Development of an LC-MS Method for the Identification of beta-Casein Genetic Variants in Bovine Milk

Bovine beta-casein is a highly polymorphic protein, with A1 and A2 representing the most frequent variants identified in Western cattle breeds. Depending on the presence of histidine or proline at position 67 of the sequence, beta-casein variants can be grouped in two families: those within the A1 family (A1-like), possessing histidine, and those within the A2 family (A2-like), with proline. Upon gastrointestinal digestion, specific peptides endowed with opioid-like activity, called beta-casomorphins, may be released from beta-casein. Interestingly, the presence of histidine at position 67 seems to favor the release of a peptide called beta-casomorphin-7, which has been proposed as a risk factor for the development of some non-communicable diseases. Based on these assumptions, the A2 milk market (i.e., a milk containing only A2 beta-casein) is spreading worldwide. In the scientific community, however, the impact of A1 beta-casein on human health remains a matter of debate. Aim of the present study was to develop an analytical method based on mass spectrometry to distinguish between bovine A2 milk and commercial milk, typically composed of a mixture of A1-like and A2-like beta-casein. By enzymatically hydrolyzing beta-casein, a specific peptide containing the critical mutation at position 67 has been identified. This peptide constituted a suitable marker to determine the presence of A1-like and/or A2-like beta-casein in bovine milk. The developed method proved appropriate for the detection of beta-casein in real milk samples; we estimated that contaminations of A2 milk with up to 1% commercial milk could be successfully detected.