Use of Glutathione, Pure or as a Specific Inactivated Yeast, as an Alternative to Sulphur Dioxide for Protecting White Grape Must from Browning.

One of the problems that most seriously affects oenology today is enzymatic browning, especially when grapes are infected by grey rot. We studied the capacity of glutathione (GSH) and a specific inactivated dry yeast rich in glutathione (IDY-GSH) to protect white grape must from browning compared to that of sulphur dioxide (SO2). The results indicate that SO2 drastically reduces the oxygen consumption rate (by around 72%), protects hydroxycinnamic acids from oxidation and prevents grape must against browning even in the presence of laccase. Specifically, the presence of SO2 reduced the colour's blue-yellow component (b*) by around 91% in control conditions and around 76% in the presence of laccase. GSH, pure or in the form of IDY-GSH, also reduces the oxygen consumption rate (by 23% and 36%, respectively) but to a lesser extent than SO2. GSH also favours the formation of grape reaction product (GRP) from hydroxycinnamic acids and effectively protects grape must against browning in healthy grape conditions. Specifically, the presence of GSH reduced b* by around 81% in control conditions. Nevertheless, in the presence of laccase, it was not effective enough, reducing b* by around 39% in the case of pure GSH and 24% in the case of IDY-GSH. Therefore, both forms of GSH can be considered as interesting alternative tools to SO2 for preventing browning in white grape must, but only when the grapes are healthy.

Characterization of Mannoprotein Structural Diversity in Wine Yeast Species.

The structure of yeast cell wall (CW) mannoproteins (MPs) influences their impact on wine properties. Yeast species produce a diverse range of MPs, but the link between properties and specific structural features has been ill-characterized. This study compared the protein and polysaccharide moieties of MP-rich preparations from four strains of four different enologically relevant yeast species, named Saccharomyces boulardii (SB62), Saccharomyces cerevisiae (SC01), Metschnikowia fructicola (MF77), and Torulaspora delbrueckii (TD70), and a commercial MP preparation. Monosaccharide determination revealed that SB62 MPs contained the highest mannose/glucose ratio followed by SC01, while polysaccharide size distribution analyses showed maximum molecular weights ranging from 1349 kDa for MF77 to 483 kDa for TD70. Protein identification analysis led to the identification of unique CW proteins in SB62, SC01, and TD70, as well as some proteins shared between different strains. This study reveals MP composition diversity within wine yeasts and paves the way toward their industrial exploitation.

The diversity of effects of yeast derivatives during sparkling wine aging.

This study shows the monitoring of the physical, chemical and sensorial changes that occur in the sparkling wine along 18 months of aging due to different typology yeast-derived products; dry inactivated yeast from Saccharomyces (Saccharomyces cerevisiae) and non-Saccharomyces (Torulaspora delbrueckii) yeast strains, yeast autolysate, and yeast protein extract tested at two different doses. The addition of 5 g/hL yeast protein extract and inactivated yeast from T. delbrueckii helped to preserve esters in wines with 9 and 18 months of aging on lees. The addition of yeast autolysate achieved greater polysaccharide enrichment and gave rise to sparkling wines with the highest antioxidant activity. Effects on foaming properties were quite different depending on the aging time. Despite this, sparkling wines treated with 10 g/hL of yeast autolysate and Optimum White™ generally exhibited the highest foamability and foam stability. Further experiments with higher doses are needed to observe clear effects on sensory profile.

The dual role of amyloid-ß-sheet sequences in the cell surface properties of FLO11-encoded flocculins in Saccharomyces cerevisiae.

Fungal adhesins (Als) or flocculins are family of cell surface proteins that mediate adhesion to diverse biotic and abiotic surfaces. A striking characteristic of Als proteins originally identified in the pathogenic Candida albicans is to form functional amyloids that mediate cis-interaction leading to the formation of adhesin nanodomains and trans-interaction between amyloid sequences of opposing cells. In this report, we show that flocculins encoded by FLO11 in Saccharomyces cerevisiae behave like adhesins in C. albicans. To do so, we show that the formation of nanodomains under an external physical force requires a threshold number of amyloid-forming sequences in the Flo11 protein. Then, using a genome editing approach, we constructed strains expressing variants of the Flo11 protein under the endogenous FLO11 promoter, leading to the demonstration that the loss of amyloid-forming sequences strongly reduces cell-cell interaction but has no effect on either plastic adherence or invasive growth in agar, both phenotypes being dependent on the N- and C-terminal ends of Flo11p. Finally, we show that the location of Flo11 is not altered either by the absence of amyloid-forming sequences or by the removal of the N- or C-terminus of the protein.

