Purification and identification of antioxidant peptides from Chinese cherry (Prunus pseudocerasus Lindl.) seeds

Journal of Functional Foods 19 (2015) 394–403

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Purification and identification of antioxidant peptides from Chinese cherry (Prunus pseudocerasus Lindl.) seeds Peng Guo a, Yijun Qi b, Chuanhe Zhu a,*, Qun Wang b,** a b

College of Food Science and Engineering, Shandong Agricultural University, Taian, Shandong 271018, China Department of Chemical and Biological Engineering, Iowa State University, Ames, IA 50011, USA

A R T I C L E

I N F O

A B S T R A C T

Article history:

Chinese cherry seed protein hydrolysate (CPH) was prepared with enzymatic hydrolysis using

Received 4 March 2015

Alcalase and Neutrase, to obtain antioxidant peptides. The CPH was gradually fractionated

Received in revised form 28 August

by ultrafiltration through 10, 5 and 3 kDa molecular weight cut-off (MWCO) membranes, then

2015

the potential sub-fraction F-IV-E(a) was gained from the active fraction F-IV (MW < 3 kDa)

Accepted 1 September 2015

using gel filtration chromatography. F-IV-E(a)-4 and F-IV-E(a)-5 with the significantly higher

Available online

(P < 0.05) antioxidant activities were obtained by reversed phase high performance liquid chromatography (RP-HPLC). F-IV-E(a)-4 and F-IV-E(a)-5 were identified as Phe–Pro–Glu–Leu–

Keywords:

Leu–Ile (731.92 Da) and Val–Phe–Ala–Ala–Leu (520.61 Da) by the protein sequencer and the

Chinese cherry (Prunus

quadrupole time-of-flight mass spectrometer (Q-TOF MS), respectively. The results sug-

pseudocerasus Lindl.) seeds

gested that F-IV-E(a)-4 and F-IV-E(a)-5 may serve as potential antioxidants and might be

Antioxidant activity

evaluated as food additives, functional food and pharmaceuticals. © 2015 Elsevier Ltd. All rights reserved.

Peptide Protein hydrolysate

1.

Introduction

Human health is continuously subjected to various free radicals and oxidative stress from exogenous or endogenous factors which can probably bring about homeostasis imbalance if free radicals are not promptly removed and undesirable impacts on cell, tissues and DNA (Kryston, Georgiev, Pissis, & Georgakilas,

2011). The occurrences of diseases is associated with excessive free radicals, such as chronic diseases, cardiovascular disorders, degenerative diseases, cancer and other major disorders (Harnedy & FitzGerald, 2012; Maulik & Kumar, 2012). Also the oxidation of biomacromolecules during food processing, transportation and storage is involved in food quality deterioration (Sarmadi & Ismail, 2010; Wardhani, Fuciños, Vázquez, & Pandiella, 2013).

* Corresponding author. College of Food Science and Engineering, Shandong Agricultural University, No. 61, Daizong Road, Tai’an, Shandong Province 271018, China. Tel.: +86 538 8246021; fax: +86 538 8242850. E-mail address: [email protected]; [email protected] (C. Zhu). ** Corresponding author. Department of Chemical and Biological Engineering, Iowa State University, 2114 Sweeney Hall, Ames, IA 50011, USA. Tel.: 001 (515) 294-8216. E-mail address: [email protected] (Q. Wang). Abbreviations: CPH, cherry seed protein hydrolysate; MWCO, molecular weight cut-off; MW, molecular weight; RP-HPLC, reversed phase high performance liquid chromatography; Q-TOF MS, quadrupole time-of-flight mass spectrometry; CPI, cherry seed protein isolate; BHT, butylated hydroxytoluene; BHA, butylated hydroxyanisole; PG, propyl gallate; TBHQ, tertiary butylhydroquinone; ESI, electrospray ionization; DPPH, 1,1-diphenyl-2-picrylhydrazyl; ABTS, 2, 2′-azinobis-3-ethylbenzthiazoline-6-sulphonate; TFA, triflouroacetic acid http://dx.doi.org/10.1016/j.jff.2015.09.003 1756-4646/© 2015 Elsevier Ltd. All rights reserved.

