Probiotic Potential and Antimicrobial Properties of Lactic Acid Bacteria Isolated from Multi-Age Piglets

Probiotic Potential and Antimicrobial Properties of Lactic Acid Bacteria Isolated from Multi-Age Piglets | 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 Probiotic Potential and Antimicrobial Properties of Lactic Acid Bacteria Isolated from Multi-Age Piglets Nattakarn Awaiwanont, Montira Intanon, Promporn Inyoo, Matsarina Kongton, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7920513/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Lactic acid bacteria (LAB) are important probiotics that support intestinal health and growth performance in pigs through modulation of gut microbiota and production of antimicrobial compounds. This study aimed to isolate and evaluate LAB with probiotic potential from feces of healthy, antibiotic- and probiotic-free piglets of different ages. Forty-two fecal samples were collected from a farm in Lamphun province, yielding 318 LAB isolates, of which 135 gram-positive, catalase-negative bacilli were characterized for probiotic traits. Only two isolates (PMvet212 and PMvet318) survived at pH 3.1 with a viability loss of less than 1 log CFU/mL; both tolerated 0.3% bile salt, and PMvet212 also survived at 0.5% bile salt. Their hydrophobicity values were 7.85% and 12.38%, respectively, indicating low adhesion capacity. Both isolates showed alpha-hemolysis. Cell-free supernatants of these isolates inhibited Escherichia coli from diarrheic piglets and Staphylococcus aureus ATCC6538, with inhibition zones classified as intermediate. These findings indicate that LAB from piglet feces, particularly PMvet212, possess moderate probiotic potential and antibacterial activity, and may serve as candidates for development as feed additives to promote swine gut health and reduce reliance on antibiotics. Piglet feces Lactic acid bacteria (LAB) Probiotic traits Antimicrobial effects Escherichia coli Figures Figure 1 Figure 2 Introduction Escherichia coli (E. coli) is one of the most important pathogens causing diarrhea in neonatal and weaned piglets. Piglet diarrhea leads to substantial economic losses in the swine industry due to increased morbidity and mortality, reduced growth performance, and higher veterinary costs (Fairbrother et al., 2005 ). Prevention is generally more effective than treatment, yet traditional control strategies have relied heavily on antibiotics (Luppi., 2017) This dependence has raised serious concerns about antibiotic residues in meat and the global spread of antimicrobial resistance (AMR) (Davis et al., 2002 ). As regulatory authorities and consumers increasingly demand reductions in antibiotic use, there is a growing need for sustainable alternatives that can maintain animal health and productivity without contributing to AMR (Moredo et al., 2015 ). Several alternative approaches have been proposed, including organic acids, copper sulfate, zinc oxide, prebiotics, herbal supplements, and pro-biotics. Among these, probiotics have attracted particular attention for their ability to promote intestinal health, reduce the incidence and severity of enteric diseases, and support growth performance in pigs, while reducing the need for antibiotics (Namkun et al., 2004). Pro-biotic supplementation has been shown to modulate the gut microbiota, enhance immune function, and improve nutrient utilization in swine. Recent studies have con-firmed that probiotics can serve as viable antibiotic alternatives for preventing bacterial diarrhea in piglets, with benefits extending to improved feed efficiency and over-all health (Bogere et al., 2019 ). Moreover, probiotic use aligns with the One Health framework by mitigating AMR risks at the animal–human–environment interface (Laird et a., 2021). At birth, the gastrointestinal tract of animals is sterile but rapidly colonized by microorganisms from the mother and the surrounding environment. This early colonization plays a critical role in the development of the digestive system, metabolic functions, and immune competence of neonates (Boaventura et al., 2012 ) The gut microbiota’s composition is shaped by factors such as delivery mode, maternal microbiota, diet, and environ-mental exposure (Binns., 2013) In pigs, a diverse and balanced gut microbiota supports resistance to pathogens, adaptation to dietary changes, and improved physiological functions (Fouhse et al., 2016 ). The porcine gastrointestinal tract is anatomically compartmentalized, with the small intestine particularly the duodenum, jejunum, and ileum being the main site for nutrient digestion and absorption. Maintaining a healthy microbiota in these regions is critical for growth and disease prevention, especially in intensive swine production systems. Probiotics are defined as live microorganisms that, when administered in adequate amounts, confer health benefits to the host by improving the microbial balance in the gastrointestinal tract (FAO/WHO, 2002). Effective probiotics must survive exposure to gastric acid and bile salts, adhere to intestinal epithelial cells, and exhibit antagonistic effects against pathogens (Kontula et al., 1998 ). In swine production, probiotics are used both to support growth and to prevent colonization by enteric pathogens such as Salmonella spp ., E. coli , and Clostridium perfringens (De Angelis et al., 2006 ). Lactic acid bacteria (LAB) are among the most widely studied probiotic groups due to their non-pathogenic nature, acid and bile tolerance, and ability to produce antimicrobial substances such as organic acids, hydrogen peroxide, and bacteriocins (Mojgani et al., 2015 ). These antimicrobial compounds are considered safe and, in some cases, have been linked to reduced pathogen load and improved gut health in pigs (Ozugul et al., 2018). The probiotic properties of LAB are highly strain-specific, even among isolates belonging to the same species (Senok et al., 2015 ). Only a limited number of strains with strong acid and bile tolerance, adhesion ability, and potent antimicrobial activity have been identified, and their functional properties often vary depending on the host and environ-mental context. Recent advances in microbiome analysis and targeted isolation techniques have enabled the discovery of novel LAB strains from indigenous pig breeds and different rearing environments, some of which show promise for improving gut integrity, modulating immunity, and reducing pathogenic E. coli colonization (Yu et al., 2024 ). However, further research is needed to characterize these strains comprehensively and assess their potential as antibiotic alternatives in commercial pig production. Therefore, the aim of the present study was to isolate and characterize LAB with probiotic potential from fecal samples of pigs at different ages raised without antibiotics or probiotics. The focus was on identifying strains that could survive under gastro-intestinal conditions, exhibit inhibitory activity against pathogenic E. coli , and demonstrate other desirable probiotic traits. The findings of this work provide a basis for selecting promising LAB strains for development as feed additives to promote swine gut health, reduce reliance on antibiotics, and contribute to the global effort to mitigate AMR in livestock production. Materials and Methods Ethical Approval Since fecal samples were collected without handling or restraining the animals, specific approval for animal use was not required. However, the experimental procedures involved the use of microbial isolates. Therefore, all microbiological work was reviewed and approved by the Institutional Biosafety Committee (IBC) of Chiang Mai University (Approval No. CMUIBC A-0763004). Sample Collection The study population comprised suckling piglets aged 7–30 days. The required sample size was estimated based on an assumed Lactobacillus detection prevalence of 50%, a total piglet population of 2,800, an effect size of 0.3, statistical power (1 – β) of 0.98, and an alpha error probability of 0.02, using G*Power software (version 3.1). The minimum calculated sample size was 40; therefore, 42 fecal samples were collected to ensure adequate statistical power. Samples were obtained from a commercial pig farm in Lamphun province where no antibiotics or probiotics had been used. To maximize diversity, no more than five samples were taken from any single farrowing pen. Freshly voided feces with normal consistency and yellow to brown coloration were col-lected from the pen floor, placed in sterile 5 mL microcentrifuge tubes (≥2 g each), and transported at 4 °C to the laboratory for processing. Isolation of Lactic Acid Bacteria (LAB) One gram of fecal sample was homogenized in 9 mL of 0.85% sterile saline and serially diluted (10⁻⁴ to 10⁻⁸). Aliquots (100 µL) were spread onto de Man, Rogosa, and Sharpe (MRS) agar supplemented with 0.5% CaCO₃. Plates were incubated anaerobi-cally at 37 °C for 48 h. Colonies producing clear zones were subcultured in MRS broth to obtain pure isolates. Gram‐positive, non‐spore‐forming bacteria were retained and tested for catalase activity; only catalase‐negative isolates were preserved at –20 °C in MRS broth with 20% (v/v) sterile glycerol (Guo et al., 2010). Evaluation of Probiotic Properties Acid Tolerance Colonies grown on MRS agar were suspended in 5 mL phosphate-buffered saline (PBS; pH 7.2) and adjusted to 0.5 McFarland standard (~1.5 × 108 CFU/mL). One milliliter of suspension was mixed with 9 mL sterile PBS adjusted to