María-Concepción Aristoy

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.

Biochemical and sensory changes in dry-cured ham salted with partial replacements of NaCl by other chloride salts.

The reduction of the content of sodium chloride in dry-cured ham was studied in to prevent the problems related to high sodium intake (i.e. the hypertension). One of the possibilities to reduce the sodium content is the partial replacement of sodium chloride by mixtures of potassium, magnesium and calcium chloride salts. The effect of two salting formulations (formulation II: 50% NaCl-50% KCl and formulation III: 55% NaCl, 25% KCl, 15 CaCl(2) and 5 MgCl(2)) on the protease activity through the dry-curing process and on the sensory characteristics of the final product was evaluated and compared to those of control hams (formulation I, 100% NaCl). Sensory attributes were all affected in the hams containing CaCl(2) and MgCl(2) while hams containing 50% KCl and NaCl (formulation II) were better valued, except for the attribute taste probably due to the potassium contribution to bitter taste.

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.

Peptide generation in the processing of dry-cured ham

Abstract High-performance liquid chromatography (HPLC) was used to separate small water-soluble peptides extracted from dry-cured ham at different processing times (from 0 to 15 months). The elution profile of the deproteinised extracts after gel filtration HPLC showed five ranges of molecular mass: I (4500-2700 Da), II (2700-1200 Da), III (1200-500 Da), IV (500-375 Da) and V (375-160 Da). A clear increase of those peptides below 2700 Da was observed as dry-curing progressed confirming an intense proteolysis especially up to 3.5 months of process. Peptide profiling of deproteinized extracts by reverse phase HPLC and free solution capillary electrophoresis showed substantial changes in peptide patterns along the dry-curing. These mappings could provide valuable information regarding time of processing.

Nucleotides and their degradation products during processing of dry-cured ham, measured by HPLC and an enzyme sensor

The aim of this work was to study how nucleotide degradation during the processing of dry-cured ham is affected when using three types of salting (100% NaCl; 50% NaCl and 50% KCl; 55% NaCl, 25% KCl, 15% CaCl₂ and 5% MgCl₂). Divalent salts in the salting mixture depressed the breakdown rate from the beginning of the process (salting and post-salting) up to the ripening stage (7 months) when the inosine (Ino), hypoxanthine (Hx) and xanthine (X) concentrations matched for the three treatments. The evolution of Hx and Hx+X were analysed by HPLC and an enzyme sensor, respectively, during processing. Time and temperature conditions during the curing time did not affect Hx stability. The usefulness of the enzyme sensor was confirmed and it is a practical tool to determine Hx + X in dry-cured ham, as an index of minimum curing time. A good correlation between enzyme sensor and HPLC data was observed.

Biochemical and sensory properties of dry-cured loins as affected by partial replacement of sodium by potassium, calcium, and magnesium.

An attempt to decrease the NaCl content in dry-cured products through the use of three different salting treatments (II: 55% NaCl, 25% KCl, 15% CaCl(2), 5% MgCl(2); III: 45% NaCl, 25% KCl, 20% CaCl(2), 10% MgCl(2); and IV: 30% NaCl, 50% KCl, 15% CaCl(2), 5% MgCl(2)) in comparison to a control (I: 100% NaCl) was assayed to evaluate the biochemical and sensory characteristics in the final product. Most proteolytic enzyme activities from the loins submitted to the experimental salting treatments, especially treatments II and IV, remained higher than those salted traditionally (control). The higher aminopeptidase activity was also reflected in a larger release of free amino acids. Finally, a sensory paired comparison test revealed that those loins salted with the treatment II were not significantly different from the loins salted traditionally (100% NaCl), so that this treatment could be successfully used for sodium reduction.

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.

Availability of essential amino acids in dry-cured ham

The proteolytic changes taking place in muscle proteins during the processing of dry-cured ham were evaluated from the point of view of its increased nutritional quality. Sodium dodecyl sulphate polyacrylamide gel electro-phoresis (SDS-PAGE) revealed the breakdown of several muscle proteins in dry-cured ham. Peptide analysis by both reverse-phase HPLC (RP-HPLC) or free solution capillary electrophoresis (FSCE) confirmed the intense proteolysis. Dry-cured ham also showed large amounts of all the free essential amino acids: valine (221.4 mg/100g muscle), methionine (90.8 mg/100g muscle), isoleucine (155.7 mg/100g muscle), leucine (246.9 mg/100g muscle), phenylalanine (37.0 mg/100g muscle), tryptophan (25.7 mg/100g muscle) and, in particular, lysine (509.0 mg/100g muscle). Thus, dry-cured ham constitutes a concentrated source of essential amino acids of great importance when the energy intake is low or in a poor nutritional quality diet.

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.