Journal of Functional Foods 19 (2015) 394–403

Therefore, antioxidants which play a critical role in scavenging free radicals and retarding oxidation-induced deterioration have been reported in several researches. Many synthetic antioxidants have been recognized, such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl gallate (PG) and tertiary butylhydroquinone (TBHQ), and are widely applied in food preservation and retarding lipid oxidation (Bernardini et al., 2011; Harnedy & FitzGerald, 2012). However, the synthetic antioxidants have a certain degree of potential risk threats, thus their usage should be under strict limitation (Chalamaiah, Kumar, Hemalatha, & Jyothirmayi, 2012; Puchalska, Marina, & García, 2014). In this regard, interests have been emerging among researchers to purify and identify natural antioxidants. Natural antioxidants that were extracted from animals and plants can enhance the nutritional value when added into food, and also protect the body from oxidative stress when consumed (El-Shourbagy & El-Zahar, 2014; Senanayake, 2013). Recently the interests in antioxidant peptides derived from various kinds of food as well as by-products of food processing have been intensified. These peptides are recognized to be composed of functional amino acid sequences with easy adsorption and low molecular weight (He, Girgih, Malomo, Ju, & Aluko, 2013; Onuh, Girgih, Aluko, & Aliani, 2014). Moreover these natural peptides exhibited different models of free radicalscavenging abilities in addition to nutritive value without significant side-effects (Samaranayaka & Li-Chan, 2011). Generally, antioxidant peptides are usually constituted by 3–20 amino acid residues per chain, and ascribe the antioxidant activities to the inherent composition and sequence of amino acids (He et al., 2013). In this sense, enzymatic hydrolysis could be employed to obtain antioxidant peptides due to its advantages over chemical hydrolysis, such as environmental friendliness, specificity and safety. Many antioxidant peptides were identified from food proteins derived from animal sources like threadfin bream surimi processing byproduct, egg white protein, milk proteins, Sphyrna lewini muscle, tilapia gelatin (Chen, Chi, Zhao, & Lv, 2012; Hogan, Zhang, Li, Wang, & Zhou, 2009; Sun, Zhang, & Zhuang, 2013; Wang, Li, Chi, Zhang, & Luo, 2012; Wiriyaphan, Chitsomboon, Roytrakul, & Yongsawadigul, 2013) and from plant origin such as Chinese leek seeds, corn gluten meal, Zizyphus jujuba fruits, mungbean meal (Hong, Chen, Hu, Yang, & Wang, 2014; Lapsongphon & Yongsawatdigul, 2013; Memarpoor-Yazdi, Mahaki, & Zare-Zardini, 2013; Zhuang, Tang, & Yuan, 2013). With the rapid growth of cherry processing industry, the cherry by-products with no retrieving nutrition becomes the main barrier limiting its further processing, and also brings environmental problems in developing countries (Chen, Yin, Zhu, & Yu, 2014). Converting by-product materials into valueadded functional ingredients by applying biotechnology is a desirable approach. The cherry seeds take the most parts of the by-products of cherry wine, which contain abundant valuable proteins that could be the precursor of antioxidant peptides by the means of enzymatic hydrolysis. In the present study, Chinese cherry seed protein hydrolysate (CPH) and their purified peptides were investigated. In addition, within our knowledge, the purification and identification of antioxidant peptides from Chinese cherry seed protein have not been reported before.

2.

Materials and methods

2.1.

Materials

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Chinese cherry (Prunus pseudocerasus Lindl.) was purchased from local market (Taian City, Shandong Province, China). Chinese cherry seeds were sorted from the residue after cherry wine fermentation, and then ground into powder (average diameter of 250 µm) prior to preparation of protein. Alcalase with activity of 200,000 U/g, Neutrase with activity of 60,000 U/g and Sephadex G-25 and G-15 were purchased from China Shanghai Yuanye Biotech Company. All other chemicals and reagents mentioned in this study were of analytical grade, or otherwise mentioned.

2.2.

Preparation of enzymatic hydrolysates

2.2.1.

