Application and evaluation of denaturing gradient gel electrophoresis to analyse the yeast ecology of wine grapes

Abstract

The performance of denaturing gradient gel electrophoresis (DGGE) for analysing yeasts associated with wine grapes was compared with cultural isolation on malt extract agar (MEA). After optimisation of PCR and electrophoretic conditions, the lower limit of yeast detection by PCR-DGGE was 10(2) cfuml(-1), although this value was affected by culture age and the relative populations of the species in mixed culture. In mixed yeast populations, PCR-DGGE detected species present at 10-100-fold less than other species but not when the ratio exceeded 100-fold. Aureobasidium pullulans was the main species isolated from immature, mature, and both damaged and undamaged grapes. It was not detected by PCR-DGGE when present at populations less than 10(3) cfug(-1). When approaching maturity, damaged grapes gave a predominance of Metschnikowia and Hanseniaspora species (10(5)-10(7) cfug(-1)), all detectable using PCR-DGGE. However, various species of Rhodotorula, Rhodosporidium and Cryptococcus were not detected by this method, even when populations were as high as 10(4) cfug(-1). PCR -DGGE was less sensitive than culture on MEA for determining the yeast ecology of grapes and could not reliably detect species present at populations less than 10(4) cfug(-1). However, this method detected a greater diversity of species than agar plating.

Yeast populations associated with Ghanaian cocoa fermentations analysed using denaturing gradient gel electrophoresis (DGGE)

“…species constituting less than 4% of the yeast microflora were not detected by the culture-based method. The lower level of detection of the DGGE method has been reported to be 1% of the yeast microflora (Prakitchaiwattana et al, 2004). Possibly H. guilliermondii was present in numbers below the detection limit of the culture-based method but above the limit of the DGGE method.…”

“…in the intestinal colon (Tannock, 1999;Nielsen et al, 2003) and soil (Muyzer et al, 1993;Muyzer and Smalla, 1998). During recent years the method has also been applied to investigate yeast populations in milk (Cocolin et al, 2002a), sourdough (Meroth et al, 2003;Gatto and Torriani, 2004) and during coffee (Masoud et al, 2004) and wine fermentations (Cocolin et al, 2000(Cocolin et al, , 2001(Cocolin et al, , 2002bHernán-Gómez et al, 2000;Mills et al, 2002;Cocolin and Mills, 2003;Prakitchaiwattana et al, 2004).…”

PCR and DGGE Detection Limits for Wine Spoilage Microbes

“…Plant materials, such as polysaccharides, plant lipids and polyphenols are known to inhibit PCR reactions, which ultimately will also influence the outcome of the DGGE detection limit results (Lodhi et al, 1994). The plant material and inhibitory substances that were extracted during DNA isolation could also have had an influence on the PCR amplification of the DNA template, and could cause a decrease in the sensitivity of this detection method (Prakitchaiwattana et al, 2004). The sensitivity of the primers used in this study differed in terms of PCR and DGGE detection limits.…”

“…Several bacterial and yeast species are present in wine during alcoholic fermentation and MLF (Prakitchaiwattana et al, 2004). Detection limits of the reference microbes inoculated into SSS and white wine therefore were determined as part of a mixed population with the universal and bacteria-specific primers.…”

Directed metabolomic approaches for the characterization and development of new yeast strains

“…It should be noted that some yeast species found in grapes and must, like Aureobasidium pullulans or Cryptococcus amylolentus, are enzymatically interesting showing a wide range and intensity of hydrolytic activities but have a low incidence on fermentation development. A. pullulans dominates the microbial consortia of grape [25,26], however, its null fermentative power and its low adaptation to fermentative environment bias their contribution to the fermentation process, making their interest in winemaking scarce [27]. There is a simple method to minimize the isolation of this yeast-like fungus by keeping the fresh grape must refrigerated overnight in order to greatly reduce the Aureobasidium population.…”