Purification and identification of an antioxidant peptide from perilla seed (Perilla frutescens) meal protein hydrolysate

Abstract This study aimed to obtain antioxidant peptides from perilla seed meal (PSM), which is normally discarded as an industrial waste during seed oil extraction. PSM protein was hydrolyzed using trypsin and fractionated by ultrafiltration. Molecular weight fraction (<3 kDa) with the highest antioxidant activity was purified using prep‐HPLC and analytical HPLC. The purification fold of the peptide (fraction V) obtained from PSM protein hydrolysate on DPPH radical scavenging activity, ABTS radical scavenging activity, and reducing power was 1.79‐, 1.59‐, and 1.81‐fold, respectively, after the three‐step purification procedure. The sequence of the purified peptide from fraction V that exhibited free radical scavenging activity and reducing power was identified as Ile‐Ser‐Pro‐Arg‐Ile‐Leu‐Ser‐Tyr‐Asn‐Leu‐Arg (1,330.77 Da). These results demonstrate that PSM protein, a by‐product from the oil seed extraction, can be used as a source of natural antioxidant peptides for food and/or nutraceutical applications.

2005). Natural-derived antioxidant peptides are 2-20 amino acid residues long, and their activities are dependent on their sequence, structure, and hydrophobicity (Rahman et al., 2018). The antioxidant peptides from animal sources have been well studied.
Perilla (Perilla frutescens var. japonica HARA), an annual plant, is cultivated throughout Asian countries. Its Korean name is "delkkae" and its leaves are widely used as a spice, garnishment, and food colorant (Ha et al., 2012). Perilla seeds are commonly subjected to oil extraction because of the presence of enriched oil content containing a high percentage of unsaturated fatty acids (approximately more than 90%) and α-linolenic acid (ranging from 52.58% to 61.97%; Ding, Hu, Shi, Chao, & Liu, 2012). Perilla seed meal (PSM), the residue from the seed oil extraction process, can be a potentially abundant source of structurally diverse bioactive compounds (Luther et al., 2007;Lutterodt, Slavin, Whent, Turner, & Yu, 2011). PSM contains relatively higher protein content compared to perilla seeds before oil extraction.
In particular, cold-pressed PSM may provide better health benefits because cold-press extraction involves no heat treatment or solvents (Parry & Yu, 2004;Yu, Haley, Perret, & Harris, 2002). Therefore, owing to its high protein content, cold-pressed PSM can be used as starting material for production of antioxidant peptides (Di Bernardini et al., 2011). Although PSM is rich in protein, most of the PSM is discarded as waste or used for animal feed owing to its low cost. Therefore, in this study, PSM was hydrolyzed to develop novel antioxidant peptides, as a way of better utilizing this valuable resource.

| Preparation of protein extract from defatted PSM
Perilla seed meal protein extraction was carried out following a modified procedure by He, Girgih, Malomo, Ju, and Aluko (2013).
PSM was dissolved in deionized water (1:10, w/v) and the slurry stirred for 1 hr at 25°C. After adjusting to pH 10 with 1 M NaOH, the slurry was stirred again for 1 hr at 25°C and centrifuged at 1,600 g for 30 min. The supernatant was collected, adjusted to pH 4.0 using 1 M HCl, and left for 30 min to allow protein precipitation. Subsequently, the mixture was centrifuged (1,600 g, 4°C) for 30 min. The resultant precipitate was re-dispersed in deionized water, adjusted to pH 7.0 with 1 M NaOH, freeze dried, and stored at −20°C. This powder was subsequently referred as the PSM protein extract.

| Preparation of hydrolysates from PSM protein
Preliminary experiments using various enzymes (alcalase, neutrase, trypsin, papain, and pepsin) showed that the most potent antioxidant activity was observed for PSM protein hydrolysate derived from a 3 hr hydrolysis using trypsin (25 units). These hydrolysis parameters were thus used in the present study. PSM protein powder was mixed with 0.1 M sodium phosphate buffer (pH 8.0) in the ratio of 1:20 (w/v), and 25 units of trypsin were added to the reaction. Hydrolysis was carried out for 3 hr at 37°C using a water bath with stirring.
Hydrolysis was terminated by heating the mixture at 100°C for 10 min, followed by centrifugation at 1,600 g for 3 hr. The supernatant (PSM protein hydrolysate) was collected, lyophilized, and stored at −40°C until analysis.

