022) Probiotic and functional properties potential of lactic acid bacteria isolated from Tilapia (Oreochromis niloticus) in Ivory Coast. https://doi.org/10.21203/rs.3.rs-1481094/v1 Trunk T, Khalil HS, Leo JC (2018) Bacterial autoaggregation. AIMS Microbiol 4(1):140. https://doi.org/10.3934/microbiol.2018.1.140 Kang CH, Han SH, Kim Y, Jeong Y, Paek NS (2017) Antibacterial activity and probiotic properties of lactic acid bacteria isolated from traditional fermented foods. KSBB J 32(3):199–205. https://doi.org/10.7841/ksbbj.2017.32.3.199 Kim Y, Choi SI, Jeong Y, Kang CH (2022) Evaluation of Safety and Probiotic Potential of Enterococcus faecalis MG5206 and Enterococcus faecium MG5232 Isolated from Kimchi, a Korean Fermented Cabbage. Microorganisms 10(10):2070. https://doi.org/10.3390/microorganisms10102070 Del Re B, Sgorbati B, Miglioli M, Palenzona D (2000) Adhesion, autoaggregation and hydrophobicity of 13 strains of Bifidobacterium longum. Lett Appl Microbiol 31(6):438–442. https://doi.org/10.1046/j.1365-2672.2000.00845.x Additional Declarations No competing interests reported. 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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-7120721","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":487081082,"identity":"6f3f7d25-b0d8-4774-8226-2521fe3d0ece","order_by":0,"name":"Hatice Ahu Kahraman","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABJ0lEQVRIiWNgGAWjYBACAwYGNhDN2MDAfABEMoAEDlQwQNn4tbAlgJXxgLScgRhCSAuPAVwLAz4t5gzMzx7zVNyT7Rc783XDxx3b5OzZjyUeOMBgI7vhAP+xD1i0WDawmRvznCk2njk7d9vNmWduG/PwpB0Aakkz3nCAmXkGNocdYDCT5m1LSNxwO3fbbd6224k9DOkNhz8wHE4EacHqlwPs36R5/yUk7r+d8+z237bb9T38zxuAtvzHo4UHaEsD0BbpHLbbjG23E3gkwA47gFvLYZ4yyTnHEoxn3E4zu9nbdtuw58azhAMHDJKNZx5mNsaq5Xj7Nok3NQmy/bOTn9342XZbnr0/zfjDgQo72b7jjY+xhjL2sAfFFy6pUTAKRsEoGAWEAQABFnGFXT3WowAAAABJRU5ErkJggg==","orcid":"","institution":"University of Burdur Mehmet Akif Ersoy","correspondingAuthor":true,"prefix":"","firstName":"Hatice","middleName":"Ahu","lastName":"Kahraman","suffix":""},{"id":487081083,"identity":"21b61d0a-37b3-4623-9817-fd54602cbc38","order_by":1,"name":"Melahat Deveci","email":"","orcid":"","institution":"University of Burdur Mehmet Akif Ersoy","correspondingAuthor":false,"prefix":"","firstName":"Melahat","middleName":"","lastName":"Deveci","suffix":""},{"id":487081084,"identity":"ca34a09d-77d3-42d9-98ef-6f36624ec5ca","order_by":2,"name":"Elif Naz Gürsel","email":"","orcid":"","institution":"University of Burdur Mehmet Akif Ersoy","correspondingAuthor":false,"prefix":"","firstName":"Elif","middleName":"Naz","lastName":"Gürsel","suffix":""},{"id":487081085,"identity":"ec474144-5786-400a-aea9-00305007e330","order_by":3,"name":"Aleyna Gacar","email":"","orcid":"","institution":"University of Burdur Mehmet Akif Ersoy","correspondingAuthor":false,"prefix":"","firstName":"Aleyna","middleName":"","lastName":"Gacar","suffix":""},{"id":487081086,"identity":"75681720-a96f-4de9-b919-cbe6153e81c1","order_by":4,"name":"Neslihan Öztürk","email":"","orcid":"","institution":"University of Burdur Mehmet Akif Ersoy","correspondingAuthor":false,"prefix":"","firstName":"Neslihan","middleName":"","lastName":"Öztürk","suffix":""}],"badges":[],"createdAt":"2025-07-14 11:38:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7120721/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7120721/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87039113,"identity":"9000a79c-91cb-4ad7-bc13-54fe07420037","added_by":"auto","created_at":"2025-07-18 13:36:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":23635,"visible":true,"origin":"","legend":"\u003cp\u003eHydrophobicity Levels (%) of the Isolates After 24 Hours. I-1:\u003cem\u003ePediococcus pentosaceus\u003c/em\u003eI-1, I-2: \u003cem\u003eLactobacillus plantarum\u003c/em\u003eI-2, I-3:\u003cem\u003eL. plantarum\u003c/em\u003e I-3, I-4:\u003cem\u003eP. pentosaceus\u003c/em\u003e I-4, I-5:\u003cem\u003eL. plantarum\u003c/em\u003e I-5, I-6:\u003cem\u003eP. pentosaceus\u003c/em\u003e I-6\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7120721/v1/6532116629ae8eddacba9344.png"},{"id":87039084,"identity":"8c6ff483-e524-4976-8ce2-19b4d6643354","added_by":"auto","created_at":"2025-07-18 13:36:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":21801,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of aggregation levels (%) of the ısolates. I-1:\u003cem\u003ePediococcus pentosaceus\u003c/em\u003e I-1, I-2: \u003cem\u003eLactobacillus plantarum\u003c/em\u003e I-2, I-3:\u003cem\u003eL. plantarum\u003c/em\u003e I-3, I-4:\u003cem\u003eP. pentosaceus\u003c/em\u003e I-4, I-5:\u003cem\u003eL. plantarum\u003c/em\u003e I-5, I-6:\u003cem\u003eP. pentosaceus\u003c/em\u003e I-6\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7120721/v1/fc80062cf67c14ad961d2e3f.png"},{"id":87040588,"identity":"f5b68c99-b6c0-43de-a2b9-b39e2a460167","added_by":"auto","created_at":"2025-07-18 13:52:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1012206,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7120721/v1/10eb8c52-81eb-4d49-9c54-3d8964640a81.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Assessment of Antimicrobial Activity and Selected Safety Attributes of Lactiplantibacillus plantarum and Pediococcus pentosaceus Isolates for Potential Probiotic Use","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLactic acid bacteria (LAB) are generally described as Gram-positive, catalase-negative, facultative anaerobic, non-spore-forming cocci or rods that produce lactic acid as the main end product during carbohydrate fermentation [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Food-associated lactic acid bacteria include species from the genera \u003cem\u003eCarnobacterium, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Oenococcus, Streptococcus, Tetragenococcus, Weissella\u003c/em\u003e, and \u003cem\u003eVagococcus\u003c/em\u003e [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. LAB hold a significant place in the food industry due to their well-documented beneficial effects on human health.