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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 E.Maskova@vupp.cz Aim Material and method Material 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 Results 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 cheese 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. Conclusion 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. Acknowledgement This work was supported by the project N. QC 1111 of the Ministry of Agriculture of the Czech Republic.
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