Citation: Nasuti, C.; Ruffini, J.; Sola, L.; Di Bacco, M.; Raimondi, S.; Candeliere, F.; Solieri, L. Sour Beer as Bioreservoir of Novel Craft Ale Yeast Cultures. Microorganisms 2023, 11, 2138. https://doi.org/10.3390/ microorganisms11092138 Academic Editor: Maurizio Ciani Received: 12 July 2023 Revised: 8 August 2023 Accepted: 20 August 2023 Published: 23 August 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). microorganisms Article Sour Beer as Bioreservoir of Novel Craft Ale Yeast Cultures Chiara Nasuti 1 , Jennifer Ruffini 1 , Laura Sola 2 , Mario Di Bacco 3 , Stefano Raimondi 2 , Francesco Candeliere 2 and Lisa Solieri 1,4, * 1 Department of Life Sciences, University of Modena and Reggio Emilia, Via Amendola, 2-Pad. Besta, 42122 Reggio Emilia, Italy; chiara.nasuti@unimore.it (C.N.); 227818@studenti.unimore.it (J.R.) 2 Department of Life Sciences, University of Modena and Reggio Emilia, Via Campi 51, 41125 Modena, Italy; laura.sola@unimore.it (L.S.); stefano.raimondi@unimore.it (S.R.); francesco.candeliere@unimore.it (F.C.) 3 Ca’ Del Brado Brewery, Via Andrea Costa, 146/2, 40065 Rastignano, Italy; quark@cadelbrado.it 4 National Biodiversity Future Center (NBFC), 90133 Palermo, Italy * Correspondence: lisa.solieri@unimore.it; Tel.: +39-0522522026 Abstract: The increasing demand for craft beer is driving the search for novel ale yeast cultures from brewing-related wild environments. The focus of bioprospecting for craft cultures is to identify feral yeasts suitable to imprint unique sensorial attributes onto the final product. Here, we integrated phy- logenetic, genotypic, genetic, and metabolomic techniques to demonstrate that sour beer during aging in wooden barrels is a source of suitable craft ale yeast candidates. In contrast to the traditional lambic beer maturation phase, during the aging of sour-matured production-style beer, different biotypes of Saccharomyces cerevisiae dominated the cultivable in-house mycobiota, which were followed by Pichia membranifaciens, Brettanomyces bruxellensis, and Brettanomyces anomalus. In addition, three putative S. cerevisiae × Saccharomyces uvarum hybrids were identified. S. cerevisiae feral strains sporulated, produced viable monosporic progenies, and had the STA1 gene downstream as a full-length promoter. During hopped wort fermentation, four S. cerevisiae strains and the S. cerevisiae × S. uvarum hybrid WY213 exceeded non-Saccharomyces strains in fermentative rate and ethanol production except for P. membranifaciens WY122. This strain consumed maltose after a long lag phase, in contrast to the phenotypic profile described for the species. According to the STA1+ genotype, S. cerevisiae partially consumed dextrin. Among the volatile organic compounds (VOCs) produced by S. cerevisiae and the S. cerevisiae × S. uvarum hybrid, phenylethyl alcohol, which has a fruit-like aroma, was the most prevalent. In conclusion, the strains characterized here have relevant brewing properties and are exploitable as indigenous craft beer starters. Keywords: sour beer; craft brewing; STA1 gene; dextrin; hybrid; Saccharomyces cerevisiae; Saccharomyces uvarum; Pichia membranifaciens 1. Introduction Craft beer production has gained increasing interest in many countries as an alternative to mainstream beer [1]. Craft brewing refers to relatively small breweries that aim to produce beers with unique and original sensory characteristics based on quality and diversity [2]. Special flavors increase perceived quality compared to commercial beer [3,4]. The sensory profile of beer is determined by the choice of malt or adjunct, hop variety, water quality and yeast, which catalyzes metabolic reactions during wort fermentation, giving the beer its flavor profile. Yeasts are responsible not only for sugar consumption and ethanol and CO 2 production but also for releasing aroma-active secondary side-products of alcoholic fermentation—higher alcohols and ethyl and acetate esters—that contribute to solvent-like and fruity notes and a sweet taste [5]. They also can transform neutral precursors in the wort and hops—glycosides, polyfunctional thiols, and terpenes—into sensorial active compounds, as reviewed by Svedlund et al. [6]. This bio-flavoring aptitude qualitatively and quantitatively varies depending on the strain and species employed [7]. Microorganisms 2023, 11, 2138. https://doi.org/10.3390/microorganisms11092138 https://www.mdpi.com/journal/microorganisms