Core Microbiota and Metabolome of Vitis vinifera L. cv. Corvina Grapes and Musts.

The composition and changes of the fungal population and of the metabolites present in grapes and in ferments of Vitis vinifera L. cv. Corvina, one of the major components of the Amarone musts, were dissected aiming at the identification of constant characteristics possibly influenced by the productive process. The fungal populations and metabolomic profiles were analyzed in three different vintages. 454-pyrosequencing on the ribosomal ITS1 region has been used to identify the fungal population present in Corvina grapes and fresh must. Samples were also subjected to metabolomics analysis measuring both free volatile compounds and glycosylated aroma precursors through an untargeted approach with comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry. Albeit strongly dependent on the climate, both the mycobiota and metabolome of Corvina grapes and fresh musts show some characteristics recursive in different vintages. Such persistent characteristics are likely determined by the method adopted to produce Amarone or other dry wines made from partially dried grapes. In particular, the harsh conditions imposed by the prolonged withering appear to contribute to the shaping of the fungal populations. The fungal genera and metabolites present in different vintages in V. vinifera L. cv. Corvina grapes and fresh musts represent core components of the peculiar technique of production of Amarone. Their identification allows the in-depth understanding and improved control of the process of production of this economically and culturally relevant wine

Effects of different stages of lactation on the raw milk for Fontina cheese manufacturing: chemical composition and microbiota analysis by 454 –pyrosequencing

In order to investigate the effect of lactation stage on the microbial quality of milk for fontina Fontina cheese production, nine cheese-making days were followed in two cheese factories over a four months period: three in the first 40 days after partum (early lactation stage S1), three in the following 40 days in the middle lactation (stage S2) and three in the last 40 days when cows were pregnant (stage S3). After plating, and basic chemical analysis, total microbial DNA was isolated from milk and used as template in Polymerase Chain Reaction (PCR) to study the hypervariable V1, V2 and V3 regions of the bacterial 16S rRNA gene and analysed by 454-pyrosequencing. A total of 683,128 sequence reads were generated by the pyrosequencing of 18 milk samples.An average of 498 OTUs were identified.The use of classical microbial approach and high-throughput sequencing allowed not only the description of the bacterial community but also to find difference for lactation stages. The milk samples collected at first lactation stage were characterised by higher counts of coliforms and enterococci and higher amounts of Enterococcus genus while the milk samples collected at later stage of lactation were characterised by higher amount of protein. Some recurrent species could be found at all times of sampling: Staphylococcus, Lactococcus and Streptococcus across Firmicutes and Mesorhizobium and Ralstonia among Proteobacteria phylum. This is the first study where the lactation stage is clearly linked to milk microbiot

Microbiota delle scalere coinvolto nella colorazione arancione-rossa del formaggio Fontina: investigazione attraverso 454-pyrosequencing

La Fontina DOP è un formaggio prodotto da latte intero crudo tramite l’aggiunta di ceppi autoctoni starter quali: Streptococcus thermophilus, Lactococcus lactis e Lactobacillus delbrueckii. Durante la maturazione può presentarsi un difetto di colorazione in particolare nella zona del sotto-crosta dove il colore della pasta del formaggio può virare da giallo ad arancione -rosso. Questa colorazione può essere generata da pigmenti prodotti da batteri presenti nel formaggio oppure nel magazzino e sulle scalere di stagionatura. In questo lavoro si è pensato di caratterizzare le specie microbiche sia dei formaggi che delle scalere su cui erano posizionati, per notare qualche correlazione tra colorazione anomala e specie batteriche e di lieviti. La caratterizzazione è stata fatta tramite 454-pyrosequencing della regione V1-V3 del gene 16S rRNA per i batteri e della regione ITS1-ITS4 per i lieviti (la caratterizzazione dei lieviti è stata fatta solo per le scalere e non per i formaggi dove i lieviti erano presenti in quantità non apprezzabili rispetto ai batteri). Sono state analizzate le comunità microbiche di 8 formaggi a colorazione anomala e 8 a colorazione “normale” dopo 3 mesi di stagionatura (il difetto di solito comincia a manifestarsi dopo almeno 1 mese di stagionatura). I formaggi sono stati campionati sia nel sotto-crosta che nel centro. Le scalere in legno di abete rosso sono state campionate in una sezione di circa 250mm2 sulla superficie a contatto con i formaggi. Sono state analizzate 570247 sequenze di batteri divise in 267754 per i formaggi e 302586 per le scalere. Nei formaggi la maggioranza dei batteri apparteneva al phylum Firmicutes (95%) sia nei formaggi colorati in maniera anomala che non, con una piccola percentuale di Proteobacteria. La comunità batterica isolata dalle scalere ha mostrato una biodiversità più grande infatti erano presenti Actinobacteria (48%), Bacteroidetes (31%) Proteobacteria (14%) e Firmuctes (7% ). Sono stati identificati 319 generi di batteri diversi: gli Streptococcus erano dominanti sia al centro che nel sotto-crosta dei formaggi (75%) e ciò era prevedibile considerando che Streptococcus thermophilus è uno degli starter. Sulle scalere le Flavobacteriaceae e le Corynebacteriaceae erano le famiglie batteriche dominanti: le Flavobacteriaceae non presentavano differenze per colorazione (26%) mentre le Corynebacteriaceae mostravano una significativa differenza e in particolare erano più alte sulle scalere che davano colorazioni anomale (28% sulle scalere che davano colorazione e 20% sulle altre). L’analisi dei lieviti ha rivelato anche in questo caso delle differenze significative ed in particolare le scalere che davano colorazioni anomale presentavano alte percentuali di Galactomyces (33% contro il 13% presente sulle scalere “normali”) e minori di Debariomyces (51% contro 63%) e Tremellomycetes (7% contro 18%). In conclusione si può dire che la composizione microbica del formaggio non ha mostrato delle differenze apprezzabili ma probabilmente perché con un formaggio dominato dallo “starter” è fondamentale analizzare molte più sequenze per avere un’idea del contributo che possono avere anche le popolazioni sub-dominanti quindi sarebbe necessaria un’analisi più approfondita. Per le scalere si può affermare che laddove si verifichino colorazioni anomale” c’è una significativa dominanza di Corynebacteriaceae e Galactomyces rispetto alle altre scalere dove invece dominano Debariomyces e Tremellomycetes. Sono da indagare possibili correlazioni di questi generi batterici e di lieviti nelle colorazioni anomale

