Article

Structural and ultrastructural changes in yeast cells during autolysis in a model wine system and in sparkling wines

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Abstract

This study shows the changes that occur during the autolysis of yeast in a model wine medium and in a sparkling wine after 12 months of aging, using Nomarsky Light Microscopy and Low Temperature Scanning Electron Microscopy (LTSEM). The size of the yeasts after 24 h of autolysis in a model medium is much smaller than when they are in the growth stage. With LTSEM. a large number of folds can be observed on the surface of the yeast and practically empty cells. Greater morphological changes, both structural and ultrastructural, can be observed in the yeast after 12 months of aging in wine than in the yeast after 24 h of induced autolysis. However, less of the cytoplasmic content of the yeast that has undergone autolysis in the wine was solubilized than that of the yeast after 24 h of autolysis in the model wine system. These findings indicate that autolysis of yeast in wine is a long-lasting process, which continues for at least 12 months.

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... [19,20] The shapes and appearances of 100 cells were observed at 7000X magnification. [21] The cell wall structures of the two yeasts were analyzed using transmission electron microscopy (TEM). Cells were fixed with 2.5% (w/v) glutaraldehyde-PBS buffer (0.1 mol/L, pH 7.2) overnight at 4 C, and then rinsed with the same buffer 6 times and post-fixed with 1% (w/v) osmium tetroxide for 2 h at 4 C. ...
... Ultrathin sections of the samples were prepared with a diamond knife and examined with a QUANTA200F TEM. [21] The thickness of the cell walls was measured in cells where the cell wall structures could be clearly observed, indicating that the cell wall was cut perpendicular to the cell surface. [22] The cell walls of 100 cells from each strain, which were unbudded, were measured at 50,000X magnification. ...
... [18] Chitins are usually found at the budding scars of the cell wall, and commonly $2% of chitin is found in the lateral walls of the cell. [37] However, under cell wall stress, chitin can be attached to b-1,3-glucan and b-1,6-glucan through Crh1 and Crh2 transglycosylases; [21,38] thus, a higher amount of chitin would be expected. [37] Consistent with our previous studies, the higher concentrations of polysaccharides of the Pilsner yeast ensured the robustness of this strain when used for more cycles. ...
Article
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Serial repitching of beer brewer’s yeast plays an important role in the beer industry as an ineluctable economic factor. In this study, the viability and vitality changes, as well as strains’ anti-autolytic abilities during serial beer fermentation with typical ale and lager yeast strains, were investigated. While measuring the survival rate of yeast cells is not sufficient for evaluating and predicting the yeast fermentation capacity, physiological status determination of brewer’s yeast reflects the vitality and quality of yeast in serial beer fermentations. Accumulation of reactive oxygen species in yeast causes cell damage, leading to a decline in cell vitality and viability. Aged yeast cells, after several repitchings, can result in leakage of intracellular compounds into the fermented liquor. The lager yeast (Pilsner) examined, which harbors the partial genome from S. eubayanus, showed better robustness and higher activity than the ale yeast strain M79 examined, during serial fermentation. A holistic approach, including more indicators, should be applied in evaluating the fermentation performance of brewer’s yeast.
... Autolysis is a term that describes the breakdown of cell constituents by action of endogenous enzymes. It can occur naturally when yeast have completed their growth cycle and entered the death phase, or it can be induced at high temperature and low pH (Martıńez-Rodrıǵuez et al., 2001;White et al., 2002). Proteases, β-glucanases and chitinases are among autolytic hydrolases that are implicated in this process, and therefore, it can be expected that this process may cause important change in cell wall composition. ...
... Proteases, β-glucanases and chitinases are among autolytic hydrolases that are implicated in this process, and therefore, it can be expected that this process may cause important change in cell wall composition. Accordingly, Martinez-Rodriguez et al (Martıńez-Rodrıǵuez et al., 2001) reported important ultrastructural changes during the autolysis of yeast cells using Low Temperature Scanning Electron Microscopy (LTSEM), whereas Aerosol Flow Tube -Fourier Transform Infrared Spectroscopy (AFT-FTIR) was used to monitor global biochemical changes during autolysis revealed hydrolysis of mannans and β-glucans (Cavagna et al., 2010). In spite of these apparent hydrolyses, microscopic observation of autolyzed cells showed that the cell shape was fully retained suggesting that the cell wall structure was not destroyed . ...
... However, the discrepancy can be explained by the difference in the autolysis process. In our study, it is an industrial process that consisted in an induced autolysis at high temperature for 20 h, whereas other studies were related to yeast autolysis during wine fermentation that lasted several days and was done at lower temperature Martıńez-Rodrıǵuez et al., 2001). As compared to living or active yeast cells, the main effect of autolysis/drying process was to increase the roughness of the cell surface, which was visualized as a change from a relatively smooth to wrinkled structure. ...
Thesis
Due to increasing yeast biomass production resulting from the expansion of the Biorefinery as an alternative to petrol-based energy, the yeast cell wall is receiving an increasing interest as an added value product targeting agro-nutrition markets, such as in animal nutrition and in wine for its probiotic and sorption properties. The purpose of this thesis was therefore to combine DNA microarrays, biochemical and biophysical approaches in order to investigate the relationships between these parameters as well as to highlight the impact of strains, growth conditions and processes on the cell wall composition and biophysical properties. To achieve this objective, an acido-enzymatic method was developed to specifically quantify each of the four components of the yeast cell wall, namely mannan, chitin, β-1,3-glucan and β-1,6-glucan. This method was validated on mutant strains and allowed highlighten various stresses effects. Then, the use of atomic force microscopy (AFM) has allowed investigating the same strains and four strains used in industrial fermentation. They demonstrated distinct nanomechanical and adhesive properties, due to differences in their cell wall structure and composition. In the last part, the effects of the autolysis and fluid-bed drying processes are presented. This industrial process does not change the composition of the cell wall but induces a modification in topography and surface properties of the cell. Moreover, using AFM we imaged on S. cerevisiae cell surface highly adhesive patches forming nanodomains.
... With the help of high-pressure freezing (HPF) technique, the yeast cells can be immediately cryoimmobilized and the ultrastructure of yeast during autolysis could be observed with TEM (Wright 2000). The two-layered structure of cell wall was clearly shown at the beginning of autolysis (Martinez-Rodriguez et al. 2001;Tudela et al. 2012). The electron-transparent inner layer of cell wall mostly consisted of β-glucans and could be divided into fibrous layer and amorphous matrix. ...
... Chitin is usually found at the budding scars of cell wall, and commonly about 2% of chitin is found in the lateral walls of cell (Levin 2011). However, under cell wall stress chitin was found to be attached to β-1,3-glucan and β-1,6glucan through Crh1 and Crh2 transglycosylases (Bourdet and Mandel 1953;Martinez-Rodriguez et al. 2001), and cells would deposit as much as 10 times more chitin in the lateral walls to compensate the compromised cell integrity (Orlean 2012). Therefore, higher amount of chitin would be discovered during the autolysis process. ...
Article
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Brewer’s yeast is used in production of beer since millennia, and it is receiving increased attention because of its distinct fermentation ability and other biological properties. During fermentation, autolysis occurs naturally at the end of growth cycle of yeast. Yeast cell wall provides yeast with osmotic integrity and holds the cell shape upon the cell wall stresses. The cell wall of yeast consists of β-glucans, chitin, mannoproteins, and proteins that cross linked with glycans and a glycolipid anchor. The variation in composition and amount of cell wall polysaccharides during autolysis in response to cell wall stress, laying significant impacts on the autolysis ability of yeast, either benefiting or destroying the flavor of final products. On the other hand, polysaccharides from yeast cell wall show outstanding health effects and are recommended to be used in functional foods. This article reviews the influence of cell wall polysaccharides on yeast autolysis, covering cell wall structure changings during autolysis, and functions and possible applications of cell wall components derived from yeast autolysis.
... Lipids, predominantly derived from yeast membranes, can also be released during autolysis [29,30] and are considered by some as markers of autolysis [31]. Lipid release during induced autolysis in model systems has been shown to be very rapid with sterol esters and triacylglycerols being the major lipid fractions present [29]. ...
... Similar to reports by Moreno-Arribas et al. [16], proline was the predominant amino acid in the wines. During secondary fermentation, free amino acids support yeast growth, and are also released during autolysis [3,30]. Preferential uptake of nitrogen compounds that can be metabolised efficiently by yeast results in depleted concentrations in wine, and subsequent accumulation of amino acids such as proline and lysine that cannot be used by yeast during anaerobic fermentation [57]. ...
Article
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Premium sparkling wine produced by the traditional method (analogous to the French méthode champenoise) is characterised by the development of aged wine character as a result of a second fermentation in the bottle with lees contact and lengthy ageing. Treatments (microwave, ultrasound, or β-glucanase enzymes) were applied to disrupt the cell wall of Saccharomyces cerevisiae and added to the tirage liquor for the second fermentation of Chardonnay-Pinot Noir base wine cuvée and compared to a control, to assess effects on the release of phenolics, proteins, amino acids, and lipids at 6, 12 and 18 months post-tirage. General responses to wine ageing included a 60% increase in the total phenolic content of older sparkling wines relative to younger wines and an increase in protein concentration from 6 to 12 months bottle age. Microwave and β-glucanase enzyme treatments of yeast during tirage preparation were associated with a 10% increase in total free amino acid concentration and a 10% increase in proline concentration at 18 months bottle age, compared to control and ultrasound treatment. Furthermore, microwave treatment was associated with elevated asparagine content in wine at 18 months bottle age, relative to the control and the other wines. The β-glucanase enzyme and ultrasound treatments were associated with significant accumulation of total lipids, which were driven by 2-fold increases in the phospholipid and monoacylglycerol components in wine at 18 months bottle age and, furthermore, the microwave treatment was associated with elevated triacylglycerol at 18 months bottle age. This study demonstrates that the use of yeast treatments at the tirage stage of sparkling wine production presents an opportunity to manipulate wine composition.
