An important step forward for the future development of an easy and fast procedure for identifying the most dangerous wine spoilage yeast, Dekkera bruxellensis, in wine environment

Dekkera bruxellensis is the main reason for spoilage in the wine industry. It renders the products unacceptable leading to large economic losses. Fluorescence In Situ Hybridisation (FISH) technique has the potential for allowing its specific detection. Nevertheless, some experimental difficulties can be encountered when FISH technique is applied in the wine environment (e.g. matrix and cells autofluorescence, fluorophore inadequate selection and probes low specificity to the target organisms). An easy and fast in-suspension RNA-FISH procedure was applied for the first time for identifying D. bruxellensis in wine. A previously designed RNA-FISH probe to detect D. bruxellensis (26S D. brux.5.1) was used and the matrix and cells fluorescence interferences, the influence of three fluorophores in FISH performance and the probe specificity were evaluated. The results revealed that to apply RNA-FISH technique in the wine environment a red-emitting fluorophore should be used. Good probe performance and specificity was achieved with 25% of formamide. The resulting RNA-FISH protocol was applied in wine samples artificially inoculated with D. bruxellensis. This spoilage microorganism was detected in wine at cell densities lower than those associated with phenolic off-flavours. Thus, the RNA-FISH procedure described in this work represents an advancement to facilitate early detection of the most dangerous wine spoilage yeast and, consequently, to reduce the economic losses caused by this yeast to the wine industry.This work was co-financed by Foundation for Science and Technology (FCT) and the European Union through the European Regional Development Fund ALENTEJO 2020 through the projects PTDC/BBB-IMG/0046/2014 and ALT20-03-0145-FEDER-000015, respectively. Marina González-Pérez acknowledges FCT for the economic support through the post-doctoral grant SFRH/BPD/100754/2014

Transcriptomics reveal an integrative role for maternal thyroid hormones during zebrafish embryogenesis

Thyroid hormones (THs) are essential for embryonic brain development but the genetic mechanisms involved in the action of maternal THs (MTHs) are still largely unknown. As the basis for understanding the underlying genetic mechanisms of MTHs regulation we used an established zebrafish monocarboxylic acid transporter 8 (MCT8) knock-down model and characterised the transcriptome in 25hpf zebrafish embryos. Subsequent mapping of differentially expressed genes using Reactome pathway analysis together with in situ expression analysis and immunohistochemistry revealed the genetic networks and cells under MTHs regulation during zebrafish embryogenesis. We found 4,343 differentially expressed genes and the Reactome pathway analysis revealed that TH is involved in 1681 of these pathways. MTHs regulated the expression of core developmental pathways, such as NOTCH and WNT in a cell specific context. The cellular distribution of neural MTH-target genes demonstrated their cell specific action on neural stem cells and differentiated neuron classes. Taken together our data show that MTHs have a role in zebrafish neurogenesis and suggest they may be involved in cross talk between key pathways in neural development. Given that the observed MCT8 zebrafish knockdown phenotype resembles the symptoms in human patients with Allan-Herndon-Dudley syndrome our data open a window into understanding the genetics of this human congenital condition.Portuguese Fundacao para Ciencia e Tecnologia (FCT) [PTDC/EXPL/MARBIO/0430/2013]; CCMAR FCT Plurianual financing [UID/Multi/04326/2013]; FCT [SFRH/BD/111226/2015, SFRH/BD/108842/2015, SFRH/BPD/89889/2012]; FCT-IF Starting Grant [IF/01274/2014]info:eu-repo/semantics/publishedVersio

Interaction of prion protein with acetylcholinesterase: potential pathobiological implications in prion diseases

The prion protein (PrP) binds to various molecular partners, but little is known about their potential impact on the pathogenesis of prion diseases. Here, we show that PrP can interact in vitro with acetylcholinesterase (AChE), a key protein of the cholinergic system in neural and non-neural tissues. This heterologous association induced aggregation of monomeric PrP and modified the structural properties of PrP amyloid fibrils. Following its recruitment into PrP fibrils, AChE loses its enzymatic activity and enhances PrP-mediated cytotoxicity. Using several truncated PrP variants and specific tight-binding AChE inhibitors (AChEis), we then demonstrate that the PrP-AChE interaction requires two mutually exclusive sub-sites in PrP N-terminal domain and an aromatic-rich region at the entrance of AChE active center gorge. We show that AChEis that target this site impair PrP-AChE complex formation and also limit the accumulation of pathological prion protein (PrPSc) in prion-infected cell cultures. Furthermore, reduction of AChE levels in prion-infected heterozygous AChE knock-out mice leads to slightly but significantly prolonged incubation time. Finally, we found that AChE levels were altered in prion-infected cells and tissues, suggesting that AChE might be directly associated with abnormal PrP. Our results indicate that AChE deserves consideration as a new actor in expanding pathologically relevant PrP morphotypes and as a therapeutic target

