Antioxidant capacity of water soluble extracts from Parmigiano-Reggiano cheese.

In this work the antioxidant capacity of water soluble extracts of Parmigiano-Reggiano cheese (Water Soluble Extracts - WSEs) at different aging time was studied, by measuring their radical scavenging capacity with a standard ABTS assay. The WSEs were also fractionated by semi-preparative HPLC-UV and for each fraction the antioxidant capacity and the molecular composition was determined by LC/ESI-MS, in order to identify the most active antioxidant compounds. The antioxidant capacity was also determined after simulated in vitro gastrointestinal digestion of WSEs. The data indicated that antioxidant capacity in WSE from Parmigiano-Reggiano cheese, quite unaffected by ripening time and gastrointestinal digestion, is mostly due to free amino acids, mainly tyrosine, methionine and tryptophan, and only in minimal part to antioxidant peptides.

Identification of novel dipeptidyl peptidase IV (DPP-IV) inhibitory peptides in camel milk protein hydrolysates.

Nine novel dipeptidyl peptidase IV (DPP-IV) inhibitory peptides (FLQY, FQLGASPY, ILDKEGIDY, ILELA, LLQLEAIR, LPVP, LQALHQGQIV, MPVQA and SPVVPF) were identified in camel milk proteins hydrolysed with trypsin. This was achieved using a sequential approach combining liquid chromatography tandem mass spectrometry (LC-MS/MS), qualitative/quantitative structure activity relationship (QSAR) and confirmatory studies with synthetic peptides. The most potent camel milk protein-derived DPP-IV inhibitory peptides, LPVP and MPVQA, had DPP-IV half maximal inhibitory concentrations (IC50) of 87.0 ±â€¯3.2 and 93.3 ±â€¯8.0 µM, respectively. DPP-IV inhibitory peptide sequences identified within camel and bovine milk protein hydrolysates generated under the same hydrolysis conditions differ. This was linked to differences in enzyme selectivity for peptide bond cleavage of camel and bovine milk proteins as well as dissimilarities in their amino acid sequences. Camel milk proteins contain novel DPP-IV inhibitory peptides which may play a role in the regulation of glycaemia in humans.

In Silico Approaches Applied to the Study of Peptide Analogs of Ile-Pro-Ile in Relation to Their Dipeptidyl Peptidase IV Inhibitory Properties.

Inhibition of dipeptidyl peptidase IV (DPP-IV) may be exploited to maintain the incretin effect during the postprandial phase. As a result, glycemic regulation and energy homeostasis may be improved. Food protein-derived peptides have been identified as natural agents capable of inhibiting DPP-IV. Ile-Pro-Ile is the most potent DPP-IV inhibitory peptide identified to date. A minimum analog peptide set approach was used to study peptide analogs of Ile-Pro-Ile. The DPP-IV half maximal inhibitory concentration (IC50) values of the 25 peptides evaluated ranged from 3.9 ± 1.0 µM (Ile-Pro-Ile) to 247.0 ± 32.7 µM (Phe-Pro-Phe). The presence of Pro at position 2 of tripeptides was required to achieve high DPP-IV inhibition. Most peptides behaved as competitive inhibitors of DPP-IV with the exception of peptides with a N-terminal Trp, which were mixed-type inhibitors. While possessing the structure of preferred DPP-IV substrates, most peptides studied were particularly stable during 30 min incubation with DPP-IV. Molecular docking revealed that Ile-Pro-Ile and its peptide analogs interacted in a very similar manner with the active site of DPP-IV. In addition, no correlation was found between the Hydropathic INTeraction score and the DPP-IV IC50 values of the peptides studied. This outcome suggests that free energy may not be directly responsible for enzyme inhibition by the peptides. Finally, novel DPP-IV inhibitory peptides were identified using the strategy employed herein. These results may be relevant for the development of food protein-derived peptides with serum glucose lowering and food intake regulatory properties in humans.

Occurrence of non-proteolytic amino acyl derivatives in dry-cured ham.