Preparation of CPI

Cherry seed protein isolate (CPI) was produced from defatted Chinese cherry seed powder according to the method reported by Ran, Lu, and Zhu (2007) with slight modifications as follows. Briefly, defatted cherry seed powder was dispersed in distilled water (1:20, w/v) and the suspension solution was adjusted to pH 10.0 with 1 N NaOH to solubilize the proteins for 20 min. During the process, the pH was maintained stable under continuously stirring at 40 °C for 40 min followed by centrifugation (3040 g, at 4 °C) for 20 min using GL-21MC (Shanghai Zhao Experiment Instrument Co., Ltd). The insoluble substance was discarded, and the supernatant was filtered with filter paper (Whatman No. 1) and adjusted to pH 3.84 with 1 N HCl to precipitate the proteins. Then the mixture was centrifuged (3040 g, at 4 °C) for 20 min. The precipitate was dispersed in distilled water again and adjusted to pH 7.0 with 1 N NaOH and 1 M HCl. Thereafter, the solution was freeze-dried (ALPHA 1–2 LD plus, Marin Christ Co., Germany) to produce CPI powder. Protein content of CPI was determined with bovine serum albumin as a standard (Weng et al., 2014).

2.2.2.

Preparation of CPH

The CPH was gained by the method that was established in our previous work. Hydrolysis processes were conducted in 250 mL glass conical flask. CPI was dissolved in distilled water with ratio of 1:25 (w/v) and placed into 80 °C water for 15 min to denature the protein and increase the active sites for enzymes. Next, the reaction system was cooled to room temperature, and then adjusted the pH and temperature to the compatible level for both Alcalase and Neutrase: pH 7.5 and 50 °C. After that, enzymes at the desired concentration were added to the solution to initiate the hydrolysis. During the hydrolysis experiments, the pH was maintained at the desirable value by continuously adding 0.05 N NaOH for 2 h in thermostat water bath vibrator at the spiral speed of 100 r/min. By the end of hydrolysis time, reactions were terminated in a boiling water bath for 10 min to inactivate enzymes. Afterwards, the hydrolysate was centrifuged at 3040 g for 20 min, the supernatant was collected and lyophilized for further use. Degree of hydrolysis (DH) analysis was performed using the method reported by Adler-Nissen (1979). For the amino acid analysis, the lyophilized protein isolate and hydrolysate were hydrolyzed in 6 N HCl at 110 °C for 24 h.

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Journal of Functional Foods 19 (2015) 394–403

All the samples were vaporized and the residues were dissolved in 25 mL citric acid buffer solution. An aliquot of 50 µL was loaded onto an automatic amino acid analyzer (L-8900 Amino Acid Analyser, Hitachi, Japan).

2.3.

Purification of the antioxidant peptides

2.3.1.

Ultrafiltration

The lyophilized CPH powder was dissolved in distilled water and fractionated by ultrafiltration using Continuous Flow Analytical System (Bonar Biotechnology Co., Ltd., Jinan, China) with the MWCO membranes of 10, 5 and 3 kDa (Bonar Biotechnology Co., Ltd., Jinan, China), respectively. The CPH was initially filtered by a 10 kDa membrane to generate a retentate (F-I, MW > 10 kDa), then the 10 kDa permeate was further filtered by 5 kDa membrane into two fractions of 5 kDa retentate (FII, 5–10 kDa) and the 5 kDa permeate. Thereafter, the 5 kDa permeate was fractionated into a 3 kDa retentate (F-III, 3–5 kDa) and permeate (F-IV, MW < 3 kDa). The fractions F-I, F-II, F-III and F-IV were collected and lyophilized for further purification and evaluating antioxidant activities.

2.3.2.

Gel filtration chromatography

The fractions with the highest antioxidant activity were redissolved in distilled water and further purified using a Sephadex G-25 gel filtration chromatography column (1.6 × 50 cm, Yuanye Biotech Co. Ltd., Shanghai, China). The sample loading quantity was 1 mL with the concentration of 50 mg/mL and then eluted with distilled water at a flow rate of 0.25 mL/min, and each fraction was collected after 3 min of elution. After that, the peak that represented the highest radical scavenging activities was subjected to a second round of gel filtration chromatography using a Sephadex G-15 column (1.0 × 60 cm, Yuanye Biotech Co. Ltd., Shanghai, China) with sample loading concentration of 25 mg/mL, and the elution was carried out at a flow rate of 0.25 mL/min, each fraction was collected after 2 min of elution. Each fraction of the eluted solution was monitored at 280 nm, then collected, pooled and lyophilized for further use.

2.3.3.

