Occurrence and Analysis of Sulfur Compounds in Wine

Sulfur compounds play an important role in the sensory characteristics of wine. These molecules can derive from the grape, in which the non-volatile forms are usually present as glycosylated molecules, the metabolic activities of yeast and bacteria, the chemical reactions taking place during the wine aging and storage, and the environment. The sulfur compounds include molecules positively correlated to the aromatic profile of wine, namely the volatile thiols, and are responsible for certain defects, imparting notes described as cabbage, onion, rotten egg, garlic, sulfur and rubber. Due to the low concentration of these molecules in wine, their high reactivity and the matrix complexity, the analytical methods which enable their detection and quantification represent a challenge. The solid phase microextraction (SPME) technique has been developed for sulfur compounds associated with off-flavors. The analysis of volatile thiols usually requires a derivatization followed by gas chromatography (GC)-MS or UPLC-MS methods. Besides the sulfur-containing aromas, another sulfur compound that deserves mention is the reduced glutathione (GSH) which has been widely studied due to its antioxidant properties. The analysis of GSH has been proposed using a liquid chromatography technique (HPLC or UPLC) coupled with fluorescence, MS and UV detectors

Strain Diversity of Pseudomonas fluorescens Group with Potential Blue Pigment Phenotype Isolated from Dairy Products

The blue discoloration in Mozzarella cheese comes from bacterial spoilage due to contamination with Pseudomonas. Fourteen Pseudomonas fluorescens strains from international collections and 55 new isolates of dominant bacterial populations from spoiled fresh cheese samples were examined to assess genotypic and phenotypic strain diversity. Isolates were identified by 16S rRNA gene sequencing and tested for the production of the blue pigment at various temperatures on Mascarpone agar and in Mozzarella preserving fluid (the salty water in which the cheese is conserved, which becomes enriched by cheese minerals and peptides during storage). Pulsed-field gel electrophoresis analysis after treatment with the endonuclease SpeI separated the isolates into 42 genotypes at a similarity level of 80%. Based on the pulsotype clustering, 12 representative strains producing the blue discoloration were chosen for the multilocus sequence typing targeting the gyrB, glnS, ileS, nuoD, recA, rpoB, and rpoD genes. Four new sequence typing profiles were discovered, and the concatenated sequences of the investigated loci grouped the tested strains into the so-called ''blue branch'' of the P. fluorescens phylogenetic tree, confirming the linkage between pigment production and a specific genomic cluster. Growth temperature affected pigment production; the blue discoloration appeared at 4 and 14°C but not at 30°C. Similarly, the carbon source influenced the phenomenon; the blue phenotype was generated in the presence of glucose but not in the presence of galactose, sodium succinate, sodium citrate, or sodium lactate

Investigating the Effect of Selected Non-Saccharomyces Species on Wine Ecosystem Function and Major Volatiles

Natural alcoholic fermentation is initiated by a diverse population of several non-Saccharomyces yeast species. However, most of the species progressively die off, leaving only a few strongly fermentative species, mainly Saccharomyces cerevisiae. The relative performance of each yeast species is dependent on its fermentation capacity, initial cell density, ecological interactions as well as tolerance to environmental factors. However, the fundamental rules underlying the working of the wine ecosystem are not fully understood. Here we use variation in cell density as a tool to evaluate the impact of individual non-Saccharomyces wine yeast species on fermentation kinetics and population dynamics of a multi-species yeast consortium in synthetic grape juice fermentation. Furthermore, the impact of individual species on aromatic properties of wine was investigated, using Gas Chromatography-Flame Ionization Detector. Fermentation kinetics was affected by the inoculation treatment. The results show that some non-Saccharomyces species support or inhibit the growth of other non-Saccharomyces species in the multi-species consortium. Overall, the fermentation inoculated with a high cell density of Starmerella bacillaris displayed the fastest fermentation kinetics while fermentation inoculated with Hanseniaspora vineae showed the slowest kinetics. The production of major volatiles was strongly affected by the treatments, and the aromatic signature could in some cases be linked to specific non-Saccharomyces species. In particular, Wickerhamomyces anomalus at high cell density contributed to elevated levels of 2-Phenylethan-1-ol whereas Starm. bacillaris at high cell density resulted in the high production of 2-methylpropanoic acid and 3-Hydroxybutanone. The data revealed possible direct and indirect influences of individual non-Saccharomyces species within a complex consortium, on wine chemical composition

