Characterization of Stress Tolerance and Probiotic Traits of Streptococcus salivarius Strains Isolated from Wildflower Honey

Abstract

Honey represents a distinctive microbial habitat that imposes physicochemical stresses on its resident microbiota while also serving as a potential source of beneficial microorganisms. Two Streptococcus salivarius strains, DOW-5A and DOW-10, isolated from Korean wildflower honey were identified through phenotypic observation and 16S rRNA sequencing. Comprehensive evaluation, including 5 safety assessments and 14 functional assays, was conducted to reveal distinct potential probiotic characteristics in the two isolates. Both strains exhibited no gelatinase or hemolytic activity and showed no significant cytotoxicity toward Caco-2 cells. While DOW-5A maintained higher viable counts during the simulated gastric phase, DOW-10 showed an increase after an initial decline during the simulated intestinal phase. Both strains also exhibited growth under stress conditions involving phenol or hydrogen peroxide. Among the cell-surface and adhesion-related characteristics, DOW-5A exhibited greater mucin adhesion and cell-surface hydrophobicity, whereas DOW-10 showed higher autoaggregation at 6 and 12 h. Both strains exhibited DPPH radical-scavenging activity, with no significant difference from that of the reference probiotic strain, Lactobacillus rhamnosus GG. Atypical and crystalline-like colony morphologies, suggestive of EPS production, were observed when sucrose was supplied as the carbon source. Furthermore, both strains formed biofilms in a crystal violet staining assay, while DOW-5A showed a marked increase in biofilm biomass upon sucrose supplementation. These findings highlight strain-specific variation in stress tolerance and probiotic-associated traits between the two honey-derived Strep. salivarius isolates and support their further investigation as probiotic candidates.