Combinations of Legume Protein Hydrolysates Synergistically Inhibit Biological Markers Associated with Adipogenesis

The objective was to investigate the anti-adipogenesis potential of selected legume protein hydrolysates (LPH) and combinations using biochemical assays and in silico predictions. Black bean, green pea, chickpea, lentil and fava bean protein isolates were hydrolyzed using alcalase (A) or pepsin/pancreatin (PP). The degree of hydrolysis ranged from 15.5% to 35.5% for A-LPH and PP-LPH, respectively. Antioxidant capacities ranged for ABTS•+ IC50 from 0.3 to 0.9 Trolox equivalents (TE) mg/mL, DPPH• IC50 from 0.7 to 13.5 TE mg/mL and nitric oxide (NO) inhibition IC50 from 0.3 to 1.3 mg/mL. LPH from PP–green pea, A–green pea and A–black bean inhibited pancreatic lipase (PL) (IC50 = 0.9 mg/mL, 2.2 mg/mL and 1.2 mg/mL, respectively) (p < 0.05). For HMG-CoA reductase (HMGR) inhibition, the LPH from A–chickpea (0.15 mg/mL), PP–lentil (1.2 mg/mL), A–green pea (1.4 mg/mL) and PP–green pea (1.5 mg/mL) were potent inhibitors. Combinations of PP–green pea + A–black bean (IC50 = 0.4 mg/mL), A–green pea + PP–green pea (IC50 = 0.9 mg/mL) and A–black bean + A–green pea (IC50 = 0.6 mg/mL) presented synergistic effects to inhibit PL. A–chickpea + PP–lentil (IC50 = 0.8 mg/mL) and PP–lentil + A–green pea (IC50 = 1.3 mg/mL) interacted additively to inhibit HMGR and synergistically in the combination of A–chickpea + PP–black bean (IC50 = 1.3 mg/mL) to block HMGR. Peptides FEDGLV and PYGVPVGVR inhibited PL and HMGR in silico, showing predicted binding energy interactions of −7.6 and −8.8 kcal/mol, respectively. Combinations of LPH from different legume protein sources could increase synergistically their anti-adipogenic potential.


Introduction
Obesity is defined as excessive fat accumulation, characterized by increased visceral white adipose tissue mass and abnormalities in lipid metabolism that present a risk to health [1,2]. However, its prevalence has doubled around the world and steadily increased over the past 50 years, reaching pandemic levels [3]. This pathological condition results from complex interactions between genes and environmental factors, such as calorie-dense food intake, sedentary lifestyle and stress [4,5].
were ground separately in a commercial blender in a 1:10 grain/water ratio. The pH of the supernatant was adjusted to 8.0 with 1 M sodium hydroxide, and protein extraction was carried out at 35 • C with stirring for 1 h. The mixture was centrifuged at 5000× g for 15 min at 25 • C. Then, the pH was adjusted to the isoelectric point of the different legumes with 1 M hydrochloric acid to precipitate proteins, followed by centrifugation at 10,000× g for 20 min at 4 • C. The supernatant was discarded and the pellet was freeze-dried in a Lab Conco Freeze Dryer 4.5 (Kansas, MO, USA). Legume protein isolates (LPI) were stored at −20 • C until further analysis.

Simulated Gastrointestinal Digestion
Legume protein hydrolysates (LPH) were obtained after simulated gastrointestinal digestion with pepsin/pancreatin (PP) following the method described by Mojica et al. (2015) [25]. Briefly, LPI was suspended in water (1:20 w/v) and autoclaved for 20 min at 121 • C to denature proteins and improve hydrolysis. Sequential enzyme digestion was carried out with pepsin/substrate 1:20 (w/w) (pH 2.0) followed by pancreatin/substrate 1:20 (w/w) at pH 7.5 at 37 • C for 2 h each. The hydrolysis was stopped by heating at 75 • C for 20 min, and the resulting LPI hydrolysates were centrifuged at 20,000× g for 15 min at 4 • C. LPH were dialyzed to eliminate salts using a 500 Da molecular weight cutoff membrane and then freeze-dried in a LabConco FreeZone Freeze Dry System. Hydrolysates were stored at −20 • C until further analysis.

Alcalase Enzymatic Digestion
Alcalase (A) was used for the hydrolysis of LPI. First, a portion of LPI was suspended in water (1:20 w/v) and autoclaved for 20 min at 121 • C. Then, enzymatic digestion was carried out using a protease/substrate ratio of 1:20 (w/w), time of hydrolysis 2 h, with pH and temperature optimal for alcalase activity (pH 7.0, T: 50 • C, respectively). Protein hydrolysis was stopped by heating at 75 • C for 20 min, and the resulting LPH were centrifuged at 20,000× g for 15 min at 4 • C. LPH were dialyzed to eliminate salts using a 500 Da molecular weight cutoff membrane and then freeze-dried in a LabConco FreeZone Freeze Dry System. Hydrolysates were stored at −20 • C until further analysis [24].

