From bioprotective effects to diversification of wine aroma: Expanding the knowledge on Metschnikowia pulcherrima oenological potential.

Microbial diseases are of major concern in vitiviniculture as they cause grape losses and wine alterations, but the prevention with chemical substances represents a risk to human health and agricultural ecosystem. A promising alternative is the biocontrol and bioprotection activity of non-Saccharomyces yeasts, such as Metschnikowia pulcherrima, which also presents positive oenological traits when used in multistarter fermentations. The aim of this study was to assess the impact of a selected M. pulcherrima strain in the post-harvest withering and vinification of Garganega grapes to produce the sweet 'passito' wine Recioto di Gambellara DOCG (Italy). M. pulcherrima was firstly inoculated on grape at the beginning of the withering process, and afterwards in must for multistarter sequential microfermentation trials with Saccharomyces cerevisiae. Microbiological, chemical, and sensory analyses were carried out to monitor the vinification of treated and control grapes. Grape bunches during withering were a suitable environment for the colonization by M. pulcherrima, which effectively prevented growth of molds. Differences in grape must composition were observed, and the diverse inoculation strategies caused noticeable variations of fermentation kinetics, main oenological parameters, wine aroma profile, and sensory perception. M. pulcherrima proved effective to protect grapes against fungal infections during withering and contribute to alcoholic fermentation generating wine with distinguished aromatic characteristics.

Finding a correct species assignment for a Metschnikowia strain: insights from the genome sequencing of strain DBT012.

Metschnikowia pulcherrima is an important yeast species that is attracting increased interest thanks to its biotechnological potential, especially in agri-food applications. Phylogenetically related species of the so-called 'pulcherrima clade' were first described and then reclassified in one single species, which makes the identification an intriguing issue. Starting from the whole-genome sequencing of the protechnological strain Metschnikowia sp. DBT012, this study applied comparative genomics to calculate similarity with the M. pulcherrima clade publicly available genomes with the aim to verify if novel single-copy putative phylogenetic markers could be selected, in comparison with the commonly used primary and secondary barcodes. The genome-based bioinformatic analysis allowed the identification of 85 consensus single-copy orthologs, which were reduced to three after split decomposition analysis. However, wet-lab amplification of these three genes in nonsequenced type strains revealed the presence of multiple copies, which made them unsuitable as phylogenetic markers. Finally, average nucleotide identity (ANI) was calculated between strain DBT012 and available genome sequences of the M. pulcherrima clade, although the genome dataset is still rather limited. Presence of multiple copies of phylogenetic markers as well as ANI values were compatible with the recent reclassification of the clade, allowing the identification of strain DBT012 as M. pulcherrima.

Reconstruction of Simplified Microbial Consortia to Modulate Sensory Quality of Kombucha Tea.

Kombucha is a fermented tea with a long history of production and consumption. It has been gaining popularity thanks to its refreshing taste and assumed beneficial properties. The microbial community responsible for tea fermentation-acetic acid bacteria (AAB), yeasts, and lactic acid bacteria (LAB)-is mainly found embedded in an extracellular cellulosic matrix located at the liquid-air interphase. To optimize the production process and investigate the contribution of individual strains, a collection of 26 unique strains was established from an artisanal-scale kombucha production; it included 13 AAB, 12 yeasts, and one LAB. Among these, distinctive strains, namely Novacetimonas hansenii T7SS-4G1, Brettanomyces bruxellensis T7SB-5W6, and Zygosaccharomyces parabailii T7SS-4W1, were used in mono- and co-culture fermentations. The monocultures highlighted important species-specific differences in the metabolism of sugars and organic acids, while binary co-cultures demonstrated the roles played by bacteria and yeasts in the production of cellulose and typical volatile acidity. Aroma complexity and sensory perception were comparable between reconstructed (with the three strains) and native microbial consortia. This study provided a broad picture of the strains' metabolic signatures, facilitating the standardization of kombucha production in order to obtain a product with desired characteristics by modulating strains presence or abundance.

Glutathione production by non-Saccharomyces yeasts and its impact on winemaking: A review.

