This study selected the best folate-producing strains in folate-free medium among five commercial starter and probiotic strains and evaluated the utilisation of fruit by-products (pitaya, passion fruit and grape), modified pectin from orange albedo (MP) and other carbohydrate sources for growth and folate production by the selected strains. The three selected strains [Lacticaseibacillus (L.) rhamnosus GG, Bifidobacterium (B.) infantis BB-02, and Streptococcus (S.) thermophilus TH-4] grew equally well in m-MRS broth containing these products or carbohydrates, reaching populations above 7 log CFU mL⁻¹ in 24 h. Production of folate was strain-dependent. L. rhamnosus GG showed the highest folate production when supplemented with fruit by-products and MP, achieving the utmost folate concentration after 24 h in the presence of grape by-product (101.2 ± 11.2 ng mL⁻¹). The MP has also influenced positively the production of folate by this strain. The study evidenced that fruit by-products can be explored as substrates for bio-enriched food product development using selected folate-producing strains.

The escalating global concern regarding bacterial antibiotic resistance in animal production has intensified the search for sustainable and effective alternatives to conventional antimicrobials. In this study, two L. reuteri strains (LBM-Ti195 and LBM-Ti173) are isolated from broiler cecal microbiota that were characterized through an integrated approach, combining phenotypic assays with comparative genomic analysis. Both strains exhibited antibacterial activity against relevant veterinary and foodborne pathogens, including Listeria monocytogenes CECT 934, Staphylococcus aureus CECT 239, Clostridium perfringens Type A, and Campylobacter jejuni CCAMP 162. The inhibitory activity anti-S. aureus increased by more than 10% modifying cultivation conditions, while comparative genomic analysis identified an M23-family metallopeptidase as a potential candidate for pathogen inhibition. Phenotypically, both strains produced folate and metabolized fructooligosaccharides (FOS) and inulin, supporting their potential compatibility with synbiotic formulations. Genome reconstruction reinforces these functional findings by revealing a complete predicted de novo folate biosynthesis pathway. In addition, CAZyme annotation identified higher copy numbers of glycosyltransferases GT2 and GT4 compared with the reference strains, suggesting differences in cell-surface carbohydrate metabolism and exopolysaccharide (EPS)-associated traits. Safety profiling revealed no hemolytic activity or conserved virulence factors under the tested conditions. However, phenotypic tetracycline resistance was detected, and in silico analysis identified an acquired tetW gene in a putative plasmid-associated context, highlighting the importance of an in-depth evaluation of strains with probiotic potential. Collectively, these findings position LBM-Ti195 and LBM-Ti173 as promising avian-derived L. reuteri candidates for next-generation zootechnical probiotic development, while highlighting antimicrobial resistance (AMR) mitigation and further functional validation as essential steps toward safe application.