Degree of Hydrolysis (DH)
An aliquot of 64 µL of sample solution was placed in 1 mL of 0.2125 M sodium phosphate buffer pH 8.2. A 0.05% TNBS (trinitrobenzene sulfonic acid) solution was sequentially added to light-protected test tubes and then the reaction mixture was placed in a water bath at 50 • C for 30 min. The reaction was stopped by adding 0.1 M sodium sulfite. After cooling for 15 min at room temperature, the absorbance was measured at 420 nm. The total hydrolysis of the sample was carried out using 6 N HCl at 110 • C for 24 h. A calibration curve was produced using leucine (0 to 10 mM) as standard. DH was calculated according to the following equation: where h is the number of free amino groups in the hydrolyzed sample and h total is the number of free amino groups after complete hydrolysis of the LPI.

Gel Electrophoresis Analysis SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis)
The freeze-dried LPI of the five legume types tested and their respective LPH were analyzed by SDS-PAGE, under reducing conditions (1:20 β-mercaptoethanol, β-ME). Precast (4% to 20%) gradient polyacrylamide, Tris-HCl gels were used with a Bio-Rad Criterion Cell under a constant voltage of 200 V for 35 min. Standards (10 to 250 kDa) were used to calculate molecular mass. After staining Foods 2020, 9, 1678 4 of 22 with Simply Blue Safe Stain overnight, and destaining with water, the gel was visualized using a Chemidoc TM XRS + System (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Identification and Characterization of Potentially Bioactive Peptides
LPH were analyzed by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS) using a Q-tof Ultima mass spectrometer (Water, Milford, CT, USA), equipped with an Alliance 2795 HPLC system. Separation of the components was performed by using a mobile phase of Solvent A (95% H 2 O, 5% ACN and 0.1% formic acid) and Solvent B (95% ACN, 5% H 2 O and 0.1% formic acid) using a flow rate of 400 µL min −1 . The elution was in a linear gradient (0 min, 100% A; 1 min, 100% A; 2 min, 90% A, 10% B; 6 min, 60% A, 40% B; 10 min, 100% B; 12 min, 100% B; 14 min, 100% A; 15 min, 100% A). The temperature was kept at 20 • C during the whole procedure. A splitter with a split ratio of 1:10 was used, where one part was used by the mass spectrometer and ten parts were used for the waste. The Q-tof Ultima mass spectrometer was equipped with a Z-spray ion source. Using the positive ion electrospray mode (+ESI), the analysis on the Q-tof was carried out in V-mode with an instrument resolution between 9000 and 10,000 based on full width at half maximum, with a flow rate of 400 µL min −1 . The source temperature was set at 80 • C and desolvation temperatures were set at 250 • C. The Q-tof was operated at a capillary voltage of 3.5 kV and a cone voltage of 35 V. The final detector was a microchannel plate with high sensitivity. The MassLynx 4.1 V software (Waters, Milford, CT, USA) was used to control the instruments and to process the data to obtain the highest probability of the peptide's sequences. Confirmation of peptide sequences in each legume protein was performed using the BLAST ® tool (http://www.blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 15 February 2019).

ABTS Radical Scavenging Activity
The ABTS •+ radical cation was produced by reacting 7 mM 2,2 -azinobis (3-ethyl-benzothiazoline -6-sulfonic acid) diammonium salt (ABTS) with 2.45 mM potassium persulfate. The mixture was left to stand in the dark at room temperature for 20 h before use (overnight). The radical was stable in this form under these conditions for more than 48 h. The ABTS •+ solution was diluted with 0.01 M phosphate buffer (pH 7.4) to an absorbance of 0.70 ± 0.02 at 734 nm. The scavenging activity was expressed as IC 50 values Trolox equivalent per mg of the sample using the following equation y = 411.91x + 8.4207, R 2 = 0.99.

DPPH Radical Scavenging Activity
Antioxidant activity of legume hydrolysates was determined using DPPH (2,2-diphenyl-1 -picrylhydrazyl) free radical scavenging assay. Protected from light, 100 µL of each sample or buffer as control was added to 1.5 mL of methanolic DPPH solution (0.1 mM) (1,1-diphenyl-2-picrylhydrazyl) and stirred by vortex (3000 rpm) for 30 s. After 30 min of incubation, the absorbance of the solution was read at 517 nm. The analytical curve was constructed using 5-205 µg/mL of the Trolox solution to determine TE (y = 0.0016 x − 0.0294; R 2 = 0.94). The results were expressed as IC 50 values of Trolox equivalent (TE) per mg of the sample.

