Physicochemical, antioxidant, antimicrobial, and in vitro cytotoxic activities of corn pollen protein hydrolysates obtained by different peptidases

Abstract The applications of protein hydrolysates as food preservatives and nutraceutical ingredients have attracted much attention because of their beneficial effects. The interest in these ingredients has shifted toward their biological activities with benefits to human health. Bioactive peptides are known as antioxidant agents that could promote health‐promoting effects and prolong food shelf‐life beyond their basic nutritional value. Thus, the aim of this study was to investigate antioxidant, antimicrobial, and in vitro cytotoxic properties of corn pollen protein (CPP) hydrolysates obtained by different enzymes. Proteolytic activity in terms of degree of hydrolysis (DH) and SDS‐PAGE analysis was measured in pancreatin (H‐Pan), pepsin (H‐Pep), and trypsin (H‐Tri) hydrolysates. Amino acid composition, antioxidant and antimicrobial activities, and cytotoxicity of hydrolysates were evaluated. DH and SDS‐PAGE revealed higher proteolytic activity of pepsin compared to other enzymes. Amino acid analysis showed that the functional amino acids such as antioxidant types were most predominant in H‐Pep compared to two other samples. Antioxidant activity of hydrolysates was found to be affected by the type of enzyme and the concentration of hydrolysates. There was a significant difference (p < 0.05) between antioxidant activity of different hydrolysates. The highest antioxidant activity in terms of Trolox equivalent antioxidant capacity (0.23–2.75 mM), DPPH (33.3%–64.8%), and hydroxyl (33.7%–63.2%) radical scavenging activities, chelation of iron (33.2%–62.5%) and copper (30.2%–50.5%) metals, and total antioxidant activity (0.65–0.85) was obtained for H‐Pep followed by H‐Pan and H‐Tri samples. Antibacterial tests showed that pepsin‐hydrolyzed protein was not significantly (P > 0.05) effective against E. coli at any concentrations, however, it showed significant (P < 0.05) concentration‐dependent effect against S. aureus (with inhibition zones of 15–25 mm). Cytotoxicity results revealed that CPP, as a nonhydrolyzed protein, did not generally show antiproliferative activity, however, a significant (P < 0.05) ability of H‐Pep hydrolysate in decreasing HT‐29 colon cancer cell line viability was seen in a concentration‐dependent manner (the lowest cell viability of 32% at 5 mg/mL). Overall, investigating the application of protein‐based hydrolysates is one of the possible strategies that govern their applied intentions as preservatives and nutraceuticals in the food and pharmaceutical industries.


