Marta Gallego

Dipeptidyl peptidase IV inhibitory peptides generated in Spanish dry-cured ham

Dipeptidyl peptidase IV (DPP-IV) inhibitors are promising new therapies for type 2 diabetes. The aim of this study was to assay DPP-IV inhibitory peptides that can be present in a water soluble extract of Spanish dry-cured ham. Such an extract was fractionated by size-exclusion chromatography and the in vitro DPP-IV inhibitory activity determined in each collected fraction. Then, several peptides previously identified in dry-cured ham extracts or known to be products of DPP IV action were synthesised and assayed for DPP-IV inhibition. Peptides KA and AAATP showed the strongest DPP-IV inhibitory activity, with IC50 values of 6.27 mM and 6.47 mM, respectively. Dipeptides AA, GP, PL, and carnosine, as well as peptides AAAAG, ALGGA, and LVSGM were also DPP-IV inhibitors, although at a lower degree. These findings suggest the potential of Spanish dry-cured ham as a natural precursor of DPP-IV inhibitory peptides. These biopeptides could also be used as ingredients for functional foods or pharmaceutical products against type 2 diabetes.

Degradation of LIM domain-binding protein three during processing of Spanish dry-cured ham

Extensive proteolysis takes place during the processing of dry-cured ham due to the action of muscle peptidases. The aim of this work was to study the degradation of LIM domain binding protein 3 (LDB3), which is located at the Z-lines of the sarcomere, at different times during the Spanish dry-cured ham processing (2, 3.5, 5, 6.5, and 9 months). A total of 107 peptides have been identified by mass spectrometry, most of them generated from the first region of the protein sequence (position 1-90) providing evidence for the complexity and variability of proteolytic reactions throughout the whole process of dry-curing. Methionine oxidation has been observed in several peptides by the end of the process. The potential of some of the identified peptides to be used as biomarkers of dry-cured ham processing has also been considered.

Small peptides hydrolysis in dry-cured meats.

Large amounts of different peptides are naturally generated in dry-cured meats as a consequence of the intense proteolysis mechanisms which take place during their processing. In fact, meat proteins are extensively hydrolysed by muscle endo-peptidases (mainly calpains and cathepsins) followed by exo-peptidases (mainly, tri- and di-peptidyl peptidases, dipeptidases, aminopeptidases and carboxypeptidases). The result is a large amount of released free amino acids and a pool of numerous peptides with different sequences and lengths, some of them with interesting sequences for bioactivity. This manuscript is presenting the proteomic identification of small peptides resulting from the hydrolysis of four target proteins (glyceraldehyde-3-phosphate dehydrogenase, beta-enolase, myozenin-1 and troponin T) and discusses the enzymatic routes for their generation during the dry-curing process. The results indicate that the hydrolysis of peptides follows similar exo-peptidase mechanisms. In the case of dry-fermented sausages, most of the observed hydrolysis is the result of the combined action of muscle and microbial exo-peptidases except for the hydrolysis of di- and tri-peptides, mostly due to microbial di- and tri-peptidases, and the release of amino acids at the C-terminal that appears to be mostly due to muscle carboxypeptidases.

Titin-derived peptides as processing time markers in dry-cured ham.

The complex proteolysis in Spanish dry-cured ham processing generates large amounts of small peptides and free amino acids which are responsible for the characteristic texture and flavour of this traditional product. The aim of this work was to study the degradation of the giant protein titin throughout the dry-curing process (2, 3.5, 5, 6.5, and 9 months) through the use of proteomic tools. A total of 320 peptides have been identified by nanoliquid chromatography coupled to tandem mass spectrometry, being some of them identified only at 9 months of processing. In order to confirm the absence of these peptides at other times of processing, MALDI-TOF MS was also employed as a fast and easier technique. Only four peptides, KDEAAKPKGPIKGVAKK, KKLRPGSGGEK, KNTDKWSECAR and ISIDEGKVL, were exclusively identified at 9 months of curing by using both methodologies so that these peptides could be used as potential biomarkers of dry-cured ham processing time.

The use of label-free mass spectrometry for relative quantification of sarcoplasmic proteins during the processing of dry-cured ham

The aim of this work was to quantify changes in the abundance of the major sarcoplasmic proteins throughout the ham dry-curing process by using a label-free mass spectrometry methodology based on the measurement of mass spectral peak intensities obtained from the extracted ion chromatogram. For this purpose, extraction of sarcoplasmic proteins was followed by trypsin digestion and analysis by nanoliquid chromatography coupled to tandem mass spectrometry (Q/TOF) for the identification and relative quantification of sarcoplasmic proteins through individual quantification of trypsinised peptides. In total, 20 proteins, including 12 glycolytic enzymes, were identified and quantified. The accuracy of the protocol was based on MS/MS replicates, and beta-lactoglobulin protein was used to normalise data and correct possible variations during sample preparation or LC-MS/MS analysis. Mass spectrometry-based proteomics provides precise identification and quantification of proteins in comparison with traditional methodologies based on gel electrophoresis, especially in the case of overlapping proteins. Moreover, the label-free approach used in this study proved to be a simple, fast, reliable method for evaluating proteolytic degradation of sarcoplasmic proteins during the processing of dry-cured ham.

Optimisation of a simple and reliable label-free methodology for the relative quantitation of raw pork meat proteins.

