Enrico Vaudano

Biogenic Amine Production by Contaminating Bacteria Found in Starter Preparations Used in Winemaking

The aim of this work was to investigate if contaminating microorganisms, eventually present in bacteria and yeast preparations used as commercial starters in winemaking, have the ability to produce the biogenic amines histamine, putrescine and tyramine. Thirty commercial starters (14 yeasts Saccharomyces cerevisiae and 16 bacteria Oenococcus oeni) were cultured in synthetic broth and analyzed by TLC to detect amine production. Oenococcus oeni commercial preparations did not contain contaminants, but some yeast preparations resulted contaminated with amine-producing bacteria. Bacterial contaminants were isolated and analyzed for their ability to produce biogenic amines using HPLC and TLC. Decarboxylase genes were identified using PCR analysis followed by sequencing. Fermentations were performed in grape juice with two yeast commercial preparations containing bacterial contaminants to check if the potential biogenic amine production could happen also during winemaking. It was found that this production is possible; in particular, in the conditions used in this work, tyramine production was detected. Therefore, the results of this study have significance in relation to the risk of biogenic amines in wine. Moreover a novel species of Lactobacillus was found to be able to produce histamine.

Application of real-time RT-PCR to study gene expression in active dry yeast (ADY) during the rehydration phase.

During the industrial production of active dry yeast (ADY) and its subsequent use in winemaking, the yeast cell is subjected to drastic environmental changes that force it to undergo extensive metabolic modifications and changes in gene expression. In this study, we describe the use of real-time reverse transcription-polymerase chain reaction (RT-PCR) to monitor gene expression in ADY Saccharomyces cerevisiae during rehydration in different media. We used three statistical approaches to investigate the expression stability of eight potential reference genes during the rehydration process. The reference system thus obtained was used to normalize the expression values of three genes codifying for the ammonium transporters -- MEP1, MEP2, and MEP3 -- and two genes involved in the osmotic response-SIP18 and GPD1. The results suggested that for the target genes tested, the yeast reacted immediately to rehydration only when a fermentable carbon source was present in the medium. Furthermore, MEP2 expression was modulated by the ammonium concentration, indicating that nitrogen catabolite repression (NCR) is active during the rehydration phase.

Quantitative expression analysis of mleP gene and two genes involved in the ABC transport system in Oenococcus oeni during rehydration

Oenococcus oeni is recognized as the principal microorganism responsible for malolactic fermentation, and the control of its activity is of primary importance in winemaking. The aim of this study was to quantify the levels of expression of the malate transporter gene (mleP) and of two genes putatively involved in the ATP-binding cassette transport system (oeoe_1651, oeoe_0550) to better understand the physiological response of bacteria during rehydration. These genes coding for transporters were studied in different rehydration media. Initially, three different statistical algorithms were used to identify suitable reference genes to be used for the normalization of expression data in O. oeni during rehydration, and to this purpose, the best genes found were ddl and gyrB. The results showed that the genes for transporters of malate and sugar (mleP, oeoe_1651) were activated immediately after a few minutes of rehydration, when specific medium compositions were used.

Identification of reference genes suitable for normalization of RT-qPCR expression data in Saccharomyces cerevisiae during alcoholic fermentation

Expression data from RT-qPCR (reverse transcription quantitative PCR) needs to be normalized to account for experimental variability among samples caused by differential yields of the transcripts in RNA extraction or in the reverse transcription. The most common method is to normalize against one or more reference genes (RG). We have selected RGs suitable for normalization of RT-qPCR raw data in Saccharomyces cerevisiae during alcoholic fermentation. The RGs were evaluated by three different statistical methods. The suitability of the selected RG sets was compared with ACT1, a commonly used non-validated single RG, by normalizing the expression of two target genes. Expression profiles of the target genes revealed the risk of misleading interpretation of expression data due to an unreliable RG.

