Changes on antioxidant activity of microwave-treated protein hydrolysates after simulated gastrointestinal digestion: Purification and identification

Accepted Manuscript Changes on antioxidant activity of microwave-treated protein hydrolysates after simulated gastrointestinal digestion: Purification and identification Sunantha Ketnawa, Malithi Wickramathilaka, Andrea M. Liceaga PII: DOI: Reference:

S0308-8146(18)30149-3 https://doi.org/10.1016/j.foodchem.2018.01.133 FOCH 22322

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

31 August 2017 11 January 2018 22 January 2018

Please cite this article as: Ketnawa, S., Wickramathilaka, M., Liceaga, A.M., Changes on antioxidant activity of microwave-treated protein hydrolysates after simulated gastrointestinal digestion: Purification and identification, Food Chemistry (2018), doi: https://doi.org/10.1016/j.foodchem.2018.01.133

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1

Full Title

2 3

Changes on antioxidant activity of microwave-treated protein hydrolysates after simulated gastrointestinal digestion: Purification and identification

4 5

Abbreviated running title

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Antioxidant activity of peptides after GI-digestion

7 8

Name(s) of Author(s)

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Sunantha Ketnawa, Malithi Wickramathilaka and Andrea M. Liceaga*

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Author Affiliation(s)

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Department of Food Science, Purdue University, 745 Agriculture Mall Dr., West Lafayette, IN 47907

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Authors email addresses

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S. Ketnawa, Email: [email protected]

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M. Wickramathilaka, Email: [email protected]

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A. M. Liceaga, Email: [email protected]

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Contact information for Corresponding Author

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*Andrea M. Liceaga, Email: [email protected], Tel. 765-496-2460

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22

Abstract

23

Two samples of trout frame protein hydrolysates were prepared by Microwave

24

Pretreatment followed by Conventional Enzymatic hydrolysis (MPCE) and Non-Pretreated

25

followed by Microwave-assisted Enzymatic hydrolysis (NPME) were subjected to simulated

26

gastrointestinal digestion. Changes on degree of hydrolysis, antioxidant activity, molecular

27

weight, and amino acid composition between undigested and after gastrointestinal digestion of

28

peptides were investigated. Comparing to undigested peptides, a breakdown of MPCE and

29

NPME into smaller molecules was observed. Degree of hydrolysis, ABTS•+radical scavenging

30

activity and reducing power increased (P<0.05) for both samples after gastrointestinal digestion.

31

A purified peptide from GI-MPCE had two possible sequences, NGRLGYSEGVM or

32

GNRLGYSWDD (1,182.65 Da). Whereas GI-NPME had two peptides IRGPEEHMHR or

33

RVAPEEHMHR (1,261.77 Da) and SAGVPRHK or SARPRHK (962.63 Da). These results

34

indicate that digested hydrolysates can be a rich source of antioxidants. Isolated peptides

35

extracted from trout frame by-products could be new food ingredients used as natural

36

antioxidants.

37 38

Keywords: simulated gastrointestinal digestion; antioxidant activity; protein hydrolysates,

39

microwave treatment; peptide sequence.

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1. Introduction

41

Scientists are constantly seeking bioactive peptides with antioxidant activity due to their

42

beneficial role in providing protection from oxidative stress without the risk of side effects that

43

are typically associated with synthetic antioxidants (Zeng, Dong, Zhao, & Liu, 2013). To exert

44

physio-biological effects in vivo, bioactive peptides must resist gastrointestinal digestion and

45

reach, in active form, their target sites after absorption (Espejo-Carpio, García-Moreno, Pérez-

46

Gálvez, Morales-Medina, Guadix, & Guadix, 2016; Wu, Fu, Sun, Zhang, Liu, Cao, et al., 2015).

47

Furthermore, the gastrointestinal tract is known to be a major oxidation site in the human body

48

(Srigiridhar, Nair, Subramanian, & Singotamu, 2001) and thereby it is important assessing

49

bioactive peptide stability after digestion. In vitro simulated gastrointestinal digestive model

50

(SGM) is a well-accepted approach to obtain preliminary observations in determining

51

bioavailability of the peptides prior to conducting in vivo studies. Previous studies report that

52

SGM results in more potent peptides compared with other types of enzymatic digestion

53

(Samaranayaka, Kitts, & Li-Chan, 2010; Teixeira, Pires, Nunes, & Batista, 2016).

54

Trout frame hydrolysates are a potential source of bioactive peptides with antioxidant

55

activity as reported by Nguyen, Jones, Kim, San Martin-Gonzalez, and Liceaga (2017) and

56

Ketnawa and Liceaga (2016). Based on these preliminary results of Ketnawa and Liceaga

57

(2016), two hydrolysates with the maximum antioxidant activity were selected to investigate

58

the stability of antioxidant activity through simulated gastrointestinal digestion. The present

59

study was undertaken to investigate changes on antioxidant activity of selected hydrolysates

60

released in a SGM. Furthermore, characterization, purification and identification of active

61

peptides after SGM were also determined to verify the possibility of application in functional

62

food materials or nutraceutical industries. 3

63

2. Material and Methods

64

2.1. Chemicals

65

Alcalase® 2.4L (≥2.4 AU/g) from Protease from Bacillus licheniformis, Subtilisin A, (EC

66

3.4.21.62), pepsin from gastric porcine mucosa (EC 3.4.23.1) and pancreatin from porcine

67

pancreas (EC 232-468-9), Trinitrobenzene sulfonic acid or picrysulfonic acid (TNBS), bovine

68

serum albumin (BSA), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (TROLOX), 3-

69

(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-4’,4’-disulfonic acid (ferrozine), 2,2-azinobis (3-ethyl-

70

benzothiazoline-6-

71

Sigma Chemical Co., Ltd (St. Louis, MO, USA). Other chemicals used were of analytical grade

72

and purchased from Fisher Scientific (Waltham, MA, USA). Double-deionized water was used

73

as needed.

sulfonic acid) (ABTS•+) and potassium ferricyanide were procured from

74 75

2.2. Raw material preparation

76

From our previous study, protein hydrolysates were prepared using frames of farmed

77

rainbow trout (Oncorhynchus mykiss) (Ketnawa & Liceaga, 2016). The two treatments with

78

highest antioxidant activity were selected for this study. One sample treatment was prepared by

79

microwave pretreatment at 90ºC for 5 min, followed by conventional hydrolysis in water bath at

80

55ºC for 4 min (MPCE). The second sample was only treated by microwave-assisted hydrolysis

81

at 55ºC for 2 min (NPME). Both samples were selected to further investigate the effect of

82

gastrointestinal digestion on residual antioxidant activity. Characterization, purification and

83

identification of active peptides after gastrointestinal digestion were also investigated.

