Probing the microbial diversity and probiotic candidates from Pakistani foods: isolation, characterization, and functional profiling

Probiotics represent beneficial living microorganisms that confer physiological, nutritional, and functional advantages to human health, holding significant potential for development of functional foods. This research aimed to isolate, identify, and characterize potential probiotic bacterial strains sourced from fermented and non-fermented foods from Pakistan. A total of 341 bacterial strains were isolated from diverse food samples (81) collected from various regions of Pakistan. Strains were identified using 16S rRNA gene sequencing and phylogenetic analysis. The identified strains belonged to genera Bacillus , Staphylococcus , Microbacterium , Shigella , Micrococcus , Enterococcus , Sporosarcina , Paenibacillus , Limosilactobacillus , Kosakonia , Dietzia , Leclercia , Lacticaseibacillus , Levilactobacillus , Kluyvera , Providencia , Enterobacter , Neisseria , Streptococcus , Acinetobacter , Corynebacterium , Pantoea , Mammaliicoccus , Pseudomonas , Burkholderia , and Alkalihalobacillus . Selected strains were chosen for probiotic assessment, employing existing literature as a guideline. Among these selections, six strains exhibited hemolytic activity, and seven strains displayed resistance to multiple antibiotics, prompting their exclusion from subsequent evaluations. The remaining strains demonstrated auto-aggregation capacities spanning 3.39–79.7%, and displayed coaggregation capabilities with reported food-borne pathogens. Furthermore, nine strains exhibited antimicrobial properties against food-borne pathogens. The assessment encompassed diverse characteristics such as cell surface hydrophobicity, survival rates under varying conditions, cholesterol reduction ability, casein digestion capability, and antioxidant activity. Phylogenomic analysis, digital-DNA DNA hybridization (digi-DDH), and average nucleotide identity (ANI) calculations unveiled novel species potentially belonging to the genera Sporosarcina and Dietzia . Based on these findings, we advocate for the consideration of Staphylococcus cohnii subsp. cohnii NCCP-2414, Lacticaseibacillus rhamnosus NCCP-2569 and Levilactobacillus brevis NCCP-2574 as prime probiotic candidates with the potential for integration into formulation of functional foods.