Optimised Extraction and Preliminary Characterisation of Mannoproteins from Non-Saccharomyces Wine Yeasts.

The exogenous application of yeast-derived mannoproteins presents many opportunities for the improvement of wine technological and oenological properties. Their isolation from the cell wall of Saccharomycescerevisiae has been well studied. However, investigations into the efficiency of extraction methods from non-Saccharomyces yeasts are necessary to explore the heterogeneity in structure and composition that varies between yeast species, which may influence wine properties such as clarity and mouthfeel. In this study, nine yeast strains were screened for cell wall mannoprotein content using fluorescence microscopy techniques. Four species were subsequently exposed to a combination of mechanical and enzymatic extraction methods to optimize mannoprotein yield. Yeast cells subjected to 4 min of ultrasound treatment applied at 80% of the maximum possible amplitude with a 50% duty cycle, followed by an enzymatic treatment of 4000 U lyticase per g dry cells weight, showed the highest mannoprotein-rich yield from all species. Furthermore, preliminary evaluation of the obtained extracts revealed differences in carbohydrate/protein ratios between species and with increased enzyme incubation time. The results obtained in this study form an important step towards further characterization of extraction treatment impact and yeast species effect on the isolated mannoproteins, and their subsequent influence on wine properties.

Antioxidant activity from inactivated yeast: Expanding knowledge beyond the glutathione-related oxidative stability of wine.

Maintaining wine oxidative stability during barrel ageing and shelf life storage remains a challenge. This study evaluated the antioxidant activities of soluble extracts from seven enological yeast derivatives (YDs) with increased glutathione (GSH) enrichment. YDs enriched in GSH appeared on average 3.3 times more efficient at quenching radical species than YDs not enriched in GSH. The lack of correlation (Spearman correlation ρ = 0.46) between the GSH concentration released from YDs and their radical scavenging activity shed light on other non-GSH compounds present. After 4-methyl-1,2-benzoquinone derivatization, UHPLC-Q-ToF MS analyses specifically identified 52 nucleophiles potentially representing an extensive molecular nucleophilic fingerprint of YDs. The comparative analysis of YD chemical oxidation conditions revealed that the nucleophilic molecular fingerprint of the YD was strongly correlated to its antiradical activity. The proposed strategy shows that nucleophiles co-accumulated with GSH during the enrichment of YDs are responsible for their antioxidant activities.

Metabolic diversity conveyed by the process leading to glutathione accumulation in inactivated dry yeast: A synthetic media study.

Glutathione-rich inactivated dry yeasts (GSH-IDY) are purported to accumulate glutathione intracellularly and then released into the must. Glutathione is beneficial for wine quality, but research has highlighted that GSH-IDYs have a synergic antioxidant effect similar to that of molecular GSH. Combination of negative mode ultra-high-resolution Fourrier-Transform Ion-Cyclotron-Resonance Mass Spectrometry ((-)FT-ICR-MS), ultra-high-performance liquid chromatography coupled to a Quadrupole-Time of Flight mass spectrometer (UHPLC-Q-ToF-MS) and HPLC/Diode Detector Array (DAD)-Fluorescence spectroscopy was applied to three inactivated dry yeasts soluble fractions, with increasing intracellular glutathione concentration, in order to explore the chemical diversity released in different synthetic media. Using the mean of size exclusion chromatography/DAD and fluorescence detection we report than most of the signals detected were below the 5-75 kDa-calibrated region of the chromatogram, indicating that most of the soluble protein fraction is composed of low molecular weight soluble peptides. In light of these results, high-resolution mass spectrometry was used to scan and annotate the low molecular weight compounds from 50 to 1500 Da and showed that GSH level of enrichment in IDYs was correlated to a discriminant chemical diversity of the corresponding soluble fractions. Our results clearly show an impact of the GSH accumulation process not only visible on the glutathione itself, but also on the global diversity of compounds. Within the 1674 ions detected by (-)FT-ICR-MS, the ratio of annotated elemental formulas containing carbon, hydrogen, oxygen, nitrogen and sulfur (CHONS) to annotated elemental formulas containing carbon, hydrogen, oxygen (CHO) increased from 0.2 to 2.1 with the increasing levels of IDYs GSH content and 36 unique CHONS annotated formulas were unique to the IDY with the highest concentration of GSH. Amongst the 1674 detected ions 193 were annotated as potential peptides (from 2 to 5 residues), 61 ions were annotated as unique amino acid combinations and 46% of which being significantly more intense in GSH-rich IDY. Thus, the process leading to the accumulation of glutathione also involves other metabolic pathways which contribute to an increase in CHONS containing compounds potentially released in wine, notably peptides.