4

84

2.3. Effects of in vitro simulated gastrointestinal digestion model (SGM)

85

Resistance of the MPCE and NPME during in vitro simulated gastrointestinal digestion

86

model (SGM) by pepsin and pancreatin was assessed as previously described by (Ketnawa,

87

Martinez-Alvarez, Benjakul, & Rawdkuen, 2016). Briefly, hydrolysates (20 mg/mL w/v of

88

protein) were dissolved in distilled water and mixed with equal amount of pepsin (4 %, w/w of

89

protein) in 0.1 M KCl-HCl (pH 2.0). The mixture was incubated at 37°C for 2 h (stomach

90

conditions) using a continuous shaking water bath. Thereafter, the pH of the reaction mixture

91

was raised to 5.3 using 1.0 M NaHCO3 and further to pH 7.5 with 1.0 M NaOH. Pancreatin (4

92

%, w/w of protein) in 0.1 M K3PO4 (pH 8.0) was then added. The mixture was incubated at 37°C

93

for 2 h (duodenal conditions) with continuous shaking. Finally, the pH of the reaction mixture

94

was adjusted to 7.0 with 1 M HCl or NaOH. Digestion was terminated by placing the mixture in

95

a water bath held at 100°C for 10 min. During SGM, aliquots were taken at 0 h (undigested

96

hydrolysates), 2 h (during gastric digestion), 3 h (during intestinal digestion) and 4 h (after total

97

digestion). Mixtures were cooled at room temperature, and centrifuged at 12,000×g for 15 min.

98

Supernatants were further lyophilized, kept in plastic tubes, and stored at -20°C until further

99

analysis. Fractions derived from SGM were referred to as undigested (0 h), gastric digestion (2

100

h), intestinal digestion (3 h) and after total digestion (4 h). Degree of hydrolysis, ABTS•+radical

101

scavenging and reducing power (RP) activities were determined. Effect of SGM was evaluated

102

by comparing the changes in degree of hydrolysis, ABTS•+radical scavenging and RP activities

103

of Gastric 2-h (during gastric digestion), Intestinal 3-h (during intestinal digestion) and GI 4-h

104

(GI-MPCE and GI-NPME) to undigested peptides (MPCE and NPME), and expressed as

105

increment of activity (fold).

106

5

107

2.4. Degree of hydrolysis (DH)

108

The extent of enzymatic hydrolysis as percent of peptide bonds cleaved, was quantified

109

by monitoring the production of free amino groups, using the trinitrobenzenesulfonic acid

110

(TNBS) method adapted from Adler-Nissen (Adler-Nissen, 1986) modified by (Liceaga-

111

Gesualdo & Li-Chan, 1999) and described in (Ketnawa & Liceaga, 2016).

112 113

2.5. Antioxidant activity determination

114

2.5.1. The 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) free radical (ABTS•+)

115

scavenging assay and Reducing power (RP)

116

The ABTS•+ radical scavenging activity was determined according to method described in

117

Ketnawa and Liceaga (2016). The reducing power activity (RP) was measured according to a

118

previously reported method described in (Ketnawa & Liceaga, 2016). Results were expressed as

119

µmol TROLOX equivalent antioxidant capacity (µmol TE)/ g of protein in the hydrolysate based

120

on a TROLOX standard curve. In simulated gastrointestinal digestion experiment, ABTS•+ and

121

RP were expressed as increment of activity (fold) of µmol TROLOX equivalent antioxidant

122

capacity (µmol TE) per gram of protein in SGM hydrolysates compared to that of undigested

123

peptides.

124

2.6. Characterization by determination of molecular weight distribution

125

The molecular weight distribution of the two hydrolysates (MPCE and NPME) obtained

126

before and after in vitro digestion were evaluated by size-exclusion chromatography using a

127

Water e2695 HPLC system equipped with Water2489 UV/Visible detector (Water Company,

128

Waters Co., Milford, MA, USA) with SuperdexTM Peptides 10/300 GL column (GE Healthcare

129

Bio-Science AB, Uppsala, Sweden) with a fractionation range between 100 and 7,000 Da. An 6

130

amount of 1 mL of 5 mg of protein/mL of hydrolysates solution was prepared by dissolving

131

hydrolysate powder in 5 mM sodium phosphate buffer (pH 7.0) and filtered at 0.22 µm PVDF

132

filter for removal of particulates before injection. The injection volume was 100 µl and the flow

133

rate was 0.40 ml/min through the column temperature of 25°C using 100 mM sodium phosphate

134

buffer (pH 7.0) as a mobile phase. Absorbance was monitored at both A215 and A280 nm.

135

Molecular weight standards including bovine serum albumin (66,400 Da), aprotinin (6,511 Da),

136

vitamin B12 (1,355 Da), hippuryl-L-histidyl-L-leucine (429 Da), cytidine (243 Da), DL-

137

dithiotheritol (154 Da) and glycine (75 Da) were run through the column at the same conditions

138

and were used for the molecular weight calculation. Plots of retention time for molecular weight

139

standards were used to construct the calibration curve, from which hydrolysate molecular

140

distributions were computed. The logarithm of molecular weight (lg MW) and the retention

141

time (Rt) were in a linear relationship and the formula was calculated as Rt = -0.2094(lg MW)

142

+ 1.2747 (R² = 0.9867, P<0.05).

143

2.7. Characterization by determination of amino acid composition

144

The total amino acid composition of freeze dried samples was analyzed by UPLC Amino

145

acid Analysis Solution using the AccQ•Tag Ultra Derivatization kit with UV detection (Water

146

Corporations, Milford, MA, USA) by the Danforth Center’s Proteomics and Mass Spectrometry

147

Facility (St Louis, Missouri, USA). The quantity (moles) of amino acids in each peptide fraction

148

was calculated using a series of standards. The relative abundance of amino acids of initial

149

protein hydrolysates as well as the hydrolysates before and after SGM were calculated by

150

dividing the quantity (moles) of each individual amino acid by the sum of all amino acid

151

concentrations. The value was converted to a percentage and expressed as the relative abundance

152

amino acid (% mole). 7

153

2.8. Isolation and purification of antioxidative peptides

154

2.8.1. Size Exclusion Chromatography (SEC)

155

Fractions with antioxidant activity were introduced to a molecular size exclusion

156

chromatography-HPLC connected to a Water e2695 HPLC system equipped with Water2489

157

UV/Visible detector (Water Company, Waters Co., Milford, MA, USA). The column was

158

washed with 100 mM sodium phosphate buffer pH 7.0 at a flow rate of 0.45 mL/min for at least

159

one column volume. The mobile phase was the same buffer and the fractions were analyzed at

160

absorbance at A215 and A280 nm. The peaks of A215 were tested for antioxidant activity by

161

ABTS•+ radical scavenging activity assay. Prior to antioxidant activity assays, the protein content

162

of each peak was quantified by bicinchonimic acid (BCA) protein assay according to the

163

manufacturer’s protocol (ThermoFisher Scientific, Waltham, MA, USA) and using bovine

164

serum albumin as standard. The molecular weight of antioxidative peptides isolated on size

165

exclusion chromatography was estimated according to aforementioned details. The above steps

166

were repeated several times until an adequate volume of each fraction were pooled to measure

167

the ABTS•+ scavenging activity and for further purification. The highest active factions collected

168

from SEC were further purified by preparative high performance liquid on an analytical C18

169

column to obtain a pure peptide (Malaypally, Liceaga, Kim, Ferruzzi, San Martin, & Goforth,

170

2015).

