Strain prevalence and killer factor only partially influence the fermentation activity of pairwise Saccharomyces cerevisiae wine strains inoculation.

Commercial Saccharomyces cerevisiae starters are single-strain cultures widely used in winemaking to optimise the fermentation process and improve the organoleptic quality of wine. Unfortunately, the worldwide extensive use of a limited number of industrial strains led to the standardisation of the sensory properties, reducing the identity of wines. Therefore, the use of multi-strain S. cerevisiae starters can be an alternative tool to alter the sensory profile of wines, increasing the diversity of wine styles. However, this strategy may be interesting only if the overall fermentation kinetics is not affected. To date, there is a lack of information regarding the influence of multi-strain starters on the overall fermentation process in wine. In this context, killer toxins, affecting the viability of sensitive strains, can play a significant role. This study aimed to evaluate the effects of pairing eight wine strains of S. cerevisiae (two sensitive, three neutral and three killer) in co-fermentations compared to single-strain fermentations. Results evidenced that, among co-fermentations where the strain prevalence was significant, the killer strains constituted 79% to 100% of the total yeast population when co-inoculated with a sensitive one. However, in most of the cases, co-fermentations kinetics were similar to those of sensitive strains or worse than both strains. Thus, the presence of a killer strain alone is not sufficient to predict the overall fermentation progress, which is an essential information in winemaking. Interestingly, the neutral strain P304.4 was always prevalent, regardless of the second strain and, in most of the co-fermentations, the overall fermentation trend was similar to the P304.4 single-strain fermentation. Regardless of killer activity, our results suggest that the effect of strains on fermentative kinetics is still unpredictable, and further studies are needed to thoroughly explore strain to strain interactions in winemaking.

Starmerella bacillaris Released in Vineyards at Different Concentrations Influences Wine Glycerol Content Depending on the Vinification Protocols.

Starmerella bacillaris is a non-Saccharomyces yeast proposed for must fermentation together with Saccharomyces cerevisiae because of its high glycerol and moderate volatile acidity production. Furthermore, it was demonstrated that the same S. bacillaris strains that possess interesting technological properties exhibited antifungal activity against Botrytis cinerea, suggesting the release of this yeast in the vineyard. To obtain a positive effect during the following winemaking process, the maintenance of suitable concentrations of S. bacillaris is essential. Therefore, to obtain information on the survival of S. bacillaris, a small-scale field trial was performed. One week before the harvest, two different concentrations of S. bacillaris (106 and 107 cells/mL) were sprayed on Pinot grigio bunches, and the strain concentration was monitored by means of qPCR during the subsequent fermentation process. In addition, the combined effect of different winemaking techniques was evaluated, i.e., the vinification of juice, juice with marc and cryomaceration treatment. Results demonstrated that, under the tested conditions, S. bacillaris released in the vineyard remained viable for one week on grape bunches and increased glycerol content during the subsequent fermentation process. Different vinification protocols influenced cell concentrations. In particular, the cryomaceration treatment, due to the use of low temperature, supported S. bacillaris growth due to its cryotolerant aptitude. The collected data open new perspectives on the control of alcoholic fermentation, involving both vineyard and cellar management.

Thermal resistance and high-performance microwave decontamination assessment of Bacillus endospores isolated from food-grade herbal extracts.

Generally, endospore contamination can occur from different sources during product manufacturing in many industries and therefore lower its quality by affecting physicochemical properties and shelf-life. Bacterial endospores can germinate inside the product and produce several enzymes, which can cause several undesirable changes. This study assessed the spores thermal resistance and applied a microwave decontamination technique toward herbal extracts (Tilia tomentosa and Centella asiatica) containing ethanol or glycerol. Based on 16S rRNA analysis, the detected contaminant endospores belonged to different Bacillus species, namely B. subtilis, B. zhangzhouensis, and B. pumilus. The thermal resistance assessment using inoculated endospores in the actual products revealed B. pumilus T2 as the most resistant endospore to the heat treatments tested in both T. tomentosa and C. asiatica extracts. Finally, a high-performance microwave technique was used to decontaminate T. tomentosa extract against the mixture of Bacillus spores. Results from the microwave technique indicate that the increase of temperature from 100°C to 105°C not only decontaminated the product but also could dramatically decrease the effective thermal treatment time (10 times), which can benefit the product quality. The results provided in this study considerably contribute to improving an original decontamination method for products containing glycerol and ethanol with the most negligible effect on product quality.

