Relation of amino acid composition, hydrophobicity, and molecular weight with antidiabetic, antihypertensive, and antioxidant properties of mixtures of corn gluten and soy protein hydrolysates

Abstract New mixed Alcalase‐hydrolysates were developed using corn gluten meal (CP) and soy protein (SP) hydrolysates, namely CPH, SPH, SPH30:CPH70, SPH70:CPH30, and SPH50:CPH50. Amino acid profile, surface hydrophobicity (H 0), molecular weight (MW) distribution, antioxidant activity, angiotensin‐converting enzyme (ACE), α‐amylase, and α‐glucosidase inhibitory activities, and functional characteristics of hydrolysates were determined. Hydrolysis changed the amount of hydrophilic and hydrophobic amino acid composition and significantly increased the H 0 values of hydrolysates, especially for CPH. The DPPH radical scavenging activity (RSA) was higher for CPH, SPH30:CPH70, and SPH50:CPH50 than SPH and SPH70:CPH30. Moreover, SPH, SPH70:CPH30, and SPH50:CPH50 showed lower MW than CPH, and this correlated with the higher hydrophilicity, and ABTS and hydroxyl RSA values obtained for SPH and the mixed hydrolysates with predominantly SPH. SPH70:CPH30 exhibited higher ACE, α‐glucosidase, and α‐amylase inhibitory activities among all samples due to its specific peptides with high capacity to interact with amino acid residues located at the enzyme active site and also low binding energy. At 15% degree of hydrolysis, both SPH and CPH showed enhanced solubility at pH 4.0, 7.0 and 9.0, emulsifying activity, and foaming capacity. Taken together, SPH70:CPH30 displayed strong antioxidant, antihypertensive, and antidiabetic attributes, emulsifying activity and stability indexes, and foaming capacity and foaming stability, making it a promising multifunctional ingredient for the development of functional food products.


