Ethanolphilic lactic acid bacterium Fructilactobacillus fructivorans as the key microorganism for fermentation of narazuke, a traditional Japanese preserved food.

Narazuke is a traditional Japanese preserved food produced by repeatedly embedding salted vegetables in sake kasu, which is a byproduct remaining after pressing sake mash. While the prototype of narazuke existed as early as the 8th century and has long been enjoyed by Japanese people, whether microbial fermentation occurs during narazuke-making remained unclear. We first analyzed the microbiota of narazuke throughout manufacturing processes, showing that the prokaryotic microbial community gradually converged and was finally dominated by a single lactic acid bacterium (LAB), Fructilactobacillus fructivorans. Laboratory-scale narazuke-making tests also demonstrated that F. fructivorans dominated the niche, with a concomitant increase in lactic acid after incubation for 2 months, revealing that the LAB was responsible for narazuke fermentation. The environment of narazuke-making is quite harsh for microbial survival because of its high ethanol content, suggesting that F. fructivorans proliferating in narazuke has potent ethanol tolerance. Thus, we isolated LAB strains from narazuke and investigated the effect of ethanol on their growth. The isolated narazuke strains grew even in the presence of 15% ethanol, whereas the type strain didn't grow. The narazuke strains exhibited significantly faster growth in media containing 5%-10% ethanol than in ethanol-free medium, which is known as ethanolphilicity. Taken together, this study identified F. fructivorans as the main fermenting microbe in narazuke. Its abundance in an ethanol-rich environment is attributed to ethanolphilicity, which is characterized by active growth in the presence of ethanol, highlighting a novel microbial strategy for adaptation to a niche with high ethanol content.IMPORTANCEEthanolphilicity is a notable microbial trait that is distinct from ethanol tolerance because ethanolphilic microorganisms exhibit more active growth in the presence of ethanol than in an ethanol-free milieu. Although the mechanisms of ethanol tolerance have been intensively investigated in various microorganisms, the molecular basis underlying the effects of ethanolphilicity remains elusive. In the current study, we isolated the ethanolphilic LAB Fructilactobacillus fructivorans from traditional Japanese preserved food. Comparative transcriptomic analysis suggested that altered fatty acid metabolism may be involved in ethanolphilicity. Ethanolphilicity is induced by ethanol as well as other alcohols, including methanol and isopropanol. Because industrial fermentation processes are usually performed in the presence of various alcohols and solvents, which often prohibit microbial growth, our findings are expected to be applicable for developing robust strains with ethanolphilicity, as well as novel enzymes working in solvents.