Tiziana Nardi

A sulphite-inducible form of the sulphite efflux gene SSU1 in a Saccharomyces cerevisiae wine yeast.

Sulphite is widely used as a preservative in foods and beverages for its antimicrobial and antioxidant activities, particularly in winemaking where SO(2) is frequently added. Thus, sulphite resistance mechanisms have been extensively studied in the fermenting yeast Saccharomyces cerevisiae. Sulphite detoxification, involving a plasma membrane protein encoded by the SSU1 gene, is the most efficient resistance mechanism in S. cerevisiae. In this study, we characterized the unusual expression pattern of SSU1 in the wine strain 71B. We provide, for the first time, evidence of SSU1 induction by sulphite. The study of SSU1 expression during fermentation and in different growth conditions showed that sulphite is the main regulator of SSU1 expression, explaining its specific pattern. Combining analyses of gene expression and growth behaviour in response to sulphite, we found that 71B displayed unique behavioural patterns in response to sulphite pre-adaptation that may be explained by changes in SSU1 expression. Examination of the genomic organization of the SSU1 locus and sequencing of the region revealed three different alleles in 71B, two of which corresponded to translocated VIII-XVI forms. The lack of differences between promoter regions suggests that this inducible SSU1 expression pattern is due to modification of regulatory/signalling pathways.

Evaluation of Red Chicory Extract as a Natural Antioxidant by Pure Lipid Oxidation and Yeast Oxidative Stress Response as Model Systems

The search for renewable and abundant sources of antioxidants has recently focused on agricultural byproducts, especially promising due to their natural origins and low costs. In particular, plant raw materials are sources of important compounds such as dietary fiber, carotenoids, tocopherols, and polyphenolics, which are mostly discarded during harvesting and processing. Among these vegetal crops, red chicory is attractive because of the large quantity of its byproducts (residues as leaves and stems); moreover, there is no information on its role as a food and feed ingredient. In this study, red chicory leaf residue was evaluated as a natural substitute for synthetic antioxidants for the food and feed industry. After lyophilization, a red chicory extract (RC) was characterized for its phenolic profile and its oxidative stability as compared to BHT. RC was shown to reduce lipid peroxidation of different oils in the Rancimat test. In addition, the antioxidant property of RC was studied in a model system by evaluating the Saccharomyces cerevisiae response to oxidative stress by means of gene expression. In this analysis, the RC extract, added to the yeast culture prior to oxidative stress induction, exhibited a pleiotropic protective effect on stress responsive genes.

Oxidative stress response and nitrogen utilization are strongly variable in Saccharomyces cerevisiae wine strains with different fermentation performances

We used RNA-sequencing (RNA-seq) to analyze the expression profile of four vineyard strains of Saccharomyces cerevisiae having different fermentation performances. The expression profiles obtained in two steps of the fermentation process were compared with those obtained for the industrial wine strain EC1118 and for the laboratory strain S288c. The two strains with low fermentation efficiency, namely, S288c and the vineyard strain R103, exhibited markedly different expression profiles when compared to the other four strains. We also found that the vineyard strains P283 and P301 are characterized by a high expression of the transcription factor Met32p in the first step of the fermentation. Met32p, in coordination with the Hap4p transcription factor, determined the over-expression of the genes involved in the respiration processes, in the response to oxidative stress and in the sulfur amino acids biosynthesis. These combined actions are likely to increase the level of antioxidants whose protective effect could contribute to improve the fermentation process. Gene expression and phenotypic data revealed that the vineyard strain P301 has low nitrogen utilization in comparison to the other wine strains, combined with high fermentation efficiency. Analysis of the genes involved in fermentation stress response revealed a lower expression in strains characterized by low fermentation efficiency, particularly in the first fermentation phase. These findings evidenced the high variability of transcriptional profiles among different wine yeast strains and clarify their connection with complex phenotypic traits, such as the fermentation efficiency and the nitrogen sources utilization.

Adaptation of yeasts Saccharomyces cerevisiae and Brettanomyces bruxellensis to winemaking conditions: a comparative study of stress genes expression.