| Determination of degree of hydrolysis
The degree of hydrolysis (DH) was estimated according to Jang et al. (2016) based on a modification of Hoyle and Merritt's (1994) method. Briefly, PSM protein hydrolysates were mixed with 20% trichloroacetic acid (TCA; 1:1 ratio v/v) and centrifuged at 1,600 g for 30 min at 4°C. The soluble protein content in the supernatant was measured using the microplate bicinchoninic acid (BCA) colorimetric method described by Smith et al. (1985). Twenty microliters of each sample was added to 160 µl of BCA reagent and incubated at 37°C for 30 min. Absorbance was measured at 560 nm using a microplate spectrophotometer (Epoch, Biotek, Winooski, VT, USA).
Bovine serum albumin (BSA) was used as the standard. The DH was expressed as following equation:

| DPPH radical scavenging activity
The methodology to measure DPPH radical scavenging activity was adopted from Jang et al. (2016)

| Reducing power
The reducing power was determined following the methodology given by Yen and Chen (1995), with modifications. Sample (100 µl) was mixed with 100 µl of 0.2 M sodium phosphate buffer (pH 6.6) and 100 µl of 1% potassium ferricyanide. The solution was incubated at 50°C for 20 min. After incubation, 100 µl of 10% TCA was added. This mixture was centrifuged at 18 g for 10 min at 4°C. Next, 100 µl of the upper layer was mixed with 100 µl of distilled water and 200 µl of 0.1% ferric chloride, and allowed to stand at room temperature for 10 min. The solution (200 µl) was transferred to a clear bottom 96-well microplate, and absorbance at 700 nm was measured using a microplate spectrophotometer (Epoch, Biotek).

| Ultrafiltration
The tryptic PSM protein hydrolysate was fractioned according

| Preparative high-performance liquid chromatography
The ultrafiltration fraction with the highest antioxidant activity was separated according to Jang et al. (2016), using a HPLC system equipped with an XBridge OST C 18 preparative column

| Analytical high-performance liquid chromatography
Fractions were collected using a preparative high-performance liquid chromatography (prep-HPLC) and loaded onto an Atlantis™ dC 18 column (5 µm, 4.6 mm × 150 mm, Waters Co.) connected to the HPLC system (Waters 2695; Waters Co.) to confirm singular peaks as described by Jang et al. (2016). Briefly, solvent A and solvent B were prepared as described in the previous section. The flow rate was 0.8 ml/min with a linear gradient of 0%-50% solvent B in 8 min. Antioxidant activity of only single peaks of the fraction was measured.

| Molecular mass distribution and amino acid sequences of purified peptides
The molecular mass distribution and amino acid sequence of the purified peptides were determined by Q-TOF mass spectrometer

| Statistical analysis
All the experiments were conducted in triplicate with results expressed as mean ± standard deviation (mean ± SD), unless otherwise indicated. A one-way analysis of variance (ANOVA) was applied using the statistical software SPSS ver. 23.0 (SPSS Inc., Chicago, IL, USA), and the Duncan's multiple range test comparisons at p < 0.05 was run to determine significant differences.

| Degree of hydrolysis
The DH estimates the extent of hydrolysis by determining the number (%) of peptide bond cleaved, where higher enzyme concentration and hydrolysis time will lead to increased DH or smaller molecular weight peptides (Mueller & Liceaga, 2016