Glutathione (GSH) is a non-protein thiol naturally present in grape berries and produced by yeasts during fermentation. It has a strong antioxidant activity; thus, the addition of pure GSH during winemaking is recommended to limit the oxidative phenomena of wine, preserving sensory characteristics and stability, ultimately promoting a healthier product by reducing the need for SO2 addition. A promising alternative approach considers the use of yeast starter cultures high producers of this compound in situ, during the fermentation process, in substitution of external GSH addition. Recent research showed that multistarter fermentations with non-Saccharomyces yeasts produce even higher concentrations of GSH compared to single Saccharomyces cerevisiae. Accumulation of GSH in yeast cells is also considered valuable during the growth and dehydration of biomass for starter production, aiding strains to overcome the stressful conditions of industrial process. Moreover, a current trend in oenology is the use during fermentation of inactivated dry yeasts preparations as a source of nutrients, and many of them contain GSH-enriched cells. The aim of this review was to assess the significance of GSH production for the exploitation of wine-related non-Saccharomyces yeasts, both in starter biomass production and during fermentations, which were until now studied in detail for S. cerevisiae. This compendium highlights an interesting new feature of non-conventional yeasts and upgrade the strategy of multistarter fermentation as a valuable tool to positively modulate wine composition.

Non-conventional yeasts for food and additives production in a circular economy perspective.

Yeast species have been spontaneously participating in food production for millennia, but the scope of applications was greatly expanded since their key role in beer and wine fermentations was clearly acknowledged. The workhorse for industry and scientific research has always been Saccharomyces cerevisiae. It occupies the largest share of the dynamic yeast market, that could further increase thanks to the better exploitation of other yeast species. Food-related 'non-conventional' yeasts (NCY) represent a treasure trove for bioprospecting, with their huge untapped potential related to a great diversity of metabolic capabilities linked to niche adaptations. They are at the crossroad of bioprocesses and biorefineries, characterized by low biosafety risk and produce food and additives, being also able to contribute to production of building blocks and energy recovered from the generated waste and by-products. Considering that the usual pattern for bioprocess development focuses on single strains or species, in this review we suggest that bioprospecting at the genus level could be very promising. Candida, Starmerella, Kluyveromyces and Lachancea were briefly reviewed as case studies, showing that a taxonomy- and genome-based rationale could open multiple possibilities to unlock the biotechnological potential of NCY bioresources.

New insights into the variability of lactic acid production in Lachancea thermotolerans at the phenotypic and genomic level.

Non-conventional yeasts are increasingly applied in fermented beverage industry to obtain distinctive products with improved quality. Among these yeasts, Lachancea thermotolerans has multiple features of industrial relevance, especially the production of l(+)-lactic acid (LA), useful for the biological acidification of wine and beer. Since few information is available on this peculiar activity, the current study aimed to explore the physiological and genetic variability among L. thermotolerans strains. From a strain collection, mostly isolated from wine, a huge phenotypic diversity was acknowledged and allowed the selection of a high (SOL13) and a low (COLC27) LA producer. Comparative whole-genome sequencing of these two selected strains and the type strain CBS 6340T showed a high similarity in terms of gene content and functional annotation. Notwithstanding, target gene-based analysis revealed variations between high and low producers in the key gene sequences related to LA accumulation. More in-depth investigation of the core promoters and expression analysis of the genes ldh, encoding lactate dehydrogenase, indicated the transcriptional regulation may be the principal cause behind phenotypic differences. These findings highlighted the usefulness of whole-genome sequencing coupled with expression analysis. They provided crucial genetic insights for a deeper investigation of the intraspecific variability in LA production pathway.

Volatile organic compounds from Starmerella bacillaris to control gray mold on apples and modulate cider aroma profile.