pH 2.0 or pH 3.1 using 1 N HCl. The mixtures were vortexed for 10 s and incubated at 37 °C for 3 h. Viable cell counts before and after incubation were determined by plate counting on MRS agar. Acid tolerance was expressed as the reduction in bacterial counts (log CFU/mL) com-pared with the initial value (Guo et al., 2010). Bile Salt Tolerance Bacterial suspensions prepared as above were inoculated (1 mL) into 9 mL MRS broth supplemented with 0.3%, 0.5%, or 1% (w/v) bile salts (Sigma-Aldrich). Control cultures contained no bile salts. Tubes were incubated anaerobically at 37 °C for 24 h. Viable cell counts were determined at 0 and 24 h, and tolerance was evaluated based on survival relative to controls. Each strain was tested in duplicate (Ren et al., 2014). Adhesion Ability to Intestinal Epithelium (Surface Hydrophobicity) Adhesion potential was assessed by the microbial adhesion to hydrocarbons (MATH) method. Bacteria grown on MRS agar were suspended in 5 mL PBS (pH 7.2) and adjusted to 6.0 McFarland standard. Optical density at 600 nm (OD₆₀₀) was adjusted to 0.6. Three milliliters of suspension was mixed with 1 mL of xylene in a glass tube, vortexed for 90 s, and left at room temperature for 30 min to allow phase separation. The aqueous phase was carefully removed, and OD₆₀₀ was measured again. Surface hydrophobicity (%H) was calculated as: %𝐻 = [(OD₆₀₀ before mixing – OD₆₀₀ after mixing)/OD₆₀₀ before mixing] × 100 100 Controls consisted of PBS and xylene without bacteria. All assays were performed in duplicate (Ekmekci., 2009) Hemolytic Activity For safety evaluation, isolates were streaked on tryptic soy agar (TSA) supplemented with 5% (w/v) sheep blood and incubated at 37 °C for 48 h. Hemolysis type was determined visually: β-hemolysis (clear zone), α-hemolysis (green/brown discoloration), and γ-hemolysis (no change). Staphylococcus aureus ATCC 6538 was used as a β-hemolysis positive (Adimpong et al., 2002). Antimicrobial Activity Against E. coli and S. aureus ATCC 6538 The agar well diffusion method was used. LAB cultures grown in MRS broth at 37 °C for 24 h were centrifuged at 4,000 × g for 5 min, and supernatants were filtered through 0.45 µm membranes to obtain cell-free supernatants (CFS). Indicator strains pathogenic E. coli (from diarrheic piglets) and Staphylococcus aureus ATCC 6538 were adjusted to 0.5 McFarland standard in PBS and spread onto nutrient agar plates. Wells (7 mm) were bored aseptically, and 80 µL CFS was added per well. Lactic acid (2% v/v) served as the positive control. Plates were incubated at 37 °C for 24 h, and inhibition zones were measured in millimeters. All tests were performed in duplicate (Sirichokchatchawan et al., 2018). Data Interpretation Probiotic Properties Acid Tolerance Survival at acidic pH was assessed by comparing bacterial counts (log CFU/mL) before and after incubation at pH 2.0 and pH 3.1. A reduction of less than 1 log CFU/mL at pH 3.1 was considered acceptable for acid tolerance (Petsuriyawong et al., 2011). Bile Salt Tolerance Bacterial counts (log CFU/mL) at 0 h and 24 h in MRS broth containing bile salts were compared. Strains were classified as tolerant if no significant reduction in viable counts was observed over the incubation period. Surface Adhesion Ability Adhesion potential was expressed as surface hydrophobicity (%H) calculated by the MATH assay. Higher %H values indicated stronger cell surface adhesion capabilities. Hemolytic Activity Hemolysis was classified into three types: β-hemolysis, indicating complete lysis of red blood cells and the presence of a clear zone around the colony; α-hemolysis, representing partial lysis with a green or brown discoloration surrounding the colony; and γ-hemolysis, showing no lysis or visible change in the medium. According to safety criteria for probiotic use, LAB strains exhibiting α- or β-hemolysis were considered un-suitable, whereas only γ-hemolytic strains were deemed acceptable (Buxton et al., 2015). Antimicrobial Activity Against E. coli and S. aureus ATCC 6538 The antimicrobial potential of the cell-free supernatants (CFS) was assessed by measuring the diameter of the inhibition zones against Escherichia coli and Staphylococ-cus aureus ATCC 6538. The results were interpreted according to the criteria summarized in Table 1. This approach allowed for a comparative evaluation of the inhibitory effects of different CFS preparations on the selected pathogenic strains. Table 1. Interpretation of antimicrobial activity based on inhibition zone diameter Symbol Interpretation Inhibition Zone Diameter (mm) - Non-inhibition 5 ++ Intermediate inhibition >10 +++ Strong inhibition >15 ++++ Very strong inhibition >20 Interpretation criteria modified from Sirichokchatchawan et al. (2018) Statistical Analysis Data were analyzed using descriptive statistics using SPSS software (IBM SPSS Statistics, 29 version). For experiments performed on a single isolate with triplicate assays, results were presented as mean values without standard deviation. Means and standard deviations were calculated and reported where appropriate to summarize the results. Results Isolation of Lactic Acid Bacteria (LAB) from Swine Feces From 42 individual fecal samples collected from pigs of different ages, a total of 318 bacterial colonies producing clear zones on MRS agar supplemented with 0.5% CaCO₃ under anaerobic incubation at 37 °C for 48 h were initially obtained. These colonies were preliminarily considered as potential lactic acid bacteria. Gram staining revealed that 296 isolates were Gram-positive, of which 146 were rod-shaped (bacilli), 136 were cocci, and 14 were coccobacilli. Nineteen isolates were identified as yeasts based on morphology, and three isolates could not be maintained during subculturing; both groups were excluded from further analysis. Catalase testing of the 296 Gram-positive isolates showed that 277 were cata-lase-negative, consistent with typical LAB characteristics, whereas 19 were cata-lase-positive and thus removed from subsequent screening. Consequently, 135 isolates were confirmed as Gram-positive, rod-shaped, catalase-negative bacteria and retained for probiotic property evaluation. For five isolates with ambiguous Gram-staining results, the test was repeated. Among these, isolates PMvet120, PMvet151, and PMvet183 exhibited budding cells and pleomorphic shapes, which were consistent with yeast morphology rather than bacteria (Figure 1). These isolates also showed poor growth in MRS broth and were therefore excluded. Probiotic Properties of Selected LAB Isolates Acid Tolerance Among the 135 LAB isolates confirmed as Gram-positive, rod-shaped, and cata-lase-negative, only five strains PMvet120, PMvet151, PMvet183, PMvet212, and PMvet318 demonstrated acid tolerance, with a reduction in viable counts of approximately 1.00 log CFU/mL or less after incubation at pH 3.1 for 3 h. No isolates survived exposure to pH 2.0 under the same conditions. The survival rates of these five isolates at pH 3.1 are summarized in Table 2. Table 2. Survival of selected LAB isolates at pH 3.1 after 3 h of incubation at 37 °C. Values are expressed as mean ± standard deviation (n = 3). Isolate ID log CFU/mL Initial Count (average) pH 3.1 PMvet120 6.37±0.5 5.35±0.4 PMvet151 6.20±0.3 PMvet183 6.15±0.7 PMvet212 5.60±0.5 PMvet318 5.56±0.6 Bile Salt Tolerance The five acid-tolerant isolates were subsequently evaluated for their ability to survive in the presence of bile salts. After 24 h of anaerobic incubation at 37 °C, all isolates maintained viable counts above 6.00 log CFU/mL in MRS broth containing 0.3% bile salts. However, when the concentration was increased to 0.5%, only isolate PMvet212 retained a viable count of 6.56 ± 0.04 log CFU/mL, indicating substantial bile tolerance. In contrast, isolates PMvet151, PMvet183, and PMvet318 exhibited counts below the detection limit (<3.00 log CFU/mL) at 0.5% bile concentration, while PMvet120 showed partial survival. At 1.0% bile salts, none of the isolates survived above the detection threshold. The survival profiles are presented in Table 3. Table 3. Survival of LAB isolates in MRS broth containing different bile salt concentrations after 24 h of incubation at 37 °C. Values are expressed as mean ± standard deviation (n = 3). Isolate ID 0% (0 h) 0% (24 h, Control) 0.3% (24 h) 0.5% (24 h) PMvet120 6.45 ± 0.4 9.34 ± 0.6 7.35 ± 0.5 3.54 ± 0.3 PMvet151 6.58 ± 0.3 9.02 ± 0.5 7.46 ± 0.4 <3.00 PMvet183 6.47 ± 0.5 9.12 ± 0.6 7.54 ± 0.4 <3.00 PMvet212 6.81 ± 0.4 9.66 ± 0.5 7.81 ± 0.3 6.56 ± 0.4 PMvet318 6.00 ± 0.6 9.08 ± 0.6 7.38 ± 0.5 <3.00 Cell Surface Hydrophobicity (MATH Assay) The hydrophobicity of the five selected LAB isolates was assessed using the microbial adhesion to hydrocarbons (MATH) assay with xylene as the hydrophobic phase. The results revealed considerable variation among isolates. PMvet318 exhibited the highest hydrophobicity (12.38 ± 0.03%), suggesting a relatively better ability to adhere to the intestinal epithelium. This was followed by PMvet212 (7.85 ± 0.02%), while the remaining isolates demonstrated values below 7%, indicating weaker adhesion potential. The hydrophobicity profiles are summarized in Table 4. Table 4. Cell surface hydrophobicity (%) of selected LAB isolates as determined by the MATH assay using xylene. Values are expressed as mean ± standard deviation (n = 3). Isolate ID OD600 Before OD After (0 min) OD After (30 min) %Hydrophobicity (mean) PMvet120 0.937 ± 0.01 1.092 ± 0.02 1.014 ± 0.02 5.93 ± 0.03 PMvet151 