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To evaluate the potential differences in the propensity of β-casein A1 (β-CNA1) and A2 (β-CNA2) from bovine milk to release health-relevant β-casomorphins (BCMs), food-derived peptides were monitored over time in the blood of eight human volunteers who consumed milk containing both protein variants. Liquid chromatography coupled with high resolution tandem mass spectrometry revealed interindividual variability of milk peptidomic profiles in human blood. BCMs were not detected, whereas BCM precursors originating from both β-CNA1 and β-CNA2 were ascertained, with β-CNA2-derived peptides showing a slightly greater susceptibility to proteolysis. Ten synthetic peptides mimicking circulating BCM precursors from β-CNA1 and β-CNA2, which were incubated ex vivo with the blood of two volunteers, showed comparable potential to generate BCMs. The formation of BCMs seemed to depend mainly on the size of the BCM precursors and less on the presence of His 67 or Pro 67 . These findings challenge the belief that BCMs are released exclusively from β-CNA1 and support the nutritional safety of conventional milk, informing health policies regarding milk consumption., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

A dedicated two-step purification procedure prior to nanoliquid chromatography-electrospray-tandem mass spectrometry analysis enabled the identification of bovine milk-derived peptides absorbed and circulating in the plasma of three healthy volunteers who received 250 mL of pasteurized milk after a 10-days washout. The appearance and clearance of milk peptides in plasma were monitored at various time points. Overall, 758, 273 and 212 unique peptides derived from 15, 15 and 18 bovine milk proteins, respectively, were identified in the plasma of these volunteers, evidencing a substantial inter-individual variability. Peptides encrypting possible bioactive and/or immunogenic molecules originating from caseins, β-lactoglobulin and minor milk proteins were detected. Peptide representation data revealed the combined action of endoproteases involved in primary hydrolysis during gastroduodenal digestion and exopeptidases that hydrolyse peptides in the small intestine. It remains to be established whether the half-life and concentration ranges of circulating milk-derived peptides may have any impacts on human health., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The aim of this study was to evaluate the dynamics of proteolysis during dough fermentation started with different lactic acid bacteria species, through the identification of intermediate and small-sized peptides generated during fermentation. Single-strain cultures of Levilactobacillus brevis, Fructilactobacillus sanfranciscensis, Companilactobacillus alimentarius, and Leuconostoc pseudomesenteroides were assayed as sourdough starters. Assays were carried out at lab-scale for 48 h of fermentation, using both unstarted and yeast-leavened dough as controls. Physicochemical and microbiological analyses were combined with peptidomic and proteomic profiling, identifying several hundreds of peptides mainly released from the water-soluble wheat proteins, including β-amylase, triticin, and serpins. Both α- and γ-gliadins were hydrolyzed, though only at the N-terminal domain, while the central protein region - encrypting celiac disease epitopes- remained unaffected. The bacterial-mediated consumption of sugars and the concomitant hydrolysis of starch degrading β-amylase could underlie improved digestibility and several nutritionally beneficial effects of sourdough baked products., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The need of controlling illegal addition of water buffalo (WB) milk from foreign countries to the Italian counterpart devoted to the production of Protected Denomination of Origin (PDO) Mozzarella di Bufala Campana (MBC) cheese has promoted the development of simple, fast and cheap isoelectrofocusing (IEF) methods for evaluating the nature of the raw material to be used according to a high-throughput sample multiplexing format, avoiding the use of dedicated mass spectrometry-based procedures. Thus, combined proteomic methods were here integrated with optimized western blotting protocols in solving the complex IEF pattern of casein (CN) mixtures observed when Italian and foreign WB milk are mixed together. Identification of internally deleted α s1 -CN hepta-phosphorylated species as well as of still unknown β-CN A hexa-phosphorylated and N-terminally-nicked β-CN A phosphorylated forms present uniquely in foreign WB milk samples, allowed recognizing these molecules as adulteration markers to be assayed in combined IEF-immunoblotting procedures; the latter ones showing optimal migration characteristics to be used in routine assays. A linear relationship between detected area of specific immunorecognized gel bands and percentage of international WB milk added to the Italian counterpart was verified, demonstrating that this method has an adulteration detection limit close to 3% v/v. Based on these results, this analytical procedure is here proposed as optimal one for evaluating the authenticity of PDO MBC cheese products., (Published by Elsevier Ltd.)