... A strain-dependent phenotype that, as a consequence of cell disruption, implies the release in the fermenting wine of several yeastassociated compounds able to influence organoleptic and foaming properties of sparkling wine [77,78]. Several reports suggested the positive effect of yeast strains with high autolytic capacity on sparkling wine quality and foaming properties [24,79,80]. This characteristic is desired at the end of the re-fermentation in sparkling wines, generally two or four months after the end of secondary fermentation. ...
... A strain-dependent phenotype that, as a consequence of cell disruption, implies the release in the fermenting wine of several yeast-associated compounds able to influence organoleptic and foaming properties of sparkling wine [77,78]. Several reports suggested the positive effect of yeast strains with high autolytic capacity on sparkling wine quality and foaming properties [24,79,80]. This characteristic is desired at the end of the re-fermentation in sparkling wines, generally two or four months after the end of secondary fermentation. ...
Article
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The sparkling wine market has expanded in recent years, boosted by the increasing demand of the global market. As for other fermented beverages, technological yeasts and bacteria selected to design commercial starter cultures represent key levers to maximize product quality and safety. The increasing economic interest in the sector of sparkling wine has also implied a renewed interest in microbial resource management. In this review, after a brief introduction, we report an overview of the main characterization criteria in order to select Saccharomyces cerevisiae strains suitable for use as starter cultures for the production of base wines and to drive re-fermentation of base wines to obtain sparkling wines. Particular attention has been reserved to the technological characterization aspects of re-fermenting phenotypes. We also analysed the possible uses of selected non-Saccharomyces and malolactic strains in order to differentiate specific productions. Finally, we highlighted the main safety aspects related to microbes of enological interest and underlined some microbial-based biotechnological applications helpful to pursue product and process innovations. Overall, the sparkling wine industry may find a relevant benefit from the exploitation of the wide resources associated with vineyard/wine microbial diversity.
... Our group has analyzed morphological aspects of wine yeast viability and autolysis, including the presence/absence of budding, vacuole size, cell size, and cytoplasm separation from the cell wall (González et al., 2003;González et al., 2008;Martínez-Rodríguez et al., 2001b;Martínez-Rodríguez et al., 2004). In those studies, we compared viable cell counts and total microscopic cell count as we had observed an increasing concentration of dead yeast cells, ranging from 10 5 cells/mL in the first week of incubation to 10 6 cells/mL after day 20 of fermentation. ...
... These cell wall changes are visible only under an electron microscope. In a study conducted by our group, Martínez-Rodríguez et al. (2001b) observed the development of folds on the yeast cell wall caused by loss of volume during autolysis (see Figure 2.2), a finding that coincides with previous reports (Charpentier & Feuillat, 1993;González et al., 2008). ...
Chapter
Traditional-method sparkling wines, which are made using particular varieties of grape, contain carbon dioxide gas as a natural consequence of the process used in their production. This gas is a byproduct of the secondary fermentation of natural or added sugars in the base wine. The fermentation takes place in closed vessels and the resulting wine has a minimum pressure of 4 atm at 20ºC. Depending on the winemaking method used, sparkling wines are classified as tank-fermented wines or bottle-fermented wines. In the tank-fermented method, secondary fermentation takes place under isobaric conditions in a sealed tank with a capacity of tens of hectoliters. These tanks are equipped with stirring mechanisms that mix the yeast uniformly into the base wine. In the transfer method, the sparkling wine is produced in bottles, which are generally magnums measuring 1.5 or 2 L to minimize storage space requirements. It is then left to age on lees for at least 2 months, after which it is transferred to a tank maintained under isobaric conditions with carbon dioxide or nitrogen to prevent loss of the gas. The wine is then cold processed at –5ºC, filtered, and sometimes transferred to a second tank.
... Yeast cells have been shown to exhibit elongated and ovoid morphologies during fermentation, compared to the more spherical shaped cells in sparkling wine lees aged for 12 months [28]. In contrast, morphologies associated with cellular degradation appeared as a reduction in cell volume during induced autolysis in a model wine whereas cells surface wrinkles/folds were more apparent on yeast cells aged in wine for 12 months. ...
... Decreased nucleic acid content, possibly due to degradation, is a potential consequence of increased cell permeability over time. It has previously been observed that natural autolysis is a slow process during ageing of sparkling wines produced by the traditional method [4] and that loss of cytoplasmic contents is a common feature of extended maturation [28]. ...
Article
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Sparkling wine made by the traditional method (Méthode Traditionelle) develops a distinct and desirable flavour and aroma profile attributed to proteolytic processes during prolonged ageing on lees. Microwave, ultrasound and addition of β-glucanase enzymes were applied to accelerate the disruption of Saccharomyces cerevisiae, and added to the tirage solution for secondary fermentation in traditional sparkling winemaking. Scanning electron microscopy and flow cytometry analyses were used to observe and describe yeast whole-cell anatomy, and cell integrity and structure via propidium iodide (PI) permeability after 6-, 12- and 18-months post-tirage. Treatments applied produced features on lees that were distinct from that of the untreated control yeast. Whilst control yeast displayed budding cells (growth features) with smooth, cavitated and flat external cell appearances; microwave treated yeast cells exhibited modifications like ‘doughnut’ shapes immediately after treatment (time 0). Similar ‘doughnut’-shaped and ‘pitted/porous’ cell features were observed on progressively older lees from the control. Flow cytometry was used to discriminate yeast populations; features consistent with cell disruption were observed in the microwave, ultrasound and enzyme treatments, as evidenced by up to 4-fold increase in PI signal in the microwave treatment. Forward and side scatter signals reflected changes in size and structure of yeast cells, in all treatments applied. When flow cytometry was interpreted alongside the scanning electron microscopy images, bimodal populations of yeast cells with low and high PI intensities were revealed and distinctive ‘doughnut’-shaped cell features observed in association with the microwave treatment only at tirage, that were not observed until 12 months wine ageing in older lees from the control. This work offers both a rapid approach to visualise alterations to yeast cell surfaces and a better understanding of the mechanisms of yeast lysis. Microwave, ultrasound or β-glucanase enzymes are tools that could potentially initiate the release of yeast cell compounds into wine. Further investigation into the impact of such treatments on the flavour and aroma profiles of the wines through sensory evaluation is warranted.
... Autolysis is not the same as cell death because it may occur while the yeast cells are still alive in beer fermentation, although cell constituents are released to the surroundings. Autolysis of wine yeasts contributes to the sensory evaluation of wine with the release of flavor compounds and foaming agents (Martinez-Rodriguez et al., 2001;Alexandre & Guilloux-Benatier, 2006;Cavagna et al., 2010). However, the autolysis of brewer's yeast leaves unpleasant flavors such as a yeasty smell, bitterness and astringency in beer. ...
... Serving as the last bulwark, cell wall plays a vital role in maintaining cell form and in controlling growth (Inouhe et al., 1997;Palomero et al., 2007). It was reported that cell wall becomes wrinkled and ridged but can still maintain its integrity after cell death (Hernawan & Fleet, 1995;Martinez-Rodriguez et al., 2001;Cavagna et al., 2010) because the damage to cell wall was partial and small-scale (Xu et al., 2013). Thus cell wall has its own place in autolysis. ...
Article
In this work, we performed DNA microarray studies on lager brewer's yeast Saccharomyces pastorianus to investigate changes in gene expression in the process of autolysis. The two strains we used were Qing2 and 5-2. The strain 5-2 is a mutant of Qing2, and it autolyzes much more slowly than its parent strain. Four samples of these two strains during different autolysis stages (0% and 15%) were tested using DNA microarray containing > 10,000 yeast's genes. Analysis of genes with the same transcription pattern (up-regulated or down-regulated in both the two strains) showed that the same 99 genes were up-regulated (transcription levels were increased), and the same 97 genes were down-regulated (transcription levels were decreased) by 5-fold or more during autolysis. Genes involved in energy production/utilization, protein anabolism, and stress response were down-regulated. Meanwhile, genes related to cell wall organization and biogenesis, starvation response and DNA damage response were up-regulated. Analysis of genes with opposite transcription patterns (up-regulated in one strain and down-regulated in the other one) showed that 246 genes were up-regulated in 5-2 (autolyzes slowly) while down-regulated in Qing2 (autolyzes rapidly). Other 18 genes had opposite transcription levels, indicating that the strain which autolyzes slowly had better cell vitality despite the same autolysis stage. These findings might further promote the global understanding of autolysis in yeast.This article is protected by copyright. All rights reserved.
... These authors observed the disappearance of chitinase from Champagne wine and attributed this observation to the likelihood that the chitinase was fixed on the cell wall of S. cerevisiae and that of the bacterium Oenococcus oeni during the aging period on lees. Furthermore, the grape chitinases have been demonstrated to maintain their activity in wine for at least a few months after the end of alcoholic fermentation (27,28), thereby explaining the reduced haze levels observed during aging on lees with no observed increase in mannoprotein levels (data not shown). ...
... Boiling cells results in a large number of wrinkles or folds on the cell walls. This is a cell structure similar to that of cells undergoing autolysis, as observed by Martínez-Rodríguez et al. (28). The reduced cell surface area in boiled cells may have consequently resulted in the slight reduction in the amount of chitinases bound to the cell wall. ...
Article
Full-text available
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.
... Since the natural autolysis is a long-lasting process, it is usual to use model systems to obtain results in shorter periods of time (Martínez-Rodríguez et al. 2001);Petruzzi et al. (2014a) tested two commercial (BM45 and RC212) and three wild S. cerevisiae strains (W13, W47 and Y28) and a commercial cell wall preparation to remove OTA. As pH, temperature and ethanol are the major factors affecting autolysis in a model system, different pHs (3.0 and 3.5) temperatures (25 and 30°C) and ethanol concentrations (5, 10 and 15 %) were used. ...