171

2.8.2. Reversed-phase high-performance liquid chromatography (RP-HPLC)

172

Waters HPLC system was used to separate the desirable fractions after size exclusion.

173

The selected fractions from SEC were further separated using reversed-phase high-performance

174

liquid chromatography (RP-HPLC) on an analytical C18 column (YMC Pack ODS AM 125058

175

2546WT, YMC America, Inc., Allentown, PA, USA). The mobile phase A, was composed of

176

0.1% TFA in distilled water (v/v), and mobile phase B, was composed of 0.1% TFA in

177

acetonitrile and a linear gradient was developed. To separate the peptides according to their

178

hydrophobicity levels, elution was performed by the following gradient conditions: 0.0-10.0 min,

179

5% B; 10.0-60.0 min, 5.0-30.0% B; 60.0-70.0 min, 100% B; 70.0-80.0 min, 100% A, at a flow

180

rate of 1.0 mL/min for a sample run at 80 minutes. The absorbance of the eluted peaks was

181

monitored at A215 nm using a UV detector. All fractions were collected and lyophilized for

182

antioxidant activity assays. The above steps were repeated several times until an adequate

183

volume, able to measure the ABTS•+ scavenging activity, was obtained. Prior to antioxidant

184

activity assays, the protein content of each peak was quantified by BCA protein assay. The same

185

fractions were pooled and concentrated to remove acetonitrile and TFA. The purified antioxidant

186

peptides used for all follow-up experiments were pooled and lyophilized.

187

2.8.3. Identification of isolated peptides

188

Peptides with the highest ABTS•+ scavenging activity were selected to identify molecular

189

mass and amino acid sequence. A 0.5 µl of sample was added to 0.5 µl of the matrix (10 mg/ml

190

a-cyano-4-hydroxycinnamic acid in 50% Acetronitrile (ACN) /0.1% Trifluoroacetic acid (TFA)

191

on a MALDI sample plate. An analyzed matrix-assisted laser desorption/ionization-time of flight

192

(MALDI-TOF) mass spectrometer using a Matrix-assisted laser desorption/ionization mass

193

spectrometry (MALDI MS/MS) on a Voyager-DE PRO (Applied Biosystems, Foster City, CA,

194

USA). MALDI MS/MS data was obtained on a 4800 Plus TOF/TOF (Applied Biosystems,

195

Foster City, CA, USA at Drug Discovery Center, Purdue University. The amino acid sequence

196

was determined by the De novo sequencing method using DeNovo Explorer -MDS-SCIEX

197

software to derive tandem mass spectrometry (MS/MS) spectra. 9

198 199

2.9. Statistical analysis

200

For statistical analysis, all the data were expressed as mean ± standard deviation of

201

triplicate determinations, unless otherwise indicated. Analysis of variance (ANOVA) using a

202

general linear model with Tukey's pairwise comparison of means (P<0.05) was used to

203

determine the statistical significance of the observed differences among means. SPSS® Version

204

16.0 software (IBM Inc, Chicogo, IL, USA) was used for the statistical analysis.

205 206

3. Results and discussion

207

3.1. Degree of hydrolysis (DH)

208

In a previous experiment (Ketnawa & Liceaga, 2016), MPCE and NPME demonstrated

209

the best antioxidant activities (ABTS•+and RP). Therefore, in this study both samples were

210

selected to further investigate their antioxidant activity changes and/or stability following

211

simulated gastrointestinal digestion. Samples were collected through each step of SGM (0, 2, 3,

212

4-h) and the DH was determined.

213

As shown in Table 1, the DH for MPCE and NPME (0 h) was 45%, and 56%,

214

respectively. After digestion by pepsin (gastric digestion) for 2 h, the percentage of peptide

215

bonds cleaved (DH) increased. Further incubation with pancreatin (intestinal digestion; from 2.0

216

to 4.0 h) produced a sharp increase in DH to 81% (GI-MPCE) and 99% (GI-NPME) after 4 h.

217

Pepsin, an endopeptidase, can cleave the C-terminal of Phe, Tyr and Trp. Pancreatin consists of

218

multiple

219

carboxypeptidases. This mixture of enzymes function together to increase the efficiency in

gastrointestinal

enzymes

including

trypsin,

elastase,

chymotrypsin

and

10

220

cleavage of the polypeptides. Thus, proteins were hydrolyzed to oligopeptides or were

221

completely broken down to amino acids (Xiao, Huang, Chen, Chen, Li, & Shi, 2014). Other

222

studies have also reported an increase on DH under a SGM; for example, fish skin gelatin, and

223

collagen hydrolysates (Ketnawa, Martinez-Alvarez, Benjakul, & Rawdkuen, 2016; Sun, Chang,

224

Ma, & Zhuang, 2016).

225

3.2. Molecular weight distribution

226

The molecular weight (MW) distribution between MPCE and NPME showed an increase

227

in smaller MW peptides when comparing molecular weight profile before and after SGM. For

228

MPCE, the molecular weight decreased from 28,708 Da (fraction 1) before digestion to 5,661 Da

229

(fraction 12) after SGM. The same trend was observed for NPME, there was an increase of

230

smaller MW peptides from 27,137 Da (fraction 1) to 8,123 Da (fraction 12) before and after

231

SGM, respectively. In addition, after 4 hours in SGM, both GI-MPCE and GI-NPME showed an

232

increase of smaller peptides content, showing MW distribution between 204 and 8,000 Da

233

(fractions 1-10) (Table 1).

234

From the data, it could be concluded that the extensive hydrolysis of the samples by

235

pepsin and pancreatin generated smaller peptides. Moreover, GI-MPCE generated more peptides

236

with small molecular weight (<1,800 Da) up to 64.96 % (summation of fractions 5-9, Table 1)

237

than those obtained from GI-NPME up to 59.18 % (summation of fractions 6-10, Table 1). This

238

result shows that microwave pretreatment enhances gastrointestinal hydrolysis of initial protein

239

hydrolysate. Furthermore, GI-tract enzymes more easily digested the microwave pretreatment

240

hydrolysate (MPCE), compared to the hydrolysate derived from non-pretreatment, microwave-

241

assisted hydrolysis (NPME). The microwave treatment likely enhanced hydrolysis by unfolding

242

or transforming the protein structure leading to more accessible target sites by enzymes that 11

243

resulted in smaller molecules (Ketnawa & Liceaga, 2016). Results from this study need to be

244

further explored to evaluate bioavailability and absorption across human intestine cells or an in-

245

vivo human digestion model.

