Biomedical Engineering Reference
In-Depth Information
Whereas probe- and primer-based identification approaches offer the specificity
and selectivity required to detect and monitor specific LAB in sourdough samples,
they were not designed to offer a complete picture of the predominant LAB species
diversity or to reveal new or unknown LAB species diversity in sourdough ecosys-
tems. In contrast, community fingerprinting methods such as denaturing gradient
gel electrophoresis (DGGE) and temporal temperature gradient gel electrophoresis
(TTGE) do not require prior knowledge of the ecosystem's diversity and are univer-
sally applicable to study the species diversity and dynamics of complex bacterial
communities in food environments [
244
]. The universal use of DGGE fingerprinting
is based on the sequence-dependent separation of a mixture of equally sized PCR
amplicons generated from a common taxonomic marker such as the 16S rRNA
gene. For the design of PCR primers, the V1, V3, and V6-V8 hypervariable regions
of the 16S rRNA gene are most commonly used. Taxonomic information on indi-
vidual members of the sample community can be obtained by band position analysis
provided that an identification database is available, clone library analysis, sequenc-
ing of excised and purified DGGE bands or hybridization using species-specific
probes. Major drawbacks of DGGE fingerprinting include its inability to detect sub-
dominant (i.e., <1%) community members and the fact that a single strain or species
may be represented by multiple bands in the DGGE profile due to heterogeneous
rRNA operons and/or heteroduplex molecules. Either using universal or group-
specific 16S rRNA gene primers, DGGE has been widely applied to inventorize
LAB communities in sourdoughs [
41,
47,
106,
128,
245,
246
] and to investigate the
dynamics, adaptation, and source of predominant sourdough LAB communities [
34,
39,
80,
107,
123,
142,
155,
185
]. Likewise, primers targeting the 26S LSU rDNA
have been used for DGGE fingerprinting analysis of sourdough yeast communities
[
29,
46
]. To maximally cover the microbial species diversity present in a sourdough
ecosystem, a number of DGGE studies have combined the use of 16S and 23S
rRNA gene primers to determine in parallel the predominant LAB and yeast compo-
sition of sourdough samples [
33,
39,
41,
47,
123,
142,
245
] . Compared to DGGE,
TTGE has been used to a much lesser extent for culture-independent analysis of the
sourdough microbiota [
112
]. In many of the cited studies, the sequence heterogene-
ity of the multicopy 16S rRNA gene is mentioned as an important limitation in
DGGE, as this may lead to an overestimation of the LAB species diversity. The
degree of overestimation can be estimated by scoring individual bands by position
analysis with a reference database and/or by band sequencing. Alternatively, single-
copy genes that do not exhibit this heterogeneity such as
rpoB
have been evaluated
for DGGE fingerprinting of LAB species during food fermentations [
247
] .
Microarray technology represents one of the most recent culture-independent
approaches to study the diversity and identify individual members of the sourdough
microbiota. Phylogenetic microarrays, containing partial 16S rRNA gene sequences
as targets, are ideally suited for this purpose but are currently not available for sour-
dough microbiota. Alternatively, a functional gene microarray can be used when the
original annotation information allows one to link the responding oligonucleotides to
the original species. Weckx and co-workers [
108,
109
] used a LAB functional gene
