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Microbiological approaches to reduce the sulphite addition in oenology
Full text linkSulphite is widely used in winemaking for its antimicrobial and antioxidant properties, although its toxic effect on human health is proven. For this reason strategies for reducing chemical preservatives in winemaking is strongly demanded. Wine yeasts can cope with SO2 by different systems, such as acetaldehyde and S-amino acids production or SO2 export.
In this study a first screening of SO2 resistance and on plate production of SO2 and H2S have been performed for autochthonous strains, isolated in Veneto vineyards to be used as starter of fermentation in the production of Prosecco di Valdobbiadene DOCG and DOC Piave wines, compared to commercial strains.
Then the oenological characteristics of 11 S. cerevisiae strains of have been evaluated. These strains are 4 autochthonous strains isolated during local selection projects in DOCG Conegliano - Valdobbiadene and DOC Piave areas, together with 6 commercial strains, which genome have been recently sequenced, and relative informations are available in the principal genomic databases, and the laboratory strain S288c, the first one that has been sequenced. Main technology and quality characters have been evaluated to determine the suitability of strains for winemaking process. In particular have been studied the production of ethanol and glycerol, the glucose consumption at 2 and 7 days, the production of hydrogen sulphide, acetaldehyde and sulphur dioxide and the resistance to various concentrations of free sulphur dioxide in synthetic must. Sulphite response in yeast has been investigated in order to elucidate factors that affect sulphite production during vinification. Moreover acetaldehyde, another compound produced by yeast, linked with sulphite metabolism or detoxification, has been analysed since it affects wine quality.
Genetic characteristics identified after genome sequencing of 4 autochthonous strains (2 from Prosecco area and 2 from Raboso area), such as oenological SNPs, strain-specific genes and important translocations, have been analyzed in Real-time PCR for a large number of autochthonous strains.
Then the behaviour towards sulphite of 4 wine yeasts has been investigated, and transcriptome analysis during fermentation has been performed by means of next generation sequencing. For all strains, fermentation rate was monitored together with sulphite production in synthetic must supplemented with different doses of SO2 (0 mg/l and 25 mg/l).
Finally, a selection of reference genes for Real-time PCR has been made, and a set of genes suitable for such conditions has been identified.
Results point out the importance of verifying strain attitudes towards sulphite at different sulphite concentrations. This study tries to clarify the complex regulative mechanisms of sulphites during fermentation, thus giving new guidelines for critic control of these fermentation parameters in order to maximize effect of sulphite added thus limiting the dose employed during vinification.I solfiti sono ampiamente utilizzati nella vinificazione per le loro proprietà antimicrobiche e antiossidanti, sebbene il loro effetto tossico sulla salute umana sia dimostrato. Per questo motivo le strategie di riduzione dei conservanti chimici nel processo di vinificazione è fortemente richiesto. I lieviti possono rispondere alla presenza di SO2 con sistemi diversi, come la produzione di acetaldeide e ammino acidi solforati o l’esporto di SO2.
In questo studio è stato fatto un primo screening sulla resistenza alla SO2 e sulla produzione di SO2 e H2S in piastra da parte di ceppi autoctoni, isolati nei vigneti del Veneto per essere utilizzato come starter di fermentazione nella produzione di Prosecco di Valdobbiadene DOCG e Vini del Piave DOC, confrontati con dei ceppi commerciali.
Inoltre sono state valutate le caratteristiche enologiche di 11 ceppi di S. cerevisiae. I ceppi in esame sono 4 ceppi autoctoni isolati durante i progetti di selezione locale nelle aree Conegliano - Valdobbiadene DOCG e Piave DOC, insieme con 6 ceppi commerciali, il cui genoma è stato recentemente sequenziato, e le informazioni relative sono disponibili nelle banche dati genomiche principali, e il ceppo di laboratorio S288c , il primo che è stato sequenziato. I principali caratteri tecnologici e di qualità sono stati valutati per determinare l'idoneità dei ceppi alla vinificazione. In particolare, sono state studiate la produzione di etanolo e glicerolo, il consumo di glucosio a 2 e 7 giorni, la produzione di idrogeno solforato, acetaldeide e biossido di zolfo e la resistenza a varie concentrazioni di biossido di zolfo libero in mosto sintetico. La risposta ai solfiti nel lievito è stata studiata al fine di chiarire i fattori che influenzano la produzione dei solfiti durante la vinificazione. Inoltre l’acetaldeide, un altro composto prodotto da lievito, collegato con il metabolismo solfito o disintossicazione, è stata analizzata in quanto influisce sulla qualità del vino.
