Barbara Bovo

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.

Biodiversity, dynamics and ecology of bacterial community during grape marc storage for the production of grappa

The Italian spirit obtained from grape marc, grappa, is produced by an extended storage of the marc which allows alcoholic fermentation. Bacterial populations can develop and are associated with off-flavour production. Grape marc acidification before storage is a common practice in distilleries to control bacterial proliferation. Few studies have been published on the microbial biodiversity in grape marc and no information exists about microbiology of acidified marcs and physiological properties needed for colonizing such an environment. The aim of this study was to investigate the composition and dynamics of grape marc bacterial populations during the long-period storage by microbiological analyses of acidified and untreated marcs. Eight bacterial species were identified by ARDRA - 16s rRNA sequencing at the beginning of the fermentation. Among them the bacterial species of Tatumella terrea, Acetobacter ghanensis and Tatumella ptyseos were identified for the first time in a wine environment. In later stages Oenococcus oeni and members of the Lactobacillus plantarum group became dominant in acidified and non-acidified grape marc, respectively. Further molecular typing of L. plantarum isolates yielded 39 strains. To explain the prevalence of L. plantarum in untreated samples, all strains were tested for potential antimicrobial activity and for biofilm formation ability. Although no antimicrobial activity was found, many strains exhibited the ability to form a biofilm, which may confer an ecological advantage to these strains and their dominance during marc storage.

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.

Whole-Genome Sequences of Streptococcus thermophilus Strains TH1435 and TH1436, Isolated from Raw Goat Milk.

ABSTRACT We report the genome sequences of two Streptococcus thermophilus strains, TH1435 and TH1436, isolated from raw goat milk devoted to the production of artisanal cheese in the Friuli-Venezia Giulia region in Italy. The genome sequences of these two quickly acidifying strains are the first available genome sequences of S. thermophilus strains isolated in Italy.

Effects of grape marcs acidification treatment on the evolution of indigenous yeast populations during the production of grappa

Aims:  Grappa is a typical Italian product obtained from the distillation of grape marcs, the main by‐product of grape crushing. One technological treatment frequently performed on marcs is their acidification, in order to contrast the development of unwanted spoilage bacteria during the storage period needed for alcoholic fermentation. A pilot‐scale experiment was set‐up to study the dynamics of yeast populations during a 30‐day fermentation of acidified and nonacidified Prosecco grape pomace.

Genome Sequences of Four Italian Streptococcus thermophilus Strains of Dairy Origin

ABSTRACT This report describes the genome sequences of four Streptococcus thermophilus strains, namely, TH982, TH985, TH1477, and 1F8CT, isolated from different dairy environments from the Campania and the Veneto regions in Italy. These data are aimed at increasing the genomic information available on this species, which is of paramount importance for the dairy industry.

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 106 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.

Whole-Genome Sequence of Streptococcus macedonicus Strain 33MO, Isolated from the Curd of Morlacco Cheese in the Veneto Region (Italy)

ABSTRACT A genetic characterization of Streptococcus macedonicus is important to better understand the characteristics of this lactic acid bacterium, frequently detected in fermented food bacteria communities. This report presents the draft genome sequence description of strain 33MO, the first publicly available genome sequence of an Italian S. macedonicus isolate.

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, β-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.