The impact of CUP1 gene copy-number and XVI-VIII/XV-XVI translocations on copper and sulfite tolerance in vineyard Saccharomyces cerevisiae strain populations.

In wine production, sulfites are widely used as antimicrobials and antioxidants, whereas copper is associated with fungicides and wine fining treatments. Therefore, wine yeasts are constantly exposed to these agents. Copper tolerance is related to the copy number of the CUP1 gene, encoding for a metallothionein involved in copper detoxification. In wine yeasts, sulfite resistance mainly depends on the presence of the translocation t(XVI;VIII) in the promoter region of the SSU1 gene. This gene encodes for a plasma membrane sulfite pump involved in sulfite metabolism and detoxification. Recently, a new translocation, t(XVI;VIII), was identified. In this work, 253 Saccharomyces cerevisiae strains, representing three vineyard populations from two different continents, were analyzed, along with 20 industrial starters. Copper and sulfites tolerance as well as distribution of CUP1 gene copy-number, t(XVI;VIII)and t(XVI;XV) of SSU1 gene were studied to evaluate the impact of these genomic variations on population phenotypes. The CUP1 gene copy-number was found to be highly variable, ranging from zero to 79 per strain. Moreover it differently impacted the copper tolerance in the populations of the two continents. The diffusion of t(XVI;VIII) and, for the first time, t(XVI;XV) was determined in the three vineyard populations. The correlation between the presence of the translocation and strain sulfite tolerance levels was significant only for the t(XVI;VIII).

Genetic variability and physiological traits of Saccharomyces cerevisiae strains isolated from "Vale dos Vinhedos" vineyards reflect agricultural practices and history of this Brazilian wet subtropical area.

Vale dos Vinhedos appellation of origin has a very recent history as industrial wine making region. In this study we investigated the genetic and phenotypic variability of Saccharomyces cerevisiae strains isolated from South-Brazilian vineyards in order to evaluate strain fermentation aptitude and copper and sulphites tolerance. Merlot grape bunches were collected from three vineyards and yeast isolation was performed after single bunch fermentation. High genotypic variability was found and most of the genotypes revealed to be vine-specific. No industrial strain dissemination was present in the sampled vineyards, although it has been wildly reported in traditional winemaking countries. From the phenotypic traits analysis these Brazilian native strains showed good fermentation performances, good tolerance to sulphites and, in particular, a high copper tolerance level. Copper is the most important metal in the formulation of fungicides against downy mildew (Plasmopara viticola), one of the most harmful disease of the vines, and other fungal pests. The high tolerance to copper suggests an environmental adaptation to the strong use of copper-based fungicides, requested by the wet subtropical climate.

The Different Physical and Chemical Composition of Grape Juice and Marc Influence Saccharomyces cerevisiae Strains Distribution During Fermentation.

During white-grape winemaking, grape marc is separated from juice immediately after crushing. Both mark and juice are obtained from the same grapes, but they differ strongly for their physical and chemical properties. Marc is mainly composed of solid residues. Its pH is usually higher than that of the juice and Saccharomyces cerevisiae strains are largely present. Therefore, it can be considered as a potential alternative environment for the selection of industrial yeasts. In order to evaluate the effect of different pH and physical state of the two matrices on grapes yeast population composition, the isolation of S. cerevisiae, from both grape juice and marc during simultaneous fermentations, was performed. After yeast identification and genotyping, strains present at high frequencies were tested in fermentation at different pH values. Biofilm production was also tested to evaluate strain ability to develop on a solid matrix. Genotype analysis showed that high-frequency strains were always more abundant in one of the two environments, suggesting the existence of a selective effect. Generally, fermentations at different pH revealed that the best fermentation performance of each strain, in terms of CO2 production, was in the pH range of its original environment. Only one strain, mostly present in grape marc, produced a high biofilm level. Therefore, biofilm production does not seem to favor strain adaptation to grape marc condition. PRACTICAL APPLICATION: These results demonstrate that grape juice and marc represent two different environments able to influence yeast strains distribution. The pH level can be included among the selection factors acting on yeast strains distribution. Grape marc can be considered a yeasts reservoir and its fermentation can be used for the development and isolation of new strains, genetically and physiologically different from those present in the grape juice.

