CENTRO DE REFERENCIA PARA LACTOBACILOS
Unidad Ejecutora - UE
Fluorescence in situ hybridization for detection of classical propionibacteria with specific 16S rRNA-targeted probes and its application to enumeration in Gruyere cheese
BABOT J.D.; HIDALGO M.; ARGAÑARAZ MARTINEZ E.; APELLA M.C.; PEREZ CHAIA A.
INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY
ELSEVIER SCIENCE BV
Año: 2011 vol. 145 p. 221 - 221
The classical or dairy propionibacteria have well-documented industrial applications and have been proposed for probiotic applications. Given their industrial importance it is necessary to employ fast and reliable techniques to monitor the growth during products elaboration, industrial fermentations or the intestinal transit. Therefore, the aimof this investigation was to design oligonucleotide probes targeting the 16S rRNA of dairy propionibacteria and optimise the ﬂuorescence in situ hybridization (FISH) protocol to detect these bacteria. Two speciﬁc probes were in silico designed to detect Propionibacterium freudenreichii and P. jensenii, named Pfr435 and Pj446 respectively. The FISH protocol was optimised for the hybridisation of propionibacteria cells with the universal probe Eub338 and the designed probes. These probes were assayed in situ for their speciﬁcity to hybridise species of propionibacteria by observation using ﬂuorescence microscopy and results were compared with the probe Pap446 previously designed for P. acidipropionici. Probes Pap446, Pfr435 and Pj446 were also evaluated by ﬂuorescence spectrophotometry to assess the inﬂuence of cells physiological state during growth in batch culture in the ﬂuorescence intensity. The maximum ﬂuorescence intensity was observed at the onset of the stationary phase of growth and was then reduced. However, changes on the cells permeability did not reduce the efﬁciency of 16S rRNA hybridisation with the ﬂuorescence-labelled probes. Propionibacteria counts obtained by FISH and plate count methods were compared in a commercial Gruyère cheese. The results showed that this method can be used as a rapid technique for the enumeration of these bacteria in cheese samples.