Advances in Food Mycology

Evaluation of Molecular Methods for the Analysis of Yeasts in Foods 87
Food / Beverage
Extract and purify DNA/RNA
PCR-amplification of yeast rDNA
DGGE/TGGE separation of DNA amplicons
DNA bands
correspond to
different species
Isolate and sequence bands to give species identity
Figure 2. A culture-independent approach for determining the yeast ecology of foods
and beverages using PCR-DGGE/TGGE analyses
ture of urea and formamide), while TGGE uses a gradient of temperature
to denature the strands of DNA amplicons. Usually, each DNA
band found in the gel corresponds to a yeast species. The band is
excised from the gel and sequenced to give the species identity. Thus,
a profile of the species associated with the ecosystem is obtained,
without the need for agar culture. It is believed that this molecular
approach overcomes the bias of culture methods, and reveals species
that might fail to produce colonies on agar media. Consequently, it is
considered that a more accurate representation of the diversity of
yeast species in the food product is obtained (Giraffa, 2004). Table 5
lists a range of studies where PCR-DGGE/TGGE have been applied
to the analysis of food and beverage yeasts.
Generally, there has been good agreement between yeast species
detected in foods and beverages by PCR-DGGE/TGGE and culture
on agar media, but some discrepancies have been noted. In some cases,
yeasts were recovered by DGGE/TGGE analyses but not by culture.
These observations have led to suggestions that viable but nonculturable
yeasts may be present (Mills et al., 2002; Meroth et al.,
2003; Masoud et al., 2004; Prakitchaiwattana et al., 2004; Nielsen
et al., 2005). However, DNA from non-viable yeast cells or DNA
released from autolysed yeast cells could account for these findings,
and caution is needed when interpreting the DGGE/TGGE data. To
address this limitation, Mills et al. (2002) and Cocolin and Mills