0.952 ± 0.02 1.146 ± 0.03 1.042 ± 0.02 9.30 ± 0.04 PMvet183 0.937 ± 0.01 1.145 ± 0.03 1.062 ± 0.03 6.46 ± 0.05 PMvet212 0.871 ± 0.02 1.014 ± 0.02 0.924 ± 0.02 7.85 ± 0.02 PMvet318 0.935 ± 0.02 1.171 ± 0.03 1.010 ± 0.02 12.38 ± 0.03 Hemolytic Activity (Safety Evaluation) Hemolytic activity of the five LAB isolates was assessed on tryptic soy agar (TSA) supplemented with 5% sheep blood. The results indicated that isolates PMvet120, PMvet151, and PMvet183 exhibited γ-hemolysis, characterized by the absence of any clear or discolored zones around colonies, thereby suggesting a non-hemolytic and potentially safe profile for probiotic application. In contrast, isolates PMvet212 and PMvet318 displayed α-hemolysis, evidenced by greenish discoloration surrounding the colonies, which indicates partial red blood cell lysis (figure 2). The presence of α-hemolysis in these isolates raises potential safety concerns and suggests that they may not be suitable candidates for direct probiotic use without further safety evaluation. Antimicrobial Activity Against Escherichia coli and Staphylococcus aureus The antimicrobial activity of the cell-free supernatants (CFS) from the five selected LAB isolates was evaluated using the agar well diffusion assay against E. coli (field iso-late from diarrheic piglet) and Staphylococcus aureus ATCC 6538. Among the tested iso-lates, only PMvet212 and PMvet318 produced distinct and measurable inhibition zones against both target pathogens. In contrast, isolates PMvet120, PMvet151, and PMvet183 demonstrated incomplete or weak inhibition against E. coli and showed no detectable inhibition against S. aureus . The measured diameters of inhibition zones are summarized in Table 5. Table 5. Antimicrobial activity of cell-free supernatants (CFS) from selected LAB isolates against Escherichia coli (field isolate) and Staphylococcus aureus ATCC 6538, determined by the agar well diffusion assay. Values are expressed as mean ± standard deviation (n = 3). Isolate ID Clear zone (mm) – E. coli Clear zone (mm) – S. aureus PMvet120 Incomplete inhibition (no clear zone) 0.0 ± 0.0 PMvet151 Incomplete inhibition (no clear zone) 0.0 ± 0.0 PMvet183 Incomplete inhibition (no clear zone) 0.0 ± 0.0 PMvet212 12.50 ± 0.4 11.75 ± 0.5 PMvet318 11.50 ± 0.3 10.50 ± 0.4 Control (2% lactic acid) 18.50 ± 0.5 15.25 ± 0.4 Discussion The present study identified two lactic acid bacteria (LAB) isolates of the Bacilli group from fecal samples of suckling piglets (7–30 days old) that demonstrated probiotic potential. These findings are in line with earlier reports by Buasai Petsuriyawong (2011) and Dowarah et al. (2018), who successfully isolated LAB from piglet feces collected around weaning age (28–35 days). The detection of LAB at this early stage supports the well-established observation that piglet intestines are naturally colonized by Lactobacillus spp . before weaning, which are among the most prevalent beneficial microbes in the small intestine (Petri et al., 2010). Colonization is strongly influenced by maternal sources, particularly sow feces, the environment, and milk, consistent with evidence from both human and porcine milk showing the transfer of probiotic microorganisms to offspring (Mustakim et al., 2019). Recent investigations further confirm that maternal microbial transfer plays a vital role in shaping early-life gut microbiota composition and disease resilience in piglets (Bogere et al., 2019). In addition to bacterial isolates, three yeast strains were also identified from the piglet feces in this study. Their ability to withstand acidic pH and bile salts is noteworthy, as yeasts are increasingly recognized as effective probiotics due to their ability to survive harsh gastrointestinal conditions. Previous works have identified Saccharomyces boulardii and related yeast genera as promising candidates due to their acid tolerance, bile salt resistance, and ability to inhibit pathogens (Czerucka et al., 2007; Elghandour et al., 2020). More recently, novel yeast strains from livestock sources have been shown to modulate immune responses and protect against enteric infections, reinforcing their potential in animal health management (Boontiam et al., 2022; Canibe et al., 2022). This suggests that while LAB remain the dominant focus of probiotic studies, cocci-shaped LAB such as Enterococcus and yeast isolates should not be over-looked in future investigations. Although these yeast isolates were excluded in this study, they will be preserved and further evaluated in our next phase of work for potential probiotic application in pigs, following recent findings highlighting the efficacy of Saccharomyces strains in modulating gut health. The acid and bile salt tolerance assays conducted here further confirmed strain-specific variability. Out of 135 confirmed LAB isolates, only five survived pro-longed exposure at pH 3.1, although none survived at pH 2.0. This is consistent with earlier findings that probiotic survival in gastric conditions is highly strain-dependent (Yeo et al., 2016). Among these five isolates, PMvet212 demonstrated superior tolerance to 0.5% bile salt, a concentration comparable to the physiological levels encountered in the small intestine. Such tolerance is crucial for probiotic viability after oral administration, as bile salts are known to disrupt microbial membranes (Gotcheva et al., 2002). Recent studies have reinforced the role of bile-tolerant LAB in reducing diarrhea incidence and supporting in-testinal health in weaning piglets (Wang et al., 2020; Wang et al., 2025). Thus, the resilience of PMvet212 under bile stress highlights its potential as a candidate strain for feed supplementation in piglets facing post-weaning stress. Other important probiotic attributes, including adhesion ability and hemolytic activity, were also evaluated. Among the isolates, PMvet318 showed the highest surface hydrophobicity (12.38%), though this value was lower than the threshold (>40%) suggested in some previous studies. This observation highlights the multifactorial nature of bacterial adhesion, which involves not only hydrophobicity but also other factors such as auto-aggregation, extracellular polysaccharide production, and mucin interactions (Garcia-Cayuela et al., 2014). Regarding safety, isolates PMvet120, PMvet151, and PMvet183 exhibited γ-hemolysis, whereas PMvet212 and PMvet318 displayed α-hemolysis. Although α-hemolysis is less severe than β-hemolysis, the presence of hemolytic activity warrants further investigation, as hemolysin genes have been identified in some Lactobacillus strains (Chokesajjawatee et al., 2020). Following current safety recommendations (FAO/WHO, 2006; Anadón et al., 2006; Zendeboodi et al., 2020), only γ-hemolytic strains are considered safe for probiotic use. Thus, while PMvet212 and PMvet318 exhibited promising functional traits, additional genome-based safety evaluations and in vivo tests are required to ensure their suitability for probiotic applications. Antimicrobial activity was detected in isolates PMvet212 and PMvet318, which inhibited both Escherichia coli and Staphylococcus aureus . These findings are noteworthy, since E. coli is a common cause of piglet diarrhea and antibiotic resistance remains a serious issue in swine production. The inhibitory patterns observed here are consistent with reports showing that LAB inhibit pathogens through the production of organic acids and bacteriocins (Sirichokchatchawan et al., 2018; Yeo et al., 2016). More recent evidence indicates that piglet-derived LAB can enhance intestinal integrity and immune function while reducing pathogen colonization (Qiao et al., 2015; Lu et al., 2018). The inhibition zones (10–12 mm) recorded in this study are comparable to those previously reported (11–13 mm) for Lactobacillus strains from piglets (Sirichokchatchawan et al., 2018), suggesting moderate antimicrobial potential typical of naturally occurring LAB. Nevertheless, further in vivo evaluation is needed to confirm probiotic efficacy, adhesion capacity, and safety under farm conditions. Moreover, as the antimicrobial activity observed here mainly reflects acid-mediated inhibition, future studies will examine both untreated and pH-neutralized cell-free supernatants to distinguish organic acid effects from bacteriocin-associated inhibition (Gao et al., 2025). These results provide a foundation for subsequent research exploring combined LAB yeast formulations for improved piglet gut health and sustainable livestock production. Conclusion In this study, two bacilli-shaped LAB isolates derived from piglet feces exhibited promising probiotic potential, demonstrating tolerance to acidic and bile conditions, moderate antimicrobial activity, and partial adhesion ability. These findings indicate their potential as feed additives in swine production, although their overall functional performance remains moderate compared with the properties expected from well-established commercial probiotics. Therefore, further comprehensive evaluations are required. Future research should extend the characterization of these isolates to include inhibition assays against a broader range of enteric pathogens, assessments of immunomodulatory functions, antibiotic resistance profiling, and in vivo performance trials. Additionally, combining LAB with other probiotic candidates such as yeast may offer synergistic benefits for gut health and growth in piglets. Such integrative approaches would contribute to reducing antibiotic