Brettanomyces is the major microbial cause for wine spoilage worldwide and causes significant economic losses. Like Saccharomyces cerevisiae, it is well adapted to winemaking, but molecular pathways involved in this acclimatization are still unknown. In this work, we report a time-scale comparison between the two yeasts coping with alcoholic fermentation. Orthologs of some well-characterized stress genes of S. cerevisiae were searched by sequence alignment in the Dekkera/Brettanomyces partial genome; nine genes were finally selected on the basis on their similarity and involvement in adaptation to wine. Transcript analysis during a model grape juice fermentation indicates that a subset of genes (i.e., ATP1, ERG6, VPS34) shows peculiar expression patterns in Brettanomyces bruxellensis but also that some common regulations of stress response exist between the two yeasts, although with different timing (i.e., for MSN4, SNF1, HSP82, NTH1). This suggests that B. bruxellensis efficient survival in such challenging conditions is due to mechanisms unique to it, together with a conserved yeast stress response. This study, although limited by the poor genetic data available on B. bruxellensis, provides first insights into its gene expression remodeling in winemaking and opens new frames for further investigations.

Biodiversity, dynamics and ecology of bacterial community during grape marc storage for the production of grappa

The Italian spirit obtained from grape marc, grappa, is produced by an extended storage of the marc which allows alcoholic fermentation. Bacterial populations can develop and are associated with off-flavour production. Grape marc acidification before storage is a common practice in distilleries to control bacterial proliferation. Few studies have been published on the microbial biodiversity in grape marc and no information exists about microbiology of acidified marcs and physiological properties needed for colonizing such an environment. The aim of this study was to investigate the composition and dynamics of grape marc bacterial populations during the long-period storage by microbiological analyses of acidified and untreated marcs. Eight bacterial species were identified by ARDRA - 16s rRNA sequencing at the beginning of the fermentation. Among them the bacterial species of Tatumella terrea, Acetobacter ghanensis and Tatumella ptyseos were identified for the first time in a wine environment. In later stages Oenococcus oeni and members of the Lactobacillus plantarum group became dominant in acidified and non-acidified grape marc, respectively. Further molecular typing of L. plantarum isolates yielded 39 strains. To explain the prevalence of L. plantarum in untreated samples, all strains were tested for potential antimicrobial activity and for biofilm formation ability. Although no antimicrobial activity was found, many strains exhibited the ability to form a biofilm, which may confer an ecological advantage to these strains and their dominance during marc storage.

Acidification of grape marc for alcoholic beverage production: Effects on indigenous microflora and aroma profile after distillation

Grappa is an Italian alcoholic beverage obtained from distillation of grape marc, the raw material derived from separation of must during the winemaking process. Marc is stored for a period lasting from few days to several weeks, when fermentation of residual sugars occurs mainly by yeast activity. Many distilleries have adopted different solutions to manage this critical phase in order to avoid spoilage microorganisms: marc acidification is the most widely diffused. In this work, Prosecco grape pomace was acidified with sulphuric acid (to pH 2.9) and stored, whereas non-acidified grape marc was used as control (pH 3.9). Samples for microbiological analysis were collected at the beginning of the storage period, after 15 and 43days. At the beginning of the ensilage (time T0) the indigenous microflora was represented both by yeasts and bacteria at a concentration of about 10(6)cfu/g. During the first 15days, when the fermentation generally takes place, yeast population grew considerably (up to 10(7)cfu/g) in acidified grape marc, where bacterial population was maintained at low levels. Moreover, yeast populations recovered at the three sampling times in both treated and untreated marc were genetically characterised. This analysis showed that the species succession lead to non-Saccharomyces species dominance (in particular Issatchenkia and Pichia genera) in both conditions although acidified marc showed a lower percentage of Saccharomyces at any sampling time analysed, this meaning that non-Saccharomyces species were favoured in this environment. Gas chromatographic analysis showed a remarkable change in the aromatic profile of distilled grape marcs at the end of the storage, thus evidencing that concentration of monitored volatile compounds usually produced by microflora was generally lowered by the acidification treatment. This work demonstrates for the first time the strong effect of a persistent acidification treatment both on the microbiota of grape pomace and on the aromatic profile of the distillate. Indeed, the lowering of the pH caused significant changes in yeast-bacteria populations ratio and in yeast species turnover. These microbiological changes determine an improvement of the aromatic profile of the distillate, due to the reduction of the main volatile products associated with potential off-flavours.