It is well established that non-Saccharomyces wine yeasts, considered in the past as undesired or spoilage yeasts, can enhance the analytical composition, and aroma profile of the wine. The contribution of non-Saccharomyces yeasts, including the ability to secret enzymes and produce secondary metabolites, glycerol and ethanol, release of mannoproteins or contributions to color stability, is species- and strain-specific, pointing out the key importance of a clever strain selection. The use of mixed starters of selected non-Saccharomyces yeasts with strains of Saccharomyces cerevisiae represents an alternative to both spontaneous and inoculated wine fermentations, taking advantage of the potential positive role that non-Saccharomyces wine yeast species play in the organoleptic characteristics of wine. In this context mixed starters can meet the growing demand for new and improved wine yeast strains adapted to different types and styles of wine. With the aim of presenting old and new evidences on the potential of non-Saccharomyces yeasts to address this market trend, we mainly review the studies focused on non-Saccharomyces strain selection and design of mixed starters directed to improve primary and secondary aroma of wines. The ability of non-Saccharomyces wine yeasts to produce enzymes and metabolites of oenological relevance is also discussed.

Abstract:Nowadays it is widely accepted that non-Saccharomyces yeasts, which prevail during the early stages of alcoholic fermentation, contribute significantly to the character and quality of the final wine. Among these yeasts, Wickerhamomyces anomalus (formerly Pichia anomala, Hansenula anomala, Candida pelliculosa) has gained considerable importance for the wine industry since it exhibits interesting and potentially exploitable physiological and metabolic characteristics, although its growth along fermentation can still be seen as an uncontrollable risk. This species is widespread in nature and has been isolated from different environments including grapes and wines. Its use together with Saccharomyces cerevisiae in mixed culture fermentations has been proposed to increase wine particular characteristics. Here, we review the ability of W. anomalus to produce enzymes and metabolites of oenological relevance and we discuss its potential as a biocontrol agent in winemaking. Finally, biotechnological applications of W. anomalus beyond wine fermentation are briefly described.

Metabolism by phase II enzymes and transport from intestinal cells back into the lumen by ATP binding cassette (ABC) transporters limits the bioavailability of the flavanone hesperetin, the aglycone of hesperidin. This study investigates to what extent other flavonoids modulate the metabolism and transport of hesperetin by characterizing the effect of co-administrating a series of flavonoids using Caco-2 cell monolayers in a two-compartment transwell system. Flavonoids may interfere with hesperetin metabolism and can also inhibit the apically located ABC transporter breast cancer resistance protein (ABCG2) which was previously shown to be responsible for the apical transport of hesperetin metabolites. Co-exposure of Caco-2 cell monolayers to hesperetin with specific flavonoids reduced the ratio of apical efflux to basolateral transport of hesperetin metabolites, and in some cases, also reduced the amount of hesperetin metabolites detected extracellularly. As intracellular accumulation of hesperetin metabolites did not account for this decrease, inhibition of metabolism of hesperetin is likely the underlying mechanism for the reduced metabolite formation and excretion. In spite of the reduction in metabolism the amount of hesperetin metabolites transported to the basolateral side significantly increased upon co-exposure with specific flavonoids and therefore co-administration of specific flavonoids could be a strategy to improve the bioavailability of hesperetin.

