Detection of the Adulteration of Goat Cheeses Using by dfgh4bnmu


									          Detection of the Adulteration of Goat Cheeses Using the PCR Method
                                                                                 Eva Mašková, Ivana Paulíčková
                                            Food Research Institute Prague, Radiová 7, 102 31 Prague 10, Czech Republic

Aim                                                                                                       Material and method
The aim of this work was the optimization and validation of the PCR method for the
                                                                                   Goat cheeses from food store chains and bio/eco food shops.
determination of cow milk in goat cheeses and the application of this method in
                                                                                   They were made by 16 different producers located in 4 European countries.
detecting of the adulteration of goat cheeses by cow milk.
                                                                                                          DNA extraction – the isolation kit Invisorb Spin Food I (Invitek, Germany) –
Introduction                                                                                                               non-chaotropic solid phase extraction
The majority of retail cheeses are made from cow milk. Pure goat cheeses are
                                                                                                          The PCR reaction mix of 50 μl contained :
considered as specialities with characteristic sensory features, primarily taste and
                                                                                                          2,5 U Platinum Taq DNA Polymerase (Invitrogen, England)
flavour. Their supply fluctuates with the season of the year depending on the
                                                                                                          1 x concentrated PCR buffer without Mg (Invitrogen, England)
reproductive cycle of goats. Goat milk is priced much higher than cow milk. This often
                                                                                                          1,5 mM MgCl2 (Invitrogen, England)
induces attempts to make these cheeses from raw material where goat milk is at least
partially replaced with undeclared cow milk. However, the consumer has the right to                       0,2 mM dNTP mix (Eppendorf,Germany)
know the species origin of the cheese, be it for nutritional or religious reasons.                        25 pmol of each primer :
                                                                                                          SIM: 5´GAC CTC CCA GCT CCA TCA AAC ATC TCA TCT TGA TGA AA 3´
For the time being the valid reference method for the detection of cow milk in dairy                      ( common forward primer)
products is based on the isoelectric focusation of milk caseins. Other electro-migration                  Cattle B: 5´CTA GAA AAG TGT AAG ACC CGT AAT ATA AG 3´ - reverse primer for cow
methods, some ELISA techniques, chromatographic methods and even a method                                 Goat G: 5´CTC GAC AAA TGT GAG TTA CAG AGG GA 3´ - reverse primer for goat
based on the principle of mass spectrometry were also tested for this purpose. The                        DNA extract
procedures mentioned above are often unsuitable for the analysis of a complex food                        Sterile water Molecular Biology Grade, DNase free
matrix and also evince lower sensitivity for thermally treated substances. This is why                    PCR amplification
the methods based on the polymerase chain reaction are increasingly employed in the                       Initial denaturation – 94oC, 1 min
recognition of respective milk kinds.                                                                     40 cycles with the following step-cycle profile : Denaturation – 94oC, 30 s
                                                                                                                                                            Annealing – 60oC, 30 s
                                                                                                                                                            Extension – 72oC, 30 s
                                                                                                          Final extension – 72oC, 5 min
                                                                                                          Electrophoretic identification of PCR product
                                                                                                          3 % agarose gel in 1 x TBE buffer, stained with ethidium bromide,
                                                                                                          horizontal electrophoresis - 110 V, 1 hr 30 min

A series of model samples of pure                        The described PCR method made it
goat cheese with cow cheese was                          possible to detect the amplified                   Table shows the results of PCR analyses of 17 goat cheeses from retail trade.
prepared     for     determining    the                  fragment of cow DNA, size 274 bp                   Undeclared presence of cow milk was detected in cheeses with No. 5,9 and 10.
detection limit of this PCR reaction.                    and goat DNA, size 157 bp.
                                                                                                                          Cheese brand                 Country of      Presence of   Presence of
  1   2   3   4   5    6   7   8   9   10                   1   2   3    4   5    6   7   8   9   10
                                                                                                           No.                                           origin         cow milk      cow milk
                                                                                                                                                                        declared      detected
                                                                                                            1    Balkan cheese                       Czech Republic        No            No
                                                                    274 bp                                  2    Fresh non-ripening natural cheese   Czech Republic        No            No
                                                                                                            3    Dessert cheese                      Czech Republic        No            No
                                                                             157 bp
                                                                                                            4    Natural cheese                      Czech Republic        No            No
          1   5   10   25 50 100                                                                            5    Natural semi-hard sliced cheese     Czech Republic        No           Yes
                                                                                                            6    Natural brynza cheese               Czech Republic        No            No
                                                                                                            7    Genuine semi-hard ripening          Czech Republic        No            No
                                                                                                            8    Tylžský cheese                      Czech Republic        No            No
                                                                                                            9    Pickled natural cheese                 Slovakia           No           Yes
Determination of detection limit of PCR reaction                                                            10 Natural cheese with red pepper           Slovakia           No           Yes
                                                         PCR analysis of goat cheeses for presence
                                                         of cow milk component                              11 Gouda                                 The Netherlands       No            No
Lane 2 – 50 bp DNA Ladder                                                                                   12 Fresh natural cheese Chavroux             France            No            No
Lane 3 – 1 % cow cheese in goat cheese                   Lane 1 – 50 bp DNA Ladder                          13 Fresh natural cheese Soignon              France            No            No
Lane 4 – 5 % cow cheese in goat cheese                   Lane 2 – Goat standard                             14 Natural cheese with surface mould         France            No            No
Lane 5 – 10 % cow cheese in goat cheese                  Lane 3 – Cheese No.10 – goat DNA                      Soignon
Lane 6 – 25 % cow cheese in goat cheese                  Lane 4 – Cheese No. 6 – goat DNA
Lane 7 – 50 % cow cheese in goat cheese                                                                     15 Fresh cheese Cabridoux                    France            No            No
                                                         Lane 5 and 6 – Cheese No.10 – cow DNA
Lane 8 – 100 % cow cheese                                Lane 7 and 8 – Cheese No. 6 – cow DNA              16 Cheese with surface mould                 France            No            No
Lane 9 – Negative control                                Lane 9 – Negative control                             Chevre du Poitou
Lane 10 – Cow standard                                   Lane 10 – Cow standard                             17 Cheese Tomme                              France            No            No
The 1 % detection limit of this
PCR reaction was found.

 An optimized PCR method for the determination of an undeclared quantity of cow milk in goat cheeses was validated.

 The 1 % detection limit for the PCR reaction was determined using model samples.

 It was found that 3 analysed goat cheeses contained an undeclared cow milk component. One of these cheeses was of Czech origin and two of them were made in Slovakia.

This work was supported by the project N. QC 1111 of the Ministry of Agriculture of the Czech Republic.

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