Isolation and characterisation of Saccharomyces cerevisiae mutants with increased cell wall chitin using fluorescence-activated cell sorting.

Yeast cell wall chitin has been shown to bind grape pathogenesis-related chitinases that are the primary cause of protein haze in wines suggesting that yeast cell walls may be applied for haze protection. Here we present a high throughput screen to identify yeast strains with high cell wall chitin using a reiterative enrichment strategy and Fluorescence-Activated Cell Sorting of cells labelled with either GFP-tagged chitinase or with Calcofluor White. To assess the validity of the strategy, we first used a pooled deletion strain library of Saccharomyces cerevisiae. The strategy enriched for deletion mutants with genes that had previously been described as having an impact on chitin levels. Genes that had not previously been linked to chitin biosynthesis or deposition were also identified. These genes are involved in cell wall maintenance and/or membrane trafficking functions. The strategy was then applied to a mutagenized population of a commercial wine yeast strain, Saccharomyces cerevisiae EC1118. Enriched mutant strains showed significantly higher cell wall chitin than the wild type and significantly reduced the activity of chitinases in synthetic model wine, suggesting that these strains may be able to reduce haze formation in wine.

French and Mediterranean-style diets: Contradictions, misconceptions and scientific facts-A review.

The determination of appropriate dietary strategies for the prevention of chronic degenerative diseases, cancer, diabetes, and cardiovascular diseases remains a challenging and highly relevant issue worldwide. Epidemiological dietary interventions have been studied for decades with contrasting impacts on human health. Moreover, research scientists and physicians have long debated diets encouraging alcohol intake, such as the Mediterranean and French-style diets, with regard to their impact on human health. Understanding the effects of these diets may help to improve in the treatment and prevention of diseases. However, further studies are warranted to determine which individual food components, or combinations thereof, have a beneficial impact on different diseases, since a large number of different compounds may occur in a single food, and their fate in vivo is difficult to measure. Most explanations for the positive effects of Mediterranean-style diet, and of the French paradox, have focused largely on the beneficial properties of antioxidants, among other compounds/metabolites, in foods and red wine. Wine is a traditional alcoholic beverage that has been associated with both healthy and harmful effects. Not withstanding some doubts, there is reasonable unanimity among researchers as to the beneficial effects of moderate wine consumption on cardiovascular disease, diabetes, osteoporosis, and longevity, which have been ascribed to polyphenolic compounds present in wine. Despite this, conflicting findings regarding the impact of alcohol consumption on human health, and contradictory findings concerning the effects of non-alcoholic wine components such as resveratrol, have led to confusion among consumers. In addition to these contradictions and misconceptions, there is a paucity of human research studies confirming known positive effects of polyphenols in vivo. Furthermore, studies balancing both known and unknown prognostic factors have mostly been conducted in vitro or using animal models. Moreover, current studies have shifted focus from red wine to dairy products, such as cheese, to explain the French paradox. The aim of this review is to highlight the contradictions, misconceptions, and scientific facts about wines and diets, giving special focus to the Mediterranean and French diets in disease prevention and human health improvement. To answer the multiplicity of questions regarding the effects of diet and specific diet components on health, and to relieve consumer uncertainty and promote health, comprehensive cross-demographic studies using the latest technologies, which include foodomics and integrated omics approaches, are warranted.

Chitinases and thaumatin-like proteins in Sauvignon Blanc and Chardonnay musts during alcoholic fermentation.

Protein precipitation, also referred to as protein instability, may lead to haziness in bottled wines and result in significant commercial losses. To avoid problems of this nature, fining finished wines with clay (bentonite) is the most commonly applied methodology. However, bentonite fining reduces yield and may affect wine quality. Protein haze has been primarily linked to grape pathogenesis-related proteins, in particular chitinases and thaumatin-like proteins. To better understand the persistence of these proteins during fermentation, reverse phase chromatography was used to monitor the evolution of total grape proteins as well as of chitinases and thaumatin-like proteins during alcoholic fermentation. The data confirm a previously reported significant decrease in total protein content during fermentation. This reduction in total protein levels was observed throughout fermentation, and was affected by factors such as fermentation temperature, yeast strain or grape cultivar. However, significant changes in the concentration of free chitinases were observed in a yeast strain-dependent manner. The data thus confirm the correlation between the levels of yeast cell wall chitin and changes in chitinase concentration, and suggest that it is primarily the amount of lateral chitin, and not the chitin in bud scars, that is responsible for this activity.

Yeast Cell Wall Chitin Reduces Wine Haze Formation.

Protein haze formation in bottled wines is a significant concern for the global wine industry, and wine clarification before bottling is therefore a common but expensive practice. Previous studies have shown that wine yeast strains can reduce haze formation through the secretion of certain mannoproteins, but it has been suggested that other yeast-dependent haze protective mechanisms exist. On the other hand, the addition of chitin has been shown to reduce haze formation, likely because grape chitinases have been shown to be the major contributors to haze. In this study, Chardonnay grape must fermented by various yeast strains resulted in wines with different protein haze levels, indicating differences in haze-protective capacities of the strains. The cell wall chitin levels of these strains were determined, and a strong correlation between cell wall chitin levels and haze protection capability was observed. To further evaluate the mechanism of haze protection, Escherichia coli-produced green fluorescent protein (GFP)-tagged grape chitinase was shown to bind efficiently to yeast cell walls in a cell wall chitin concentration-dependent manner, while commercial chitinase was removed from synthetic wine in quantities that also correlated with the cell wall chitin levels of the strains. Our findings suggest a new mechanism of reducing wine haze, and we propose a strategy for optimizing wine yeast strains to improve wine clarification.IMPORTANCE In this study, we establish a new mechanism by which wine yeast strains can impact the protein haze formation of wines, and we demonstrate that yeast cell wall chitin binds grape chitinase in a chitin concentration-dependent manner. We also show that yeast can remove this haze-forming protein from wine. Chitin has in the past been shown to efficiently reduce wine haze formation when added to the wine in high concentration as a clarifying agent. Our data suggest that the selection of yeast strains with high levels of cell wall chitin can reduce protein haze. We also investigate how yeast cell wall chitin levels are affected by environmental conditions.