| INTRODUC TI ON
Reactive oxygen species (ROS) such as OH radicals, singlet O, and H 2 O 2 play important roles in modulating intracellular signal transduction pathways in biological systems. These ROS can be naturally blocked or eliminated by endogenous antioxidant defense systems in living organisms (Lorenzo et al., 2018). However, in some cases such as extreme environmental conditions, excessive ROS can be often generated, and if they are accumulated for a prolonged period, they will overwhelm the cellular natural antioxidant defense capacity resulting in oxidative damage to critical biological and vital macromolecules such as proteins, lipids, and DNA (Ravash et al., 2022).
Likewise, accumulation of ROS free radicals in cells can lead to oxidative stresses resulting in disturbance of cell homeostasis and damage to the cell. Thus, an additional exogenous antioxidant protection system is necessary to maintain the oxidative balance status.
Antioxidants, especially those supplied by dietary natural sources, are important to maintain redox homeostasis in cells of the human body. These natural-based antioxidants show no undesirable adverse effects compared to chemically synthesized antioxidants (Nottagh et al., 2020;Wang et al., 2021).
Most of the peptides can offer antioxidant action similar to that of other well-known antioxidants. Bioactive peptides obtained from plant protein sources have been well demonstrated to exhibit excellent antioxidant properties that could promote health-promoting effects and prolong food shelf-life beyond their basic nutritional value. The importance of characteristics of peptides derived from different food sources has been extensively discussed (Lorenzo et al., 2018). The outer layer of cereal grains contains secondary metabolites such as bioactive substances, which provide positive nutritional and health-promoting properties in human body. Most of these bioactive compounds are concentrated in bran-or germ-rich milling fractions, which can be separated, purified, and used as nutraceuticals. Different health-promoting and functional properties including antioxidant, antihypertensive, and antitumor activities of bioactive peptides have been outlined in cereal protein hydrolysates (Peighambardoust et al., 2006. Proteins present in different sections of cereals are reported to contribute to biological functions such as antioxidant activities (Esfandi et al., 2019). Protein hydrolysates from cereal pollens also possess significant antioxidant and angiotensin-converting enzyme (ACE) inhibitory activities (Maqsoudlou et al., 2019). Plant-derived bioactive peptides are important functional ingredients that provide many health-promoting effects, among which is preventing oxidative damages in living organisms (Akbarbaglu et al., 2021). Corn pollen is considered a nutritionally valuable resource, due to its rich sources of protein and fatty acids that are concentrated in the protective layer of pollen called "kitt zone" and this makes it play a significant role in preventing diseases such as hypertension, cancer, and diabetes (Chakrabarti et al., 2018). Health-promoting and nutritional effects of corn pollen have led to its use as a natural additive in a wide range of foods (Albenzio et al., 2017). In this study, it is hypothesized that corn pollen is a rich source to produce bioactive peptides due to its different types of proteins.
Enzymatic hydrolysis is one of the most common methods used on a laboratory scale to produce bioactive compounds (Karami, Peighambardoust, Hesari, & Akbari-Adergani, 2019). There are many studies reporting the hydrolysis of food proteins from different sources such as dairy, meat, egg, and plant byproducts proteins using trypsin (Deng et al., 2018), pepsin and papain (Xu et al., 2009), and alcalase (Maqsoudlou et al., 2019). Most of the studies confirm antioxidant and ACE inhibitory properties of functional peptides obtained from vegetable source proteins such as wheat germ proteins (Karami, Peighambardoust, Hesari, Akbari-Adergani, & Andreu, 2019b). However, to the best of our knowledge, there is no study on properties of hydrolysates obtained from corn pollen protein (CPP) by different proteolytic enzymes yet. In this study, it is hypothesized that different proteases may affect functional properties of protein hydrolysates from CPP. Thus, the main objective of this study was to compare antioxidant and antibacterial potential of functional peptides obtained by enzymatic hydrolysis of CPP using different enzymes such as pancreatin, pepsin, and trypsin.

| Materials
The floral corn (Zea mays L. saccharata) pollen was gathered from maize farms in Gorgan (Golestan, Iran) in June 2020. Pollens were dehydrated at a temperature of below 40°C, then stored until used.

| Corn pollen protein (CPP) extraction
Corn pollen was defatted using N-hexane (at a ratio of 1:4 w/v) for 48 h at room temperature. The pollen solids were vacuum filtered from the suspension. Defatted pollen solids were then dispersed in distilled water (at 10% w/v) at pH = 10. Protein extraction was car-

| Enzymatic hydrolysis
Enzymatic hydrolysis can be affected by a variety of factors, such as temperature, pH, and concentration. Enzymes work best within specific temperature and pH ranges, and suboptimal conditions can cause an enzyme to lose its ability to bind to a substrate. An increase in temperature generally speeds up enzymatic reactions.
However, extreme high temperatures can cause an enzyme to denature. Moreover, every enzyme has an optimum pH range for best performance. Changing the pH outside of this range will slow enzyme activity. Increasing enzyme concentration will speed up the reaction, as long as there is substrate available to bind to. In this study, pancreatin, trypsin, and pepsin enzymes were used for enzymatic hydrolysis of corn pollen protein. The conditions of the hydrolysis process were selected based on the optimal activity point of each enzyme previously tested and optimized in our laboratory (Akbarbaglu et al., 2022).

