3.1. Evaluation of Antihypertensive Activity (ACE-I) in Hams with Different Curing Losses (33% and 38%)
Angiotensin-I-converting enzyme (ACE-I) is one of the key enzymes in the regulation of blood pressure, given its participation in the renin angiotensin–aldosterone system (RAAS) [
41].
Figure 2 indicates the evolution of ACE inhibitory activity throughout the assay as a function of peptide concentration in hams with different curing loss.
Table 1 and
Table 2 indicate the effect of pig genetic line and processing on ACE inhibitory activity, represented as the concentration of peptides necessary (mg/mL) to inhibit 50% of this activity (IC
50). All samples showed ACE inhibitory activity, which increased with increasing concentration of the peptides. This could be due to the presence of small peptides smaller than 3 kDa [
15,
42,
43]. The ham that showed the highest ACE-I activity (IC
50) was RIB
33, having a greater potential to control diseases associated with the cardiovascular system [
44].
The IC
50 in the final product was lower (higher activity) in Iberian hams than in white hams (
Table 1), probably due to the longer curing time used in Iberian hams, consistent with what has been observed in other studies [
15]. However, when the weight loss is 33%, no significant differences were observed between genetic lines (
Table 2).
The processing method did not significantly influence ACE-I activity in Iberian hams (
p ≥ 0.05), although it was slightly higher in salt-reduced hams (RIB
38) (
Table 1).
A study conducted at the Catholic University of Murcia (UCAM) showed that the consumption of cured ham rich in bioactive peptides has a positive influence on the regulation of glycaemia and cholesterolemia in healthy patients, so that far from being a restricted food, its regular consumption has a positive effect on modifiable risk factors associated with premature cardiovascular disease [
20].
Table 3 presents the results of the effect of processing time on the production of ACE inhibitory activity. In RWC, hams with a weight loss of 33% have greater antihypertensive activity than those with a 38% weight loss (
p ≤ 0.05). However, in Iberian hams, processing time does not imply greater ACE-I activity. In Serrano and Panxian hams, some have observed that this activity increases significantly in the last curing phase [
30,
42]. Furthermore, other authors have also observed this behavior for dipeptide AA, which increases its activity by 40% from 6 months to 12 months of ham curing [
45]. Because ACE-I has been detected in the hams studied, it could counteract the harmful effects of sodium in the body [
46].
3.2. Antioxidant Activity
The DPPH radical study to evaluate the antioxidant activity of samples has been described as a suitable procedure for this purpose [
47].
Cured ham has been identified as a source of peptides with antioxidant activity [
48]. Despite this, no studies have evaluated antioxidant activity in salt-reduced hams. DPPH scavenging activity is also a commonly used technique to evaluate antioxidant capacity. This activity is directly associated with hydrophobic AA in peptides, so these AA will exist in antioxidant peptides [
49,
50].
Figure 3 indicates the evolution of in vitro antioxidant activity as a function of the peptide concentration of the hams with different processing; all samples show higher antioxidant activity as the concentration of peptides increases. RWC
38 has higher antioxidant activity, reaching 75% inhibition. In RIB
38, we also observed an increase in antioxidant activity as the curing process progressed, higher than the healing process, higher than in RIB
33. TIB
38 shows lower antioxidant activity than RWC
38 and is like RIB
38. The ham with the lowest antioxidant activity was RIB
33 in all the peptide concentrations we studied.
Table 4 and
Table 5 indicate the in vitro DPPH radical (antioxidant) scavenging activity of the ham in the drying and final phases, respectively. The concentration (mg/mL) of each NPN needed to inhibit 50% of the antioxidant activity (IC
50) was evaluated. All the samples studied showed antioxidant activity both in the drying phase and in the final product.