Culturable Yeast Diversity of Grape Berries from Vitis vinifera ssp. sylvestris (Gmelin) Hegi

Vitis vinifera L. ssp. sylvestris (Gmelin) Hegi is recognized as the dioecious parental generation of today's cultivars. Climatic change and the arrival of pathogens and pests in Europe led it to be included on the International Union for Conservation of Nature (IUCN) Red List of Threatened Species in 1997. The present work focused on the study of culturable yeast occurrence and diversity of grape berries collected from wild vines. Sampling was performed in 29 locations of Azerbaijan, Georgia, Italy, Romania, and Spain. In total, 3431 yeast colonies were isolated and identified as belonging to 49 species, including Saccharomyces cerevisiae, by 26S rDNA D1/D2 domains and ITS region sequencing. Isolates of S. cerevisiae were also analyzed by SSR-PCR obtaining 185 different genotypes. Classical ecology indices were used to obtain the richness (S), the biodiversity (H'), and the dominance (D) of the species studied. This study highlights the biodiversity potential of natural environments that still represent a fascinating source of solutions to common problems in winemaking

The Role of Yeasts as Biocontrol Agents for Pathogenic Fungi on Postharvest Grapes: A Review

In view of the growing concern about the impact of synthetic fungicides on human health and the environment, several government bodies have decided to ban them. As a result, a great number of studies have been carried out in recent decades with the aim of finding a biological alternative to inhibit the growth of fungal pathogens. In order to avoid the large losses of fruit and vegetables that these pathogens cause every year, the biological alternative's efficacy should be the same as that of a chemical pesticide. In this review, the main studies discussed concern Saccharomyces and non-Saccharomyces yeasts as potential antagonists against phytopathogenic fungi of the genera Penicillium and Aspergillus and the species Botrytis cinerea on table grapes, wine grapes, and raisins

Identifying the Main Drivers in Microbial Diversity for Cabernet Sauvignon Cultivars from Europe to South Africa: Evidence for a Cultivar-Specific Microbial Fingerprint

Microbial diversity in vineyards and in grapes has generated significant scientific interest. From a biotechnological perspective, vineyard and grape biodiversity has been shown to impact soil, vine, and grape health and to determine the fermentation microbiome and the final character of wine. Thus, an understanding of the drivers that are responsible for the differences in vineyard and grape microbiota is required. The impact of soil and climate, as well as of viticultural practices in geographically delimited areas, have been reported. However, the limited scale makes the identification of generally applicable drivers of microbial biodiversity and of specific microbial fingerprints challenging. The comparison and meta-analysis of different datasets is furthermore complicated by differences in sampling and in methodology. Here we present data from a wide-ranging coordinated approach, using standardized sampling and data generation and analysis, involving four countries with different climates and viticultural traditions. The data confirm the existence of a grape core microbial consortium, but also provide evidence for country-specific microbiota and suggest the existence of a cultivar-specific microbial fingerprint for Cabernet Sauvignon grape. This study puts in evidence new insight of the grape microbial community in two continents and the importance of both location and cultivar for the definition of the grape microbiome.The YeSViTE project (FP7-IRSES-2013-GA612441) supported the secondments of J.T. and F.V. to the Stellenbosch University (South Africa) and R.F. to the Agrarian University of Georgia (Georgia), and the grape sampling in Tuscany carried out by D.F. This work was also supported by Winetech grant SU IWBT 16-02

Sustainability of food side streams: a case study of fermented blends made with sour whey and sunflower press cake powder using the back-slopping technique

