Oleic acid and ergosterol supplementation mitigates oxidative stress in wine strains of Saccharomyces cerevisiae

During fermentation of high-sugar-containing medium lacking lipid nutrients, wine yeasts undergo oxidative stress and oxidative damage to cell membranes and proteins. Considering that cell membranes are important stress sensors, and that under hypoxic conditions wine yeasts modulate cell membranes composition by incorporating lipids available in the growth medium, in the present work, the effects of lipid nutrition on wine yeast oxidative stress response were evaluated on two strains of Saccharomyces cerevisiae. Biomarkers of oxidative stress, oxidative damage and antioxidant response were evaluated together with viability and acetic acid production during fermentation of a synthetic must lacking lipid nutrients as compared to added oleic acid and ergosterol. The results show that the availability of lipid nutrients causes a significant reduction in the intracellular content of reactive oxygen species and in the oxidative damage to membranes and proteins, as indicated by flow cytometry of cells stained with dihydroethidum (DHE) and propidium iodide (PI) and by Western blot of protein carbonyls. Accordingly, lipid nutrients feeding results in the increase in cell viability and superoxide activity, and the reduction in trehalose accumulation, proteinase A activity and production of acetic acid. In summary, these results are compatible with the hypothesis that the supplementation of lipid nutrients mitigates oxidative stress and oxidative damage in wine strains of S. cerevisiae during growth under unfavourable conditions.     

Effect of growth temperature on yeast lipid composition and alcoholic fermentation at low temperature

The loss of viability of wine yeast strains due to low-temperature fermentations could be overcome by increasing their stress tolerance and adaptability. Changes in membrane lipid composition are one of the first responses to cold stress. The aim of this study was to analyze the various adaptation mechanisms to low temperatures by comparing the better adapted Saccharomyces species. The viability, vitality, fermentation capacity, and lipid composition of different Saccharomyces species (S. cerevisiae, S. bayanus, S. uvarum, and a hybrid S. cerevisiae/S. uvarum) with different fermentative origins (wine, beer, and baker’s strains together with a laboratory strain) were compared after culturing at low (13 °C) and optimal (25 °C) temperatures. In spite of specific responses of the different strains/species, the results showed that at low temperature, the medium-chain fatty acid and the triacylglyceride content increased, whereas the phosphatidic acid content and the phosphatidylcholine/phosphatidylethanolamine ratio decreased. Only the laboratory strain was not able to ferment the sugars, and after growing at both temperatures, its lipid composition was very different from that of the other strains. The hybrid strain showed the highest sugar consumption at 13 °C and the best vitality whatever the preculture temperature used. The rest of the species needed a preadaptation at low temperature involving a change in their lipid composition to improve their fermentation rate at 13 °C.     

Effect of fermentation temperature and culture media on the yeast lipid composition and wine volatile compounds

The temperature of a wine fermentation strongly affects lipid metabolism and thus, aromatic profiles. Most of the metabolic studies are done in well-controlled laboratory conditions, yet wine is produced in less-reproducible industrial conditions. The aim of this study is to analyse the effect of fermentation temperature (13 degrees C and 25 degrees C) and culture media (synthetic media and grape must) on yeast lipid composition and volatile compounds in wine. Our results show that yeast viability was better at 13 degrees C than at 25 degrees C whichever growth medium is used, but that the complexity of the grape must enabled cells to reach higher viable population size. Viability was also related to the incorporation of linoleic acid and beta-sitosterol, which were present in the grape must. A lower temperature modified the cellular lipid composition of yeast, increasing the degree of unsaturation at the beginning of fermentation and decreasing the chain length as fermentation progressed. We also found that medium-chain fatty acids, mainly dodecanoic acid, were present in the cell phospholipids. Wines produced from grape must were more aromatic and had a lower volatile acidity content than those derived from a synthetic medium. Fermentations that were performed at the lower temperature also emphasized this feature.     

Influence of pre-fermentative treatment on the fatty acid content of Saccharomyces cerevisiae (M(3)30-9) during alcoholic fermentation of grape must

The effect of skin maceration and must filtration on the growth and fermentative capacity of one strain of Saccharomyces cerevisiae was studied. Skin maceration increased the fatty acid concentration of the must, thus affecting the lipid composition of the yeast cell membranes. Sterile filtration resulted in a reduction of the fatty acid content of the must extracted during maceration, but increased the fatty acid content of the yeast membranes, thus increasing the fermentative capacity without affecting either growth or cell viability.     