Nitric Oxide (NO) Radical Scavenging
Nitric oxide production was estimated by the accumulation of nitrite (NO 2 ), a stable product of the nitric oxide (NO) reaction with oxygen in Griess reagent (1% sulfanilamide and 0.1% N-1-(naphtha-yl) ethylenediamine dihydrochloride in 2.5% H 3 PO 4 ). Sodium nitroprusside (SNP) was used as the NO Foods 2020, 9, 1678 5 of 22 donor. NO reacts with oxygen to produce nitrate and nitrite. Briefly, SNP (10 mM) in phosphate-buffered saline was mixed with different concentrations of each LPH sample (0.1-5 mg/mL freeze-dried hydrolysate). The samples were incubated at 25 • C. After 120 min, 0.5 mL incubated solution was mixed with 0.5 mL of Griess reagent. Results were expressed as % inhibition related to PBS treatment [26].  [27]. Briefly, to measure the formation of free fatty acids upon hydrolysis of TG (4:0) or TG (8:0), each substrate was prepared with one volume of the substrate (50 mM dissolved in tert-butanol), also containing the pH indicator, which was mixed vigorously on a vortex with nine volumes of buffer solution to reach a final substrate concentration of 5 mM. Buffer solution included 2.5 mM 3-Morpholinopropane-1-sulfonic acid (MOPS), 0.5 mM sodium taurodeoxycholate hydrate (NaTDC), 150 mM NaCl, 6 mM CaCl 2 . Then, 20 µL of enzyme solution at appropriate dilution in buffer was added in each microplate well and 100 µL of substrate emulsion was quickly added using an eight-channel pipette. Subsequently, the plate was placed in a microtiter plate scanning spectrophotometer (x-Mark™, Bio-rad, Hercules, CA, USA) and shaken for 5 s before each reading. The decrease in absorbance at a wavelength corresponding to the λmax of the pH indicator was recorded every 30 s at 37 • C. Blanks without enzyme were performed and data were collected at least in triplicate for 15 min.

Hydroxy-3-methylglutaryl Coenzyme a Reductase (HMG-CoA Reductase) Activity Assay
The HMG-CoA reductase assay kit CS-1090 from Sigma-Aldrich (St. Louis, MO, USA) was carried out under conditions recommended by the manufacturer at 37 • C. Statin drug (pravastatin) was employed as a positive control. In summary, aliquots containing NADPH (4 µL), HMG-CoA substrate (12 µL) and a buffer pH 7.4 were placed into a UV compatible 96-well plate. The analyses were initiated by the addition of HMG-CoA reductase (2 µL) in each well and incubated in the presence or absence of pravastatin (250 nM) or 5-0.1 mg/mL hydrolysate concentration of each LPH. The rate of NADPH consumed was monitored by reading the decrease in absorbance at 340 nm by microtiter plate scanning spectrophotometer (x-Mark™, Bio-rad, Hercules, CA, USA). The HMG-CoA-dependent oxidation of NADPH and the inhibition properties of LPH were measured by the absorbance reduction, which is directly proportional to the enzyme activity.

Isobolographic Analysis of PL Inhibitory Activity by LPH
The interactions were validated by isobolographic analysis in which the combinations were comprised of equieffective doses of the individual components. Using the IC 50 values of each LPH, the additive line was plotted and the equieffective dose was calculated. Subsequently, a dose-response curve of PL inhibition was obtained in a fixed-ratio for the mixture of LPH (1:1) that was based on the IC 50 values of each protein hydrolysate. The experimental IC 50 values for the LPH combinations were calculated. In an isobologram, when the protein hydrolysate combination IC 50 lies on the theoretical IC 50 add line, then the mixture is considered to be synergistic. An interaction index (γ) was calculated according to the following formula: IC 50 combination/IC 50 theoretical. Gamma values around 1 (γ = 1) indicated additive interaction; γ > 1 implied and antagonistic interaction and γ < 1 indicated a synergistic interaction.

Molecular Docking (In Silico Analysis)
The structural mechanism by which the peptides present in LPH interact with the enzymes, pancreatic lipase and HMG-CoA reductase was evaluated by in silico analysis through molecular Foods 2020, 9, 1678 6 of 22 docking, as described by   [28]. Molecular docking analysis was performed to predict individual peptide biological potential using DockingServer ® [29]. Peptides were designed using the software Instant MarvinSketch (Chem Axon Ltd., Budapest, Hungary). The MMFF94 force field [30] was used for the energy minimization of ligand molecules, peptides, orlistat and pravastatin. Gasteiger partial charges were added to the peptide ligand atoms (peptides). Non-polar hydrogen atoms were merged, and rotatable bonds were defined. Docking calculations were carried out on PL (1LPB) and HMG-CoA reductase (1DQ9) protein crystal structures. Essential hydrogen atoms, Kollman united atom type charges and solvation parameters were added with the aid of AutoDock tools [31]. Affinity maps and spacing were generated using the Autogrid program. AutoDock parameter set-and distance-dependent dielectric functions were used in the calculation of the van der Waals and the electrostatic terms, respectively. Initial position, orientation and torsions of the ligand molecules were set randomly. Each docking experiment was derived from 100 different runs that were set to terminate after a maximum of 250,000 energy evaluations. The population size was set up to 150. During the search, a translational step of 0.2 Å, and quaternions and torsion steps of 5 were applied.