246 247

3.3. Change in antioxidant activities

248

The assay of ABTS•+radical scavenging activity can be applied to both lipophilic and

249

hydrophilic compounds, and has been widely used as an antioxidant activity assay (Miliauskas,

250

Venskutonis, & van Beek, 2004). Strong ABTS•+scavenging activity for the water-soluble

251

ABTS•+ free radicals, expressed as Trolox equivalent antioxidant capacity (TEAC), was

252

demonstrated by SGM digest samples (Fig. 1A). Following gastric or pepsin digestion (2-h),

253

TEAC increased (P<0.05) around 94-fold and 24-fold for Gastric-MPCE (2,072.99 µmol TE/g

254

protein) and Gastric-NPME (372.87 µmol TE/g protein), respectively, compared to undigested (0

255

h)-MPCE (21.97 µmol TE)/g protein) and undigested (0 h)-NPME (16.07 µmol TE)/g protein).

256

Most increment of TEAC can be observed during intestinal digestion (Fig. 1A). TEAC

257

continuously increased during intestinal digestion (3-h) for Intestinal-MPCE (4,116.27 µmol

258

TE/g protein) and intestinal-NPME (2,149.41 µmol TE/g protein), respectively (P<0.05).

259

However, after intestinal/pancreatin digestion, TEAC increased (P<0.05) approximately 68.94

260

and 47.48-fold for GI-MPCE (1,514.50 µmol TE/g protein) and GI-NPME (762.87 µmol TE/g

261

protein), respectively. This means that an extensive increment of TEAC was found during pepsin

262

(Gastric-) to pancreatin (Intestinal-) digestion in the first 3 h, the subsequent digestion with

263

pancreatin (up to 4 h) resulted in a slight loss in ABTS•+scavenging activity around 100-fold.

264

When the pepsin digest was hydrolyzed with pancreatin, additional peptide bond cleavages lead

265

to the accumulation of shorter peptides (tri- and di-peptides) and free amino acids, thus, 12

266

becoming more hydrophilic. The digests with increased polarity (amino acids, small peptides)

267

could readily react with water-soluble ABTS•+ (Zhu, Chen, Tang, & Xiong, 2008). The

268

conceivable structural changes resulting from pepsin digestion may also favor trapping of

269

ABTS•+radicals, thus further enhancing the quenching by the sample digest. Peptide structural

270

changes at this stage would hinder the access by ABTS•+. Therefore, lower TEAC can observed

271

after completion of the pancreatin digestion.

272

For the reducing power assay, the presence of antioxidants in the tested samples results in

273

reducing the Fe3+/ferricyanide complex to the ferrous form. The results also expressed as trolox

274

equivalent antioxidant capacity (TEAC) are presented in Fig.1B. Following gastric digestion (2

275

h), the TEAC value increased by approximately 8-fold and 10-fold for Gastric-MPCE (13.44

276

µmol TE)/g protein) and Gastric-NPME (13.44 and 24.98 µmol TE)/g protein, respectively

277

(P<0.05) compared to undigested-MPCE and undigested-NPME (1.77 and 2.47 µmol TE)/g

278

protein, respectively). Furthermore, TEAC continuously increased during intestinal digestion up

279

to 62-fold and 31-fold for Intestinal-MPCE (109.17 µmol TE)/g protein) and Intestinal-NPME

280

(76.92 µmol TE)/g protein), respectively (P<0.05). Despite increasing TEAC activity in the stage

281

of intestinal digestion, the value was decreased approximately 44-fold and 10-fold for GI-MPCE

282

(77.50 µmol TE)/g protein) and GI-NPME (24.49 µmol TE)/g protein), respectively (P<0.05)

283

after the final gastrointestinal digestion.

284

The increase in the reducing power of SGM-digests shows that fish frame hydrolysates

285

obtained by microwave pretreatment followed by conventional enzymatic hydrolysis (MPCE)

286

can be more effective hydrogen or electron donors after in vitro digestion (Figure 1). Zhu, Chen,

287

Tang, & Xiong, (2008) reported the same trend, where the reducing power of zein hydrolysate

288

was increased after treatment by pepsin for 1 h and pancreatin for 2 h. The increased reducing 13

289

power of sample digests can be attributed to a number of factors. With the increase in hydrolysis

290

(DH), electron-dense amino acid side residue chain groups, containing polar or charged moieties

291

become more exposed (Zhu, Chen, Tang, & Xiong, 2008; Zhu, Zhang, Zhou, & Xu, 2016).

292

Furthermore, peptide bond scission and an increased availability of certain amino acid residues

293

during digestion provide an additional source of protons and electrons to maintain a high redox

294

potential. These physicochemical changes can also explain the enhanced radical scavenging

295

capacity of protein hydrolysate digests. A number of studies have demonstrated a good

296

correlation between certain amino acids residues or MW peptides with radical scavenging ability

297

(Zhu, Chen, Tang, & Xiong, 2008; You, Zhao, Regenstein, & Ren, 2010; Xiao, Huang, Chen,

298

Chen, Li, & Shi, 2014; Zhu, Zhang, Zhou, & Xu, 2016). Thus, we can infer that trout frame

299

protein hydrolysates contained antioxidant peptides that can donate hydrogen/electron to free

300

radicals contributing to the radical-scavenging properties and terminate the radical chain

301

reactions. Results also indicate that antioxidative peptides were modified by gastro-intestinal

302

digestion to enhance their radical-scavenging and reducing power activities.

303

The time dependence for these two antioxidant activities are similar, all of which showed

304

an increase during the gastric digestion, and then a decrease after the pancreatin incubation

305

treatment. Both antioxidant activities significantly increased (P<0.05) after 2 h of gastric

306

digestion. Significant increase was observed over the next 2 h (P<0.05). After GI-digestion, both

307

antioxidant activities decreased compared to those during pancreatin (intestinal) digestion (at 4

308

h), but increased (P<0.05) when compared to the undigested peptides (at 0 h). You, Zhao,

309

Regenstein, & Ren, (2010) showed that there was a 5% increase in ABTS•+ radical scavenging

310

activity and 77% increase in reducing power of the final GI-digest of loach (Misgurnus

311

anguillicaudatus) protein hydrolysates. Intestinal digestion of salmon (Salmo salar) protein 14

312

hydrolysates showed the highest value of ABTS•+scavenging activity and ferric-reducing power

313

(Borawska, Darewicz, Pliszka, & Vegarud, 2016). Several studies have also reported an increase

314

in the antioxidant activity of protein hydrolysates after being digested in a simulated model

315

system (Ketnawa, Martinez-Alvarez, Benjakul, & Rawdkuen, 2016; Senphan & Benjakul, 2014;

316

Teixeira, Pires, Nunes, & Batista, 2016).

317

3.4. Relationship among molecular weight, amino acid composition and their antioxidative

318

activities

319

Generally, there is no direct relationship between antioxidant activity and molecular

320

weight. However, one previous study indicated that the peptides with smaller molecular

321

weight have stronger antioxidant activities, are more resistant to the gastrointestinal digestion,

322

and are easier to cross the intestinal barrier to exert biological activities, allowing a rapid

323

absorption (Sun, Chang, Ma, & Zhuang, 2016). Smaller size peptides could be the cause of the

324

antioxidative activity seen in this study.