| Pancreatin and trypsin
To perform enzymatic hydrolysis, defatted CPP powder (15.2% crude protein) was dissolved in 100 mL of 0.2 M potassium phosphate buffer (pH = 7.4) at a concentration of 10% (w/v) inside an Erlenmeyer flask. An amount of 0.2 g (2% w/v) pancreatin or trypsin in powder form was individually added and allowed to completely hydrate on a magnetic stirrer (250 rpm) keeping at a constant temperature of 37°C. The reaction was performed over a period of 120 min. At the end of the enzymatic hydrolysis process, the thermal process was performed in a water bath at 90°C for 20 min to inactivate peptidases. The samples were then cooled down to room temperature. The obtained suspension was then centrifuged at 5000 × g for 30 min and the supernatants were then lyophilized (Free Zone 4.5, Labconco, Kansas City, MO, USA) at −60°C for 72 h, and stored at −18°C inside zip-lock bags until use.

| Pepsin
To perform pepsin hydrolysis, the same procedure was used as explained in Section 2.3.1, except that 0.1 M acetate buffer (pH = 3) was used to provide optimal conditions for enzymatic hydrolysis.

| Determining the degree of hydrolysis
To measure DH, CPP hydrolysate suspension and trichloroacetic acid (TCA, 0.45 M) were mixed at a ratio of 1:1 (v/v). The mixture was then incubated at 4°C for 10 min and centrifuged (5000 × g) for 15 min. The soluble proteins were estimated using Coomassie brilliant blue dye-binding methods, according to the standard curve plotted based on different concentrations of bovine serum albumin.
The degree of hydrolysis (DH) was calculated based on the following equation:

| FT-IR spectroscopy
FT-IR spectroscopy was used to assess possible changes in chemical structure of native pollen protein compared to its hydrolysates as action of different enzymes (Peighambardoust et al., 2007). For this purpose, protein or hydrolysates powders were mixed with KBr (1:100 w/w) and compressed to make pellet disks. Infrared spectrum was recoded using spectrophotometer (Bruker-Tensor 27, Bremen, Germany) covering wavenumber range 4000-400 cm −1 at 4 cm −1 resolution (Soltanzadeh et al., 2021a(Soltanzadeh et al., , 2021b).

| Amino acid composition of CPP and hydrolysates
The effect of proteolytic action of different proteases on amino acid composition of CPP and its hydrolysate was studied by reversedphase (RP) HPLC (Young Lin Acme 9000, YL Instruments, Anyang, Korea) equipped with an RP column (150 mm × 4.6 mm × 0.5 mm, Teknokroma, RP-C18 ODS-A, Barcelona, Spain) and a fluorescence detector (LC305. Lab Alliance. State College, PA, USA) van der Goot et al., 2008). Acetate buffer was run as mobile phase with a flow rate of 1.3 mL min −1 . First, the hydrolyzed samples were diluted with solution A (125 mM borate, pH = 9.4) at a ratio of 1:20 followed by adding methanol and L-homoserine. The present mixture was blended with solution B (O-Phthaldialdehyde + Borate + Mercaptoethanol + MeOH) and C (1) DH ( % ) = Protein (TCA + Supernatant) Protein (corn pollen hydrolysate suspension) × 100 (0.75 M HCl) and was injected into the RP-HPLC with a Hamilton syringe. Total amino acid residues were determined after hydrolysis with 6 N HCl at 110°C for 24 h. CPP samples previously hydrolyzed by the enzymes did not undergo the hydrolysis process and the amino acid composition was expressed as mg g −1 dry sample (Ngamsuk et al., 2020).
Amino acid composition can be used as an indication of nutritional and functional groups of AA in CPP hydrolysates compared to its native protein according to Equations (2-5):