Statistical Analysis
The results were expressed as the mean ± SD of at least two independent experiments with three repetitions each and analyzed through ANOVA. Statistical significance (p < 0.05) was determined using Student's t-test for comparing mean pairs and Tukey's test for multiple mean comparisons using software JMP version 8.0 (SAS Institute, Cary, NC, USA).

Protein Profile for LPI and LPH by Gel Electrophoresis Analysis
The SDS-PAGE analysis was conducted to observe the effect of enzymatic hydrolysis with alcalase and pepsin/pancreatin in the molecular weight distribution of LPI ( Figure 1A). Three lanes per legume are depicted. The first lane represents LPI before hydrolysis; the second lane contains the LPI after hydrolysis with the commercial enzyme alcalase (A); and the third lane contains the LPI after simulated gastrointestinal digestion with pepsin and pancreatin (PP). Protein components of the LPI ranged from 10 to 100 kDa. Enzymatic hydrolysis exerted an effect on molecular weight distribution, mostly to the high molecular weight fractions. Black bean protein isolate (first lane) reveals several major bands which are 47 and 44 kDa phaseolin bands [25] ( Figure 1A). Phytohemagglutinin is also visible, which corresponds to the 31 kDa band. Major identified proteins in common black bean were phaseolin, lectin, protease and α-amylase inhibitors, Kunitz trypsin inhibitor and Bowman-Birk inhibitor [32]. Green pea LPI revealed convicilin (72 kDa), legumin (25, 39 kDa) and vicilin (44, 32, 16 kDa) fractions [33]. In the case of lentil LPI, the most intense bands observed in the SDS-PAGE correspond to subunits of vicilin (48 kDa) and convicilin (63 kDa). Other lower molecular mass bands were observed which can represent gamma-vicilin and a mixture of albumin polypeptides [34]. The protein profile of chickpea and fava bean was also influenced by the hydrolysis process.

Degree of Hydrolysis
The effect of the digestion conditions on the degree of hydrolysis (DH) was evaluated. Figure 1B shows the comparison between the LPI hydrolyzed with alcalase and by a simulation of gastrointestinal digestion with PP. The average yield of hydrolysis ranged from 2.72% to 26.61% and 21.50% to 45.26% for A-LPH and PP-LPH, respectively. In general, across all the legume hydrolysates, DH showed great variability; however, significant differences between the enzymes alcalase and PP were observed in the black bean, chickpea and fava bean hydrolysates. In the case of green pea and lentil hydrolysates, no statistical differences were found using alcalase and PP enzyme. The lowest values of DH were observed in chickpea and fava bean LPI hydrolyzed with alcalase.

Degree of Hydrolysis
The effect of the digestion conditions on the degree of hydrolysis (DH) was evaluated. Figure 1B shows the comparison between the LPI hydrolyzed with alcalase and by a simulation of gastrointestinal digestion with PP. The average yield of hydrolysis ranged from 2.72% to 26.61% and 21.50% to 45.26% for A-LPH and PP-LPH, respectively. In general, across all the legume hydrolysates, DH showed great variability; however, significant differences between the enzymes alcalase and PP were observed in the black bean, chickpea and fava bean hydrolysates. In the case of green pea and lentil hydrolysates, no statistical differences were found using alcalase and PP enzyme. The lowest values of DH were observed in chickpea and fava bean LPI hydrolyzed with alcalase.

Peptide Sequences and Predicted Bioactivity
The peptide sequences resulting from the PP and alcalase digestion were identified by highperformance-liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS). Twenty-seven peptide sequences were obtained by alcalase enzymatic digestion and thirty-four peptides were sequenced from PP. Peptide identity was confirmed by Blast tool and most peptides were identified in legume parental proteins. Moreover, the physicochemical properties of the Protein profile of black bean, green pea, chickpea, lentil and fava bean, before and after hydrolysis with alcalase and simulated gastrointestinal digestion. Each sample is presented in 3 wells: 1st well belongs to LPI profile (1), 2nd belongs to protein isolate after hydrolysis with alcalase (2), and the 3rd belongs to protein isolate after pepsin/pancreatin digestion (3). STD: standard. (B) Degree of hydrolysis (%) of legume protein isolates hydrolyzed with alcalase (A-LPH) and after simulated gastrointestinal digestion with pp (PP-LPH). Different uppercase letters indicate significant differences between alcalase and pepsin/pancreatin digestion (p < 0.05); different lowercase letters indicate significant differences (p < 0.05) among legume protein hydrolysates. Results represent the mean ± SD of at least two independent experiments.