325

frame protein hydrolysates were cleaved into small peptides and free amino acids by pepsin and

326

pancreatin. The molecular weight distribution of GI-digests (Table 1) confirmed that samples

327

were further hydrolyzed into small polypeptides and free amino acids during SGM. These

328

observations proved that trout frame hydrolysates maintained antioxidant activity through the

329

SGM (Fig. 1A and 1B). The findings of this study show that trout frame protein oligopeptides

330

below 1,800 Da exhibited the best ABTS•+ scavenging activity and reducing power. These

331

antioxidant peptides are most likely to be stable in a digestion system under proteolytic activities,

332

acidic and alkaline pH conditions. These results are in agreement with other studies with salmon,

333

sardinelle, yellow fin tuna, flathead fish and cape hake; where antioxidative peptides ranging

334

from 500-3,000 Da, showed the dependence of antioxidant properties on molecular weight 15

During in vitro digestion, microwave-treated trout

335

(Malaypally, Liceaga, Kim, Ferruzzi, San Martin, & Goforth, 2015; Nurdiani, Vasiljevic,

336

Yeager, Singh, & Donkor, 2017; Sila & Bougatef, 2016; Sun, Chang, Ma, & Zhuang, 2016;

337

Teixeira, Pires, Nunes, & Batista, 2016; Xiao, Huang, Chen, Chen, Li, & Shi, 2014). Several

338

studies have reported peptides smaller than 1,800 Da after digestion in SGM. For example,

339

Senphan & Benjakul (2014) and Karnjanapratum, Benjakul, O'Callaghan, O'Keeffe, FitzGerald,

340

& O'Brien (2016) reported that peptides from fish skin hydrolysates, with molecular weight of

341

364-550 Da, showed the highest ABTS•+radical scavenging activity after digestion in a SGM.

342

The antioxidant activity of peptides depends not only on their molecular weight, but also on

343

other factors such as amino acid composition, sequence and configuration of peptides (Jin, Zhou,

344

Li, Lai, & Li, 2015; Sila & Bougatef, 2016). In addition, the mechanism of action of

345

antioxidants in various test systems and the localization of antioxidants in various phases of food

346

or biological systems could affect the results of antioxidant assays (Ketnawa, Martinez-Alvarez,

347

Benjakul, & Rawdkuen, 2016).

348

Even though amino acid composition is not the only criteria to assess the antioxidant

349

ability of peptides, determination of changes through SGM is also important. The levels and

350

composition of free amino acids and peptides released during SGM, may give further

351

information regarding the antioxidant activities of protein hydrolysates (Sabeena Farvin,

352

Andersen, Otte, Nielsen, Jessen, & Jacobsen, 2016). Amino acid composition of hydrolysates is

353

related to many factors such as the starting material and DH. During the enzymatic hydrolysis

354

and GI-digestion process, some amino acids can lose their bioavailability. For example, loss of

355

lysine via Maillard reactions, isopeptide and cross-link formation, and racemization of amino

356

acyl- residues can occur when proteins are exposed to heat and strongly alkaline conditions

357

(Schwass & Finley, 1984; Jang, Liceaga and Yoon, 2016). Influencing factors could be the type 16

358

of amino acid, pH, temperature and reaction time (Chi, Hu, Wang, Li, & Luo, 2015). Antioxidant

359

activity of peptides was based on the molecular weight, the presence of specific amino acids and

360

their specific positioning in the sequence. Therefore, amino acid composition of the starting

361

material is important for producing antioxidant peptides (Nguyen, Jones, Kim, San Martin-

362

Gonzalez, & Liceaga 2017). A review by (Sila & Bougatef, 2016) presents several studies which

363

proved that antioxidant peptides generally contain 2-20 amino acid residues per molecule. It has

364

been reported that peptides rich in Pro, Leu, Ala, and aromatic amino acids (AAA) Phe, Trp, Tyr

365

and His show an scavenging effect of free radicals through direct electron transfer, and inhibit

366

the propagation of oxidized lipid by-products (Wiriyaphan, Xiao, Decker, & Yongsawatdigul,

367

2015). In previous work on Alaska Pollock (Gadus chalcogrammus) frame protein hydrolysate,

368

aromatic amino acids were absent; a considerable small amount (around 4%) of these amino

369

acids were present in the rainbow trout frame protein hydrolysates (Hou, Li, Zhao, Zhang, & Li,

370

2011). In addition, the presence of hydrophobic amino acids (HAA) residues in the hydrolysates

371

offers structural properties that can enhance interactions with lipids in foods and enhance

372

antioxidant entry into target organs through hydrophobic interactions with membrane lipid

373

bilayers (Wu, Cai, Zhang, Mi, Cheng, & Li, 2015). In this study (Table 2), antioxidant amino

374

acids such as Tyr, Phe, Pro, Ala, His and Leu accounted for 14.33-14.94 % of the total amino

375

acids. The increment in AAA was found in undigested (0 h) MPCE and NPME; however, AAA

376

content slightly decreased in GI-MPCE and GI-NPME. A similar trend was observed for the

377

relative amount of HAA where the total relative abundance of Gly, Ala, Val, Ile, Leu, Phe and

378

MetS in all samples (MPCE, GI-MPCE, NPME and GI-NPME) was high (Table 2). Before

379

SGM, the highest HAA levels were observed in MPCE and NPME (39.31 % and 41.94 %,

380

respectively). Interestingly, HAA in GI-MPCE and GI-NPME remained unchanged. Decreasing 17

381

of AAA and unchanging of HAA might be due to the digestive enzymes used. Pepsin is most

382

effective at cleaving peptide bonds between hydrophobic and preferably aromatic amino acids

383

such as Phe, Trp, and Try (Dunn, 2001). Pancreatin exhibited the activities of trypsin (cleavage

384

of peptide bonds at Arg and Lys sites), chymotrypsin (cleavage of peptide bonds at Phe, Trp,

385

Tyr, and Leu sites), and elastase (cleavage of peptide bonds at Ala and other aliphatic amino

386

acids) (Hou, Wu, Dai, Wang, & Wu, 2017). The presence of higher HAA residue content in the

387

separated fractions (MPCE, GI-MPCE, and NPME, GI-NPME) of the hydrolysates can facilitate

388

the entry of antioxidant peptides into target organs through hydrophobic interactions with

389

membrane lipid bilayers, which results in enhanced antioxidant effects (Girgih, He, Hasan,

390

Udenigwe, Gill, & Aluko, 2015). This needs to be further explored for the peptides derived from

391

this study.