\u003c/p\u003e\u003cp\u003eProbiotics are defined as live microorganisms that confer health benefits to the host when consumed in adequate amounts [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. LAB hold an important place among microorganisms used as probiotics. LAB and \u003cem\u003eBifidobacteria\u003c/em\u003e are among the most commonly utilized probiotic bacteria. Today, probiotic bacteria are the subject of extensive research due to their beneficial effects, particularly in the prevention and treatment of common diseases [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Probiotic microorganisms are defined as live microbial cells that confer health benefits to the host when administered in adequate amounts [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Today, probiotic bacteria are widely studied for their beneficial effects on various common diseases. LAB are part of the natural microflora of food, while they can also be used as starters or probiotic cultures for fermented foods [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. LAB produce lactic acid as a result of fermentation, and their byproducts contribute to significantly to the discrict flavor, aroma, and overall sensory profile of fermented food products. Moreover, many pathogenic bacteria are sensitive to various antimicrobial compounds produced by LAB, including hydrogen peroxide (H₂O₂), diacetyl, alcohol, carbon dioxide (CO₂), and bacteriocins. Owing to these properties LAB are commonly employed for the biocontrol of foodborne pathogens. These organisms have a wide range of applications, including nutrient production, disease treatment, and the production of macromolecules, enzymes, and metabolites [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eNaturally fermented foods without added starter cultures serve as sources of natural probiotics [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Therefore, this study aims to evaluate to some probiotic properties of isolated and identified six LABs (three of \u003cem\u003eLactobacillus plantarum\u003c/em\u003e and three of \u003cem\u003ePediococcus pentosaceus\u003c/em\u003e), including antibacterial potential against to pathogenic bacteria, their tolerance to low pH, pancreatin and bile salts, cell surface hydrophobicity, autoaggregation as well as determining the antibiotic resistance and to determine the resistance of these isolates to various environmental conditions and to identify the most promising probiotic candidate among them.\u003c/p\u003e"},{"header":"Material and metod","content":"\u003cp\u003eThe six lactic acid bacteria (LAB) strains (three \u003cem\u003ePediococcus pentosaceus\u003c/em\u003e and three \u003cem\u003eLactobacillus plantarum)\u003c/em\u003e used in this study were previously isolated from raw milk, white cheese and Turkish sucuk [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Briefly, ten gram of each samples were homogenized with 90 mL peptone water (Merck, Darmstadt, Germany), and serial dilutions were prepared. A 100µL from each sample was spread onto MRS agar (Merck, Darmstadt, Germany), then incubated at 30°C for 48 h anaerobically. Then, colonies were identified colony morphology, gram staining and catalase tests. Gram-positive, and catalase-negative colonies were selected and stored in MRS broth with %15 glycerol at -20°C. Selected potential LAB colonies identified using Matrix Supported Laser Desorption/Ionization Flight Time Mass Spectrometry (MALDI-TOF MS) for species identification with the method previously described by Uysal et al. [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Then, six isolates identified using 16S rDNA gene sequencing following the method by Klindworth et al. [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] with using the 8F (5' AGAGTTTGATCMTGGCTCAG 3') and 1387R (5' GGGCGGWGTGTACAAGGC 3') primers. Isolates I-1, I-4, and I-6 were exhibited 100% similarity to the reference sequence of \u003cem\u003ePediococcus pentosaceus\u003c/em\u003e and registered to GenBank with the accession numbers PV345696, PV345694 and PV345692, respectively, while isolates I-2, I-3 and I-5 showed 100% similarity to the reference sequence of \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e and registered to GenBank with accession numbers PV345698, PV345700 and PV345699, respectively. These identified isolates are given in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, and these were used in the following analyses for the study.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\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\u003eIsolates used in the study [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIsolate\u003c/p\u003e\u003cp\u003eCode\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eProduct\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMALDI-TOF MS Results\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16 sRNA Sequencing Results\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eGenBank No\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eI-1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWhite cheese\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003ePediococcus pentosaceus\u003c/em\u003e 091015_01 LBK\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003ePediococcus pentosaceus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePV345696\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eI-2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWhite cheese\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eLactobacillus plantarum\u003c/em\u003e DSM 20246 DSM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePV345698\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eI-3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWhite cheese\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eLactobacillus plantarum\u003c/em\u003e DSM 2601 