Article
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Biological decontamination of mycotoxins using microorganisms is one of the well-known strategies for the management of mycotoxins in foods and feeds. Yeasts are an efficient biosorbant, used in winemaking to reduce the concentration of harmful substances from the must which affect alcoholic fermentation (medium-chain fatty acids) or which affect wine quality in a negative way (ethyl phenols and sulphur products). In recent years, several studies have demonstrated the ability of yeasts to remove ochratoxin A (OTA) by live cells, cell walls and cell wall extracts, yeast lees. In spite of the physical and chemical methods applied to remove the toxin, the biological removal is considered a promising solution, since it is possible to attain the decontamination without using harmful chemicals and without losses in nutrient value or palatability of decontaminated food. In addition, adsorption is recognized as economically viable, technically feasible and socially acceptable. This paper intends to review the current achievements of OTA removal mediated by yeasts, the recent updates in the selection of strains acting at the same time as starters and as biological tools to remove OTA and the factors affecting the removal process.
... Different strategies have been suggested for accelerating yeast autolysis. Enzymes able to hydrolize B-glucans from yeast cell walls and thermolysis are the most widely proposed tools (Martínez-Rodríguez et al., 2001;Comuzzo et al., 2012;Bzducha-Wróbel et al., 2014). Recently the potential of non-thermal processing technologies such as high pressure homogenization (Comuzzo et al., 2015) and ultrasound (Martín et al., 2013) have been also investigated to induce autolysis of wine yeasts. ...
Article
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The potential of the application of pulsed electric fields (PEF) to induce accelerate autolysis of a commercial strain of Saccharomyces cerevisiae for winemaking use was evaluated. The influence of PEF treatments of different intensity (5–25 kV/cm for 30–240 μs) on cell viability, cytoplasmic membrane permeabilization and release of mannoproteins and compounds absorbing at 260 and 280 nm has been investigated. After 8 days of incubation at 25°C the Abs600 of the suspension containing the control cells was kept constant while the Abs600 of the suspension containing the cells treated by PEF decreased. The measurement of the absorbance at 260 and 280 nm revealed no release of UV absorbing material from untreated cells after 8 days of incubation but the amount of UV absorbing material released drastically increased in the samples that contained cells treated by PEF after the same storage period. After 18 days of storage the amount of mannoproteins released from the untreated cell was negligible. Conversely, mannoprotein concentration increased linearly for the samples containing cells of S. cerevisiae treated by PEF. After 18 days of incubation the concentration of mannoproteins in the supernatant increased 4.2 times for the samples containing cells treated by PEF at 15 and 25 kV/cm for 45 and 150 μs. Results obtained in this study indicates that PEF could be used in winemaking to accelerate the sur lie aging or to obtain mannoproteins from yeast cultures.
... Yeast autolysis is a very complex and slow process which occurs after fermentation when yeasts begin to die and disintegrate [1,2]. It is characterized by the hydrolysis of biopolymers under the action of endogenous hydrolytic enzymes such as proteases, nucleases, lipases and β-glucanases [3][4][5]. ...
Article
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Over recent years, ageing on lees has acquired a great distinction in winemaking as an interesting technique for improving wine quality. The aim of the present work was to evaluate the effectiveness of two novel processing techniques, ultrasounds and microwaves (both combined with stirring and abrasion treatments), in order to accelerate the autolytic process in wine yeast. Furthermore, the effects of ultrasonic treatment on the organoleptic properties of a red wine aged on lees were also studied. The content of polysaccharides (HPLC–RI), anthocyanins (HPLC–PDAD/ESI–MS), volatile compounds (GC–FID), colour parameters (spectrophotometry) and sensory analysis was periodically evaluated. Results showed that ultrasound treatment is a reliable technique for shortening the ageing on lees process by strongly increasing the concentration of polysaccharides released into the wine after only 2 weeks of treatment and without adversely affecting the sensory quality of the wine. Furthermore, the addition of glass beads stood out as a good abrasive agent increasing the polysaccharides release up to 10 times the control when coupled with ultrasound treatment. Nevertheless, both stirring and microwave treatments were less effective, possibly due to the short period of application used. The impact of the alcoholic degree on the yeast autolysis was also evaluated, and, moreover, it was confirmed that moderate heating promotes a higher release of polysaccharides into the wine.
... After autolysis, yeast cells are much smaller and show wrinkles or folds and ridges. These wrinkles are attributed to plasmolysis, which is supported by an increase in the size of the vacuole because of solubilization of the cytoplasmic content [5]. These authors compared structural and ultrastructural changes in yeast cells during autolysis in a model wine system and in sparkling wines. ...
Chapter
Autolysis of yeast cells occurs after they have completed their life cycle and entered the death phase. It is characterized by a loss of cell membrane permeability, alteration of cell wall porosity, hydrolysis of cellular macromolecules by endogenous enzymes, and subsequent leakage of the breakdown products into the extracellular environment. Although a naturally occurring event, autolysis can be induced by exposing yeasts to elevated temperatures (40–60 °C), organic solvents, or detergents. Yeast autolysis occurs in many foods and beverages, where it may affect their sensory quality and commercial acceptability.
... Additionally, the cell changes that occur in S. cerevisiae during fermentation (autoproteolysis) and aging (autolysis) are responsible for the organoleptic properties of wine. Accordingly, the amount of nitrogen in autolysates together with free amino acid concentrations, which differ greatly depending on the yeast strain, can have a considerable influence on the flavor, composition, and quality of the final product (Martínez-Rodríguez et al., 2001a, 2001b. Proteolytic enzymes might be involved in the turnover of nitrogenous compounds before and during autolysis in winemaking conditions. ...
Article
Saccharomyces cerevisiae is the main microorganism involved in wine fermentation. It has been used as a model organism in molecular biology for many years and is the only wine yeast species for which abundant genomic and proteomic information is available. Most of the techniques currently used in functional genomics and proteomics were initially developed in this yeast. The fact that S. cerevisiae was the first microorganism to be widely used in the development of genome technology allowed other phylogenetically related yeasts to be analyzed subsequently in global sequencing projects, and the use of comparative genomics has since led to important conclusions regarding gene functionality. S. cerevisiae has also been used in the development of the more recent field of proteomics. Proteomic studies have generated vast amounts of data on protein expression profiles and variability in laboratory strains of S. cerevisiae, and these have recently been extended to include wine strains. Important advances have also been made in metabolomics, a new field in which S. cerevisiae is practically the only eukaryote to have been studied to date. The integration of different types of omic data into predictive models has provided the basis for new research strategies in systems biology.
... To each tube, 0.1 mL of yeast biomass suspension (viable or non--viable cells) in phosphate buffer were added to give a final concentration of 0.0088 g/mL. After 7 days of contact between the biomass and the medium (21,22,(35)(36)(37), the solution was centrifuged and the non-adsorbed amounts of pesticides in the medium were measured. Non-viable cells were prepared by the addition of sodium azide (Sigma-Aldrich) to the biomass suspended in phosphate buffer (0.025 % final volume fraction in the media) in order to exclude metabolic action and preserve the structural integrity of the cells (21,38). ...
Article
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The capacity for the removal of pyrimethanil and fenhexamid, two fungicides commonly used for the control of Botrytis cinerea in vineyards, has been evaluated during an alcoholic fermentation process in batch system. Commercial and wild strains of Saccharomyces cerevisiae were used. Batch fermentations were carried out in yeast extract-malt extract medium (YM) with 18.0 % (by mass) glucose, and the fungicides were added separately at three concentrations: 0.1, 1.0 and 10.0 mg/L. The removal capacity of yeast strains was also examined in stationary phase cultures of Saccharomyces cerevisiae. Stationary assays were performed with yeast biomass harvested from the stationary phase of an anaerobic fermentation process, with separate additions of 0.1, 1.0 and 10.0 mg/L of both fungicides. Removal studies with stationary phase cells were performed with viable and non-viable cells inactivated with sodium azide. This study clearly shows that both Saccharomyces cerevisiae strains were able to remove fenhexamid and pyrimethanil in stationary and fermentative assays. The removal potential is shown to be strain dependent in stationary but not in fermentative assays. However, the removal potential is dependent on the type of fungicide in both stationary and fermentative assays. In stationary phase cultures no significant difference in fungicide removal potential between viable and non-viable cells was observed, indicating that both pesticides were not degraded by metabolically active cells. However, the presence of both pesticides influenced fermentation kinetics and only pyrimethanil at 10.0 mg/L increased the production of volatile acidity of both strains.
... After the completion of fermentation, the yeast biomass was maintained in contact with the media for seven days by daily mixing. During this time, the major proportion of mannose was released from the yeast cell walls into the media (Nunez et al., 2007) due to accelerated autolysis triggered by the lack of nutrients (Martínez-Rodriguez et al., 2001a;2001b;2002). However, during the alcoholic fermentation conducted by both yeast strains, no significant reduction was found in the concentration of OTA. ...
Article
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The capacity for removal of ochratoxin A (OTA) during alcoholic fermentation was evaluated in batch systems with one commercial strain and one wild strain of Saccharomyces cerevisiae. Batch alcoholic fermentations were carried out in yeast extract-malt extract broth (YM) medium, with 18.0% glucose and OTA added to final concentrations of 3.48 and 4.95 ng/mL respectively. The removal capacity of each yeast strain was examined after completion of fermentation in batch culture and after extended contact with yeast biomass. The removal capacity of the yeast strains was also examined in stationary phase cultures. Stationary phase yeasts were studied with biomass harvested from the stationary phase of anaerobic fermentation, by incubation in phosphate buffer, with the addition of 5.00 ng/mL of OTA. Removal studies with stationary phase cells were performed with viable and non-viable cells inactivated with Na-azide. The study showed that in growing phase cultures, OTA removal was significant only after extended contact with yeast biomass; up to 29.7% and 25.4% for wild yeast ZIM 1927 and commercial yeast Lalvin EC-1118 respectively, but not during alcoholic fermentation. In stationary phase cultures, viable and non-viable cells were not significantly different in OTA removal from the medium. This demonstrated that OTA was not metabolised, but possibly adsorbed by the yeast cells. The presence of OTA in synthetic media influenced yeast metabolism, causing the production of higher volatile acidity by 0.08 and 0.13 g/L for Lalvin EC-1118 and ZIM 1927 respectively, and lower concentrations of reducing sugar, by 0.32 g/L, but only for ZIM 1927.
