Strain specific Starmerella bacillaris and Saccharomyces cerevisiae interactions in mixed fermentations.

AIMS: Yeast interactions have a key role in the definition of the chemical profile of the wines. For this reason, winemakers are increasingly interested in mixed fermentations, employing Saccharomyces cerevisiae and non-Saccharomyces strains. However, the outcome of mixed fermentations is often contradictory because there is a great variability among strains within species. Previously, it was demonstrated that the loss of culturability of Starmerella bacillaris in mixed fermentations with S. cerevisiae was due to the physical contact between cells. Therefore, to further explore previous observations, the interaction mechanisms among different strains of Starm. bacillaris and S. cerevisiae during mixed fermentations were investigated. METHODS AND RESULTS: Fermentations were conducted under conditions that allow physical contact between cells (flasks) but also using a double-compartment fermentation system in which cells of both species were kept separate. The role of competition for nutrients and antimicrobial compounds production on yeast-yeast interaction mechanisms was also investigated. Three Starm. bacillaris and three S. cerevisiae strains were used to investigate if interaction mechanisms are modulated in a strain-specific way. Both species populations were affected by physical contact, particularly Starm. bacillaris that lost its culturability during fermentation. In addition, loss of culturability of Starm. bacillaris strains was observed earlier in flasks than in the double-compartment system. The phenomena observed occurred in a strain couple-dependent way. Starm. bacillaris disappearance seemed to be independent of nutrient depletion or the presence of inhibitory compounds (which were not measured in this study). CONCLUSION: Overall, the results of the present study reveal that cell-to-cell contact plays a role in the early death of non-Saccharomyces but the extent to which it is observed depends greatly on the Starm. bacillaris/S. cerevisiae strains tested.

Survey of the yeast ecology of dehydrated grapes and strain selection for wine fermentation.

In this study we investigated the yeast population present on partially dehydrated Nebbiolo grapes destined for 'Sforzato di Valtellina', with the aim to select indigenous starters suitable for the production of this wine. Yeasts were enumerated, isolated, and identified by molecular methods (5.8S-ITS-RFLP and D1/D2 domain sequencing). A genetic, physiological (ethanol and sulphur dioxide tolerance, potentially useful enzymatic activities, hydrogen sulphide production, adhesive properties, and killer activity) and oenological (laboratory pure micro-fermentations) characterization was also carried out. Based on relevant physiological features, seven non-Saccharomyces strains were chosen for laboratory-scale fermentations, either in pure or in mixed-culture (simultaneous and sequential inoculum) with a commercial Saccharomyces cerevisiae strain. Finally, the best couples and inoculation strategy were further tested in mixed fermentations in winery. In both laboratory and winery, microbiological and chemical analyses were conducted during fermentation. The most abundant species on grapes were Hanseniaspora uvarum (27.4 % of the isolates), followed by Metschnikowia spp. (21.0 %) and Starmerella bacillaris (12.9 %). Technological characterization highlighted several inter- and intra-species differences. The best oenological aptitude was highlighted for species Starm. bacillaris, Metschnikowia spp., Pichia kluyveri and Zygosaccharomyces bailli. The best fermentation performances in laboratory-scale fermentations were found for Starm. bacillaris and P. kluyveri, due to their ability to reduce ethanol (-0.34 % v/v) and enhance glycerol production (+0.46 g/L). This behavior was further confirmed in winery. Results of this study contribute to the knowledge of yeast communities associated with a specific environment, like those of Valtellina wine region.

Effect of alternative fungicides and inoculation strategy on yeast biodiversity and dynamics from the vineyard to the winery.

Fungi and oomycetes found in vineyards cause diseases such as powdery and downy mildew. Consequently, conventional and alternative agronomical practices are widely used prior to harvest to protect grapes. Alternative products are considered more eco-friendly and environmentally sustainable in comparison to conventional chemical products. However, the effect of these alternative products on yeast ecology, from the vineyard to the winery, is poorly understood. This study compared the effect of alternative and conventional chemical antifungal compounds (copper and sulphur based) on grapes' mycobiota in the vineyard and during subsequent winery fermentation using culture-dependent and -independent approaches. Culture-dependent data indicated a treatment-dependent effect on the load and diversity of yeast populations on grapes. It was found that the population of Hanseniaspora uvarum was higher on grapes previously treated with laminarin and copper, compared to the other levels registered on grapes previously treated with the rest of antifungal products tested in this study (including the untreated and conventional treatment controls). Concerning, wine quality, the chemical composition was not correlated to the application of antifungal treatment in the vineyard. Understanding the effect of different antifungal products on grape and wine microbial communities may help in setting up guidelines for wine grape production. These guidelines, can be used to guarantee quality in the pursuit of a sustainable competitive advantage in the market.