Table 4 indicates the IC
50 values obtained for each sample and the effect of pig genetic line and processing on the antioxidant activity of the TIB, RIB, and RWC hams. Genetic line significantly influenced the uptake of the DPPH radical in these samples (
p ≤ 0.05), as did RIB
33 and RWC
33 (
Table 5). However, salt reduction and deboning did not influence the antioxidant activity of the samples, although it was higher in TIB
38. The RWC
38 hams have the highest antioxidant activity because they reached the IC
50 with a lower peptide concentration (0.155 ± 0.013 mg/mL). These data coincide with the higher proteolysis index obtained in white hams in a previous study [
3] due to the higher activity of cathepsins and calpains of this breed [
4]. In Serrano hams, peptides have been identified with an IC
50 at a concentration of 1.5 mg/mL [
46]. Furthermore, Jinhua hams in eastern China, managed an IC
50 at a lower concentration of 1 mg/mL [
51]. However, in subsequent studies, Jinhua hams achieved an IC
50 at 2.5 mg/mL, whereas Xuanwei hams required a concentration of 4.5 mg/mL [
52]. In contrast to this study, others have shown that meat from purebred and Duroc-crossed Iberian pigs would be less predisposed to oxidation than those from white pig breeds [
53]. Others claim that meat products such as Iberian ham have a greater antioxidant capacity than fresh ham products before being cured, or other foods such as red wine [
54].
Table 6 shows that, for both salt-reduced Iberian and white hams, the increase in curing time significantly affects the antioxidant capacity of the samples (
p ≤ 0.05), being higher in RIB
38 and RWC
38. This could be due to the increase observed in proteolytic activity in the later stages of curing [
3], often related to the increase in temperature [
23].
The results show that the antioxidant capacity of the hams increases as the curing process progresses and is not affected by the reduction in the Iberian ham. Therefore, cured hams would be a good source of antioxidant activity despite containing pro-oxidant agents such as salt and heme and even reactive oxygen species (ROS), which can cause cell damage [
55,
56].
3.3. Bioactive Peptide Sequencing
The peptides present in the samples of the hams from the five batches studied (RIB
38, RIB
33, RWC
38, RWC
33, and TIB
38) were sequenced by LC-MS/MS analysis.
Table 7 indicates the number of sequenced peptides per sample. The ham with the highest number of sequenced peptides was RWC
38, RIB
33 had the lowest number of peptides sequenced, and RIB
38 presented a greater number of peptides than TIB
38. This coincides with the values of non-protein nitrogen and the proteolysis index (PI) obtained in a previous study in hams with a loss of 38%, where the highest and lowest NPN and PI were found in salt-reduced white hams (RWC
38) and traditionally cured hams (TIB
38), respectively [
3].
In this study, no peptides already obtained from the database were found among the peptides obtained in the proteomic study. Therefore, their bioactivity has not been demonstrated in previous studies.
In other studies, identical sequences were found in cured ham, for example, KAAAAP, AAPLAP, and KPVAAP, with origin in different types of myosin protein, were identified as the peptides with the highest ACE-I activity in Teruel PDO ham [
30], and are also present in Serrano ham [
20].
Their stability and their retention of bioactivity during processing and after in vitro digestion were examined. In vivo studies showed that the AAATP peptide had the highest antihypertensive activity, lowering systolic blood pressure with a short-term effect [
46]. Furthermore, other sequences with antihypertensive activity were identified, such as ASGPINFT and DVITGA (both also derived from myosin protein). In another study, AAATP with the KA dipeptide had DPP4 inhibitory activity that would contribute to improving the concentration of glucose in the blood [
20].
The antioxidant power is another bioactivity studied in traditional Serrano ham [
46]. The SAGNPN peptide has been identified to have the greatest capacity to donate electrons, neutralizing the oxidative capacity, even more than the peptides synthesized [
46,
57]; furthermore, the peptide GLAGA had the highest reducing power [
58]. Moreover, SNAAC and AEEEYPDL, identified in the cured ham, had high antioxidant activity [
59].
Numerous bioactive peptides with a high antihypertensive power have been identified in Iberian ham, which are higher than those in Serrano ham. The sequences that are repeated most frequently, which coincide with the BIOPEP database, are PPK, PAP, and AAP [
60]. However, the following dipeptides, such as EA, with ACE-I activity, or PP and VE, which showed ACE- and DPP4-inhibitory activity have also been sequenced [
61].
Dipeptides with anti-inflammatory and cardiovascular protective activity (PA, GA, VG, EE, ES, DA, and DG) have been identified in hams with reduced salt content, besides contributing to the product aroma and flavor [
62]. However, no studies have been found in fresh deboned and salt-reduced Iberian or white ham.
