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

Antiradical and antimicrobial properties of fermented red chicory (Cichorium intybus L.) by-products

Discarded leaves of red chicory (Radicchio \u201cRosso di Chioggia\u201d IGP) were fermented with one Saccharomyces yeast and four lactic acid bacteria chosen on the basis of their ability to grow on plant material without any need of supplements. Antiradical and antimicrobial activities of the resulting products were assessed. Among the strains tested, Lactobacillus plantarum and L. hilgardii gave the best performances and also provided fermented substrates with antiradical and antimicrobial activities. In particular the latter compounds were found only in fermented samples, confirming that the choice of appropriate microorganisms for fermentation could be useful when the aim is to target specific functional foods starting from by-products or waste material

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

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

Genome Sequences of Four Italian Streptococcus thermophilus Strains of Dairy Origin.

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

Genome Sequences of Streptococcus thermophilus Strains MTH17CL396 and M17PTZA496 from Fontina, an Italian PDO Cheese.

Here is presented the whole-genome sequences of Streptococcus thermophilus strains MTH17CL396 and M17PTZA496, isolated from fontina protected designation of origin (PDO) cheese in the Valle d\u2019Aosta Region (Italy). S. thermophilus is a lactic acid bacterium widely present in dairy products, and these are the first publicly available genome sequences of S. thermophilus strains isolated from cheese

MytiBase: a knowledgebase of mussel (M. galloprovincialis) transcribed sequences

<p>Abstract</p> <p>Background</p> <p>Although Bivalves are among the most studied marine organisms due to their ecological role, economic importance and use in pollution biomonitoring, very little information is available on the genome sequences of mussels. This study reports the functional analysis of a large-scale Expressed Sequence Tag (EST) sequencing from different tissues of <it>Mytilus galloprovincialis </it>(the Mediterranean mussel) challenged with toxic pollutants, temperature and potentially pathogenic bacteria.</p> <p>Results</p> <p>We have constructed and sequenced seventeen cDNA libraries from different Mediterranean mussel tissues: gills, digestive gland, foot, anterior and posterior adductor muscle, mantle and haemocytes. A total of 24,939 clones were sequenced from these libraries generating 18,788 high-quality ESTs which were assembled into 2,446 overlapping clusters and 4,666 singletons resulting in a total of 7,112 non-redundant sequences. In particular, a high-quality normalized cDNA library (Nor01) was constructed as determined by the high rate of gene discovery (65.6%). Bioinformatic screening of the non-redundant <it>M. galloprovincialis </it>sequences identified 159 microsatellite-containing ESTs. Clusters, consensuses, related similarities and gene ontology searches have been organized in a dedicated, searchable database <url>http://mussel.cribi.unipd.it</url>.</p> <p>Conclusion</p> <p>We defined the first species-specific catalogue of <it>M. galloprovincialis </it>ESTs including 7,112 unique transcribed sequences. Putative microsatellite markers were identified. This annotated catalogue represents a valuable platform for expression studies, marker validation and genetic linkage analysis for investigations in the biology of Mediterranean mussels.</p

Selezione e caratterizzazione di microrganismi destinati alla produzione di distillati

