Integrated Evaluation of the Multifunctional DPP-IV and ACE Inhibitory Effect of Soybean and Pea Protein Hydrolysates

Nowadays, notwithstanding their nutritional and technological properties, food bioactive peptides from plant sources garner increasing attention for their ability to impart more than one beneficial effect on human health. Legumes, which stand out thanks to their high protein content, represent valuable sources of bioactive peptides. In this context, this study focused on the characterization of the potential pleotropic activity of two commercially available soybean (SH) and pea (PH) protein hydrolysates, respectively. Since the biological activity of a specific protein hydrolysate is strictly correlated with its chemical composition, the first aim of the study was to identify the compositions of the SH and PH peptides. Peptidomic analysis revealed that most of the identified peptides within both mixtures belong to storage proteins. Interestingly, according to the BIOPEP-UWM database, all the peptides contain more than one active motive with known inhibitory angiotensin converting enzyme (ACE) and dipeptidyl-dipeptidases (DPP)-IV sequences. Indeed, the results indicated that both SH and PH inhibit DPP-IV and ACE activity with a dose-response trend and IC50 values equal to 1.15 ± 0.004 and 1.33 ± 0.004 mg/mL, and 0.33 ± 0.01 and 0.61 ± 0.05 mg/mL, respectively. In addition, both hydrolysates reduced the activity of DPP-IV and ACE enzymes which are expressed on the surface of human intestinal Caco-2 cells. These findings clearly support that notion that SH and PH may represent new ingredients with anti-diabetic and hypotensive effects for the development of innovative multifunctional foods and/or nutraceuticals for the prevention of metabolic syndrome.


Introduction
Food bioactive peptides are short protein fragments (2-20 amino acid residues in length) that, in addition to their known nutritional value, are able to modulate physiological pathways, thereby exerting a positive impact on human health [1]. Hence, both animal and plant foods or by-products with high protein content represent valuable sources of functional bioactive peptides, which can be produced either by enzymatic hydrolysis (using proteolytic enzymes from either plants or microbes), hydrolysis with digestive enzymes (simulated gastrointestinal digestion), or by fermentation. Some studies also used a combination of these methods to produce peptides with a biological activity [2].
In this panorama, legumes, which stand out thanks to their high protein content, are a cheap, sustainable, and a healthy source of nutrition. Notably, soybeans (Glycine max) are on average composed of~35-40% protein [7]. Clinical studies have linked the consumption of soy-based food with a reduced risk of developing a number of chronic diseases, such as obesity, hypercholesterolemia, and insulin-resistance/type II diabetes. As for the active substance in soy foods, protein plays a role in cardiovascular disease prevention [3,8], and some cholesterol-lowering and anti-diabetic peptides have already been singled out in glycinin and β-conglycinin sequences, respectively [9].
In addition to soybean, pea (Pisum sativum L.) represents one of the major legumes in the world and it is composed of~26% protein [7]. Thanks to its excellent yields, availability, and its low production costs, pea is most widely used as a source of commercial proteins for different purpose [10]. Many studies have highlighted the health benefits associated with the consumption of pea protein. In particular, pea protein and its hydrolysates exert antioxidant, antihypertensive, and hypocholesterolemic activities [11][12][13][14].
In general, most previous studies in this area have focused on the consumption of pea and soybean proteins, but the health-promoting activity does not lie in the protein but in the peptides, which are encrypted within the protein and released upon digestion/hydrolysis. Hence, with a more mature perception of the phenomenon, owing to the presence of numerous bioactive peptides, these protein hydrolysates may provide more than one biological activity, therefore eliciting multiple health benefits [15][16][17][18]. For this reason, the production of hydrolysates with multifunctional behavior represents a valid strategy in the development of new generations of functional foods and nutraceuticals [19].
Indeed, this study focused on the deep characterization of the potential pleotropic health-promoting behavior of two commercially available soybean (SH) and pea (PH) protein hydrolysates, respectively. Both SH and PH are obtained by industrial processes, beginning with the selection of soybean and pea protein sources (isolated proteins or enriched protein flours) in combination with a hydrolysis process which is able to deliver an optimized performance in term of structure and, thanks to the resulting taste being almost neutral, allows for a nutritional contribution to food and beverage formulations. SH and PH are then filtered and concentrated before spray drying to obtain a purified product for different food applications.
In addition to, and in part thanks to, their nutritional and technological properties (as a smoothing and whipping agent), SH and PH are successfully used in every formulation to replace nutritional content of animal origin, and they may also be exploited as new valuable and pleotropic ingredients for making innovative multifunctional foods and/or nutraceuticals.
Thus, since the biological activity of a specific protein hydrolysate is strictly correlated with its chemical composition, the first aim of this study was to identify the compositions of SH and PH peptides by peptidomic analysis. Then, their potential hypotensive and antidiabetic activities were evaluated by initially measuring the ability of both hydrolysates to inhibit, in vitro, angiotensin converting enzyme (ACE) and dipeptidyl-dipeptidases (DPP)-IV activity and, afterwards, by carrying out experiments using Caco-2 cells that express both enzymes on their membrane surfaces.