RP-HPLC

Reversed phase high performance liquid chromatography (RPHPLC) (Shimadzu Technologies Co. Ltd., Japan) on a Kromasil 100-5-C18 semipreparation column (4.6 × 250 mm, Kromasil Technologies Co. Ltd., Sweden) was used for further purification. The elution was performed with a linear gradient of acetonitrile/water (solvent system was as followings: 5–100% in 60 min, containing 0.1% TFA) at a flow rate of 1.0 mL/min and elution peaks were detected at 220 nm. The active fraction of eluted peak was collected by an automated fraction collector and concentrated using a vacuum rotary evaporator (R-3 HB, BUCHI Labortechnik, Switzerland), then freeze-dried before the amino acid sequence and molecular mass of purified peptides were analyzed.

2.4. Identification of amino acid sequence and molecular mass After the purification of RP-HPLC, amino acid sequences of purified peptides representing higher antioxidant activity were

measured based on the method of N-terminal amino acid sequencing using a protein sequencer (Applied Bio-systems Inc., USA) and the molecular mass was determined using quadrupole-time of flight mass spectrometry (Q-TOF MS) (Micromass, Altrincham, UK) coupled with electrospray ionization (ESI) source. The conduction was performed in positive ion mode and the spectrum was recorded by charged [M + H]+ state over the mass/charge (m/z) range of 100–1000. ESI conditions were as follows: fragmentation voltage: 200 V; nozzle voltage: 200 V; nebulization pressure: 50 psi; capillary voltage: 4.0 kV; quadrupole temperature: 100 °C; drying air temperature: 350 °C; dry gas and aerosol: N2; flow rate: 6.0 L/min, respectively. Auto MS/MS whose collision energy is 35 V was employed. The spectra were analyzed by the Masslynx software 4.1 (Waters Corp.).

2.5.

Determination of the antioxidant activity

2.5.1.

Hydroxyl radical scavenging activity

Hydroxyl radical scavenging activity was determined according to the method described by Wang et al. (2013). The reaction system containing 1.0 mL of 1.865 mM 1, 10-phenanthroline solution and 2.0 mL of sample solution was mixed with 1.0 mL of 1.865 mM FeSO4·7H2O solution in a capped tube, then the Fenton reaction was initiated by adding 1.0 mL of H2O2 (0.03%, v/v) to generate hydroxyl radicals. Then the reaction system was incubated at 37 °C in a water bath for 60 min. In the end, the absorbance of reaction mixture was measured at 536 nm using a spectrophotometer. Ascorbic acid was used as a positive control. All tests were performed in triplicate. The hydroxyl radical scavenging activity was calculated using the following equation:

hydroxyl radical scavenging activity ( % ) =

As − An × 100% Ab − An

where: As is the absorbance of sample; An is the absorbance of negative control with no antioxidant; Ab is the absorbance of blank without H2O2. A half elimination ratio (IC50) defined as the concentration of the sample required to inhibit 50% of hydroxyl radical can be calculated by linear regression.

2.5.2.

DPPH radical scavenging activity

The DPPH free radical scavenging activity was measured by following the method described by Girgih et al. (2014) with following modifications: A 100 µL of the sample solution was added to 100 µL of methanol and 100 µL of 0.6 mM of DPPH solution (prepared daily in methanol) in a 96-well microplate, then the reaction solution was well mixed by a microvibration plateoscillator for 10 s before incubating in 37 °C water bath for 1.5 h. The absorbance was then measured by a microplate reader (SpectraMax M2 & M2e Multi-Mode, Molecular Devices Co., Ltd., USA) at 515 nm (As). Reduced glutathione (GSH) was used as a positive control. All tests were performed in triplicate. The DPPH radical scavenging activity was calculated according to the following equation:

DPPH radical scavenging activity ( % ) =

Ab − As × 100% Ab

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Journal of Functional Foods 19 (2015) 394–403

where Ab is the absorbance of blank using the methanol replacing the sample, As is the absorbance of sample solution. A percent inhibition versus concentration curve was gained and the concentration of sample required for 50% inhibition was expressed as IC50.

2.5.3.

