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UDC 637.352:577.213.38 scientific note
ISSN 1330-9862
(FTB-1356)
Detection of Adulteration in Italian Mozzarella Cheese Using
Mitochondrial DNA Templates as Biomarkers
Maria Feligini1*, Ivan Bonizzi1, Vlatka Cubric Curik2, Pietro Parma1,
Gian Franco Greppi1and Giuseppe Enne1
1Istituto Sperimentale Italiano »Lazzaro Spallanzani«, Viale Papa Giovanni XXIII 7,
I-26900 Lodi, Italy
2Dairy Science Department, Faculty of Agriculture, University of Zagreb,
Svetosimunska 25, HR-10 000 Zagreb, Croatia
Received: July 23, 2004
Revised version: November 4, 2004
Accepted: February 28, 2005
Summary
Considering the importance of monitoring adulterations of genuine cheeses in the
dairy industry, a polymerase chain reaction–based method was developed to detect bo-
vine-specific mitochondrial DNA sequence in Italian water buffalo Mozzarella cheese.
DNA was isolated from cheese matrix and governing liquid by organic extractions and kit
purifications. Amplifications of a 134-bp fragment were performed with a bovine–specific
set of primers designed on the sequence alignment of bovine and buffalo mitochondrial
cytochrome oxidase subunit I. The specificity of the primers was tested using DNA from
the blood of two species (water buffalo and bovine), which are present together in adulte-
rated Italian Mozzarella cheese. This method reliably detected a content of 0.5 % of bovine
milk, making it suitable for routine fraud monitoring.
Key words: Italian Mozzarella cheese, mitochondrial DNA, adulteration
Introduction
Fraudulent addition of bovine milk during the man-
ufacturing of water buffalo Mozzarella cheese has in-
creased in recent years, due to the growing market de-
mands in EU (European Union). Several analytical
methods have been developed in order to protect con-
sumers and producing countries from fraud originated
by the addition of bovine milk in water buffalo Mozza-
rella cheese. Methods based on protein analysis by chro-
matographic (1), electrophoretic (2,3) and immunologi-
cal (4) techniques have been optimized and recently
bovine milk has also been detected in water buffalo
Mozzarella cheese by mass spectrometry (5). All tech-
niques were based on strategies suited to evaluate the
protein patterns originating from the major whey pro-
teins or casein fraction. All these analytical methods are
able to detect bovine milk proteins in water buffalo Mo-
zzarella cheese to the minimum level of 0.5–1 %.
Identification of animal species using DNA analysis
has become more and more effective to detect adulter-
ations in commercial dairy products (6,7). DNA from so-
matic milk cells, principally represented by leucocytes
(8), persists in cheese and may be analysed for species
discrimination. Several PCR-based techniques (DNA hy-
bridization assay; restriction enzyme analysis, RFLP;
single-stranded conformation polymorphism analysis,
SSCP; duplex polymerase chain reaction, duplex-PCR)
91
M. FELIGINI et al.: Detection of Adulteration in Mozzarella Cheese, Food Technol. Biotechnol. 43 (1) 91–95 (2005)
* Corresponding author; Phone: ++39 0371 351 18; Fax: ++39 0371 437 070; E-mail: maria.feligini@isils.it
were performed to amplify nuclear genome obtained
from milk and ripened cheese (9–11). An adapted DNA
extraction procedure and an estimation of the DNA
quality, carried out by amplifying a large 3622-bp b-ca-
sein sequence (10), confirmed that DNA is not com-
pletely degraded after cheese manufacturing processes.
Specific polymerase chain reaction (PCR) amplify-
ing a fragment of the cytochrome b gene has been de-
veloped for discrimination of species in food ingredi-
ents. DNA targets in the mitochondrial genome (12)
have several advantages over nuclear ones; they are
generally more abundant in any given sample than sin-
gle-copy nuclear genes and contain a greater number of
point mutations which can be used for better definition
of species differences. By using appropriate primer pairs,
mitochondrial sequences were amplified in many spe-
cies (13–18) and the resulting differences were used for
species authentication.