Chemicals
All the commercial chemicals which were used are listed in the Supplementary Materials.

SH and PH Samples Description
A. Costantino & C. S.p.a (Italy), supplied the soybean and pea hydrolysates as spray dried samples (Soy Peptone FM batch 221/00351, 100 g; Pea Protein Hydrolysate GT Plus batch 221F0001, 100 g) directly from the production process. See the Supplementary Materials for the Technical data sheet of both samples.

SH and PH Ultrafiltration
Before proceeding to the assessment of biological activity, SH and PH were passed through ultrafiltration (UF) membranes with a 3 kDa cut-off, using a Millipore UF system (Millipore, Bedford, MA, USA). The recovered peptides solutions (SH (F3) and PH (F3)) were lyophilized and stored −80 • C until use. The experiments were carried out in a half-volume 96-well solid plate (white) with SH and PH at final concentration range of 0.01-2.0 mg/mL and using conditions previously optimized [20]. For further details, see the Supplementary Materials.

In Vitro Measurement of ACE Inhibitory Activity
In order to assess their ACE-inhibitory activity, the SH and PH hydrolysates were tested as previously reported [21,22]. The detailed procedures are available in the Supplementary Materials.

Cell Culture Conditions
Caco-2 cells, obtained from INSERM (Paris, France), were routinely sub-cultured following conditions which have been already optimized [16] and detailed reported in the Supplementary Materials.

Evaluation of Caco-2 Cell Viability by MTT Experiments
In order to assess the safe range of concentrations of the SH and PH hydrolysates on Caco-2 cells, preliminary cell viability experiments were carried out using MTT assay. The detailed procedure is shown in the Supplementary Materials.

Evaluation of the Inhibitory Effect of SH and PH on Cellular DPP-IV Activity
A total of 5 × 10 4 Caco-2 cells/well were seeded in black 96-well plates with a clear bottom. The second day after seeding, the spent medium was discarded and cells were treated with 1.0, 2.5, and 5.0 mg/mL of SH and PH for 1, 3, and 6 h at 37 • C. Experiments were carried out following previously optimized conditions [23]. More details are available in the Supplementary Materials.

Evaluation of the Inhibitory Effect of SH and PH on Cellular ACE1 Activity
Caco-2 cells were seeded on 96-well plates at a density of 5 × 10 4 cells/well and treated with SH and PH (from 0.1 to 5.0 mg/mL) or vehicle in growth medium for 6 h at 37 • C. The ACE inhibitory activity was measured using the ACE1 Activity Assay Kit (Biovision, Milpitas Blvd., Milpitas, CA, USA). The experimental method is detailed in the Supplementary Materials.

Statistical Analysis
All the data sets were checked for normal distribution by the D'Agostino and Pearson test. Since they are all normally disturbed with p-values < 0.05, statistical analyses were carried out by one-way ANOVA (Graphpad Prism 9, GraphPad Software, La Jolla, CA, USA) followed by Tukey's multiple comparison test. For each assay, at least four independent experiments were performed, and each experiment were performed in triplicate. Values were expressed as means ± SD; p-values < 0.05 were considered to be significant.

Peptidomic Characterization of SH and PH
The compositions of the peptides of SH and PH hydrolysates were analyzed by HPLC-ESI-MS/MS. Figure 1 reports the total ion current (TIC) of the MS/MS of eluted peptides, while Table 1 shows the peptide molecular weight distribution of each sample, and the overall peptides, which were identified, are reported in Table S1.   Table 2 lists the identified peptides from the most abundant proteins. Among these, six and nine peptides were identified in the SH and PH samples, respectively. The length of those peptides ranged from 9 to 25 amino acids with a molecular weight in the range 1055 -2422.3 KDa, and in both samples, most of the identified peptides belong to storage proteins. Indeed, 50% of the identified soybean peptides belong to Glycinin G1, whereas in the case of the pea derived peptides, 55% and 33% belong to the Vicilin and Legumin A2 proteins, respectively. Interestingly, according to BIOPEP-UWM database (https:// biochemia.uwm.edu.pl/biopep-uwm/, accessed on 20 May 2022), all the peptides contain more than one active motive with both known inhibitory ACE and DPP-IV sequences.
Indeed, both SH (F3) and PH (F3) reduced the ACE activity with a dose-response trend and IC 50 values equal to 0.40 ± 0.01 and 0.43 ± 0.01 mg/mL, respectively, clearly suggesting that the short peptides which are contained within SH and PH hydrolysates are those responsible for ACE inhibitory activity.
Finally, Table 3 summarizes the comparison of IC 50 values of both samples against DPP-IV and ACE.