Effect of additives on the rehydration of Saccharomyces cerevisiae wine strains in active dry form: influence on viability and performance in the early fermentation phase

The effect of five additives on the rehydration process of three commercial Saccharomyces cerevisiae wine strains, in the form of active dry yeast, was tested using the approach of design of experiments. The response to additives was monitored by observing the viability, measured using fluorescence methodology at the end of the rehydration experiments, and fermentative performance, focusing attention on the first phase of fermentation, assessed based on ethanol production and the total number of cells after 48 h from the time of inoculation in synthetic must. The results showed a correlation between viability and the presence of magnesium in the rehydration medium. However, improvement in viability was not associated with better fermentation performance, which was not affected or in some cases significantly reduced. Among the additives examined, only rehydration with inactive dry yeast and ammonium showed a positive effect on the subsequent fermentations, but not for all of the strains tested. In general, considering all of the additives tested, no relationship was found between viability at the end of rehydration and fermentation performance. These findings suggest that the viability of the rehydrated yeast cells may not be a good index of fitness in the subsequent fermentation. Copyright © 2014 The Institute of Brewing & Distilling     

Electrospray Ionization Mass Spectrometry Characterization of Musts and Alembic Brazilian Cachaças Using Selected Yeast Strains

The choice of fermentation system during cachaça production can greatly influence the chemical composition of the beverage. In this work, Saccharomyces cerevisiae strains were selected based on fermentative properties and used as starters to produce alembic cachaça. In distillery scale production, the selected yeast strains exhibited greater adaptiblity to the fermentation environment and hence remained predominant throughout the process. Electrospray ionization mass spectrometry in the negative ion mode revealed that most of the compounds present in the must are different from those formed in the distillate for both cachaças obtained from spontaneous and selected strains. However, beverages produced using selected strains showed greater similarity in chemical profiles than those produced from spontaneous strain fermentation. Moreover, a smaller number of ions were detected in beverages produced by selected strain than from spontaneous strain fermentation. Our results indicate that the selected S. cerevisiae strains evaluated are able to produce cachaças less subject to variation in chemical composition. This could potentially improve brand consistency and thus commercial viability, particularly in the international market.     

Effect of oxygen and lipid supplementation on the volatile composition of chemically defined medium and Chardonnay wine fermented with Saccharomyces cerevisiae

Oxygen or lipids are required to complete stressful alcoholic fermentation. Lack of these nutrients can inhibit sugar uptake and growth, which leads to incomplete or ‘stuck’ fermentation. Oxygen or lipids supplementation not only restores yeast fermentative activity and also affects formation of yeast volatile metabolites. To clarify the effect of oxygen and lipid supplementation on the formation of flavour active metabolites during wine fermentation, we evaluated the addition of these two nutrients to chemically defined grape juice and filter clarified Chardonnay must. Lipid addition increased the concentration of esters, higher alcohols and volatile acids, whereas oxygen increased the concentration of higher alcohols and altered the proportion of acetate to ethyl esters and the proportion of branch-chain acids to medium-chain fatty acids. Combined addition of lipids and oxygen showed an additive effect on concentration of higher alcohols whereas oxygen suppressed the enhancing effect of lipids on formation of esters and volatile acids. Our results demonstrate the potential of lipid and oxygen supplementation for the manipulation of wine aroma in white wine fermentation.     

Effect of grape indigenous Saccharomyces cerevisiae strains on Montepulciano d'Abruzzo red wine quality

The growth of selected, indigenous Saccharomyces cerevisiae added as starters (SRS1, MS72 and RT73) was monitored during Montepulciano d'Abruzzo wine production. In all the fermentations the addition of the starter, caused a decrease of the non-Saccharomyces yeasts. When strains MS72 and RT73 were used as starters they were detected in the first phases of fermentations, while strain SRS1 competed successfully with native yeasts during all the process. Wines obtained by fermentation with the indigenous starters showed some different characteristics, according to the chemical and sensory analyses. This study highlighted that among selected starters with high fermentative capacity, some are able to dominate better than other natural wine yeast biota, whereas some strains can interact and survive besides native yeast populations during the fermentation. As a consequence, the dominance character can have a positive or negative effect on wine quality and has to be considered in the frame of yeast selection in order to improve or characterize traditional wines. Winemakers could choose among different degrees of yeast dominance to modulate the interaction among starter and native wine yeast population.     