The European reference method (ERM) recognises the fraudulent addition of bovine (B) milk in water buffalo (WB) milk/dairy products based on concomitant isoelectric focusing (IEF) detection of B γ 2 - and γ 3 -CN fragments after corresponding plasminolysis. We here used proteomics to characterise false positive results occurring in the ERM as being due to WB β-CN(f100-209), which is also formed after plasminolysis of genuine WB milk/dairy products and comigrates in IEF with B γ 2 -CN. These ERM limitations were overcome by a dedicated proteomic procedure based on loading of B/WB milk/cheese CN extracts on a hydroxyapatite column, in situ trypsinolysis and elution of B β-CN(f1-25)4P and WB β-CN(f1-28)4P proteotypic peptides. Based on their similar ionisation properties and resolution in MALDI-TOF-MS, these phosphopeptides were identified as suitable markers for detection of B material in WB milk/dairy products to a detection limit of 0.8% v/v. This proteomic procedure is here proposed as integrative/alternative to the ERM., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

For the purpose of a varietal assessment, the berry skin anthocyanin profiles of 11 ancient native red grape varieties, sampled within the Irpinian area (Southern Italy), were compared to those of three reference Vitis vinifera cultivars and of a Kober 5BB rootstook hybrid (Vitisberlandieri×Vitisriparia). The 3,5-O-diglucoside anthocyanins and their acylated derivatives were monitored as signature compounds of non-V. vinifera grapes, using both reversed phase-high performance liquid chromatography (RP-HPLC) and matrix assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry (MS). One variety (i.e. Tenta) was demonstrated to be an interspecific hybrid cross. Three additional varieties, namely Lacrima Nera, Aglianicone and a yet-unnamed variety, were classified as "late generation hybrids" (or non-V. vinifera×V. vinifera hybrids) because of a very diluted hybrid character, that was revealed only by MS methods. Five cultivars, i.e. Aglianico Lasco, Cannella, Coda di Volpe Rossa, Mentuonico, Olivella Nera, were catalogued as purebred V. vinifera. Due to the peculiar anthocyanin profile one variety (Tuccanese) remained unassigned. The methodology is of general applicability to support the process of varietal discrimination on a molecular basis with the objective of classifying autochthonous old grapevine varieties., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

A novel genetic variant at the αs1-casein locus of water buffalo (WB), 8-residue shorter than its wild-type has been found and sequenced. The internal deletion of the peptide E(35)KVNELsT(42) was confirmed by the isolation of the junction peptide. The 8-residue deletion mutant has a molecular weight that is 919 Da less than that of the wild-type. The novel isoform with a unique f35-42 deletion could be the result of the skipping of exon 6, generating an exon 6-deleted variant of αs1-casein. The wild-type and its shortened αs1-casein forms were found to co-exist in many individual milk samples. In contrast, the 8-residue, internally deleted αs1-casein variant did not occur in water buffaloes of the Mediterranean breed reared in Italy. Wild-type αs1-casein has 6 to 8 phosphate groups (P) while the internally deleted form 6 and 7P per molecule., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The entire panel of peptides produced from caseins (CN) and whey proteins (WP) that survive in vitro sequential gastro-pancreatic digestion and translocate across monolayers of Caco-2 cells, used as a model of the intestinal epithelium, has been characterised by HPLC and mass spectrometry. Among the milk-derived bioactive peptides, only minor amounts of mono-phosphorylated peptides arising from αs1- and β-CN were detected. The absorption behaviour of two resistant β-lactoglobulin (β-Lg) domains, β-Lg 125-135 and β-Lg 40-60, was studied in detail using synthetic peptides. The IgE-binding properties of the digests recovered from the apical and basolateral monolayer compartments were evaluated by dot-blot, using the sera of milk allergic children (N=5). Outcomes indicated β-Lg 127-135 as a possible "immune sensitising factor"in vivo. The almost complete loss of the IgE-affinity of CN and WP after digestion points out the need to design in vivo experiments to track the metabolic fate of dietary proteins., (Copyright © 2013 Elsevier Ltd. All rights reserved.)