Validation of a Standard Protocol to Assess the Fermentative and Chemical Properties of Saccharomyces cerevisiae Wine Strains.

This paper reports on a common experiment performed by 17 Research Units of the Italian Group of Microbiology of Vine and Wine (GMVV), which belongs to the Scientific Society SIMTREA, with the aim to validate a protocol for the characterization of wine strains of Saccharomyces cerevisiae. For this purpose, two commercial S. cerevisiae strains (EC 1118 and AWRI796) were used to carry out inter-laboratory-scale comparative fermentations using both synthetic medium and grape musts and applying the same protocol to obtain reproducible, replicable, and statistically valid results. Ethanol yield, production of acetic acid, glycerol, higher alcohols, and other volatile compounds were assessed. Moreover, the Fourier transform infrared spectroscopy was also applied to define the metabolomic fingerprint of yeast cells from each experimental trial. Data were standardized as unit of compounds or yield per gram of sugar (glucose and fructose) consumed throughout fermentation, and analyzed through parametric and non-parametric tests, and multivariate approaches (cluster analysis, two-way joining, and principal component analysis). The results of experiments carried out by using synthetic must showed that it was possible to gain comparable results from three different laboratories by using the same strains. Then, the use of the standardized protocol on different grape musts allowed pointing out the goodness and the reproducibility of the method; it showed the main traits of the two yeast strains and allowed reducing variability amongst independent batches (biological replicates) to acceptable levels. In conclusion, the findings of this collaborative study contributed to the validation of a protocol in a specific synthetic medium and in grape must and showed how data should be treated to gain reproducible and robust results, which could allow direct comparison of the experimental data obtained during the characterization of wine yeasts carried out by different research laboratories.

Bioprotection strategies in winemaking.

Worldwide the interest for biological control of food spoilage microorganisms has significantly increased over the last decade. Wine makes no exception to this trend, as consumer demands for wines free of preservatives that are considered negative for human health, increase. Biological control during wine fermentation aims at producing high quality wines, while minimizing, or even eliminating, the use of chemical additives. Its success lies in the inoculation of microorganisms to prevent, inhibit or kill undesired microbes, therefore maintaining wine spoilage at the lowest level. The food industry already makes use of this practice, with dedicated commercial microbes already on the market. In winemaking, there are commercial microbes currently under investigation, particularly with the aim to reduce or replace the use of sulphur dioxide. In this review, the potential of wine yeasts and lactic acid bacteria as bioprotection agents and their mechanisms of action during wine fermentation are presented.

Influence of single nitrogen compounds on growth and fermentation performance of Starmerella bacillaris and Saccharomyces cerevisiae during alcoholic fermentation.