Grappa is an alcoholic beverage obtained from the distillation of grape pomace derived from grapes produced exclusively in Italy. The process of production of Grappa starts in the winery where winemaking takes place: after harvesting and grape crushing, the must is separated from grape marcs mainly composed by grape skins and seeds, with or without rasp. In the case of red winemaking, separation of marcs takes place after maceration when fermentation has already started, so that they contain alcohol and a low quantity of sugar. On the contrary, marcs obtained from white winemaking contain sugars not yet fermented. For this reason in this second case the raw vegetable material conferred to the distillery is stored for a variable period to allow alcohol production to occur. Marcs can be conserved in different manners, using silos or smaller containers such as plastic tunnels, for period lasting from few days to several weeks. During this period, the main chemical process is sugar degradation and alcohol production, carried out by yeasts, although a large number of other molecules with positive (aromatic substances) or negative (off-flavours and toxic compounds) effect can rise from the activity of the indigenous microflora. Among the technological supports (pH improvement, temperature control, anaerobic environment) developed by modern distilleries to avoid spoiling of the raw material due to unsuitable storing conditions the management of microbial populations by means of selected yeast introduction is pursued. Concerning the case of grappa, international studies are almost absent, as its production is limited by the legislation to our national territory. This study is part of a wider project, supported by Regione Veneto, concerning the selection of an yeast to be inoculated in grape marcs destined to the production of grappa. The first objective to be achieved was about the study of the composition and population dynamics of the yeast microflora of grape marcs using conventional and molecular methods present in literature or studied by the Microbiology Unit working at the Department of Agricultural Biotechnology. Two white grape varieties, namely Moscato and Prosecco, from two distinct areas of the Veneto Region, were used. 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. Subsequentely, the effects of refrigeration and acidification treatments of grape pomace on dynamics of yeast populations during the storage period have been studied. A total of 200 isolates were collected from Prosecco grape marcs acidified and non acidified, after 4 and 15 days from the beginning of storage. Identification of enological yeasts belonging to Saccharomyces sensu stricto group was performed by means of a multiplex PCR, and their characterization at strain level was achieved by mitochondrial DNA restriction analysis. Genetic analyses evidenced the development of different microbial strains in the acidified environment and in the non-acidified control. Acidification treatment, applied to inhibit bacterial microflora, disclosed to be able to influence yeast population from a qualitative rather than quantitative point of view. pH decreasing caused a great effect of selection, indeed only 18 out of 121 identified strains were found in both conditions. A total of 104 isolates identified as belonging to Saccharomyces cerevisiae group were chosen from a pool of about 1000 yeasts collected in various experimentations, from marcs of different origin or submitted to technological treatments during the ensilage period. Such isolates were grown in a synthetic must formulation in order to investigate their fermentation performances. Yeast strains isolated from Moscato grape marcs showed the best features in terms of vigour, expressed as sugar consumption after 2 and 7 days of fermentation. The level of foam production was also investigated and a sensory evaluation was conducted at the end of fermentation, mainly to evaluate possible off-flavour production. Other technological traits such as the level of resistance to the antioxidant sulphur dioxide and the hydrogen sulphide production were tested. Considering SO2 resistance all isolates are able to tolerate the concentration of 50 mg/l, commonly used in the winery. At the intermediate dose of 100 mg/l the most resistant strains come from non acidified Prosecco marc. Moreover, it is stated that microbial glycosidase activity contributes to wines and distillates aroma through the hydrolysis of non-volatile flavourless glycoconjugate precursors, which releases free terpenols and other aromatic compounds. Glycosides, composed of a terpenic hydrocarbon (aglycon) and a carbohydrate moiety, are concentrated in grape berries hence contribution of glycosidase activity is determinant for the expression of Grappa aroma. So, from all these isolates ?