DSM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePV345700\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eI-4\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRaw milk\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003ePediococcus pentosaceus\u003c/em\u003e AL908877 BK14924 UKH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003ePediococcus pentosaceus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePV345694\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eI-5\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSucuk\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eLactobacillus plantarum\u003c/em\u003e DSM 1055 DSM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePV345699\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eI-6\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSucuk\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003ePediococcus pentosaceus\u003c/em\u003e DSM 20206 DSM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003ePediococcus pentosaceus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePV345692\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eDetermination of Probiotic Properties\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eHemolytic Activity\u003c/b\u003e\u003c/p\u003e\u003cp\u003eHemolytic activity of the isolates was evaluated on Tryptic Soy Agar (TSA; Difco) supplemented with 5% sheep blood, as described by Diguță et al. [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Each bacterial isolate was inoculated onto the surface of the agar using the streaking method, and the plates were incubated at 37°C for 48 hours.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTolerance to Low pH, Pancreatin, and Bile Salts\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe isolation of LAB was evaluated under low pH and simulated gastrointestinal conditions Low pH, pancreatin, and bile salts were used to simulate these conditions. Initially, overnight (18-hour) incubated cultures were centrifuged (4000 × g, 10 minutes, 4°C), and the pellets were washed twice with 0.9% saline solution. Bacterial cells were suspended in MRS broth and adjusted to pH 3 using 1 N hydrochloric acid. Simultaneously, bacteria were transferred to media containing 0.1% pepsin (pH 3, Sigma-Aldrich), 0.3% bile salts (pH 4.8, Oxgall, Thermo Fisher), and 0.1% pancreatin (pH 8, Sigma-Aldrich). Then, all samples were incubated anaerobically at 37°C for 3 hours. The number of viable bacterial cells (CFU/mL) was determined at the beginning and after 3 hours by inoculation on MRS agar. All tests were performed in triplicate [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eAntibacterial Activity of Cell-Free Supernatants\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe antibacterial activity of LAB isolates was evaluated using cell-free culture supernatants by the agar well diffusion method, as described by Gharib [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The isolates were inoculated into MRS broth and incubated at 37°C for 48 hours. After the incubation, the cultures were centrifuged at 4000 × g for 10 minutes to separate the bacterial cells. The resulting supernatants were filtered through a 0.22 µm pore size filter to obtain fresh, sterilized, cell-free supernatants. Antibacterial activity was tested against four pathogenic bacteria: \u003cem\u003eSalmonella\u003c/em\u003e Typhimurium (ATCC 14088), \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (ATCC 27853), \u003cem\u003eEnterococcus faecalis\u003c/em\u003e (ATCC 29212), and \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e (ATCC 700603). Target bacteria were swabbed onto Mueller-Hinton agar plates, and then 100 µL of cell-free supernatant was added to wells punched into agar plates. The presence of a clear inhibition zone around the wells indicated antibacterial activity, whereas the absence of a zone indicated no inhibitory effect. The diameter of inhibition zones was measured using a digital caliper.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAntibiotic Resistance\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe antibiotic resistance of LAB isolates was determined using the disk diffusion method according to Diguță et al. [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. All isolates were adjusted to a turbidity of 0.5 McFarland standard and spread onto MRS agar plates using sterile swab. Antibiotic discs were then placed on the agar surface, and the plates were incubated at 37°C for 48 hours. The isolates were tested for their susceptibility to six antibiotics: Cefoxitin (30 µg), Danofloxacin (5 µg), Lincomycin (15 µg), Kanamycin (5 µg), Amoxicillin–Clavulanic acid (30 µg), and Neomycin (30 µg). After incubation, inhibition zones were measured using a digital caliper. The isolates were categorized based on the diameter of the inhibition zones as follows: Resistant (R): no inhibition zone; Semi-susceptible (SS): inhibition zone between 7–16 mm; Susceptile (S): inhibition zone \u0026gt; 16 mm [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell Surface Hydrophobicity\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe LAB isolates were evaluated for the hydrophobicity according to the method described by Rokana et al. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Overnight cultures grown in MRS broth were centrifuged at 20,000 rpm for 15 minutes at 4°C. The resulting pellets were washed twice with PBS and resuspended in PBS buffer. The optical density (H₀) of the suspension was adjusted to 0.84–0.89 at 600 nm. Then, 1 mL of n-hexane or chloroform was added to 3 mL of the bacterial suspension, and the mixture was vortexed for 1 minute. After stabilization, the test tubes were incubated at 37°C for 1 hour. The aqueous phase was carefully collected, and its absorbance (H₁) was measured at 600 nm. The percentage of cell surface hydrophobicity was calculated using the following formula:\u003c/p\u003e\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\%\\:Cell\\:Surface\\:Hydrophobicity\\:=\\:(1\\:-\\:H₁\\:/\\:H₀)\\:\\times\\:\\:100$$\u003c/div\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eAutoaggregation Assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo determine the level of autoaggregation, bacterial suspensions prepared as described above were adjusted to an optical density of 0.80 at 600 nm (OD₆₀₀). A 4 mL aliquot of each cell suspension was transferred into clean glass test tubes and incubated at 37°C for 24 hours under static conditions. After the incubation, 1 mL of the upper phase was carefully collected, and its absorbance (ODₜ) was measured at 600 nm. The percentage of autoaggregation was calculated using the following formula [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]:\u003c/p\u003e\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:Autoaggregation\\:\\left(\\%\\right)\\:=\\:(1\\:-\\:ODₜ\\:/\\:ODᵢ)\\:\\times\\:\\:100\\:$$\u003c/div\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eAll experiments were performed in triplicate, and the results were expressed as mean (M) ± standard deviation (SD). The paired t-test was used to determine differences (p \u0026lt; 0.05).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eDetermination of Probiotic Properties\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe hemolytic activity of the strains was tested on blood agar plates supplemented with 5% sheep blood. None of the isolates exhibited β-hemolysis, indicating that none of the tested LAB strains demonstrated pathogenicity. Tolerance and resistance to low pH, pancreatin, and bile salts are essential for the survival of probiotic LAB in the intestinal tract [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Therefore, tolerance tests are widely used as preliminary screening tools for identifying potential probiotic strains [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In this study, the survival rates of LAB isolates were evaluated under the following conditions: 0.1% pepsin (pH 3), 0.1% pancreatin (pH 8) and 0.3% bile salts (pH 8). The variation in the survival rates of LAB strains under these stress conditions is presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\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\u003eSurvival of LAB ısolates under simulated gastrointestinal conditions (log₁₀ CFU/m)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e0.1% Pepsin\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e0.1% Pancreatin\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u003cp\u003e0.3% Bile Salts\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIsolate\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eStrains\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eInitial\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3 h\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eInitial\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3 h\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eInitial\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003e3 h\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eI-1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003ePediococcus pentosaceus\u003c/em\u003e I-1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e5.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e6.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e5.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e5.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eI-2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eLactobacillus plantarum\u003c/em\u003e I-2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e6.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e6.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e6.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e6.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e7.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eI-3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eLactobacillus plantarum\u003c/em\u003e I-3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e5.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e6.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e6.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e6.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e5.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eI-4\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003ePediococcus pentosaceus\u003c/em\u003e I-4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e5.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e5.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e5.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e5.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e5.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eI-5\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eLactobacillus plantarum\u003c/em\u003e I-5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e6.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e6.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e6.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e5.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e6.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eI-6\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003ePediococcus pentosaceus\u003c/em\u003e I-6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e5.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e6.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e5.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e5.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e4.