Nitrogen is among the essential nutriments that govern interactions between yeast species in the wine environment. A thorough knowledge of how these yeasts assimilate the nitrogen compounds of grape juice is an important prerequisite for a successful co- or sequential fermentation. In the present study, we investigated the efficiency of 18 nitrogen sources for sustaining the growth and fermentation of two Starm. bacillaris strains displaying metabolic properties, compared to the reference yeast S. cerevisiae The analysis of growth and fermentation parameters provided a comprehensive picture of Starm. bacillaris preferences with respect to nitrogen sources for sustained growth and fermentation. Important differences were observed in S. cerevisiae regarding rates, final population and CO2 production. In particular, Lys and His supported substantial Starm. bacillaris growth and fermentation contrary to S. cerevisiae, while only 3 nitrogen sources, Arg, NH4+ and Ser, promoted S. cerevisiae growth more efficiently than that of Starm. bacillaris strains. Furthermore, Starm. bacillaris strains displayed a higher fermentative activity than S. cerevisiae during the first phase of culture with Gly or Thr, when the former species consumed solely fructose. Finally, no correlation has been shown between the ability of nitrogen sources to support growth and their fermentation efficiency. The specificities of Starm. bacillaris regarding nitrogen sources preferences are related to its genetic background, but further investigations are needed to elucidate the molecular mechanisms involved. These data are essential elements to be taken into account in order to make the best use of the potential of the two species.IMPORTANCE Mixed fermentations combining non-Saccharomyces and S. cerevisiae strains are increasingly implemented in the wine sector as they offer promising opportunities to diversify the flavour profile of end-products. However, competition for nutrients between species can cause fermentation problems, which is a severe hindrance to the development of these approaches. With the knowledge provided in this study on the nitrogen preferences of Starm. bacillaris, winemakers will be able to set up a nitrogen nutrition scheme adapted to the requirement of each species during mixed fermentation, through must supplementation with relevant nitrogen compounds. This will prevent nitrogen depletion or competition between yeasts for nitrogen sources, and consequently potential issues during fermentation. The data of this study highlight the importance of an appropriate nitrogen resource management during co- or sequential fermentation for fully exploiting the phenotypic potential of non-Saccharomyces yeasts.

Can a Corn-Derived Biosurfactant Improve Colour Traits of Wine? First Insight on Its Application during Winegrape Skin Maceration versus Oenological Tannins.

In winemaking, oenological tannins are used to preserve wine colour by enhancing the antioxidant activity, taking part in copigmentation, and forming polymeric pigments with anthocyanins. As a novel processing aid, in this study, a biosurfactant extract was evaluated as a solubilizing and stabilizing agent of anthocyanins in red wine. The biosurfactant extract under evaluation was obtained from a fermented residual stream of the corn milling industry named corn steep liquor (CSL). Two red winegrape varieties (Vitis vinifera L. cv. Aglianico and Cabernet sauvignon) were studied for anthocyanin content and profile, and colour traits, during simulated skin maceration for 7 days at 25 °C, as well as polymerization and copigmentation at the end of maceration. A model wine solution was used as a control, which was added either with the CSL biosurfactant or with four different oenological tannins (from grape skin, grape seed, quebracho, and acacia). The results showed that CSL biosurfactant addition improved the colour properties of skin extracts by the formation of more stable compounds mainly through copigmentation interactions. These preliminary results highlighted that the effectiveness of CSL biosurfactant is variety-dependent; however, there is no significant protection of individual anthocyanin compounds as observed for delphinidin and petunidin forms using quebracho tannin.

Chloroanisoles occurrence in wine from grapes subjected to electrolyzed water treatments in the vineyard.

Recently the use of electrolyzed water (EW) attracted much attention as a high-performance, new technology for its potential use in the food industry. The aim of this work was to investigate the impact of grape EW treatments, applied at different time intervals prior to harvest, on the indigenous yeast populations of grape surface (Chenin blanc and Cabernet franc) and the occurrence of 2,4,6-trichloroanisole (TCA) in Cabernet franc wine. In addition, the evolution of inoculated and spontaneous fermentations on treated and non-treated grapes was also considered. The yeast population present on grape berries surface was influenced in a grape variety and EW treatment time-dependent way, since only Chenin blanc grapes treated with EW 7 days prior to harvest had significantly lower yeast population levels, compared to the respective control. Concerning the yeast diversity in the grape samples, a dominance of Aureobasidium pullulans was observed in treated grapes, independently of the grape variety. At the end of alcoholic fermentation, 2,4,6-trichloroanisole was detected in wine when the EW solution was applied at one or two weeks before harvest time. After wine storage, 2,4,6-trichloroanisole and chlorophenols contents generally exhibited a loss relative to initial values. The results showed that EW treatments tended to slightly increase the TCA concentration in final wine and did not affect the fermentation performances and chromatic properties of resulting wine. On the other hand, absorption or desorption phenomena by wine lees could be involved in the change of 2,4,6-trichloroanisole concentration in wine during storage time.

Impact of Chemical and Alternative Fungicides Applied to Grapevine cv Nebbiolo on Microbial Ecology and Chemical-Physical Grape Characteristics at Harvest.