-glucosidase activity was rapidly screened both by using a newly developed fluorimetric method and by growing the cells in a minimal medium containing the disaccharide cellobiose as the only carbon source. Both the technological and quality traits found for all the isolates were submitted to a statistical analysis in order to detect yeast strains with the best overall characteristics, to be used as starter for distillate production. Recently the ?-glucosidase activity was suggested to be related to the major exo- ?-1,3-glucanase EXG1 of Saccharomyces cerevisiae, which is involved in processes like growth, budding and mating. For this reasons the molecular approach based on construction of mutant strains in exoglucanases genes was recently considered. In particular two deletion mutant for EXG1 and EXG2 genes respectively has been constructed, following a protocol essentially based on homology recombination. The effect of gene knock-out on ?-glucosidase activity was ascertained through fluorimetric assays, showing a little reduction in exg1 null mutant. Growth in Prosecco must was also performed, in 1L bioreactors, but fermentation curves did not reveal significative differences among strains, showing that probably deletion of exoglucanase genes do not influence yeast growth. All the showed results indicate that grape marcs of different origins or submitted tocdifferent technological treatments are a variable source of yeasts that can be selected to be inoculated as starter for grappa production.La grappa è una bevanda alcolica ottenuta dalla distillazione di vinacce derivate da uve prodotte esclusivamente in Italia. Il processo produttivo comincia in cantina dove ha luogo la vinificazione: dopo la raccolta e la pigiatura dell’uva, il mosto è separato dalle vinacce, costituite principalmente dalle bucce e dai vinaccioli, con o senza raspi. Le vinacce ottenute dalla vinificazione in bianco contengono zuccheri non ancora fermentati. Per questo motivo il materiale vegetale conferito alla distilleria è conservato per un periodo variabile per consentire lo svolgimento della fermentazione alcolica. Le vinacce possono essere conservate da pochi giorni ad alcune settimane in diversi modi, in silos o in tunnel di plastica. Durante questo periodo, il principale processo chimico che avviene ad opera dei lieviti è la degradazione degli zuccheri con produzione di alcol, anche se un numero elevato di altre molecole con effetti positivi (sostanze aromatiche) e negativi (composti tossici), può originare dalla microflora indigena. Con lo scopo di evitare il deterioramento del materiale vegetale dovuto a inopportune condizioni di stoccaggio, le moderne distillerie hanno introdotto l’uso di alcuni trattamenti tecnologici (miglioramento del pH, controllo della temperatura, assenza di ossigeno), e recentemente ha trovato interesse anche l’impiego di lieviti selezionati per il controllo della popolazione microbica. A questo proposito, studi internazionali nel campo della grappa sono pressocchè assenti, essendo la sua produzione limitata per legge al nostro territorio nazionale. Questo studio si inserisce nell’ambito di un più ampio progetto, finanziato dalla Regione Veneto, di selezione di lieviti da usare come starter da inoculare nella vinaccia destinata alla produzione di grappa. Il primo obiettivo da perseguire era lo studio della composizione e delle dinamiche di popolazione della microflora di lieviti, mediante metodi convenzionali e molecolari presenti in letteratura o studiati dal gruppo di Microbiologia del Dipartimento di Biotecnologie Agrarie. Sono state scelte due varietà di grappa, Moscato e Prosecco, provenienti da due distinte aree della Regione Veneto. Le conte della popolazione di lieviti sono state effettuate all’inizio, dopo 4 e 15 giorni di stoccaggio in condizioni di anaerobiosi. Una settantina di isolati per ogni campionamento sono stati identificati a livello di specie mediante analisi RAPD-PCR e successivo sequenziamento della regione ITS. All’inizio dello stoccaggio si è osservata una buona biodiversità in entrambe le vinacce, con un’alta percentuale di Hanseniaspora opuntiae, ma assenza di ceppi di Saccharomyces, che invece diventano dominanti dopo soli 4 