Elizabeth Escudero

Antihypertensive effect and antioxidant activity of peptide fractions extracted from Spanish dry-cured ham

This study examined the antihypertensive and antioxidant activities of water soluble fractions of a Spanish dry-cured ham extract. Antihypertensive activity of a fractionated peptide extract, by size-exclusion chromatography was determined by measuring changes in systolic blood pressure of spontaneously hypertensive rats after oral administration. Every sample exhibited antihypertensive activity, pooled fractions corresponding to 1700 Da or lower were the most antihypertensive with a decrease of 38.38 mm Hg in systolic blood pressure. In vitro experiments revealed marked in vitro angiotensin I-converting enzyme inhibitory activity in fractions corresponding to these elution volumes. Some of the fractions exhibited great 1,1-diphenyl-2-picrylhydrazyl radical-scavenging activity, ranging from 39% to 92% as well as superoxide ion extinguishing ability with values ranging from 41.67% to 50.27% of the antioxidant activity, suggesting the presence of peptides with antioxidant activity. These findings suggest that Spanish dry-cured ham contains peptides with antioxidant and antihypertensive activities.

Angiotensin I-Converting Enzyme Inhibitory Peptides Generated from in Vitro Gastrointestinal Digestion of Pork Meat

The main purpose of this work was to study the generation of Angiotensin I-converting enzyme inhibitory (ACEI) peptides after gastrointestinal digestion of pork meat by the action of pepsin and pancreatin at simulated gut conditions. The hydrolysate was further subjected to reverse phase chromatography in order to separate the fractions with ACEI activity. Using MALDI-TOF/TOF mass spectrometry, 12 peptides were identified in these fractions. It is worth highlighting the novel peptides ER, KLP, and RPR with IC(50) values of 667 microM, 500 microM, and 382 microM, respectively. Results obtained by MALDI-TOF/TOF mass spectrometry were complemented by a second approach consisting of the analysis of the hydrolysate directly by nanoLC-ESI-MS/MS followed by a study of the obtained sequences and comparison with known ACEI peptide sequences. By using these two approaches, a total of 22 peptides were selected for its synthesis and further in vitro assay of ACEI activity. The strongest ACE inhibition was observed for peptide KAPVA (IC(50) = 46.56 microM) followed by the sequence PTPVP (IC(50) = 256.41 microM). Sequence similarity searches revealed that these two peptides derive from muscle titin, constituting the first identified ACEI peptides coming from this protein. This is also the first time that ACEI sequences MYPGIA and VIPEL have been reported. Other identified and synthesized sequences showed less ACEI activity. The obtained results evidence the potential of pork meat proteins as a source of antihypertensive peptides after gastrointestinal digestion.

Purification and Identification of antihypertensive peptides in Spanish dry-cured ham.

Novel sequences exhibiting in vitro ACE inhibitory activity as well as in vivo antihypertensive activity were identified from Spanish dry-cured ham. Water soluble peptide extracts from dry-cured ham were purified by size-exclusion chromatography and reversed-phase high performance liquid chromatography and then, further identification of sequences was carried out by nano-liquid chromatography coupled to tandem mass spectrometry. A total of 73 peptide sequences were identified from active fractions presenting 100% homology with different Sus scrofa skeletal muscle proteins. All identified peptides showed Mr between 374 and 1610 and amino acid sequences between 5 and 14 amino acids in length. Considering the low molecular mass and structural requirements for ACE inhibition some of the identified peptides were synthesised and their IC(50) calculated. The most potent peptide was found to be AAATP (IC(50) value of 100 μM). This peptide also showed good in vivo activity because it decreased systolic blood pressure by -25.62 ± 4.5 mmHg (p<0.05) in spontaneous hypertensive rats after 8 h administration. Other sequences yielded a moderate ACE inhibition. Results from this study show that Spanish dry-cured ham may represent a source of natural peptides with potential benefit for human health.

Identification of novel antioxidant peptides generated in Spanish dry-cured ham

The objective of this study was to purify and identify antioxidant peptides present in a water soluble extract of Spanish dry-cured ham. The initial extract was loaded into a Sephadex G25 column and fractions showing antioxidant activity were collected, pooled together and subjected to reversed-phase chromatography for further purification. Using a nano-LC-MS/MS analysis, 27 peptides were identified in these fractions. Several key peptides were selected for synthesis and the determination of their antioxidant properties using the DPPH radical-scavenging assay and reducing power analysis. The strongest radical-scavenging activity was observed with peptide SAGNPN which showed 50% antioxidant activity at a concentration of 1.5mg/ml. On the other hand, the peptide GLAGA showed the higher reducing power with 0.5 units of absorbance at 700 nm at a concentration of 1mg/ml. Other synthesised sequences showed lower antioxidant activity. The results indicate the potential of Spanish dry-cured ham as a source of antioxidant peptides naturally generated during the dry-curing process.

Stability of ACE inhibitory ham peptides against heat treatment and in vitro digestion.