... According to Martínez-Rodríguez et al. [32], progressing process of yeast cell autolysis results in a decreased volume of the cells, which leads to the leakage of cytoplasmic material being an effect of wall-membrane complex of yeast cell damage. The study presented by Rajashree and Muthukumar [18] demonstrated that S. cerevisiae NCYC 1026 yeasts cultured in Sabouraud medium (SDB) enriched with sodium selenite at various doses exhibited distinct metabolic changes. ...
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This paper presents the results of microscopic examinations of the yeast cells cultured in yeast extract-peptone-dextrose (YPD) media supplemented with sodium selenite(IV). The analysis of the morphological changes in yeast cells aimed to determine whether the selected selenium doses and culturing time may affect this element accumulation in yeast cell structures in a form of inorganic or organic compounds, as a result of detoxification processes. The range of characteristic morphological changes in yeasts cultivated in experimental media with sodium selenite(IV) was observed, including cell shrinkage and cytoplasm thickening of the changes within vacuole structure. The processes of vacuole disintegration were observed in aging yeast cells in culturing medium, which may indicate the presence of so-called ghost cells lacking intracellular organelles The changes occurring in the morphology of yeasts cultured in media supplemented with sodium selenite were typical for stationary phase of yeast growth. From detailed microscopic observations, larger surface area of the cell (6.03 μm(2)) and yeast vacuole (2.17 μm(2)) were noticed after 24-h culturing in the medium with selenium of 20 mg Se(4+)/L. The coefficient of shape of the yeast cells cultured in media enriched with sodium selenite as well as in the control YPD medium ranged from 1.02 to 1.22. Elongation of cultivation time (up to 48 and 72 h) in the media supplemented with sodium selenite caused a reduction in the surface area of the yeast cell and vacuole due to detoxification processes.
... Proteases, β-glucanases and chitinases are among autolytic hydrolases that are implicated in this process, and therefore, it can be expected that this process may cause important change in cell wall composition. Accordingly, Martinez-Rodriguez, Polo and Carrascosa (2001) reported ultrastructural changes during the autolysis of yeast cells using lowtemperature scanning electron microscopy (LTSEM) and aerosol flow tube-Fourier transform infrared spectroscopy, and revealed some release of mannan and β-glucans during this process. In spite of this apparent loss of polysaccharides, microscopic observation of autolysed cells showed that the cell shape was fully retained upon rehydration, suggesting that the cell wall structure was not destroyed (Hernawan and Fleet 1995). ...
Article
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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. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.
... On the other hand, proteolytic yeast enzymes are mainly aspartyl endoproteases, saccharopepsin [19], which are a highly specific family of proteases that tends to cleave peptide bonds involving hydrophobic residues as well as having a beta-methylene group [20]. Therefore, it seems that a systematic degradation of oligopeptides in smaller peptides [21,22] occurred in sparkling wine as base wine lees content increased. ...
Conference Paper
Quality branded (PDO) soft "pasta filata" cheese like "Mozzarella di bufala Campana" are of both scientific and economic interest because of the growing sale trend and because of problems related to protection of their quality traits. The use of the natural whey starter cultures is required by the standard of identity of the PDO cheese. Although it is one of the main factors affecting the typicality of the product, it is also responsible for the variability of its sensory quality. Commercial defined starter cultures used for other varieties of soft Mozzarella cheese may significantly improve the hygienic quality of the product and reduce its variability but they also result in a flattening of important sensory properties with a significant reduction of consumer acceptance. In the framework of a research project whose main objectives where the standardization of the quality, the increase of the shelf‐life of water‐buffalo Mozzarella cheese, whilst protecting its typical flavour characteristics, we developed a multiple strain starter containing Lactobacillus delbrueckii ssp. bulgaricus, Streptococcus thermophilus and Lb. rhamnosus. The use of the MSS allowed to obtain an excellent product reproducibility from both the rheological and sensory standpoint. Proteolysis and aroma profile of Mozzarella cheese produced using the MSS and of control produced using the traditional technology was compared. Primary proteolysis was evaluated by UREA‐PAGE while gas‐chromatography was used to evaluate volatile organic compounds and organic acids. The control and the MSS Mozzarella cheese did not show any significant difference in the proteolytic profile, while minor differences where found in organic acids. Acetic acid content was 4 times lower in the control, in which lactic acid was slightly higher compared to the MSS cheese. Both formic and citric acid content were similar in the two cheeses. We conclude that the MSS can be used to control the variability of water‐buffalo Mozzarella cheese without negatively affecting its typical flavour.
... On the other hand, proteolytic yeast enzymes are mainly aspartyl endoproteases, saccharopepsin [19], which are a highly specific family of proteases that tends to cleave peptide bonds involving hydrophobic residues as well as having a beta-methylene group [20]. Therefore, it seems that a systematic degradation of oligopeptides in smaller peptides [21,22] occurred in sparkling wine as base wine lees content increased. ...
Article
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In this paper, we explored an innovative and sustainable technology to improve the sensorial characteristics of traditional sparkling wine. Different volumes of lees, recovered from the first fermentation, were included into the “liqueur de tirage” for the second fermentation of Bombino grapes base wine. Proteolysis evolution was compared to that of traditional Bombino grapes sparkling wine over 18 months after “dégorgement”, using combined liquid chromatography techniques and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of peptides in the size of 1500–3000 Da range. The addition of lees exhibited a clear impact on proteolysis, most likely due to the hydrolytic enzymes released by yeast autolysis. The profile of volatile organic compounds was assessed by gas chromatography–MS, foam stability, and the sensory traits were both evaluated by a panel of trained testers. The sensory traits were positively influenced by the addition of lees up to 60 mL/L base wine. Therefore, changing the amount of lees addition can be used to modulate the aromatic composition of sparkling wine in an attempt to improve it, and this also allows the shortening of ageing time.
... It is likely that autolysis of yeast cells in iQhilika is an important part of the production process as far as flavour is concerned. This is similar in many ways to sparkling wines (Pretorius 2000, Martinez-Rodriguez et al. 2001, Gonzalez et al. 2003) and sherry (Charpentier et al. 2004) where autolysis of yeast contributes organoleptic qualities unique to the product. ...
... H/K strains are considered important during vinification since they produce aromatic compounds of interest and modify the chemical composition of wines [9][10][11]18,112]. Two species stand out for producing high amounts of β-phenylethyl acetate, H. guilliermondii [113], and H. vineae [18,26,54,58,61,62,114]. However, this compound is not found at such high levels in the other species, as discussed above. ...
Article
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Apiculate yeasts of the genus Hanseniaspora/Kloeckera are the main species present on mature grapes and play a significant role at the beginning of fermentation, producing enzymes and aroma compounds that expand the diversity of wine color and flavor. Ten species of the genus Hanseniaspora have been recovered from grapes and are associated in two groups: H. valbyensis, H. guilliermondii, H. uvarum, H. opuntiae, H. thailandica, H. meyeri, and H. clermontiae; and H. vineae, H. osmophila, and H. occidentalis. This review focuses on the application of some strains belonging to this genus in co-fermentation with Saccharomyces cerevisiae that demonstrates their positive contribution to winemaking. Some consistent results have shown more intense flavors and complex, full-bodied wines, compared with wines produced by the use of S. cerevisiae alone. Recent genetic and physiologic studies have improved the knowledge of the Hanseniaspora/Kloeckera species. Significant increases in acetyl esters, benzenoids, and sesquiterpene flavor compounds, and relative decreases in alcohols and acids have been reported, due to different fermentation pathways compared to conventional wine yeasts.
... During this aging period, several compounds (including lipids, carbohydrates, nucleotides, amino acids, peptides, mannoproteins and volatile com- pounds) are released in the autolysis process (Alexandre & GuillouxBenatier, 2006). These molecules influence the characteristics of the wine and its sensorial quality (Alexandre & Guilloux-Benatier, 2006;Martínez-Rodríguez, Polo, & Carrascosa, 2001). Sparkling wine quality depends on several parameters, such as foam characteristics (foam- ability, persistence, in mouth aggressiveness, and bubble size), color, acidity and aroma. ...
Article
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One of the strongest factors that affects the volatile profile of sparkling wine is the winemaking process. Here we focus on determining the effects of the second fermentation and aging on lees of sparkling wine from País grape variety combining different analysis techniques for the first time in sparkling wine: gas chromatography/mass spectrometry/olfactometry and sensorial analysis. During the second fermentation and aging, there was a significant loss of esters that might be related to the adsorption on lees and ester volatility and chemical hydrolysis. The concentration of several compounds such as some esters (diethyl succinate, ethyl lactate, and ethyl isovalerate) increased during aging and could be used as aging markers. Vitispiranes were identified as the best norisoprenoids aging markers for young sparkling wines (12 months of aging). Also, PCA showed that time of aging on lees affected mostly esters and terpenes. On the other hand, the diminution of fruity/floral impact odorants during aging was not perceived in sensorial trials. Our results suggest that the responsibility for fruity/floral nuances in sparkling wine might reside in a few high-impact aromatic compounds, such as ethyl isobutyrate, isoamyl acetate, ethyl hexanoate, β-phenylethanol and diethyl succinate.
... After several pieces of research trying to select better autolytic yeasts and to accelerate the autolytic process in different ways [4,14,[16][17][18] and with the aim of improving this ageing over lees, several attempts have been made by adding different components of the yeasts to the wine, among them are yeast autolysates, cell walls, mannoproteins and b-glucanases-all of them accepted by the European union as oenological additives [19]. The use of these additives is supported by the development of the autolysis itself, which involves the hydrolysis of the cell walls and the release of their biopolymers and other molecules into the wine [20][21][22]. ...