全天然黑莓果酒加工工艺的研究

以黑莓为原料,经复合酵母进行单桶发酵,发酵过程中不需添加SO2,而根据菌种强势生长的原理及控制发酵条件来抑制杂菌繁殖,生成具有黑莓果香和酒的醇香的全天然干红黑莓酒。研究了复合菌种比例、原料处理方式、初始糖度、发酵温度、接种量、下胶用量等因素对产品质量影响,确定了黑莓果酒的最佳加工工艺。     

Adaptive evolution of wine yeast

Alcoholic fermentation is one of the main phases in wine production. It is usually conducted by yeasts belonging to the species Saccharomyces cerevisiae. Industrial S. cerevisiae strains are highly specialized organisms, which have evolved to utilize to their full potential the different environments or ecological niches. So, during the alcoholic fermentation, the yeast has been adapted to different kinds of stress conditions; this adaptation is call "domestication". In this review, we describe the different mechanisms involved in the adaptive evolution of wine yeast strains.     

Lipid composition of wine strains of Saccharomyces kudriavzevii and Saccharomyces cerevisiae grown at low temperature

Some species of the Saccharomyces genus have shown better adaptation at low temperature than the wine yeast Saccharomyces cerevisiae. That is the case of the cryophilic yeast Saccharomyces kudriavzevii. Several studies have revealed the importance of the lipid composition in the yeast adaptive response at different environmental temperatures. Thus we analysed the lipid composition of three S. kudriavzevii strains during growth at optimum (28°C) and low temperature (12°C), and compared them with different commercial strains; one S. cerevisiae strain and two hybrids between S. cerevisiae and S. kudriavzevii. Our results show a general increase in the medium-chain fatty acid, triacylglyceride, sterol esters and squalene and a decrease in the chain length of the fatty acids, in phosphatidic acid and in the ratio phosphatidylcholine/phosphatidylethanolamine at low temperatures. The S. kudriavzevii strains had higher percentages of medium-chain fatty acids and squalene and shorter chain lengths regardless of the growth temperature. This differential lipid composition may partially explain the better adaptation of S. kudriavzevii at low temperatures. We have also confirmed the better fermentation performance of the strains of this species at low temperature, being an appealing alternative to S. cerevisiae for cold fermentations.     

Release of cell wall polysaccharides from Saccharomyces cerevisiae thermosensitive autolytic mutants during alcoholic fermentation

In order to increase the release of cell wall polysaccharides during alcoholic fermentation, a wine strain of Saccharomyces cerevisiae was subjected to UV mutagenesis to obtain thermosensitive autolytic mutants affected in cell wall integrity. Five mutants and the parental strain were utilized in fermentation trials conducted at 28, 32 and 34 degrees C. Results showed that at all temperatures the mutant strains released into the medium a higher polysaccharide quantity than the parental strain. In particular, at 28 degrees C there was a doubling of these macromolecules. At the end of alcoholic fermentation, all strains showed at 28 degrees C elevated and similar levels of viable cells; at 32 degrees C this parameter remained high for mutant strains ts16 and ts39 and the parental strain; at 34 degrees C all strains underwent a drop in cell viability, which was less intense in the case of strain ts16. As a relationship between cell viability and the quantity of polysaccharides released by the yeast strain was not found, it can be assumed that the mutation led to cells with a less stable wall and thus an easier release of macromolecules into the medium.     

Quality control method based on quartz crystal microbalance and WGA for flour milled from germinated wheat

Plasma membrane is the initial sensor of different stress conditions and its composition is modified with response to environmental changes. In the present study, we have modified the lipid composition of the membrane by growing Saccharomyces cerevisiae in the presence of different fatty acids and ergosterol. All supplemented fatty acids were incorporated into the cell and this incorporation produced significant changes in the lipid composition. The incubation with ergosterol also modified the lipid composition of the cells; however, these cells presented a strong reduction in the content of this sterol. The different cellular lipid composition has been related to viability and fermentation performance at low temperature (13 °C). The cells incubated with palmitoleic acid (C16:1) showed higher viability and significant reduction in the fermentation length. These cells presented higher C16:1 and ergosterol content, shorter chain length of the fatty acids and higher ratio of sterols/phospholipids. Therefore redesigning the composition of cellular membranes during industrial yeast propagation seems to be a promising strategy for improving fermentation performance in the winery.     