| INTRODUC TI ON
Plant proteins have been widely considered as sustainable ingredients for the development of bioactive peptides and hydrolysates with antidiabetic, antihypertensive, and antioxidant activities (Das et al., 2022;Jin, Liu, et al., 2016). Enzymatic hydrolysis is a mild process that does not damage the amino acid composition compared with acidic hydrolysis, which could break down amino acids to toxic substances, such as 3-chloropropane-1,2-diol with carcinogenic effects (Adler-Nissen, 1979, 1984Lee & Khor, 2015;Nikoo et al., 2022;Wong et al., 2020). Thus, enzymatic hydrolysis is widely used in food industry as a controllable process for limited hydrolysis of proteins (Rezvankhah et al., 2021a(Rezvankhah et al., , 2021bRezvankhah, Yarmand, et al., 2022).
The resulting glucose is absorbed at a higher rate into the blood, resulting in increased blood glucose level (Chandrasekaran & Gonzalez de Mejia, 2022;Tacias-Pascacio et al., 2020). Moreover, hypertension is associated with angiotensin I-converting enzyme (ACE), an enzyme of the renin-angiotensin system pathway and important target of antihypertensive agents (Gharibzahedi & Smith, 2021;Guo et al., 2020;Ozón et al., 2022;Wang et al., 2019). Plant protein hydrolysates have shown strong inhibitory activities against α-glucosidase, α-amylase, and ACE toward the prevention and management of diabetes and hypertension (Karimi et al., 2020(Karimi et al., , 2021Liu et al., 2020;Qiao et al., 2020). These inhibitory activities have been attributed to the presence of specific peptides, predominantly those composed of highly hydrophobic amino acids, released by commercial proteases (Das et al., 2022). The interaction between these peptides and amino acid residues at the active site of enzymes leads to inhibition of enzymatic activity (Quintero-Soto et al., 2021).
Corn gluten meal (CGM) is a major by-product of the corn wet milling process . It contains 62%-71% protein with zein as the prominent protein fraction, accounting for 68% of the total protein, and glutelin as the residual part (~28% of zein weight) (Hu et al., 2022;Ren et al., 2018). Zein limits the application of CGM in various foods due to its poor water solubility (Yang et al., 2007). Furthermore, CGM contains several hydrophobic amino acid residues, which are buried inside the protein structure (Shen et al., 2020). Corn gluten meal is deficient in lysine and tryptophan, limiting its use in human nutrition (Zhu et al., 2019). Some studies have reported that enzymatic modification of CGM improved its solubility and bioavailability Jin, Liu, et al., 2016;Jin, Ma, et al., 2016;Liu et al., 2015). Corn protein hydrolysates (CPH) consist of small peptides with different molecular weight (MW) profiles, including di-and tripeptides, which can be effectively absorbed into blood circulation (Jin, Liu, et al., 2016;Jin, Ma, et al., 2016). Antioxidant and ACE-inhibitory activities of CPH have been reported (Li et al., 2019;Ren et al., 2018;Wang et al., 2020;Yang et al., 2007). When subjected to gastrointestinal digestion, CPH showed 12.9% increased antioxidant activity while retaining 77.5% of peptides compared with the undigested hydrolysates (Ren et al., 2018). Moreover, Yang et al. (2007) reported that peptide Ala-Tyr in Alcalase-hydrolyzed CGM at 50 mg/kg body weight decreased the systolic blood pressure of rats by 9.5 mmHg at 2 h after oral administration. Furthermore, corn germ protein hydrolysates (CGPH) and associated peptidic fraction (F1) with MW <2 kDa showed higher radical scavenging and α-glucosidase inhibitory activities than F2 fraction with MW of 2-10 kDa (Karimi et al., 2020).
These inhibitory activities can be attributed to the different amino acid sequences, which determine the interactions with the active site residues of the enzyme (Quintero-Soto et al., 2021).
Soy proteins (SPs) are one of the most utilized plant proteins in foods due to their nutritional quality, availability, and affordability compared with other sources of plant proteins (Xu et al., 2021).
Native SPs are composed of a mixture of globulins and albumins (Tian et al., 2020). Ninety percent of the proteins are storage proteins with a globular structure consisting mainly of 7S (β-conglycinin) and 11S (glycinin) globulins (Chen et al., 2011a(Chen et al., , 2011b. Soy proteins have been considered for their emulsifying activity and gelling potential compared with other plant proteins (Bessada et al., 2019). However, native SP has compact globular structures, leading to low molecular flexibility, relatively low emulsifying properties, and antioxidant activities compared with its modified states .

Moreover, dietary SPs have shown antidiabetic activity in humans,
indicating potential involvement of α-amylase and α-glucosidase inhibition (Das et al., 2022;Wang et al., 2019;Zhang et al., 2021;Zhao et al., 2021). Enzymatic hydrolysis of SP has been shown to increase solubility, antioxidant and α-glucosidase inhibitory, and antihypertensive activities of SPH (Jiang et al., 2018;Tian et al., 2020;Wang et al., 2019). α-Glucosidase inhibitory activity of SPH was reported to be higher than that of flaxseed, rapeseed, sunflower, and sesame protein hydrolysates (Han et al., 2021). Furthermore, novel peptides IY, YVVF, LVF, WMY, LVLL, and FF were identified from ACE-inhibiting Alcalase-derived SPH (Xu et al., 2021). The high hydrophobicity scores of the peptides might be the main contributor to the activity of SPH. The C-terminal hydrophobic residues showed important interactions that may have contributed to ACE inhibition (Xu et al., 2021).
A combination of plant protein hydrolysates can compensate for the deficiency of individual hydrolysates (Akharume et al., 2021). For instance, CGM has a low amount of lysine but is rich in methionine and cysteine and hydrophobic amino acids (Hu et al., 2022). Conversely, SP has a high content of lysine but limited sulfur-containing and hydrophobic amino acids (Tian et al., 2020). The combination of hydrolysates not only improves the deficiencies and balances the amino acid composition but also can augment biological (i.e., antioxidant, antidiabetic, and antihypertensive activities), and functional properties such as emulsifying activity. Since CGM and CPH demonstrate weaker ACE inhibitory activity than SP and SPH, it is postulated that combinations of SPH and CPH can produce improved hypertension property (Xu et al., 2021).
Hence, the objective of this study was to investigate the influence of combinations of CGM and SP hydrolysates on the bioactivities, including in vitro antidiabetic, antihypertensive and antioxidant activities, and functional properties. There is a dearth of studies on the combination of complementary bioactive hydrolysates; thus, the mixed protein hydrolysates can be explored as novel plant-based ingredients with augmented antioxidant, antihypertensive, antidiabetic, and techno-functional properties.