Peptide Sequences and Predicted Bioactivity
The peptide sequences resulting from the PP and alcalase digestion were identified by high-performance-liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS). Twenty-seven peptide sequences were obtained by alcalase enzymatic digestion and thirty-four peptides were sequenced from PP. Peptide identity was confirmed by Blast tool and most peptides were identified in legume parental proteins. Moreover, the physicochemical properties of the peptides are listed in Table 1. A-hydrolysates and PP-hydrolysates showed peptides with around six and eight amino acids, respectively. Most of their amino acids were aliphatic (glycine, proline, valine and alanine), which are non-polar and hydrophobic. Molecular weight for both A and PP peptides ranged from 440 (SPPE) to 1408 Da (VNPDPAGGPTSGRAL). The isoelectric point ranged from the acidic pI 2.78 (DLVLDVPS) to alkaline pI 11.52 (KPSSAAGAVR). The net charge was neutral in 46% of the peptides sequenced; 36% of the peptides presented negative charge and 18% were positively charged. Hydrophobicity ranged from 4.88 (TKAGGTAF) to 22.78 kcal/mol (VELVGPK).
The biological potential of peptide sequences generated by A and PP is shown in Figure 2. The percentage of biological potential is relative to the total bioactive peptides produced by each enzymatic system used. Most peptides in all the legumes evaluated potentially exert activity by blocking dipeptidyl peptidase-IV (DPP-IV) and angiotensin-converting enzyme (ACE). Potential biological activities such as stomach mucosa regulator, antiamnestic, antithrombotic, antioxidative and glucose uptake promoters were also observed. The biological potential of peptide sequences generated by A and PP is shown in Figure 2. The percentage of biological potential is relative to the total bioactive peptides produced by each enzymatic system used. Most peptides in all the legumes evaluated potentially exert activity by blocking dipeptidyl peptidase-IV (DPP-IV) and angiotensin-converting enzyme (ACE). Potential biological activities such as stomach mucosa regulator, antiamnestic, antithrombotic, antioxidative and glucose uptake promoters were also observed.

ABTS Radical Scavenging
The antioxidant capacity of the whole hydrolysate (LPH) was measured spectrophotometrically by the disappearance of the blue/green stable ABTS •+ radical, caused by scavenging ( Figure 3A). The concentration of inhibitor required to produce 50% inhibition was calculated (IC 50 ) and the results of ABTS •+ radical assay were presented as Trolox equivalent (TE) antioxidant capacity using Trolox as standard. PP-lentil hydrolysate was demonstrated to have the highest ability to scavenge the ABTS •+ radical (IC 50 = 0.30 ± 0.13 TE/mL), which was followed by PP-black bean (IC 50 = 0.60 ± 0.06 TE/mL) and both PP and A-green pea hydrolysates (IC 50 = 0.61 ± 0.03 and 0.60 ± 0.03 TE mg/mL, respectively) (p < 0.05).

Pancreatic Lipase Inhibitory Activity
LPH were tested for their ability to inhibit PL. All the hydrolysates demonstrated to inhibit the enzyme in a dose-dependent manner, as shown in Figure 4A. There were no significant differences between PP and A-black bean hydrolysates and PP and A-chickpea, (p < 0.05). PP-green pea hydrolysate presented the lowest IC 50 value (0.8 ± 0.06 mg/mL) among all the legume PP hydrolysates. Nevertheless, the IC 50 values of the PP-lentil hydrolysate (7.7 ± 0.64 mg/mL) and A-fava bean hydrolysate (11.8 ± 1.75 mg/mL) were significantly higher (less potent inhibition) compared to the rest of the hydrolysates.  Orlistat synthetic inhibitor IC50 = 2.73 µg/mL [31]. Different capital letters indicate significant differences between alcalase and pepsin/pancreatin digestion (p < 0.05); different lowercase letters indicate significant differences (p < 0.05) among legume protein hydrolysates. (B) Inhibition potential of LPH to HMG-CoA (IC50 mg/mL). Simvastatin synthetic inhibitor IC50 = 0.08 µg/mL. Different capital letters indicate significant differences between alcalase and pepsin/pancreatin digestion (p < 0.05); different lowercase letters indicate significant differences (p < 0.05) among legume protein hydrolysates. Results represent the mean ± SD of at least two independent experiments.

Isobolograms of the LPH Interactions
The results of the isobolographic PL assay are presented in Figure 5A,B. It was observed that three combinations of LPH showed synergistic potential, namely PP-green pea and A-black bean, PP-green pea and A-green pea, A-black bean and A-green pea, with IC50 values of 0.40 ± 0.00 mg/mL, 0.95 ± 0.02 mg/mL and 0.65 ± 0.04 mg/mL, respectively.  [31]. Different capital letters indicate significant differences between alcalase and pepsin/pancreatin digestion (p < 0.05); different lowercase letters indicate significant differences (p < 0.05) among legume protein hydrolysates. (B) Inhibition potential of LPH to HMG-CoA (IC 50 mg/mL). Simvastatin synthetic inhibitor IC 50 = 0.08 µg/mL. Different capital letters indicate significant differences between alcalase and pepsin/pancreatin digestion (p < 0.05); different lowercase letters indicate significant differences (p < 0.05) among legume protein hydrolysates. Results represent the mean ± SD of at least two independent experiments.