Impact of yeast starter formulations on the production of volatile compounds during wine fermentation

The most diffused starter formulation in winemaking is actually represented by active dry yeast (ADY). Spray‐drying has been reported as an appropriate preservation method for yeast and other micro‐organisms. Despite the numerous advantages of this method, the high air temperatures used can negatively affect cell viability and the fermentative performance of dried cells. In the present study, 11 wine S. cerevisiae strains (both indigenous and commercial) were submitted to spray‐drying; different process conditions were tested in order to select the conditions allowing the highest strain survival. The strains exhibited high variability for tolerance to spray‐drying treatment. Selected strains were tested in fermentation at laboratory scale in different formulations (free fresh cells, free dried cells, immobilized fresh cells and immobilized dried cells), in order to assess the influence of starter formulation on fermentative fitness of strains and aromatic quality of wine. The analysis of volatile fraction in the experimental wines produced by selected strains in different formulations allowed identification of > 50 aromatic compounds (alcohols, esters, ketones, aldehydes and terpenes). The results obtained showed that the starter formulation significantly influenced the content of volatile compounds. In particular, the wines obtained by strains in dried forms (as both free and immobilized cells) contained higher numbers of volatile compounds than wines obtained from fresh cells. Copyright © 2014 John Wiley & Sons, Ltd.     

Interactions between yeast, oxygen and polyphenols during alcoholic fermentations: Practical implications

During alcoholic fermentation, even when Saccharomyces cerevisiae cells have used the required oxygen for lipid synthesis, they can consume much more oxygen with no detrimental effect on the fermentation process. Under these conditions, most of the superfluous oxygen is consumed by yeasts by the partial functioning of several nonrespiratory oxygen consumption pathways, which are characterized by the production of reactive oxygen species (ROS). When excess oxygen is added to yeast cells, cell sterol content decreases, following the strong oxidation of intracellular sterols. During aging of fermented products in the presence of nonviable yeast lees (harvested at the end of alcoholic fermentation), the lees can consume oxygen for at least 3 years of the aging process. This oxygen consumption by yeast lees is related to moderate oxidation of yeast membrane lipids by the action of free radicals, strongly decreasing sterols in the yeast lees. The biochemical reactions involved in the oxygen consumption pathways during alcoholic fermentation may be the same as those responsible for oxygen consumption observed in yeast lees. Therefore, the cross-reactivity of complex plant polyphenols, tannins and yeast towards oxygen can easily occur during technological processes (alcoholic fermentation and wine aging). The micro-oxygenation of yeasts releases ROS during alcoholic fermentation and may favour the oxidation of wine phenolic compounds. As yeasts have much higher affinities for oxygen than plant polyphenols, viable yeast and yeast lees compete with phenolic compounds and then hinder the wine aging process. Also, the partial adsorption of plant polyphenols on yeast occurs during alcoholic fermentation, which modifies the overall reactivity of yeast and polyphenols towards oxygen.     

Yeast viability during fermentation and sur lie ageing of a defined medium and subsequent growth of Oenococcus oeni

Interactions between the yeast strain used for primary oenological fermentation and the bacterium used to conduct subsequent malolactic fermentation were studied under model winemaking conditions. A commercial Saccharomyces cerevisiae wine yeast (strains, EC 1118, AWRI 835 and CY-3079) was grown in a defined medium whose composition approximated grape juice. Fermentations by all strains reached dryness, and retained a cell viability of greater than 90% upon completion of fermentation. Highest total viable cell number and percentage of viable cells were recorded for EC 1118. A sur lie ageing of the fermented medium over a 12 week period revealed a bi-phasic decay of culture viability for all strains. Thus 99% of cells had died within 2 weeks post-fermentation. Viabilities were then stable for the subsequent 4–6 week period before a second decline phase ensued and ended in either a minimal ( ca 100 CFU/mL, EC 1118) or no viable cells being detected at 12 weeks of ageing. The growth response of an Oenococcus oeni inoculum to yeast culture supernatants, previously aged for up to 12 weeks in the presence or absence of yeast lees, was evaluated in a bio-assay. In this way, yeast strains could be designated as being either inhibitory, neutral or stimulatory to the growth of O. oeni (strain Lc5p). Inhibition by supernatants of strain EC 1118 was evident, but found to be reduced by ageing the supernatant (with or without lees). Conversely, longer ageing on yeast lees increased the magnitude of the stimulatory response in O. oeni (strain Lc5p) to the supernatant from the wine yeast (strain CY-3079).     