dependence, mitigating antimicrobial resistance, and promoting sustainable livestock production. Despite the limitations of this preliminary work, it provides a valuable foundation for probiotic strain selection. Upcoming investigations will focus on genome-based safety screening, adhesion assays using intestinal epithelial cell models, and in vivo validation to confirm the efficacy and safety of these potential probiotic strains under practical farming conditions. Declarations Acknowledgements The authors also thank Faculty of Veterinary Medicine, Chiang Mai university, Thailand for supporting the budget. Statement of Animal Rights This study did not involve the use of live animals. All bacterial experiments were conducted under biosafety approval from the Institutional Biosafety Committee (IBC) of Chiang Mai University (Approval No. CMUIBC A-0763004). Data availability The authors emphasize that the raw data used for this work will be available from the corresponding author only upon a reasonable request. Conflict of Interest Statement The authors have no relevant financial or non-financial interests to disclose. Author contribution Nattakarn Awaiwanont and Montira Intanon: Methodology, Laboratory examination, Writing-original draft, Writing-review and editing, Promporn Inyoo1 and Matsarina Kongton: Sample collection, Panuwat Yamsakul: Sample collection, Methodology, Formal analysis Writing-original draft, Writing-review and editing. 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Qiao J, Li H, Wang Z, Wang W (2015) Effects of Lactobacillus acidophilus dietary supplementation on performance, intestinal barrier function, rectal microflora, and serum immune function in weaned piglets challenged with Escherichia coli lipopolysaccharide. Antonie Van Leeuwenhoek 107:883–891. https://doi.org/10.1007/s10482-015-0380-z Ren D, Li C, Qin Y, Yin R, Du S, Ye F, Liu C, Liu H, Wang M, Li Y, Sun Y, Li X, Tian M, Jin N (2014) In vitro evaluation of the probiotic and functional potential of Lactobacillus strains isolated from fermented food and human intestine. Anaerobe 30:1–10. Senok AC, Ismaeel AY, Botta GA (2015) Probiotics: Facts and myths. Clin Microbiol Infect 11:958–966. Sirichokchatchawan W, Pupa P, Praechansri P, Am-in N, Tanasupawat S, Sonthayanon P, Prapasarakul N (2018) Autochthonous lactic acid bacteria isolated from pig feces in Thailand show probiotic properties and antibacterial activity against enteric pathogenic bacteria. Microb Pathog 119:208–215. Wang H, Xu R, Zhang H, Su Y, Zhu W (2020) Swine gut microbiota and its interaction with host nutrient metabolism. Anim Nutr 6:410–420. Wang M, Zhou X, Birch Hansen LH, Sheng Y, Yu B, He J, Yu J, Zheng P (2025) Complex probiotics can reduce diarrhea by boosting immunity and balancing gut microbiota in weaned piglets. Front Immunol 16. https://doi.org/10.3389/fimmu.2025.1629044 Yeo S, Lee S, Park H, Shin H, Holzapfel W, Huh CS (2016) Development of putative probiotics as feed additives: Validation in a porcine-specific gastrointestinal tract model. Appl Microbiol Biotechnol 100:10043–10054. Yu J, Zuo B, Li Q, Zhao F, Wang J, Huang W, Sun Z, Chen Y (2024) Dietary supplementation with Lactiplantibacillus plantarum P-8 improves the growth performance and gut microbiota of weaned piglets. Microbiol Spectr 12:2. https://doi.org/10.1128/spectrum.02345-22 Zendeboodi F, Khorshidian N, Mortazavian AM (2020) Probiotic: Conceptualization from a new approach. Curr Opin Food Sci 32:103–123. https://doi.org/10.1016/j.cofs.2020.03.009 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7920513","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":542195714,"identity":"5d68bb40-826f-48f3-a38c-61f6f9d46c98","order_by":0,"name":"Nattakarn Awaiwanont","email":"","orcid":"","institution":"Chiang Mai University","correspondingAuthor":false,"prefix":"","firstName":"Nattakarn","middleName":"","lastName":"Awaiwanont","suffix":""},{"id":542195715,"identity":"75212207-5f2a-4e3e-abcd-9690514099f6","order_by":1,"name":"Montira Intanon","email":"","orcid":"","institution":"Chiang Mai 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06:25:39","extension":"html","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":116556,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7920513/v1/44b38ee707510819a1a0eae1.html"},{"id":95800096,"identity":"f5330668-84b0-4f60-8261-594309a37eec","added_by":"auto","created_at":"2025-11-13 08:21:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":752199,"visible":true,"origin":"","legend":"\u003cp\u003eMicroscopic images of representative isolates after Gram staining, observed under a light microscope at 1,000 × magnification: (a) PMvet120; (b) PMvet151; (c) PMvet183; (d) PMvet212; and (e) PMvet318.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7920513/v1/45fe2c95c15dab1f5542a2bf.png"},{"id":95704963,"identity":"b8c84a7b-eeb4-4ea6-a3b0-0f452d771cec","added_by":"auto","created_at":"2025-11-12 06:25:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":517798,"visible":true,"origin":"","legend":"\u003cp\u003eFigure 3. Hemolytic patterns of the five LAB isolates on tryptic soy agar supplemented with 5% sheep blood after 48 h of incubation at 37 °C. Isolates PMvet120 (b), PMvet151 (c), and PMvet183 (d) exhibited γ-hemolysis (no lysis), whereas PMvet212 (e) and PMvet318 (f)showed α-hemolysis (partial lysis, greenish discoloration). \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC 6538 (a) was included as a positive control, displaying β-hemolysis (complete lysis with a clear zone).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7920513/v1/315b29f7da20dde406157655.png"},{"id":99313760,"identity":"fb15bbe7-c449-4a70-9480-1282a252ebdb","added_by":"auto","created_at":"2025-12-31 16:20:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1917854,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7920513/v1/1f395839-86d4-4424-bc87-109d1e78f5f8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Probiotic Potential and Antimicrobial Properties of Lactic Acid Bacteria Isolated from Multi-Age Piglets","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003eEscherichia coli (E. coli)\u003c/em\u003e is one of the most important pathogens causing diarrhea in neonatal and weaned piglets. Piglet diarrhea leads to substantial economic losses in the swine industry due to increased morbidity and mortality, reduced growth performance, and higher veterinary costs (Fairbrother et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Prevention is generally more effective than treatment, yet traditional control strategies have relied heavily on antibiotics (Luppi., 2017) This dependence has raised serious concerns about antibiotic residues in meat and the global spread of antimicrobial resistance (AMR) (Davis et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). As regulatory authorities and consumers increasingly demand reductions in antibiotic use, there is a growing need for sustainable alternatives that can maintain animal health and productivity without contributing to AMR (Moredo et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Several alternative approaches have been proposed, including organic acids, copper sulfate, zinc oxide, prebiotics, herbal supplements, and pro-biotics. Among these, probiotics have attracted particular attention for their ability to promote intestinal health, reduce the incidence and severity of enteric diseases, and support growth performance in pigs, while reducing the need for antibiotics (Namkun et al., 2004). Pro-biotic supplementation has been shown to modulate the gut microbiota, enhance immune function, and improve nutrient utilization in swine. Recent studies have con-firmed that probiotics can serve as viable antibiotic alternatives for preventing bacterial diarrhea in piglets, with benefits extending to improved feed efficiency and over-all health (Bogere et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Moreover, probiotic use aligns with the One Health framework by mitigating AMR risks at the animal\u0026ndash;human\u0026ndash;environment interface (Laird et a., 2021).\u003c/p\u003e\u003cp\u003eAt birth, the gastrointestinal tract of animals is sterile but rapidly colonized by microorganisms from the mother and the surrounding environment. This early colonization plays a critical role in the development of the digestive system, metabolic functions, and immune competence of neonates (Boaventura et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) The gut microbiota\u0026rsquo;s composition is shaped by factors such as delivery mode, maternal microbiota, diet, and environ-mental exposure (Binns., 2013) In pigs, a diverse and balanced gut microbiota supports resistance to pathogens, adaptation to dietary changes, and improved physiological functions (Fouhse et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The porcine gastrointestinal tract is anatomically compartmentalized, with the small intestine particularly the duodenum, jejunum, and ileum being the main site for nutrient digestion and absorption. Maintaining a healthy microbiota in these regions is critical for growth and disease prevention, especially in intensive swine production systems.