The objective of this study was to isolate, identify, and assess the safety and functionality in vitro of putative probiotic bacterial strains. Isolation procedures were based on standard methods using elective and selective media. The isolates were identified by comparative 16S rRNA sequencing analysis while their safety was determined according to the safety tests recommended by the FAO/WHO such as antibiotic resistance, hemolysin, and biogenic amine production. Most of the isolates did not pass the in vitro safety tests; therefore, only Lactiplantibacillus plantarum (from ant intestine and cheese), Lacticaseibacillus paracasei (from goat milk and kimchi), Enterococcus faecium (from chili doenjang and vegetables with kimchi ingredients), Limosilactobacillus fermentum (from saliva), and Companilactobacillus alimentarius (from kimchi) were identified and selected for further studies. The isolates were further differentiated by rep-PCR and identified to the strain level by genotypic (16S rRNA) and phenotypic (Gen III) approaches. Subsequently, the strain tolerance to acid and bile was evaluated resulting in good viability after simulated gastrointestinal tract passage. Adhesion to mucin in vitro and the presence of mub, mapA, and ef-tu genes confirmed the adhesive potential of the strains and the results of features associated with adhesion such as hydrophobicity and zeta potential extended the insights. This study reflects the importance of fermented and non-fermented food products as a promising source of lactic acid bacteria with potential probiotic properties. Additionally, it aims to highlight the challenges associated with the selection of safe strains, which often fail in the in vitro tests, thus hindering the possibilities of "uncovering" novel and safe probiotic strains.

CISAS Center for Research and Development in Agrifood Systems and Sustainability Escola Superior de Tecnologia e Gestão Instituto Politécnico de Viana do Castelo Viana do Castelo Portugal

Food Research Center Departamento de Alimentos e Nutrição Experimental Faculdade de Ciências Farmacêuticas Universidade de São Paulo São Paulo SP 05508 000 Brazil

Human Effective Microbes Department of Advanced Convergence Handong Global University Pohang 37554 Gyeongbuk South Korea

Laboratory of Infection Oncology Institute of Clinical Molecular Biology Christian Albrechts University Kiel University Hospital Schleswig Holstein Schwanenweg 20 24105 Kiel Germany

ProBacLab Department of Advanced Convergence Handong Global University Pohang 37554 Gyeongbuk South Korea

ProBacLab Laboratório de Microbiologia de Alimentos Departamento de Alimentos e Nutrição Experimental Faculdade de Ciências Farmacêuticas Universidade de São Paulo São Paulo SP 05508 000 Brazil

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