3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Food Chemistry & Biotechnology
P95 IDENTIFICATION OF bACTERIAL STRAINS
OF lACTOCOCCus lACTis SPECIES IN hARD
ChEESES uSING PCR
ALEnA ŠPAnOVá, JITKA HERZOGOVá, PETR
PTáČEK 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
Lactococci are the most prominent group of lactic acid
bacteria applied in dairy fermentations. Strains of L. lactis
ssp. lactis have been used in the manufacture of different
types of cheese. Moreover, some lactococcal strains produce
bacteriocins which show activity against food pathogens.
Differentiation of these strains on the basis of phenotypic
tests is time-consuming and can lead to misclassification.
Due to the wide use in dairy industry and different technological
properties, fast and reliable PCR-based methods were
developed which enable identification of L. lactis 1 and distinction
between the two subspecies lactis and cremoris 2 . Falsenegative
results can occur due to the presence of extracellular
PCR inhibitors in real tested samples of dairy products. 3–5
The problem of pure DnA preparation can be resolved by
means of various isolation and purification methods. Solid
phase systems based on non-selectively adsorbing DnA have
been developed. It was shown that PCR-ready DnA can be
isolated using magnetic microspheres P(HEMA-co-EDMA)
containing carboxyl groups 4,5 in the presence of high concentrations
of PEG 6000 and naCl.
The aim of this work was to develop a method for
PCR-ready DnA isolation from different hard cheese samples.
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 amplification.
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 6 in the Institute of Macromolecular
Chemistry (Academy of Sciences of the Czech Republic)
in Prague. Magnetic particles were separated on a Dynal
MPC-M magnetic particle concentrator (Oslo, norway).
s791
M e t h o d s
The type strain Lactococcus lactis subsp. lactis CCM
1877 T from the Czech Collection of Microorganisms was
used as a control strain. It was cultivated on MRS agar
with 0.5 % of glucose 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 ) was added and incubated at 55 °C overnight.
DnA was extracted using phenol methods 7 , precipitated with
ethanol, and dissolved in TE buffer (10 mM Tris-HCl, 1mM
EDTA, pH 7.8).
The DnA from hard cheese samples was isolated from
crude cell lysates from cheese filtrates by the phenol extraction
procedure 7 (control) and by magnetic microspheres (see
later). Magnetic microspheres P(HEMA-co-EDMA) containing
carboxyl groups (100 µl, 2 mg ml –1 ) 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 PALA4 and PALA14 (targeted
on acm A gene encoding N-acetylmuramidase specific
to Lactococcus lactis, 1131 bp long PCR products) 1 were
used for the identification of Lactococcus lactis species.
The PCR mixture contained 1 µl of each 10mM 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 (1.5mM), 1–3 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
94 °C (hot start), 60 s of denaturation at 94 °C, 60 s of primer
annealing at 45 °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.
Results and Discussion
Pre-PCR processing procedures have been developed
to remove or reduce the effects of PCR inhibitors from hard
cheese samples. Ten different cheese samples were used. The
method of DnA isolation using magnetic microspheres was
evaluated. Different amounts of cheese and different procedures
of their homogenisation were tested at first. The best
results were achieved with cheese samples (1 g of cheese
1.5 ml –1 of sterile water) homogenised in a grinding mortar.
The hard pieces of cheese samples were removed using filtration
through sterile gauze. The fat layer was removed from
the filtrates by pipetting. The cells in the filtrates (1.5 or 3 ml)
were centrifuged (10,000 g 5 min –1 ), washed with 1 ml of ste-