Dynamic transformation of the sulfur metabolome during natto fermentation: Supersulfide omics study

Supersulfides are sulfur species with catenated sulfur atoms, such as cysteine hydropersulfide (CysSSH). Although supersulfides are biologically important metabolites owing to their unique chemical properties, their transformation in plants via microbial fermentation remains unknown. Natto is a traditional Japanese food prepared from soybeans fermented using Bacillus subtilis var. natto and is an excellent model for investigating this transformation. Compared to unfermented soybeans, natto contains higher supersulfide contents; however, the specific molecular changes that occur during fermentation remain unclear. Herein, we investigated the molecular profiles of supersulfides in natto using mass spectrometry-based supersulfide omics. Quantitative supersulfide profiling revealed an increase in soybean supersulfide content during fermentation using B. subtilis var. natto. However, the total sulfur content did not significantly change, suggesting that microorganisms may play a role in the biotransformation of sulfur-containing molecules into supersulfides. Furthermore, quantitative supersulfide metabolomics and untargeted polysulfide omics revealed time-dependent fermentation increases in the levels of both reduced supersulfides, including CysSSH and cysteine hydrotrisulfide, and oxidized supersulfides, such as cystine trisulfide. Moreover, high-molecular-weight supersulfides were detected. Thus, the supersulfide content significantly increased during B. subtilis var. natto fermentation. Supersulfidated proteins were not detected in natto, likely because soybean-derived proteins were degraded by B. subtilis var. natto. Conversely, supersulfide proteomics revealed the presence of various supersulfide-modified proteins in soybeans, particularly the supersulfidation of 11S glycinin. This is the first study to reveal that microbial fermentation significantly transforms the supersulfide profile in plants. Moreover, the diverse supersulfides abundantly present in natto may contribute to its health-promoting properties.