Angiotensin I-converting enzyme (ACE) inhibitory peptides derived from Spanish dry-cured ham have been examined for their stability during processing and after in vitro digestion. Results indicate that peptides preserved almost the same ACE inhibitory activity before and after applying diverse heating (from 50 to 117°C), times of processing (from 3 to 60min) and simulated in vitro digestion with gastrointestinal proteases. Peptides KAAAAP, AAPLAP, KPVAAP, IAGRP, and KAAAATP were the most potent peptides with IC50 values ranging from 12.37 to 25.94μM. Peptides IAGRP and PTPVP have also been identified in the processed sample (6min at 117°C), and in the in vitro digested sample. This study proves the high stability of ACE inhibitory peptides derived from Spanish dry-cured ham against temperature of processing and gastrointestinal digestion as well as the powerful ACE inhibitory activity of some of the peptides identified in Spanish dry-cured ham.

Characterization of Peptides Released by in Vitro Digestion of Pork Meat

The main objective of this work was to identify and characterize the peptides generated by simulated gastrointestinal digestion of pork meat (longissimus dorsi) by the sequential action of pepsin and pancreatin. The obtained hydrolysate was analyzed by liquid chromatography coupled to a quadrupole time-of-flight mass spectrometer equipped with a nanoelectrospray ionization source (nano LC-ESI-MS/MS). Using this technique 51 different peptides were identified in the hydrolysate, corresponding to fragments of the main structural muscle proteins and some well-known sarcoplasmic proteins. To the best of our knowledge, this constitutes the highest number of peptides identified in pork meat digests. Peptide fragment size ranged from six to sixteen amino acids, being rich in proline residues and thus making them more resistant to further degradation by digestive enzymes. The present study constitutes a clear evidence of the extensive degradation that pork muscle proteins would undergo after gastrointestinal digestion, giving rise to a wide variety of short peptides. So, the use of in vitro digestion contributes to a better knowledge about the generation of peptides from diets with high protein quality.

Antihypertensive activity of peptides identified in the in vitro gastrointestinal digest of pork meat.

This study investigated the in vivo antihypertensive activity of three novel peptides identified in the in vitro digest of pork meat. These peptides were RPR, KAPVA and PTPVP and all of them showed significant antihypertensive activity after oral administration to spontaneously hypertensive rats, RPR being the peptide with the greatest in vivo activity. To our knowledge, this is the first report showing the in vivo antihypertensive action of the three peptides from nebulin (RPR) and titin (KAPVA and PTPVP), thus confirming their reported in vitro angiotensin I-converting enzyme (ACE) inhibitory activity. These findings suggest that pork meat could constitute a source of bioactive constituents that could be utilized in functional foods or nutraceuticals.

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.

Antihypertensive effect of peptides naturally generated during Iberian dry-cured ham processing

Iberian dry-cured ham is considered a product of excellence in Spain and also highly valued by consumers overseas. Its processing conditions include a minimum time of ripening of 24 months which results on a very intense proteolysis of muscular proteins due to the action of endogenous enzymes and the generation of numerous peptides and free amino acids responsible for its characteristic flavour properties. Previous studies in Spanish dry-cured ham of 10 months of ripening identified the amino acid sequences of many of the peptides generated during the proteolysis as well as described their potential as bioactive peptides. However, differences in genetics and processing conditions such as the time of ripening, could result on differences in the action of endogenous enzymes and the generated peptides. In this study, differences in the generated peptides between Iberian dry-cured (24 months of ripening) and traditional Spanish dry-cured ham (14 months of ripening) have been detected. Iberian dry-cured ham extract was separated using size-exclusion chromatography and the ACE-inhibitory activity of the obtained fractions was evaluated in vitro showing up to 97.7% of inhibition in some of the fractions. The antihypertensive effect of the Iberian ham extract was also assayed in vivo using spontaneously hypertensive rats. A significant decrease of 12 mm Hg in SBP after 8 h of ingestion (p < 0.05) that returned to values similar to the control after 24 h of the treatment was observed. The analysis by mass spectrometry in tandem of the peptide extract revealed a total of 2632 peptides which contained the ACE inhibitory Pro–Pro–Lys, Pro–Ala–Pro, and Ala–Ala–Pro repeated in their sequences a total of 322, 302 and 119 times, respectively. This study reveals Iberian dry-cured ham as a source for natural antihypertensive peptides with very high potential in comparison with Spanish dry-cured ham, probably due to the differences in genetics and time of ripening which influence proteolysis and thus the generation of peptides.

A peptidomic approach to study the contribution of added casein proteins to the peptide profile in Spanish dry-fermented sausages

Peptidomics is a necessary alternative in the analysis of naturally generated peptides in dry-fermented processing. The intense proteolysis occurred during the processing of dry-fermented sausages is due to the action of endopeptidases and exopeptidases from both, endogenous muscle origin and lactic acid bacteria (LAB) added in the starter. Sodium caseinate is frequently used as an additive in this type of products because of its emulsifying properties, and consequently influences the protein profile available during the proteolysis. In this study, a mass spectrometry approach has been used to determine the impact of added sodium caseinate in the final peptide profile as well as to analyse its possible influence in the presence of certain previously described casein-derived bioactive peptides.