392

The positively charged amino acids (PCAA) levels are similar to the negatively charged

393

amino acids (NCAA). All samples showed that they were considerably high in PCAA and

394

NCAA. However, the content of negatively charged acidic amino acid residues (NCAA) was the

395

highest in GI-digest fractions. Previous reports have shown that NCAA had a strong antioxidant

396

effect because their excess electrons can easily be donated to quench free radicals (Girgih, He,

397

Hasan, Udenigwe, Gill, & Aluko, 2015; Shan He, Franco, & Zhang, 2013). Another important

398

amino acid is histidine. Histidine in particular has strong radical-scavenging activity because of

399

the presence of an imidazole ring (Samaranayaka & Li-Chan, 2011). Histidine increased in

400

undigested microwave-treated trout frame protein hydrolysates (both MPCE and NPME), but

401

decreased after SGM (both GI-MPCE and GI-NPME). The antioxidant activity of microwave-

402

treated trout frame hydrolysates appeared to be caused by these amino acids in the peptide

403

fragments. Therefore, the radical scavenging activity of microwave treatment trout frame protein 18

404

hydrolysate was presumed to be due to the content of particular amino acids. On the contrary,

405

fish protein hydrolysates have been reported to exhibit variations in their amino acid composition

406

depending on several factors such as raw material, enzyme source and hydrolysis conditions

407

(Chalamaiah, Dinesh kumar, Hemalatha, & Jyothirmayi, 2012). Additional compositional and

408

structural information of the peptides in these GI-digest fractions was obtained after size-

409

exclusion fractionation, from determination of their peptide profiles, and amino acid sequences.

410

Fish frame peptides can be used as food additives and dietary nutrients capable of resisting

411

digestive proteases.

412

3.5. Purification and identification of antioxidant peptide

413

The fraction of GI-digest obtained from size exclusion that showed the highest radical

414

scavenging activity was further separated using reverse-phase high performance liquid

415

chromatography (RP-HPLC) on a C18 column.

416

Moreover, compositions and the specific position of amino acids in the peptide may

417

play an important role in its antioxidant activities. High content of HAA, especially at the N-

418

or C-terminus of peptides, could enhance the activities of antioxidative peptides by interacting

419

with lipid molecules and donating protons into radicals to scavenge radicals (Li & Li, 2013).

420

As shown in Fig. 2A and 2B, the ABTS•+ scavenging activity was observed in eluting parts. Five

421

fractions (fractions 7-11) with different elution times were selected (Fig.2A-2, GI-MPCE).

422

Figures 2A-2 and 2B-2 show that all five fractions exhibited ABTS•+ scavenging activities. The

423

ABTS•+ scavenging activity of active fraction (fraction 7) of both GI-MPCE and GI-NPME

424

showed 190.55 and 200.50 µmol TE/ mg protein content whereas other fractions showed a lower

425

scavenging activity. Thus, the fraction 7 was lyophilized and the active fraction corresponding to

426

the highest peak from RP-HPLC was further purified by a C18 column (Fig. 2A-3 and 2B-3). 19

427

The active fraction number 75 showed the highest ABTS•+ scavenging activity with 337.51 and

428

716.08 µmol TE/ mg protein content for MPCE and NPME, respectively. As shown in Fig. 3A

429

and 3B, the active peak was identified, and the primary sequence of the purified peptide was

430

determined. The analysis of mass spectra (m/z) of daughter ions obtained from the parent ion and

431

their chromatograms allowed peptides to be identified. The purified peptide obtained for

432

GI-MPCE had two possible sequences derived from MALDI MS/MS: Asp-Gly-Arg-Leu-Gly-

433

Tyr-Ser-Glu-Gly-Val-Met (NGRLGYSEGVM) or Gly-Asp-Arg-Leu-Gly-Tyr-Ser-Trp-Asp-Asp

434

(GNRLGYSWDD) with molecular weight of 1,182.65 Da. For GI-NPME, two purified peptides

435

and two possible sequences were obtained. The first peptide was Iso-Arg-Gly-Pro-Glu-Glu-His-

436

Met-Arg (IRGPEEHMHR) or Arg-Val-Ala-Pro-Glu-Glu-His-Met-Arg (RVAPEEHMHR) with

437

molecular weight of 1,261.77 Da, and the second one was Ser-Ala-Gly-Val-Pro-Arg-His-Lys

438

(SAGVPRHK) or Ser-Ala-Arg-Pro-Arg-His-Lys (SARPRHK), molecular weight of 962.63 Da

439

(Figure not shown). These peptides could potentially resist human gastrointestinal tract

440

digestion, as identified from fraction 75 (Fig. 3A and 3B).

441

Antioxidant activity of the selected fractions of GI-MPCE and GI-NPME could be based

442

on HAA, Pro and Try residues present in sequence. Some studies have reported that peptide

443

sequences containing Tyr exhibit strong antioxidant activity, especially when the presence of

444

Tyr was present at both terminals of the peptide sequence (Chalamaiah, Dinesh kumar,

445

Hemalatha, & Jyothirmayi, 2012; Fan, He, Zhuang, & Sun, 2012). The antioxidant activity of

446

Tyr is thought to be from the capability of the phenolic groups to serve as hydrogen donors,

447

which is one mechanism of inhibiting the radical-mediated peroxidizing chain reaction (Fan,

448

He, Zhuang, & Sun, 2012). The presence of HAA (Leu, Val and Phe), hydrophilic and basic

449

amino acids (His, Pro and Lys), and aromatic amino acids (Phe and Tyr) in the peptide sequence 20

450

are believed to contribute to its overall high antioxidant activity. Based on these, GI-NPME

451

showed higher ABTS•+ activity than GI-MPCE due to presence of those amino acids in the

452

sequence. Moreover, polar/charged amino acids such as Arg at the C-terminus position can

453

also contribute to the antioxidant activity (Sun, Chang, Ma, & Zhuang, 2016). Our results

454

were similar to previous reports, where HAA or arginine existed in the terminus of one of the

455

peptides. (Sun, Chang, Ma, & Zhuang, 2016) found ABTS•+ scavenging active peptides with

456

15

457

1,337.51 Da) in GI-digested hydrolysates of Alaska pollock (Theragra chalcogramma) skin

458

collagen. Furthermore, two peptides purified from Flathead (Platycephalus fuscus) protein

459

hydrolysates were Try-Gly-Cys-Cys and Asp-Ser-Ser-Cys-Ser-Gly, with molecular weight of

460

444.11 and 554.16 Da, respectively showed 2,2-diphenyl-1-pycryl-hydrazyl (DPPH) and

461

ABTS•+ scavenging activities as 94.03 and 82.89 %, respectively (Nurdiani, Vasiljevic,

462

Yeager, Singh, & Donkor, 2017). The electron properties of amino acid residues are very

463

important, and bulky hydrophobicity at the C-terminal is also closely related to the antioxidant

464

activity. The results suggest that the electronic, hydrogen-bonding properties and location of the

465

amino acids, along with the steric properties of the amino acid residues at the C- and N-termini

466

may be the root cause of the antioxidant activity of peptides (Zou, He, Li, Tang, & Xia, 2016).

467

Further studies on the quantitative analysis of key peptides will be required.