Development of reliable analytical tools for evaluating the influence of reductive winemaking on the quality of Lugana wines

This paper presents methods for the definition of important analytical tools, such as the development of sensitive and rapid methods for analysing reduced and oxidised glutathione (GSH and GSSG), hydroxycinnamic acids (HCA), bound thiols (GSH-3MH and Cys-3MH) and free thiols (3MH and 3MHA), and their first application to evaluate the effect of reductive winemaking on the composition of Lugana juices and wines. Lugana is a traditional white wine from the Lake Garda region (Italy), produced using a local grape variety, Trebbiano di Lugana. An innovative winemaking procedure based on preliminary cooling of grape berries followed by crushing in an inert environment was implemented and explored on a winery scale. The effects of these procedures on hydroxycinnamic acids, GSH, GSSG, free and bound thiols and flavanols content were investigated. The juices and wines produced using different protocols were examined. Moreover, wines aged in tanks for 1, 2 and 3 months were analysed. The high level of GSH found in Lugana grapes, which can act as a natural antioxidant and be preserved in must and young wines, thus reducing the need of exogenous antioxidants, was particularly interesting. Moreover, it was clear that polyphenol concentrations (hydroxycinnamic acids and catechins) were strongly influenced by winemaking and pressing conditions, which required fine tuning of pressing. Above-threshold levels of 3-mercaptohexan-1-ol (3MH) and 3-mercaptohexyl acetate (3MHA) were found in the wines and changed according to the winemaking procedure applied. Interestingly, the evolution during the first three months also varied depending on the procedure adopted. Organic synthesis of cysteine and glutathione conjugates was carried out and juices and wines were subjected to LC–MS/MS analysis. These two molecules appeared to be strongly affected by the winemaking procedure, but did not show any significant change during the first 3 months of post-bottling ageing. This supports the theory, already proposed in the literature, that there are other synthetic pathways for free thiol formation

Phylogenomics of the world’s otters

Comparative whole-genome analyses hold great power to illuminate commonalities and differences in the evolution of related species that share similar ecologies. The mustelid subfamily Lutrinae includes 13 currently recognized extant species of otters,1–5 a semiaquatic group whose evolutionary history is incompletely understood. We assembled a dataset comprising 24 genomes from all living otter species, 14 of which were newly sequenced. We used this dataset to infer phylogenetic relationships and divergence times, to characterize patterns of genome-wide genealogical discordance, and to investigate demographic history and current genomic diversity. We found that genera Lutra, Aonyx, Amblonyx, and Lutrogale form a coherent clade that should be synonymized under Lutra, simplifying the taxonomic structure of the subfamily. The poorly known tropical African Aonyx congicus and themore widespread Aonyx capensis were found to be reciprocallymonophyletic (having diverged 440,000 years ago), supporting the validity of the former as a distinct species. We observed variable changes in effective population sizes over time among otters within and among continents, although several species showed similar trends of expansions and declines during the last 100,000 years. This has led to different levels of genomic diversity assessed by overall heterozygosity, genome-wide SNV density, and run of homozygosity burden. Interestingly, there were cases in which diversity metrics were consistent with the current threat status (mostly based on census size), highlighting the potential of genomic data for conservation assessment. Overall, our results shed light on otter evolutionary history and provide a framework for further in-depth comparative genomic studies targeting this grou