\u003c/p\u003e\u003cp\u003eProbiotics are defined as live microorganisms that, when administered in adequate amounts, confer health benefits to the host by improving the microbial balance in the gastrointestinal tract (FAO/WHO, 2002). Effective probiotics must survive exposure to gastric acid and bile salts, adhere to intestinal epithelial cells, and exhibit antagonistic effects against pathogens (Kontula et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). In swine production, probiotics are used both to support growth and to prevent colonization by enteric pathogens such as \u003cem\u003eSalmonella spp\u003c/em\u003e., \u003cem\u003eE. coli\u003c/em\u003e, and \u003cem\u003eClostridium perfringens\u003c/em\u003e (De Angelis et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Lactic acid bacteria (LAB) are among the most widely studied probiotic groups due to their non-pathogenic nature, acid and bile tolerance, and ability to produce antimicrobial substances such as organic acids, hydrogen peroxide, and bacteriocins (Mojgani et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). These antimicrobial compounds are considered safe and, in some cases, have been linked to reduced pathogen load and improved gut health in pigs (Ozugul et al., 2018).\u003c/p\u003e\u003cp\u003eThe probiotic properties of LAB are highly strain-specific, even among isolates belonging to the same species (Senok et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Only a limited number of strains with strong acid and bile tolerance, adhesion ability, and potent antimicrobial activity have been identified, and their functional properties often vary depending on the host and environ-mental context. Recent advances in microbiome analysis and targeted isolation techniques have enabled the discovery of novel LAB strains from indigenous pig breeds and different rearing environments, some of which show promise for improving gut integrity, modulating immunity, and reducing pathogenic \u003cem\u003eE. coli\u003c/em\u003e colonization (Yu et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). However, further research is needed to characterize these strains comprehensively and assess their potential as antibiotic alternatives in commercial pig production.\u003c/p\u003e\u003cp\u003eTherefore, the aim of the present study was to isolate and characterize LAB with probiotic potential from fecal samples of pigs at different ages raised without antibiotics or probiotics. The focus was on identifying strains that could survive under gastro-intestinal conditions, exhibit inhibitory activity against pathogenic \u003cem\u003eE. coli\u003c/em\u003e, and demonstrate other desirable probiotic traits. The findings of this work provide a basis for selecting promising LAB strains for development as feed additives to promote swine gut health, reduce reliance on antibiotics, and contribute to the global effort to mitigate AMR in livestock production.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSince fecal samples were collected without handling or restraining the animals, specific approval for animal use was not required. However, the experimental procedures involved the use of microbial isolates. Therefore, all microbiological work was reviewed and approved by the Institutional Biosafety Committee (IBC) of Chiang Mai University (Approval No. CMUIBC A-0763004).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSample Collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study population comprised suckling piglets aged 7–30 days. The required sample size was estimated based on an assumed \u003cem\u003eLactobacillus\u003c/em\u003e detection prevalence of 50%, a total piglet population of 2,800, an effect size of 0.3, statistical power (1 – β) of 0.98, and an alpha error probability of 0.02, using G*Power software (version 3.1). The minimum calculated sample size was 40; therefore, 42 fecal samples were collected to ensure adequate statistical power. Samples were obtained from a commercial pig farm in Lamphun province where no antibiotics or probiotics had been used. To maximize diversity, no more than five samples were taken from any single farrowing pen. Freshly voided feces with normal consistency and yellow to brown coloration were col-lected from the pen floor, placed in sterile 5 mL microcentrifuge tubes (≥2 g each), and transported at 4 °C to the laboratory for processing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation of Lactic Acid Bacteria (LAB)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOne gram of fecal sample was homogenized in 9 mL of 0.85% sterile saline and serially diluted (10⁻⁴ to 10⁻⁸). Aliquots (100 µL) were spread onto de Man, Rogosa, and Sharpe (MRS) agar supplemented with 0.5% CaCO₃. Plates were incubated anaerobi-cally at 37 °C for 48 h. Colonies producing clear zones were subcultured in MRS broth to obtain pure isolates. Gram‐positive, non‐spore‐forming bacteria were retained and tested for catalase activity; only catalase‐negative isolates were preserved at –20 °C in MRS broth with 20% (v/v) sterile glycerol (Guo et al., 2010).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEvaluation of Probiotic Properties\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAcid Tolerance\u003c/p\u003e\n\u003cp\u003eColonies grown on MRS agar were suspended in 5 mL phosphate-buffered saline (PBS; pH 7.2) and adjusted to 0.5 McFarland standard (~1.5 × 108 CFU/mL). One milliliter of suspension was mixed with 9 mL sterile PBS adjusted to pH 2.0 or pH 3.1 using 1 N HCl. The mixtures were vortexed for 10 s and incubated at 37 °C for 3 h. Viable cell counts before and after incubation were determined by plate counting on MRS agar. Acid tolerance was expressed as the reduction in bacterial counts (log CFU/mL) com-pared with the initial value (Guo et al., 2010).\u003c/p\u003e\n\u003cp\u003eBile Salt Tolerance\u003c/p\u003e\n\u003cp\u003eBacterial suspensions prepared as above were inoculated (1 mL) into 9 mL MRS broth supplemented with 0.3%, 0.5%, or 1% (w/v) bile salts (Sigma-Aldrich). Control cultures contained no bile salts. Tubes were incubated anaerobically at 37 °C for 24 h. Viable cell counts were determined at 0 and 24 h, and tolerance was evaluated based on survival relative to controls. Each strain was tested in duplicate (Ren et al., 2014).\u003c/p\u003e\n\u003cp\u003eAdhesion Ability to Intestinal Epithelium (Surface Hydrophobicity)\u003c/p\u003e\n\u003cp\u003eAdhesion potential was assessed by the microbial adhesion to hydrocarbons (MATH) method. Bacteria grown on MRS agar were suspended in 5 mL PBS (pH 7.2) and adjusted to 6.0 McFarland standard. Optical density at 600 nm (OD₆₀₀) was adjusted to 0.6. Three milliliters of suspension was mixed with 1 mL of xylene in a glass tube, vortexed for 90 s, and left at room temperature for 30 min to allow phase separation. The aqueous phase was carefully removed, and OD₆₀₀ was measured again. Surface hydrophobicity (%H) was calculated as:\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;%𝐻 = [(OD₆₀₀ before mixing – OD₆₀₀ after mixing)/OD₆₀₀ before mixing] × 100 \u003csub\u003e100\u003c/sub\u003e\u003c/p\u003e\n\u003cp\u003eControls consisted of PBS and xylene without bacteria. All assays were performed in duplicate (Ekmekci., 2009)\u003c/p\u003e\n\u003cp\u003eHemolytic Activity\u003c/p\u003e\n\u003cp\u003eFor safety evaluation, isolates were streaked on tryptic soy agar (TSA) supplemented with 5% (w/v) sheep blood and incubated at 37 °C for 48 h. Hemolysis type was determined visually: β-hemolysis (clear zone), α-hemolysis (green/brown discoloration), and γ-hemolysis (no change). \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC 6538 was used as a β-hemolysis positive (Adimpong et al., 2002).\u003c/p\u003e\n\u003cp\u003eAntimicrobial Activity Against \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e ATCC 6538\u003c/p\u003e\n\u003cp\u003eThe agar well diffusion method was used. LAB cultures grown in MRS broth at 37 °C for 24 h were centrifuged at 4,000 × g for 5 min, and supernatants were filtered through 0.45 µm membranes to obtain cell-free supernatants (CFS). Indicator strains pathogenic \u003cem\u003eE. coli\u003c/em\u003e (from diarrheic piglets) and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC 6538 were adjusted to 0.5 McFarland standard in PBS and spread onto nutrient agar plates. Wells (7 mm) were bored aseptically, and 80 µL CFS was added per well. Lactic acid (2% v/v) served as the positive control. Plates were incubated at 37 °C for 24 h, and inhibition zones were measured in millimeters. All tests were performed in duplicate (Sirichokchatchawan et al., 2018).\u003c/p\u003e\n\u003cp\u003eData Interpretation\u003c/p\u003e\n\u003cp\u003eProbiotic Properties\u003c/p\u003e\n\u003cp\u003eAcid Tolerance\u003c/p\u003e\n\u003cp\u003eSurvival at acidic pH was assessed by comparing bacterial counts (log CFU/mL) before and after incubation at pH 2.0 and pH 3.1. A reduction of less than 1 log CFU/mL at pH 3.1 was considered acceptable for acid tolerance (Petsuriyawong et al., 2011).\u003c/p\u003e\n\u003cp\u003eBile Salt Tolerance\u003c/p\u003e\n\u003cp\u003eBacterial counts (log CFU/mL) at 0 h and 24 h in MRS broth containing bile salts were compared. Strains were classified as tolerant if no significant reduction in viable counts was observed over the incubation period.\u003c/p\u003e\n\u003cp\u003eSurface Adhesion Ability\u003c/p\u003e\n\u003cp\u003eAdhesion potential was expressed as surface hydrophobicity (%H) calculated by the MATH assay. Higher %H values indicated stronger cell surface adhesion capabilities.