468

4. Conclusions

amino

acid

bases

(Met-Gly-Pro-Pro-Gly-Leu-Ala-Gly-Ala-Pro-Gly-Glu-Ala-Gly-Arg;

469

In this study, the effect of simulated gastrointestinal digestion on the stability of

470

antioxidant capacity of microwave-treated protein hydrolysates was evaluated. The digest

471

fractions exhibited noticeable antioxidant potential especially after simulated gastrointestinal

472

digestion. The ABTS•+ radical scavenging activities of peptides were related to degree of 21

473

hydrolysis, molecular weight, amino acid composition and amino acid sequence. Low

474

molecular size peptides (<1,800 Da) showed the highest free radical scavenging activity in

475

vitro. One single purified peptide with two possible sequences derived for GI-MPCE whilst two

476

purified peptides with two possible sequences each were obtained from GI-NPME. Natural

477

antioxidants derived from trout by-products treated by a microwave pretreatment and

478

conventional hydrolysis (MPCE) or by microwave-assisted enzymatic hydrolysis (NPME), can

479

maintain the antioxidant activity following in vitro gastrointestinal digestion and thus have

480

potential to be applied in food or nutraceutical industries.

481

Acknowledgements

482

Funding for this research was provided by Hatch Act formula funds by the College of

483

Agriculture, Purdue University. The authors would like to thank Bell Aquaculture™ for kindly

484

supplying the rainbow trout frames used in this study. We are also appreciate for Dr. Bernard

485

Tao Biochemical-engineering laboratory providing for instrument support. Authors give special

486

thanks to Dr. Connie Bonham instrument specialist Drug Discovery center for assistance in

487

performing TOF MS/MS. Authors greatly appreciate Dr. Lloyd Fricker, Department of

488

Molecular Pharmacology Albert Einstein College of Medicine, Dr. Karl Wood, Department of

489

chemistry and Dr. Mark Hall, Department of Biochemistry, Purdue University for providing

490

technical expertise.

491

Conflict of Interest

492

Authors have declared that no competing interests exist. We confirm that we have given due

493

consideration to the protection of intellectual property associated with this work and that there

494

are no impediments to publication, including the timing of publication, with respect to

495

intellectual property. We understand that the Corresponding Author is the sole contact for the 22

496

Editorial process (including Editorial Manager and direct communications with the office).

497

He/she is responsible for communicating with the other authors about progress, submissions of

498

revisions and final approval of proofs.

499 500

References

501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535

Adler-Nissen, J. (1986). Enzymic Hydrolysis of Food Proteins: Elsevier Applied Science Publishers. Borawska, J., Darewicz, M., Pliszka, M., & Vegarud, G. E. (2016). Antioxidant properties of salmon (Salmo salar L.) protein fraction hydrolysates revealed following their ex vivo digestion and in vitro hydrolysis. Journal of the Science of Food and Agriculture, 96(8), 2764-2772. Chalamaiah, M., Dinesh kumar, B., Hemalatha, R., & Jyothirmayi, T. (2012). Fish protein hydrolysates: Proximate composition, amino acid composition, antioxidant activities and applications: A review. Food Chemistry, 135(4), 3020-3038. Chi, C.-F., Hu, F.-Y., Wang, B., Li, Z.-R., & Luo, H.-Y. (2015). Influence of amino acid compositions and peptide profiles on antioxidant capacities of two protein hydrolysates from skipjack tuna (Katsuwonus pelamis) dark muscle. Marine Drugs, 13(5), 2580-2601. Dunn, B. M. (2001). Overview of Pepsin-like Aspartic Peptidases. In Current Protocols in Protein Science): John Wiley & Sons, Inc. Espejo-Carpio, F. J., García-Moreno, P. J., Pérez-Gálvez, R., Morales-Medina, R., Guadix, A., & Guadix, E. M. (2016). Effect of digestive enzymes on the bioactive properties of goat milk protein hydrolysates. International Dairy Journal, 54, 21-28. Fan, J., He, J., Zhuang, Y., & Sun, L. (2012). Purification and identification of antioxidant peptides from enzymatic hydrolysates of tilapia (Oreochromis niloticus) frame protein. Molecules, 17(11). Girgih, A. T., He, R., Hasan, F. M., Udenigwe, C. C., Gill, T. A., & Aluko, R. E. (2015). Evaluation of the in vitro antioxidant properties of a cod (Gadus morhua) protein hydrolysate and peptide fractions. Food Chemistry, 173, 652-659. He, S., Franco, C., & Zhang, W. (2013). Functions, applications and production of protein hydrolysates from fish processing co-products (FPCP). Food Research International, 50(1), 289-297. Hou, H., Li, B., Zhao, X., Zhang, Z., & Li, P. (2011). Optimization of enzymatic hydrolysis of Alaska pollock frame for preparing protein hydrolysates with low-bitterness. LWT - Food Science and Technology, 44(2), 421-428. Hou, Y., Wu, Z., Dai, Z., Wang, G., & Wu, G. (2017). Protein hydrolysates in animal nutrition: Industrial production, bioactive peptides, and functional significance. Journal of Animal Science and Biotechnology, 8, 24. Jang H.L., Liceaga, A. and Yoon, K.Y . (2016). Purification, characterization and stability of an antioxidant peptide derived from sandfish (Arctoscopus japonicus) protein hydrolysates. Journal of Functional Foods, 20: 433-442.