Procedures based on mitochondrial cytochrome b
gene amplification were also described in order to dis-
criminate bovine milk in buffalo cheese. DNA was re-
covered from cheese and analyzed by RFLP-PCR (19)
and duplex PCR (20). These methods currently repre-
sent valid complements to protein electrophoretic and
immunochemical analyses.
On the basis of various studies demonstrating that
DNA is not completely degraded after thermal and en-
zymatic processes involved in Mozzarella cheese pro-
duction, we propose a new strategy for the detection of
low amounts of bovine milk in water buffalo Mozzarella
cheese by means of polymerase chain reaction. Our me-
thod is based on the amplification of a 134-bp stretch of
mitochondrial cytochrome oxidase subunit I gene isola-
ted from cheese matrix and, for the first time, from go-
verning liquid of Mozzarella cheese.
Materials and Methods
Samples
Three experimental lots of Mozzarella cheese were
manufactured mixing water buffalo and bovine milk in
ratios of 70:30, 80:20, 90:10, 95:5, 99:1 and 99.5:0.5. The
cheese and governing liquid samples were preserved at
4–6 °C. Six hours, one week and two weeks after the
cheese-making, each lot was frozen at –20 °C to inter-
rupt enzymatic processes. The lots of Mozzarella cheese
were made in dairy by traditional manufacturing.
Ten Protected Designation of Origin (PDO) water
buffalo Mozzarella cheeses made by several producers
in Campania region were purchased in Italian super-
markets and were subjected to two different DNA ex-
traction techniques.
DNA extraction from governing liquid
DNA was isolated from the mixes (six hours, one
week and two weeks after the cheese-making), commer-
cial samples, bovine and water buffalo blood by a phe-
nol/chloroform method, followed by ethanol precipita-
tion. Aliquots (40 mL) of governing liquid were centri-
fuged for 30 min at 3000 rpm and 4 °C, and the pellets
were resuspended in 1 mL of lysis buffer according to
Sambrook et al. (21). A volume of 100 mLofa10mg/mL
proteinase K solution (Sigma, St. Louis, MO) was added
to the suspensions, which were then incubated on a linear
shaker at 42 °C overnight. An equal volume of phenol
was added to 1 mL of the digested suspensions, then
mixed for 3 min and centrifuged for 30 min at 14 000
rpm and 4 °C. Supernatant was transferred in a new tu-
be and solvent extraction was repeated twice more us-
ing phenol and chloroform in 1:1 volume ratio. DNA
was pelleted by adding sodium acetate (3 M, pH=5.2)
and ethanol and centrifuging for 30 min at 14 000 rpm
and 4 °C. The pellets were finally suspended in 200 mL
of double distilled water. DNA quality was controlled
by agarose gel electrophoresis and spectrophotometric
measurement.
DNA extraction from cheese matrix
Samples of mixes (two weeks after cheese-making)
were cut into small pieces with sterile disposable cutters
and weighed in order to obtain 5-g aliquots. Aliquots
were transferred into sterile 12-mL tubes along with
1-mL of lysis buffer (21) and 100 mL of a 10 mg/mL pro-
teinase K solution.
DNA from cheese matrix was obtained by perform-
ing phenol/chlorophorm as described above, followed
by Nucleospin Food kit purification (Macherey-Nagel,
Düren, Germany) in accordance with manufacturer’s in-
structions.
Primers
Primers for PCR amplification of bovine mitochon-
drial DNA were designed in a region of cytochrome
oxidase subunit I (COI gene), which was selected by
aligning bovine and buffalo mitochondrial sequences
(GenBank accession No. AY488491).
Forward and reverse primers span the positions
134–153 and 246–267, respectively. The following prim-
ers were used: BT3 forward, 5’-GAACTCTGCTCGGA-
GACGAC-3’; BT4 reverse 5’-AGCACCAATTATTAGG-
GGAAC-3’.
