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.

Parmigiano–Reggiano (PR) is a worldwide known Italian, long ripened, hard cheese. Its inclusion in the list of cheeses bearing the protected designation of origin (PDO, EU regulation 510/2006) poses restrictions to its geographic area of production and its technological characteristics. To innovate the Parmigiano–Reggiano (PR) cheese manufacturing chain from the health and nutritional point of view, the output of defatted PR is addressed. Two defatting procedures (Soxhlet, and supercritical CO₂ extraction) were tested, and the obtained products were compared in the composition of their nitrogen fraction, responsible for their nutritional, organoleptic, and bioactive functions. Free amino acids were quantified, and other nitrogen compounds (peptides, proteins, and non-proteolytic aminoacyl derivatives) were identified in the extracts and the mixtures obtained after simulated gastrointestinal digestion. Moreover, antioxidant and angiotensin converting enzyme (ACE) inhibition capacities of the digests were tested. Results obtained from the molecular and biofunctional characterization of the nitrogen fraction, show that both the defatted products keep the same nutritional properties of the whole cheese.

Non-specific lipid transfer proteins (nsLTP) were shown to be among the most significant allergens, in particular in several fruits belonging to the Rosaceae family. The molecular features of LTPs, such as the presence of eight cysteine residues forming four disulfide bridges, confer a compact structure, decreasing the probability of degradation due to cooking or digestion, thereby increasing the chance of systemic absorption and severe allergic reactions. Few studies on LTP-induced allergies regarding almond (Prunus dulcis L) are available in the literature. In the present work, we describe for the first time the extraction and purification of an almond LTP, achieving its full characterisation by using liquid chromatography and exact mass spectrometry; the full sequence was identified by means of LC-ESI-Orbitrap-MS applying a bottom-up approach. The characterised protein consists of 92 amino acids and has a calculated exact MW of 9579.0. The presence of four disulfide bridges was confirmed after reduction, as shown by a mass increment of 8 Da. Finally, its potential allergenicity was confirmed via an in silico approach. The results presented here demonstrate the enormous potential of advanced MS techniques for obtaining high-quality structural and functional data of allergenic proteins in a short time.