| Materials
Soy protein isolate (SPI, ~90% protein) and CGM (~62% protein) were supplied by Shansong Industrial Chinese Co. Ltd. and a grain processing refinery, Golshahd Co. Ltd., respectively. Alcalase 2.4 L from Bacillus licheniformis, with the activity of 2.4 Anson Units (AU)/g, and a density of 1.18 g/ml was purchased from Novozymes.

| Enzymatic hydrolysis of proteins
Protein solutions of SPI (~90% protein) and CGM (~62% protein) were prepared at 5% w/w. Soy protein isolate solution was heated at 90°C for 15 min to unfold the protein while the CGM solution was heated at 100°C for 30 min due to its low water solubility, thus the intense thermal treatment for denaturation (Tian et al., 2020;Yang et al., 2007). Enzymatic hydrolysis using Alcalase was conducted at temperature 60°C, pH 8.0, and enzyme/substrate ratio of 2.5% w/w. This condition was optimized in preliminary studies.
Hydrolysis time was considered based on DH reaching 15%; thus, 90 and 210 min were obtained for hydrolysis of SPI and CGM, respectively. Corn gluten meal have zein and glutelin as the main proteins (heat resistant) and thus required longer hydrolysis time to reach DH of 15% (Yang et al., 2007). After the reaction, CPH and SPH solutions were heated at 95°C for 10 min to terminate enzymatic activity. Then, the protein/peptide solutions were centrifuged at 15,000 g for 10 min and the obtained supernatants (rich in soluble peptides) were collected and adjusted to pH 7.0 using 1 M HCl, and spray-dried (DORSA tech) through a drying air of 180°C (the exhausting temperature of 75-80°C) and an air flow of 0.3-0.4 MPa (Akbarbaglu et al., 2019;Rezvankhah, Emam-Djomeh, et al., 2022;Rezvankhah, Yarmand, et al., 2022;Sarabandi et al., 2019). The powdered hydrolysates were stored at −18°C until the next experiments.

| Determination of the degree of hydrolysis (DH)
Degree of hydrolysis was determined using the pH-stat protocol reported by Adler-Nissen (1986) and calculated using the equation: where B is the volume (ml) of NaOH needed to maintain the pH constant; N b is the normality of the consumed base; M p is the mass of protein in CP and SP; h tot is the total number of peptide bonds in the protein substrates (considered 9.2 for CP and 7.75 for SP) (Adler-Nissen, 1979, 1986Jin et al., 2015;Tian et al., 2020;Xu et al., 2021); and α is the amount of α-NH 2 released during the proteolysis reaction.

| Amino acid composition
The amino acid profiles were determined using reversed-phase high-performance liquid chromatography (RP-HPLC, Agilent 1100 HPLC; Agilent Ltd.), as described by Liu et al. (2012). First, the samples were hydrolyzed in the glass tubes using 6 M HCl at 120°C for 12 h. Thereafter, the digests were filtered through 0.22 μm pore size filter. The separation was performed using a Zorbax analytical column (C18, 4 × 250 mm, 5 μm particle size; Agilent) at the temperature of 40°C with a UV detector spectra monitored at 338 nm. The elution of column with the flow rate of 1 ml/min was conducted with mobile phases comprising 7.40 mmol/L of sodium acetate/triethylamine/tetrahydrofuran (400:0.10:2, v/v/v), set at pH 7.1 using acetic acid and 7.40 mmol/L of sodium acetate/ methanol/acetonitrile (1.5:2.5:2.5, v/v/v), set at pH 7.1. A standard solution comprising of 17 amino acids was used as an external standard. (1)

| Surface hydrophobicity (H 0 )
The protein and hydrolysate samples were investigated for the H 0 using 1-anilinonaphthalene-8-sulfonic (ANS) according to the method of He et al. (2021). The samples were diluted to 0.01-0.02 mg/ml in 10 mM phosphate buffer (pH 7). The fluorescence intensity was measured at a wavelength of 390 nm (excitation) and 470 nm (emission) using a fluorescence spectrophotometer (LS-55, Perkin Elmer). The slope of the plot of fluorescence vs. concentration of samples was expressed as the surface hydrophobicity (H 0 ).

| SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
SDS-polyacrylamide gel electrophoresis was used to estimate the MW profile of CP, SP, and their hydrolysates following the method of Rezvankhah et al. (2021b). Briefly, a sample solution (5 mg/ml) of proteins and respective hydrolysates was mixed with an equal amount of Laemmli sample buffer (960 μl of 66 mM Tris-HCl, pH 6.9, 27.3% w/v glycerol, 2.2% SDS, 0.01% bromophenol blue). Then, the prepared samples were combined with 2-mercaptoethanol and heated for denaturation at 95°C for 5 min before the electrophoresis. The concentration of 12% Mini-Protean™ precast gels (Bio-Rad) was used to run the electrophoresis. Thereafter, 10 μl of cooled samples was loaded on the gels, and then subjected to a constant voltage of 150 V. Additionally, a marker with MW standards (Bio-Rad Broad Range Marker) was run alongside the samples. When the process finished, the gels were stained with 0.1% Coomassie Brilliant Blue R-250 in a mixture of 10% acetic acid and 40% methanol for 2 h. The protein/peptide bands were visualized by discoloring the gels using a mixture of 40% methanol and 10% acetic acid solutions.

| MW distribution
Gel permeation chromatography (GPC) was applied to determine MW distribution of the CP, SP, and hydrolysate samples following the method of Rezvankhah et al. (2021b). The Waters Breeze HPLC system (Waters Corporation) equipped with a Waters UV detector and Superdex Peptide HR column (30 cm × 10 mm and 13-15 μm particle size) was used to evaluate the protein/peptide sizes. The analytes were dissolved in ultrapure water and stirred for 30 min at 25°C. The solution was centrifuged at 12,000 g for 10 min, and the supernatant was filtered through a 0.22 μm membrane filter. The 2.9 | Antioxidant activity 2.9.1 | DPPH radical scavenging activity Antioxidant activity was determined according to the method of Zheng et al. (2015). 2,2 Diphenyl-1-picrylhydrazyl solution at 0.2 mM in 95% ethanol and the protein/peptide solution at 7 mg/ml were prepared. Then, 2 ml of DPPH solution was combined with 2 ml of samples and stored for 30 min in the dark. The absorbance of the mixtures was read at 517 nm using a spectrophotometer. To compare the antioxidant activity of the analytes, ascorbic acid (0.01 mg/ml) was used as a positive control. The RSA% was calculated using the equation: where the absorbance values were for control (A C ), sample (A S ), and blank (A B ).

| ABTS radical cation scavenging activity
The ABTS ·+ scavenging activity of hydrolysates was evaluated according to the protocol reported by Amini Sarteshnizi et al. (2021).
The ABTS solution (940 μl) was combined with 60 μl of samples (7 mg/ml) and vigorously shaken, and then incubated at 25°C for 10 min in the dark. The absorbance was read at 734 nm using a spectrophotometer, and ascorbic acid (0.01 mg/ml) was used as a positive control.

| Hydroxyl radical scavenging activity (RSA)
The hydroxyl RSA was evaluated using the protocol reported by Zheng et al. (2015). Two milliliter of samples (7 mg/ml), 2 ml of FeSO 4 (6 mM), and 2 ml of H 2 O 2 (6 mM) were thoroughly mixed and kept for 10 min at room temperature. Then, 2 ml of salicylic acid (6 mM) was added and incubated for 30 min. The absorbance was then read at 510 nm (A S ). Distilled water was used as the blank (instead of salicylic acid solution) and the control (instead of sample solution). Ascorbic acid (0.01 mg/ml) was used as a positive control. The RSA was calculated using equation 2.

