Gajami-sikhae is a traditional Korean fermented fish food made by naturally fermenting flatfish (Glyptocephalus stelleri) with other ingredients. This study was the first to investigate the diversity and dynamics of lactic acid bacteria in gajami-sikhae fermented at different temperatures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). A total of 4824 isolates were isolated from the fermented gajami-sikhae. These findings indicated that Latilactobacillus, Lactiplantibacillus, Levilactobacillus, Weissella, and Leuconostoc were the dominant genera during fermentation, while the dominant species were Latilactobacillus sakei, Lactiplantibacillus plantarum, Levilactobacillus brevis, Weissella koreensis, and Leuconostoc mesenteroides. At all temperatures, L. sakei was dominant at the early stage of gajami-sikhae fermentation, and it maintained dominance until the later stage of fermentation at low temperatures (5 °C and 10 °C). However, L. plantarum and L. brevis replaced it at higher temperatures (15 °C and 20 °C). The relative abundance of L. plantarum and L. brevis reached 100% at the later fermentation stage at 20 °C. These results suggest that the optimal fermentation temperatures for gajami-sikhae are low rather than high temperatures. This study could allow for the selection of an adjunct culture to control gajami-sikhae fermentation.

Unlike Enterococcus faecium strains, some Enterococcus lactis strains are considered potential probiotic strains as they lack particular virulence and antibiotic resistance genes. However, these closely related species are difficult to distinguish via conventional taxonomic methods. Here, for the first time, we used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with BioTyper and in-house databases to distinguish between E. faecium and E. lactis. A total of 58 reference and isolated strains (89.2%) were correctly identified at the species level using MALDI-TOF MS with in-house databases. However, seven strains (10.8%) were not accurately differentiated as a single colony was identified as a different species with a similar score value. Specific mass peaks were identified by analyzing reference strains, and mass peaks at 10,122 ± 2 m/z, 3650 ± 1 m/z, and 7306 ± 1 m/z were unique to E. faecium and E. lactis reference strains, respectively. Mass peaks verified reproducibility in 60 isolates and showed 100% specificity, whereas 16S rRNA sequencing identified two different candidates for some isolates (E. faecium and E. lactis). Our specific mass peak method helped to differentiate two species, with high accuracy and high throughput, and provided a viable alternative to 16S rRNA sequencing.