Viticulture is a cropping system in which treatment against fungal diseases (in particular powdery and downy mildews) can be extremely frequent. Accordingly, a reduction in antimicrobial treatments and the application of environmentally-friendly compounds are becoming increasingly important for a more sustainable viticulture. In addition to their effect against pathogens, the impact of these products on the quality of the grapes is very important for the oenological industries, but unfortunately at present few data are available. We evaluated the effect of the application of biocontrol products and resistance inducers in the vineyard on the mechanical properties, microbial ecology, technological and phenolic maturity of Vitis vinifera "Nebbiolo" grapes at harvest. The yield and vigor of vines were not influenced by the treatments, nor were the production of primary and secondary metabolites. However, the active ingredients influenced the mechanical properties of the skin (hardness and thickness). A significant hardening of the skin was detected when laminarin and chito-oligosaccharides were used, and sulfur induced a thickening of the skin with potential consequences for wine quality. Furthermore, the yeast community present on grape berries was influenced by the treatments. The abundance of Aureobasidium pullulans, the dominant species on the grape berry, changed in response to the compounds used. In addition, Alternaria sp. was reduced in some treatments with a potentially positive effect on the quality and the safety of the grapes. This study provides an overview of the effect of biocontrol products and resistance inducers on microbial ecology and "Nebbiolo" grape quality, contributing to the establishment of more sustainable and effective defense strategies in viticulture.

Effect of mixed fermentations with Starmerella bacillaris and Saccharomyces cerevisiae on management of malolactic fermentation.

This work aims to improve the management of the malolactic fermentation (MLF) in red wines by elucidating the interactions between Starmerella bacillaris and Saccharomyces cerevisiae in mixed fermentations and malolactic bacteria. Two Starm. bacillaris strains were individually used in mixed fermentations with a commercial S. cerevisiae. MLF was performed using two autochthonous Lactobacillus plantarum and one commercial Oenococcus oeni inoculated following a simultaneous (together with S. cerevisiae) or sequential (at the end of alcoholic fermentation) approach. The impact of yeast inoculation on the progress of MLF was investigated by monitoring the viable microbial populations and the evolution of the main oenological parameters, as well as the volatile organic composition of the wines obtained in mixed and pure micro-scale winemaking trials. Our results indicated that MLF was stimulated, inhibited, or unaffected in mixed fermentations depending on the strains and on the regime of inoculation. O. oeni was able to perform MLF under all experimental conditions, and it showed a minimal impact on the volatile organic compounds of the wine. L. plantarum was unable to perform MLF in sequential inoculation assays, and strain-depending interactions with Starm. bacillaris were indicated as factor affecting the outcome of MLF. Moreover, uncompleted MLF were related to a lower aromatic complexity of the wines. Our evidences indicate that tailored studies are needed to define the appropriate management of non-Saccharomyces and malolactic starter cultures in order to optimize some technological parameters (i.e. reduction of vinification time) and to improve qualitative features (i.e. primary and secondary metabolites production) of red wines.

Effect of mixed species alcoholic fermentation on growth and malolactic activity of lactic acid bacteria.

In recent years, there is an increasing interest from the winemaking industry for the use of mixed fermentations with Starmerella bacillaris (synonym Candida zemplinina) and Saccharomyces cerevisiae, due to their ability to modulate metabolites of oenological interest. The current study was carried out to elucidate the effect of this fermentation protocol on the growth and malolactic activity of lactic acid bacteria (LAB) used for malolactic fermentation (MLF) and on the chemical and volatile profile of Nebbiolo wines and their chromatic characteristics. To this end, two LAB species, namely Lactobacillus plantarum and Oenococcus oeni, were inoculated at the beginning and at the end of the alcoholic fermentation (AF) performed by pure and mixed yeast using the abovementioned yeasts. The different yeast inoculation protocols and the combination of species tested influenced greatly the interactions and behavior of the inoculated yeasts and LAB during AF and MLF. For both LAB species, inoculation timing was critical to how rapidly MLF started and finished. Fermentation inoculated with L. plantarum, at the beginning of the AF, completed MLF faster than those inoculated with O. oeni. The presence of Starm. bacillaris in mixed fermentation promoted LAB growth and activity, in particular, O. oeni. Furthermore, LAB species choice had a greater impact on the volatile and chromatic profile of the wines than inoculation time. These findings reveal new knowledge about the importance of LAB species choice and inoculation time to ensure fast MLF completion and to improve wine characteristics in mixed fermentation with Starm. bacillaris and S. cerevisiae.

