Profiling of Multiphosphorylated Peptides in Kefir and Their Release During Simulated Gastrointestinal Digestion

Abstract

Casein phosphopeptides are multiphosphorylated milk peptides, which can have anticariogenic activity and improve mineral absorption by binding bivalent metal ions. The present study investigated phosphopeptides in kefir because fermentation may lead to their enhanced release from milk proteins. After selective enrichment by hydroxyapatite extraction, phosphopeptides and their phosphorylation degree were identified by matrix-assisted desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) before and after enzymatic dephosphorylation. Peptide structures were determined by ultrahigh-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (UHPLC–ESI–MS/MS) revealing 27 phosphopeptides in kefir, including nine peptides containing the motif pSpSpSEE, which binds minerals most efficiently. The majority (18) of phosphopeptides were derived from β-casein, but only three were derived from the most abundant milk protein α s1 -casein. After simulated gastrointestinal digestion, MALDI-TOF-MS analysis detected eight putative phosphopeptides in kefir, four of which were assigned by UHPLC–ESI–MS/MS to α s2 -casein 124–133 , α s2 -casein 137–146 , β-casein 30–40 , and κ-casein 147–161 . These results indicate that kefir is a good dietary source of multiphosphorylated peptides.

Proteolysis and Process-Induced Modifications in Synbiotic Yogurt Investigated by Peptidomics and Phosphopeptidomics

“…Some evidences suggested that CPPs, acting as mineral carriers in vivo that survive at least in part the simulated gastrointestinal digestion of milk proteins, pass through the bloodstream and are detected in human plasma after ingestion of milk and dairy products . The cluster of phosphorylated seryl residues -SerP-SerP-SerP-Glu-Glu-, which appears as a final product of proteolysis in long-ripened cheeses, was partly dephosphorylated in yogurt samples, especially in synbiotic yogurt, confirming previous results obtained in kefir . The progressive decline of native CPPs present in a pH 4.6 soluble fraction of yogurt can be easily inferred from Tables S2–S6 and visualized in Figure , in which peptides are represented by pink-purple shaded colors, according to the degree of phosphorylation.…”

“…29 The cluster of phosphorylated seryl residues -SerP-SerP-SerP-Glu-Glu-, which appears as a final product of proteolysis in long-ripened cheeses, 27 was partly dephosphorylated in yogurt samples, especially in synbiotic yogurt, confirming previous results obtained in kefir. 30 The progressive decline of native CPPs present in a pH 4.6 soluble fraction of yogurt can be easily inferred from Tables S2−S6 and visualized in Figure 4, in which peptides are represented by pink-purple shaded colors, according to the degree of phosphorylation. The control milk base, in fact, already contained intact β-CN (f1−28) 3P and 4P (SerP at 15, 17, 18, 19 and 17, 18, 19 sites, respectively) as dominant primary peptides and a few other derived fragments in lower amounts.…”

Proposal of the Annotation of Phosphorylated Amino Acids and Peptides Using Biological and Chemical Codes

“…In texts designed as human-readable, amino acid residues containing the phosphate group may be highlighted in sequences (underlined, displayed using color or bold fonts). Recent examples of this way of phosphate annotation may be found in the articles published by Savastano et al [ 41 , 42 ], Pourjoula et al [ 43 ], and Bekker-Jensen et al [ 44 ]. The phosphorylation of amino acid residues is recently annotated using the letter “p” before one letter symbol of the amino acid [ 45 , 46 , 47 , 48 ].…”

Antioxidant Properties of Casein Phosphopeptides (CPP) and Maillard-Type Conjugated Products

“…In particular, specific casein hydrolysates from bovine milk demonstrate antioxidant activity via radical scavenging properties in both aqueous and lipid model systems [8][9][10][11][12][13]. Casein phosphopeptides (CPPs) derived from tryptic digests of casein also have a strong affinity to sequester divalent metal ions, such as non-heme iron and copper, two potential food pro-oxidants under specific conditions [14][15][16]. Bovine milk casein-derived CPPs may therefore serve to remove pro-oxidative metal catalysts such as iron from an environment of oxidizable lipids to prevent lipid oxidation [17].…”

“…heme iron and copper, two potential food pro-oxidants under specific conditions [14][15][16]. Bovine milk casein-derived CPPs may therefore serve to remove pro-oxidative metal catalysts such as iron from an environment of oxidizable lipids to prevent lipid oxidation [17].…”