Yeasts and wine off-flavours: a technological perspective

Review article. Part of the special issue "Wine microbiology and safety: from the vineyard to the bottle (Microsafety Wine)", 19-20 Nov. 2009, ItalyIn wine production, yeasts have both beneficial and detrimental activities. Saccharomyces cerevisiae is the yeast mainly responsible for turning grape juice into wine but this species and several others may also show undesirable effects in wines. Among such effects, technologists are particularly concerned with the production of offflavours that may occur during all stages of winemaking. Typical spoiling activities include the production of ethyl acetate by apiculate yeasts before fermentation, hydrogen sulphide by S. cerevisiae during fermentation phases, acetaldehyde by film-forming yeasts during bulk storage, and volatile phenols by Dekkera bruxellensis during storage or after bottling. The occurrence of these hazards depends on the technological operations designed to obtain a given type of wine and most can be avoided by current preventive or curative measures. On the contrary, good manufacturing practices must be strengthened to deal with the problem of volatile phenol production in red wines. Appropriate monitoring of D. bruxellensis populations and quantification of 4-ethylphenol is advised during storage, particularly when oak barrels are used, and absence of viable cells must be guaranteed in bottled wines. This work, which is based on our experience at winery level, aims to provide information on appropriate technological strategies to deal with the problem of off-flavours produced by yeasts

Impact of volatile phenols and their precursors on wine quality and control measures of Brettanomyces/Dekkera yeasts

Volatile phenols are aromatic compounds and one of the key molecules responsible for olfactory defects in wine. The yeast genus Brettanomyces is the only major microorganism that has the ability to covert hydroxycinnamic acids into important levels of these compounds, especially 4-ethylphenol and 4-ethylguaiacol, in red wine. When 4-ethylphenols reach concentrations greater than the sensory threshold, all wine’s organoleptic characteristics might be influenced or damaged. The aim of this literature review is to provide a better understanding of the physicochemical, biochemical, and metabolic factors that are related to the levels of p-coumaric acid and volatile phenols in wine. Then, this work summarizes the different methods used for controlling the presence of Brettanomyces in wine and the production of ethylphenols

Effect of kaolin silver complex on the control of populations of Brettanomyces and acetic acid bacteria in wine

In this work, the effects of kaolin silver complex (KAgC) have been evaluated to replace the use of SO2 for the control of spoilage microorganisms in the winemaking process. The results showed that KAgC at a dose of 1 g/L provided effective control against the development of B. bruxellensis and acetic acid bacteria. In wines artificially contaminated with an initial population of B. bruxellensis at 104 CFU/mL, a concentration proven to produce off flavors in wine, only residual populations of the contaminating yeast remained after 24 days of contact with the additive. Populations of acetic bacteria inoculated into wine at concentrations of 102 and 104 CFU/mL were reduced to negligible levels after 72 h of treatment with KAgC. The antimicrobial effect of KAgC against B. bruxellensis and acetic bacteria was also demonstrated in a wine naturally contaminated by these microorganisms, decreasing their population in a similar way to a chitosan treatment. Related to this effect, wines with KAgC showed lower concentrations of acetic acid and 4-ethyl phenol than wines without KAgC. The silver concentration from KAgC that remained in the finished wines was below the legal limits. These results demonstrated the effectiveness of KAgC to reduce spoilage microorganisms in winemaking.info:eu-repo/semantics/acceptedVersio

Reuse of oak chips for modification of the volatile fraction of alcoholic beverages