| ACE inhibition assay
The potential antihypertensive activity of CP, SP, and hydrolysates was assessed by the in vitro inhibition of angiotensin I-converting enzyme (ACE) based on the method of Boye et al. (2010). ACE inhibition (%) was computed by the equation: where the absorbance values were for control (A C ), sample (A S ), and blank (A B ). Also, the IC 50 value, the concentration of sample that inhibited 50% of ACE activity, was determined using sample concentrations of 0.1-2 mg/ml.

| Determination of in vitro antidiabetic properties
2.11.1 | α-Glucosidase inhibition assay The inhibitory activity of CP, SP, and their hydrolysates against rat intestinal α-glucosidase was assessed following the method of Karimi et al. (2020). Briefly, the enzyme was extracted from acetone powder from rat intestine, and the obtained solution was diluted to 90 mU/ml. Then, 150 μl of different concentra- where the absorbance values were for control (A C ) and sample (A S ).
Sample concentrations of 10-500 μg/ml were used to determine the IC 50 values.

| α-Amylase inhibition assay
The α-amylase inhibitory activity of CP, SP, and hydrolysates was determined using the method reported by Rahimi et al. (2022). Briefly, 100 μl of different concentrations of the samples (10-500 μg/ml) was combined with 120 μl of α-amylase solution (0.6 U/ml) and incubated at 37°C for 5 min. Then, 120 μl of 0.5% (w/v) starch solution was added. The enzyme activity was terminated by heating the reaction mixture at 100°C for 10 min followed by cooling to ambient temperature. The undigested starch was separated by centrifugation at 15,000 g for 2 min. Then, 20 μl of the supernatant was mixed with 1 ml of PAHBAH and the solution was heated to 70°C for 10 min. The sample solutions were cooled, and absorbance values were read at 410 nm. The inhibitory activity was determined using the following equation: where the absorbance values were for sample (A S ), blank (A B , phosphate buffer, enzyme, sample), and control (A C , starch, buffer, enzyme).
Furthermore, IC 50 values were determined as previously described.
Acarbose, at its IC 50 value (0.125 mg/ml), was used as a positive control. The emulsion (100 μl) was taken from the container bottom immediately after production to determine EAI. Also, ESI was determined by taking 100 μl of the emulsion from the container bottom after 10 min.

|
The aliquots were combined with 5 ml of SDS (0.1%), and the absorbance of the diluted solutions was read at 500 nm using a UV-Vis spectrophotometer. EAI and ESI were computed using the equations: where A 0 is the absorbance of diluted emulsion at 500 nm immediately after homogenization, DF is the dilution factor (50), I is the path length of the cuvette (m), is the oil volume fraction (0.25), C is the protein concentration in the aqueous phase (g/m 3 ), Δ A = A 0 − A 10 , and Δ t = 10 min.

| Foaming properties
Two foaming properties including the foaming capacity (FC) and foaming stability (FS) of the CP, SP, and hydrolysates were assessed Protein content in the supernatant Total protein content in the sample × 100 following the procedure reported by Rezvankhah, Emam-Djomeh, et al. (2022) and Rezvankhah, Yarmand, et al. (2022). The 10 mg/ ml sample solutions in a 50 ml measuring cylinder was whipped at 19,000 rpm for 2 min using a laboratory-scale homogenizer (IKA, T25). The total volume (ml) of the initial foam was determined. Also, the foam volume was recorded after storage time of 30 min at room temperature. Foaming capacity and FS were calculated using the following equations: where the volume before whipping (ml), the volume immediately after whipping (ml), and the volume after standing for 30 min (ml) are denoted by A, B, and C, respectively.

| Statistical analysis
The experimental data were reported as means ± standard deviation. One-way analysis of variance (ANOVA) was used to analyze the obtained data. The Duncan test was applied to evaluate the comparison of mean difference using the SPSS software (version 26, IBM software).