Saccharomyces cerevisiae-Starmerella bacillaris strains interaction modulates chemical and volatile profile in red wine mixed fermentations.

The use of Starmerella bacillaris in combination with Saccharomyces cerevisiae is considered as a state-of-the-art biological application to modulate wine composition. This application implies a detailed understanding of yeast-yeast interactions during mixed fermentations and their effect on the composition of the resulting wines. In this context, ten commercial S. cerevisiae strains were used as partners of an indigenous, previously characterized Starm. bacillaris strain in order to get a better insight into the impact of S. cerevisiae strain employed. The different combinations of strains tested influenced the growth dynamics, the fermentation behavior and, as a consequence, wine composition in a couple-dependent manner. In addition, wines produced from mixed fermentations had significantly lower levels of ethanol, acetic acid and ethyl acetate, and showed higher amounts of glycerol, higher alcohols and esters compared to pure S. cerevisiae control fermentations. This study reveals the importance of S. cerevisiae strain choice on the chemical composition of the wines produced from mixed culture fermentations with Starm. bacillaris.

Cell-to-cell contact mechanism modulates Starmerella bacillaris death in mixed culture fermentations with Saccharomyces cerevisiae.

The use of mixed culture fermentations with selected Starmerella bacillaris and Saccharomyces cerevisiae strains is gaining winemaking attention, mainly due to their ability to enhance particular characteristics in the resulting wines. In this context, yeast interspecies interactions during fermentation have a fundamental role to determine the desired product characteristics, since they may modulate yeast growth and as a consequence metabolite production. In order to get an insight into these interactions, the growth and death kinetics of the abovementioned species were investigated in pure and mixed culture fermentations, using cv. Nebbiolo grape must. Trials were conducted in flasks but also in a double-compartment fermentation system in which cells of the two species were kept separate by a filter membrane. Although the two species had similar growth pattern during the first days of fermentation, Starm. bacillaris died earlier when tested in the flask than in the double-compartment fermentor. The early death of Starm. bacillaris seemed to be not caused by nutrient limitation nor by accumulation of growth inhibitory compounds (which were not measured in the present study). Rather, cell-to-cell contact mechanism, dependent on the presence of viable S. cerevisiae cells, appears to be responsible for the observations made. These results contribute to better understand the factors that influence Starm. bacillaris death during wine fermentations.

Specific Phenotypic Traits of Starmerella bacillaris Related to Nitrogen Source Consumption and Central Carbon Metabolite Production during Wine Fermentation.

Over the last few years, the potential of non-Saccharomyces yeasts to improve the sensory quality of wine has been well recognized. In particular, the use of Starmerella bacillaris in mixed fermentations with Saccharomyces cerevisiae was reported as an appropriate way to enhance glycerol formation and reduce ethanol production. However, during sequential fermentation, many factors, such as the inoculation timing, strain combination, and physical and biochemical interactions, can affect yeast growth, the fermentation process, and/or metabolite synthesis. Among them, the availability of yeast-assimilable nitrogen (YAN), due to its role in the control of growth and fermentation, has been identified as a key parameter. Consequently, a comprehensive understanding of the metabolic specificities and the nitrogen requirements would be valuable to better exploit the potential of Starm. bacillaris during wine fermentation. In this study, marked differences in the consumption of the total and individual nitrogen sources were registered between the two species, while the two Starm. bacillaris strains generally behaved uniformly. Starm. bacillaris strains are differentiated by their preferential uptake of ammonium compared with amino acids that are poorly assimilated or even produced (alanine). Otherwise, the non-Saccharomyces yeast exhibits low activity through the acetaldehyde pathway, which triggers an important redistribution of fluxes through the central carbon metabolic network. In particular, the formation of metabolites deriving from the two glycolytic intermediates glyceraldehyde-3-phosphate and pyruvate is substantially increased during fermentations by Starm. bacillaris This knowledge will be useful to better control the fermentation process in mixed fermentation with Starm. bacillaris and S. cerevisiaeIMPORTANCE Mixed fermentations using a controlled inoculation of Starmerella bacillaris and Saccharomyces cerevisiae starter cultures represent a feasible way to modulate wine composition that takes advantage of both the phenotypic specificities of the non-Saccharomyces strain and the ability of S. cerevisiae to complete wine fermentation. However, according to the composition of grape juices, the consumption by Starm. bacillaris of nutrients, in particular of nitrogen sources, during the first stages of the process may result in depletions that further limit the growth of S. cerevisiae and lead to stuck or sluggish fermentations. Consequently, understanding the preferences of non-Saccharomyces yeasts for the nitrogen sources available in grape must together with their phenotypic specificities is essential for an efficient implementation of sequential wine fermentations with Starm. bacillaris and S. cerevisiae species. The results of our study demonstrate a clear preference for ammonium compared to amino acids for the non-Saccharomyces species. This finding underlines the importance of nitrogen sources, which modulate the functional characteristics of inoculated yeast strains to better control the fermentation process and product quality.