HMG-CoA Reductase Inhibitory Activity
HMGR catalyzes the rate-limiting step of the mevalonate pathway that leads to cholesterol biosynthesis; thus, inhibition of its enzymatic activity plays a significant role in lowering endogenous cholesterol levels during hypercholesterolemia [35]. Figure 4B shows the inhibition of HMGR activity by PP and A hydrolysates. A-chickpea was the most effective hydrolysate with an IC 50 = 0.15 ± 0.04 mg/mL, followed by PP-lentil (IC 50 = 1.17 ± 0.52 mg/mL), A-green pea (IC 50 = 1.45 ± 0.25 mg/mL) and PP-green pea (IC 50 = 1.50 ± 0.07 mg/mL). By contrast, the highest IC 50 value was observed in PP-chickpea (IC 50 = 8.84 ± 0.74 mg/mL).

Isobolograms of the LPH Interactions
The results of the isobolographic PL assay are presented in Figure 5A,B. It was observed that three combinations of LPH showed synergistic potential, namely PP-green pea and A-black bean, PP-green pea and A-green pea, A-black bean and A-green pea, with IC 50 values of 0.40 ± 0.00 mg/mL, 0.95 ± 0.02 mg/mL and 0.65 ± 0.04 mg/mL, respectively.
For the inhibition of HMGR, interactions among LPH presented synergistic and additive potential ( Figure 5B). A synergistic effect was observed with the combination of A-chickpea and PP-black bean (IC 50 = 1.33 ± 0.07), while additive interactions were observed between A-chickpea and PP-green pea (IC 50 = 0.84 ± 0.04 mg/mL) and PP-lentil and A-green pea (IC 50 = 1.34 ± 0.00 mg/mL).

Figure 5. (A) Synergistic interaction of PP-green pea and A-black bean hydrolysates to block
pancreatic lipase (IC50 mg/mL). Bar plots represent the mean ± standard deviation of at least two independent experiments, with only the expected and combined values compared statistically. Different number of stars means significant difference (p < 0.05). The lower the values, the more potency. Results represent the mean ± SD of at least two independent experiments. (B) Interactions between legume hydrolysate combinations to block pancreatic lipase and 3-hydroxy-3methylglutaryl coenzyme A reductase HMGR. Results are expressed in IC50 mg/mL; γ value < 1 synergism, γ value = 1 additive effect, γ value > 1 antagonism.

Molecular Docking Study of Peptides Inhibiting Pancreatic Lipase and HMG-CoA Reductase
Molecular docking analysis was performed to predict the potential of the LPH to interact with PL and HMG-CoA reductase enzymes. Peptide and macromolecular target docking shows theoretical affinity, type of interactions and distances [36,37]. Table 2 shows the minimum estimated free energy for peptides sequenced from LPH, with PL and HMGR. Estimated free energy indicates that compounds with the most negative value present higher potential to interact with the target enzyme. The peptides studied had free energy values ranging from −5.5 (CSSSSG) to −7.6 (FEDGLV) kcal/mol for PL. Peptide FEDGLV was obtained from A-lentil and CSSSSG from PP-chickpea. In the case of Figure 5. (A) Synergistic interaction of PP-green pea and A-black bean hydrolysates to block pancreatic lipase (IC 50 mg/mL). Bar plots represent the mean ± standard deviation of at least two independent experiments, with only the expected and combined values compared statistically. Different number of stars means significant difference (p < 0.05). The lower the values, the more potency. Results represent the mean ± SD of at least two independent experiments. (B) Interactions between legume hydrolysate combinations to block pancreatic lipase and 3-hydroxy-3-methylglutaryl coenzyme A reductase HMGR. Results are expressed in IC 50 mg/mL; γ value < 1 synergism, γ value = 1 additive effect, γ value > 1 antagonism.

Molecular Docking Study of Peptides Inhibiting Pancreatic Lipase and HMG-CoA Reductase
Molecular docking analysis was performed to predict the potential of the LPH to interact with PL and HMG-CoA reductase enzymes. Peptide and macromolecular target docking shows theoretical affinity, type of interactions and distances [36,37]. Table 2 shows the minimum estimated free energy for peptides sequenced from LPH, with PL and HMGR. Estimated free energy indicates that compounds with the most negative value present higher potential to interact with the target enzyme. The peptides studied had free energy values ranging from −5.5 (CSSSSG) to −7.6 (FEDGLV) kcal/mol for PL. Peptide FEDGLV was obtained from A-lentil and CSSSSG from PP-chickpea. In the case of HMGR, binding affinities of peptides ranged from −6.1 (CSSSSG and GPPVDVPQ) to −8.8 (PYGVPVGVR) kcal/mol, obtained from PP-chickpea and PP-fava bean, respectively. Orlistat and pravastatin were also evaluated as a control, presenting free energy values of −5.6 and −6.2 kcal/mol for pancreatic lipase and HMGR, respectively. The most stabilized pose of the peptide bonds with PL and HMG-CoA reductase can be observed in Figure 6A,B, respectively. All the peptides identified in the different legume protein hydrolysates were able to interact with amino acid residues of PL and HMGR catalytic site. These peptides interacted with the enzymes mainly through hydrogen bonds and hydrophobic, polar and cation π interactions.
Foods 2020, 9, x FOR PEER REVIEW 16 of 24 HMGR, binding affinities of peptides ranged from −6.1 (CSSSSG and GPPVDVPQ) to −8.8 (PYGVPVGVR) kcal/mol, obtained from PP-chickpea and PP-fava bean, respectively. Orlistat and pravastatin were also evaluated as a control, presenting free energy values of −5.6 and −6.2 kcal/mol for pancreatic lipase and HMGR, respectively. The most stabilized pose of the peptide bonds with PL and HMG-CoA reductase can be observed in Figure 6A,B, respectively. All the peptides identified in the different legume protein hydrolysates were able to interact with amino acid residues of PL and HMGR catalytic site. These peptides interacted with the enzymes mainly through hydrogen bonds and hydrophobic, polar and cation π interactions.

