β-casein gene polymorphism permits identification of bovine milk mixed with bubaline milk in mozzarella cheese

Mozzarella cheese is traditionally prepared from bubaline (Bubalus bubalis) milk, but product adulteration occurs mainly by addition of or full substitution by bovine milk. The aim of this study was to show the usefulnes of molecular markers to identify the admixture of bovine milk to bubaline milk during the manufacturing process of mozzarella cheese. Samples of mozzarella cheese were produced by adding seven different concentrations of bovine milk: 0%, 1%, 2%, 5%, 8%, 12% and 100%. DNA extracted from somatic cells found in cheese were submitted to PCR-RFLP analysis of casein genes: α-s1-CN – CSN1S1 that encompasses 954 bp from exon VII to intron IX (Alu I and Hinf I), β-CN – CSN2 including 495 bp of exon VII (Hae III and Hinf I), and κ-CN – CSN3, encompassing 373 bp of exon IV (Alu I and Hind III). Our results indicate that Hae III-RFLP of CSN2 exon VII can be used as a molecular marker to detect the presence of bovine milk in “mozzarella” cheese.

Mozzarella cheese has its origin in Italy where it is traditionally manufactured from bubaline (Bubalus bubalis) milk.The most commonly employed production process of mozzarella cheese is a traditional technique where bacterial fermentation of milk induces pH reduction and curd precipitation (Chapman et al., 1981).Nowadays, many countries that have a considerable number of buffalo cows widely use the milk of these animals for mozzarella cheese production.
Some dairy products can be adulterated by milk admixture from different species in order to maximize profit.Certification is, thus, a way to guarantee cheese quality and to protect consumers against fraudulent producers.Bubaline breeders demand high precision technology to validate milk origin to assure that only bubaline milk is present in the composition of the manufactured product.The most common type of adulteration in the manufacture of mozzarella cheese is the addition or full replacement of bubaline milk by bovine milk.Consequently, several methods have been developed to detect milk mixture in these products.Methods based on electrophoresis and chromatography in-clude isoelectric focusing (Moio et al., 1989), high-performance liquid chromatography (Visser et al., 1991;Veloso et al., 2002;Enne et al., 2005), nuclear magnetic resonance spectroscopy (Andriotti et al., 2000), and also hydrophobic interaction chromatography (Bramanti et al., 2003).However, these methods present limitations due to time intensive protocols and/or high costs.
An alternative way to detect milk mixtures is the use of molecular markers to identify the DNA of different species (Bardin et al. 1994;Branciari et al., 2000;Rea et al., 2001;Bottero et al., 2002;Leoparelli et al., 2007).Here we describe a relatively rapid and simple method to identify admixtures of bovine milk to bubaline milk, by extracting DNA directly from Mozzarella cheese and analyzing a β-casein gene polymorphism.
Samples of "pasta filata" mozzarella cheese were produced using 7 L of milk according to the methodology developed by Kuo et al. (2001).The samples were prepared in the "Laticínio White Milk" facility (Marilia, SP, Brazil), using bovine milk mixed with bubaline milk at concentrations of 0%, 1%, 2%, 5%, 8%, 12% and 100% (T1-T7).Bovine milk was obtained from a herd of different milky breeds (Holstein, Brown-Swiss, Jersey, and the Brazilian native breeds: Gir, Guzerá and Girolanda) with the intention to have as many β-casein gene variants as possible.
As predicted, only CSN2 Hae III-RFLP distinguished between the bovine and bubaline casein genes after electrophoresis on a 3% agarose gel (Figure 1).The intensity of the bands allowed the identification of even very low concentrations of bovine milk added in the process of manufacturing mozzarella cheese.After electrophoresis on the Otaviano et al.
We also considered other bovine CSN2 gene variants (described by Bonsing et al., 1988; GenBank accession number X14711) and bubaline CSN2 gene variants (Singh and co-workers, GenBank; accession numbers: A-DQ191172.1, B-DQ191171.1 and C-DQ191170.1) and performed virtual analyses using pDRAW32 software (Figure 3).The bubaline variants showed 95% sequence similarity with the bubaline variant described here, but they could be distinguished from the bovine variant by the smaller Hae III fragments.
The In this report we show that the Hae III-RFLP pattern of CSN2 exon VII is capable of distinguishing the bubaline and bovine genes, providing a simple method for detecting the presence of bovine milk in mozzarella cheese.
other RFLPs studied, CSN1S1 (Alu I and Hinf I), CSN2 (Hinf I) and CSN3 (Alu I, Hind III) did not show any difference among bovine and bubaline fragments.The CSN1S1 954 bp fragment resulting from digestion with Alu I generated fragments of 651 and 303 bp, and the one resulting from digestion with Hinf I generated fragments of 298, 248, 213 and 195 bp at all concentrations of bovine milk (experiments T1 to T7).The same occurred with the 373 bp fragment of CSN3 digested by Alu I, which generated fragments of 222, 139 and 12 bp, and by the enzyme Hind III, which generated fragments of 224 and 149 bp.The CSN2 495 bp fragment,digested by Hinf I also yielded fragments of 249, 152 and 94 bp in all samples, at the seven bovine milk concentrations.These results confirmed our previous data, showing that bovine and bubaline genes cannot be differentiated by these RFLPs.Various methods based on DNA analysis have been previously described for identifying bovine milk addition to pure buffalo cheese products.Branciari et al. (2000) reported a PCR-RFLP method on DNA isolated from Italian mozzarella by sequential organic extraction and resin purification.Rea (2001) proposed a duplex-PCR technique to identify bovine and water buffalo DNA in a single PCR assay of milk and mozzarella cheese.Bottero et al. (2002) also developed a duplex-PCR assay and distinguished between fragments of bovine and bubaline mitochondrial cytochrome b genes.More recently,Loparelli et al. (2007)   reported a real-time PCR method capable of quantifying bovine milk addition, based on analysis of the bovine mitochondrial cytochrome b gene and the nuclear growth hormone gene.

Figure 2 -
Figure 2 -Bovine and bubaline Hae III-RFLP of CSN2 exon VII visualized after electrophoresis on a 12% acrylamide gel: A, 373 bp, 59 bp and 63 bp fragments from cheese prepared with pure bovine milk; B, 436 bp and 59 bp fragments from cheese containing pure bubaline milk; C: 50 bp DNA Ladder.

Figure 3 -
Figure 3 -Virtual analysis of Hae III-RFLP products of the CSN2 exon VII, using the software pDRAW32 on the sequences of (A) bovine and (B) bubaline variants obtained from GenBank; (C) bubaline restriction pattern obtained in this study.