| SDS-PAGE (sodium dodecyl sulfatepolyacrylamide gel electrophoresis)
Variation in molecular weight (Mw) of the chemical components of CPP hydrolysates was characterized by SDS-PAGE according to the method described by Mechmeche et al. (2017) with alteration as explained below. Polyacrylamide (PA) gel with a concentration of 5 and 12.5% was used for stacking and running gels, respectively.
Dispersions of hydrolysates in DW (0.2% w/v) and a buffer containing bromophenol blue at a ratio of 1:1 were prepared, which was then heated at 90°C for 10 min followed by cooling to ambient temperature. Aliquots of 20 μL of sample and 5 μL of a protein marker (Mw of 20-120 kDa) were injected into electrophoresis wells. The gel was then placed in the set-up. SDS-PAGE was carried out at a constant voltage of 70 and 120 V for stacking and running gels, respectively. When the reagent line reached the bottom of the gel, the process was ended and the gel was then removed and stained by Coomassie brilliant blue (R-250) (10% w/v) overnight to observe the protein bands.

| DPPH free radical scavenging
To evaluate DPPH free radical scavenging activity (RSA), 1.5 mL hydrolysate solution at a concentration of 10 mg mL −1 was blended with 1.5 mL of 0.2 mM alcoholic solution of DPPH. The obtained solution was then stored in dark for 40 min. The mixture was then centrifuged (5000 × g, 15 min) and the supernatant was taken and the adsorption was measured by a UV-vis spectrophotometer (Varian Cary 500, Agilent Technologies, Santa Clara, CA, USA) at 517 nm.
DPPH free radical scavenging was estimated from the following ABTS + radical scavenging activity was measured using Equation (6)

| Hydroxyl radical scavenging activity
Evaluation of hydroxyl radical scavenging activity was performed using α-deoxyribose oxidation. For this purpose, 0.2 mL of 10 mM Fe 2 SO 4 -EDTA solution was added to 0.5 mL of 10 mM α-deoxyribose.
Then, hydrolysate sample (0.2 mL), sodium phosphate buffer (0.2 M, 0.9 mL), and hydrogen peroxide (10 mM, 0.2 mL) were mixed in a reaction mixture and incubated at 37°C for 1 h. An aliquot of 1 mL trichloroacetic acid (2.8% w/v) was added to the mixture. Then, 1 mL of thiobarbituric acid (1% w/v) was added to stop the reaction. The resulting mixture was heated to boiling for 15 min followed by cooling to ambient temperature. The mixture was centrifuged (5000 × g for 10 min) and the absorption of the supernatant was recorded at 532. In the blank sample, the equivalent volume of water was used.
The hydroxyl radical inhibition was calculated using Equation (6) at absorbance of 532 nm.

| Reducing power activity assay
To evaluate antioxidant activity of peptides, the reduction of Fe +3 / ferricyanide complex to the ferrous form can be used as reducing power activity. For this purpose, hydrolysate sample (0.5 mL), phosphate buffer (0.5 mL, pH = 6.6), and potassium ferricyanide (0.5 mL at 10 mg mL −1 ) were mixed and incubated for 20 min at 50°C. Then, 0.5 mL of 10% trichloroacetic acid (10% w/v) was added and the resulting mixture was centrifuged (5000 × g) for 10 min. The obtained supernatant was diluted with 1 mL distilled water and 0.2 mL ferric chloride (0.1% w/v) and kept at room temperature for 10 min and its (2) Essential amino acids (EAA) = Thr + Met + Val + Leu + Ile + Trp absorption was obtained at 700 nm. As a blank sample, the equivalent volume of water was used instead of the original sample (Shahidi & Zhong, 2015). The reducing power activity was calculated using Equation (6) at absorbance of 700 nm.