PCR amplification and analysis of results
PCR amplification was performed in a 50 mL reac-
tion volume containing 50 ng DNA, 200 mM dNTPs, 10
mM Tris-HCl, pH=8.3, 50 mM KCl, 1.5 mM MgCl2, 100
nM primers, 1.5 U DNA polymerase (AmpliTaq Gold,
Perkin Elmer). The PCR reaction was carried out in a
GeneAmp 9600 thermal cycler (Perkin Elmer), using the
following conditions: an initial denaturation step at 95
°C for 10 min followed by 35 cycles of 95 °C for 30 s, 56
°C for 30 s and 72 °C for 30 s; final extension step was
performed at 72 °C for 10 min. Amplicons were stored
at 15 °C before electrophoresis. Positive (DNA from bo-
vine blood) and negative (DNA from water buffalo
blood) control samples were run in each amplification.
Results and Discussion
The extraction method used to isolate DNA from
Mozzarella cheese matrix and governing liquid showed
good DNA yield and quality. Extracted DNA was used
as a template to amplify the bovine cytochrome oxidase
92 M. FELIGINI et al.: Detection of Adulteration in Mozzarella Cheese, Food Technol. Biotechnol. 43 (1) 91–95 (2005)
subunit I specific fragment. Species discrimination was
based on a new 134 bp amplification fragment (22).
Primers' functionality and specificity were tested
with samples of DNA isolated from water buffalo and
bovine blood (Fig. 1). In addition, a nucleotide-nucleotide
BLAST similarity search (http://www.ncbi.nlm.nih.gov/
BLAST) was conducted with the bovine-specific primer
sequences to check the absence of unintended matches
with buffalo’s genome.
A calibration curve was built using cheese matrix
samples containing known amounts of bovine milk,
ranging from 0.5 up to 30 %. The fluorescence intensity
of PCR products is shown in Fig. 2; a direct relation be-
tween bovine milk proportion and band intensity was
observed, according to the data by Maudet and Taberlet
(15). Image analysis was performed using an image cap-
ture device and dedicated software (Bio-Rad Quantity
One®) in order to confirm the linear trend (data not
shown).
After these initial assays, the test was applied to the
corresponding samples of Mozzarella’s governing liq-
uid. Fig. 3 shows the results obtained from the amplifi-
cation of the DNA extracted from the governing liquids
of the mixes six hours after the cheese-making (Fig. 3a),
93
M. FELIGINI et al.: Detection of Adulteration in Mozzarella Cheese, Food Technol. Biotechnol. 43 (1) 91–95 (2005)
Fig. 1. Sequence comparison between the bovine mitochondrial DNA amplified region (134 bp) and the water buffalo orthologous one
(GenBank Accession No. AY488491). Primers’ sequences are highlighted
Fig. 2. Separation of PCR products after the amplification of a
partial sequence of mtDNA control region (134-bp) extracted
from cheese matrix. Amplicons were separated in a 3 % agarose
gel containing ethidium bromide. Lane M, 100 bp DNA Ladder
(BioLABs, New England); lanes 1 to 6 correspond to the mixtures
of water buffalo Mozzarella cheeses containing 0.5, 1, 5, 10, 20
and 30 % of bovine milk, respectively; lane 7, positive control;
lane 8, negative control; lane 9, PCR master mix; lane 10, double
distilled water
Fig. 3. Polymerase chain reaction of mtDNA from the governing
liquids of the mixes analysed six hours (A), one week (B) and two
weeks (C) after cheese-making. Lane M, 100 bp DNA Ladder
(BioLABs, New England); lanes 1 to 6 correspond to the gover-
ning liquids of water buffalo Mozzarella cheese mixtures contai-
ning 0.5 to 30 % of bovine milk respectively; lane 7, positive con-
trol; lane 8, negative control; lane 9, PCR master mix; lane 10,
double distilled water
one week after the cheese-making (Fig. 3b) and two
weeks after the cheese-making (Fig. 3c), respectively.