Superoxide anion radical (O2−·) scavenging activity

Superoxide anion radical (O2−·) scavenging activity was measured according to the method reported by Jiang et al. (2014). One millilitre of hydrolysate samples was combined with 1.8 mL of 50 mM Tris–HCl buffer (pH 8.2). After the reaction mixture was incubated at 25 °C for 10 min, 0.1 mL of 10 mM pyrogallol (dissolved in 10 mM HCl) was added to initiate the reaction. The absorbance of the solution at 320 nm was measured and recorded every 30 s for 4 min. The slope of the absorbance line represented the oxidation rate of pyrogallol for sample (ΔAs), while the control was carried out with 1.0 mL of distilled water replacing the sample whose slope of the absorbance line represented the autoxidation rate of pyrogallol for control (ΔAc). Ascorbic acid was used as a positive control. All tests were performed in triplicate. The capability for scavenging superoxide anion radical was calculated as:

Superoxide anion radical scavenging activity ( % ) =

ΔAc − ΔAs × 100% ΔAc

where: ΔAs is the absorbance of sample; ΔAc is the absorbance of control. The inhibition concentration at 50% inhibition (IC50) was calculated by linear regression.

2.5.4.

ABTS radical cation (ABTS•+) scavenging activities

ABTS radical cation scavenging activity was determined according to the ABTS radical cation decolourization assay reported by Re, Pellegrini, Proteggente, Yang, and Riceevans (1999) and Chang, Ismail, Yanagita, Esa, and Baharuldin (2015) with a slight modification. The ABTS radical cations were generated by the combined solution containing ABTS stock solution (7 mM dissolved in deionized water) and potassium persulphate at final concentration of 2.45 mM. The mixture was incubated in the dark at room temperature for 16 h to allow the cation radical development before use. Then the radical cation solution was diluted in deionized water to obtain the absorbance of around 0.70–0.72 at 734 nm. After that, 1.0 mL of diluted ABTS radical solution was mixed with 1.0 mL of samples. Ten minutes later, the absorbance was measured at 734 nm against the corresponding blank. Trolox was employed as positive control. All tests were performed in triplicate. The ABTS radical cation scavenging activity was calculated using the following equation:

ABTS radical cation scavenging activity ( % ) =

Ac − As × 100% Ac

where: As is the absorbance of sample (ABTS•+ solution plus sample); Ac is the absorbance of control (ABTS•+ solution). Concentration of the substance required for 50% reduction in absorbance (IC50) was calculated from the percent inhibition versus concentration curve.

2.6.

Statistical analysis

ANOVA test (using SPSS 19.0 software) was applied to find statistically significant differences among results, when three replicates of every sample were done. Differences between the means of parameters were determined by using a level of significance equal to 0.05.

3.

Results and discussion

3.1.

Preparation of CPI and CPH

The extraction ratio of CPI extracted from defatted Chinese cherry seed powder reached 42.27 ± 0.84% using the method reported by Ran et al. (2007). The components and DPPH radical scavenging ability for CPI were analyzed and the results were shown Table 1. The DPPH radical scavenging ability was 6.79 ± 0.38 at 5 mg/mL, and the content of protein was up to 82.16 ± 0.58%. The CPH was gained according to the above method (2.5.5: preparation of CPH), and the DH was 14.56 ± 0.29% (n = 3). Analysis of amino acid composition was carried out by applying an automatic amino acid analyzer. The results (data were not shown) indicate that there are no difference between CPI and CPH in terms of amino acid composition (essential amino acid: Leu, Ile, Lys, Met, Thr, Val, His, Phe; nonessential amino acid: Pro, Ser, Tyr, Glu, Gly, Ala, Arg, Asp, Cys). Additionally, the result was in accord with the previous study that hydrolysis did not change the amino acid composition of protein hydrolysate (Zhou, Wang, & Jiang, 2012).

3.2.

Purification of antioxidant peptides

3.2.1.

Fractionation of CPH by ultrafiltration

MW of peptide plays a critical role in the antioxidant activity (Guo, Kouzuma, & Yonekura, 2009; Wu, Pan, Chang, & Shiau, 2005). The effects of molecular size on bioactivity through fractionation by ultrafiltration have been investigated by many scientists (Kim, Je, & Kim, 2007; Liu, Kong, Xiong, & Xia, 2010; Zhuang et al., 2013). It has been reported that hydrolysates of smaller MW exhibited higher antioxidant activity than those of larger MW (Dávalos, Miguel, Bartolomé, & López-Fandiño, 2004). The freeze-dried CPH was dissolved in distilled water to

Table 1 – Compositions and DPPH radical scavenging ability of cherry seed isolated protein. Samples

Defatted cherry seed powder

Cherry seed isolated protein

Moisture (%) Protein (%) Crude fat (%) Ash (%) Carbohydrates (%) DPPH radical scavenging ability (%)

3.44 ± 0.21 44.38 ± 0.67 2.62 ± 0.49 6.41 ± 0.42 43.15 ± 0.84 N.D.