The Geographic Distribution of Saccharomyces cerevisiae Isolates within three Italian Neighboring Winemaking Regions Reveals Strong Differences in Yeast Abundance, Genetic Diversity and Industrial Strain Dissemination.

In recent years the interest for natural fermentations has been re-evaluated in terms of increasing the wine terroir and managing more sustainable winemaking practices. Therefore, the level of yeast genetic variability and the abundance of Saccharomyces cerevisiae native populations in vineyard are becoming more and more crucial at both ecological and technological level. Among the factors that can influence the strain diversity, the commercial starter release that accidentally occur in the environment around the winery, has to be considered. In this study we led a wide scale investigation of S. cerevisiae genetic diversity and population structure in the vineyards of three neighboring winemaking regions of Protected Appellation of Origin, in North-East of Italy. Combining mtDNA RFLP and microsatellite markers analyses we evaluated 634 grape samples collected over 3 years. We could detect major differences in the presence of S. cerevisiae yeasts, according to the winemaking region. The population structures revealed specificities of yeast microbiota at vineyard scale, with a relative Appellation of Origin area homogeneity, and transition zones suggesting a geographic differentiation. Surprisingly, we found a widespread industrial yeast dissemination that was very high in the areas where the native yeast abundance was low. Although geographical distance is a key element involved in strain distribution, the high presence of industrial strains in vineyard reduced the differences between populations. This finding indicates that industrial yeast diffusion it is a real emergency and their presence strongly interferes with the natural yeast microbiota.

Whole-Genome Sequence of Starmerella bacillaris PAS13, a Nonconventional Enological Yeast with Antifungal Activity.

Starmerella bacillaris is a fermentative yeast commonly found in vineyards. Here, we present the draft genome sequence of S. bacillaris PAS13, a nonconventional enological yeast with a potential role as a biocontrol agent. This gene sequence will provide insights into the genetic basis of yeast activity against gray mold disease (Botrytis cinerea).

Corrigendum: Biocontrol Ability and Action Mechanism of Starmerella bacillaris (Synonym Candida zemplinina) Isolated from Wine Musts against Gray Mold Disease Agent Botrytis cinerea on Grape and Their Effects on Alcoholic Fermentation.

[This corrects the article on p. 1249 in vol. 7, PMID: 27574517.].

Biocontrol Ability and Action Mechanism of Starmerella bacillaris (Synonym Candida zemplinina) Isolated from Wine Musts against Gray Mold Disease Agent Botrytis cinerea on Grape and Their Effects on Alcoholic Fermentation.