| Amino acid composition
Amino acid composition of the hydrolysates is presented in Table 1.
The RP-HPLC amino acid profile of CP showed higher hydrophobic amino acid contents, while the SP exhibited higher hydrophilic amino acid contents (Table 1). Similar findings have been reported in earlier investigations (Reyes Jara et al., 2018;Zhou et al., 2012). Enzymatic hydrolysis by Alcalase significantly changed the amino acid profiles of both proteins (Fadimu et al., 2021;Rezvankhah, Emam-Djomeh, et al., 2022;Rezvankhah, Yarmand, et al., 2022). CPH had higher content of hydrophilic amino acids, while SPH showed higher content of hydrophobic amino acids than their respective native proteins (CP and SP) ( Table 1).
The variation in amino acid profiles could be related to the unfolding of the protein structures and exposure of the hydrophobic segments during enzymatic hydrolysis (Jin et al., 2015;Liu et al., 2012). This variation could also be due to the separation of some unhydrolyzed polypeptides during the hydrolysis and removal by centrifugation (Fadimu et al., 2021;Rezvankhah, Emam-Djomeh, et al., 2022;Rezvankhah, Yarmand, et al., 2022). Furthermore, the amino acid composition varied among all mixed hydrolysates including SPH70:CPH30, SPH50:CPH50, and SPH30:CPH70. Indeed, the contents of hydrophilic and hydrophobic amino acids of the mixed hydrolysates were determined by the dominant hydrolysate portion.
CPH was the major constituent in SPH30:CPH70, and the addition of SPH increased the quantity of hydrophilic amino acids when compared to CPH alone (Table 1). Previous studies have reported high hydrophobic and sulfur-containing amino acid contents for CGM (CP) and CPH, and high hydrophilic amino acid and lysine contents for SP and SPH (Jin, Liu, et al., 2016;Jin, Ma, et al., 2016;Reyes Jara et al., 2018).

| Surface hydrophobicity
Surface hydrophobicity (H 0 ) has an important effect on the macromolecular structural stability, surface, and biological properties of proteins (Rezvankhah, Emam-Djomeh, et al., 2022;Rezvankhah, Yarmand, et al., 2022;Wang et al., 2020). As shown in Figure 1, enzymatic hydrolysis of CP and SP significantly increased the H 0 values, as previously reported by others . The noncovalent, particularly the hydrophobic interactions, and disulfide bonds (SS) are abundantly present in CP Wang et al., 2020). Although not considered the prevalent driving force for aggregation, hydrophobic interactions influence the aggregation tendency . For CPH, the fluorescence intensity with ANS remarkably increased, indicating a higher H 0 value than CP. The hydrophobic patches are buried inside the zein and glutelin structures . When the proteins are hydrolyzed, the hydrophobic segments are exposed to the surface. Albeit, it did not lead to aggregation. The insoluble aggregates may have been separated by centrifugation, while the soluble aggregates were maintained . According to a previous study, CPH had an emulsion-like appearance, which indicates that hydrolysis of CP not only increased its surface hydrophobicity but also decreased the MW and disulfide bonds of the protein, thereby transforming the insoluble aggregates into soluble aggregates .
Hydrolysis also increased the H 0 of SP, but this increase in SPH was remarkably lower than CPH; this may be related to the amino acid composition of SP and CP or their corresponding hydrolysates (Rezvankhah, Emam-Djomeh, et al., 2022;Rezvankhah, Yarmand, et al., 2022;Wang et al., 2016;Zheng et al., 2015) ( Table 1). SPH had lower hydrophobic amino acid composition (28.42 g/100 g) than CPH (39.74 g/100 g), and this is likely due to the dominant hydrophobic amino acid portion in CP. As shown in Figure 1, the combination of SPH with CPH, depending on the dominant part, resulted in different H 0 values. Therefore, the order of H 0 values for combined hydrolysates of SP and CP was SPH30:CPH70 > SPH50:CPH50 > SPH 70:CPH30. Therefore, the higher the content of CPH in the mixture, the higher the H 0 value achieved (Figure 1).