| Fe 2+ chelating activity
To measure Fe 2+ chelating activity of protein hydrolysates, the sample (1 mL), iron (ІІ) chloride solution (0.05 mL at 2 mM), double-distilled water (1.85 mL), and ferrosine (0.1 mL at 5 mM) were combined and stirred vigorously at room temperature for 10 min. The absorbance of the resulting mixture was read at 562 nm (Shahi et al., 2020). The Fe 2+ chelating activity was calculated from Equation (6)  was transferred to the microtube tube (5 mL) followed by adding 1 mL of 10% pyridine and 20 μL of 0.1% pyrocatechol. The mixture was then incubated for 5 min and the adsorption was read at 632 nm.

| Total antioxidant activity (TAA)
This method is based on the reduction of molybdenum (VI) to molybdenum (V). It is a capacity that is associated with the formation of the green complex of phosphomolybdenum in acidic pH. To measure TAA, a mixture containing 0.1 mL sample solution (10-50 mg mL −1 ) and 1 mL of reagent (0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate) was prepared and incubated in a water bath set to 90°C for 90 min. After cooling to ambient temperature, the absorbance was measured at 695 nm. Double distilled water was used as a control (Sarabandi et al., 2018). TAA activity was calculated using Equation (6) considering absorbance at 695 nm ).

| Cytotoxic and antiproliferative activity
The MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) is a colorimetric method for assessing cell metabolic activity against bioactive compounds. In this study, MTT test was carried out were seeded in a 96-well plate and exposed to different serial con-

| FTIR spectroscopy
The chemical structure of a biological compound can be deter-

| Amino acid composition
The amino acid composition (AAC) of proteins or polypeptides plays an important role in their both nutritional and technofunctional properties.  As can be seen in Figure 3c, the ratio of EAA to TAA in H-Pep and H-Pan hydrolysates was higher than those of other samples. This value of hydrolysate samples accounts for almost 30%, which is close to that recommended by FAO/WHO (32%).

| DPPH and ABTS + radical scavenging activity
The effect of enzyme type and different peptide concentrations on antioxidant activity in terms of DPPH (anionic and lipophilic radicals,

| Hydroxyl radical scavenging
Hydroxyl radicals are important chemicals with the ability to adversely affect biological molecules such as DNA. The effect of hydrolysate type and concentration on OH radicals scavenging is shown in Figure 3c. The highest OH radical inhibition activity belonged to H-Pep followed by H-Pan and H-Tri hydrolysates, particularly at their higher concentrations. These results are parallel to those obtained for DPPH and ABTS + radical scavenging activity TA B L E 1 Amino acid composition (mg g −1 DM) of corn pollen protein (CPP) and its hydrolysates obtained by pancreatin (H-Pan), pepsin (H-Pep) and trypsin (H-Tri). tests. As explained before, hydrolysates with lower Mw (higher DH) could expose more hydrophobic residues in the peptides, providing more reactive sites to scavenge free radicals, including OH radicals. Furthermore, antioxidant amino acids (Trp, Met hydrolyzed products. In this study, the effects of the type of proteases on the degree of hydrolysis and antioxidant indices of primary protein were investigated although proteases do not have much effect on polysaccharides due to their specific activity. Hence, the changes in these properties after enzymatic hydrolysis can be neglected.

| Morphologic observations
The overall appearance of corn pollen protein before and after hy-

| Antibacterial properties
In previous sections, it was shown by DH ( Figure 1a) (Aghamirzaei et al., 2015). The antibacterial activity was assessed by evaluating the diameter of inhibition zone (DIZ) (Peighambardoust et al., 2016 Figure 4f, where no inhibition zones were seen. These results are in accordance with several works reporting that Gram-negative bacteria are more resistant than Gram-positive bacteria (Jemil et al., 2014).

| Cytotoxic activity
In this study, MTT test was used to study the effect of H-Pep hydro-

ACK N OWLED G M ENTS
This study was part of PhD study carried out at the University of Tabriz (Tabriz, Iran).

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors confirm that they have no conflicts of interest with respect to the work described in this manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data will be available on request.