Article
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Sparkling winemaking is an increasing industry in Castilla y León region of Spain. Several trials have been made to improve the techniques in order to offer better products to the market. This study focuses on the evaluation of the influence of different co-adjuvants in the sparkling winemaking process (β-glucanases, autolysated yeasts, yeasts cell walls and purified mannoproteins) by measuring different oenological parameters at 3, 6 and 9 months of ageing after the secondary fermentation. Both physicochemical and sensorial analyses are considered in order to reveal the incidence of these co-adjuvants in the final product. A consumers study has also been carried out with the aim of knowing the possible impact of the co-adjuvants in the market of the so elaborated sparkling wines. β-Glucanases seem to increase the ageing characteristics of the sparkling wines, while yeast derivatives (yeast cell walls and yeast autolysates) improve their fruity and flowery character.
... After autolysis, the yeast cells are much smaller and have wrinkles or folds and ridges (Avakyants 1982, Charpentier et al. 1986, Kollar et al. 1993). These wrinkles are thought to be due to plasmolysis, with the increased vacuole size due to solubilisation of the cytoplasmic content supporting this suggestion (Martinez-Rodriguez et al. 2001a). In these studies, the structural and ultrastructural changes occurring in yeast cells during autolysis were compared in a model wine system and in sparkling wines. ...
... Along the second fermentation, the content of glucanases and vacuolar proteases increased more under PC in flor yeast (Table 1), whereas nucleases in general were more abundant in P29. The presence of glucanases and vacuolar proteases with high content in flor yeast at T2 may be explained due to the structural and morphological changes during autolysis [26]. Since this yeast strain is not adapted to pressure conditions, the requirement of proteins involved in cell wall remodeling could be more relevant in flor yeast than in P29. ...
Article
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Apoptosis and later autolysis are biological processes which take place in Saccharomyces cerevisiae during industrial fermentation processes, which involve costly and time-consuming aging periods. Therefore, the identification of potential cell death biomarkers can contribute to the creation of a long-term strategy in order to improve and accelerate the winemaking process. Here, we performed a proteomic analysis based on the detection of possible apoptosis and autolysis protein biomarkers in two industrial yeast strains commonly used in post-fermentative processes (sparkling wine secondary fermentation and biological aging) under typical sparkling wine elaboration conditions. Pressure had a negatively effect on viability for flor yeast, whereas the sparkling wine strain seems to be more adapted to these conditions. Flor yeast strain experienced an increase in content of apoptosis-related proteins, glucanases and vacuolar proteases at the first month of aging. Significant correlations between viability and apoptosis proteins were established in both yeast strains. Multivariate analysis based on the proteome of each process allowed to distinguish among samples and strains. The proteomic profile obtained in this study could provide useful information on the selection of wine strains and yeast behavior during sparkling wine elaboration. Additionally, the use of flor yeasts for sparkling wine improvement and elaboration is proposed.
... Despite a high production yield, acidic or alkaline hydrolysis is an expensive method, which produces products with high salt contents and possibly carcinogenic effects (Nagodawithana 1992). Enzymatic hydrolysis can be carried out by either microbial enzymes or other exogenous cell wall proteolytic enzymes, which produces yeast lysates low in salt content (Martıńez-Rodrıǵuez et al. 2001). Enzymatic hydrolysis using digestive enzymes, which will decompose the cell wall, is a mild strategy compared to the acid hydrolysis leading to production of peptides with high nutritional values and diverse biological activities (Jung et al. 2011;Bekatorou et al. 2006). ...
Article
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Saccharomyces cerevisiae is being used for long as a rich source of proteins, sugars, nucleotides, vitamins and minerals. Autolyzed and hydrolyzed yeast biomass has found numerous applications in the health food industry as well as livestock feeds. Here, we have compared three lysis methods for production of yeast lysates using autolysis, plasmolysis (ethyl acetate 1.5%), and enzymatic hydrolysis (Alcalase 0.2%). The efficiency of each process was compared according to soluble solid and protein contents, cell lysis monitoring, and release of intracellular materials, cell viability and microscopic analysis. Results showed that plasmolysis by ethyl acetate was found to be more efficient compared to autolysis, with a higher recovery of yeast extract (YE) content. In comparison, the content of released solids and proteins were higher during the enzymatic hydrolysis using Alcalase compared to autolysis and plasmolysis treatments. The highest decrease in optical density of 600 nm was monitored for the hydrolyzed cells. Besides, we defined “Degree of Leakage (DL)” as a new index of the lysis process, referring to the percentage of total released proteins from the cells and it was estimated to about 65.8%, which represents an appropriate indicator of the cell lysis. The biochemical and biophysical properties of the hydrolyzed yeast product as well as its biological activity (free radical scavenging activity and bacterial binding capacity) suggest that Alcalase could be used to accelerate the lysis of yeast cells and release the valuable intracellular components used for foodstuffs, feed and fermentation media applications. Graphic abstract Production of baker’s yeast lysates using autolysis, plasmolysis, and enzymatic hydrolysis methods.
... The yeast cell wall is a dynamic macromolecular complex in which components (β-1,3 and β-1,6-glucans, mannoproteins, and chitin) are linked, forming a molecular network with several functions [5]. Numerous studies have reported the structural and morphological changes of the cell wall, mainly during aging [6][7][8][9]. These authors confirmed that although the cell wall structure showed folds and morphological changes, it remained unbroken in yeast cells at long-term aging. ...
Article
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In this study, a first proteomic approach was carried out to characterize the adaptive response of cell wall-related proteins to endogenous CO2 overpressure, which is typical of second fermentation conditions, in two wine Saccharomyces cerevisiae strains (P29, a conventional second fermentation strain, and G1, a flor yeast strain implicated in sherry wine making). The results showed a high number of cell wall proteins in flor yeast G1 under pressure, highlighting content at the first month of aging. The cell wall proteomic response to pressure in flor yeast G1 was characterized by an increase in both the number and content of cell wall proteins involved in glucan remodeling and mannoproteins. On the other hand, cell wall proteins responsible for glucan assembly, cell adhesion, and lipid metabolism stood out in P29. Over-represented proteins under pressure were involved in cell wall integrity (Ecm33p and Pst1p), protein folding (Ssa1p and Ssa2p), and glucan remodeling (Exg2p and Scw4p). Flocculation-related proteins were not identified under pressure conditions. The use of flor yeasts for sparkling wine elaboration and improvement is proposed. Further research based on the genetic engineering of wine yeast using those genes from protein biomarkers under pressure alongside the second fermentation in bottle is required to achieve improvements.
... After the second fermentation, the wine is subjected to a long aging period in contact with the yeast lees for at least 9 months (Spanish sparkling wine, Cava) at low temperatures (12-16 • C). During this last stage, the organoleptic properties of wine are influenced by the release of certain secondary metabolites, such as glycerol, acetate, succinate, pyruvate, and esters [1][2][3][4]. Numerous studies attempt to relate the change in the concentration of aromatic compounds produced during aging and their effect on the final aroma of sparkling wines [5][6][7][8][9]. A recent study, conducted by our research group, focused on the study of the effect of endogenous CO 2 overpressure during the second fermentation in the production of sparkling wines. ...
Article
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Sparkling wines elaboration has been studied by several research groups, but this is the first report on analysis of biological processes according to the Gene Ontology terms (GO terms) and related to proteins expressed by yeast cells during the second fermentation of sparkling wines. This work provides a comprehensive study of the most relevant biological processes in Saccharomyces cerevisiae P29, a sparkling wine strain, during the second fermentation under two conditions (without and with endogenous CO2 overpressure) in the middle and the end of second fermentation. Consequently, a proteomic analysis with the OFFGEL fractionator and protein identification with LTQ Orbitrap XL coupled to HPLC were performed. The classification of biological processes was carried out using the tools provided by the Saccharomyces Genome Database. Results indicate that a greater number of biological processes were identified under condition without CO2 overpressure and in the middle of the fermentation versus the end of the second fermentation. The biological processes highlighted under condition without CO2 overpressure in the middle of the fermentation were involved in the carbohydrate and lipid metabolic processes and catabolic and biosynthetic processes. However, under CO2 overpressure, specific protein expression in response to stress, transport, translation, and chromosome organization and specific processes were not found. At the end of fermentation, there were higher specific processes under condition without CO2 overpressure; most were related to cell division, growth, biosynthetic process, and gene transcription resulting in increased cell viability in this condition. Under CO2 overpressure condition, the most representative processes were related to translation as tRNA metabolic process, chromosome organization, mRNA processing, ribosome biogenesis, and ribonucleoprotein complex assembly, probably in response to the stress caused by the hard fermentation conditions. Therefore, a broader knowledge of the adaptation of the yeast, and its behavior under typical conditions to produce sparkling wine, might improve and favor the wine industry and the selection of yeast for obtaining a high-quality wine.
... The study by Babayan et al. (1981) dealt with the issue of dynamic hydrolysis product accumulation (amino nitrogens and nucleic compounds) in the extracellular space of baker's yeast suspension. In addition, a number of studies involve the investigation of structural and ultra-structural changes in the yeast cells during yeast autolysis in a model wine system and in actual wines (Polo and Carrascosa, 2001;Pozo-Dengra et al., 2006). Recently, vibrational spectroscopic and microspectroscopic technologies have also been developed for structural and molecular characterization of microbial cells and have been applied as rapid and nondestructive techniques to monitor autolysis of yeast cells in base wine, providing information useful to the understanding of the process (Burattini et al., 2008;Cavagna et al., 2010). ...
Article
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A two-step autolysis process was proposed to enhance amino acid production from spent brewer's yeast. The technique was developed based on comparative study of the dynamics of production and release of proteins and amino acids during the autolysis of a concentrated suspension (22 wt.%) and a dilute yeast cell suspension (11.25 wt.%). The results suggest that, in the concentrated yeast suspension, proteins are more effectively broken down into amino acids, but the product release rate was lower due to a lower concentration gradient across the cell membrane. Thus, a two-step process, in which a high protein conversion occurred in a concentrated cell suspension during the first 13 h period, followed by a 26 h autolysis process within a dilute cell suspension, provided a higher overall yield of amino acids compared than the single-step process. The two-step process was found to result in a 25% higher amino acid yield with a weight fraction increase from 0.4 to 0.5 g/g dry wt. Other than these findings, the effect of adding NaCl to the suspension during autolysis was also investigated. It was found that, for the autolysis conditions employed in this study, the addition of NaCl did not significantly affect the production of protein but inhibited the production of amino acids.
