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Thermal treatments of milk induce changes in the properties of milk whey proteins. The aim of this study was to investigate the specific changes related to nutrients in the whey proteins of dairy cow milk after pasteurization at 85 °C for 15 s or ultra-high temperature (UHT) at 135 °C for 15 s. A total of 223 whey proteins were confidently identified and quantified by TMT-based global discovery proteomics in this study. We found that UHT thermal treatment resulted in an increased abundance of 17 proteins, which appeared to show heat insensitivity. In contrast, 15 heat-sensitive proteins were decreased in abundance after UHT thermal treatment. Some of the heat-sensitive proteins were connected with the biological immune functionality, suggesting that UHT thermal treatment results in a partial loss of immune function in the whey proteins of dairy cow milk. The information reported here will considerably expand our knowledge about the degree of heat sensitivity in the whey proteins of dairy cow milk in response to different thermal treatments and offer a knowledge-based reference to aid in choosing dairy products. It is worth noting that the whey proteins (lactoperoxidase and lactoperoxidase) in milk that were significantly decreased by high heat treatment in a previous study (142 °C) showed no significant difference in the present study (135 °C). These results may imply that an appropriately reduced heating intensity of UHT retains the immunoactive proteins to the maximum extent possible.

The high temperatures used in the production of milk may induce modifications in proteins structure. Due to occurrence of the Maillard reaction, lactose binds lysine residues in proteins, affecting the nutritional value. Milk is also an important source of allergenic proteins (i.e., caseins, β-lactoglobulin and α-lactalbumin). Thus, this modification may also affect the allergenicity of these proteins. Focusing on milk whey proteins, a screening on different Ultra High Temperatures (UHT) and pasteurized milk samples was performed to identify lactosylation sites, in particular in protein known epitopes, and to verify the correlation between lactosylation and the harshness of the treatment. Whey proteins were extracted from milk samples after caseins precipitations at pH 4.6 and, after chymotryptic and tryptic in solution digestion, peptides were analysed by UPLC-MS and LTQ-Orbitrap. Results show the presence of lactosylated lysine residues in several known epitopes. Then, a β-lactoglobulin epitope was selected and synthesized by solid phase synthesis followed by in solution lactosylation, obtaining high reaction yields and purities. The synthesis of lactosylated allergenic epitopes, described here for the first time, is a useful tool for further studies on the technological impacts on food allergenicity.

Staphylococcus aureus is the most common contagious pathogen associated with bovine subclinical mastitis. Current diagnosis of S. aureus mastitis is based on bacteriological culture of milk samples and somatic cell counts, which lack either sensitivity or specificity. Identification of milk proteins that contribute to host defense and their variable responses to pathogenic stimuli would enable the characterization of putative biomarkers of subclinical mastitis. To accomplish this, milk whey samples from healthy and mastitic dairy cows were analyzed using a label-free quantitative proteomics approach. In total, 90 proteins were identified, of which 25 showed significant differential abundance between healthy and mastitic samples. In silico functional analyses indicated the involvement of the differentially abundant proteins in biological mechanisms and signaling pathways related to host defense including pathogen-recognition, direct antimicrobial function, and the acute-phase response. This proteomics and bioinformatics analysis not only facilitates the identification of putative biomarkers of S. aureus subclinical mastitis but also recapitulates previous findings demonstrating the abundance of host defense proteins in intramammary infection. All mass spectrometry data are available via ProteomeXchange with identifier PXD007516 ., Competing Interests: The authors declare no conflict of interest.