3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Food Chemistry & Biotechnology
P101 IDENTIFICATION OF VIAbLE
lACTObACillus CELLS IN FERMENTED
DIARy PRODuCTS
ŠTěPánKA TRACHTOVá, ALEnA ŠPAnOVá and
BOHUSLAV RITTICH
Brno University of Technology, Faculty of Chemistry, Department
of Food Chemistry and Biotechnology
Purkyňova 118, 612 00 Brno
trachtova@fch.vutbr.cz
Introduction
Lactobacillus and Bifidobacterium species are commonly
found in foods and are members of the gastrointestinal
tract of humans and animals. They are the most commonly
used group of lactic acid bacteria (LAB) in the production of
human probiotics. Methods for qualitative and quantitative
control of probiotic products are required due to the growing
interest in their commercial exploitation. Differentiation of
viable and non-viable cells of LAB from a product is still
problematic. Culture-dependent enumeration is relatively
time-consuming and the results may be influenced by poor
viability or low densities of the target organisms 1 . Rapid and
reliable methods are needed for routine determination of initial
cell counts in the inoculum or for viable cell estimation
during the time period of storage. Therefore, culture-independent
analysis as an alternative and/or complementary method
for quality control measurements of probiotic products was
developed. The combination of PCR and the ethidium monoazide
(EMA-PCR) dye is a new method for selective distinction
between viable and dead bacterial cells 2-4 . The general
application of EMA is based on EMA penetration into dead
cells with compromised cell-membrane (cell-wall) integrity.
EMA is covalently linked to DnA by photoactivation and this
DnA cannot be amplified in PCR. Viable cells remain intact
and only DnA from these cells can be amplified and gives a
PCR product.
The aim of this work was to optimise and use EMA-
PCR for distinction between viable and dead Lactobacillus
cells in real samples of dairy products (yoghurts).
Materials and Methods
C h e m i c a l s a n d E q u i p m e n t
The primers for PCR were synthesised by Generi-
Biotech (Hradec Králové, Czech Republic); TaqI DnA
polymerase was from Bio-Tech (Prague, Czech Republic);
DnA ladder 100 bp (Malamité, Moravské Prusy), and EMA
from Sigma (St. Louis, USA). The Lactobacillus paracasei
CCDM strain (obtained from the Czech Collection of Dairy
Microorganisms, CCDM, Tábor, Czech Republic) was used
for DnA isolation. The dairy products (white yoghurts before
and after expiration date) were obtained from the market. The
PCR reaction mixture was amplified on an MJ Research Programme
Cycler PTC-100 (Watertown, USA).
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M e t h o d s
Bacterial cells of Lactobacillus paracasei were cultivated
at 37 °C aerobically in liquid MRS medium for 24 h
or on MRS agar up to 48 hours, respectively. Altogether 1 ml
of the cells was washed by water and resuspended in 500 μl
lysis buffer (10 mM Tris-HCl, pH 7.8, 5 mM EDTA, pH 8.0,
lysozyme 3 mg ml –1 ), and incubated at laboratory temperature
for 1 h; 10 μl proteinase K (1 µg ml –1 ) and 2.5 μl SDS
(20 %) (end concentration 0.5 %) was added and the mixture
was incubated at 55 ºC for 18 h. DnA was extracted from
crude cell lysates with phenol 5 and dissolved in TE buffer
(10 mM Tris-HCl, pH 7.8; 1 mM EDTA, pH 8.0). The concentration
of DnA was estimated spectrophotometrically and
DnA was dissolved to a concentration of 10 µg ml –1 .
Yoghurt samples (Klasik white yoghurt OLMA Olomouc
from the trade network before and after expiration date, 1 g)
was resuspended in 1 ml sterile water. The cells were washed
twice with sterile water and treated with EMA (0.1 mg ml –1 )
for 10 min at laboratory temperature. Photoactivation was
performed using light exposure (halogen lamp, 500 W) for
5 minutes on ice. Then the cells were washed with 1 ml of
water to remove EMA from the sample. The cells without
EMA treatment were used as control. Afterwards the cells
were lysed by boiling (10 min) and crude cell lysates (5 μl)
were used as DnA matrix in EMA-PCR.
PCR was performed with LBLMA 1 and R16 primers
specific to the Lactobacillus genus 6 , which enabled the amplification
of a 250 bp long amplicon. Briefly: the PCR mixture
contained 0.5 μl of each 10 mM dnTP, 0.5 μl (10 pmol μl –1 )
of each primer, 0.5 μl of Taq 1.1 polymerase (1 U μl –1 ), 2.5 μl
of buffer (1.5 mM), 1–5 μl of DnA matrix, and PCR water
was added up to a volume of 25 μl. The amplification reactions
were carried out using the following cycle parameters:
5 min of the initial denaturation period at 94 °C (hot start),
60 s of denaturation at 94 °C, 60 s of primer annealing at
55 °C, and 60 s of extension at 72 °C. The final polymerisation
step was prolonged to 10 min, the number of cycles
was 30. The EMA-PCR products (250 bp) were detected
using agarose gel electrophoresis (1.8 %) in 0.5 × TBE buffer
(45 mM boric acid, 45 mM Tris-base, 1 mM EDTA, pH 8.0).
The DnA was stained with ethidium bromide (0.5 μg ml –1 ),
observed on a UV transilluminator (305 nm), and photographed
on a TT667 film using a Polaroid CD34 camera.
Results and Discussion
The ability of EMA to covalently bind to DnA and to
inhibit PCR was confirmed using purified DnA isolated from
Lactobacillus paracasei cells. As a result of EMA activity,
DnA treated with EMA was not amplified in PCR in contrast
to DnA without EMA treatment. The results are shown in
Fig. 1. The method developed was applied for the discrimination
of viable and dead Lactobacillus cells from dairy products
(yoghurt). The results are shown in Fig. 2. and Table I.
non-expired or shortly expired (12 days) yoghurts contained
both dead and viable Lactobacillus cells because intensities
of EMA-PCR products amplified from EMA treated and