The reagents and chemicals used in this study include M17 broth (115029), MRS broth (110661), and Brain-Heart Infusion (BHI) agar (110493), all purchased from Merck. Sigma-Aldrich provided the sugars for fermentation tests (sucrose, fructose, glucose, maltose, galactose, lactose, mannitol, and sorbitol), MRS broth, and glucose for EPS quantification. Nutrient agar was obtained from HiMedia Laboratories. Hydrogen peroxide (3%), phenol red, trichloroacetic acid [80% weight/volume (w/v)], ethanol (90%), and glycerol (20%) were all sourced from VWR International. The API 20 Strep kit was supplied by bioMérieux. All reagents were prepared and used according to the manufacturer’s instructions.

Twenty-six yoghurt samples were collected randomly from households and local markets in Rawalpindi, Pakistan (Table S1). Samples were stored in sterile glass bottles at 4°C and processed within two hours under hygienic conditions. LAB was isolated using M17 and MRS media. M17 broth (1.96 g) was mixed with 1 g lactose and 0.92 g agar in 50 mL distilled water, and MRS broth (2.6 g) was similarly prepared. Both media were sterilized at 121°C using traditional autoclaving for 15 minutes. After cooling down, they were poured into Petri plates in a laminar flow hood. After ensuring no contamination, samples were streaked on MRS media and incubated at 37°C for 48 hours. Aerobic conditions were maintained by placing the plates in a standard incubator. Anaerobic conditions were maintained using an Oxoid Anaerobic Jar HP0011 with an atmosphere generation system AN0035 (Thermo Fisher Scientific), which created an anaerobic environment suitable for the growth of anaerobic bacteria. The BHI agar medium was used to grow pathogenic bacteria E. coli and S. aureus, and nutrient agar media for the antimicrobial evaluation.

Morphological identification of colonies included observing colour, shape, size, and structure. Gram staining was done following the basic protocol [20]. Catalase activity was tested by placing isolates on a slide and treating them with 3% hydrogen peroxide, observing for bubble formation [21]. Sugar fermentation tests used eight sugars (sucrose, fructose, glucose, maltose, galactose, lactose, mannitol, and sorbitol) to identify microorganisms at the species level. Each sugar (1 g) was dissolved in 10 mL distilled water, sterilised by syringe filtration, and mixed with nutrient broth containing phenol red as an indicator. Tubes were inoculated with isolates and incubated at 37°C for 48 hours.

Further molecular analysis, such as 16S ribosomal RNA (rRNA) sequencing, is recommended to confirm species-level identification. While this study primarily focused on screening isolates for EPS production and their probiotic properties, future work will include 16S rRNA sequencing and alignment for precise taxonomic classification of the isolated strains.

Isolates were preserved in MRS and M17 broth with 20% glycerol at 4°C. Cultures were incubated for 24 hours at 37°C, centrifuged, and stored. Isolated strains were screened for EPS production by streaking on MRS and M17 agar plates containing 5% lactose and incubating anaerobically at 37°C for 48 hours. EPS-producing strains exhibited mucoid or ropy colonies and were preserved in MRS broth with 10% glycerol at –20°C.

The production of EPS from the isolated strains was achieved by using sucrose and glucose (5% w/v) as carbon sources. Samples were incubated at 37°C for 24–48 hours, and cells were removed by centrifugation at 13,000 rpm for 10 minutes. The supernatant was treated with 2.5% (v/v) of 80% (w/v) trichloroacetic acid to remove proteins, followed by centrifugation at 12,000 rpm for 15 minutes. The resulting supernatant was mixed with three volumes of 90% cold ethanol and incubated overnight at 4°C to precipitate the EPS. The EPS was recovered by centrifugation, dried at 42°C, and resuspended in distilled water.

EPS was quantified using the phenol-sulfuric acid method [22]. Absorbance was measured at 490 nm with a spectrophotometer (Thermo Scientific Evolution 201). A standard curve for glucose was made for quantification. Strains with a high amount of EPS were compared with the help of a graph of standard sugar solutions. Total EPS was estimated in each sample by the phenol-sulphuric acid method using glucose as a standard. Higher-producing EPS strains were identified using the API Strep system. Strains were inoculated into API Strep kits and incubated at 36°C for 24 hours, and results were recorded based on colour changes.

Antagonistic effects of probiotics against foodborne pathogens were tested. Preserved cultures were activated in MRS and M17 broth with 1% tryptone, streaked on MRS and M17 agar plates, and incubated at 37°C for 24 hours. Bacteriocin production was evaluated by inoculating isolated LAB in MRS and M17 broth. Foodborne pathogens were grown on nutrient agar, and bacteriocin production was tested using the disc diffusion method. The antimicrobial activity was evaluated by placing sterile discs impregnated with bacteriocin solution onto the agar plates inoculated with the test pathogens. After incubation at 37°C for 24 hours, the clear inhibition zones around the discs were measured using a digital calliper. The diameter of each inhibition zone was recorded in millimeters. Each experiment was performed in triplicate to ensure reproducibility.