Discussion
Vicilin and legumin-like proteins comprise more than 70% of the total proteins found in the legumes used for this study. However, differences in the amino acid sequence of homologous proteins contained in the legume species used can lead to changes in the protein profile obtained after the hydrolysis treatment.
A higher DH is indicative of more peptide bonds cleaved, resulting in lower molecular weight peptides [38]. The application of the enzymatic hydrolysis with A and PP notably changed the structure of the legume native proteins. These changes can be noted in the disappearance of protein bands higher than 10 kDa after treatment and the formation of polypeptides with lower molecular mass. Similar findings were obtained in a study of legume isolate proteins [39].
The enzymatic action of pepsin and pancreatin in a sequential way to perform the simulated gastrointestinal digestion increases the enzyme selectivity and specificity, enabling the production of a larger number of small protein fractions or free amino acids [40]. Increasing cleavage sites are available for the second enzyme after the first one has acted, thus forming a more diverse mixture of amino acid residues in comparison to alcalase. According to these results, the action of the gastrointestinal enzymes was more effective to cleave distinct peptide bonds in black bean, chickpea and fava bean LPI. Even though enzymatic hydrolysis with PP treatment exhibited higher DH in the mentioned legumes, the action of alcalase showed the same effect as PP in green pea and lentil. These results suggest that fractions of vicilin commonly found in plants such as peas or lentils were susceptible to digestion with the different enzymatic treatments [41,42]. On the other hand, due to the very broad substrate specificity, alcalase can hydrolyze most peptide bonds within a protein molecule. Therefore, its use on substrates like legume proteins causes a high degree of hydrolysis, producing many peptides of small sizes [43]. The low DH values obtained in fava bean and chickpea LPH with alcalase could be related to vicilin, as this protein is known for its emulsifying, foaming and gelling properties [44,45]. Moreover, legumes rich in glutamic acid could have led to lower degradation by alcalase. This enzyme has a negative charge in physiological pH, which may diminish the DH due to the net negative charge present in glutamic acid [46]. In addition, resistance toward enzymatic activity may be a consequence of structural differences and the compactness of proteins [33]. Similar results were found by Ghribi et al. (2015) [47] in chickpea isolates hydrolyzed with alcalase, where it was found that a small degree of chickpea hydrolysis (DH = 4%) could enhance the emulsifying properties of chickpea protein. In another study performed with fava bean by Liu et al. (2019) [48], this moderate alcalase hydrolysis suggests the production of suitable lower molecular mass that could result in a more flexible peptide structure, increased surface charge and hydrophobicity, which could positively affect the emulsifying activity [41,49].
All the hydrolysates demonstrated the potential to inhibit pancreatic lipase and HMG-CoA reductase in a dose-dependent manner. According to the literature, Lee et al. (2015) [50] performed an inhibition assay for pancreatic lipase with phenolic compounds from methanolic extracts of seven selected legumes. The results showed pancreatic lipase inhibition in a dose-dependent manner, with no significant differences among red bean, chickpea, black soybean, yellow soybean and black-eyed pea extracts. The lowest reported IC 50 values were for red bean (5.90 ± 0.59 mg/mL), chickpea (6.97 ± 2.19 mg/mL) and black soybean (6.65 ± 0.62 mg/mL). These values are higher (less potent) compared to most of the hydrolysates obtained in this work. The variation may be attributed to the differences in the extraction system, the varieties used and the bioactive compounds present in legume protein hydrolysates. Another report associated the peptide fragments released after protein enzymatic hydrolysis with the potential to act as HMGR inhibitors. For instance, the authors identified peptide sequences below 3 kDa from amaranth protein with hypocholesterolemic potential (GGV, IVG and VGVL) [51]. Recent studies performed on soybean glycinin and β-Conglycinin protein-derived peptides have also reported that these peptides could inhibit HMGR activity in vitro and in silico studies [52,53].
From all evaluated legume protein hydrolysate combinations used to block PL and HMG-CoA reductase activities, four combinations acted synergistically. PP-green pea with A-black bean, PP-green pea with A-green pea and A-black bean with A-green pea were demonstrated to inhibit more efficiently the activity of PL. Furthermore, the A-chickpea and PP-black bean combination acted synergistically against HMG-CoA reductase activity. Previous studies have demonstrated the efficacy of legume protein hydrolysates to inhibit markers related to obesity (black bean, green pea and chickpea hydrolysates) [19,[54][55][56]. Several reports have demonstrated synergistic interactions among diverse bioactive compounds. For instance, synergistic and additive interactions of peptides produced from black bean for ACE inhibition were reported by Luna-Vital et al. (2015) [57]. The combination of peptides GLTSK and MTEEY showed a synergistic interaction, reducing the concentration needed to inhibit half of the enzymatic activity by approximately 30%. Further studies focusing on each peptide sequence are essential to confirm the possible effects of their interaction. These interactions can be significant and lead to lower effective dosages associated with the effects of pure compounds [58][59][60]. There is evidence that legume protein hydrolysates could interact with enzymes related to obesity. Exploring the possibilities to improve their performance, along with increasing the desired effect, is important.