| Molecular weight profile
Approximate MW of CP, SP, CPH, SPH, SPH30:CPH70, SPH70:CPH30, and SPH50:CPH50 was determined using SDS-PAGE (Figure 2a). The   Figure 2a). The combination of SPH and CPH is hypothesized to present stronger biological activities and functional properties than their respective hydrolysates.
It has been reported that lower MW peptides are not visible on SDS-PAGE gels, associating with heating effects on the protein conformation Rezvankhah, Emam-Djomeh, et al., 2022;Rezvankhah, Yarmand, et al., 2022). Heating is performed for various aims (denaturation/unfolding of the protein structures and terminating of enzyme) during the hydrolysis stage or preparation of analytes for SDS-PAGE analysis. Most of the SDS-PAGE gels have been designed to determine molecules/peptides with MW above 10 kDa; thus, visualizing molecules with lower MW is possible using techniques such as gel permeation chromatography Rezvankhah, Emam-Djomeh, et al., 2022;Rezvankhah, Yarmand, et al., 2022).

| Molecular weight distribution
Changes in MW that could not be detected by SDS-PAGE, especially MW below 10 kDa, were determined by GPC, given the high potential of GPC for accurate determination of MW distribution .

Molecular weight distribution of the proteins and their hydrolysates
is shown in Figure 2b. Peptide size is one of the most important factors that influence the functional properties, bioavailability, and bioactivities of peptides (Rezvankhah et al., 2021a;Rezvankhah, Emam-Djomeh, et al., 2022;Rezvankhah, Yarmand, et al., 2022).

| Antioxidant activity
As shown in Figure 3a, among the proteins and hydrolysates, CPH (46.25%), SPH30:CPH70 (46.70%), and SPH50:CPH50 (47.30%) showed the highest DPPH RSA with no significant difference (p > .05), followed by SPH70:CPH30 (33.50%), CP (19.30%), SPH (12.40%), and SP (9.30%). Ascorbic acid, however, exhibited higher DPPH RSA at a much lower concentration than the hydrolysates. CP and its hydrolysates had high content of hydrophobic amino acids, which may be related to their higher reactivity with DPPH radicals (Jin et al., 2015;Karimi et al., 2020Karimi et al., , 2021. Conversely, SP and SPH, which have high content of hydrophilic amino acids, showed lower antioxidant activity (Figure 3a). Enzymatic hydrolysis significantly increased the antioxidant activity of the proteins, and this is associated with the liberation of medium-and small-sized peptides with exposed hydrophobic and reactive groups with antioxidant power (Zhou et al., 2017). CPH with higher hydrophobicity (Figure 1 and  showing lower ABTS RSA. The production of peptides with MW <1 kDa remarkably influences the antioxidant power of hydrolysates (Tian et al., 2020). Also, SPH had higher hydrophilic amino acids that are known to have high interaction with the hydrophilic radical (ABTS) (Hu, Chen, et al., 2020;Hu, Dunmire, et al., 2020). ABTS radical has hydrophilic affinity, while DPPH radical has hydrophobic affinity; thus, SPH indicated higher ABTS RSA, while CPH exhibited higher DPPH RSA, similar to previous findings (Hu, Chen, et al., 2020;Rezvankhah, Yarmand, et al., 2022).
Based on the hydroxyl RSA results in Figure 3c, the lower the MW of the hydrolysates, the higher the antioxidant power obtained.
SPH due to its higher hydrophilic amino acids and containing lower MW peptides (Figure 2a,b) exhibited higher hydroxyl RSA, while CP due to its higher hydrophobic amino acid profiles and larger peptides exhibited lower hydroxyl RSA (Rezvankhah, Emam-Djomeh, et al., 2022;Rezvankhah, Yarmand, et al., 2022). MW of peptides can substantially affect their antioxidant activity. The small-and medium-sized peptides have shown stronger antioxidant power due to their ability to interact with the radicals (Bu et al., 2020;Rezvankhah et al., 2021a;Singh et al., 2014;Tian et al., 2020;Zhao et al., 2021).  (Liu et al., 2022). Also, CPH indicated potent hydroxyl RSA compared with nonhydrolyzed protein .