Effect of SH and PH Peptides on Caco-2 Cell Viability
The MTT assay was used for assessing the safe range of concentrations of the SH and PH hydrolysates on Caco-2 cells. After a 48 h treatment, any effect on the Caco-2 cell viability was observed in the range of 1.0-5.0 mg/mL versus untreated cells (C) (Figure 4). The ability of SH and PH to inhibit DPP-IV was then investigated in cell-based conditions using Caco-2 cells, which express high levels of this protease on their membranes. Briefly, these cells were treated with SH and PH (1.0-5.0 mg/mL) and their DPP-IV inhibitory effects were assessed in a kinetic mode after 1, 3, and 6 h ( Figure S3A,C). Figure S3A shows that, after 1 h, SH inhibited cellular DPP-IV activity by 25.07 ± 8.07%, 36.91 ± 2.37%, and 40.32 ± 2.46 at 1.0, 2.5, and 5 mg/mL, respectively, and by 25.07 ± 6.78%, 42.46 ± 6.49%, and 48.69 ± 7.29% at the same concentrations after 3 h. The maximum reductions in DPP-IV activity were observed at 6 h ( Figure S3A and Figure 5A), where SH inhibited cellular DPP-IV activity by 37.9 ± 10.0%, 56.6 ± 3.6%, and 59.4 ± 2.4% at 1.0, 2.5, and 5 mg/mL, respectively, versus untreated cells, following a dose-response trend, confirming the in vitro results.

