Identification and characterization of non-saccharomyces spoilage yeasts isolated from Brazilian wines

The industry of fine wines and also locally consumed table wines is emerging in Brazil with an increasing volume and economic impact. Enologists in this region currently lack information about the prevalence and characteristics of spoilage yeasts, which may contaminate and potentially undervalue Brazilian wines. Herein, we analyzed 50 local red wines including 27 fine wines (V. vinifera) and 23 table wines (V. labrusca). Presumptive spoilage yeasts were isolated on differential medium, and classified by RFLP-PCR and sequencing of the ITS1-5.8S-ITS2 and D1/D2 26S rDNA loci. The prevalence of spoilage yeasts in fine wines (11 %) was comparable to that reported in European and US wines, and significantly lower than that observed for local table wines (70 %). The majority of isolates belonged to Brettanomyces bruxelliensis, followed by Pichia guillermondii, and more rarely Candida wickerhamii and Trigonopsis cantarelli. The Brettanomyces isolates varied greatly in off-flavor production, displayed ethanol tolerance (>10 % by volume), tolerated sulfite (≥0.68 mg/l mSO₂), and 39 % of them grew on ethanol as sole carbon source. We discuss the causes and consequences of spoilage yeasts in relation to the Brazilian wine industry.     

Variation in phenolic compounds,anthocyanins, and color in red wine treated with enzymatic extract of Kluyveromyces marxianus

The effect of the addition of enzymatic extract of Kluyveromyces marxianus NRRL-Y-7571 during the maceration and fermentation steps of Cabernet Sauvignon wine production was evaluated. The results obtained in the analytical determinations of the wines showed levels within the limits established by legislation and similar to values found in other studies. The results show that by adding the enzyme to the red wines these showed color characteristics considered to be superior to those of the control wine and accelerated the extraction of phenolic compounds and anthocyanins. It was observed that by using the commercial enzyme preparation there was an increase of 15 % in polyphenol content compared to the control wine and an increase of 28 % when the crude enzyme extract was used. Anthocyanin content in the wine increased after treatment with the commercial enzyme preparation (10 %) and with the use of the crude enzymatic extract (22 %). Considering all comparison criteria, the K. marxianus enzymatic extract showed results statistically similar or superior to those obtained with the commercial enzyme preparation.     

Aeromonas detection and characterization using genus-specific PCR and single-strand conformation polymorphism (SSCP)

Based on sequence alignment, oligonucleotide primers targeting the Aeromonas extracellular lipase gene were developed for PCR detection of member of the genus. A pair of primers designed for conserved regions of the gene amplified a 276?bp sequence in all Aeromonas species and tested strains, but did not have a positive result with other Gram-positive and Gram-negative bacteria, showing high specificity and sensitivity. Selective enrichment in alkaline peptone water, followed by centrifugation, and direct usage of cells suspension as template, detected initial populations of 10?c.f.u.?ml(-1). Single-strand conformation polymorphism analysis of the PCR products allowed the characterization of Aeromonas strains with a high discriminatory power (Simpson's index?=?0.988). The method presented here provides a useful tool for the rapid detection of Aeromonas and the characterization of Aeromonas isolates.     

Diversity of extracellular proteases among Aeromonas determined by zymogram analysis

Aims: The current research was aimed at comparing extracellular proteolytic activities and zymogram profiles among Aeromonas spp. Methods and Results: Extracellular proteases of 47 strains of Aeromonas were analyzed by substrate (casein and gelatin) co‐polymerized SDS‐PAGE, and caseinolytic activity was determined using azocasein. Large variation on caseinolytic activity was evidenced. In general, the caseinolytic activity of Aeromonas hydrophila strains was significantly higher than that of other Aeromonas species. Several caseinolytic and gelatinolytic profiles were detected in Aeromonas. Cluster analysis allowed separating Aeromonas strains in four and three groups, based on their caseinolytic and gelatinolytic profiles, respectively. Although not specific patterns were evident, most Aer. hydrophila strains were clustered together and differed from Aeromonas caviae strains. The main caseinases of Aer. hydrophila were a serine protease with an apparent molecular weight (AMW) of 56 kDa and a metalloprotease with AMW of 22 kDa. Gelatinase profiles were characterized by the presence of high molecular weight metalloproteases (84 and 93 kDa), although the most active enzyme was a serine protease with AMW of 56 kDa. Other new caseinases and gelatinases were detected in specific Aeromonas strains. Conclusions: Aeromonas strains exhibited several extracellular proteolytic profiles, with a larger inter than intraspecific variation. Moreover, zymogram analyses allowed identifying new caseinases and gelatinases in Aeromonas. Significance and Impact of the Study: This is the first report on the intra‐ and interspecific variation of proteolytic profiles in Aeromonas determined by zymogram analysis, including the detection of new caseinases and gelatinases in this genus.