Study of Putative Activity Peptide Sequences
A search has been conducted for peptide precursors that may contain biopeptides in their sequence and could theoretically be activated after digestion. This technique is useful for very small sequences (less than seven Amino Acids) and by using the proteomics procedure, it is impossible to detect them.
To contextualize the type of bioactivity of the samples, a Z-scoring was performed to plot the variation between samples regarding the mean of the different activities (heatmap). The results are shown in
Figure 4; a higher intensity red color means that this activity will be over-represented regarding the mean of the five samples. Bioactivities are grouped according to the intensity of occurrence in each sample. In addition, the succession of rows and columns is rearranged to avoid intersection of the dendrogram lines. Blue lines represent the value of the coefficient. Individually, we have represented in which sample each group of activities stands out for each group or clusters (
Figure 5), each corresponding to a group of bioactivities. In RWC
33, the main activity is immunostimulatory. No studies have been found on the presentation of this bioactivity in cured ham.
RWC
38 showed the highest antioxidant activity. These results coincide with the activity observed in vitro using DPPH (
Table 4). Other bioactivities that stand out in this sample are those of neuropeptide activation, hypolipemic, anti-inflammatory, anti-cancer, and hypotensive activities. Antioxidant and hypotensive activity have also been well studied in white pig hams.
Several studies confirm the occurrence of these bioactivities in ham [
48]. Recently, peptides with anti-inflammatory activity have been identified in Xuanwei hams, showing reduced symptoms of inflammatory bowel disease in mice, and it has been pro-posed that these peptides could be a functional drug in patients suffering from this disease [
63].
In RIB
33 hams, the predominant activities are stimulatory, immunomodulatory, a CaMPDE inhibitor, a DPP4 inhibitor, antithrombotic, and ACE-I, consistent with our results for antihypertensive activity (
Table 2), where RIB
33 had the highest ACE-I. Likewise, the Iberian ham showed greater ACE-I activity compared to the traditional Serrano hams [
60].
Antihypertensive activity is well studied in ham [
20,
21,
60]. There are studies that would claim that Serrano ham would be a good source of DPP4 and that these peptides could be an adjunct in the treatment of type 2 diabetes [
64].
RIB
38 hams have HMG-CoA reductase inhibitory, regulatory, and immunological activity. HMG-CoA reductase inhibitors play an important role in the control of hyper-cholesterolemia and, indirectly, in the control of the onset of cardiovascular disease. Other studies have found dipeptides such as DA, DD, EE, ES, and LL in cured ham, which have been identified as the main inhibitors of this coenzyme [
65]. Furthermore, TIB
38 hams stand out for their binding, ubiquitin mediator protein activator, renin inhibitor, dipeptidyl peptidase III inhibitor, and embryotoxic activity, bioactivities that have not yet been studied.
Each group of bioactivities was represented by a color (
Figure 4). In
Figure 5, we can observe six clusters, one for each group of bioactivities, where the values of that group of bioactivities are quantified for each sample.
Because bioactive sequence fragments are found in the samples, a spider web plot with normalized quantification of the peptide precursors of the five hams is shown in
Figure 6. This distribution allows differentiation between hams according to activity. The potential bioactivity of the peptides identified in each sample is reflected by using the same scale and amplitude and the same scale and width of the axis, allowing comparison between them.
Cured ham is considered a good source of different bioactive peptides that have important functional activities, such as the inhibition of the angiotensin converting enzyme, hypoglycemic, and anti-inflammatory activities [
29].
3.4. Bioactivity Analysis Based on Amino Acid Composition
The composition of Amino Acid (AA) used to analyze the bioactivity of the samples was conducted on 38% cured hams, as these had the best organoleptic characteristics and, therefore, would be destined for the end consumer.
In bioactivity studies, it is important to consider the structural properties of sequences [
66]. Certain characteristics, such as size, hydrophobicity, and composition, may influence the stability or bioavailability of the peptides. Approximately 20 sequences were selected from those identified in each ham with less than 1.5 kDa and with a maximum of 12 AA in their chain. Processing time causes the size of the peptides to decrease and increases the antioxidant activity of the peptides [
49], as short AA sequences are more likely to be bioactive [
62,
67]. In addition, over 50% of the AAs in the chain should be hydrophobic, as this contributes to antioxidant activity [
68]. The presence of AAs A, D, E, G, L, P, and V confers antioxidant and antihypertensive activity on the peptide sequence [
68,
69,
70], and this activity is directly related to the molecular weight of the peptide sequence [
71]. However, the presence of H, Y, W, F, M, and C could inhibit free radicals by direct electron transfer [
67]. The amino acid sequences of the peptides identified from salt-reduced Iberian hams (RIB) are shown in
Table 7.