| PSM hydrolysate fractionation by ultrafiltration
In this study, ultrafiltration using three different MWCO membranes (10, 5, and 3 kDa) was employed to separate the PSM hydrolysate into four molecular size fractions (>10 kDa, 5-10 kDa, 3-5 kDa, and <3 kDa). The degree of molecular weight distribution is shown in and showed highest antioxidant activity relative to the higher molecular weight fractions. It is known that peptides will have improved antioxidant activities than native proteins, mostly due to an increased availability of the functional side chain to the reactive species, and the electron-dense peptide bonds generated by enzymatic hydrolysis (Udenigwe & Aluko, 2011). In our study, the resultant ultrafiltrates were tested for antioxidant activity. Figure 1 shows the antioxidant activities of ultrafiltered fractions and native PSM protein hydrolysate. As shown in Figure 1a, DPPH radical scavenging activities of peptide fractions (at 0.1 mg/ml) with molecular weight <3 kDa was estimated to be 36.89%, significantly higher (p < 0.05) than the PSM hydrolysate without ultrafiltration. In contrast, the DPPH radical scavenging activities of fractions with a molecular weight of 3-5 kDa, 5-10 kDa, and >10 kDa were significantly lower than the PSM hydrolysate. These results are in agreement with those by Li, Jiang, Zhang, Mu, and Liu (2008) (Zhuang, Tang, & Yuan, 2013). Figure 1c shows the reducing power of orig-  weight, as this enhances easy reduction of radical-mediated lipid peroxidation and the ability to react with lipid radicals (Ranathunga et al., 2006). Consequently, the < 3 kDa fraction, which showed the highest antioxidant activities, was chosen and purified for further studies. To further purify the antioxidant peptides from these fractions (IV and V), they were loaded on a dC 18 column using analytical HPLC.

| Purification of antioxidant peptides
The elution profile of the peptides is given in Figure 4, demonstrating that the fractionated peptides were relatively pure. As shown in Table 2, the purification fold of the purified antioxidant peptide (PAP1) derived from fraction IV on DPPH radical scavenging activity, ABTS radical scavenging activity, and reducing power was 2.18-, 1.38-, and 1.40-fold throughout the three-step purification procedure, respectively. The purification fold of the purified antioxidant peptide (PAP2) derived from fraction V on DPPH radical scavenging activity, ABTS radical scavenging activity, and reducing power was 1.79-, 1.59-, and 1.81-fold, respectively, after the three-step purification procedure.

| Molecular mass distribution and amino acid sequences of purified peptides
The purified peptides were analyzed by Q-TOF mass spectroscopy for identification of peptides and molecular mass distribution. The amino acid sequence of PAP1 could not be verified.
This was probably because of the incorporation of unexpected to an increase in their lipid solubility, which facilitates access to hydrophobic radical species. Moreover, hydrophobic amino acids are able to promote entry of the peptide into target organs through hydrophobic association, which is achieved favorably due to antioxidant properties (He et al., 2013;Sarmadi & Ismail, 2010). In this study, hydrophobic amino acid residues Ile, Pro, and Leu were present in the purified fraction PAP2, which could explain its high antioxidant activity. Wang, Camp, and Ehlenfeldt (2012)   while keeping their own stability through resonating structure . Memarpoor-Yazdi, Mahaki, and Zare-Zardini (2013) indicated that the presence of basic amino acids such as Arg within the peptide sequence was effective on its metal ion chelating activity. Therefore, based on the results obtained in this study, we assume that Leu, Ile, Ser, Tyr, and Arg in PAP2 played an important role in enabling antioxidant peptides to function as potent radical scavengers.

| CON CLUS ION
In the present study, the proteins of perilla seed by-products (PSM) were hydrolyzed by trypsin to obtain antioxidant peptides. The PSM protein hydrolysate was purified through ultrafiltration and reversephase HPLC (preparative and analytical). The amino acid sequence of the purified antioxidant peptide (PAP1) derived from fraction IV could not be confirmed. For PAP2 from fraction V, the amino acid sequence was identified as Ile-Ser-Pro-Arg-Ile-Leu-Ser-Tyr-Asn-Leu-Arg, with a molecular weight of 1,330.77 Da. The purified peptides exhibited good antioxidant activities, such as reducing power, DPPH radical scavenging activity, and ABTS radical scavenging activity.
The high activity of purified antioxidant peptides was due to their low molecular weight (<3 kDa) and the presence of specific amino acids including Leu, Ile, Pro, and Ser. These results suggest that hydrolysates from PSM can be used as natural antioxidants. However, further studies on antioxidant activities in vivo are needed.

ACK N OWLED G M ENTS
This work was supported by the 2018 Yeungnam University Research Grant.

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.

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