| In vitro antihypertensive property
Results of the ACE inhibitory activity of the proteins and their hydrolysates are presented in Figure 4a. respectively. It was observed that hydrolysis of SP increased the ACE inhibitory activity as previously reported Xu et al., 2021). Although proteins (SP and CP) and their hydrolysates (SPH and CPH) exhibited high ACE-inhibitory activity (higher than 70%), the combined SPH and CPH also showed strong ACEinhibitory activity. The hydrophobic amino acids positioned at the Cterminal residues have been shown to contribute to ACE-inhibitory activity of peptides (Ambigaipalan et al., 2015). CP demonstrated weaker ACE inhibitory activity because the active peptide is locked in within the protein primary structure. Therefore, the activity obtained for the intact protein (CP and SP) could be due to unknown molecules co-isolated with the proteins. Liu et al. (2020) identified 12 peptides from active fractions of Alcalase-hydrolysate (CPH) obtained from CGM with good ABTS radical scavenging and ACE inhibitory activities . Similar findings were reported for SPH (Xu et al., 2021). Hence, the combination of CPH and SPH led to alterations in amino acid profiles of the new mixed hydrolysates, which influenced the bioactivities.
Angiotensin-converting enzyme such as other enzymes has binding sites that could interact with peptide inhibitors (Quintero-Soto
It was observed that the combination of SPH and CPH (SPH70:CPH30) led to mixed hydrolysates with higher α-glucosidase inhibitory activity than CPH. Acarbose, a synthetic compound, at the same concentration exhibited much higher α-glucosidase inhibitory activity than the crude hydrolysates. However, it has several side effects (Das et al., 2022), making the natural hydrolysates promising safer alternatives. The strongest α-glucosidase activity of SPH could be associated with the higher content of smaller peptides produced at the same DH compared with CPH (Figure 2a,b). Also, the composition of amino acid residues influences the activity (Quintero-Soto et al., 2021). The presence of the basic amino acids (lysine and arginine) at the end of the peptide chains and amino acids with hydroxyl groups (serine, threonine, and tyrosine) contributes to αglucosidase inhibition through the interaction with the active site of the enzyme (Karimi et al., 2020(Karimi et al., , 2021. The prevalent interactions are electrostatic and hydrogen bonds which lead to suppression of the enzyme activity (Rezvankhah, Emam-Djomeh, et al., 2022;Rezvankhah, Yarmand, et al., 2022). Incorporation of SPH increased the α-glucosidase inhibitory activity of CPH, likely due to augmentation of the amino acid composition.
As illustrated in Figure 4c, hydrolysis significantly increased the α-amylase inhibitory activity of the samples.  (Ngoh & Gan, 2016). Indeed, calcium ions participate in structure formation, functions, and regulation of the stability of α-amylase (Admassu et al., 2018). Moreover, amino acid residues including glycine, leucine, serine, aspartic and glutamic acids, proline, phenylalanine, tryptophan, and tyrosine have been shown to bind the active site of the enzyme, thus increasing the potential to achieve inhibition (Karimi et al., 2020). SPH and SPH70:CPH30 exhibited higher potential in inhibiting α-amylase than the other hydrolysates.

| Functional properties
Solubility, emulsifying, and foaming properties of the samples are provided in Table 2. The solubility of proteins and their hydrolysates was determined at pH 5, 7, and 9. Hydrolysis of CP and SP significantly improved the protein solubility. At pH 4, CP and SP were not soluble in water (0%) due to the insolubility of CP, zero net charges of the proteins (CP and SP), or lack of electrostatic repulsions (Rezvankhah et al., 2020;Zheng et al., 2015). Conversely, CPH, SPH, and their combinations exhibited higher solubility ranging from 92% to 96%. This result is related to the small-sized peptides released during the hydrolysis process (Chen et al., 2011a(Chen et al., , 2011b (Jin et al., 2015). FS showed a slightly different result (

ACK N OWLED G M ENT
The authors would like to acknowledge Tarbiat Modares University for their financial support.

FU N D I N G I N FO R M ATI O N
This study was supported by the office of vice chancellor for research at Tarbiat Modares University (Grant Number: 9830422005).

CO N FLI C T O F I NTE R E S T
None of the authors declare a conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used in this paper are available in case of reviewer or editor request.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.