\u003c/p\u003e\n\u003cp\u003eHemolytic Activity\u003c/p\u003e\n\u003cp\u003eHemolysis was classified into three types: β-hemolysis, indicating complete lysis of red blood cells and the presence of a clear zone around the colony; α-hemolysis, representing partial lysis with a green or brown discoloration surrounding the colony; and γ-hemolysis, showing no lysis or visible change in the medium. According to safety criteria for probiotic use, LAB strains exhibiting α- or β-hemolysis were considered un-suitable, whereas only γ-hemolytic strains were deemed acceptable (Buxton et al., 2015).\u003c/p\u003e\n\u003cp\u003eAntimicrobial Activity Against \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e ATCC 6538\u003c/p\u003e\n\u003cp\u003eThe antimicrobial potential of the cell-free supernatants (CFS) was assessed by measuring the diameter of the inhibition zones against \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eStaphylococ-cus aureus\u003c/em\u003e ATCC 6538. The results were interpreted according to the criteria summarized in Table 1. This approach allowed for a comparative evaluation of the inhibitory effects of different CFS preparations on the selected pathogenic strains.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 1. Interpretation of antimicrobial activity based on inhibition zone diameter\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eSymbol\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eInterpretation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eInhibition Zone Diameter (mm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eNon-inhibition\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026lt;5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eWeak inhibition\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026gt;5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eIntermediate inhibition\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026gt;10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e+++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eStrong inhibition\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026gt;15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e++++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eVery strong inhibition\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026gt;20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eInterpretation criteria modified from Sirichokchatchawan et al. (2018)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData were analyzed using descriptive statistics using SPSS software (IBM SPSS Statistics, 29 version). For experiments performed on a single isolate with triplicate assays, results were presented as mean values without standard deviation. Means and standard deviations were calculated and reported where appropriate to summarize the results.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eIsolation of Lactic Acid Bacteria (LAB) from Swine Feces\u003c/p\u003e\n\u003cp\u003eFrom 42 individual fecal samples collected from pigs of different ages, a total of 318 bacterial colonies producing clear zones on MRS agar supplemented with 0.5% CaCO₃ under anaerobic incubation at 37 \u0026deg;C for 48 h were initially obtained. These colonies were preliminarily considered as potential lactic acid bacteria. Gram staining revealed that 296 isolates were Gram-positive, of which 146 were rod-shaped (bacilli), 136 were cocci, and 14 were coccobacilli. Nineteen isolates were identified as yeasts based on morphology, and three isolates could not be maintained during subculturing; both groups were excluded from further analysis.\u003c/p\u003e\n\u003cp\u003eCatalase testing of the 296 Gram-positive isolates showed that 277 were cata-lase-negative, consistent with typical LAB characteristics, whereas 19 were cata-lase-positive and thus removed from subsequent screening. Consequently, 135 isolates were confirmed as Gram-positive, rod-shaped, catalase-negative bacteria and retained for probiotic property evaluation. For five isolates with ambiguous Gram-staining results, the test was repeated. Among these, isolates PMvet120, PMvet151, and PMvet183 exhibited budding cells and pleomorphic shapes, which were consistent with yeast morphology rather than bacteria (Figure 1). These isolates also showed poor growth in MRS broth and were therefore excluded.\u003c/p\u003e\n\u003cp\u003eProbiotic Properties of Selected LAB Isolates\u003c/p\u003e\n\u003cp\u003eAcid Tolerance\u003c/p\u003e\n\u003cp\u003eAmong the 135 LAB isolates confirmed as Gram-positive, rod-shaped, and cata-lase-negative, only five strains PMvet120, PMvet151, PMvet183, PMvet212, and PMvet318 demonstrated acid tolerance, with a reduction in viable counts of approximately 1.00 log CFU/mL or less after incubation at pH 3.1 for 3 h. No isolates survived exposure to pH 2.0 under the same conditions. The survival rates of these five isolates at pH 3.1 are summarized in Table 2.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Table 2. Survival of selected LAB isolates at pH 3.1 after 3 h of incubation at 37 \u0026deg;C. Values are expressed as mean \u0026plusmn; standard deviation (n = 3).\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003eIsolate ID\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\"\u003e\n \u003cp\u003elog CFU/mL\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eInitial Count (average)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003epH 3.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePMvet120\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"5\"\u003e\n \u003cp\u003e6.37\u0026plusmn;0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.35\u0026plusmn;0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePMvet151\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e6.20\u0026plusmn;0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePMvet183\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e6.15\u0026plusmn;0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePMvet212\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.60\u0026plusmn;0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePMvet318\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.56\u0026plusmn;0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eBile Salt Tolerance\u003c/p\u003e\n\u003cp\u003eThe five acid-tolerant isolates were subsequently evaluated for their ability to survive in the presence of bile salts. After 24 h of anaerobic incubation at 37 \u0026deg;C, all isolates maintained viable counts above 6.00 log CFU/mL in MRS broth containing 0.3% bile salts. However, when the concentration was increased to 0.5%, only isolate PMvet212 retained a viable count of 6.56 \u0026plusmn; 0.04 log CFU/mL, indicating substantial bile tolerance. In contrast, isolates PMvet151, PMvet183, and PMvet318 exhibited counts below the detection limit (\u0026lt;3.00 log CFU/mL) at 0.5% bile concentration, while PMvet120 showed partial survival. At 1.0% bile salts, none of the isolates survived above the detection threshold. The survival profiles are presented in Table 3.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 3. Survival of LAB isolates in MRS broth containing different bile salt concentrations after 24 h of incubation at 37 \u0026deg;C. Values are expressed as mean \u0026plusmn; standard deviation (n = 3).\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eIsolate ID\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0% (0 h)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0% (24 h, Control)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0.3% (24 h)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0.5% (24 h)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePMvet120\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e6.45 \u0026plusmn; 0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e9.34 \u0026plusmn; 0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e7.35 \u0026plusmn; 0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3.54 \u0026plusmn; 0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePMvet151\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e6.58 \u0026plusmn; 0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e9.02 \u0026plusmn; 0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e7.46 \u0026plusmn; 0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026lt;3.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePMvet183\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e6.47 \u0026plusmn; 0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e9.12 \u0026plusmn; 0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e7.54 \u0026plusmn; 0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026lt;3.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePMvet212\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e6.81 \u0026plusmn; 0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e9.66 \u0026plusmn; 0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e7.81 \u0026plusmn; 0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e6.56 \u0026plusmn; 0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePMvet318\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e6.00 \u0026plusmn; 0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e9.08 \u0026plusmn; 0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e7.38 \u0026plusmn; 0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026lt;3.