Proteolysis is the most important event occurring during maturation of dry-cured hams: it strongly influences the flavour and the texture of the aged ham by the accumulation of peptides and free amino acids released by protein hydrolysis. Apart from compounds of proteolytic origin, it has been demonstrated that also non-proteolytic amino acyl derivatives (γ-glutamyl amino acids, pyroglutamyl-amino acids and lactoyl-amino acids) may accumulate during ripening of cheese, and they can be also found in fermented soy sauce, where they contribute to the umami taste of the products. Using a semi-quantitative analysis, in this paper we report the occurrence of significant amounts of γ-glutamyl amino acids and, for the first time, pyroglutamyl-amino acids and lactoyl-amino acids, in aged ham. The amino acid counterparts were mainly found to be hydrophobic amino acids. The amount of these compounds was found to increase with time, because they are not degraded by proteolytic activity. They were also found to be stable to simulated gastrointestinal digestion. Angiotensin Converting Enzyme inhibitory activity was also tested, but they were not found to be characterized by significant ACE-inhibitory activity.

Milk protein isolate (MPI) as a source of dipeptidyl peptidase IV (DPP-IV) inhibitory peptides.

A multifactorial [temperature (40, 50 and 60°C), hydrolysis time (60, 150 and 240min) and enzyme to substrate ratio (E:S; 1.0, 1.5 and 2.0%)] design of experiments (DOE) was used to optimise the release of dipeptidyl peptidase IV (DPP-IV) inhibitory peptides during hydrolysis of bovine milk protein isolate (MPI) with Neutrase 0.8L™, yielding 15 hydrolysates (H1-H15). Variation in temperature and time had a significant effect on DPP-IV inhibitory properties (p<0.05) in contrast with E:S (p>0.05). The DPP-IV half maximal inhibitory concentration (IC50) of H4, a potent sample, was maintained following simulated gastrointestinal digestion (SGID, DPP-IV IC50=0.60±0.06vs. 0.58±0.09mgml-1, p>0.05). Several peptides with DPP-IV inhibitory features or known activity were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) within the hydrolysates. MPI hydrolysates may have potential for use as dietary ingredients with serum glucose lowering activity in humans.

Release of dipeptidyl peptidase IV (DPP-IV) inhibitory peptides from milk protein isolate (MPI) during enzymatic hydrolysis.

The release of dipeptidyl peptidase IV (DPP-IV) inhibitory peptides from bovine milk protein isolate (MPI) during trypsin hydrolysis was studied using a design of experiments (DOE) approach. A 3 factor×3 level DOE including temperature (40, 50 and 60°C), enzyme to substrate ratio (E:S; 0.50, 1.25 and 2.00% (w/w)) and hydrolysis time (60, 150 and 240min) was used during the generation of 15 hydrolysates (H1-H15). The degree of hydrolysis (DH) varied between 6.98±0.31 (H8) to 12.75±0.62% (H10). The DPP-IV half maximal inhibitory concentration (IC50) ranged from 0.68±0.06 (H11)/0.68±0.10 (H4) to 1.59±0.11mgmL-1 (H8). Temperature had no effect (p>0.05) on the DPP-IV IC50 value, while an increase in E:S or time significantly decreased DPP-IV IC50 value (p<0.05). The DPP-IV IC50 value of 0.69mgmL-1, predicted by response surface methodology (RSM), to be obtained with an hydrolysate generated at 50.5°C, 2% ES and 231min (H16) was similar to the experimentally obtained value (DPP-IV IC50=0.66±0.10mgmL-1, p>0.05, n=3). Following simulated gastrointestinal digestion (SGID) of H16 (H16_CorPP), the DPP-IV IC50 value increased (p<0.05) to 0.90±0.07mgmL-1. There was no significant difference between the DPP-IV IC50 value of the SGID of MPI (MPI_CorPP, 0.89±0.11mgmL-1) and that of H16_CorPP. Potent known DPP-IV inhibitory peptide sequences were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) within H16, some of which were also present within H16_CorPP. MPI hydrolysates may be of interest for serum glucose regulation in humans.

Hybrid in Silico/in Vitro Approach for the Identification of Angiotensin I Converting Enzyme Inhibitory Peptides from Parma Dry-Cured Ham.