2.21 ± 0.19 82.16 ± 0.58 1.26 ± 0.23 5.46 ± 0.37 8.91 ± 0.72 6.79 ± 0.38

All the values were mean ± SD (n = 3). SD, standard deviation; N.D., not determined.

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Fig. 1 – Radical scavenging abilities of fractions isolated by ultrafiltration and their parent hydrolysates at 5 mg/mL. All the values were mean ± SD (n = 3). SD, standard deviation. Values with same superscripts indicated no significant difference (P > 0.05).

obtain the concentration of 25 mg/mL, and was fractionated by ultrafiltration with MWCO membranes (10, 5 and 3 kDa) to obtain four fractions which were named as F-I (MW > 10 kDa), F-II (5–10 kDa), F-III (3–5 kDa) and F-IV (MW < 3 kDa), respectively. As shown in Fig. 1, all the fractions of F-I, F-II, F-III and F-IV exhibited higher radical scavenging abilities than their parent hydrolysates at the concentration of 5 mg/mL, among which the F-IV showed the strongest scavenging ability in terms of DPPH radical, hydroxyl radical and superoxide anion radical, while both F-III and F-IV showed the same highest ABTS radical cation scavenging activity. From above, we concluded that the F-IV was relatively rich in peptides of smaller MW. This result was similar to other reports showing that the antioxidant activity of hydrolysates relied on their MW distributions, and peptides and hydrolysates with lower MW could interact with free radicals interfering in oxidative process more effectively (Li et al., 2013; Mendis, Rajapakse, Byun, & Kim, 2005; Zarei et al., 2014).

3.2.2.

Gel filtration chromatography of F-IV

To obtain higher antioxidant sub-fractions, F-IV was loaded onto a Sephadex G-25 gel filtration column. As shown in Fig. 2A, F-IV was separated into five sub-fractions (F-IV-A to F-IV-E). These sub-fractions were pooled in terms of their peak values and freeze-dried for evaluating antioxidant activities. Among the five sub-fractions, the F-IV-E showed the higher DPPH radical scavenging ability (55.04 ± 0.87%), hydroxyl radical scavenging activity (53.25 ± 0.96%), ABTS radical cation scavenging activities (56.27 ± 0.86%) and superoxide anion radical scavenging activity (51.89 ± 0.92%) than the other sub-fractions at the concentration of 3 mg/mL, as shown in Fig. 2B. Thus the freeze-dried F-IV-E is chosen to perform a second round gel filtration for further purification, and the elution profile was

shown in Fig. 2C. Three sub-fractions with good resolution were separated by Sephadex G-15. They were collected and pooled according to the corresponding tube numbers, and freezedried for investigation of their radical scavenging abilities, as shown in Fig. 2D. From the results, the F-IV-E(a) had higher radical scavenging abilities than the other two sub-fractions in terms of DPPH radical (65.74 ± 0.84%), hydroxyl radical (58.21 ± 0.89%), ABTS radical cation (55.58 ± 0.91%) and superoxide anion radical (62.23 ± 0.87%) at the concentration of 2 mg/ mL, while the sub-fraction (c) had almost no radical scavenging abilities.

3.2.3.

Purification of F-IV-E(a) using RP-HPLC

Throughout the gel filtration chromatography, the potential subfraction F-IV-E(a) was preliminary purified, then the RP-HPLC was applied, which was widely considered to be the final step in the peptide purification with its advantages of high sensitivity, resolution and column efficiency. The F-IV-E(a) was loaded onto a semi-preparation column and five major peaks (1–5) were obtained, as shown in Fig. 3. According to the RP-HPLC elution properties, the large polar or hydrophilic peptides eluted first then the non-polar or hydrophobic peptides followed (Zhang, Mu, & Sun, 2014). E(a)-5 should have the strongest hydrophobic properties among the five peptides, and their radical scavenging abilities were measured (Table 2). The IC50 values of these five antioxidant peptides were 0.31 ± 0.0.03 mg/mL (E(a)-5), 0.57 ± 0.02 mg/mL (E(a)-4), 1.02 ± 0.0.3 mg/mL (E(a)-3), 10.08 ± 0.03 mg/mL (E(a)2), and 14.56 ± 0.02 mg/mL (E(a)-1), respectively. The IC50 of DPPH radical scavenging activity, ABTS radical scavenging activity and superoxide anion radical for fraction E(a)-5 was 0.22 ± 0.01 mg/mL, 0.38 ± 0.02 mg/mL, 0.51 ± 0.02 mg/mL, respectively. Meanwhile the fraction E(a)-4 also exhibited the similar but higher superoxide anion radical scavenging.