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eResults are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Different superscript letters (a\u0026ndash;d) indicate statistically significant differences between groups at the same time point (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003eSurvival of LAB isolates under low pH conditions is critically important for their resistance to gastric acid. The initial bacterial load was adjusted to 10\u003csup\u003e6\u003c/sup\u003e CFU/mL in all samples (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). After 3 hours of exposure to 0.1% pepsin (pH 3), the highest survival rate was observed in \u003cem\u003eL. plantarum\u003c/em\u003e I-2 (6.91 log₁₀ CFU/mL) and followed by \u003cem\u003eL. plantarum\u003c/em\u003e I-3, while the lowest was recorded in isolate \u003cem\u003eP. pentosaceus\u003c/em\u003e I-6 (5.62 log₁₀ CFU/mL) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The highest resistance was determined for isolate \u003cem\u003eP. pentosaceus\u003c/em\u003e I-1 for pancreatin (7.75log₁₀ CFU/mL) and followed by I-2 (6.55 log₁₀ CFU/mL). The lowest resistant was determined in \u003cem\u003eP. pentosaceus\u003c/em\u003e I-4 (5.70 log₁₀ CFU/mL) and \u003cem\u003eP. pentosaceus\u003c/em\u003e I-6 (5.64 log₁₀ CFU/mL). The isolates were tested at 0.3% bile salt concentration to reflects physiological levels in the human gastrointestinal tract. All isolates exhibited survival levels ranging from 4.99 to 7.60 log₁₀ CFU/mL under bile salt exposure. After 3 hours of exposure, the highest viability was observed in isolate \u003cem\u003eL. plantarum\u003c/em\u003e I-2 (7.60 log₁₀ CFU/mL), while the lowest was detected in isolate \u003cem\u003eP. pentosaceus\u003c/em\u003e I-6 (4.99 log₁₀ CFU/mL).\u003c/p\u003e\u003cp\u003e\u003cb\u003eAntimicrobial Activity\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe diameters of inhibition zones formed by the cell-free supernatants of LAB isolates against \u003cem\u003eEnterococcus faecalis, Pseudomonas aeruginosa, Klebsiella pneumoniae\u003c/em\u003e, and \u003cem\u003eSalmonella\u003c/em\u003e Typhimurium are presented in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Enrofloxacin (40 \u0026micro;g/mL) was used as a positive control for the antimicrobial evaluation. The LAB isolates exhibited varying levels of antimicrobial activity. All isolates demonstrated inhibition zones against \u003cem\u003eE. faecalis\u003c/em\u003e, with ranging from 11 to 12 mm, and against \u003cem\u003eP. aeruginosa\u003c/em\u003e, with zones of 8 to 10 mm. Isolates \u003cem\u003eL. plantarum\u003c/em\u003e I-2, \u003cem\u003eL. plantarum\u003c/em\u003e I-3, and \u003cem\u003eL. plantarum\u003c/em\u003e I-5 formed 8 mm inhibition zones against \u003cem\u003eS\u003c/em\u003e. Typhimurium, whereas the remaining isolates (\u003cem\u003eP. pentosaceus\u003c/em\u003e I-1, \u003cem\u003eP. pentosaceus\u003c/em\u003e I-4, and \u003cem\u003eP. pentosaceus\u003c/em\u003e I-6) showed no inhibitory effect. Also, none of the isolates showed antimicrobial activity against \u003cem\u003eK. pneumoniae\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAntimicrobial activity of cell-free supernatants against some pathogenic microorganisms\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eKlepsiella pneumonia\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eSalmonella Typhimurium\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eEnterococcus\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003efaecalis\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\u003e\u003cem\u003ePediococcus pentosaceus\u003c/em\u003e I-1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eLactobacillus plantarum\u003c/em\u003e I-2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eLactobacillus plantarum\u003c/em\u003e I-3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ePediococcus pentosaceus\u003c/em\u003e I-4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eLactobacillus plantarum\u003c/em\u003e I-5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ePediococcus pentosaceus\u003c/em\u003e I-6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003edwell\u0026thinsp;=\u0026thinsp;6 mm, NI- no inhibition\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003cb\u003eAntibiotic Susceptibility of the Isolates\u003c/b\u003e\u003c/td\u003e\u003c/tr\u003e\u003cp\u003eThe antibiotic susceptibility of the LAB isolates was evaluated using the disc diffusion method on MRS agar and results are shown in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. With a few exceptions, the isolates exhibited similar resistance profiles. All isolates were resistant to Kanamycin (5 \u0026micro;g) and susceptible to Amoxicillin\u0026ndash;Clavulanic acid (30 \u0026micro;g). Excluding \u003cem\u003eL. plantarum\u003c/em\u003e I-3, all isolates were susceptible to Lincomycin (15 \u0026micro;g) and except \u003cem\u003eL. plantarum\u003c/em\u003e I-5, the other isolates were semi-susceptible to Cefoxitin (30 \u0026micro;g) and resistant to Danofloxacin (5 \u0026micro;g). Isolates \u003cem\u003eP. pentosaceus\u003c/em\u003e I-1, \u003cem\u003eP. pentosaceus\u003c/em\u003e I-4, and \u003cem\u003eP. pentosaceus\u003c/em\u003e I-6 were resistant to Neomycin (30 \u0026micro;g), while \u003cem\u003eL. plantarum\u003c/em\u003e I-2, \u003cem\u003eL. plantarum\u003c/em\u003e I-3, and \u003cem\u003eL. plantarum\u003c/em\u003e I-5 were found to be semi-susceptible.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAntibiotic Susceptibility Profiles of LAB Isolates\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIsolate\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCefoxitin\u003c/p\u003e\u003cp\u003e(30\u0026micro;g)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDanofloxacin\u003c/p\u003e\u003cp\u003e(5\u0026micro;g)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLincomycin\u003c/p\u003e\u003cp\u003e(15 \u0026micro;g)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eKanamycin\u003c/p\u003e\u003cp\u003e(5\u0026micro;g)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eAmoxyclin Clavulonic acid (30\u0026micro;g)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNeomycin\u003c/p\u003e\u003cp\u003e(30\u0026micro;g)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ePediococcus pentosaceus\u003c/em\u003e I-1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eLactobacillus