Changes in the Lipid Composition of Saccharomyces cerevisiae Race Capensis (G-1) During Alcoholic Fermentation and Flor Film Formation

The cellular lipid composition of one flor-forming strain of Saccharomyces cerevisiae during fermentation and the subsequent period of film formation with different oxygen levels was studied. Irrespective of fermentation conditions, only those yeasts which came into contact with oxygen after fermentation formed a flor film. After the fermentation, these yeasts entered an adaptation phase in which the percentage of oleic acid increased considerably at the expense of other long-chain fatty acids. Their phospholipid contents remained high, as well as the unsaturation index of their fatty acids and the ergosterol/phospholipids ratio was maintained below 1. These changes allowed an increased viability of yeasts in the wine of up to 80% and the acquisition of sufficient hydrophobicity and floatability to reach the surface and form flor film.     

The effect of nitrogen addition on the fermentative performance during sparkling wine production

The transformation of must to wine is influenced by several factors, including the nitrogen composition of the grape must, which has an important impact on yeast growth, fermentation kinetics and the organoleptic properties of the final product. In the production of sparkling wine by the traditional method, a second fermentation takes place inside the bottle, followed by yeast autolysis. Before their inoculation, yeasts cells must be adapted to the wine by the process known as pied-de-cuve. The aim of this study is to determine how nitrogen composition both in the pied-de-cuve and the base wine, affects the development of the second fermentation. This effect was analyzed in three different strains and at two different fermentation temperatures (12 and 16 °C). The results indicate that the nitrogen intake during the pied-de-cuve is crucial for the development of the second fermentation, with strain-dependent nutrient preferences during this phase. The addition of organic nitrogen in this phase can enhance the second fermentation. However, the addition of nitrogen to the base wine, had little effect on fermentation kinetics, indicating that either the residual nitrogen of the wines (< 30 mg N/l), or the nitrogen taken up during the pied-de-cuve, was sufficient to cover the low nitrogen requirements during the second fermentation, and to ensure the good development of this process. The base wine and the temperature had also strong effects on the fermentation length and development. To optimize sparkling wine production, all factors involved in the second fermentation should be considered, although the interactions between base wine, temperature and yeast strain have the strongest effect on fermentation kinetics.     

Saccharomyces cerevisiae and Hanseniaspora uvarum mixed starter cultures: Influence of microbial/physical interactions on wine characteristics

The growing trend in the wine industry is the revaluation of the role of non-Saccharomyces yeasts, promoting the use of these yeasts in association with Saccharomyces cerevisiae. Non-Saccharomyces yeasts contribute to improve wine complexity and organoleptic composition. However, the use of mixed starters needs to better understand the effect of the interaction between these species during alcoholic fermentation. The aim of this study is to evaluate the influence of mixed starter cultures, composed by combination of different S. cerevisiae and Hanseniaspora uvarum strains, on wine characteristics and to investigate the role of cell-to-cell contact on the metabolites produced during alcoholic fermentation. In the first step, three H. uvarum and two S. cerevisiae strains, previously selected, were tested during mixed fermentations in natural red grape must in order to evaluate yeast population dynamics during inoculated fermentation and influence of mixed starter cultures on wine quality. One selected mixed starter was tested in a double-compartment fermentor in order to compare mixed inoculations of S. cerevisiae/H. uvarum with and without physical separation. Our results revealed that physical contact between S. cerevisiae and H. uvarum affected the viability of H. uvarum strain, influencing also the metabolic behaviour of the strains. Although different researches are available on the role of cell-to-cell contact-mediated interactions on cell viability of the strains included in the mixed starter, to our knowledge, very few studies have evaluated the influence of cell-to-cell contact on the chemical characteristics of wine.     

Saccharomyces cerevisiae-Oenococcus oeni interactions in wine: current knowledge and perspectives

Winemaking can be summarized as the biotransformation of must into wine, which is performed principally by Saccharomyces cerevisiae strains during the primary or alcoholic fermentation. A secondary fermentation, the so-called malolactic fermentation (MLF) is a biodeacidification that is often encouraged, since it improves wine stability and quality. Malolactic fermentation usually occurs either spontaneously or after inoculation with selected bacteria after alcoholic fermentation. The main organism responsible for MLF, the lactic acid bacterium Oenococcus oeni, develops in physicochemically harsh conditions, which may lead to MLF failure. Furthermore, yeast that ferment must before or together with O. oeni can prevent or stimulate the progress of MLF. These phenomena are part of the interactions observed between yeast and bacteria. The mechanisms that govern yeast bacteria interaction are reviewed and the consequences for winemaking are discussed. In the light of recent advances, future prospects are also presented.