... Furthermore, on the basis of our previous research we can assume that wrinkled or folded cells, are the same cells, which during computer image analysis were characterized by an irregular cell wall area. Martinez-Rodriguez et al. [2001] observed wrinkled or folded yeast cells after few months of aging in wine, using scanning electron microscopy. The above authors report that during autolysis, the cell membrane was destroyed. ...
Article
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The purpose of this work was to compare the influence of long-term continuous wine fermentation on two yeast strains ( Saccharomyces bayanus S.o./1 and S. bayanus S.o./1AD) immobilized on foam glass. Fermentation was carried out in the 4-columns fermentor for 3.5 months, and the must contained approximately 320 g·dm -3 total sugar. During the course of the process, the number of cells decreased both with the column number and with every subsequent month of the fermentor's work. The number of cells of the S.o./1AD strain was higher than that of the S.o./1 strain. After the end of fermentation, the number of viable S.o./1AD cells on the surface of carrier was higher than those of the S.o./1 strain. Yeast isolated from the carrier from the first column was characterized by the highest part of viable cells (43%). In the third and fourth columns, the amount of vi- able cells was similar for both strains (adequately 20% and 10%). Moreover, various shapes of cells isolated from the carrier after the end of fermentation were observed, for example: elongated, in the shape of a "pear", wrinkled and in the form of few connected cells. Yeast cells S.o./1 were more distorted.
... Enzymes partially destroy yeast cell wall, so that soluble cytoplasm leaks to the surface. This group of cell degradation can be divided into two subgroups: 1. Autolysis is self-digestion of a cell through the action of its own enzymes; the process has been defined as the hydrolysis of intracellular biopolymers under the effect of hydrolytic enzymes associated with cell death (Martinez-Rodriguez et al., 2001). Autolysis is initiated by the cell lysosomes which release digestive enzymes into the cytoplasm. ...
Article
β-glucan is a polymer of glucose which is produced by many different organisms – bacteria, fungi, plants. In this natural polymer glucose monomers are linked by β glycosidic bonds. Nowadays non-cellulosic, fungal β-glucans are being broadly investigated. The main reason being that these biological polymers are recognized as potent immunological stimulators for human immune system. Experimental data show that β-glucans protect mammals from different infections and increase immune system cells cytotoxicity against cancer. One of these immunomodulating substances is yeast β-glucan extracted from Saccharomyces cerevisiae. A large part of fungal cells mass consists of β-glucans. β-glucan is a major structural component of various yeast cells and is vital for cell functions. In the present paper we describe a simple β-glucan extraction procedure using Actinomyces rutgersensis 88 yeast lysing enzymatic complex for yeast cell lysis and soft alkaline β-glucan extraction in further steps.
Article
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Scientific researches on characterization of the commercial enological preparation of Lallzyme MMX® containing β-glucanase and its influence on autolysis of different yeast strains typically employed in the production of sparkling wine are lacking. The aim of the present work was to plug a gap in this field, studying the β-glucanase activity of Lallzyme MMX® and its interactions with BCS103® and EC1118® yeast strains. The results showed that β-glucanase was slightly inhibited by ethanol, but its residual activity at wine pH was sufficient for the purposes. Kinetic parameters showed a better enzyme–substrate complex formation for the EC1118® strain. The influence on yeast lysis during 12 months of bottle-aging was monitored, demonstrating that enzyme addition did not substantially influence either the content and progression of total proteins, or foam characteristics. However, scanning and transmission electron microscopy images and free amino acid analysis indicated β-glucanase improved cell wall degradation of both selected yeasts, evidencing a lower autolytic capacity of the BCS103® strain. Our study demonstrated that addition of β-glucanase catalyzed cell disorganization and promoted release of yeast components into sparkling wine, with strain-dependent effects. Therefore, employment of β-glucanase rich Lallzyme MMX® might effectively accelerate some aging characteristics of traditional sparkling wines.
Article
This study shows the morphological changes of Saccharomyces cerevisiae EC1118 during the second fermentation of Spanish cava wines, in relation with progression of fermentation and aging. In the first stages of active fermentation, and associated with the increase in viable counts, budding cells and a relative homogeneity in cell size were observed. Close to the moment of sugar exhaustion cells acquired the morphology of stationary phase, to finally enter in a death phase with cell size reduction, and cytoplasm alterations including inhomogeneity, refringency, and detachment of the cell wall. At the beginning of this step structures reminiscent to autophagosomes are observed. This is in accordance with the appearance of molecular markers of autophagy described elsewhere in similar winemaking conditions.
Article
Light lees that spent more than one year in barrels were used for ultrasound-assisted yeast lysis (22W/L, 18°C) in a model wine. For comparison, a classical yeast autolysis at mild temperature (25°C) was performed. The effect of ultrasound on lees was evaluated by analysing the release of proteins and polysaccharides to the model wine, and the viability of the yeasts contained in the lees. Under conditions tested, ultrasound-assisted yeast lysis increased the concentrations of proteins and polysaccharides in the model wine due to the release of these compounds from yeasts. Unlike the classical autolysis, ultrasound led to a high cell disruption, and after 20h of ultrasonic treatment, viable cells were hardly found. Furthermore, the final cell concentration for the ultrasound-assisted yeast lysis was much lower than that for the classical autolysis. The inactivation rate constant of ultrasound-assisted yeast lysis was 2.54×10(-5)s(-1). Finally, the morphological changes in cells were examined by scanning electron microscopy to verify the effect of ultrasound on yeast cells.
Article
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Chapter
Wine aging on lees is a traditional winemaking practice used during wine processes. After alcoholic fermentation, the wines are placed in barrels or in stainless steel tank with their yeasts for several months. Lees can be periodically stirred (bâtonnage). In Burgundy, most of white wines are traditionally aged on lees, especially those from “Côte de Beaune.” However, this technique is now used worldwide. Lees aging increases complexity, aroma and flavor compounds, palate weight, and texture in a wine. Taking into account the properties of the lees, new products have been developed for the wine industry called inactive dry yeast, which has gained interest in the oenological industry. Indeed, these preparations could be used for multiple purposes such as improving alcoholic fermentation or malolactic fermentation, enhance organoleptic characteristics of wine, or ensure wine safety.
Article
High-pressure homogenization (HPH) was applied at 90 MPa to different yeasts prior to use or prior to their use in the preparation of tirage solutions for sparkling wines. The effects on yeast cell viability, fermentation, and death kinetics during secondary fermentation and aging were determined. Saccharomyces bayanus L951 and Saccharomyces cerevisiae ML692 and commercial strains S. bayanus Lalvin CH14, S. bayanus IOC 18-2007, S. bayanus Lalvin EC1118, and S. bayanus IT 1818 were used as starters. Chemical characteristics and volatile compound profiles of the final sparkling wines produced with either HPH-treated or HPH-nontreated yeast were assessed. Results showed that sensitivity to HPH treatment was strain dependent, and after 40 days of secondary fermentation all samples reached pressure levels of at least 0.60795 MPa, independent of the HPH treatment, with the exception of those inoculated with strain ML692. Scanning electron microscope microphotographs of the samples aged for 40 days indicated for all strains an acceleration of autolysis due to HPH treatment. SPME-GC-MS and electronic nose data indicated significant changes due to HPH treatment. According to partial least squares analysis, the sparkling wines produced using HPH-treated cells, except strain L951, were significantly different from the control wines. High-pressure homogenization seems to be a versatile approach for modulation of the autolytic phenomena of starter tirage cultures of sparkling wines.
Article
The chapter begins by describing the lees and autolysis phenomena: mechanism and released products. It then reviews the influence of ageing '. sur lies' on the organoleptic and hygienic properties and the stability of white, red and sparkling wines. The chapter includes a description of different yeast preparations mimicking the lees and their use in winemaking.
Article
Background and Aims Yeast autolysis is a key step in the production of sparkling wines using the traditional method and a source of polysaccharides, mannoproteins and proteins. The evidence, however, of the enrichment of these macromolecules during ageing is not evident in the literature. In this study, autolysis of yeast lees in sparkling wines (Cava) was monitored over nine consecutive vintages. Methods and Results The concentration of polysaccharides and proteins did not clearly increase over time, probably because these macromolecules are simultaneously released from lees and removed by other processes. In a new approach, the autolytic process was reproduced, and involved recovering the lees and maintaining them in a model wine solution for 1 year. The lees released polysaccharides and proteins, but only a small proportion of that found in the sparkling wines, especially in younger wines. Conclusions This study confirmed that yeast enrich sparkling wines in key macromolecules but in low proportion in comparison with the usual concentration present in sparkling wine. This aspect should be considered by wineries that aim to achieve autolysis attributes, since the majority of sparkling wines are commercialised after short periods of ageing. Significance of the Study This study determined for the first time the true contribution of yeast autolysis to the total polysaccharide and protein concentration of sparkling wines from nine consecutive vintages.