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eCell Surface Hydrophobicity (MATH Assay)\u003c/p\u003e\n\u003cp\u003eThe hydrophobicity of the five selected LAB isolates was assessed using the microbial adhesion to hydrocarbons (MATH) assay with xylene as the hydrophobic phase. The results revealed considerable variation among isolates. PMvet318 exhibited the highest hydrophobicity (12.38 \u0026plusmn; 0.03%), suggesting a relatively better ability to adhere to the intestinal epithelium. This was followed by PMvet212 (7.85 \u0026plusmn; 0.02%), while the remaining isolates demonstrated values below 7%, indicating weaker adhesion potential. The hydrophobicity profiles are summarized in Table 4.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 4. Cell surface hydrophobicity (%) of selected LAB isolates as determined by the MATH assay using xylene. Values are expressed as mean \u0026plusmn; standard deviation (n = 3).\u0026nbsp;\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"614\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eIsolate ID\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eOD\u0026lt;sub\u0026gt;600\u0026lt;/sub\u0026gt; Before\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eOD After\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(0 min)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eOD After\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(30 min)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e%Hydrophobicity\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(mean)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePMvet120\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.937 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1.092 \u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1.014 \u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5.93 \u0026plusmn; 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePMvet151\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.952 \u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1.146 \u0026plusmn; 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1.042 \u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e9.30 \u0026plusmn; 0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePMvet183\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.937 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1.145 \u0026plusmn; 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1.062 \u0026plusmn; 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e6.46 \u0026plusmn; 0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePMvet212\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.871 \u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1.014 \u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.924 \u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e7.85 \u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePMvet318\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.935 \u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1.171 \u0026plusmn; 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1.010 \u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e12.38 \u0026plusmn; 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eHemolytic Activity (Safety Evaluation)\u003c/p\u003e\n\u003cp\u003eHemolytic activity of the five LAB isolates was assessed on tryptic soy agar (TSA) supplemented with 5% sheep blood. The results indicated that isolates PMvet120, PMvet151, and PMvet183 exhibited \u0026gamma;-hemolysis, characterized by the absence of any clear or discolored zones around colonies, thereby suggesting a non-hemolytic and potentially safe profile for probiotic application. In contrast, isolates PMvet212 and PMvet318 displayed \u0026alpha;-hemolysis, evidenced by greenish discoloration surrounding the colonies, which indicates partial red blood cell lysis (figure 2). The presence of \u0026alpha;-hemolysis in these isolates raises potential safety concerns and suggests that they may not be suitable candidates for direct probiotic use without further safety evaluation.\u003c/p\u003e\n\u003cp\u003eAntimicrobial Activity Against \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe antimicrobial activity of the cell-free supernatants (CFS) from the five selected LAB isolates was evaluated using the agar well diffusion assay against \u003cem\u003eE. coli\u003c/em\u003e (field iso-late from diarrheic piglet) and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC 6538. Among the tested iso-lates, only PMvet212 and PMvet318 produced distinct and measurable inhibition zones against both target pathogens. In contrast, isolates PMvet120, PMvet151, and PMvet183 demonstrated incomplete or weak inhibition against \u003cem\u003eE. coli\u003c/em\u003e and showed no detectable inhibition against \u003cem\u003eS. aureus\u003c/em\u003e. The measured diameters of inhibition zones are summarized in Table 5.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 5. Antimicrobial activity of cell-free supernatants (CFS) from selected LAB isolates against \u003cem\u003eEscherichia coli\u003c/em\u003e (field isolate) and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC 6538, determined by the agar well diffusion assay. Values are expressed as mean \u0026plusmn; standard deviation (n = 3).\u0026nbsp;\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" class=\"fr-table-selection-hover\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIsolate ID\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eClear zone (mm) \u0026ndash; \u003cem\u003eE. coli\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eClear zone (mm) \u0026ndash; \u003cem\u003eS. aureus\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003ePMvet120\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003eIncomplete inhibition (no clear zone)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e0.0 \u0026plusmn; 0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003ePMvet151\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003eIncomplete inhibition (no clear zone)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e0.0 \u0026plusmn; 0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003ePMvet183\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003eIncomplete inhibition (no clear zone)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e0.0 \u0026plusmn; 0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003ePMvet212\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003e12.50 \u0026plusmn; 0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e11.75 \u0026plusmn; 0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003ePMvet318\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003e11.50 \u0026plusmn; 0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e10.50 \u0026plusmn; 0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eControl (2% lactic acid)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003e18.50 \u0026plusmn; 0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e15.25 \u0026plusmn; 0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study identified two lactic acid bacteria (LAB) isolates of the Bacilli group from fecal samples of suckling piglets (7\u0026ndash;30 days old) that demonstrated probiotic potential. These findings are in line with earlier reports by Buasai Petsuriyawong (2011) and Dowarah et al. (2018), who successfully isolated LAB from piglet feces collected around weaning age (28\u0026ndash;35 days). The detection of LAB at this early stage supports the well-established observation that piglet intestines are naturally colonized by \u003cem\u003eLactobacillus spp\u003c/em\u003e. before weaning, which are among the most prevalent beneficial microbes in the small intestine (Petri et al., 2010). Colonization is strongly influenced by maternal sources, particularly sow feces, the environment, and milk, consistent with evidence from both human and porcine milk showing the transfer of probiotic microorganisms to offspring (Mustakim et al., 2019). Recent investigations further confirm that maternal microbial transfer plays a vital role in shaping early-life gut microbiota composition and disease resilience in piglets (Bogere et al., 2019).\u003c/p\u003e\n\u003cp\u003eIn addition to bacterial isolates, three yeast strains were also identified from the piglet feces in this study. Their ability to withstand acidic pH and bile salts is noteworthy, as yeasts are increasingly recognized as effective probiotics due to their ability to survive harsh gastrointestinal conditions. Previous works have identified \u003cem\u003eSaccharomyces boulardii\u003c/em\u003e and related yeast genera as promising candidates due to their acid tolerance, bile salt resistance, and ability to inhibit pathogens (Czerucka et al., 2007; Elghandour et al., 2020). More recently, novel yeast strains from livestock sources have been shown to modulate immune responses and protect against enteric infections, reinforcing their potential in animal health management (Boontiam et al., 2022; Canibe et al., 2022). This suggests that while LAB remain the dominant focus of probiotic studies, cocci-shaped LAB such as \u003cem\u003eEnterococcus\u003c/em\u003e and yeast isolates should not be over-looked in future investigations. Although these yeast isolates were excluded in this study, they will be preserved and further evaluated in our next phase of work for potential probiotic application in pigs, following recent findings highlighting the efficacy of \u003cem\u003eSaccharomyces\u003c/em\u003e strains in modulating gut health.