Con il sequenziamento del genoma di 4 ceppi autoctoni (2 da zona del Prosecco e 2 dalla zona Raboso) è stato possibile individuare delle caratteristiche genetiche, come ad esempio SNPs enologiche, geni ceppo-specifici e traslocazioni importanti, che sono stati analizzati in Real-time PCR per un gran numero di ceppi autoctoni .
Inoltre il comportamento di 4 lieviti enologici nei confronti dei solfiti è stato studiato, ed è stata effettuata l'analisi del trascrittoma durante la fermentazione per mezzo di next generation sequencing. Per tutti i ceppi la velocità di fermentazione è stata monitorata, insieme alla produzione di SO2, in mosto sintetico con differenti dosi di SO2 (0 mg / l e 25 mg / l).
Infine, è stata fatta una selezione di geni reference da usare in Real-time PCR, e una serie di geni adatti a queste condizioni è stato identificato.
I risultati sottolineano l'importanza di verificare l'atteggiamento del ceppo nei confronti dei solfiti a diverse concentrazioni di SO2. Questo studio cerca di chiarire i complessi meccanismi di regolazione dei solfiti durante la fermentazione, dando così nuove linee guida per il controllo critico di questi parametri di fermentazione, al fine di massimizzare l'effetto dei solfiti aggiunto limitando in tal modo la dose impiegata durante la vinificazione
Transcriptome analysis of wine yeast strains under sulphite-induced stress conditions
No full textSulphite is widely used in winemaking for its antimicrobial and antioxidant properties,
although its toxic effect on human health is proven. For this reason strategies for reducing
chemical preservatives in winemaking is strongly demanded. Wine yeasts can cope with SO2
by different systems, such as acetaldehyde production, sulphite uptake and reduction or SO2
export.
The aim of this work was to study the genes involved in sulphites response in S. cerevisiae
and see how different strains respond to SO2 addition in the early stage of fermentation.
In this study 10 strains has been chosen among those whose genome has been sequenced: 6
commercial yeasts, EC1118, AWRI796, AWRI1631, VIN13, QA23, VL3, and 4 isolated
directly from vineyard, R008, R103, P301, P283. The strain S288C was added to the
analysis, as reference. Fermentation trials in synthetic must were conducted at laboratory
scale to assess the main technological and quality traits and to investigate strain behaviours
towards sulphite. The 4 strains showing the strongest differences in terms of response to SO2
were selected. To clarify the genetic basis of this complex enological trait we performed
transcription profiling using SOLID technology. The analysis was conducted during
fermentation process mimicking winemaking condition, in synthetic must supplemented with
25 mg/l of SO2 in 1l-capacity bioreactors. For all strains, fermentation rate was determined
overall the process, together with sulphite and acetaldehyde production. RNA seq was
performed at early exponential phase (6 g/l of CO2 produced) to investigate yeast adaptation
under sulphite conditions. Gene expression analysis suggested that both specific gene
activities and more general genetic pathways are involved in sulphite response
Diffusion of FSY1 fructose transporter in a population of Saccharomyces cerevisiae isolated in vineyard and its effect on sugar conversion
Full text linkSelection and validation of reference genes for quantitative real-time PCR studies during Saccharomyces cerevisiae alcoholic fermentation in presence of sulfite
No full textSulfur dioxide is extensively used during industrial fermentations and contributes to determine the harsh conditions of winemaking together with low pH, high sugar content and increasing ethanol concentration. Therefore the presence of sulfite has to be considered in yeast gene expression studies to properly understand yeast behavior in technological environments such as winemaking. A reliable expression pattern can be obtained only using an appropriate reference gene set that is constitutively expressed regardless of perturbations linked to the experimental conditions. In this work we tested 15 candidate reference genes suitable for analysis of gene expression during must fermentation in the presence of sulfite. New reference genes were selected from a genome-wide expression experiment, obtained by RNA sequencing of four Saccharomyces cerevisiae wine strains grown in enological conditions. Their performance was compared to that of the most common genes used in previous studies. The most popular software based on different statistical approaches (geNorm, NormFinder and BestKeeper) were chosen to evaluate expression stability of the candidate reference genes. Validation was obtained using other wine strains by comparing normalized gene expression data with transcriptome quantification both in the presence and absence of sulfite. Among 15 reference genes tested ALG9, FBA1, UBC6 and PFK1 appeared to be the most reliable while ENO1, PMA1, DED1 and FAS2 were the worst. The most popular reference gene ACT1, widely used for S. cerevisiae gene expression studies, showed a stability level markedly lower than those of our selected reference genes. Finally, as the expression of the new reference gene set remained constant over the entire fermentation process, irrespective of the perturbation due to sulfite addition, our results can be considered also when no sulfite is added to the must. © 2015 Elsevier B.V