Gray mold is one of the most important diseases of grapevine in temperate climates. This plant pathogen affects plant growth and reduces wine quality. The use of yeasts as biocontrol agents to apply in the vineyard have been investigated in recent years as an alternative to agrochemicals. In this work, fermenting musts obtained from overripe grape berries, therefore more susceptible to infection by fungal pathogens such as Botrytis cinerea, were considered for the selection of yeasts carrying antifungal activity. Thirty-six isolates were identified as Starmerella bacillaris, a species recently proven to be of enological interest. Among them 14 different strains were studied and antifungal activity against B. cinerea was demonstrated, for the first time, to be present in S. bacillaris species. The production of volatile organic compounds (VOCs), tested in vitro, was found to be the main responsible of S. bacillaris antifungal effects. All the strains were able to reduce B. cinerea decay on wounded grape berries artificially inoculated with gray mold. The colonization level of wound was very high reaching, after 5 days, a concentration of 10(6) cells per ml of grape juice obtained after berry crushing. At this cell concentration S. bacillaris strains were used to ferment synthetic and natural musts. The sequential yeast inoculation, performed by adding S. cerevisiae 48 h after S. bacillaris, was needed to complete sugar consumption and determined a significant increase in glicerol content and a reduction of ethanol and acetic acid concentrations. The high wound colonization ability, found in this work, together with the propensity to colonize grape berry and the interesting enological traits possessed by the selected S. bacillaris strains allow the use of this yeast as biocontrol agent on vine and grape berries with possible positive effects on must fermentation, although the presence of S. cerevisiae is needed to complete the fermentation process. This work introduces new possibilities in wine yeast selection programs in order to identify innovative wine yeasts that are simultaneously antifungal agents in vineyards and alternative wine starters for grape must fermentation and open new perspective to a more integrated strategy for increasing wine quality.

Aptitude of Saccharomyces yeasts to ferment unripe grapes harvested during cluster thinning for reducing alcohol content of wine.

Among the viticultural techniques developed to obtain wine with reduced alcohol content, the use of unripe grapes with low sugar and high malic acid concentration, harvested at cluster thinning, was recently explored. So far, no studies have evaluated the fermentation performances of Saccharomyces in unripe grape musts, in terms of fermentation ability and reducing malic acid contents, to improve the quality of this low-alcohol beverage. In this work, we evaluated 24 S. cerevisiae strains isolated from Italian and Croatian vineyards with different fermentation aptitudes. Moreover, four S. paradoxus were considered, as previous works demonstrated that strains belonging to this species were able to degrade high malic acid amounts in standard musts. The industrial strain S. cerevisiae 71B was added as reference. Sugar and malic acid contents were modified in synthetic musts in order to understand the effect of their concentrations on alcoholic fermentation and malic acid degradation. S. cerevisiae fermentation performances improved when glucose concentration decreased and malic acid level increased. The conditions that simulate unripe grape must, i.e. low glucose and high malic acid content were found to enhance S. cerevisiae ability to degrade malic acid. On the contrary, S. paradoxus strains were able to degrade high amounts of malic acid only in conditions that resemble ripe grape must, i.e. high glucose and low malic acid concentration. In fermentation trials when low glucose concentrations were used, at high malic acid levels S. cerevisiae strains produced higher glycerol than at low malic acid condition. Malic acid degradation ability, tested on the best performing S. cerevisiae strains, was enhanced in fermentation trials when unripe grape must was used.

Outlining a selection procedure for Saccharomyces cerevisiae isolated from grape marc to improve fermentation process and distillate quality.

Nowadays grape marc represents one of the main by-product of winemaking. Many South Europe countries valorize this ligno-cellulosic waste through fermentation and distillation for industrial alcoholic beverage production. The storage of marcs is a crucial phase in the distillation process, due to the physicochemical transformations ascribed to microbial activity. Among the methods adopted by distillers to improve the quality of spirits, the use of selected yeasts has not been explored so far, therefore in this work we evaluated the selection criteria of Saccharomyces cerevisiae strains for grape marc fermentation. The proposed selection procedure included three steps: characterization of phenotypical traits, evaluation of selected strains on pasteurised grape marc at lab-scale (100 g) and pilot-scale fermentation (350 kg). This selection process was applied on 104 strains isolated from grape marcs of different origins and technological treatment. Among physiological traits, ß-glucosidase activity level as quality trait seems to be only partially involved in increasing varietal flavour. More effective in describing yeast impact on distillate quality is the ratio higher alcohols/esters that indicates strain ability to increase positive flavours. Finally, evaluating grape marc as source of selected yeasts, industrial treatment rather than varietal origin seems to shape strain technological and quality traits.

Exploring the use of Saccharomyces cerevisiae commercial strain and Saccharomycodes ludwigii natural isolate for grape marc fermentation to improve sensory properties of spirits.