The bioactivity assessment of foodborne peptides is currently a research area of great relevance, and, in particular, several studies are devoted to the antihypertensive effects through the inhibition of angiotensin I converting enzyme (ACE). In the present work, a straightforward workflow to identify inhibitory peptides from food matrices is proposed, which involves a hybrid in vitro/in silico tandem approach. Parma dry-cured ham was chosen as case study. In particular, the advantage of using the hybrid approach to identify active sequences (in comparison to the experimental trials alone) has been pointed out. Specifically, fractions obtained by in vitro gastrointestinal digestion of ham samples of 18 and 24 months of aging have been assessed for ACE inhibition. At the same time, the released peptidomic profiles, which cannot be entirely evaluated by using in vitro assays, have been screened for the inhibition by using an in silico model. Then, to identify novel inhibitory sequences, a series of strong candidates have been synthesized and assessed for their inhibitory activity through in vitro assay. On the one hand, the use of computational simulations appeared to be an effective strategy to find active sequences, as confirmed by in vitro analysis. On the other hand, strong inhibitory sequences were identified for the first time in Parma dry-cured ham (e.g., LGL and SFVTT with IC50 values of 145 and 395 µM, respectively), which is a product of international dietary and economic relevance. Therefore, these findings demonstrate the usefulness of in silico methodologies coupled to in vitro tests for the identification of potentially bioactive peptides, and they give an important contribution to the study of the overall nutritional value of Parma ham.

Identification and quantification of different species in animal fibres by LC/ESI-MS analysis of keratin-derived proteolytic peptides.

In the present paper, a proteomic method for species determination in fibres has been developed. Keratin was extracted from yak, wool and cashmere fibres and digested by trypsin, providing peptide mixtures that were analyzed by liquid chromatography coupled with electrospray mass spectrometry (LC/ESI-MS) in order to identify peptidic species-specific markers able to differentiate the fibres. Several suitable peptide markers were identified and validated in different fibres of different origin and having undergone different technological treatments, showing 100% specificity and 100% selectivity. Most of the peptide markers were also identified by means of high-resolution mass spectrometry, confirming the origin from species-specific keratin sequences. Some peptides were also used for the quantification of the different species in mixed fibres by LC/ESI-MS. Validation experiments and blind tests confirmed their ability to act as very specific quantitative and qualitative markers. The method here developed is a valid complement to the standard benchmark methods for fibre identification and quantification and will be very useful for assessing the authenticity of textile products.

Tolerability of a fully maturated cheese in cow's milk allergic children: biochemical, immunochemical, and clinical aspects.

BACKGROUND: From patients' reports and our preliminary observations, a fully maturated cheese (Parmigiano-Reggiano; PR) seems to be well tolerated by a subset of cow's milk (CM) allergic patients. OBJECTIVE AND METHODS: To biochemically and immunologically characterize PR samples at different maturation stage and to verify PR tolerability in CM allergic children. Seventy patients, with suspected CM allergy, were enrolled. IgE to CM, α-lactalbumin (ALA), ß-lactoglobulin (BLG) and caseins (CAS) were tested using ImmunoCAP, ISAC103 and skin prick test. Patients underwent a double-blind, placebo-controlled food challenge with CM, and an open food challenge with 36 months-maturated PR. Extracts obtained from PR samples were biochemically analyzed in order to determine protein and peptide contents. Pepsin and trypsin-chymotrypsin-pepsin simulated digestions were applied to PR extracts. Each PR extract was investigated by IgE Single Point Highest Inhibition Achievable assay (SPHIAa). The efficiency analysis was carried out using CM and PR oral challenges as gold standards. RESULTS: The IgE binding to milk allergens was 100% inhibited by almost all PR preparations; the only difference was for CAS, mainly α(S1)-CAS. Sixteen patients sensitized to CM tolerated both CM and PR; 29 patients tolerated PR only; 21 patients, reacted to both CM and PR, whereas 4 patients reactive to CM refused to ingest PR. ROC analysis showed that the absence of IgE to BLG measured by ISAC could be a good marker of PR tolerance. The SPHIAa using digested PR preparations showed a marked effect on IgE binding to CAS and almost none on ALA and BLG. CONCLUSIONS: 58% of patients clinically reactive to CM tolerated fully maturated PR. The preliminary digestion of CAS induced by PR maturation process, facilitating a further loss of allergenic reactivity during gut digestion, might explain the tolerance. This hypothesis seems to work when no IgE sensitization to ISAC BLG is detected.