Chapter
The process of winemaking is the result of biochemical transformations brought about by the action of several enzymes from various microorganisms, including yeasts, especially Saccharomyces cerevisiae, resulting in alcoholic fermentation producing ethanol as the main ingredient of alcoholic beverages like wines. Lactic acid bacteria also play a very significant role in carrying out a secondary process, known as malolactic fermentation, though most bacteria are involved in the spoilage of wines, as is the case with acetic acid bacteria. Furthermore, the fruit must is also exposed to numerous enzymes originating from sources other than yeasts and bacteria that contribute to the reactions involved in winemaking. Most wines are produced from grapes, but fruits other than grapes are also used to produce wines. In recent years, however, nongrape fruit wines have been gaining consumer interest mainly due to the scientific evidence of various health benefits of habitual consumption of a wide variety of fruits and their products. The emerging fruit wines include those of blueberries, blackberries, strawberries, and cherries. The focus of this chapter is to provide a review of the chemistry behind fruit wine manufacturing; discuss the nutritional composition, antioxidant capacity, and biological activity of these wines; and compare their composition with that of traditional grape wines. In general, fruit wines are a good dietary source of minerals, such as potassium, calcium, and phosphorus; antioxidants and phytonutrients, such as carotenoids (carotene and lutein); and phenolic compounds (anthocyanins, flavonols, flavan-3-ols, proanthocyanidins, ellagitannins, and phenolic acids). In contrast to traditional red wines, limited studies have been conducted on human health benefits of fruit wines, and thereby in-depth investigations using human clinical trials are needed to better understand the potential health benefits of specific fruit wines. In discussing the chemistry, the significance of chemical reactions in winemaking is included, with illustrative pathways.
Article
The aim of this study was to discuss the functions of FKS family genes which encode β-1, 3-glucan synthase regarding the viability and autolysis of yeast strain. Loss of FKS1 gene severely influences the viability and anti-autolytic ability of yeast. Mutation of FKS1 and FKS2 genes led to cell reconstruction, resulting in a sharp shrinkage of cell volume and decreased stress resistance, viability, and anti-autolytic ability. Deletion of FKS3 gene did not clearly influence the synthesis of β-1, 3-glucan of yeast but increased the strain's stress resistance, viability, and anti-autolytic ability. It is suggested that FKS3 would be the potential target for improving the stress resistance of yeast. The results revealed the relationship among FKS family genes and demonstrated their functions on yeast cell wall construction and anti-autolytic ability.
Article
The aim of this study was to demonstrate the potential of PEF for inducing autolysis of R. glutinis, with the purpose of designing a more efficient and ecofriendly carotenoid extraction process: an extraction from fresh biomass, using cheaper, non-toxic, environmental-friendly solvents. Propidium iodide uptake and release of intracellular components revealed the irreversible electroporation of R. glutinis by PEF. Flow cytometry measurements detected morphological changes in PEF-treated R. glutinis cells during incubation caused by the autolysis triggering effect of electroporation. After submitting the fresh biomass to a PEF treatment (15 kV/cm, 150 μs) that irreversibly electroporated more than the 90% of the cells, ethanol proved ineffective for extracting carotenoids from fresh biomass of R. glutinis. However, after incubating the PEF-treated fresh biomass for 24 h at 20 °C in a pH 7 buffer, ca. 240 μg/g d.w. of carotenoids were recovered after 1 h of extraction in ethanol. The highest amount of carotenoids extracted (375 μg/g d.w.) from the PEF-treated cells of R. glutinis was obtained after having incubated them at 25 °C for 24 h in a medium of pH 8.0. The improvement in carotenoid extraction by incubating the R. glutinis cells after PEF treatment seems to be caused by PEF-triggered autolysis, which tends to disrupt the association of carotenoids with other molecules present in the cytoplasm, and causes a degradation of the cell wall.
Article
Because gluten‐free type brewing with unmalted sorghum does not provide adequate nitrogen for complete fermentation, wort supplementation with ammonia (as diammonium phosphate, DAP) or lysine on yeast performance was investigated. By Phenotype Microarray, under aerobic conditions, greater yeast growth was indicated with DAP than lysine both as single source and combined with sorghum wort amino acids. With sorghum fermentation, both DAP and lysine improved maltose and maltotriose uptake. However, DAP supplementation also maintained yeast numbers (24.0–21.3 x 106 cells/ml) whereas there was a decline with lysine supplementation. Lysine supplementation also resulted in adverse effects on yeast cell morphology. Neither DAP nor lysine supplementation resulted in evident genetic change to the yeast but the change in substrate from barley malt wort to unmalted sorghum wort slightly altered the yeast genetically. Therefore, ammonia as DAP has potential as a nitrogen supplement for improving yeast fermentation performance in sorghum gluten‐free brewing. This article is protected by copyright. All rights reserved.
Chapter
Sparkling wines are produced in many different countries. Among sparkling wines, some are produced from Charmat or bottle fermentation. In this chapter, the practical methods and principles of producing quality sparkling wine is given in details including each step of the production from viticultural parameters, base wine production up to secondary fermentation. After the second fermentation in bottle, the wine is aged on lees. During aging, yeast autolysis occurred. The mechanisms of yeast autolysis and the compounds released are presented. The consequences of yeast autolysis on wine quality are detailed. Finally the riddling and disgorging operation are presented
Article
El objetivo del estudio fue evaluar el efecto de dietas con alta concentración de Saccharomyces cerevisiae sobre la proliferación de hemocitos en camarones Cryphiops caementarius machos. Los camarones (5.3 ± 1.2 cm de longitud total y 7.4 ± 2.9 g de peso total) se colectaron del río Pativilca (Perú). Cada camarón se mantuvo en un recipiente instalado dentro del acuario (seis camarones por acuario). Se empleó una dieta control (3% de levadura) y tres dietas experimentales (6, 9 y 12% de levadura) con dos repeticiones por tratamiento durante 28 días de cultivo. El número total de hemocitos fue mayor (p<0.05) con 6% de levadura (134.75 x 105 cél/ml), así mismo el número de granulocitos (31.44 x 105 cél/ml) y semigranulocitos (102.44 x 105 cél/ml). El número de hialinocitos disminuyó en todos los tratamientos y se mantuvo entre 0.31 y 1.56 x 105 cél/ml. El número de hemocitos atípicos se mantuvieron bajos en todos los tratamientos y sin diferencias con el basal (0.31 x 105 cél/ml). La dieta con 6% de levadura incrementó (p<0.05) el número total de hemocitos y los hemocitos granulocitos y semigranulocitos en los camarones machos C. caementarius. En cambio, las dietas con 9 y 12% de levadura afectaron (p<0.05) la proliferación de hemocitos totales y diferenciales.
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Yeasts are becoming popular as novel ingredients in fish feeds because of their potential to support better growth and concomitantly ensure good fish health. Here, three species of yeasts ( Cyberlindnera jadinii , Blastobotrys adeninivorans and Wickerhamomyces anomalus ), grown on wood sugars and hydrolysates of chicken were subjected to two down-stream processes, either direct heat-inactivation or autolysis, and the feed potential of the resulting yeast preparations was assessed through a feeding trial with Atlantic salmon fry. Histological examination of distal intestine based on widening of lamina propria, showed that autolyzed W. anomalus was effective in alleviating mild intestinal enteritis, while only limited effects were observed for other yeasts. Our results showed that the functionality of yeast in counteracting intestinal enteritis in Atlantic salmon was dependent on both the type of yeast and the down-stream processing method, and demonstrated that C. jadinii and W. anomalus have promising effects on gut health of Atlantic salmon.
Article
Full-text available
Yeasts are becoming popular as novel ingredients in fish feeds because of their potential to support better growth and concomitantly ensure good fish health. Here, three species of yeasts (Cyberlindnera jadinii, Blastobotrys adeninivorans and Wickerhamomyces anomalus), grown on wood sugars and hydrolysates of chicken were subjected to two down-stream processes, either direct heat-inactivation or autolysis, and the feed potential of the resulting yeast preparations was assessed through a feeding trial with Atlantic salmon fry. Histological examination of distal intestine based on widening of lamina propria, showed that autolyzed W. anomalus was effective in alleviating mild intestinal enteritis, while only limited effects were observed for other yeasts. Our results showed that the functionality of yeast in counteracting intestinal enteritis in Atlantic salmon was dependent on both the type of yeast and the down-stream processing method, and demonstrated that C. jadinii and W. anomalus have promising effects on gut health of Atlantic salmon.
Article
In this study, we evaluated the potentialities of ATR-FTIR microspectroscopy coupled to PCA in monitoring the major biochemical changes that occur during the autolysis of yeasts used for sparkling wine production. For this purpose, mid-infrared measurements were made on cells of the model strain Saccharomyces cerevisiae EC1118 in the course of autolysis induced at 30 degrees C for five days in a model and in a base wine. By relating principal component loadings to the corresponding absorption bands, it was shown that they well describe compositional modifications induced by autolytic process on yeast cells, such as partial hydrolysis of proteins, increase of peptides, free nucleotides, lipids, mannans, and beta-1,3 glucans. The corresponding score-score plots allowed us to monitor the different kinetics and to distinguish among faster, intermediate, and slower processes. ATR-FTIR microspectroscopy coupled with PCA is proposed as a sensitive method that can provide useful information to select efficient yeast strains, capable of accelerated autolysis, to be used in the second fermentation and aging of sparkling wines.
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For determination of the physiological role and mechanism of vacuolar proteolysis in the yeast Saccharomyces cerevisiae, mutant cells lacking proteinase A, B, and carboxypeptidase Y were transferred from a nutrient medium to a synthetic medium devoid of various nutrients and morphological changes of their vacuoles were investigated. After incubation for 1 h in nutrient-deficient media, a few spherical bodies appeared in the vacuoles and moved actively by Brownian movement. These bodies gradually increased in number and after 3 h they filled the vacuoles almost completely. During their accumulation, the volume of the vacuolar compartment also increased. Electron microscopic examination showed that these bodies were surrounded by a unit membrane which appeared thinner than any other intracellular membrane. The contents of the bodies were morphologically indistinguishable from the cytosol; these bodies contained cytoplasmic ribosomes, RER, mitochondria, lipid granules and glycogen granules, and the density of the cytoplasmic ribosomes in the bodies was almost the same as that of ribosomes in the cytosol. The diameter of the bodies ranged from 400 to 900 nm. Vacuoles that had accumulated these bodies were prepared by a modification of the method of Ohsumi and Anraku (Ohsumi, Y., and Y. Anraku. 1981. J. Biol. Chem. 256:2079-2082). The isolated vacuoles contained ribosomes and showed latent activity of the cytosolic enzyme glucose-6-phosphate dehydrogenase. These results suggest that these bodies sequestered the cytosol in the vacuoles. We named these spherical bodies "autophagic bodies." Accumulation of autophagic bodies in the vacuoles was induced not only by nitrogen starvation, but also by depletion of nutrients such as carbon and single amino acids that caused cessation of the cell cycle. Genetic analysis revealed that the accumulation of autophagic bodies in the vacuoles was the result of lack of the PRB1 product proteinase B, and disruption of the PRB1 gene confirmed this result. In the presence of PMSF, wild-type cells accumulated autophagic bodies in the vacuoles under nutrient-deficient conditions in the same manner as did multiple protease-deficient mutants or cells with a disrupted PRB1 gene. As the autophagic bodies disappeared rapidly after removal of PMSF from cultures of normal cells, they must be an intermediate in the normal autophagic process. This is the first report that nutrient-deficient conditions induce extensive autophagic degradation of cytosolic components in the vacuoles of yeast cells.
