Microbial and Metabolic Dynamics during Fermentation of Vinegar with Rye Bread

Vinegar is a widely consumed acidic fermented product prepared from diverse raw materials. In this study, we investigated vinegar fermentation using rye bread as a substrate, focusing on the comparative dynamics of spontaneous and backslopping fermentation. Two fermentation batches were produced, each in triplicate. One batch was subjected to spontaneous fermentation, while the other was inoculated with a starter culture via backslopping. The pH, microbial communities, and metabolite profiles were analyzed at various stages of fermentation to characterize the fermentation process. In both batches, the pH decreased to below 3.5, and the microbial abundance increased, with greater changes observed in the inoculated batch. Community analysis revealed that Acetobacter dominated the late fermentation stage in both batches. Clostridium and Gluconacetobacter were more abundant in the spontaneous batch, whereas Leuconostoc, Lactobacillus, Gluconobacter, along with Pichia, and Wickerhamomyces were dominant in the inoculated batch. Ethanol was a major early stage metabolite, particularly in the inoculated batch, whereas acetic acid became predominant in both batches during the later stages of fermentation. There were batch-specific variations in lactic and butyric acids. The main amino acids identified in both batches were glycine, serine, and proline. Statistical analysis revealed distinct volatile profiles between the batches, with butyric acid, ethanol, 2,4,5-trimethyl-1,3-dioxolane, and ethyl butanoate enriched in the spontaneous batch, while mesitylene, ethyl acetate, α-terpineol, and phenethyl acetate were more abundant in the inoculated batch. This study explored vinegar fermentation using rye bread, which may serve as a model for utilizing upcycled or surplus bread in sustainable fermentation processes.