giorni di fermentazione, attestandosi allo stesso livello fino alla fine della fermentazione. Successivamente sono stati studiati gli effetti dei trattamenti di refrigerazione e acidificazione sulle dinamiche delle popolazioni di lieviti durante lo stoccaggio. Sono stati collezionati un totale di 200 isolati da vinacce di Prosecco acidificate e non acidificate, dopo 4 e 15 giorni di stoccaggio. L’identificazione di ceppi enologici appartenenti al gruppo Saccharomyces sensu stricto è stata effettuata mediante multiplex PCR, e la loro caratterizzazione a livello di ceppo è stata ottenuta con l’analisi del DNA mitocondriale. Le analisi genetiche hanno evidenziato lo sviluppo di ceppi diversi nell’ambiente acidificato e nel controllo non acidificato. Il trattamento di acidificazione, applicato per inibire la microflora batterica, si è rivelato in grado di influenzare la popolazione di lieviti dal punto di vista qualitativo piuttosto che quantitativo. La diminuzione del pH ha provocato un grosso effetto di selezione, infatti solo 18 dei 121 ceppi identificati sono stati trovati in entrambe le condizioni. Un totale di 104 isolati identificati come appartenenti al gruppo Saccharomyces cerevisiae sono stati scelti da un pool di circa 1000 lieviti, collezionati nel corso delle diverse sperimentazioni, da vinacce di diversa origine o sottoposta a trattamenti tecnologici durante il periodo di insilamento. Gli isolati sono stati cresciuti in mosto sintetico per verificare le loro performance di fermentazione. I lieviti isolati da vinacce di Moscato mostrano le caratteristiche migliori in termini di vigore fermentativo, espresso come consumo di zucchero dopo 2 e 7 giorni di fermentazione. E’ stato indagato anche il livello della produzione di schiuma, e alla fine della fermentazione è stata condotta una valutazione sensoriale per individuare i possibili odori sgradevoli. Sono stati esaminati anche altre caratteristiche tecnologiche, quali la resistenza all’antiossidante e antisettico anidride solforosa e la produzione di idrogeno solforato. Considerando la resistenza alla SO2 tutti gli isolati sono in grado di tollerare una concentrazione di 50 mg/l, comunemente usata in cantina. Alla dose intermedia di 100 mg/l i ceppi più resistenti provengono da vinacce di Prosecco non acidificate. Inoltre, è noto che l’attività glicosidasica microbica contribuisce all’aroma di vini e distillati attraverso l’idrolisi di precursori glicoconiugati non volatili e inodori, che rilasciano terpenoli e altri composti aromatici. I glicosidi, composti di un’idrocarburo terpenico (aglicone) e di un carboidrato, sono concentrati nella buccia, perciò il contributo dell’attività glicosidasica è determinante per l’espressione dell’aroma della grappa. L’attività ?-glucosidasica pertanto, è stata rapida mente testata in tutti gli isolati sia utilizzando un metodo fluorimetrico appositamente messo a punto, sia crescendo le cellule in un terreno minimo contenente il disaccaride cellobiosio come sola fonte di carbonio. I caratteri tecnologici e di qualità monitorati per tutti gli isolati sono stati sottoposti ad analisi statistica con lo scopo di individuare i lieviti con le caratteristiche complessive migliori per essere utilizzati come starter nella produzione di distillati. Recentemente è stato suggerito che l’attività ?-glucosidasica sia correlata alla maggiore eso- ?-1,3- glucanasi EXG1 di Saccharomyces cerevisiae, coinvolta in processi come crescita, gemmazione e mating. Per questi motivi è stato preso in considerazione un approccio basato sulla costruzione di mutanti per i geni codificanti per esoglucanasi. In particolare sono stati costruiti due mutanti di delezione per i geni EXG1 ed EXG2 rispettivamente, seguendo un protocollo basato essenzialmente sul principio della ricombinazione omologa. L’effetto della delezione sull’attività ?-glucosidasica è stato provato con il saggio fluorimetrico, evidenziando una lieve diminuzione nel mutante nullo exg1. Inoltre i ceppi mutati sono stati cresciuti in mosto naturale di Prosecco, in fermentatori da 1L, ma le curve di fermentazione non hanno rivelato differenze significative tra i ceppi. E’ possibile concludere perciò che probabilmente la delezione dei geni per le esoglucanasi non influenza la crescita dei lieviti. I risultati ottenuti complessivamente indicano che le vinacce di diversa origine o sottoposte a trattamenti tecnologici rappresentano una fonte ampia di lieviti che possono essere selezionati come starter nel campo della produzione della grappa