Short-term response of different Saccharomyces cerevisiae strains to hyperosmotic stress caused by inoculation in grape must: RT-qPCR study and metabolite analysis

During the winemaking process, glycerol synthesis represents the first adaption response of Saccharomyces cerevisiae to osmotic stress after inoculation in grape must. We have implemented an RT-qPCR (Reverse Transcription-quantitative PCR) methodology with a preventive evaluation of candidate reference genes, to study six target genes related to glycerol synthesis (GPD1, GPD2, GPP2 and GPP1) and flux (STL1 and FPS1), and three ALD genes coding for aldehyde dehydrogenase involved in redox equilibrium via acetate production. The mRNA level in three strains, characterized by different metabolite production, was monitored in the first 120xa0min from inoculation into natural grape must. Expression analysis shows a transient response of genes GPD1, GPD2, GPP2, GPP1 and STL1 with differences among strains in term of mRNA abundance, while FPS1 was expressed constitutively. The transient response and different expression intensity among strains, in relation to the intracellular glycerol accumulation pattern, prove the negative feedback control via the HOG (High Osmolarity Glycerol) signalling pathway in S.xa0cerevisiae wine strains under winery conditions. Among the ALD genes, only ALD6 was moderately induced in the hyperosmotic environment but not in all strains tested, while ALD3 and ALD4 were drastically glucose repressed. The intensity of transcription of ALD6 and ALD3 seems to be related to different acetate production found among the strains.

An RT-qPCR approach to study the expression of genes responsible for sugar assimilation during rehydration of active dry yeast

A short reactivation period in aqueous media is required for active dry yeast (ADY) to be utilised in winemaking. Rehydration restores the active metabolic conditions necessary for good fermentative and competitive abilities. We used a reverse transcription-quantitative PCR (RT-qPCR) method with relative quantification to investigate the expression of seven hexose transporter genes (HXT1-7) and one invertase-encoding gene (SUC2) during ADY rehydration in water with or without sucrose. For this, seven candidate reference genes were evaluated, and the three most stably expressed genes were selected and used for mRNA normalisation. The results show that, during the rehydration in the presence of sucrose, yeast cells are able to immediately hydrolyse this sugar into glucose and fructose as soon as they are introduced in the medium. Subsequently, differential glucose/fructose uptake occurs, which is mediated by hexose transporters. At the transcriptomic level, there is a strong induction of the high-affinity transporters, HXT2 and HXT4, and the low-affinity transporters, HXT3 and HXT1, when ADY is rehydrated with sucrose, while HXT5 and HXT6/7 are expressed at high levels with a moderate tendency to decrease. In water, the HXT2 gene was the only one of the transporter genes studied that showed significant variations. These results suggest that during rehydration, expression is not simply regulated by the affinity to hexose but is also controlled by other mechanisms that allow the cell to bypass glucose control. Moreover, the expression of SUC2 showed little variation in media with sucrose, suggesting that other invertases and/or posttranscriptional controls exist.

Aroma compounds and sensory characteristics of Arneis Terre Alfieri DOC wines: the concentration of polyfunctional thiols and their evolution in relation to different ageing conditions

AbstractnArneis is an Italian autochthonous white grape cultivar. This study involved investigating the chemicophysical, aromatic composition and sensory evaluation of Arneis wine in relation to different oenological practices during winemaking and ageing. For the first time, polyfunctional thiols related aromas were identified above their perception threshold in Arneis wine. Moreover, citrus, grapefruit and tropical fruit notes characterized this wine during the first month of ageing, thus suggesting a strong correlation between chemical and sensory results. The concentration of thiols decreased rapidly during the first month of ageing, together with the tropical fruit notes. In particular, the perception of the grapefruit note of the wine decreased in association with the 3-mercaptohexyl acetate content in both the barrel and stainless steel (ST) aged wines. Moreover, the ageing technique clearly affected wine composition and sensory profile; barrel aged wines were characterized both by vanilla and honey notes, and a weaker perception of thiols despite containing a similar concentration to ST aged wines. Within this study, the ability of glutathione to decrease the oxidation of volatile thiols in Arneis wine was confirmed, but under experimental conditions employed, the addition of reduced glutathione did not lead to any statistically significant differences in sensory results.