Journal of Functional Foods 19 (2015) 394–403

399

Fig. 2 – A. Gel filtration chromatography of F-IV on a Sephadex G-25 column. B. Radical scavenging abilities of five subfractions. All the values were mean ± SD (n = 3). SD, standard deviation. Values with same superscripts indicated no significant difference (P > 0.05). C. Gel filtration chromatography of F-IV-E on a Sephadex G-15 column. D. Radical scavenging abilities of five sub-fractions. All the values were mean ± SD (n = 3). SD, standard deviation. Values with same superscripts indicated no significant difference (P > 0.05).

However, in comparison with the positive control (GSH, Trolox, ascorbic acid), the free radical scavenging activities of all fractions were significantly lower (P < 0.05). A significant difference (P < 0.05) in radical scavenging abilities was observed between

the E(a)-4, 5 and other peptides (E(a)-1, 2, 3). Therefore, the E(a)-4 and E(a)-5 were selected and collected, pooled according to their own retention time by repeating elution performance.

Fig. 3 – RP-HPLC profile of active peak F-IV-E(a) on a C18 column.

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Table 2 – The value of IC50 for F-IV-E(a)-1–5 peptides. Peptides

E(a)-1 E(a)-2 E(a)-3 E(a)-4 E(a)-5 Ascorbic acid GSH Trolox

IC50 (mg/mL) Hydroxyl radical

DPPH radical

ABTS radical cation

Superoxide anion radical

14.56 ± 0.02 10.08 ± 0.03 1.02 ± 0.03 0.57 ± 0.02 0.31 ± 0.03 0.05 ± 0.01 N.D. N.D.

10.12 ± 0.03 7.29 ± 0.04 0.95 ± 0.03 0.35 ± 0.01 0.22 ± 0.01 N.D. 0.31 ± 0.02 N.D.

11.91 ± 0.03 16.58 ± 0.05 0.98 ± 0.04 0.40 ± 0.01 0.38 ± 0.02 N.D. N.D. 0.10 ± 0.01

9.89 ± 0.02 13.19 ± 0.04 1.01 ± 0.02 0.25 ± 0.01 0.51 ± 0.02 0.08 ± 0.01 N.D. N.D.

All the values were mean ± SD (n = 3). SD, standard deviation; N.D., not determined.

3.3. Determination of amino acid sequence and molecular mass Antioxidative properties of peptides are related to composition, structure, molecular weight, amino acid sequence and hydrophobicity. Considering the radical scavenging ability values, the amino acid sequences and molecular mass of peptides E(a)-4 and E(a)-5 were analyzed using protein sequencer and Q-TOF MS, respectively. The mass spectra of peptides E(a)-4 and E(a)-5 were shown in Fig. 4. E(a)-4 and E(a)-5 were identified as Phe–Pro–Glu–Leu–Leu–Ile (731.92 Da, error mDa = −1.0) and Val–Phe–Ala–Ala–Leu (520.61 Da, error mDa = −0.8). The deviations between the theoretical mass of Phe–Pro–Glu–Leu– Leu–Ile (730.89 Da), Val–Phe–Ala–Ala–Leu (519.62 Da) and experimental mass of peptides E(a)-4 (731.92 Da [M + H]+), E(a)-5 (520.61 Da [M + H]+) were considered to be acceptable when re-

quired. The results indicated ESI-Q-TOF can be used to identify the peptides in most active fractions, which are potentially responsible for the observed bioactivities. This similar result was reported by María, Jochan, Estefanía, and María (2015). Phe– Pro–Glu–Leu–Leu–Ile (730.89 Da) and Val–Phe–Ala–Ala–Leu (519.62 Da) further confirmed with the general finding that short peptides (2–10 amino acids) present greater antioxidant activity than their parent proteins or polypeptides (Gu et al., 2015; Megias et al., 2004). This similar result was also gained in other reports that the antioxidant activity of hydrolysates relied on their MW distributions, and peptides and hydrolysates with lower MW exhibit greater antioxidant activity (Li et al., 2013; María et al., 2015; Rao & Sun, 2012; Zarei et al., 2014). The hydrophobic amino acids (Val, Leu etc.) and aromatic amino acids (Phe, His, Tyr and Trp) can enhance to the radical scavenging abilities of peptides (Aluko & Monu, 2003; Chen,

Fig. 4 – Mass spectra of peptides E(a)-4 and E(a)-5.

