The classic techniques for determining the viability of probiotics are .doc

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					Simultaneous detection and enumeration of viable lactic acid bacteria and
bifidobacteria in fermented milks by using propidium monoazide and real time

T. García-Cayuela, R. Tabasco, P. Fernández de Palencia*, C. Peláez, T. Requena

Departamento de Ciencia y Tecnología de Productos Lácteos. Instituto del Frío-CSIC.
José Antonio Nováis 10, 28040 Madrid, Spain,

Fermented milk products are the most popular means of delivering probiotic bacteria in
food. The presence of multiple and closely related species in these products makes the
differential enumeration of probiotic and yoghurt starter bacteria difficult due to
similarity in growth requirements and overlapping biochemical profiles of the species.
In this study we have developed a procedure that allows specific detection and
enumeration of viable bacteria in four species of lactic acid bacteria (Streptococcus
thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus casei subsp.
casei, and Lactobacillus acidophilus) and Bifidobacterium lactis, mixed in fermented
milks. The procedure is based on the combined use of propidium monoazide (PMA),
able to distinguish between viable and irreversibly damaged cells, with the speed and
sensitivity of quantitative real-time PCR (RTi-PCR) using specific primers designed
from the 16S rRNA gene for lactic acid bacteria species and the transaldolase gene for
bifidobacteria. The results of molecular enumeration were compared with those
obtained using selective plate count methods. Loss of viability of the species in
fermented milks through incubation at 4 °C was similarly (P<0.05) detected by both
methods. Furthermore, comparison of results obtained by RTi-PCR and plate counts
showed a Pearson linear correlation of 0.995. Main difference between both methods
was that enumeration of viable bacteria by RTi-PCR could be performed in 3 h, whereas
enumeration by plate count methods required 3 days. The procedure developed is a fast
method for the detection, enumeration and discrimination of viability of lactic acid
bacteria and bidifobacteria mixed in fermented milks.

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