Determination of wine microbiota using classical method, polymerase chain method and Step One Real-Time PCR during fermentation process

Abstract

The aim of our study was the identification of grape, must and wine microbiota during the fermentation process using a classical microbiological method and Real-Time PCR. The changes in different groups of microorganisms were monitored in total counts of bacteria, lactobacilli and yeasts. Microbiological parameters were observed during the current collection and processing of grapes in 2009. Samples were taken during the fermentation process in wine enterprises and a private vineyard. During this period 30 samples of wine among Müller Thurgau, Cabernet Sauvignon, Chardonnay, Tramin and Red Bio-wine were examined. Samples were collected from stages of grape-must unfiltered, grape-must filtered, the beginning of fermentation, fermentation, late fermentation and young wine. The highest total counts of bacteria ranged from 0.00 to 176 ± 15 CFU.mL⁻¹ in the wine of Müller Thurgau, the highest number of yeast ranged from 0.00 to 150 ± 9 CFU.mL⁻¹ in the wine of Müller Thurgau and the number of Lactobacillus spp. ranged from 0.00 to 92 ± 5 CFU.mL⁻¹ in the sample of Cabernet Sauvignon wine. The presence and sensitivity of Gram-positive and Gram-negative bacterial species Enterococcus faecium, Lactobacillus acidophilus, Lactobacillus crispatus and Lactobacillus salivarius were detected using Real-Time PCR (RTQ PCR). Susceptibility of Enterococcus faecium varied in different isolates from 1 to 10⁶ CFU.mL⁻¹, the sensitivity of the species Lactobacillus acidophilus in different isolates of the wine samples ranged from 1 to 10⁵ CFU.mL⁻¹. We also monitored representation of species Lactobacillus crispatus, which were captured by RTQ PCR sensitivity and ranged from 1 to 10⁵ CFU.mL⁻¹ _. Identification of the species Lactobacillus salivarius in each of isolates by RTQ PCR method showed the presence of these bacteria in the range of 1 to 10⁴ CFU.mL⁻¹.

Keywords:

• Wine
• bacteria
• yeasts
• plate diluting method
• PCR method
• Real-Time PCR

Acknowledgment

This study was supported by KEGA Cultural and Educational Grant Agency no. 053SPU-4/2011 and 013SPU-4/2012.