The presence of DNA in governing liquid arises
from the cheese matrix’s slow flaking that occurs during
the whole preservation period. This process seems to
take place in a different fashion for each sample, affect-
ing DNA quantity in each governing liquid at all times.
Therefore, although the bovine/buffalo ratio is well
known for cheese matrix, a direct quantitative relation-
ship between bovine milk percentage and band intensity
could not be observed in the governing liquid. This not-
withstanding, a clear amplification at the chosen detec-
tion limit of 0.5 % was achieved in all three lots exam-
ined.
Even if at present bovine milk in water buffalo
Mozzarella is tolerated up to 1 % (23), due to involun-
tary cross-contaminations during cheese-making that oc-
cur more frequently than deliberate adulteration of the
cheese, we chose the detection limit of 0.5 % in order to
demonstrate the reliability of the method.
Despite the lack of an evident relation between bo-
vine milk content and band intensity, the latter clearly
increases through time (i.e. from six hours to two weeks
after the cheese-making) for all samples, confirming the
link between cheese flaking and DNA quantity in the
governing liquid.
DNA from ten commercial PDO water buffalo Moz-
zarella samples’ governing liquids was extracted. Bovine
DNA was detected in one sample; the presence of bo-
vine DNA in the positive sample was confirmed by a
second extraction from the governing liquid and dupli-
cate amplification; further confirmation was achieved by
amplifying DNA extracted from the cheese matrix.
Conclusions
The need to protect both producers and consumers
from this fraud prompted the development of several
chromatographic (1), electrophoretic (2,3), immunologi-
cal (4) and mass spectrometry (5) analytical techniques,
most of which rely on the protein analysis to discrimi-
nate the two species. To date, methods based on the
isoelectrofocusing of g-caseins after plasminolysis (2)
and on HPLC (1), which are the official methods in EU
and in Italy respectively (23,24), have a minimum detec-
tion limit of 1 % of bovine milk.
Modern molecular techniques based on DNA analy-
sis have found good applicability in detecting adultera-
tion, and they represent valid complements to the meth-
ods relying on protein analysis for the identification of
animal species. DNA-based techniques have become ef-
fective and reliable also for commercial dairy products
(6,7).
The PCR assay described here is suitable for routine
testing of commercial water buffalo Mozzarella cheese,
it is fast, easy and applicable for the detection of DNA
from bovine milk, frequently used in adulteration, up to
the limit of 0.5 %. This limit was chosen considering the
current European law (23) tolerating 1 % contamination
level. As it is appropriate for testing the cheese matrix,
it is also suitable for bovine DNA detection in govern-
ing liquid.
Acknowledgments
Authors acknowledge the Caresana dairy for the
production of experimental Mozzarellas.
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Detektiranje patvorenja talijanskog sira Mozzarella upotrebom
mitohondrijalne DNA kao biomarkera
Sa`etak
U mljekarskoj se industriji velika va`nost pridaje pra}enju patvorenja izvornog talijan-
skog sira Mozzarella kravljim mlijekom. U ovom je radu cilj bio razviti metodu polimerazne
lan~ane reakcije kako bi se mogla detektirati specifi~na sekvencija mitohondrijalne DNA
goveda u siru Mozzarella. DNA je izolirana iz sira i iz sirutke ekstrakcijom organskim ota-
palima i zatim pro~i{}ena. Umno`en je fragment dug 134-bp pomo}u specifi~nih »prime-
ra« goveda dizajniranih prema sekvenciji gove|e i bivolje mitohondrijalne citokrom oksi-
daze, podjedinice 1. Specifi~nost »primera« ispitana je kori{tenjem DNA iz krvi bivola i
goveda, jer su njihove DNA prisutne u patvorenim sirevima Mozzarella. Metodom se mo-
`e pouzdano detektirati udjel od ~ak 0.5 % kravljeg mlijeka, {to ju ~ini prikladnom i za ru-
tinske analize.
95
M. FELIGINI et al.: Detection of Adulteration in Mozzarella Cheese, Food Technol. Biotechnol. 43 (1) 91–95 (2005)