182 M.V. Moreno-Arribas and M.C. Polo
However, some false negatives have been reported for lactic acid bacteria as a
consequence of excess acid production or the generation of other alkaline com-
pounds (Joosten and Northolt 1989) and also in some cases false-positives (Moreno-
Arribas et al. 2003). Some loss of activity by these bacteria has also been described
during successive steps and prolonged storage times (Izquierdo-Pulido et al. 1997),
which could be disadvantageous when applying these methods in microbiological
analysis.
6A.3.4.5 Thin-Layer Chromatography
Thin-Layer Chromatography (TLC) is a rapid and simple technique to detect several
amines. This was one of the first techniques used to determine biogenic amines in
foods (H ́alasz et al. 1994). Recently, a simple and rapid qualitative TLC method to
determine the ability of bacteria to produce biogenic amines in liquid culture media
containing the amino acid precursor was described by Garc ́ıa-Moruno et al. (2005).
6A.3.4.6 Polymerase Chain Reaction Methods
The molecular biology methods described to detect biogenic amine-producing bac-
teria are based on the polymerase chain reaction or PCR technique. With PCR,
multiple copies of a specific DNA sequence (target) can be obtained in vitro. PCR
techniques have been developed to detect bacterial amino acid decarboxylases in
a rapid, sensitive and accurate way. They cannot determine quantitative (or quali-
tative) amounts of biogenic amines, but can only be used to estimate the potential
risk of amine formation. Molecular methods for the detection of biogenic amine-
producing bacteria have been reviewed recently by Landete et al. (2007a).
Since some DNA sequences of selected genes are highly conserved, PCR can
be applied to detect specific genes in different organisms. Some conserved gene
regions that code for histidine decarboxylase have been detected in different bacte-
ria, such asLactobacillus30a,Clostridium perfringes, Lactobacillus buchneriand
Micrococcussp., and this has been used to design the oligonucleotides CL1, CL2,
JV16HC and JV17HC (Le Jeune et al. 1995). By using these oligonucleotides
and the PCR technique, lactic acid bacteria strains with the histidine decarboxy-
lase gene can be used, therefore resulting in histamine production (Torres Alves
and Teia dos Santos 2002). Another way to detect these strains is by hybridisa-
tion with DNA primers amplified with these oligonucleotides (Coton et al. 1998,
Le Jeune et al. 1995). To sequence the gene that codes tyrosine decarboxylase of
Lactobacillus brevisIOEB 9809 oligonucleotides P1 and P2 were designed from
peptides obtained from the protein (Lucas and Lonvaud-Funel 2002). By a simi-
lar approach, universal primers were designed for the early detection of potential
tyramine-producing bacteria in wine (Coton et al. 2004). Marcobal et al. (2005a)
designed the first complete set of primers to amplify the gene coding for ornithine
decarboxylase by aligning amino acid sequences of ornithine decarboxylases from
Gram-negative bacteria.