Vasileios Englezos

Exploitation of the non-Saccharomyces yeast Starmerella bacillaris (synonym Candida zemplinina) in wine fermentation: Physiological and molecular characterizations

Nowadays, the use of non-Saccharomyces yeasts in combination with Saccharomyces cerevisiae is a state-of-the-art strategy to improve complexity and enhance the analytical composition of the wines. This application has stimulated the interest of understanding how the non-Saccharomyces yeasts can contribute to the quality of the wines. The study presented here explores the potential use of Starmerella bacillaris (synonym Candida zemplinina) under winemaking conditions. Physiological and genetic characterizations of sixty-three isolates of Starm. bacillaris, previously isolated from four different varieties of grapes, were carried out. Both analyses revealed a low level of diversity between the isolates of Starm. bacillaris, while the fermentation trials in laboratory scale demonstrated the good enological performance of this species. The strong fructophilic character of this species and its ability to produce low quantities of ethanol and acetic acid and high amounts of glycerol were confirmed. The results, presented here, demonstrated a potential application of this non-Saccharomyces species in mixed wine fermentations with S. cerevisiae.

The yeast Starmerella bacillaris (synonym Candida zemplinina) shows high genetic diversity in winemaking environments

The yeast Candida zemplinina (Starmerella bacillaris) is frequently isolated from grape and wine environments. Its enological use in mixed fermentation with Saccharomyces cerevisiae has been extensively investigated these last few years, and several interesting features including low ethanol production, fructophily, glycerol and other metabolites production, have been described. In addition, molecular tools allowing the characterization of yeast populations have been developed, both at the inter- and intraspecific levels. However, most of these fingerprinting methods are not compatible with population genetics or ecological studies. In this work, we developed 10 microsatellite markers for the C. zemplinina species that were used for the genotyping of 163 strains from nature or various enological regions (28 vineyards/wineries from seven countries). We show that the genetic diversity of C. zemplinina is shaped by geographical localization. Populations isolated from winemaking environments are quite diverse at the genetic level: neither clonal-like behaviour nor specific genetic signature were associated with the different vineyards/wineries. Altogether, these results suggest that C. zemplinina is not under selective pressure in winemaking environments.

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.

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.

Modeling of the fermentation behavior of Starmerella bacillaris

Starmerella bacillaris has been proposed as a non-Saccharomyces species candidate to be used in mixed fermentations with Saccharomyces cerevisiae for the production of wine. Among the prospective applications, reductions in ethanol content and the acetic acid produced from high sugar musts have attracted particular attention. In this study, we described the fermentation behavior of six strains of S. bacillaris in grape must with initial sugar concentration ranging from 200 to 330 g/L. Time (days of fermentation) was a second variable that was monitored for its influence on fermentation. Response surface methodology was employed to model the behavior of the strains. The six strains generally behaved uniformly. Residual sugar concentration and production of ethanol, glycerol, and acetic acid mainly depended on time. Initial sugar concentration in the must was positively associated with residual glucose in musts with higher initial sugar concentration. Similarly, malic acid consumption showed a dependence on time and sugar concentration and was inhibited in musts with a higher sugar content. The behavior of S. bacillaris strains can be considered compatible with enological practices that may involve mixed fermentation with S. cerevisiae.

Mycobiota of Barbera Grapes from the Piedmont Region from a Single Vintage Year

In this study, a description of the fungal populations present on grapes of the Barbera cultivar (Vitis vinifera L.) is reported. Grapes from 15 vineyards in Montferrat, an area located in the Piedmont region in northwest Italy, were sampled at maturation during vintage 2012. The grapes were analyzed by culture-dependent and independent methods. Using plate counts on Wallerstein Laboratory Nutrient agar, we determined a yeast load of about 100 colony forming units (cfu)/mL on the grapes. In total, 206 fungal isolates were identified by two different molecular approaches. Highly reproducible and comparable results were obtained with two identification techniques, that is, PCR-based RFLP analysis of the 5.8S internal transcribed spacer (ITS) rRNA region and PCR–DGGE coupled with sequencing of the 26S rRNA gene, confirming that these two methods reliably identify wine yeasts. Both techniques identified a mycobiota mainly composed of Aureobasidium pullulans (73% of the isolates), followed by Rhodotorula glutinis (12%), Hanseniaspora spp. (8%), Issatchenkia terricola (5%), Torulaspora delbrueckii (1%), and Cryptococcus carnescens (1%). Moreover, PCR–DGGE based on DNA and RNA extracted directly from grapes confirmed the presence of A. pullulans, H. uvarum, and Starmerella bacillaris (synonym Candida zemplinina).

Alcohol reduction in red wines by technological and microbiological approaches: a comparative study

Background and Aims The aim of this work was to assess and compare the chemical composition and colour characteristics of Barbera red wines obtained after partial alcohol reduction using three promising methodologies for implementation at the industrial level. Methods and Results Alcohol reduction was achieved by: (i) pre-fermentation addition of liquid derived from grape must (reverse osmosis by-product); (ii) mixed fermentations with strains of Starmerella bacillaris and Saccharomyces cerevisiae; and (iii) dealcoholisation of wine post-fermentation with a polypropylene membrane. The microbiological approach enabled the production of wines with a slightly lower alcohol concentration (−0.2 to −0.3% v/v), while facilitating the release of anthocyanin and some esters of fatty acids (ethyl hexanoate and ethyl dodecanoate) that could contribute positively to wine aroma with pleasant nuances. The low impact of the partial replacement of grape juice on the chemical composition and chromatic characteristics of Barbera wines makes this technique a good option for reducing the ethanol concentration by up to 1.0–2.0% v/v. In contrast, the use of a polypropylene membrane influenced negatively the composition of red wines by reducing significantly the concentration of esters (−60%) and anthocyanin (−17%), independently of the dealcoholisation level (up to −2% v/v). Conclusions The alcohol reduction strategies can greatly influence the volatile and phenolic composition of the wine. The choice of either a technological or microbiological approach in the wine industry is dependent on the alcohol reduction required. Significance of the Study This is the first comparative study of three strategies to reduce the alcohol concentration on the same batch of must/wine.

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.

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.