The wine spoilage yeast species Dekkera bruxellensis, after inoculation in red wines, displayed three survival patterns characterized by: i) initial lag phase followed by growth and sequential death; ii) initial death phase leading to reduced viable counts followed by growth and sequential death; and iii) death phase leading to complete loss of viability. These survival patterns were observed for the same strain in different dry red wine blends with 12% (v/v) ethanol and pH 3.50, in the absence of free sulphur dioxide. For the same wine blend, these patterns also varied with the tested strain. Under laboratory conditions the addition of 150 mg/l of potassium metabisulphite (PMB) to dry red wine with 12% (v/v) ethanol and pH 3.50 reduced initial cell counts by more than 6 logarithmic cycles, inducing full death within less than 24 h. Winery trials showed that D. bruxellensis blooms were only prevented in the presence of about 40 mg/l of free sulphur dioxide in dry red wine, with 13.8% (v/v) ethanol and pH 3.42, matured in oak barrels. These different amounts of PMB and sulphur dioxide corresponded to about 1 mg/l of molecular sulphur dioxide. Our results therefore demonstrate that the control of populations of D. bruxellensis growing in red wine can only be achieved under the presence of relatively high doses of molecular sulphur dioxide.
In this work, we studied the ecological interactions between grape berry microorganisms and Drosophila sp. flies involved in sour rot disease during grape ripening. After veráison the total microbial counts of grape berries affected by sour rot increased from about 2 log CFU/g of berries to more than 7 log CFU/g. Berry damage provoked a clear shift in yeast diversity from basidiomycetes to ascomycetous fermentative species. The latter were mostly Pichia terricola, Hanseniaspora uvarum, Candida zemplinina, and Zygoascus hellenicus. However, these species were not able to produce the metabolites characteristic of sour rot (gluconic and acetic acids) in inoculated berries. On the contrary, the acetic acid bacteria Gluconacetobacter saccharivorans produced high levels of these acids, mainly when berries were incubated in the presence of the insect Drosophila sp. Sour rot was not observed when grape bunches were physically separated from insects, even when berries were artificially injured. The wounds made in berry skin healed in the absence of insects, thus preventing the development of sour rot. Therefore, in the vineyard, the induction of sour rot depends on the contamination of wounded berries by a microbial consortium--yeasts and acetic acid bacteria--transported by drosophilid insects which disseminate sour rot among damaged berries. In the absence of these insects, plant defense mechanisms are effective and lead to skin healing, preventing disease spread. Thus, we showed that Drosophila sp. act as a vector for microorganisms associated with grape sour rot disease.
A total of 63 strains of Dekkera bruxellensis and 32 strains of Pichia guilliermondii isolated from wine related environments were identified by restriction analysis of the 5.8S-ITS region of the rDNA. These strains were subjected to intraspecific discrimination using mtDNA restriction and RAPD-PCR analysis. The isolates identified as D. bruxellensis yielded 3 different molecular patterns of mtDNA restriction using the endonuclease HinfI. The pattern A was the most frequent (58 strains) among strains from different sources, regions and countries. Pattern B (4 strains) and C (one strain) were determined in isolates from Portuguese wines. The discrimination among the pattern A strains was achieved by a RAPD-PCR assay with 3 primers (OPA-2, OPA-3 and OPA-9). A total of 12 haplotypes were obtained with the combination of the patterns provided by the 3 OPAs. The pattern 2 was the most frequent and extensively distributed being found in strains from different countries and from different sources like wine, barrique wood and insects. The strains of P. guilliermondii were characterized with restriction of mtDNA using the endonuclease HinfI yielding 7 different restriction patterns. These patterns were associated with different efficiencies of 4-ethylphenol production. Patterns A to D corresponded to 19 strains producing low levels of 4-ethylphenol (<1 mg/l) while patterns F and G grouped 13 strains producing high levels of 4-ethylphenol (>50 mg/l), when grown in synthetic media supplemented with 100 mg/l of p-coumaric acid. The high degree of polymorphism observed shows that intraspecific typing is essential for accurate yeast dissemination studies in wine related environments.
Several microbial species associated with wine were challenged against increasing concentrations of dimethyl dicarbonate (DMDC). The concentration inducing complete cell death upon addition to red wine was regarded as the minimum inhibitory concentration (MIC). In dry red wines with 12% (v/v) ethanol and pH 3.50, the inactivation depended on the initial cell concentration. For an initial inoculum of 500 CFU/ml, the MIC of the yeasts species Schizosaccharomyces pombe, Dekkera bruxellensis, Saccharomyces cerevisiae and Pichia guilliermondii was 100mg/l. The most sensitive strains belong to Zygosaccharomyces bailii, Zygoascus hellenicus and Lachancea thermotolerans, with MIC of 25mg/l DMDC. For inoculation rates of about 10(6)CFU/ml, the maximum dose of DMDC legally authorized (200mg/l) was not effective against the most resistant species. The addition of 100mg/l potassium metabisulphite (PMB), equivalent to 1mg/l molecular sulphur dioxide, increased the inactivation effect of 100mg/l DMDC over initial yeast populations of 10(6)CFU/ml but did not fully kill S. pombe and S. cerevisiae. Lactic acid and acetic acid bacteria were not killed by the addition of 300 mg/l of DMDC. Trials performed in wines before bottling showed that in most samples indigenous bacterial populations were not affected by 200mg/l DMDC. Therefore, under winery practice, DMDC at the maximum dose legally permitted may be regarded as an efficient preservative to control low contamination rates of yeasts but ineffective against lactic acid and acetic acid bacteria.
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