Out of 29 bacterial isolates, 12 species belonging to the cocci family and 17 species belonging to the rods family were identified from 26 yoghurt samples (Table S2). Twelve isolates were divided into three main groups of cocci according to their morphological and biochemical behaviour. Five Enterococcus faecium isolates, four were Lactococcus lactis, and three were Streptococcus thermophilus. On the other hand, according to their biochemical evaluation, seventeen Lactobacillus species showed their belongingness to 5 different groups. Five Lactobacillus groups include Lactobacillus delbrueckii (specie delbrueckii), Lactobacillus plantarum, Limosilactobacillus fermentum, Lacticaseibacillus helveticus, and Lacticaseibacillus paracasei subsp. paracasei.

LAB was isolated by directly streaking raw samples on autoclaved, solidified, and selective M17 and MRS media. After incubation for 24 hours at 37°C, visible colonies were selected visually, as shown in Figure 1a and 1b, and picked for further purification. The pH of the media was adjusted to 6.5–6.9 for the optimum growth of LAB.

The isolated colonies on Petri plates were mostly circular and creamy white. Most colonies have a smooth, margined, and shiny appearance. All the isolates were Gram-positive, making Gram-positive rods and cocci-making chains and clusters, as shown in Figure 2a and 2b, when observed under a 100× microscope lens. The rods were in the form of long threads, and cocci were in chains and pairs. Furthermore, isolated species showed negative behaviour during the catalase test when treated with 3% H₂O₂ because they lacked catalase enzyme, as shown in Figure 2c. Table S3 shows the details of the colony shape, colour, margins, cell shape, and Gram staining results for various LAB species.

Out of twenty-nine identified strains of LAB, only twelve strains that showed mucoidity (Table S4) were subjected to screening for EPS production, as shown in Table 1. This includes three strains of S. thermophilus, two strains each of L. lactis, E. faecium, and L. fermentum, and one strain each of L. plantarum, L. paracasei subsp. paracasei, and L. helveticus. These strains were then subjected to a sugar fermentation test for their identification according to their fermentation pattern of 8 sugars: fructose, glucose, galactose, maltose, sucrose, lactose, sorbitol, and mannitol. E. faecium showed 100% fermentation with some variability for fructose, while S. thermophilus showed fermentation of sucrose sorbitol, glucose, and lactose.

Table 2 gives the EPS for each strain. The highest values of EPS were observed in two strains of S. thermophilus and L. lactis, 62 and 52 µg/mL, respectively. In contrast, the lowest value of EPS was observed in the strain of E. faecium. The difference in EPS amount might be the time coupled with a suitable carbon source and incubation time and temperature at which the culture was tested for their analysis.

Standardized identification of higher-producing EPS strain was performed using API Strep. Results were obtained based on colour change after 24 hours of incubation at 34°C. API Strep was used to identify streptococci and test the fermentation ability of 10 carbohydrates. After 24 hours of incubation, all the tubes showed positive results (Table 3). API Strep tests were positive for the fermentation of ribose. Arabinose, mannitol, sorbitol, lactose, trehalose, inulin, raffinose, starch, and glycogen. Moreover, positive results were also recorded for acetoin production, β-glucuronidase, α- and β-galactosidase, alkaline phosphatase, leucine arylamidase, and arginine dihydrolase production. Test results concluded that there was 99.9% homology with the S. thermophilus strain, an essential strain for yoghurt starter culture.

LAB isolated from yoghurt were subjected to test their anti-microbial activity against food-borne pathogens, mainly E. coli and S. aureus, as shown in Table 4. All investigated isolates showed mostly antagonistic activity against E. coli and S. aureus. Our findings suggest that wild strains of EPS-producing LAB isolated from local yoghurt samples with antimicrobial properties are essential. When they are used for starter culture in fermented dairy products, they show effective control against E. coli and S. aureus grown in products.

The study demonstrated the probiotic potential of EPS-producing LAB from yoghurt, specifically S. thermophilus, L. lactis, and L. fermentum, showing significant antimicrobial activity against E. coli and S. aureus. Their EPS production suggests these LAB strains can act as natural preservatives, enhancing food safety and improving the texture, stability, and health benefits of fermented products. Additionally, EPS offers health benefits like anti-ulcer, cholesterol-lowering, and immunomodulatory effects, making them ideal for functional foods. The study highlights EPS-producing LAB as a natural preservative alternative to synthetic chemicals, meeting the demand for safer, health-promoting ingredients. Future research should optimize EPS production and test these strains in various food matrices to evaluate long-term stability, aiding the development of novel functional foods and preservatives that promote healthier choices.