In Mediterranean countries the most diffuse practice to obtain the valorization of grape marc, the main by-product from winemaking, is the production of spirits. During this process, marc storage for sugar fermentation represents a crucial step, since side-fermentations leading to off-flavours production can very easily occur. In this study we evaluated the effect of the addition of two yeast strains, inoculated separately at the beginning of the storage period, into marcs from two Italian grape varieties with the aim to control the development of autochthonous microbiota and to improve spirit quality. The presence of the inoculated strains was monitored by means of PCR-based approaches. A commercial Saccharomyces cerevisiae strain, chosen as this species is notably the best ethanol producer, showed excellent ability to dominate the autochthonous microflora and to reduce off-flavours as demonstrated by chemical analysis and sensory evaluation. A Saccharomycodes ludwigii strain, chosen for increasing varietal compounds thus enhancing spirit aroma, showed a level of implantation not sufficient to assure a clear beneficial effect on quality. The implantation level of this strain was affected by S. cerevisiae competition since the highest level was found in grape marc with lower sugar content, where indigenous S. cerevisiae were less persistent.

Acidification of grape marc for alcoholic beverage production: effects on indigenous microflora and aroma profile after distillation.

Grappa is an Italian alcoholic beverage obtained from distillation of grape marc, the raw material derived from separation of must during the winemaking process. Marc is stored for a period lasting from few days to several weeks, when fermentation of residual sugars occurs mainly by yeast activity. Many distilleries have adopted different solutions to manage this critical phase in order to avoid spoilage microorganisms: marc acidification is the most widely diffused. In this work, Prosecco grape pomace was acidified with sulphuric acid (to pH 2.9) and stored, whereas non-acidified grape marc was used as control (pH 3.9). Samples for microbiological analysis were collected at the beginning of the storage period, after 15 and 43days. At the beginning of the ensilage (time T0) the indigenous microflora was represented both by yeasts and bacteria at a concentration of about 10(6)cfu/g. During the first 15days, when the fermentation generally takes place, yeast population grew considerably (up to 10(7)cfu/g) in acidified grape marc, where bacterial population was maintained at low levels. Moreover, yeast populations recovered at the three sampling times in both treated and untreated marc were genetically characterised. This analysis showed that the species succession lead to non-Saccharomyces species dominance (in particular Issatchenkia and Pichia genera) in both conditions although acidified marc showed a lower percentage of Saccharomyces at any sampling time analysed, this meaning that non-Saccharomyces species were favoured in this environment. Gas chromatographic analysis showed a remarkable change in the aromatic profile of distilled grape marcs at the end of the storage, thus evidencing that concentration of monitored volatile compounds usually produced by microflora was generally lowered by the acidification treatment. This work demonstrates for the first time the strong effect of a persistent acidification treatment both on the microbiota of grape pomace and on the aromatic profile of the distillate. Indeed, the lowering of the pH caused significant changes in yeast-bacteria populations ratio and in yeast species turnover. These microbiological changes determine an improvement of the aromatic profile of the distillate, due to the reduction of the main volatile products associated with potential off-flavours.

Consequences of relative cellular positioning on quorum sensing and bacterial cell-to-cell communication.

Cell-to-cell bacterial communication via diffusible signals is addressed and the conceptual framework in which quorum sensing is usually described is evaluated. By applying equations ruling the physical diffusion of the autoinducer molecules, one can calculate the gradient profiles that would occur either around a single cell or at the center of volumes of increasing size and increasing cell densities. Water-based matrices at 25 degrees C and viscous biofilms at colder temperatures are compared. Some basic consequences relevant for the field of microbial signalling arise. As regards induction, gradient-mixing dynamics between as little as two cells lying at a short distance appears to be sufficient for the buildup of a concentration reaching the known thresholds for quorum sensing. A straight line in which the highest concentrations occur is also created as a consequence of the gradient overlap geometry, providing an additional signal information potentially useful for chemotactic responses. In terms of whole population signalling, it is shown how the concentration perceived by a cell in the center is critically dependent not only on the cell density but also on the size of the biofilm itself. Tables and formulas for the practical prediction of N-acyl homoserine lactones concentrations at desired distances in different cell density biofilms are provided.

