Some non-Saccharomyces yeasts, including Metschnikowia pulcherrima, have been proposed as selected starters due to their contribution for the overall aroma and chemical profiles of wines. In this work, we aimed to evaluate the genetic and phenotypic diversity of Metschnikowia pulcherrima strains isolated from different locations of Douro Wine Region, and to explore their potential as co-adjuncts of S. cerevisiae in alcoholic fermentation. For that purpose, a set of 64 M. pulcherrima isolates were used. Polymerase chain reaction (PCR) fingerprinting with M13 primers demonstrated to be an efficient tool in intraspecific discrimination of M. pulcherrima strains. No significant associations were found between genotypic profiles and either geographical origin or winery. The isolates were screened for their stress resistance ability (ethanol, SO 2 , chitosan, copper, H 2 O 2 , and Grape Juice Medium), aroma-related activities (resistance to 5, 5 , 5 -trifluor-D, L-leucine and cerulenin and β-glycosidase, β-lyase and sulfite-reductase activities) as well as other relevant technological proprieties (protease activity and biogenic amines production). M. pulcherrima response to the different enological traits evaluated was greatly strain-dependent. The most discriminant features were the ability of the strains to grow in Grape-Juice Medium (GJM) and sulfite-reductase, and their β-lyase and protease activities. The enological potential of a selected M. pulcherrima strain in mixed-culture with S. cerevisiae was also assessed in natural grape-juice of a local variety, under two nitrogen regimes. M. pulcherrima proved to be promising for future industrial application as a co-starter, lowering ethanol, acetic acid and, reported here for the first time, lowering hydrogen sulfide levels in the wines.

Non-Saccharomyces yeasts have received increased attention by researchers and winemakers, due to their particular contributions to the characteristics of wine. In this group, Saccharomycodes ludwigii is one of the less studied species. In the present study, a native S. ludwigii strain, UTAD17 isolated from the Douro wine region was characterized for relevant oenological traits. The genome of UTAD17 was recently sequenced. Its potential use in winemaking was further evaluated by conducting grape-juice fermentations, either in single or in mixed-cultures, with Saccharomyces cerevisiae, following two inoculation strategies (simultaneous and sequential). In a pure culture, S. ludwigii UTAD17 was able to ferment all sugars in a reasonable time without impairing the wine quality, producing low levels of acetic acid and ethyl acetate. The overall effects of S. ludwigii UTAD17 in a mixed-culture fermentation were highly dependent on the inoculation strategy which dictated the dominance of each yeast strain. Wines whose fermentation was governed by S. ludwigii UTAD17 presented low levels of secondary aroma compounds and were chemically distinct from those fermented by S. cerevisiae. Based on these results, a future use of this non-Saccharomyces yeast either in monoculture fermentations or as a co-starter culture with S. cerevisiae for the production of wines with greater expression of the grape varietal character and with flavor diversity could be foreseen.

This work describes, for the first time, the genome sequence of a Saccharomycodes ludwigii strain. Although usually seen as a wine spoilage yeast, S. ludwigii has been of interest for the production of fermented beverages because it harbors several interesting properties, including the production of beneficial aroma compounds.

Grapes’ infection by phytopathogenic fungi may often lead to rot and impair the quality and safety of the final product. Due to the concerns associated with the extensive use of chemicals to control these fungi, including their toxicity for environment and human health, bio-based products are being highly preferred, as eco-friendlier and safer alternatives. Specifically, yeasts have shown to possess antagonistic activity against fungi, being promising for the formulation of new biocontrol products.In this work 397 wine yeasts, isolated from Portuguese wine regions, were studied for their biocontrol potential against common grapes phytopathogenic fungal genera: Aspergillus, Botrytis, Mucor and Penicillium. This set comprised strains affiliated to 32 species distributed among 20 genera. Time-course monitoring of mold growth was performed to assess the inhibitory activity resulting from either diffusible or volatile compounds produced by each yeast strain. All yeasts displayed antagonistic activity against at least one of the mold targets. Mucor was the most affected being strongly inhibited by 68% of the tested strains, followed by Botrytis (20%), Aspergillus (19%) and Penicillium (7%). More notably, the approach used allowed the detection of a wide array of yeast-induced mold response profiles encompassing, besides the decrease of mold growth, the inhibition or delay of spore germination and the complete arrest of mycelial extension, and even its stimulation at different phases. Each factor considered (taxonomic affiliation, mode of action and fungal target) as well as their interactions significantly affected the antagonistic activity of the yeast isolates. The highest inhibitions were mediated by volatile compounds. Total inhibition of Penicillium was achieved by a strain of Metschnikowia pulcherrima, while the best performing yeasts against Mucor, Aspergillus and Botrytis, belong to Lachancea thermotolerans, Hanseniaspora uvarum and Starmerella bacillaris, respectively. Notwithstanding the wide diversity of yeasts tested, only three strains were found to possess a broad spectrum of antagonistic activity, displaying strong or very strong inhibition against the four fungal targets tested. Our results confirm the potential of wine yeasts as biocontrol agents, while highlighting the need for the establishment of fit-for-purpose selection programs depending on the mold target, the timing, and the mode of application.