Volatile profiles and chromatic characteristics of red wines produced with Starmerella bacillaris and Saccharomyces cerevisiae.

The use of mixed fermentations with Starmerella bacillaris and Saccharomyces cerevisiae is gaining attention in recent years due to their ability to modulate the metabolites production of enological interest. In the present study, four of the most popular planted red grape varieties (Cabernet sauvignon, Merlot, Pinot noir and Shiraz) were fermented using the aforementioned species and two different inoculation protocols (inoculation of S. cerevisiae after 24 and 48 h from the Starm. bacillaris inoculation), in order to evaluate their impact on the volatile composition and chromatic characteristics of wines. Analysis from chemical composition showed that titratable acidity and glycerol content exhibited marked differences among wines after fermentation. For volatile compounds, mixed fermented wines using an inoculation delay of 48 h led to reduction of volatile compounds (mainly esters). A shorter 24 h delay produced wines with higher values of color intensity than pure fermented wines. The differences observed between the inoculation protocols can be explained by the growth dynamics of both species during fermentation. These findings suggest that mixed fermentations posed a great potential in reducing metabolites which are considered negative for wine quality (mainly ethyl acetate and volatile fatty acids) and with an improvement of the chromatic profile of the wines.

Volatile profile of white wines fermented with sequential inoculation of Starmerella bacillaris and Saccharomyces cerevisiae.

Mixed fermentations with Starmerella bacillaris and Saccharomyces cerevisiae affect the chemical composition of wines, by modulating various metabolites of oenological interest. The current study was carried out to elucidate the effect of sequential inoculation of the above mentioned species on the production of white wines, especially on the chemical and aromatic characteristics of Chardonnay, Muscat, Riesling and Sauvignon blanc wines. Titratable acidity and glycerol content exhibited evident differences among the wines after fermentation. For volatile compounds, mixed fermentations led to a reduction of the total esters, including ethyl acetate, which is a compound responsible for wine deterioration. However, Sauvignon blanc wines fermented by mixed cultures contained significantly higher levels of esters and thiols, both associated with positive sensory attributes. These findings suggest that sequential inoculations possess great potential in affecting and modulating the chemical and aromatic profile of white wines, especially those produced from Sauvignon blanc grapes.

Oxygen availability and strain combination modulate yeast growth dynamics in mixed culture fermentations of grape must with Starmerella bacillaris and Saccharomyces cerevisiae.

Starmerella bacillaris (synonym Candida zemplinina) is a non-Saccharomyces yeast that has been proposed as a co-inoculant of selected Saccharomyces cerevisiae strains in mixed culture fermentations to enhance the analytical composition of the wines. In order to acquire further knowledge on the metabolic interactions between these two species, in this study we investigated the impact of oxygen addition and combination of Starm. bacillaris with S. cerevisiae strains on the microbial growth and metabolite production. Fermentations were carried out under two different conditions of oxygen availability. Oxygen availability and strain combination clearly influenced the population dynamics throughout the fermentation. Oxygen concentration increased the survival time of Starm. bacillaris and decreased the growth rate of S. cerevisiae strains in mixed culture fermentations, whereas it did not affect the growth of the latter in pure culture fermentations. This study reveals new knowledge about the influence of oxygen availability on the successional evolution of yeast species during wine fermentation.