New or used barrels can be applied in ageing of alcoholic beverages. Compounds adsorbed in wood migrate between beverages along with wood extractives. As barrel ageing is costly and time-consuming, processes using wood fragments have been gaining interest. These generate wood residues for which the reuse is still not well established. This work aims at the reuse of oak fragments for the additive ageing of alcoholic beverages. Oak chips, previously immersed in fortified wine, were applied to beer, wine and grape marc spirit. Wood compounds and adsorbed wine volatiles were extracted, with more impact and satisfactory yields on beer composition. Also, wood adsorbed beverages compounds in a subtractive ageing phenomena. Beer formulations using different binomial wood concentration/temperature combinations were generated and presented to trained tasters. Higher temperatures and wood concentrations led to prominence of wood descriptors and lower perception of fruity and floral aromas, reflecting the changes in chemical composition.Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 – Programa Operacional Regional do Norte. Fermentum – Engenharia das Fermentações Lda. also participated in co-funding and provided beer samples used in this work, which authors would like to acknowledge. Lastly, authors would like to thank Mr. Benoît Verdier and Seguin Moreau for supplying the woods and Mr. Paulo Coutinho and Quinta do Portal for supplying the fortified wineinfo:eu-repo/semantics/publishedVersio

Oenological characterisation of indigenous strains of S. cerevisiae isolated in a biodynamic winery in the Cortona DOC area

Genotypic and technological characterisation of the S. cerevisiae population isolated in a biodynamic winery in the Cortona DOC area was performed to gain better knowledge of the variables that influence winemaking. The oenological performance of 11 S. cerevisiae strains was evaluated with physiological tests; strain typing was performed through analysis of interdelta sequences and 26S rDNA sequencing. The analysis revealed a remarkable variability in terms of S. cerevisiae strains, despite the homogeneity of wine features, underlining the high levels of biodiversity characterising biodynamic agriculture. Some strains were found in wines of different vintages, suggesting the presence of an established microbiota in the winery. Oenological tests demonstrated that while some yeasts provided reliable oenological performance, other strains were not able to accomplish prompt and effective alcoholic fermentation, or were characterised by spoilage characteristics, such as excessive production of volatile phenols or acetic acid. Indigenous strains of S. cerevisiae could be a useful instrument for reliable winemaking without altering the native microbiota of each oenological environment. However, characterisation of their oenological suitability, and the application of practices able to drive the evolution of microbiota, must be employed to reduce the risk of wine spoilage

Consecutive alcoholic fermentations of white grape musts with yeasts immobilized on grape skins : effect of biocatalyst storage and SO2 concentration on wine characteristics

Abstract Saccharomyces cerevisiae yeasts, immobilized by natural adsorption on grape skins, were used to carry out the alcoholic fermentation step of a winemaking process. The viability of the immobilized cells was evaluated by the implementation of 7 successive fermentations of a white grape must containing 30 mg/L of SO2. The time to complete alcoholic fermentation, the physicochemical characteristics of the produced wines (ethanol, glycerol, organic acids, volatile compounds, color) and sensory properties were evaluated. A traditional fermentation with free cells was used as control. Three other fermentations were conducted after storage of the immobilized biocatalyst (30 d, 4 oC), the first one in the same conditions of the earlier assays, and the other two with higher amounts of SO2 (60 mg/L, 90 mg/L). Wines produced with immobilized cells presented physicochemical and sensory characteristics similar to those traditionally produced with free cells. After three consecutive fermentations, chromatic characteristics became similar to those of traditional wines, but the fermentation time had been reduced from 7 d to 4 d. The fermentative process and the characteristics of the produced wines were not significantly affected by the use of higher amounts of SO2. Immobilized biocatalysts could be stored at least one month without losing its activity.Zlatina Genisheva gratefully acknowledges FCT (Contract/grant number: SFRH/BD/48186/2009) and the Project "BioInd - Biotechnology and Bioengineering for improved Industrial and Agro-Food processes", REF. NORTE-07-0124-FEDER-000028 Co-funded by the Programa Operacional Regional do Norte (ON.2 - O Novo Norte), QREN, FEDER, for the financial support of this work

Genome-Wide Search Reveals the Existence of a Limited Number of Thyroid Hormone Receptor Alpha Target Genes in Cerebellar Neurons

Thyroid hormone (T3) has a major influence on cerebellum post-natal development. The major phenotypic landmark of exposure to low levels of T3 during development (hypothyroidism) in the cerebellum is the retarded inward migration of the most numerous cell type, granular neurons. In order to identify the direct genetic regulation exerted by T3 on cerebellar neurons and their precursors, we used microarray RNA hybridization to perform a time course analysis of T3 induced gene expression in primary cultures of cerebellar neuronal cell. These experiments suggest that we identified a small set of genes which are directly regulated, both in vivo and in vitro, during cerebellum post-natal development. These modest changes suggest that T3 does not acts directly on granular neurons and mainly indirectly influences the cellular interactions taking place during development