plantarum\u003c/em\u003e I-2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eSR\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eLactobacillus plantarum\u003c/em\u003e I-3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eSR\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ePediococcus pentosaceus\u003c/em\u003e I-4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eLactobacillus plantarum\u003c/em\u003e I-5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eSR\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ePediococcus pentosaceus\u003c/em\u003e I-6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003eR \u0026ndash; Resistant; SR \u0026ndash; Intermediate (zone diameter 7\u0026ndash;16 mm); S \u0026ndash; Sensitive (zone diameter\u0026thinsp;\u0026gt;\u0026thinsp;16 mm); disc diameter\u0026thinsp;=\u0026thinsp;6 mm\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003cb\u003eHydrophobicity and Autoaggregation of LAB Isolates\u003c/b\u003e\u003c/td\u003e\u003c/tr\u003e\u003cp\u003eThe surface hydrophobicity of the isolates was assessed using two different solvents: n-hexane and chloroform. The results are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Isolate \u003cem\u003eL. plantarum\u003c/em\u003e I-5 demonstrated the highest hydrophobicity ratio after 24 hours in the presence of chloroform, and followed by \u003cem\u003eL. plantarum\u003c/em\u003e I-3 and \u003cem\u003eP. pentosaceus\u003c/em\u003e I-6. In the presence of n-hexane, isolate \u003cem\u003eL. plantarum\u003c/em\u003e I-2 showed the highest hydrophobicity, while the lowest values were observed in \u003cem\u003eP. pentosaceus\u003c/em\u003e I-4 (chloroform) and \u003cem\u003eP. pentosaceus\u003c/em\u003e I-1 (n-hexane), respectively. Autoaggregation rates of the isolates are given in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The highest autoaggregation was observed in \u003cem\u003eL. plantarum\u003c/em\u003e I-3, followed by \u003cem\u003eL. plantarum\u003c/em\u003e I-5, \u003cem\u003eP. pentosaceus\u003c/em\u003e I-1, \u003cem\u003eL. plantarum\u003c/em\u003e I-2, and \u003cem\u003eP. pentosaceus\u003c/em\u003e I-6. The lowest autoaggregation value was recorded in isolate \u003cem\u003eP. pentosaceus\u003c/em\u003e I-4.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eLactic acid bacteria (LAB), widely recognized as probiotics in the food industry, have attracted significant attention due to their health-promoting effects. Accordingly, there is growing interest in isolating and evaluating potential probiotic LAB strains from various food [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In the present study, six LAB isolates which were previously isolated and identified by 16 S rRNA gene sequencing evaluated the probiotic potential by examining their hemolytic activity, tolerance in the gastrointestinal tract, antimicrobial properties, antibiotic resistance, hydrophobicity, and autoaggregation. The ability of probiotic strains to survive and grow under low pH and in the presence of bile salts is considered a desirable characteristic [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eProbiotic strains must resist low pH, bile salts, and pancreatic enzymes to survive passage through the upper gastrointestinal tract [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In this study, all isolates remained viable after 3 hours of incubation under simulated gastrointestinal conditions, (low pH, pancreatin, and bile salts) indicating strong tolerance to gastric and intestinal environments. \u003cem\u003eL. plantarum\u003c/em\u003e I-2 has the highest survival rates of the pepsin, pancreatin and bile salts condition. The resistance of \u003cem\u003eLactobacillus\u003c/em\u003e species to bile salts is largely attributed to the presence of the bsh-1 and bsh-2 genes, which encode bile salt hydrolase enzymes that hydrolyze bile salts [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Similar results have been seen with other \u003cem\u003eL. plantarum\u003c/em\u003e strains, known for their robust resistance to tough gastrointestinal conditions [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. This aligns with previous studies highlighting the strong acid resistance of \u003cem\u003eL. plantarum\u003c/em\u003e strains [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Similar findings regarding the probiotic properties of LAB have been reported in other studies, consistent with the results of the present study [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe resistance to pancreatin, which simulates intestinal protease activity, varied among the isolates. \u003cem\u003eP. pentosaceus\u003c/em\u003e I-1 showed the highest survival rate under pancreatin condition. In contrast, strains \u003cem\u003eP. pentosaceus\u003c/em\u003e I-4 and \u003cem\u003eP. pentosaceus\u003c/em\u003e I-6 had reduced survival, likely due to sensitivity to enzymes or differences in cell wall composition. When exposed to 0.3% bile salts, which represent a normal concentration of bile in the intestine, all isolates showed varying levels of tolerance. \u003cem\u003eL. plantarum\u003c/em\u003e I-2 demonstrated the highest tolerance, suggesting better adaptability to intestinal conditions, On the other hand, \u003cem\u003eP. pentosaceus\u003c/em\u003e I-6 showed the lowest resistance, which may indicate a susceptibility of the membrane to bile-induced destabilization [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eLAB are capable of inhibiting pathogen colonization and growth in the gastrointestinal tract. This inhibitory effect may be due to the production of antimicrobial agents or by preventing pathogen adhesion to the intestinal mucosa [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In the present study, LAB isolates were tested against major foodborne pathogens, including Enterococcus faecalis, \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, Salmonella \u003cem\u003eTyphimurium\u003c/em\u003e, and \u003cem\u003eKlebsiella pneumonia\u003c/em\u003e. The isolates exhibited varying degrees of inhibition zones against \u003cem\u003eE. faecalis\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e while only \u003cem\u003eL. plantarum\u003c/em\u003e strains (I-2, I-3, I-5) inhibited Salmonella Typhimurium and none of the isolates showed inhibitory effect on \u003cem\u003eK. pneumoniae\u003c/em\u003e. The lack of antibacterial activity against \u003cem\u003eK. pneumoniae\u003c/em\u003e suggests the antimicrobial effects are specific to certain pathogens and might relate to differences in cell wall structure or sensitivity to LAB-derived substances [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSimilarly, Fossi and Ndjouenkeu identified thermotolerant LAB strains (\u003cem\u003eL. plantarum\u003c/em\u003e and \u003cem\u003eL. acidophilus\u003c/em\u003e). These selected strains showed strong antimicrobial activity against major foodborne pathogens such as \u003cem\u003eSalmonella Enteritidis\u003c/em\u003e, \u003cem\u003eSalmonella\u003c/em\u003e Typhimurium, \u003cem\u003eEscherichia coli, Staphylococcus aureus\u003c/em\u003e, and \u003cem\u003eListeria monocytogenes\u003c/em\u003e, indicating their potential for use in industrial probiotic production. Previous studies on the antibacterial effects of LAB strains are consistent with our findings, confirming that the isolated strains exhibit strong antagonistic activity against the selected pathogens [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eExamining the antibiotic resistance profile of potential probiotic strains is a crucial step, as the a risk of transferring resistance genes to the gut microbiota [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. All isolates were found resistant to kanamycin, which is common for LAB due to their intrinsic resistance due to low membrane permeability and the presence of aminoglycoside-modifying enzymes [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Many \u003cem\u003eLactobacillus\u003c/em\u003e species are naturally resistant to aminoglycosides (gentamicin, kanamycin, streptomycin, and neomycin), ciprofloxacin, and trimethoprim, while they are generally sensitive to penicillin and β-lactam antibiotics, as well as chloramphenicol, tetracycline, erythromycin, linezolid, and quinupristin-dalfopristin [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In a recent study, Duche et al. [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] evaluated the antibiotic resistance profiles of LAB strains. The resistance rates were approximately 100% for ceftazidime, cefoxitin, kanamycin, nalidixic acid, vancomycin, teicoplanin, methicillin, and norfloxacin. Resistance to cefmetazole, polymyxin B, tobramycin, and moxifloxacin ranged from 91.2\u0026ndash;97.2%. For ciprofloxacin, gentamicin, fusidic acid, and gatifloxacin, resistance rates were 76.5%, 76.5%, 79.4%, and 82.4%, respectively. Intermediate resistance was observed against penicillin G (52.9%) and clindamycin (58.5%). In contrast, high sensitivity (70.6\u0026ndash;100%) was recorded for amoxicillin-clavulanate (amoxiclav), tetracycline, tigecycline, meropenem, imipenem, trimethoprim, co-trimoxazole, and azithromycin. However, the susceptibility to amoxicillin-clavulanic acid and lincomycin (except \u003cem\u003eL. plantarum\u003c/em\u003e I-3), indicating a low risk of contributing to the spread of antibiotic resistance and suggesting positive safety profiles. The differing reactions to neomycin and danofloxacin reveal strain-specific variations in resistance patterns. Intermediate resistance to cefoxitin and neomycin in particular Pediococcus strains points to the need for further genetic testing to confirm the absence of transferable resistance genes [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCell surface hydrophobicity is an important trait that facilitates the adhesion of probiotic bacteria to epithelial cells and promotes colonization [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In the present study, the hydrophobicity of the isolates was evaluated in the presence of two different solvents. Among all tested isolates, the highest hydrophobicity was observed in L. plantarum I-5 in chloroform, while I-2 showed a strong affinity for n-hexane. This probably suggests potential differences in cell wall composition, especially between protein and lipid structures [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Similarly, studies by Coulibaly et al. [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] and Rokana et al. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] also reported the highest hydrophobicity when chloroform was used. Autoaggregation is another key factor contributing to adhesion and colonization on epithelial surfaces and is often used to assess the probiotic potential of strains [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. According to Kang et al. [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] and Kim et al. [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], autoaggregation levels can be classified as low (16\u0026ndash;35%), moderate (35\u0026ndash;50%), or high (\u0026ge;\u0026thinsp;50%). After 24 hours of incubation, the highest autoaggregation level was observed in \u003cem\u003eL. plantarum\u003c/em\u003e I3, with a value of 22%, which falls within the low aggregation category. High autoaggregation in \u003cem\u003eL. plantarum\u003c/em\u003e I-3 and \u003cem\u003eL. plantarum\u003c/em\u003e I-5 may support their colonization and survival in the gastrointestinal mucosa, by the way of the competitive exclusion of pathogens [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In contrast, \u003cem\u003eP. pentosaceus\u003c/em\u003e I-4's low autoaggregation could reduce its probiotic potential [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In contrast, \u003cem\u003eP. pentosaceus\u003c/em\u003e I-4 has minimal hydrophobicity and autoaggregation, which may limit its ability to adhere to mucosal surfaces.