Article
The term “autolysis” was introduced into biological literature by SALKOVSKY [1]. Ever since it began to be used to designate self-digestion of cells under the action of their own intracellular enzymes. This definition is sufficiently satisfactory with regard to bacteria, and there are published lots of original and review papers dealing specifically with bacteria. Considerably less material has been accumulated on eukaryotes, apparently due to the absence of the fact of the cell self-digestion in most yeasts.
Article
Cell suspensions ofSacharomyces cerevisiae, Kloeckera apiculata andCandida stellata were autolyzed in phosphate buffer, pH 4.5, for up to 10 days. Cell dry weights decreased by 25–35% after 10 days. Based on initial cell dry weight, the soluble autolysate consisted of: carbohydrate (principally polysaccharide) 3–7%; organic acids 3–6%; protein 12–13%; free amino acids 8–12%; nucleic acid products 3–5%; and lipids 1–12%. The main organic acids in autolysates were propionic, succinic and acetic and the main amino acids were phenylalanine, glutamic acid, leucine, alanine and arginine. Approximately 85–90% of cellular RNA and 25–40% of cellular DNA were degraded during autolysis. Both neutral lipid and phospholipid components were degraded, with neutral lipids but not phospholipids being found in autolysates. Scanning and transmission electron micrographs showed retention of cell wall structure and shape during autolysis, but there was extensive intracellular disorganization withinS. cerevisiae andC. stellata. There were differences in the autolytic behavior ofK. apiculata compared withS. cerevisiae andC. stellata.
Article
After culture in a synthetic and in a wine medium, the autolysis of Saccharomyces cerevisiae and Saccharomyces bayanus produced typical cell wall alterations depending on the yeast growth conditions. After growth in a wine medium, cell wall thickness did not change in either of the two yeasts even when there is an important loss of amino acids and glucans. This loss of wall material and especially of glucan involved a slackening of wall structures. The thickness of cell wall of yeast grown in a synthetic medium decreased by 50% after autolysis. This change was the consequence of a loss of amino acids and sugars which more specifically were constituents of the peripheral layer of the wall.
Article
A study was made of the composition of free and total amino acids, amino acids in peptides, and amino acids in proteins in four base nines and the sparkling wines obtained from them following the Champenoise method. Samples were taken of the sparkling wines after 9, 12, 15, 18, 24, and 31 months of aging with yeasts. The results were evaluated using different multivariate analysis techniques. It was observed that the release of amino acids during aging does not occur in all wines at the same time. The free amino acid composition of sparkling nines depends on that of the base wines from which they originate, while the distribution of amino acids in peptides and proteins depends, at least partly, on the aging time with yeasts. The findings indicate the existence of a relationship between the aging time of the wine with yeasts and the amino acid composition of peptides with a molecular weight lower than 700 Da. Castellblanch, S.A. (Sant Sadurnı´d’Anoia, Spain), for preparing the wines especially for the purpose of this research. Peer reviewed
Article
The freeze-etching technique, which is a special kind of freeze-drying, allows electron microscopic investigation of cells and tissues in the frozen state. In regard to yeast cells (Saccharomyces cerevisiae) a freeze-fixation technique has been developed which does not kill the object. The electron micrographs therefore are considered to impart an image of high fidelity. The cutting of the frozen object, which actually consists of a fine splintering, produces not only cross-sectional views (cross-fractures) of the structures but also surface views of the membranes and organelles. Many surface structures are described which have not been shown by the usual sectioning techniques. The cytoplasmic membrane contains hexagonal arrangements of particles which are apparently involved in the production of the glucan fibrils of the cell wall. Alterations of the distribution of nuclear pores are shown in cells of different ages. Freeze-etching enables a clear distinction of endoplasmic reticulum and vacuoles in yeast cells. The membranes of the vesicular systems are covered by ribosomes arranged in circular patterns. The mitochondrial envelope shows small perforations which could allow the exchange of macromolecules. The storage granules consist of concentric layers of lipid, presumably phosphatide. A Golgi apparatus has been detected which may be involved in the storage of lipid. The structure of the unit membrane and the membrane structures of all organelles as revealed by chemical fixation are confirmed in principle. Glycogen agglomerations are identified in the ground plasm of older cells. Insight into artifacts introduced by common chemical fixation and embedding techniques is obtained and discussed.
Article
Abstract By means of the freeze-etching technique ultrastructural alterations in Saccharomyces cerevisiae cells undergoing autolysis at elevated temperature were studied. Wall surfaces of intact cells were smooth. During autolysis wall surfaces became rough with granules of 20–40 nm diameter. This alteration occurred after extensive disintegration of cytoplasmic organelles and after functional and ultrastructural impairments of the plasma membrane, but well before the rupture of the plasma membrane.
Cell suspensions of Sacharomyces cerevisiae, Kloeckera apiculata and Candida stellata were autolyzed in phosphate buffer, pH 4.5, for up to 10 days. Cell dry weights decreased by 25-35% after 10 days. Based on initial cell dry weight, the soluble autolysate consisted of: carbohydrate (principally polysaccharide) 3-7%; organic acids 3-6%; protein 12-13%; free amino acids 8-12%; nucleic acid products 3-5%; and lipids 1-2%. The main organic acids in autolysates were propionic, succinic and acetic and the main amino acids were phenylalanine, glutamic acid, leucine, alanine and arginine. Approximately 85-90% of cellular RNA and 25-40% of cellular DNA were degraded during autolysis. Both neutral lipid and phospholipid components were degraded, with neural lipids but not phospholipids being found in autolysates. Scanning and transmission electron micrographs showed retention of cell wall structure and shape during autolysis, but there was extensive intracellular disorganization within S. cerevisiae and C. stellata. There were differences in the autolytic behavior of K. apiculata compared with S. cerevisiae and C. stellata.
Article
Morphological and cytochemical observation of Saccharomyces cerevisiae undergoing of induced autolysis were done in response to various chemical inducers of autolysis (NaCl, ethanol, fresh autolyzate). Changes in the inner structure of yeast cells were monitored by transmission electron microscopy and the surface of the cell wall was observed by scanning electron microscopy during autolysis. Cytochemical characterization of autolyzed cells was performed using four synthetic substrates for determination of proteinase activities but only carboxypeptidase Y could be detected in the vacuolar membranes. The morphological studies supported the data obtained from biochemical studies and confirmed that optimized conditions of autolysis have a significant effect on the structural changes of autolyzed yeast.
Article
The release of lipids during the aging of sparkling wines in contact with yeast can influence wine sensory attributes and, especially, foam characteristics. Model systems allow study of the autolysis process in a reasonable period of time compared to natural conditions, at which it can last several months. In this paper, the release of the different classes of lipids during the autolysis of three commercial yeast strains in a model wine medium has been monitored. Due to the absence of accurate quantitative methods, an HPLC method for separating and quantifying the different neutral and polar yeast lipid classes was developed. Lipids were eluted through a YMC PVA-Sil column with a complex solvent mixture. Detection was carried out with a light-scattering detector. The yeasts were suspended in the model wine buffer and incubated at 30 degrees C for up to 12 days. A release of triacylglycerols, 1,3-diacylglycerols, 2-monoacylglycerols, free fatty acids, sterol esters, and sterols was observed over the first 2 days, a period that corresponded to the maximum loss of yeast viability. A decrease in most of these lipids was observed from day 2, possibly indicative of the release of yeast hydrolytic enzymes due to the breakdown of the cell wall. Phospholipids were not detected in any of the autolysates. The mean lipid content in the autolysates as a percentage of the total lipid content in the yeasts was 8.6% for sterol esters, 3.8% for sterols, 2% for triacylglycerols, and <2% for 1,3-diacylglycerols and free fatty acids.
Article
The nitrogen composition of wines aged with yeast for a long period of time, as in the case of sparkling wines, depends on the composition of the base wine and on the compounds released by the yeast. In this paper, the release of the different classes of nitrogen compounds during autolysis of one of the strains of yeast used in the manufacture of sparkling wines has been studied. The yeast, Saccharomyces bayanus, was suspended in a model wine buffer, pH 3.0 and 10% ethanol, and incubated at 30 degrees C. Samples of the autolysate were taken after 4, 24, 48, 72, 168, and 360 h of autolysis. An electrophoretic and chromatographic study was conducted of the proteins, peptides with molecular weights higher and lower than 700 Da, and amino acids released during the autolysis. Using SDS-PAGE, it was observed that it was predominantly polypeptides with molecular weights lower than 10 000 that were released. Through HPLC of the fraction lower than 10 000 Da, it was observed that it is polypeptides with molecular weights of between 10 000 and 700 Da that are released first and that these later break up to give rise to peptides with molecular weights lower than 700 Da, which in turn break down into amino acids. This indicates that the nature of the nitrogen compounds present in wines aged with yeast depends on the aging time, being less polymerized as the aging time increases.
Study of wine yeast autolysis by electron microscopy
  • Avakiants
Avakiants, S., 1982. Study of wine yeast autolysis by electron microscopy. Vinodel. Vinograd. 2, 55-59.
Préparation d'autolysats de levures et addition dans les vins effervescents élaborés selon la méthode champenoise
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