Gray mold caused by Botrytis cinerea is a fungal disease that can determine significant economic losses of apple during the storage phase. An alternative to reduce the use of traditional synthetic fungicides is to employ the yeast Starmerella bacillaris as biological control agent (BCA), also with positive effect on apple juice fermentation for the production of cider. Thus, we aimed to evaluate the safety of 16 S. bacillaris strains and their ability to control B. cinerea. In addition, the fermentation performances in apple juice and the volatile organic compounds (VOCs) profile were assessed, both in single-strain and in sequential fermentations with Saccharomyces cerevisiae. The in vitro assays showed that all S. bacillaris strains can be considered safe from the analyzed virulence factors, and were able to significantly constrain the growth of B. cinerea, reducing mycelial growth of 50% in dual-culture and of 90% through VOCs. Moreover, in vivo antagonistic assays revealed a visible decrease of gray mold rot symptoms on apples confirming the potential of S. bacillaris as BCA. GC-MS analysis of the ciders obtained showed increased concentrations in the sequential fermentation of some higher alcohols and terpenes, positively correlated with the cider aromatic quality, and suggested the involvement of benzyl alcohol, known for its antimicrobial action, in the biocontrol efficacy.

Contribution of non-Saccharomyces yeasts to wine volatile and sensory diversity: A study on Lachancea thermotolerans, Metschnikowia spp. and Starmerella bacillaris strains isolated in Italy.

Saccharomyces cerevisiae starter cultures are largely used in winemaking to repress the wild microorganisms and achieve more predictable and desired outcomes. Notwithstanding, alternative microbial resources received increasing attention for their potential to produce wines with more distinctive and typical features. Our previous survey revealed a great inter- and intra-species diversity in an extensive collection of non-Saccharomyces wine yeasts from multiple regions of Italy. This study aimed to explore the detected biodiversity evaluating the quality of wines obtained by sequential inoculation of specific selected strains of the collection (Lachancea thermotolerans or Metschnikowia spp. or Starmerella bacillaris), and S. cerevisiae EC 1118. Fermentations of natural grape must at laboratory scale were followed by microbiological, chemical and sensorial analysis of the wines. The results indicated that each yeast species and strain exerted a distinctive impact on the wine, giving final products clearly separated with Principal Component Analysis. In particular, L. thermotolerans contributed producing relevant amounts of lactic acid and had the highest potential to reduce ethanol content; the presence of S. bacillaris increased the level of glycerol, and, remarkably, reduced acetaldehyde and total SO2; Metschnikowia spp. promoted the formation of higher alcohols and esters, and reduced volatile phenols. The sensory analysis based on the orthonasal aroma confirmed the separation between the wines obtained with the sequential fermentations and the control with single inoculation of EC 1118, although the three non-Saccharomyces species used could not be clearly distinguishable by the panelists. This study indicates that the use of selected native non-Saccharomyces strains in conjunction with S. cerevisiae positively modulates some relevant chemical parameters, and improves the aromatic intensity of wine, therefore justifying investments in non-conventional yeasts as co-starter cultures.

Exploring the diversity of a collection of native non-Saccharomyces yeasts to develop co-starter cultures for winemaking.

The inoculation of Saccharomyces cerevisiae starter cultures in grape musts is a common practice in wineries worldwide; however, native non-Saccharomyces yeast species are increasingly investigated as co-starters to augment the complexity and regionality of wine. In this study, an extensive collection of non-Saccharomyces yeasts from high-sugar matrices was created and screened with the aim to discover new strains with potentially positive oenological traits. After mining >400 yeasts from 167 samples collected across multiple Italian regions, the isolates were identified based on RAPD-PCR analysis and ITS sequencing. About one quarter of them, belonging to the genera Starmerella, Lachancea and Metschnikowia, were picked up for an in-depth molecular and physiological characterization, since these yeasts were well strewed and have a good oenological reputation. Following the genotyping, stress tolerance assays, enzymatic activity trials and single inoculum fermentations, a huge diversity was acknowledged within and between the species. Strains of S. bacillaris showed a high tolerance to ethanol and increased glycerol production, L. thermotolerans reduced volatile acidity while increasing total acidity with lactic acid, and Metschnikowia spp. exhibited remarkable aroma-related enzymatic activities, which are all prized features in winemaking. Since most of the characteristics analyzed were species and strain dependent, the obtained results are valuable for the selection of a new generation of co-starters for attempting mixed fermentation strategies aimed to improve the overall quality of regional wine.