3.5. Identification of Peptides Present in RIB Hams
The AA sequences of the peptides identified from the hydrolysates of salt-reduced Iberian hams (RIB) are shown in
Table 8.
Antioxidant activity is highly present among the selected sequences. Some have over 50% of the peptides that provide antioxidant activity. The LDLALEKD, AAFPPDVGGN, AGNPDLVLPV, and AFGPGLEGGL peptides stand out for having over 80% of AAs that would favor antioxidant activity, with AFGPGLEGGL having the highest antioxidant activity (90% of its AAs).
The AAFPPDVGGN and AFPPDVGGN have been identified as present in pork [
72] and six sequences containing them have been found (AFPPDVGGN, AAFPPDVGGN, AFPPDVGGNV, AAFPPDVGGGGNV, AFPPDVGGGGNVD, and AAFPPDVGGGGNVD). The peptides FPPDVGGN and FPPDVGGNVD originating from the protein could also be derived from these sequences, identified as myosin [
46]. From the action of the enzyme, dipeptidyl peptidase [
73] could be released from some sequences as the VD dipeptide, which would have DPP4 inhibitory activity and, therefore, anti-diabetic activity [
64,
74].
The most prominent sequence is CLFVCR, as it has 83% of hydrophobic AAs, 67% of AAs conferring ACE-I activity, and 50% of AAs scavenging free radicals. ACE-I activity would be more present in sequences containing hydrophobic AA residues in the three C-terminal positions [
75]. For this sample, the sequence AGNPDLVLPV has three hydrophobic AAs at the C-terminus. The dipeptide WK could be extracted from longer peptides originating from β-enolase, such as DGADFAKW (
Table 8). This dipeptide has been identified as an inhibitor of DPP4 [
76,
77]. Likewise, the sequences LIGIEVPH, IDLIEKPM, FDKIEDMA, WNDEIAPQ, and DLDISAPQ originate from the IE and SI dipeptides of the α-enolase protein; they have been described as ACE- and DPP4-inhibitory peptides, respectively [
74,
78]. These dipeptides could be responsible for the high antihypertensive activity observed in this study for sample RIB
38 (
Table 1).
Recently, some dipeptides related to anti-inflammatory activity, which could confer cardiovascular protection, have been identified in salt-reduced cured hams [
62]. These dipeptides are PA, GA, DA, and DG and could be derived from sequences found in RIB
38 (ALQPALKF, WNDEIAPQ, MADTFLEH, DLDISAPQ, DGADFAKW, MADTFLEH, and AGNPDLVLPV), with GA being mainly identified in the study.
Table 9 indicates the prominent peptide sequences detected in the ham samples of traditionally cured Iberian ham (TIB). In these samples, six of the selected sequences presented over 80% of the AAs that could provide antioxidant activity to the product (AFPPDVGGNV, AAFPPDVGGN, DVVLPGGNL, VAVGDKVPAD, DIAVDGEPLG AGNPDLVLPV, and AFGPGLEGGL). RIB
38 has the highest antioxidant activity. However, the sequence that stands out for having the highest amount of hydrophobic peptides is ILPGPAPW. This peptide comprises the Pro-Ala-Pro sequence, one of the most repeated sequences among the bioactive peptides described in the literature [
15], which would confer good antioxidant activity to the sample [
68]. Furthermore, these sequences could contribute to the bioactivity described for TIB
38 ham (
Figure 4).
Four sequences (ILPGPAPW, VMGAPGAPM, GDLGIEIPA, and AGNPDLVLPV) have three hydrophobic AAs at the C-terminus, and are therefore more likely to develop ACE-I activity [
75]. The AGNPDLVLPV sequence matches that found in RIB
38. In the GDLGIEIPA and IELIEKPM sequences, we can find the dipeptide IE dipeptide related to ACE-I bioactivity [
74]. The same six sequences identified in RIB
38 have also been found in TIB
38 (AAFPPDVGGNV, AAFPPDVGGN, AFPPDVGGNVD, AAFPPDVGG-NVD, AFPPDVGGN, and AFPPDVGGNV), have been identified in pork, and could have inhibited DPP4 derived from the dipeptide DV [
61].
