Multiple competitive PCR-DGGE as a tool for quantifying and profiling
defined mixed cultures of lactic acid bacteria during production of
probiotics from complex polysaccharides
J.
Pintado, J.P. Guyot, F. Ampe-2003
Journal
of Applied Microbiology, 95(5):
921-933
Abstract
Aims:
To apply a denaturing gradient gel electrophoretic (DGGE) method to quantify
and profile individual strains during a mixed culture fermentation.
Methods and
Results: DNA was extracted during the culture of lactic acid bacteria
(LAB) and amplified in a multiple competitive PCR (cPCR) using general
primers targeting 16S rDNA and DNA from Streptococcus salivarius as
competitive DNA. Subsequently the 200-kb amplified fragments were separated
by DGGE. The method was validated in pure cultures and used to profile a
mixture of three LAB grown on glucose, soluble starch and glycogen from
mussel processing waste. The inclusion of a starch- and glycogen-degrading
strain (Lactobacillus plantarum) and a weakly amylotic
nisin-resistant strain (Lact. paracasei) allowed proliferation of the
nisin producing Lactococcus lactis which in itself is unable to grow
on complex carbohydrates. cPCR-DGGE permitted the monitoring of a different
strain succession on the different carbohydrates, related to amylolytic
activity and substrate consumption, acid production and nisin production.
Conclusions:
cPCR-DGGE
is a useful tool for profiling defined mixed cultures of bacteria and hence
allows their interaction to be studied.
Significance and Impact of the Study:
Provided
validation of the method for each specific case, it may be appropriate to
monitor and control the reproducibility of any defined mixed culture of
bacteria, with the advantage that an increase in the strain numbers to be
monitored does not yield an increase in the labour of the procedure.
(Mariņas, CSIC, Eduardo Cabello 6, 36208
Vigo, Galicia, Spain, e-mail: pintado@iim.csic.es)