A quantitative, real-time PCR method was developed to
enumerate Lactobacillus plantarum IWBT B 188 during
the malolactic fermentation (MLF) in Grauburgunder
wine. The qRT-PCR was strain-specific, as it was based on
primers targeting a plasmid DNA sequence, or it was L.
plantarum-specific, as it targeted a chromosomally located
plantaricin gene sequence. Two 50 l wine fermentations
were prepared. One was inoculated with 15 g/hl Saccharomyces
cerevisiae, followed by L. plantarum IWBT B 188 at 3.6 ×
106 CFU/ml, whereas the other was not inoculated
(control). Viable cell counts were performed for up to 25
days on MRS agar, and the same cells were enumerated
by qRT-PCR with both the plasmid or chromosomally
encoded gene primers. The L. plantarum strain survived
under the harsh conditions in the wine fermentation at
levels above 105/ml for approx. 10 days, after which cell
numbers decreased to levels of 103 CFU/ml at day 25, and
to below the detection limit after day 25. In the control, no
lactic acid bacteria could be detected throughout the
fermentation, with the exception of two sampling points
where ca. 1 × 102 CFU/ml was detected. The minimum
detection level for quantitative PCR in this study was 1 ×
102 to 1 × 103 CFU/ml. The qRT-PCR results determined
generally overestimated the plate count results by about 1
log unit, probably as a result of the presence of DNA from
dead cells. Overall, qRT-PCR appeared to be well suited
for specifically enumerating Lactobacillus plantarum starter
cultures in the MLF in wine.

Keywords: Lactobacillus plantarum, malolactic fermentation, LAB, wine, starter cultures, qRT-PCR