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eBased on the results obtained, \u003cem\u003eP. pentosaceus\u003c/em\u003e I-1, \u003cem\u003eL. plantarum\u003c/em\u003e I-2, \u003cem\u003eL. plantarum\u003c/em\u003e I-3, and \u003cem\u003eL. plantarum\u003c/em\u003e I-5, demonstrated the most promising probiotic traits, including strong gastrointestinal tolerance, antimicrobial activity, and favorable cell surface characteristics. The variability seen between strains highlights the importance of thorough phenotypic and genotypic evaluation in probiotic screening. However, further studies, including in vivo trials, are required to confirm their health-promoting effects and to investigate their potential application in food systems.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e \u003cstrong\u003eand Funding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no potential conflicts of interest. This research was supported by TUBİTAK 2209A Student Scientific Research Grant (grant no. 1919B012220737).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAuthorship Contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHAK designed the experiments. HAK, MD, ENG, AG and N\u0026Ouml; carried out the analyses. HAK contributed to interpreting the results and took the lead in writing the manuscript. Both authors provided critical feedback and helped shape the research, analysis, and manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by TUBİTAK 2209A Student Scientific Research Grant (grant no. 1919B012220737).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting this study\u0026apos;s findings are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no potential conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study did not involve animal or human subjects.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAxelsson L (2004) In: Salminen S, Von Wright A (eds) Lactic acid bacteria: microbiological and functional aspects, 3rd edn. 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Microorganisms 10(10):2070. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/microorganisms10102070\u003c/span\u003e\u003cspan address=\"10.3390/microorganisms10102070\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDel Re B, Sgorbati B, Miglioli M, Palenzona D (2000) Adhesion, autoaggregation and hydrophobicity of 13 strains of Bifidobacterium longum. Lett Appl Microbiol 31(6):438\u0026ndash;442. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1046/j.1365-2672.2000.00845.x\u003c/span\u003e\u003cspan address=\"10.1046/j.1365-2672.2000.00845.x\" 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":"european-food-research-and-technology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [European Food Research and Technology](https://link.springer.com/journal/217)","snPcode":"217","submissionUrl":"https://submission.springernature.com/new-submission/217/3","title":"European Food Research and Technology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Lactiplantibacillus plantarum, Pediococcus pentosaceus, Probiotic potential, Safety attributes","lastPublishedDoi":"10.21203/rs.3.rs-7120721/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7120721/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe aim of this study is to investigate the some probiotic properties of isolated and identified six LABs (three of \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e and three of \u003cem\u003ePediococcus pentosaceus)\u003c/em\u003e, including antibacterial potential against \u003cem\u003eSalmonella\u003c/em\u003e Typhimurium (ATCC 14088), \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (ATCC 27853), \u003cem\u003eEnterococcus faecalis\u003c/em\u003e (ATCC 29212), and \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e (ATCC 700603), their tolerance to low pH, pancreatin and bile salts, cell surface hydrophobicity, autoaggregation as well as determining the antibiotic resistance. While \u003cem\u003eL. plantarum\u003c/em\u003e I-2 and \u003cem\u003eP. pentosaceus\u003c/em\u003e I-4 showed the highest and lowest resistance in the presence of pepsin and bile salt, respectively, \u003cem\u003eP. pentosaceus\u003c/em\u003e I-1 and \u003cem\u003eP. pentosaceus\u003c/em\u003e I-4 exhibited the highest and lowest resistance in the presence of pancreatin. Isolates \u003cem\u003eP. pentosaceus\u003c/em\u003e I1, \u003cem\u003eL. plantarum\u003c/em\u003e I-3, and \u003cem\u003eL. plantarum\u003c/em\u003e I-5 formed highest antibacterial activity against \u003cem\u003eP. aeruginosa, S. Typhimurium\u003c/em\u003e and \u003cem\u003eE. fecalis.\u003c/em\u003e All isolates were susceptible to Amoxicillin\u0026ndash;Clavulanic acid and were resistant to Kanamycin. Isolate \u003cem\u003eL. plantarum\u003c/em\u003e I-5 (73%) and \u003cem\u003eL. plantarum\u003c/em\u003e I-2 (33%) demonstrated the highest hydrophobicity ratio after 24 hours in the presence of chloroform and n-hexane, respectively. The highest and lowest autoaggregation was observed in \u003cem\u003eL. plantarum\u003c/em\u003e I-3 (21%) and \u003cem\u003eP. pentosaceus\u003c/em\u003e I-4 (2.5%). In conclusion, \u003cem\u003eP. pentosaceus\u003c/em\u003e I1, \u003cem\u003eL. plantarum\u003c/em\u003e I-2, \u003cem\u003eL. plantarum\u003c/em\u003e I-3, and \u003cem\u003eL. plantarum\u003c/em\u003e I-5 had desirable in vitro probiotic properties and strong inhibitory activity against the tested pathogens, and they appear to be promising candidates for probiotic bacteria to be used in the food industry.\u003c/p\u003e","manuscriptTitle":"Assessment of Antimicrobial Activity and Selected Safety Attributes of Lactiplantibacillus plantarum and Pediococcus pentosaceus Isolates for Potential Probiotic Use","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-18 13:36:27","doi":"10.21203/rs.3.rs-7120721/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision 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