The exploitation of by-products is a key factor to increase the sustainability of the agri-food chain and fermentation is a simple and eco-friendly process for achieving safe and suitable food materials. In this study, we investigated the possibility to manage a spontaneous fermentation of blends made with different proportions of two food side streams (bovine acid whey and sunflower press cake powder) through the application of a back-slopping technique of the mixed material incubated at 26°C in static conditions. A full-factorial 2-factor 3-level design of experiment was applied to infer the effect of the percent (w/w) of press cake powder in the mixture (20, 25, and 30%) and the rate of back-slopping inoculum (15, 30, and 45%). The pH value, titratable acidity, content of sugars, organic acids, and phenolic acids, enumeration of lactic acid bacteria, yeasts and molds, bacterial contaminants, presumptive Bacillus cereus, and Escherichia coli were measured for each fermentation step at 0, 24, 48, and 72 h. On the same samples, a metataxonomics analysis, targeted on bacterial 16S rRNA gene and fungal ITS region, was performed by using the Illumina MiSeq platform. Acidification of the blends (on average, starting pH = 5.45 ± 011, final pH = 4.61 ± 0.11; starting acidity =13.68 ± 1.02 °SH/50 mL, final acidity = 28.17 ± 2.92°SH/50 mL) and high LAB counts (on average, 9.39 log CFU/g ± 0.25) were observed at the end of each refreshment. In all fermented mixtures, B. cereus, E. coli, and molds counts were lower than the detection limit (<2 log CFU/g), whereas bacterial contaminants, overall spore-formers, were always present (3.74 log CFU/g ± 0.27). After 72 h, the dropping of pH value was maximum, yielding significant differences compared to previous fermentation steps (p < 0.01); particularly, the lowest pH (4.45 ± 0.06) was achieved in the central points of DoE (25% of press cake powder and 30% of back-slopping rate), representing the most suitable condition. Results from both culture-dependent and -independent techniques were consistent; although Lactococcus lactis, continuously deriving from the acid whey, was the main LAB, Pediococcus pentosaceus appeared and, in some cases, became the dominant species. Finally, a long-term trial (about 1 month), using the best condition previously pointed out, was performed with an extension of the incubation time to 84 h for each refreshment. The increase in acidity forced the natural selection toward acid-tolerant microbial strains confirming the former results. Although preliminary, these findings can be useful for developing innovative operations to manage these two relevant side streams implementing the circularity of food resources

Transcriptomics unravels the adaptive molecular mechanisms of Brettanomyces bruxellensis under SO2 stress in wine condition

CITATION: Valdetara, F. et al. 2020. Transcriptomics unravels the adaptive molecular mechanisms of Brettanomyces bruxellensis under SO2 stress in wine condition. Food Microbiology, 90. doi:10.1016/j.fm.2020.103483.The original publication is available at https://www.sciencedirect.com/journal/food-microbiologySulfur dioxide is generally used as an antimicrobial in wine to counteract the activity of spoilage yeasts, including Brettanomyces bruxellensis. However, this chemical does not exert the same effectiveness on different B. bruxellensis yeasts since some strains can proliferate in the final product leading to a negative sensory profile due to 4-ethylguaiacol and 4-ethylphenol. Thus, the capability of deciphering the general molecular mechanisms characterizing this yeast species’ response in presence of SO2 stress could be considered strategic for a better management of SO2 in winemaking. A RNA-Seq approach was used to investigate the gene expression of two strains of B. bruxellensis, AWRI 1499 and CBS 2499 having different genetic backgrounds, when exposed to a SO2 pulse. Results revealed that sulphites affected yeast culturability and metabolism, but not volatile phenol production suggesting that a phenotypical heterogeneity could be involved for the SO2 cell adaptation. The transcriptomics variation in response to SO2 stress confirmed the strain-related response in B. bruxellensis and the GO analysis of common differentially expressed genes showed that the detoxification process carried out by SSU1 gene can be considered as the principal specific adaptive response to counteract the SO2 presence. However, nonspecific mechanisms can be exploited by cells to assist the SO2 tolerance; namely, the metabolisms related to sugar alcohol (polyols) and oxidative stress, and structural compounds.https://www.sciencedirect.com/science/article/pii/S0740002020300721?via%3DihubPublishers versio

Genetic Improvement of Wine Yeasts

In recent years, wine market is undergoing a change due to the ever-growing request to improve the sensory features and nutritional properties of the final product. Most wine production is based on the use of starter cultures consisting of selected strains of Saccharomyces cerevisiae, able to ensure quick and controlled fermentations. However, the reduced number of really different starters can lead to a wine standardization resulting in flattening of taste. Moreover, there is a still growing request of winemaking process innovation of the and, in this sense, yeasts can play a central role. In order to gain innovative characteristics, the research relies on the isolation and selection of new oenological strains of S. cerevisiae and non-Saccharomyces species showing interesting metabolic or technological features, or on the improvement of wine yeasts at genetic level. In the case of the latter approach, examples to obtain both non-Genetically Modified (GM) and GM organisms (GMO) are available in literature. In this chapter we discuss the significant developments of the genetic engineering based on standard homologous integration, the inter and intraspecific hybridization in wine yeasts, the use of random mutagenesis, the foundation of the experimental evolution strategy and we describe the CRISPR/Cas9 genome editing approach that has been revolutionizing the field of biotechnology