Lactic acid bacteria have been considered safe and commonly used in foods and fermentation processes (Bernardeau et al., 2006; Zielinska & Kolonzyn-Krajewska, 2018). Typically, they are Grampositive, mostly nonmotile, nonspore-forming, facultative anaerobic (or microaerophilic), and rod-(or cocci-) shaped bacteria that are utilized in fermented dairy and nondairy products such as fermented vegetables, meats, and beverages (Nuraida, 2015). Although the majority of probiotics contain Lactobacillus and Bifidobacterium, Streptococcus genus is one of the most widely used probiotic strain. Streptococcus thermophilus has been generally isolated from traditional fermented milk and yogurt products (Vendramin et al., 2017; Zhang et al., 2019). In particular, S. thermophilus has been used as a starter for dairy products because of rapid acidifying capacity during fermentation process (Naumenko et al., 2019; Santos et al., 2019). It has been also reported to exhibit several health-beneficial effects such as growth inhibition of many pathogenic bacteria and strong adherence to the gastrointestinal tract (Braun et al., 2020; Iyer et al., 2010; Wu et al., 2014). Although probiotics have been regarded as GRAS (generally recognized as safe) strains, recent significant developments in the field

Purpose Lactobacillus reuteri is one of the most largely studied human-commensal bacteria and widely used as a form of probiotics. Safety of probiotics has become increasingly important for human consumption due to increasing health-concerns in food industry. In this study, the safety of L. reuteri IDCC 3701 isolated from human breast milk was thoroughly investigated. Methods Whole-genome sequence analysis was performed to identify antibiotic resistance and toxigenicity of L. reuteri IDCC 3701. Phenotypic analysis such as minimal inhibitory concentration, β-hemolysis, extracellular enzyme activity, and the production of biological amines and L/D-lactate, was investigated. Finally, acute oral toxicity test was performed to access L. reuteri IDCC 3701 safety. Results Although multiple resistances to gentamicin and kanamycin were observed in L. reuteri IDCC 3701, it was revealed that these resistances are intrinsic and not transferable through whole-genome analysis. In addition, various phenotypic analysis concerning hemolysis, enzyme activity, and D-lactate production did not show any negative results. Although L. reuteri IDCC 3701 harbors a histidine decarboxylase gene, no biogenic amines were detected. Finally, L. reuteri IDCC 3701 exhibited no evidence of acute toxicity according to an in vivo study. Conclusion Our findings demonstrate that L. reuteri IDCC 3701 is considered to be safe for human consumption as probiotics based on the in silico, in vitro and in vivo studies.