Correction: Del Barrio-Galán, R., et al. Evaluation of Yeast Derivative Products Developed as an Alternative to Lees: The Effect on the Polysaccharide, Phenolic and Volatile Content, and Colour and Astringency of Red Wines. Molecules 2019, 24, 1478.

The authors wish to make the following corrections to this paper [...].

Evaluation of Yeast Derivative Products Developed as an Alternative to Lees: The Effect on the Polysaccharide, Phenolic and Volatile Content, and Colour and Astringency of Red Wines.

Due to the increase of the use of yeast derivatives (YDs) in winemaking to improve the technological and sensory properties in wines, in this work we evaluated the effect of the post-fermentation application of different yeast derivative products on the physical and chemical properties and astringency of red wines during two consecutive harvests. A commercial and two experimental new yeast derivatives were applied at a medium‒high dosage (30 g/hL). The addition of different yeast derivatives in red wine increased the concentration of different polysaccharide fractions and, therefore, the total polysaccharide content, producing a decrease in the duration of the wine astringency perception over time. The use of yeast derivatives could produce an adsorption/clarification and/or protective effect on the phenolic compounds. However, it did not produce an important modification of the colour parameters. An intensification or a lower decrease of the most volatile compound groups was produced, but it depended on the YDs and yeast strain used in fermentation and post-fermentation processes.

AFM dendritips functionalized with molecular probes specific to cell wall polysaccharides as a tool to investigate cell surface structure and organization.

The yeast cell wall is composed of mannoproteins, ß-1,3/ß-1, 6-glucans and chitin. Each of these components has technological properties that are relevant for industrial and medical applications. To address issues related to cell wall structure and alteration in response to stress or conditioning processes, AFM dendritips were functionalized with biomolecules that are specific for each of the wall components, which was wheat germ agglutinin (WGA) for chitin, concanavalin A (ConA) for mannans and anti-ß-1,3/anti-ß-1,6-glucan antibodies for ß-1,3/ß-1,6-glucans. Binding specificity of these biomolecules were validated using penta-N-acetylchitopentaose, α-mannans, laminarin (short ß-1,3-glucan chain) and gentiobiose (2 glucose units linked in ß 1→6) immobilized on epoxy glass slides. Dynamic force spectroscopy was employed to obtain kinetic and thermodynamic information on the intermolecular interaction of the binary complexes using the model of Friddle-Noy-de Yoreo. Using this model, transition state distance xt, dissociate rate koff and the lowest force (feq ) required to break the intermolecular bond of the complexes were approximated. These functionalized dendritips were then used to probe the yeast cell surface treated with a bacterial protease. As expected, this treatment, which removed the outer layer of the cell wall, gave accessibility to the inner layer composed of ß-glucans. Likewise, bud scars were nicely localized using AFM dendritip bearing the WGA probe. To conclude, these functionalized AFM dendritips constitute a new toolbox that can be used to investigate cell surface structure and organization in response to a wide arrays of cultures and process conditions.

Integration of Biochemical, Biophysical and Transcriptomics Data for Investigating the Structural and Nanomechanical Properties of the Yeast Cell Wall.

