2 Biochemical Transformations Produced by Malolactic Fermentation 35
sequence is inserted into the databases available online, and by the similarity
obtained with other sequences it is possible to identify the unknown bacterium.
The sequence analysis method is very good to identify the organisms at genus
and species level but it does not differentiate at the subspecies level.
2.3.2.2 G+C Content and DNA Hybridization
Estimation of the DNA nitrous base ratio (or G+C molar percent) is a classical
genotypic method, constituting an integral part of a standard description of a bacte-
rial taxon (Botina et al. 2006). These values vary from 24% to 76% among various
bacteria (Schleifer and Kilpper-Balz 1987). It has been demonstrated, with high
statistical significance, that among strains of a single species, the variation in the
G+C ratio does not exceed 3%, compared with 10% in congeneric bacteria. LAB
have a low (less than 50%) content of G+C pairs. In particular,Oenococcushas
38–44%;Pediococcushas 34–42% andLactobacillushas 36–47%. This method
does not allow the discrimination of species with similar GC values.
DNA-DNA hybridization is a method that provides more resolution than 16S
rDNA sequencing, and has been used to describe bacterial species (Wayne et al.
1987). The 16S-23S rRNA spacer region has been suggested as a suitable region of
the bacterial genome from which to derive useful taxonomic information, particu-
larly with regard to identification at the species level (Whiley et al. 1995) and probes
have been synthesized on its sequencesto characterize bacterial species.
Lonvaud-Funel et al. (1989, 1991a) described the identification of LAB during
vinification and wine storage by DNA-DNA hybridization. Genomic DNA of the
strain to identify was hybridized with total genomic DNA probes extracted from
reference strains. They found that this method was particularly efficient when used
in colony hybridization to study mixed populations: at least five different species
can be detected in a mixture with this system (Lonvaud-Funel et al. 1991b).
In spite of these values, the method is not popular. Major disadvantages include
the laborious nature of pairwise cross-hybridizations and the impossibility of estab-
lishing a central database. Another disadvantage of the method is its high sensi-
tivity to physiological parameters. Moreover, the data on DNA homology obtained
in different laboratories are often discordant because of using different technical
approaches or not complying with standard experimental conditions.
2.3.2.3 PCR-Based Methods
RAPD: This technique has been described as a useful technique for both identi-
fication and typing (Cocconcelli et al. 1995; Nigatu et al. 2001; Du Plessis and
Dicks 1995; Sohier et al.1999). Although variability has been observed in RAPD
fingerprints, reproducibility can be achieved under carefully controlled conditions.
The main advantage of the proposed system lies in the fact that, once a high repro-
ducibility is reached, the method is fast, practical, easy to perform and inexpensive
(Rossetti and Giraffa 2005). Figure 2.3 shows an example of RAPD analysis of
different LAB with primer M13 (Rossetti and Giraffa 2005).