| Summary: | <i>Nono</i>, an important traditional fermented dairy food produced from cow’s milk in Nigeria, was studied for microbial diversity and for starter culture development for industrial production. On the basis of a polyphasic approach, including phenotypic and genotypic methods such as 16S rRNA gene sequencing, repetitive element PCR (rep-PCR) fingerprinting metagenomics, and whole genome sequencing, we identified <i>Lactobacillus</i> (<i>Lb.</i>) <i>helveticus</i>, <i>Limosilactobacillus (L.) fermentum</i>, <i>Lb. delbrueckii</i>, and <i>Streptococcus</i> (<i>S.</i>) <i>thermophilus</i> as predominant bacterial species involved with milk fermentation during traditional <i>nono</i> production in Nigeria, while the predominant yeast species in <i>nono</i> was identified as <i>Saccharomyces cerevisiae</i>. Using metagenomics, <i>Shigella</i> and potential pathogens such as enterobacteria were detected at low levels of abundance. Strains of the predominant lactic acid bacteria (LAB) were selected for starter cultures combination on the basis of their capacities for rapid growth in milk and reduction of pH below 4.5 and their gelling characteristic, which was demonstrated noticeably only by the <i>S. thermophilus</i> strains. Whole genome sequence analysis of selected bacterial strains showed the largest assembled genome size to be 2,169,635 bp in <i>Lb. helveticus</i> 314, while the smallest genome size was 1,785,639 bp in <i>Lb. delbrueckii</i> 328M. Genes encoding bacteriocins were not detected in all the strains, but all the LAB possessed genes potentially involved in diacetyl production and citrate metabolism. These bacteria isolated from <i>nono</i> can thus be used to improve the microbial safety quality of <i>nono</i> in Nigeria, in addition to improving technological parameters such as gelling viscosity, palatability, and product consistency.
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