Cloning and sequencingof rDNA ampli

Cloning and sequencing
of rDNA amplicons revealed that meso-
D. Ercolini / Journal of Microbiological Methods 56 (2004) 297–314 303
philic bacteria, including leuconostocs, L. lactis, and
Macrococcus caseolyticus were dominant in raw milk,
while S. thermophilus prevailed during the fermentation.
Other rod-shaped LAB, especially L. fermentum
and L. delbrueckii, were also found during ripening.
The technique has also been profitably used by
Cocolin et al. (2001c) to monitor the dynamic changes
in bacterial community during the ripening of natural
fermented Italian sausages by using DNA and RNA
directly extracted from the food matrix. The region V1
of the 16S rDNA was taken into account for the
analysis of the meat samples, and PCR and RT-PCR
were used to obtain amplicons to be separated by
DGGE. Lactic acid bacteria were found in the early
stage of the ripening along with other organisms,
mainly members of the family Micrococcaceae and
meat contaminants, such as Brochothrix thermosphacta
and Enterococcus spp. LAB represented the
main population, especially Lactobacillus sake and L.
curvatus, and were responsible for the acidification
and proteolysis that determined the organoleptic features
of the fermented sausages, while Micrococcaceae
were shown to have a restricted importance
compared to LAB.
The microbial community occurring during vanilla
curing in Indonesia was also studied by this approach
(Ro¨ling et al., 2001). The authors found a significant
occurrence of Bacillus isolates and also highlighted
the presence of unculturable microorganisms during
the high-temperature phases of the bean curing.
Drinks may also benefit from this type of investigation
for both fermented and nonfermented beverages.
LAB communities existing during the
fermentation of Malt whisky were monitored by
van Beek and Priest (2002) by PCR-DGGE of V3
and RT-PCR-DGGE of V6–V8 regions of 16 rDNA
coupled with traditional isolation procedure, viable
staining counts, and scanning electron microscopy.
The authors showed that mainly lactobacilli played
an important role in the fermentation and that
homofermentative Lactobacillus acidophilus and L.
crispatus dominated the last phase of the fermentation.
van Beek and Priest (2002) also optimized the
separation of lactobacilli in DGGE by adopting the
V6–V8 region of 16S rDNA as a target, giving
better resolution of several species due to higher
heterogeneity in sequence among species of the
genus Lactobacillus.
Among microbial food processes, dough leavening
by using sourdoughs as starter is often employed,
yielding appreciated bakery products. Recently, sourdough
fermentation performed by different starters
was monitored by using Lactobacillus group-specific
16S rDNA primers in PCR followed by DGGE
analysis (Meroth et al., 2003). Shifts of Lactobacillus
population during the fermentation of each dough
could be determined and the dominant species carrying
out the process up to the end were identified as
Lactobacillus sanfranciscensis, Lactobacillus pontis,
Lactobacillus mindensis, L. crispatus, Lactobacillus
johnsonii, Lactobacillus frumenti, and Lactobacillus
reuteri, depending on the type of starter used.
6. Identification of members of the cultivable
community: an alternative to isolation
The PCR-DGGE can be also used to rapidly check
the diversity of the bacterial community after cultivation
in liquid or solid and specific or nonspecific
culture media. Briefly, after colony counting has been
performed, the colonies from the plates can be collected
in ‘‘bulks’’ and subjected to DNA extraction
and PCR-DGGE analysis (Ercolini et al., 2001b).
Consequently, a DGGE fingerprint can be obtained
for each plate, each dilution, and culture medium.
This method to investigate the cultivable microbial
community has been shown to have good potential in
food microbiology. Firstly, it provides an alternative
to traditional tools for the identification of dominant
species. Qualification of the dominant species could
be achieved by sequencing the DGGE bands arising
from the patterns corresponding to the highest dilutions
in spite of the isolation of single colonies
followed by purification and biochemical identification.
The bulk from countable plates can be thus used
to identify dominant microbial species from the food
matrix as performed by Ercolini et al. (2003a) in
analysing the Stilton cheese. The advantage is that,
while sequencing of partial 16S rDNA fragments
represents a sound tool for identifying species, identification
of species using phenotypic methods such as
sugar fermentation patterns may sometimes be uncertain,
complicated, and time-consuming, particularly
within LAB, due to an increasing number of species
that vary in a few traits (Quere et al., 1997).