The yeast cell is surrounded by a cell wall conferring protection and resistance to environmental conditions that can be harmful. Identify the molecular cues (genes) which shape the biochemical composition and the nanomechanical properties of the cell wall and the links between these two parameters represent a major issue in the understanding of the biogenesis and the molecular assembly of this essential cellular structure, which may have consequences in diverse biotechnological applications. We addressed this question in two ways. Firstly, we compared the biochemical and biophysical properties using atomic force microscopy (AFM) methods of 4 industrial strains with the laboratory sequenced strain BY4743 and used transcriptome data of these strains to infer biological hypothesis about differences of these properties between strains. This comparative approach showed a 4-6-fold higher hydrophobicity of industrial strains that was correlated to higher expression of genes encoding adhesin and adhesin-like proteins and not to their higher mannans content. The second approach was to employ a multivariate statistical analysis to identify highly correlated variables among biochemical, biophysical and genes expression data. Accordingly, we found a tight association between hydrophobicity and adhesion events that positively correlated with a set of 22 genes in which the main enriched GO function was the sterol metabolic process. We also identified a strong association of ß-1,3-glucans with contour length that corresponds to the extension of mannans chains upon pulling the mannosyl units with the lectin-coated AFM tips. This association was positively correlated with a group of 27 genes in which the seripauperin multigene family was highly documented and negatively connected with a set of 23 genes whose main GO biological process was sulfur assimilation/cysteine biosynthetic process. On the other hand, the elasticity modulus was found weakly associated with levels of ß-1,6-glucans, and this biophysical variable was positively correlated with a set of genes implicated in microtubules polymerization, tubulin folding and mitotic organization.

Hypotheses on the effects of enological tannins and total red wine phenolic compounds on Oenococcus oeni.

Lot of articles report on the impact of polyphenols on wine lactic acid bacteria, but it is clear that the results still remain confusing, because the system is complicated both in term of chemical composition and of diversity of strains. In addition, red wines polyphenols are multiple, complex and reactive molecules. Moreover, the final composition of wine varies according to grape variety and to extraction during winemaking. Therefore it is nearly impossible to deduce their effects on bacteria from experiments in oversimplified conditions. In the present work, effect of tannins preparations, currently considered as possible technological adjuvants, was assessed on growth and malolactic fermentation for two malolactic starters. Experiments were conducted in a laboratory medium and in a white wine. Likewise, impact of total polyphenolic extracts obtained from different grape variety red wines was evaluated in the white wine as culture medium. As expected growth and activity of both strains were affected whatever the additions. Results suggest some interpretations to the observed impacts on bacterial populations. Influence of tannins should be, at least partly, due to redox potential change. Results on wine extracts show the need for investigating the bacterial metabolism of some galloylated molecules. Indeed, they should play on bacterial physiology and probably affect the sensory qualities of wines.

Interactions of Condensed Tannins with Saccharomyces cerevisiae Yeast Cells and Cell Walls: Tannin Location by Microscopy.

Interactions between grape tannins/red wine polyphenols and yeast cells/cell walls was previously studied within the framework of red wine aging and the use of yeast-derived products as an alternative to aging on lees. Results evidenced a quite different behavior between whole cells (biomass grown to elaborate yeast-derived products, inactivated yeast, and yeast inactivated after autolysis) and yeast cell walls (obtained from mechanical disruption of the biomass). Briefly, whole cells exhibited a high capacity to irreversibly adsorb grape and wine tannins, whereas only weak interactions were observed for cell walls. This last point was quite unexpected considering the literature and called into question the real role of cell walls in yeasts' ability to fix tannins. In the present work, tannin location after interactions between grape and wine tannins and yeast cells and cell walls was studied by means of transmission electron microscopy, light epifluorescence, and confocal microscopy. Microscopy observations evidenced that if tannins interact with cell walls, and especially cell wall mannoproteins, they also diffuse freely through the walls of dead cells to interact with their plasma membrane and cytoplasmic components.

Effects of the strain background and autolysis process on the composition and biophysical properties of the cell wall from two different industrial yeasts.

The Saccharomyces cerevisiae cell surface is endowed with some relevant technological properties, notably antimicrobial and biosorption activities. For these purposes, yeasts are usually processed and packaged in an 'autolysed/dried' formula, which may have some impacts on cell surface properties. In this report, we showed using a combination of biochemical, biophysical and molecular methods that the composition of the cell wall of two wine yeast strains was not altered by the autolysis process. In contrast, this process altered the nanomechanical properties as shown by a 2- to 4-fold increased surface roughness and to a higher adhesion to the atomic force microscope tips of the autolysed cells as compared to live yeast cells. Besides, we found that the two strains harboured differences in biomechanical properties that could be due in part to higher levels of mannan in one of them, and to the fact that the surface of this mannan-enriched strain is decorated with highly adhesive patches forming nanodomains. The presence of these nanodomains could be correlated with the upregulation of flocculin encoding FLO11 as well as to higher expression of few other genes encoding cell wall mannoproteins in this mannan-enriched strain as compared to the other strain.