Yeast distribution in Grignolino grapes growing in a new vineyard in Piedmont and the technological characterization of indigenous Saccharomyces spp. strains

The aim of this study was to characterize the yeast consortium isolated from Grignolino grapes in a newly planted vineyard in Piedmont (Italy) via analysis of the intra-vineyard yeast distribution of grape samples from single rows. A two-phase approach allowed the identification of culturable yeasts present on grape skins and, through an enriching procedure via grape fermentation, the isolation of low frequency non-Saccharomyces and Saccharomyces spp. fermentative species, including S. paradoxus, which is highly unusual during grape fermentation, along with the intra-specific characterization of S. cerevisiae isolates. Culture-based molecular techniques revealed a grape yeast microbiota formed by (in order of abundance) Hanseniaspora uvarum, the yeast-like fungus Aerobasidium pullulans, Candida zemplinina, Pichia kluyveri, Candida californica, Curvibasidium cygneicollum, Meyerozima caribbica, Rhodotorula babjevae, Metschnikowia pulcherrima and Cryptococcus flavescens. Technological properties of isolated Saccharomyces spp. strains were analysed, identifying strains, including S. paradoxus, potentially suitable as an ecotypical starter for territorial wines.

Enhanced arginine biosynthesis and lower proteolytic profile as indicators of Saccharomyces cerevisiae stress in stationary phase during fermentation of high sugar grape must: A proteomic evidence

A strain of Saccharomyces (S) cerevisiae (ISE19), which displayed an initial good adaptation to a high sugar medium with increased acetate and glycerol production but weak overall growth/fermentation performances, was selected during the alcoholic fermentation of Cortese grape must. To obtain insights into the metabolic changes that occur in the must during growth in particular conditions (high ethanol, high residual sugars and low nitrogen availability) leading to a sluggish fermentation or even fermentation arrest, comparative in-gel proteomic analyses were performed on cells grown in media containing 200g/L and 260g/L of glucose, respectively, while the YAN (Yeast Assimilable Nitrogen) concentration was maintained as it was. Two post-translationally different arginine synthases (pIs 5.6 and 5.8) were found in higher abundances in the high glucose-grown cells, together with an increased abundance of a glycosyltransferase involved in cell-wall mannans synthesis, and of two regulatory proteins (K7_Bmh1p and K7_Bmh2p) that control membrane transport. In parallel, a proteinase K-like proteolytic enzyme and three other protein fragments (Indolepyruvate decarboxylase 1, Fba1p and Eno1p) were present in lower abundances in the high glucose condition, where oxidative stress and cell cycle involved enzymes were also found to be less abundant. The overall results suggest that in stationary phase stress conditions, leading to stuck fermentation, S. cerevisiae ISE19 decreases cell replication, oxidative stress responses and proteolytic activity, while induces other metabolic modifications that are mainly based on cell-wall renewal, regulation of the solute transport across the cell membrane and de novo arginine synthesis.

Key norisoprenoid compounds in wines from early-harvested grapes in view of climate change

In view of climate change, the scheduling of an early harvest may be an agronomic option to limit wine alcohol, provided that a satisfactory content of secondary metabolites can be ensured in grapes. To better understand the link between grape ripening, seasonal trend and wine aroma, the aromatic expression of Barbera and Pinot Noir wines produced with early harvested grapes was assessed. Attention was focused on C13 norisoprenoids during both alcoholic fermentation and after three months of storage. At the end of fermentation, the highest β-damascenone content was detected in wines obtained from less ripe grapes, the content subsequently increased significantly after three months of storage; however, the levels of β-ionone decreased significantly during the same period. The reduction of wine alcohol as a result of harvesting earlier, especially for Barbera, was associated with optimal aromatic levels as well as good technological parameters.