Impact of post-harvest ozone treatments on the skin phenolic extractability of red winegrapes cv Barbera and Nebbiolo (Vitis vinifera L.).

Recently the use of ozone as sanitizing agent has been proposed on winegrapes in order to control mycobiota after harvest. The aim of this work was to investigate possible indirect physico-chemical effects of ozone treatment on berry skin phenolic composition and extractability. Vitis vinifera L. cv Nebbiolo and Barbera, chosen for their different anthocyanin profiles, were post-harvest treated for 24 and 72h with gaseous ozone (30µL/L). Skin anthocyanin and flavanol extractability was assessed during maceration (6, 24, 48, 96, 168 and 240h) using a wine-like solution. In our experimental conditions, ozone did not affect significantly the final extraction yield of anthocyanins (TA), proanthocyanidins (PRO), and flavanols reactive to vanillin (FRV) in Barbera, although TA and FRV extractabilities were higher in control samples than in ozone-treated samples during the first stages of maceration. In Nebbiolo, the final TA extraction yield was positively influenced by the ozone treatment (68.6, 64.2, and 59.9% for 24h ozone-treated berries, 72h ozone-treated berries and control samples, respectively). Final PRO and FRV extractability also increased in both ozone-treated samples compared to the control (+8.6-9.1% for PRO and +7.3-11.7% for FRV). No significant differences were found among treatments for individual anthocyanins in both cultivars at the end of maceration. Therefore, the use of ozone as sanitizing agent in red varieties prior to winemaking process can be considered because it did not negatively affect the extractability of skin anthocyanins and flavanols.

Starmerella bacillaris and Saccharomyces cerevisiae mixed fermentations to reduce ethanol content in wine.

Decreasing the ethanol content in wine is a current challenge, mainly due to the global climate change and to the consumer preference for wines from grapes with increased maturity. In this study, a central composite design (CCD) and response surface methodology (RSM) approach was used to investigate the potential application of Starmerella bacillaris (synonym Candida zemplinina) in combination with Saccharomyces cerevisiae, in mixed (co-inoculated and sequential) cultures, to understand better the mechanism of co-habitation and achieve the objective of reducing the ethanol in wines. Laboratory scale fermentations demonstrated a decrease up to 0.7 % (v/v) of ethanol and an increase of about 4.2 g/L of glycerol when S. cerevisiae was inoculated with a delay of 48 h with respect to the inoculation of S. bacillaris. Pilot-scale fermentations, carried out in winemaking conditions, confirmed the laboratory results. This study demonstrates that the combination of strains and inoculation protocol could help to reduce the ethanol content in wines.

Ozone treatments of post harvested wine grapes: Impact on fermentative yeasts and wine chemical properties.

Ozone represents a potent antimicrobial compound that is already proposed as a possible sanitizing agent, especially for surface decontamination of fruits and vegetables. The main objective of this study was to evaluate the effect of ozone, either in aqueous or gaseous form, on wine grape mycobiota and its impact during spontaneous and inoculated fermentations. Gaseous (32±1µL/L, 12 and 24h) and aqueous (5±0.25mg/L, 6 and 12min) ozone were tested as sanitizing treatments. A multiphasic approach was used employing culture-dependent (traditional plate counts) and -independent techniques, based on DNA and RNA amplification (PCR-denaturing gradient gel electrophoresis [DGGE] and reverse transcription PCR [RT-PCR]-DGGE), respectively. Microbiological analysis data highlighted a reduction of >0.5LogCFU/mL of the total yeasts present on grape berry surfaces after ozone treatments, mainly due to the reduction of apiculate yeasts. The chemical analysis of the wines, produced from the treated grapes, showed higher acetic acid content in the untreated spontaneous fermentations (0.52g/L) compared to the treated (ranged from 0.16 to 0.38g/L), while all fermentation-inoculated wines contained higher amounts of pleasant volatile compounds.