Effects of yeast inoculation on volatile compound production by grape marcs

Grape marcs are a by-product of winemaking that can be used for production of Grappa, a traditional Italian spirit. This plant material needs to undergo a fermentation process before distillation to allow production of ethanol from residual sugar and volatile aromatic compounds. To study the possible effect of selected yeast inocula on the fermentation process and subsequent quality of the distillate, a laboratory-scale fermentation of grape marcs was performed. The inoculated yeasts proved to be good colonizers of grape pomace and contributed markedly to rapid sugar consumption, which was completed within 5 days. Fermentation products were analysed by gas-chromatography. A time course analysis performed to determine the impact of the inoculated strains on the main aromatic compounds that are concentrated in the product after distillation provided evidence of a positive effect of yeast inoculation. \ua9 2010 Springer-Verlag and the University of Milan

Fragility Curves of Existing RC Buildings Accounting for Bidirectional Ground Motion

In recent decades, the considerable number of worldwide earthquakes caused considerable damage and several building collapses, underlining the high vulnerability of the existing buildings designed without seismic provisions. In this regard, this work analyses the seismic performance of a reinforced concrete building designed without any seismic criteria, characterized by a seismically-stronger and a seismically-weaker direction, such as several existing reinforced concrete-framed structures designed for vertical load only. The case study building was modelled in OpenSees considering a non-linear three-dimensional model, also accounting for the contribution of joint panel deformability on the global behavior. Thirty bidirectional ground motions have been applied to the structure with the highest component alternatively directed along the two principal building directions. Time-history analyses have been performed for eight increasing hazard levels with the aim of evaluating the influence of bidirectional ground motion on structural response and estimating the seismic vulnerability of the building. The seismic performance of the structures are provided in terms of fragility curves for the two principal directions of the building and for different damage states defined according to the European Macroseismic Scale

Attivit\ue0\ua0 \u3b2-glucosidasica di lieviti isolati da vinacce per la produzione di Grappa

Microbial glycosidase activity contributes to the aroma of wines and distillates through the hydrolysis of flavourless glycosilated compounds, present in grape skins, that release free terpenols and other aromatic compounds. Grappa, a typical Italian alcoholic product, is obtained from the distillation of grape marcs, that are mainly composed of grape skins, after a variable period of storage. The present work was aimed at analysing the \u3b2-glucosidase activity of yeasts isolated during the insilage period from grape marcs with different characteristics, such as origin (Moscato or Prosecco grapes), or technological treatments carried out during this period. \u3b2-glucosidase activity was detected using a fluorimetric method based on the emission of a fluorescent signal from a molecule containing a glycoside bond. Assays were conducted reproducing typical conditions of the oenological environment, and this method allowed us to rapidly and precisely quantify the enzymatic activity, so that a large number of yeasts could be examined. L\u2019attivit\ue0 glucosidasica dei microrganismi contribuisce all\u2019aroma dei vini e dei distillati attraverso l\u2019idrolisi dei composti glicoconiugati inodori, concentrati negli acini, che rilasciano terpenoli liberi ed altri composti aromatici. La Grappa, tipica bevanda alcolica italiana, si ottiene dalla distillazione della vinaccia, composta principalmente dalle bucce d\u2019uva, dopo un periodo di stoccaggio variabile. Questo lavoro ha avuto lo scopo di analizzare l\u2019attivit\ue0 \u3b2-glucosidasica di ceppi di lievito isolati da vinacce di diversa origine (Moscato e Prosecco) o sottoposte a trattamenti tecnologici. L\u2019attivit\ue0 \u3b2-glucosidasica \ue8 stata saggiata utilizzando un metodo, appositamente messo a punto, basato sull\u2019emissione di un segnale fluorescente da parte di una molecola contenente un legame glicosidico. I saggi sono stati condotti riproducendo le condizioni tipiche dell\u2019ambiente enologico e hanno permesso di quantificare rapidamente l\u2019attivit\ue0 enzimatica di un numero elevato di lieviti