The peptide profile of the protein hydrolysates obtained was different due to the legume source and enzyme used. As a result of the enzymatic hydrolysis and the specificity of the enzymes used, the biological activity of the legume protein hydrolysates was conferred by the specific combination of both protease used and source of proteins. The amino acid profile in peptide sequences with higher biological activity was associated with aliphatic amino acids, sulfur-containing and aromatic amino acids. Through molecular docking, it was possible to predict the binding affinity to the target enzymes (LP and HMG-CoA reductase). Peptide sequences presented lower predicted free energy values compared to the available drugs orlistat and pravastatin. These results suggest that they have a higher affinity for the catalytic site, thus exerting significant potential anti-adipogenesis activity. Non-polar interactions among peptide sequences and evaluated enzymes were more common due to the frequency of the aliphatic amino acids.
In addition to the effect of the enzymes related to lipid metabolism (LP and HMG-CoA reductase), obesity is associated with the production of reactive oxygen species and increased oxidative stress [8]. Peptides are able to contribute to the antioxidant defense in the body, being able to rapidly scavenge reactive oxygen species before cellular damage, therefore inactivating them [61]. The antioxidant capacity of the legume hydrolysates was measured using free radicals generated by ABTS and DPPH. Even though the meaning of the results obtained with these assays is limited as they use no physiological radicals, it is still possible to achieve representative data evaluating antioxidant activity [62]. Lentil protein hydrolysate generated with PP showed higher potential to scavenge the radical ABTS • compared to the lentil protein hydrolysate generated using alcalase. Different legumes have demonstrated antioxidant capacities through this method. For instance, Ngoh et al. (2016) [63] determined the antioxidant activity presented in pinto bean peptides; in this study, peptide fractions < 3 kDa exhibited 42.2% inhibition of ABTS •+ scavenging activity. The results for DPPH • suggest that lower DH in A-LPH may enhance the potential to scavenge the radical DPPH • . This is consistent with previous reports by Evangelho et al. (2017) [64], where it was observed that alcalase protein hydrolysates obtained from black bean showed higher DPPH • scavenging capacities at lower DH. Likewise, similar findings were observed in a study performed by Kou et al. (2013) [65] showing that chickpea peptides obtained by alcalase exhibited 41.3% DPPH • radical scavenging activity. Another free radical scavenging evaluation was performed with NO. This radical plays an important role in inflammatory processes; high levels of NO and its oxidized derivatives are known to be toxic, resulting in vascular damage and other ailments [66]. Legume protein hydrolysates showed the potential to scavenge NO, with important results. In the case of the NO scavenging capacity, the most potent legume hydrolysates were the ones hydrolyzed with PP. Pepsin/pancreatin protein hydrolysates presented a more extensive degree of hydrolysis, leading to more diversity of peptides in the protein hydrolysate. In a study performed by Oseguera- Toledo et al. (2015) [26], potent hydrolysate fractions of 5-10 kDa were obtained with the enzyme alcalase. These hydrolysates from black bean demonstrated the capacity to scavenge the radical NO with a range of inhibition from 57.46% to 68.26%.
Protein hydrolysates from selected legumes could participate in the inhibition of the enzymatic activity of LP and HMG-CoA reductase. Besides this, legume protein hydrolysates show the potential to exert antioxidant activity.

Conclusions
Legumes are an important source of ingredients for the formulation of healthy foods. Legume protein hydrolysates were able to block pancreatic lipase and HMG-CoA reductase using in silico and biochemical assays. Furthermore, legume protein hydrolysates showed important radical scavenging activities against ABTS, DPPH and NO radicals. These results shed light on the potential antioxidant activity of the peptides. More importantly, the combination of different legume protein hydrolysates inhibited synergistically the adipogenesis-related enzymes evaluated. Further studies are needed to determine the anti-obesity potential and the antioxidant capacity of pure synthesized peptides and their combinations. Mixtures of legume protein hydrolysates could be used as functional ingredients in the formulation of foods with the potential to prevent or treat non-communicable diseases such as obesity.