\u003c/p\u003e\n\u003cp\u003eThe acid and bile salt tolerance assays conducted here further confirmed strain-specific variability. Out of 135 confirmed LAB isolates, only five survived pro-longed exposure at pH 3.1, although none survived at pH 2.0. This is consistent with earlier findings that probiotic survival in gastric conditions is highly strain-dependent (Yeo et al., 2016). Among these five isolates, PMvet212 demonstrated superior tolerance to 0.5% bile salt, a concentration comparable to the physiological levels encountered in the small intestine. Such tolerance is crucial for probiotic viability after oral administration, as bile salts are known to disrupt microbial membranes (Gotcheva et al., 2002). Recent studies have reinforced the role of bile-tolerant LAB in reducing diarrhea incidence and supporting in-testinal health in weaning piglets (Wang et al., 2020; Wang et al., 2025). Thus, the resilience of PMvet212 under bile stress highlights its potential as a candidate strain for feed supplementation in piglets facing post-weaning stress.\u003c/p\u003e\n\u003cp\u003eOther important probiotic attributes, including adhesion ability and hemolytic activity, were also evaluated. Among the isolates, PMvet318 showed the highest surface hydrophobicity (12.38%), though this value was lower than the threshold (\u0026gt;40%) suggested in some previous studies. This observation highlights the multifactorial nature of bacterial adhesion, which involves not only hydrophobicity but also other factors such as auto-aggregation, extracellular polysaccharide production, and mucin interactions (Garcia-Cayuela et al., 2014). Regarding safety, isolates PMvet120, PMvet151, and PMvet183 exhibited \u0026gamma;-hemolysis, whereas PMvet212 and PMvet318 displayed \u0026alpha;-hemolysis. Although \u0026alpha;-hemolysis is less severe than \u0026beta;-hemolysis, the presence of hemolytic activity warrants further investigation, as hemolysin genes have been identified in some Lactobacillus strains (Chokesajjawatee et al., 2020). Following current safety recommendations (FAO/WHO, 2006; Anad\u0026oacute;n et al., 2006; Zendeboodi et al., 2020), only \u0026gamma;-hemolytic strains are considered safe for probiotic use. Thus, while PMvet212 and PMvet318 exhibited promising functional traits, additional genome-based safety evaluations and in vivo tests are required to ensure their suitability for probiotic applications.\u003c/p\u003e\n\u003cp\u003eAntimicrobial activity was detected in isolates PMvet212 and PMvet318, which inhibited both \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e. These findings are noteworthy, since \u003cem\u003eE. coli\u003c/em\u003e is a common cause of piglet diarrhea and antibiotic resistance remains a serious issue in swine production. The inhibitory patterns observed here are consistent with reports showing that LAB inhibit pathogens through the production of organic acids and bacteriocins (Sirichokchatchawan et al., 2018; Yeo et al., 2016). More recent evidence indicates that piglet-derived LAB can enhance intestinal integrity and immune function while reducing pathogen colonization (Qiao et al., 2015; Lu et al., 2018). The inhibition zones (10\u0026ndash;12 mm) recorded in this study are comparable to those previously reported (11\u0026ndash;13 mm) for \u003cem\u003eLactobacillus\u003c/em\u003e strains from piglets (Sirichokchatchawan et al., 2018), suggesting moderate antimicrobial potential typical of naturally occurring LAB. Nevertheless, further \u003cem\u003ein vivo\u003c/em\u003e evaluation is needed to confirm probiotic efficacy, adhesion capacity, and safety under farm conditions. Moreover, as the antimicrobial activity observed here mainly reflects acid-mediated inhibition, future studies will examine both untreated and pH-neutralized cell-free supernatants to distinguish organic acid effects from bacteriocin-associated inhibition (Gao et al., 2025). These results provide a foundation for subsequent research exploring combined LAB yeast formulations for improved piglet gut health and sustainable livestock production.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, two bacilli-shaped LAB isolates derived from piglet feces exhibited promising probiotic potential, demonstrating tolerance to acidic and bile conditions, moderate antimicrobial activity, and partial adhesion ability. These findings indicate their potential as feed additives in swine production, although their overall functional performance remains moderate compared with the properties expected from well-established commercial probiotics. Therefore, further comprehensive evaluations are required.\u0026nbsp;Future research should extend the characterization of these isolates to include inhibition assays against a broader range of enteric pathogens, assessments of immunomodulatory functions, antibiotic resistance profiling, and in vivo performance trials. Additionally, combining LAB with other probiotic candidates such as yeast may offer synergistic benefits for gut health and growth in piglets. Such integrative approaches would contribute to reducing antibiotic dependence, mitigating antimicrobial resistance, and promoting sustainable livestock production. Despite the limitations of this preliminary work, it provides a valuable foundation for probiotic strain selection. Upcoming investigations will focus on genome-based safety screening, adhesion assays using intestinal epithelial cell models, and \u003cem\u003ein vivo\u003c/em\u003e validation to confirm the efficacy and safety of these potential probiotic strains under practical farming conditions.\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors also thank Faculty of Veterinary Medicine, Chiang Mai university, Thailand for supporting the budget.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eStatement of Animal Rights\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study did not involve the use of live animals. All bacterial experiments were conducted under biosafety approval from the Institutional Biosafety Committee (IBC) of Chiang Mai University (Approval No. CMUIBC A-0763004).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors emphasize that the raw data used for\u0026nbsp;this work will be available from the corresponding author only upon\u0026nbsp;a reasonable request.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eConflict of Interest Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNattakarn Awaiwanont\u0026nbsp;and Montira Intanon: Methodology, Laboratory examination, Writing-original draft, Writing-review and editing, Promporn Inyoo1 and Matsarina Kongton: Sample collection, Panuwat Yamsakul: Sample collection, Methodology,\u0026nbsp;Formal analysis Writing-original draft, Writing-review and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors gratefully acknowledge Faculty of Veterinary Medicine, Chiang Mai university, Thailand.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAdimpong DB, Nielsen DS, Sorensen KI, Derkx PMF, Jespersen L (2002) Genotypic characterization and safety assessment of lactic acid bacteria from indigenous African fermented food products. 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Curr Opin Food Sci 32:103\u0026ndash;123. https://doi.org/10.1016/j.cofs.2020.03.009\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Piglet feces, Lactic acid bacteria (LAB), Probiotic traits, Antimicrobial effects, Escherichia coli","lastPublishedDoi":"10.21203/rs.3.rs-7920513/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7920513/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLactic acid bacteria (LAB) are important probiotics that support intestinal health and growth performance in pigs through modulation of gut microbiota and production of antimicrobial compounds. This study aimed to isolate and evaluate LAB with probiotic potential from feces of healthy, antibiotic- and probiotic-free piglets of different ages. Forty-two fecal samples were collected from a farm in Lamphun province, yielding 318 LAB isolates, of which 135 gram-positive, catalase-negative bacilli were characterized for probiotic traits. Only two isolates (PMvet212 and PMvet318) survived at pH 3.1 with a viability loss of less than 1 log CFU/mL; both tolerated 0.3% bile salt, and PMvet212 also survived at 0.5% bile salt. Their hydrophobicity values were 7.85% and 12.38%, respectively, indicating low adhesion capacity. Both isolates showed alpha-hemolysis. Cell-free supernatants of these isolates inhibited \u003cem\u003eEscherichia coli\u003c/em\u003e from diarrheic piglets and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC6538, with inhibition zones classified as intermediate. These findings indicate that LAB from piglet feces, particularly PMvet212, possess moderate probiotic potential and antibacterial activity, and may serve as candidates for development as feed additives to promote swine gut health and reduce reliance on antibiotics.\u003c/p\u003e","manuscriptTitle":"Probiotic Potential and Antimicrobial Properties of Lactic Acid Bacteria Isolated from Multi-Age Piglets","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-12 06:25:34","doi":"10.21203/rs.3.rs-7920513/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"92a11b6a-40fc-4b98-84e0-e8d0c8943756","owner":[],"postedDate":"November 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-26T12:39:25+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-12 06:25:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7920513","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7920513","identity":"rs-7920513","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}