Discussion
In recent decades, many studies have clearly demonstrated the ability of food protein hydrolysates to modulate ACE or DPP-IV activity [20,24,25]. In particular, hydrophobic medium-length and/or shorter peptides from different food sources are considered to be mostly responsible for the inhibition of both enzymes [24,25]. In this panorama, the main limitation of most of previous studies lies in the fact that the characterization of the DPP-IV or ACE inhibitory property of food hydrolysates was performed by analyzing each bioactivity without taking into account that the same hydrolysate may be endowed with both activities. In addition, most works available in the literature rely exclusively on in vitro tests for assessing the biological activity: this is particularly true in the case of the inhibition of DPP-IV and ACE activity. In light of these observations, a standout feature of this study is that the multifunctional DPP-IV and ACE inhibitory activities of two commercially available soybean (SH) and pea (PH) protein hydrolysates were evaluated together using in vitro tests which were implemented by cellular assays that permitted deeper insights into the mechanism of action and, contextually, a consideration of other relevant issues, such as metabolism (this is particularly true when Caco-2 cells were employed).
Overall, the present study demonstrates that SH and PH are effective at reducing DPP-IV and ACE activity in both cell-free and cell-based conditions. More specifically, as indicated in the Table 3, when comparing the calculated IC 50 values, it is clear that SH and PH display the same ability to reduce DPP-IV activity, with SH being about 2-fold more potent than PH in terms of ACE inhibition (***, p < 0.001). In addition, the results indicated that SH and PH are 3-and 2-fold more potent as ACE than DPP-IV inhibitors, respectively (****, p < 0.0001). Moreover, comparing the IC 50 values of each total hydrolysate with those corresponding to each low molecular weight fraction (<3 kDa) ( Table 3), it is clear that the medium-length and shorter peptides, which are abundant in each total hydrolysate (Table 1), are responsible for the biological activities, even though, in the case of SH and SH (F3), the same IC 50 values were observed against ACE enzyme. Interestingly, Table 2 indicates that all the most abundant peptides identified within both SH and PH contained at least one motive with DPP-IV and ACE inhibitory activity that has already been demonstrated, explaining why these hydrolysates are active on two different targets, such as ACE and DPP-IV.
As reported in the Table 3, in the case of PH, the low molecular weight fractions (<3 kDa) are about 0.8-and 1,5-fold more potent than total PH as DPP-IV and ACE inhibitors, respectively (****, p < 0.0001). This result might be explained considering that the bioactivity of total hydrolysate depends strictly on its total composition, including the inactive and active species and possible synergistic or antagonist effects [26,27]. Therefore, it is reasonable to conclude that longer peptides of PH (57.2%) may affect the activity exerted by the medium-length and shorter ones (42.8%) when present in PH hydrolysate. A similar trend was observed for SH. In fact, the SH (F3) is more active than the total hydrolysate against DPP-IV (***, p < 0.001), whereas a similar IC 50 value against the ACE target was calculated (Table 3).
Recently, it was demonstrated that soybean hydrolysates obtained using pepsin and trypsin reduced in vitro DPP-IV activity by 16.3% and 31.4%, and by 15.3% and 11.0%, respectively, at 1.0 and 2.5 mg/mL [18]. Other recent studies demonstrated that the protein hydrolysates from germinated and non-germinated soybean, obtained after simulated gastrointestinal digestion, show a modest ability to inhibit the DPP-IV [28,29]. In addition, soybean hydrolysates obtained using Corolase L10, Promod 144 MG, or Protamex reduced the enzyme, with IC 50 values of 2.5, 0.86, and 0.96 mg/mL, respectively [28]. Indeed, SH is more active than hydrolysates obtained using pepsin, trypsin, and Corolase, whereas its activity is very similar to those of peptide mixtures obtained using both Promod and Protamex.
Pea proteins digested with Corolase L10 and Promod 144 MG inhibited DPP-IV activity with IC 50 values higher than 2.5 mg/mL, whereas the pea hydrolysate obtained using Protamex dropped the enzyme activity with an IC 50 value of 0.96 mg/mL [28]. In this case, PH displays a DPP-IV activity totally in line with those of hydrolysates obtained using Protamex, whereas it is more active than the peptide mixtures obtained using Corolase and Promod.
Soybean proteins extracted with microwave-assisted technology and hydrolysate using Alcalase inhibited the ACE enzyme, and peptides belonging to the low molecular weight fraction were responsible for the biological activities. Moreover, it was also demonstrated that pea proteins digested using a thermolysin-generated peptide mixture exerted ACE inhibitory properties in a spontaneous hypertensive rat (SHR) model and in a clinical study [12,14].
Interestingly, among the peptides belonging to the low molecular weight fraction (<3 kDa) of pea hydrolysate, peptide LTFPG was isolated, whose hypotensive activity has been demonstrated both in vitro and in vivo in the SHR model [12]. Notably, LTFPG is a conserved active peptide that has also been identified and characterized in a lupin sample [15].
DPP-IV and ACE are important membrane peptidases which are physiological expressed by many tissues; i.e., intestine [30,31]. Indeed, human intestinal Caco-2 cells represent a reliable model which has been already developed and validated for the study of peptides with DPP-IV or ACE inhibitory properties [23,32,33]. In this study, it was clearly demonstrated that both SH and PH maintain their ability to reduce the activity of both DPP-IV and ACE on Caco-2 cells, even though both hydrolysates are active at a concentration ranging between 0.1 and 5 mg/mL, indicating that SH and PH are less active in cell-based than in cell-free conditions, respectively. Similar results have been previously obtained on peptic and tryptic hydrolysates of spirulina and chlorella proteins, respectively [32,34,35]. Also, in those cases, it was observed that all the tested hydrolysates were more active in cell-free than in cell-based assays, respectively. The reduced activity in the cellular assays may be explained considering the metabolic ability of Caco-2 cells [32]. Indeed, the intestinal brush border expresses many active proteases and peptidases that might actively hydrolyze food peptides modulating their bioactivity through the production of new breakdown fragments. Therefore, the intestine plays an important role not only in the process of valuable nutrient absorption, but also in actively modulating the physico-chemical and biological profiles of food protein hydrolysates. All these biochemi-cal and cellular results represent an important starting point for future investigations of SH and PH through in vivo and clinical studies on suitable animal models and human volunteers in order to obtain a "proof of concept" with respect to their multifunctional and pleotropic behavior.

Conclusions
In conclusion, our results indicate that SH and PH are multifunctional hydrolysates endowed with both anti-diabetic and hypotensive activity. It is doubtless that they are among the most potent DPP-IV and ACE inhibitor hydrolysates reported in the literature, suggesting that they may be successfully used as new valuable ingredients for the development of innovative functional foods and or dietary supplements for the prevention of cardiovascular disease and metabolic syndrome. Therefore, future in vivo and clinical studies need to be undertaken for their benefits to be better exploited in the nutraceutical sector.