Recent advances in proteomics have become an indispensable tool for a fast, precise and sensitive analysis of proteins in complex biological samples at both, qualitative and quantitative level. In this study, a label-free quantitative proteomic methodology has been optimised for the relative quantitation of proteins extracted from raw pork meat. So, after the separation of proteins by one-dimensional gel electrophoresis and trypsin digestion, their identification and quantitation have been done using nanoliquid chromatography coupled to a quadrupole/time-of-flight (Q/ToF) mass spectrometer. Relative quantitation has been based on the measurement of mass spectral peak intensities, which have been described that are correlated with protein abundances. The results obtained regarding linearity, robustness, repeatability and accuracy show that this procedure could be used as a fast, simple, and reliable method to quantify changes in protein abundance in meat samples.

Evidence of peptide oxidation from major myofibrillar proteins in dry-cured ham.

In this study, a peptidomic approach has been used in the identification of naturally generated peptides during a dry-curing process, showing methionine (Met) oxidation in their sequence. A total of 656 peptides derived from major myofibrillar proteins in Protected Designation of Origin (PDO) Teruel dry-cured ham have been identified by nanoliquid chromatography coupled to tandem mass spectrometry (nLC-MS/MS), including 120 peptides showing methionine oxidation. The percentage of oxidised peptides in the studied proteins ranged from 6% to 35%, being peptides derived from nebulin, titin, myosin heavy chains, and troponin I proteins, those showing the highest number of oxidised methionine. The identification of the peptide sequence incorporating the oxidised amino acid provides valuable information of neighbouring amino acids, degree of hydrolysis of the sample, and characteristics of the peptide, which might be very useful for a future better understanding of the oxidation mechanisms occurring in dry-curing processing.

Peptidomics as a tool for quality control in dry-cured ham processing

Spanish dry-cured ham is a high quality product whose economic value is mainly given by its curing time. An intense proteolysis takes place throughout the dry-cured processing, which results in the generation of a high amount of peptides and free amino acids responsible for the final quality of dry-cured hams. In this work, a peptidomics approach has been used to study the evolution of peptides throughout the ham dry-curing process, identifying and quantifying the generated peptides in order to define potential quality biomarkers. For this purpose, dry-cured ham extracts at different processing times (0, 2, 3.5, 5, 6.5 and 9months) were fractionated by size-exclusion chromatography and analysed by nanoliquid chromatography coupled to tandem mass spectrometry. Differences obtained in the relative quantification of peptides by using a label-free methodology were useful to establish differences between processing times, being peptides generated by the degradation of myosin light chain 1 protein mainly responsible for the observed differences during the last stages of curing. In particular, APAPAPAPPKEEKI and PAPAPAPAPAPAPAPPKE, exclusively identified at 9months of curing, would be potential markers to control the time of curing and thus the final quality of dry-cured hams. Biological significance A peptidomics approach has been used to study the evolution of peptides throughout the ham dry-curing process, identifying peptides APAPAPAPPKEEKI and PAPAPAPAPAPAPAPPKE, which were only detected at 9months of process. So, they would constitute good potential markers to control the time of processing and thus the final quality of dry-cured hams.

Effect of cooking and in vitro digestion on the antioxidant activity of dry-cured ham by-products

Dry-cured ham by-products have been traditionally used in Mediterranean household cooking of broths and stews. The aim of this work was to evaluate the effect of cooking treatments and in vitro gastrointestinal digestion on the antioxidant activity of natural peptides found in bones from Spanish dry-cured hams. The antioxidant activity was tested using five different assays and results demonstrated that cooking using conventional household methods increased the antioxidant activity of ham by-products when assessed using different antioxidant assays with the exception of the ABTS radical scavenging measurement assay. Simulated gastrointestinal digestion showed no significant effect on the antioxidant activity of ham by-products and antioxidant activity decreased when assessed using the ORAC and β-carotene bleaching assays. Analysis by MALDI-TOF MS revealed a considerable breakdown of peptides due to the action of gastrointestinal enzymes, mainly in samples cooked at 100°C for 1h. In addition, 459 peptides derived from 57 proteins were identified and quantified using mass spectrometry in tandem, evidencing that peptides derived from collagen protein were responsible for the differences in antioxidant activities observed between the uncooked and cooked samples after digestion. The results show the potential of dry-cured ham bones as a source of antioxidant peptides that retain their bioactivity after household cooking preparations and gastrointestinal digestion.

Stability of the potent antioxidant peptide SNAAC identified from Spanish dry-cured ham

Antioxidant peptides positively regulate oxidative stress in the human body as well as delay, retard or prevent protein and lipid oxidation in food products. Spanish dry-cured ham has been reported as a good source of bioactive peptides, being SNAAC the most active antioxidant peptide identified to date. In this work, the stability of this peptide against in vitro digestion, heat treatments and different salt concentrations was evaluated using three methods for measuring antioxidant activity: β-carotene bleaching assay, ABTS radical scavenging capacity and ORAC assay. The results evidenced a marked decrease in the antioxidant activity of SNAAC after gastrointestinal digestion, and the MALDI-ToF MS analysis revealed the degradation of the peptide after the process, the generation of the fragment SNAA and the presence of a peptide dimer throughout the in vitro digestion. On the other hand, the peptide SNAAC showed good heat stability after exposure to different temperatures (50°C, 72°C, and 90°C), but its antioxidant activity evaluated by ORAC assay decreased substantially when exposed to 100°C as compared with the control at 37°C. SNAAC remained stable in the presence of salt at concentrations ranging from 0 to 8% NaCl as well as it was able to inhibit about 40% of lipid oxidation in an emulsion system. These results reported the stability of the antioxidant peptide SNAAC to several conditions used in meat industry for the processing of dry-cured hams and ham-derived products and its effectiveness to partially prevent the lipid oxidation in these products. However, some strategies would be needed in order to increase the stability of the peptide during gastrointestinal digestion and thus improve its bioavailability to be used as functional food ingredient.