foods Article Effect of a Multistarter Yeast Inoculum on Ethanol Reduction and Population Dynamics in Wine Fermentation Xiaolin Zhu, María-Jesús Torija , Albert Mas , Gemma Beltran * and Yurena Navarro   Citation: Zhu, X.; Torija, M.-J.; Mas, A.; Beltran, G.; Navarro, Y. Effect of a Multistarter Yeast Inoculum on Ethanol Reduction and Population Dynamics in Wine Fermentation. Foods 2021, 10, 623. https://doi.org/ 10.3390/foods10030623 Academic Editors: Carmine Foschino and Ileana Vigentini Received: 5 February 2021 Accepted: 12 March 2021 Published: 15 March 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2021 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/). Department of Biochemistry and Biotechnology, Faculty of Oenology, University Rovira i Virgili (URV), Marcel·lí Domingo 1, 43007 Tarragona, Catalonia, Spain; xiaolin.zhu@estudiants.urv.cat (X.Z.); mjesus.torija@urv.cat (M.-J.T.); albert.mas@urv.cat (A.M.); yurenadelosangeles.navarro@urv.cat (Y.N.) * Correspondence: gemma.beltran@urv.cat; Tel.: +34-977-558442 Abstract: Microbiological strategies are currently being considered as methods for reducing the ethanol content of wine. Fermentations started with a multistarter of three non-Saccharomyces yeasts (Metschnikowia pulcherrima (Mp), Torulaspora delbrueckii (Td) and Zygosaccharomyces bailii (Zb)) at different inoculum concentrations. S. cerevisiae (Sc) was inoculated into fermentations at 0 h (coinoculation), 48 h or 72 h (sequential fermentations). The microbial populations were analyzed by a culture-dependent approach (Wallerstein Laboratory Nutrient (WLN) culture medium) and a culture- independent method (PMA-qPCR). The results showed that among these three non-Saccharomyces yeasts, Td became the dominant non-Saccharomyces yeast in all fermentations, and Mp was the minority yeast. Sc was able to grow in all fermentations where it was involved, being the dominant yeast at the end of fermentation. We obtained a significant ethanol reduction of 0.48 to 0.77% (v/v) in sequential fermentations, with increased concentrations of lactic and acetic acids. The highest reduction was achieved when the inoculum concentration of non-Saccharomyces yeast was 10 times higher (10 7 cells/mL) than that of S. cerevisiae. However, this reduction was lower than that obtained when these strains were used as single non-Saccharomyces species in the starter, indicating that interactions between them affected their performance. Therefore, more combinations of yeast species should be tested to achieve greater ethanol reductions. Keywords: wine; PMA-qPCR; Metschnikowia pulcherrima; Torulaspora delbrueckii; Zygosaccharomyces bailii; mixed fermentation; coinoculation; sequential fermentation 1. Introduction In recent years, the average ethanol concentration in wine has increased, mainly due to climate change and consumer preference for wine styles [1–3]. Different strategies have been applied to reduce ethanol production in wine, such as decreasing the leaf area to lower the sugar content in grape berries, reducing the maturity of grapes, removing sugar from the grape must, developing or screening low-alcohol wine yeasts and removing alcohol from wine (reviewed by [4]). Compared with microbiological strategies, other strategies might have negative effects on wine, such as delaying maturity, reducing the yield of berries, causing a significant reduction in anthocyanins, soluble solids and volatile compounds, and decreasing the wine color [5–8]. Therefore, microbiological strategies were considered to be effective and accompanied by smaller impacts on the wine sensory profile and quality. In particular, the use of non-Saccharomyces yeast strains for reducing the alcohol content of wines has been proven to improve the wine aroma complexity and has become a consistent proposal [9–11]. Non-Saccharomyces yeasts have been applied in fermentations to reduce ethanol using different inoculation strategies. For example, several studies have shown that single- culture fermentations with Hanseniaspora uvarum, Lachancea thermotolerans, Metschnikowia pulcherrima, Starmerella bombicola, Starmerella bacillaris, Zygosaccharomyces bailii, Zygosac- charomyces bisporus, and Zygosaccharomyces sapae species are able to reduce the ethanol Foods 2021, 10, 623. https://doi.org/10.3390/foods10030623 https://www.mdpi.com/journal/foods