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Muramoto, Yamauchi, & Nokihara, 1996; Ren et al., 2008; Riisom, Sims, & Fiorti, 1980; Sarmadi & Ismail, 2010). The antioxidant peptides (less than 10 amino acids) from cherry (Prunus cerasus L.) in fractions contain high number of hydrophobic and aromatic amino acids (María et al., 2015). Leila and Abdul (2014) reported the presence of the hydrophobic amino acid valine, aspartic acid in the peptide sequences contribute to the high antioxidant activity in the SPH. The results of amino acid analysis indicate that peptides E(a)-4 and E(a)-5 mostly consisted of the hydrophobic amino acids Phe, Phe, Ala, Val, Ile, Leu, Pro. The results further prove that hydrophobic amino acid residues and aromatic amino acids can enhance the radical scavenging abilities of peptides. Many researches indicated that sequences of the peptides and the amino acids positioning within the sequences are important to their antioxidant properties (Chen, Muramoto, Yamaguchi, Fujimoto, & Nokihara, 1998; Zhang et al., 2014). The presence of non-polar aliphatic amino acid Val at the N-terminal of the sequence has been reported to exert better antioxidant activities and retard lipid peroxidation effects (Chen, Muramoto, & Yamauchi, 1995), which is supposed to have the critical mechanisms to interrupt the radical mediated chain reaction. The results from Elias, Kellerby, and Decker (2008) indicated that antioxidative peptides often contain hydrophobic amino acid including Val or Leu at the N-terminus of the peptides. The large proportion of hydrophobic amino acid residues in the purified peptides also played an important role in facilitating scavenging radicals, because the hydrophobicity of peptides has easy accessibility to hydrophobic targets and hydrophobic peptides can easily got through the cell membrane in the living cells (Lhor et al., 2014), as well as to enhance the solubility of peptide in lipid, facilitating the contacts with hydrophobic radical species. The sequence was also found to contain dual amino acid residues, such as Ala–Ala observed in E(a)-5. The dual amino acid residues were also observed in the hydrolysates of flounder fish muscle by digestive protease (Ko, Lee, Samarakoon, Kim, & Jeon, 2013). In the present study, among these amino sequences, Leu and Ile contributed a lot to the antioxidant activity probably because their long aliphatic side-chain groups could also interrupt the radical reaction chain (Wang et al., 2014). Moreover, Glu in the sequence of E(a)-4 was conceivably capable of quenching unpaired electrons or radicals, supporting the results reported previously (Chi, Wang, Wang, Zhang, & Deng, 2015; Zhang et al., 2009). Therefore, above all, the antioxidant activity of purified Chinese cherry seed peptides seems to have close relationship with the presence of special dual amino acid sequences and hydrophobic amino acid residues.

4.

Conclusions

This study is the first to report the purification and identification of antioxidant peptides derived from Chinese cherry seed enzymatic hydrolysate. Two peptides with highest antioxidant activity were purified and were identified, which were Phe– Pro–Glu–Leu–Leu–Ile (731.92 Da) and Val–Phe–Ala–Ala–Leu (520.61 Da). Both of the peptides exhibited good radical scavenging activity on DPPH radical, hydroxyl radical, ABTS radical

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and superoxide radical. These results suggested that Phe–Pro– Glu–Leu–Leu–Ile and Val–Phe–Ala–Ala–Leu had the potential to be used as natural antioxidants and provide a theoretical basis for the application of the Chinese cherry seed antioxidant peptides in exploring functional foods and pharmaceuticals. However, further studies should be done to investigate their antioxidant activities.

Acknowledgements The research was financially supported by the Wan Defu Food Co., Ltd. Dr. Wang thanks to the support from Iowa State University (ISU) President’s Initiative on Interdisciplinary Research (PIIR) program and McGee-Wagner Interdisciplinary Research Foundation. REFERENCES

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