Unraveling yeast diversity in food fermentation using ITS1-2 amplicon-based metabarcoding

A detailed characterization of the microbial ecosystem involved in the production processes of fermented foods is essential. Although fermented foods are an important part of the human diet and have an increasing interest nowadays, some challenges still need to be solved. Specifically, yeast identification through culture-independent methodologies is still limited to the genus level. Unlike for bacterial species identifications, long-read sequencing technologies have barely been used for yeast species identification, and, to the best of the authors' knowledge, it has not been validated with mock communities reflecting food fermentation processes yet. Therefore, in the current study, we present an amplicon-based metabarcoding approach targeting the full-length internal transcribed spacer (ITS) region comprising the ITS1, 5.8S rRNA gene, and ITS2 using the PacBio HiFi sequencing platform. This approach was validated using mock communities composed of yeast species involved in sourdough, lambic beer, and cocoa fermentation processes. Accurate species-level identification was achieved for most of the species. However, special attention should be given to Saccharomyces-rich niches, as accurate species-level identification for this genus is still challenging. Furthermore, underestimation of the relative abundance of species with short ITS regions, such as Pichia and Brettanomyces, occurred. In addition, the method was successfully applied to describe the yeast diversity present in two sourdough and two lambic beer samples. Overall, the current approach provides an unprecedented way of determining the species-level yeast composition of complex ecosystems present in fermented food products.