Different mechanisms of resistance modulate sulfite tolerance in wine yeasts
No full textFrom a technological point of view, yeast resistance to sulfite is of great interest and represents an important technological character for winemaking. Several mechanisms are involved, and strain-dependent strategies to obtain SO2 resistance can deeply influence wine quality, although this choice is less relevant in determining the technological performance of the strain during fermentation. In this study, to better understand the strain-specific mechanisms of resistance, 11 Saccharomyces cerevisiae strains, whose genomes have been previously sequenced, were selected. Their attitude towards sulfites, in terms of resistance and production, was evaluated, and RNA-sequencing of four selected strains was performed during fermentation process in synthetic grape must in the presence of SO2. Results demonstrated that at molecular level, the physical effect of SO2 triggered multiple stress responses in the cell and high tolerance to general enological stressing condition increased SO2 resistance. Adaptation mechanism due to high basal gene expression level rather than specific gene induction in the presence of sulfite seemed to be responsible in modulating strain resistance. This mechanism involved higher basal gene expression level of specific cell wall proteins, enzymes for lipid biosynthesis, and enzymes directly involved in SO2 assimilation pathway and efflux
Chemical and microbiological assessment of early wine fermentation phase can predict yeast cell viability during post-fermentation process
Full text linkThe management of post-fermentation phase is essential for the protection of wine oxidation. The prolonged contact of yeast lees and wine can help to limit this problem, although off-flavours can originate. It is known that some cellular components (mannoproteins, lipids, glutathione, etc.) released into the wine influence oxygen protection; however, still active cells could contribute to maintaining protection against oxidation. To date, in the literature there is a lack of data that evaluates cell viability, especially in the post-fermentation phase, particularly using methods different by plate count that identifies only a small part of the viable population. The aim of the work was to investigate the yeast viability of 12 wine Saccharomyces cerevisiae strains during 45 days after the fermentation in natural grape juice. The major fermentation parameters were measured at early phase (40 h) and at the end of the process, and were correlated with total and viable cells in the post-fermentation phase. Contrary to what has been observed in the literature, this work demonstrates that cell viability in the post-fermentation phase is very high and dependent on the yeast strain. A predictive model that can estimate viability in the post-fermentation phase, based on parameters measured at the early fermentation phase, was successfully set up. This approach can be adopted by wineries or winemakers as it uses fermentation data (sugar and nitrogen residues, ethanol and glycerol production, total cell count) obtained through simple chemical and microbiological analyses
Comparative genomic and transcriptomic study on ecotypical Saccharomyces cerevisiae strains
No full textEffetti dell'anidride solforosa sul metabolismo dei lieviti e modifiche nel profilo trascrizionale
No full textLa grappa viene ottenuta dalle vinacce sottoposte ad acidificazione e conservate per tempi prolungati in modo da favorire la fermentazione alcoolica. Questo processo determina lo sviluppo di una microflora batterica che ha una notevole rilevanza per determinare le caratteristiche aromatiche del prodotto finale.
Lo scopo di questo studio è quello di analizzare la biodiversità microbica nelle vinacce e le modificazioni cui essa è soggetta durante il periodo di insilamento tramite sequenziamento dell’ rRNA 16S con metodiche di next generation sequencing (Roche 454 GS-FLX System).
E’ stata utilizzata uva di Prosecco proveniente dalla zona di Fregona (Treviso), i campionamenti sono stati fatti dopo 8 ore dalla pigiatura (T0) e dopo 30 giorni (T30). Per avere una miglior caratterizzazione delle specie batteriche presenti, quattro diverse regioni ipervariabili del gene 16S (V3, V4, V5, V6) sono state amplificate con due diverse coppie di primers universali e sottoposte a sequenziamento ottenendo in media 11500 sequenze per campione.
I risultati ottenuti dall’analisi delle due regioni ipervariabili del 16S sono molto simili, e mettono in luce una modificazione estremamente profonda della composizione microbica durante la maturazione della vinaccia. Accanto a specie molto abbondanti di batteri acetici e lattici descritti in precedenza, il sequenziamento massivo rivela anche la presenza di una microflora composta da generi a bassa abbondanza
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