3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Food Chemistry & Biotechnology
P96 IDENTIFICATION OF bACTERIAL STRAINS
OF sTrePTOCOCCus THerMOPHilus
SPECIES uSING PCR
ALEnA ŠPAnOVá, MARTInA TYCOVá, ŠTěPánKA
TRACHTOVá and BOHUSLAV RITTICH
Brno University of Technology, Faculty of Chemistry, Department
of Food Chemistry and Biotechnology, Purkyňova
118,612 00 Brno, Czech Republic,
spanova@fch.vutbr.cz
Introduction
Most molecular diagnostic studies of lactic acid bacteria
have been focused on the strains of Lactobacillus and Bifidobacterium
1–4 genera due to their probiotic potential and
commercial exploitation in food industry. Undeservedly less
attention has been devoted to the identification of Streptococcus
thermophilus in the literature. Streptococcus thermophilus
is namely one of the most widely used lactic acid bacteria
in the dairy industry as a starter culture component in the
manufacture of fermented dairy products, such as yoghurt
and cheese, and in probiotic preparations. Identification of
Streptococcus thermophilus is therefore important. Classical
methods based on biochemical characterisation are in many
cases insufficient. For this reason, specific PCR primers
for identification of Streptococcus thermophilus cells were
described in the literature 5,6 .
The aim of this work was to isolate PCR-ready DnA
from yoghurt and milk drink samples containing PCR inhibitors.
Carboxyl-functionalised magnetic poly(2-hydroxyethyl
methacrylate-co-ethylene dimethacrylate) microspheres
(P(HEMA-co-EDMA)) were used for DnA isolation. The
quality of extracted DnA was checked by PCR specific to
Streptococcus thermophilus.
Material and Methods
C h e m i c a l s a n d E q u i p m e n t
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 from Malamité (Moravské Prusy). The PCR reaction
mixture was amplified on an MJ Research Programme Cycler
PTC-100 (Watertown, USA).
Magnetic nonporous poly(2-hydroxyethyl methacrylateco-glycidyl
methacrylate) P(HEMA-co-GMA) microspheres
containing carboxyl groups were prepared according to the
previously described procedure 7 in the Institute of Macromolecular
Chemistry (Academy of Sciences of the Czech
Republic) in Prague. Magnetic microspheres were separated
on a Dynal MPC-M magnetic particle concentrator (Oslo,
norway).
M e t h o d s
The strain Streptococcus thermophilus CCM 4757 from
the Czech Collection of Microorganisms was used as a control
strain. It was cultivated on MRS agar with 0.5 % of glu-
s793
cose at 30 ºC for 48 h. The cells from 1.5 ml culture were
centrifuged (14,000 g 5 min –1 ), washed in water, and resuspended
in 500 µl of lysis buffer (10mM Tris, pH 7.8, 5mM
EDTA pH 8.0, lysozyme 3 mg ml –1 ). After 1 hour, 12.5 µl of
20% SDS and 5 µl of proteinase K (10 μg ml –1 ) were added
and incubated at 55 °C overnight. DnA from these crude
cell lysates was extracted using phenol method 8 , precipitated
with ethanol and dissolved in TE buffer (10mM Tris-HCl,
1mM EDTA, pH 7.8).
Altogether 2 g of yoghurt (milk drink) samples resuspended
in 2 ml water was centrifuged (12,000 g 5 min -1 ) to
sediment the bacteria cells. The cells in the sediments were
washed with 1 ml of sterile water and lysed. DnA from
yoghurt samples was isolated from crude cell lysates by the
phenol extraction procedure (control) (see above) and by
magnetic microspheres (see later). Magnetic microspheres
P(HEMA-co-EDMA) (100 µl, 2 mg ml –1 ) containing carboxyl
groups were added to the crude cell lysates (100 µl) together
with 5M naCl (400 µl), 40% PEG 6000 (200 µl) and
water to a volume of 1,000 µl (200 µl). After 15 minutes of
incubation at laboratory temperature, the microspheres with
bound DnA were separated using magnet, washed in 70%
ethanol, and DnA was eluted into 100 µl of TE buffer.
Species-specific PCR primers were used for the identification
of Streptococcus thermophilus species 9 . The PCR mixture
contained 1 µl of each 10 mM dnTP, 1 µl (10 pmol µl –1 )
of each primer, 1 µl of Taq 1.1 polymerase (1 U µl –1 ), 2.5 µl
of buffer (containing 1.5 mM Mg 2+ ions), 1–5 µl of DnA
matrix, and PCR water was added up to a 25 µl volume. The
amplification reactions were carried out using the following
cycle parameters: 5 min of the initial denaturation period at
95 °C (hot start), 60 s of denaturation at 95 °C, 60 s of primer
annealing at 58 °C, and 60 s of extension at 72 °C. The
final polymerisation step was prolonged to 10 min. PCR
was performed in 30 cycles. The PCR products were separated
and identified using electrophoresis in 1.5% agarose
gel. The DnA on the gel was stained with ethidium bromide
(0.5 μg ml –1 ), observed on a UV transilluminator (305 nm),
and documented on a TT667 film using a CD34 camera.
Results and Discussion
Fermented diary products (8 white and fruit yoghurts
and 2 milk drinks) from the market were used for the analysis.
Pre-PCR processing procedures have been developed to
remove or reduce the effects of PCR inhibitors from tested
samples. The quality of DnA in crude cell lysates of bacteria
from yoghurt (milk drink) samples was proved (see Material
and methods) using agarose gel electrophoresis. DnA
in amounts from approximately 70 to 350 ng was detected.
Then, the method of DnA isolation using magnetic microspheres
was evaluated. The amounts of eluted DnA in all
samples tested were smaller than the detection limit for gel
electrophoresis, but these amounts were sufficient for PCR.
Detection of specific 968 bp long PCR products is given in
Fig. 1. in which PCR product intensities for different amounts
of DnA matrix are compared. PCR products of higher inten-