Climate, soil, and grape varieties are the primary characteristics of terroir and lead to the definition of various appellations of origin. However, the microbiota associated with grapes are also affected by these conditions and can leave a footprint in a wine that will be part of the characteristics of terroir. Thus, a description of the yeast microbiota within a vineyard is of interest not only to provide a better understanding of the winemaking process, but also to understand the source of microorganisms that maintain a microbial footprint in wine from the examined vineyard. In this study, two typical grape varieties, Grenache and Carignan, have been sampled from four different vineyards in the DOQ Priorat winegrowing region. Afterward, eight spontaneous alcoholic fermentations containing only grapes from one sampling point and of one variety were conducted at laboratory scale. The fermentation kinetics and yeast population dynamics within each fermentation experiment were evaluated. Yeast identification was performed by RFLP-PCR of the 5.8S-ITS region and by sequencing D1/D2 of the 26S rRNA gene of the isolates. The fermentation kinetics did not indicate clear differences between the two varieties of grapes or among vineyards. Approximately 1,400 isolates were identified, exhibiting high species richness in some fermentations. Of all the isolates studied, approximately 60% belong to the genus Hanseniaspora, 16% to Saccharomyces, and 11% to Candida. Other minor genera, such as Hansenula, Issatchenkia, Kluyveromyces, Saccharomycodes, and Zygosaccharomyces, were also found. The distribution of the identified yeast throughout the fermentation process was studied, and Saccharomyces cerevisiae was found to be present mainly at the end of the fermentation process, while Aureobasidium pullulans was isolated primarily during the first days of fermentation in three of the eight spontaneous fermentations. This work highlights the complexity and diversity of the vineyard ecosystem, which contains yeasts from different species. The description of this yeast diversity will lead to the selection of native microbiota that can be used to produce quality wines with the characteristics of the Priorat.

26The yeasts present during the ripening process of ewes' and goats' cheeses 27 produced in a small traditional dairy in Mediterranean Spain were isolated and 28 identified. Five hundred and thirty strains pertaining to eleven yeast species 29 representing eight genera were identified using molecular methods.

The contribution of Debaryomyces hansenii, Kluyveromyces lactis and Kluyveromyces marxianus strains to the typical flavour of traditional ewes' and goats' cheeses was assessed. Fourteen yeast strains were grown in liquid medium mimicking cheese composition and volatile compounds were identified by GC-MS. Yeasts were able to produce key volatile compounds characteristic of the cheeses from which they were isolated. Inter-species and inter-strain variations were observed. Under the conditions tested D. hansenii produced the lowest levels of volatile compounds, with large intra-strain variations. Kluyveromyces strains primarily produced esters and alcohols. K. marxianus strains were associated with the production of acids, ethyl decanoate, 1propanol and benzaldehyde, whereas K. lactis was correlated with the presence of ketones, ethyl acetate and secondary alcohols. In conclusion, this study shows the heterogeneous potential of dairy yeasts to contribute to final cheese flavour. b Linear retention indices (LRI) of the compounds eluted from the GC-MS using a DB-624 595 capillary column (J&W Scientific 30 m×0.25 mm i.d.×1.4 m film thickness). 596 c Reliability of identification (RI): A, mass spectrum and retention time identical with an authentic 597 standard; B, tentative identification by mass spectrum. 598 d Compounds previously reported in ewes' and goats' raw milk cheeses. Reference numbers are 599

The use of non-Saccharomyces yeast for wine making is becoming a common trend in many innovative wineries. The application is normally aimed at increasing aromas, glycerol, reducing acidity, and other improvements. This manuscript focuses on the reproduction of the native microbiota from the vineyard in the inoculum. Thus, native selected yeasts (Hanseniaspora uvarum, Metschnikowia pulcherrima, Torulaspora delbrueckii, Starmerella bacillaris species and three different strains of Saccharomyces cerevisiae) were inoculated sequentially, or only S. cerevisiae (three native strains together or one commercial) was used. Inoculations were performed both in laboratory conditions with synthetic must (400 mL) as well as in industrial conditions (2000 kg of grapes) in red winemaking in two different varieties, Grenache and Carignan. The results showed that all the inoculated S. cerevisiae strains were found at the end of the vinifications, and when non-Saccharomyces yeasts were inoculated, they were found in appreciable populations at mid-fermentation. The final wines produced could be clearly differentiated by sensory analysis and were of similar quality, in terms of sensory analysis panelists’ appreciation.

We report 5-substituted uridine derivatives as novel, uncharged inhibitors of β-1,4-galactosyltransferase and chemical tools for cellular applications. The new inhibitors reduce P-selectin glycoprotein 1 (PSGL-1) expression in human monocytes. Our results also provide novel insights into a unique mode of glycosyltransferase inhibition.