Yeast population dynamics during pilot-scale storage of grape marcs for the production of Grappa, a traditional Italian alcoholic beverage.

The composition and population dynamics of the yeast microflora of grape marcs were investigated during a pilot scale fermentation study using two white grape varieties, namely Moscato and Prosecco, from two distinct areas of the Veneto Region. Yeast counts were made at the beginning, after 4 and after 15 days of marc storage under anaerobic conditions. Seventy isolates from each sampling time were identified to species by RAPD-PCR analysis and subsequent ITS region sequencing. A good biodiversity of yeasts occurred in both marcs at the beginning of fermentation, with high presence of Hanseniaspora opuntiae, but without detectable presence of Saccharomyces strains, which instead became the dominant yeast after just 4 days of fermentation, remaining at that level until the end of fermentation. Colonization of Moscato marc by S. cerevisiae resulted better, in relation to its higher sugar content. Characterization of S. cerevisiae isolates by mitochondrial DNA restriction analysis revealed the presence of 66 different strains in the marc from the Moscato grapes, without the occurrence of a clearly dominant strain, while in the marc from the Prosecco grapes only 23 different profiles were scored, with a dominant strain that accounted for 62.7% of the Saccharomyces population after 4 days of fermentation.

Long term evaluation of field-released genetically modified rhizobia.

This is the report of the first open field release of genetically modified microorganisms (GMMs) in Italy. It covers ten years of monitoring, and follows in-field GMM dynamics from strain release to disappearance below detection limits, as well as assessment of impact on resident microorganisms. The bacteria released belong to the nitrogen fixing legume endosymbiont Rhizobium leguminosarum bv. viciae, and were engineered with non-agronomically-proficient traits, in order to assess their behavior and fate without GMM-specific positive feedback from the plant. A DNA cassette containing mercury resistance and ss-galactosidase genes was introduced in either plasmid-borne or chromosomally integrated versions, in order to test the resulting strain stability. A synthetic promoter was used to drive the lacZ gene, conferring high catabolic activity to the GMM. Two different wild-type Rhizobium backgrounds were tested, comparing a non-indigenous vs. an indigenous, highly competitive strain. The latter had much greater persistence, since it was able to survive and establish at technically detectable levels for over four years after release. Selection factors, such as reiterated presence of the plant host, or lactose substrate supply, enhanced long-term survival to different extents. The lactose treatment showed that even a single trophic supplementation can surpass the benefits of symbiotic interaction for a period of several years. Concerning impact, the GMMs did not alter substantially the other soil community general microbiota. However, there were some significant differences in microbiota as a consequence of the Rhizobium inoculation. This effect was observed with either the WT or GMM, and was more evident in the release of the indigenous Rhizobium. Moreover, as the indigenous GMM had its parental, dominant wild-type in the same soil, it was possible to evaluate to what extent the GMM version could result in parent displacement ("self-impact"), and how much the two rhizobia would additively contribute to nodulation.

A rapid method for differentiating Saccharomyces sensu stricto strains from other yeast species in an enological environment.

During programs for the selection of enological yeasts, several hundred natural isolates are usually screened. The scope of these operations is to isolate strains possessing good fermentative properties without necessarily arriving at a precise species designation: in other words, to detect strains belonging to the Saccharomyces sensu stricto complex. In the present study, a pair of primers, designed within the variable D1/D2 region of the 26S subunit of ribosomal yeast RNA, have been constructed. These generate an amplification fragment of 471 bp that is specific for the seven Saccharomyces sensu stricto species, while no signal was obtained for Saccharomyces sensu lato strains (17 species) or for another 18 selected species commonly found in enological environments. A second pair of primers was also constructed, within the 18S rRNA gene, composed of perfectly conserved sequences common for all 42 yeast species examined, which generate a 900 bp (c.) band for all strains. This was used as a positive experimental control in multiplex PCR analysis using all four primers.