From a comparison study between traditional and salt-reduced cured hams, di-peptides such as DA, PA, and VG would be present in a higher proportion in traditional cured hams [
62]. The last two sequences of
Table 9 could derive from the peptides found in sample TIB
38 and could contribute to its anti-inflammatory and antihypertensive activity. Other sequences, which were identified in this study, are GA (ACE and DPP4 inhibitory activities) and DG (ACE-I activity).
The selected AA sequences of salt-reduced white hams (RWC) are shown in
Table 10. The sequences that stand out for having over 80% of AAs and confer antioxidant activity are DLAEDAPW and AEVIALPVE. The latter sequence is also present in RIB
38, with the highest antioxidant activity.
The sequence that stands out for having the highest amount of hydrophobic AAs is ILPGPAPW, the same as TIB
38, and has one of the most repeated sequences among bio-active peptides (PAP) [
15]. Furthermore, this sequence has 75% of AAs that confer antioxidant activity, three hydrophobic AAs at the C-terminus that confer ACE-I activity, and 13% of AAs that could inhibit free radicals. However, six peptide sequences with three hydrophobic AAs at the C-terminus (ILPGPAPW, AVIGPSLPL, VMGAPGAPM, ISAPSADAPM, DLAEDAPW, and GDLGIEIPA) were found in the RWC
38 samples, which conferred ACE-I activity. However, in RIB
38, of the sequences selected, none had over two hydrophobic AAs at the C-terminus. The sequences ILPGPAPW, VMGAP-GAPM, and GDLGIEIPA match TIB
38. Furthermore, the LKGADPEDVITGA and GADPEDVITGA would contain the bioactive peptide DVITGA in their chain, related to high ACE-I activity due to the presence of AA alanine at the C-terminus [
39,
46]. Despite this, the RWC
38 ham showed the least antihypertensive activity (
Figure 4); it would be necessary to study if these peptides confer ACE-I activity and in what quantity they are present. There are more sequences from which the dipeptide IE could be derived, already described as a precursor of this bioactivity [
74].
The sequence identified in RWC
38, FKAEEEYPDLS, once digested, could cause the peptide AEEEYPDL, derived from protein creatine kinase and identified as a potent antioxidant [
59]. Using multiple reaction monitoring (MRM), it was quantified at a concentration of 0.148 fg/g in cured ham [
79]. This could explain why the RWC
38 hams showed the highest antioxidant activity (
Figure 4) and the highest rate of proteolysis obtained in white hams [
3].
In RWC
38 hams, the same six sequences described in RIB
38 and TIB
38 (AAFPPDVGGNV, AAFPPDVGGNV, AFPPDVGGNVD, AAFPPDVGGNVD, AFPPDVGGNV, and AFPPDVGGNV) have been identified. However, this would explain the difference in proteolytic activity [
3] between different pig genetic lines and influence of processing, because salt-reduced Iberian hams (RIB
38 and RIB
33) had the highest DPP4 inhibitory activity, lower than white hams (RWC
38 and RWC
33) and traditional Iberian ham (TIB
38).
In a recent study of salt-reduced white ham, hydrophobic PA dipeptides (related to bitter taste and with ACE-I and anti-inflammatory activity) and VG (related to bitter and umami taste and with ACE-I activity) were identified that could be derived from the sequences identified in our sample [
62].
Different unique and common sequences that could act as peptide precursors and that have been identified in the samples would be responsible for the bioactivities found in the different types of ham. The results show that in RIB38 ham, the precursors found could be responsible for its high antihypertensive capacity, noting that the change in processing varies the sequences identified in both samples (RIB38 and TIB38). Furthermore, peptides already referenced in the literature have been found in the RWC38 ham, including a sequence that gives rise to a potent antioxidant peptide (AEEEYPDL) that would explain its increased bioactivity. However, none of these co-inciding peptides are found in Iberian hams.