23

536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580

Jin, B., Zhou, X. S., Li, B., Lai, W. T., & Li, X. Y. (2015). Influence of in vitro digestion on antioxidative activity of coconut meat protein hydrolysates. Tropical Journal of Pharmaceutical Research, 14(3), 441-447. Karnjanapratum, S., Benjakul, S., O'Callaghan, Y. C., O'Keeffe, M. B., FitzGerald, R. J., & O'Brien, N. M. (2016). Purification and identification of antioxidant peptides from gelatin hydrolysates of unicorn leatherjacket skin. Italian Journal of Food Science, 29(1). Ketnawa, S., & Liceaga, A. M. (2016). Effect of microwave treatments on antioxidant activity and antigenicity of fish frame protein hydrolysates. Food and Bioprocess Technology, 110. Ketnawa, S., Martinez-Alvarez, O., Benjakul, S., & Rawdkuen, S. (2016). Gelatin hydrolysates from farmed Giant catfish skin using alkaline proteases and its antioxidative function of simulated gastro-intestinal digestion. Food Chemistry, 192, 34-42. Li, Y.-W., & Li, B. (2013). Characterization of structure–antioxidant activity relationship of peptides in free radical systems using QSAR models: key sequence positions and their amino acid properties. Journal of theoretical biology, 318, 29-43. Liceaga-Gesualdo, A. M., & Li-Chan, E. C. Y. (1999). Functional properties of fish protein hydrolysate from herring (Clupea harengus). Journal of Food Science, 64(6), 1000-1004. Malaypally, S. P., Liceaga, A. M., Kim, K.-H., Ferruzzi, M., San Martin, F., & Goforth, R. R. (2015). Influence of molecular weight on intracellular antioxidant activity of invasive silver carp (Hypophthalmichthys molitrix) protein hydrolysates. Journal of Functional Foods, 18, 1158-1166. Miliauskas, G., Venskutonis, P. R., & van Beek, T. A. (2004). Screening of radical scavenging activity of some medicinal and aromatic plant extracts. Food Chemistry, 85(2), 231-237. Nguyen, E., Jones, O., Kim, Y. H. B., San Martin-Gonzalez, F., & Liceaga, A. M. (2017). Impact of microwave-assisted enzymatic hydrolysis on functional and antioxidant properties of rainbow trout Oncorhynchus mykiss by-products. Fisheries Science, 1-15. Nurdiani, R., Vasiljevic, T., Yeager, T., Singh, T. K., & Donkor, O. N. (2017). Bioactive peptides with radical scavenging and cancer cell cytotoxic activities derived from Flathead (Platycephalus fuscus) by-products. European Food Research and Technology, 243(4), 627-637. Sabeena Farvin, K. H., Andersen, L. L., Otte, J., Nielsen, H. H., Jessen, F., & Jacobsen, C. (2016). Antioxidant activity of cod (Gadus morhua) protein hydrolysates: Fractionation and characterisation of peptide fractions. Food Chemistry, 204, 409-419. Samaranayaka, A. G. P., Kitts, D. D., & Li-Chan, E. C. Y. (2010). Antioxidative and angiotensin-I-converting enzyme inhibitory potential of a Pacific hake (Merluccius productus) fish protein hydrolysate subjected to simulated gastrointestinal digestion and caco-2 cell permeation. Journal of Agricultural and Food Chemistry, 58(3), 1535-1542. Samaranayaka, A. G. P., & Li-Chan, E. C. Y. (2011). Food-derived peptidic antioxidants: A review of their production, assessment, and potential applications. Journal of Functional Foods, 3(4), 229-254. Schwass, D. E., & Finley, J. W. (1984). Heat and alkaline damage to proteins: racemization and lysinoalanine formation. Journal of Agricultural and Food Chemistry, 32(6), 1377-1382. Senphan, T., & Benjakul, S. (2014). Antioxidative activities of hydrolysates from seabass skin prepared using protease from hepatopancreas of Pacific white shrimp. Journal of Functional Foods, 6, 147-156. 24

581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620

Sila, A., & Bougatef, A. (2016). Antioxidant peptides from marine by-products: Isolation, identification and application in food systems. A review. Journal of Functional Foods, 21, 10-26. Srigiridhar, K., Nair, K. M., Subramanian, R., & Singotamu, L. (2001). Oral repletion of iron induces free radical mediated alterations in the gastrointestinal tract of rat. Molecular and cellular biochemistry, 219(1-2), 91-98. Sun, L., Chang, W., Ma, Q., & Zhuang, Y. (2016). Purification of antioxidant peptides by high resolution mass spectrometry from simulated gastrointestinal digestion hydrolysates of Alaska pollock (Theragra chalcogramma) skin collagen. Marine Drugs, 14(10), 186. Teixeira, B., Pires, C., Nunes, M. L., & Batista, I. (2016). Effect of in vitro gastrointestinal digestion on the antioxidant activity of protein hydrolysates prepared from Cape hake byproducts. International Journal of Food Science & Technology, 51(12), 2528-2536. Wiriyaphan, C., Xiao, H., Decker, E. A., & Yongsawatdigul, J. (2015). Chemical and cellular antioxidative properties of threadfin bream (Nemipterus spp.) surimi byproduct hydrolysates fractionated by ultrafiltration. Food Chemistry, 167, 7-15. Wu, Q., Fu, X. P., Sun, L. C., Zhang, Q., Liu, G. M., Cao, M. J., & Cai, Q. F. (2015). Effects of physicochemical factors and in vitro gastrointestinal digestion on antioxidant activity of R-phycoerythrin from red algae Bangia fusco-purpurea. International Journal of Food Science and Technology, 50(6), 1445-1451. Wu, X., Cai, L., Zhang, Y., Mi, H., Cheng, X., & Li, J. (2015). Compositions and antioxidant properties of protein hydrolysates from the skins of four carp species. International Journal of Food Science & Technology, 50(12), 2589-2597. Xiao, P., Huang, H., Chen, Y., Chen, J., Li, X., & Shi, H. (2014). Nutritional evaluation, characterization and antioxidant activity of Radix tsatidis protein hydrolysates under simulated gastrointestinal digestion. Journal of Food and Nutrition Research, 2(11), 831838. You, L., Zhao, M., Regenstein, J. M., & Ren, J. (2010). Changes in the antioxidant activity of loach (Misgurnus anguillicaudatus) protein hydrolysates during a simulated gastrointestinal digestion. Food Chemistry, 120(3), 810-816. Zeng, M., Dong, S., Zhao, Y., & Liu, Z. (2013). Antioxidant Activities of Marine Peptides from Fish and Shrimp. In Marine Proteins and Peptides, (pp. 449-466): John Wiley & Sons, Ltd. Zhu, Chen, J., Tang, X., & Xiong, Y. L. (2008). Reducing, radical scavenging, and chelation properties of in vitro digests of alcalase-treated zein hydrolysate. Journal of Agricultural and Food Chemistry, 56(8), 2714-2721. Zhu, Zhang, W.-G., Zhou, G.-H., & Xu, X.-L. (2016). Identification of antioxidant peptides of Jinhua ham generated in the products and through the simulated gastrointestinal digestion system. Journal of the Science of Food and Agriculture, 96(1), 99-108. Zou, T.-B., He, T.-P., Li, H.-B., Tang, H.-W., & Xia, E.-Q. (2016). The structure-activity relationship of the antioxidant peptides from natural proteins. Molecules, 21(1), 72.

621

25

622

List of Figures

623

Fig. 1.

624

hydrolysates during simulated gastrointestinal digestion (SGM). Results were expressed as

625

increment of activity (fold) of hydrolysates during simulated gastrointestinal digestion (SGM)

626

Increment of activity (fold) was calculated by divided ABTS•+radical scavenging or reducing

627

power (RP) activity value of SGM fractions including Gastric 2-h (during gastric digestion for 2

628

hours), Intestinal 3-h (during intestinal digestion for 3 hours) and GI 4-h (after SGM at 4 hours)

629

by the value of undigested protein hydrolysates (0 hours). ABTS•+radical scavenging or reducing

630

power activity (RP) activity of undigested hydrolysates were 1-fold. Bars represent the standard

631

deviation from triplicate determinations.

Increments in the antioxidant activity ABTS (A); Reducing Power (B) of the

632 633

Fig. 2A. Size exclusion chromatography-high performance liquid chromatography (SEC-HPLC)

634

profiles of simulated gastrointestinal digestion (SGM) of microwave pretreatment followed by

635

conventional enzymatic hydrolysis (MPCE; 2A-1) at sample concentration (100 µL injection of 5

636

mg/mL solution). Retention time ranges for the HPLC fractions collected (i.e., peaks 7-11) are

637

34-47 min for GI-MPCE and ABTS scavenging activity of the collected fractions (2A-2). C18

638

HPLC profiles of in vitro gastrointestinal (GI) of MPCE at sample concentration (20 µL injection

639

of 2 mg/mL solution). Retention time ranges for the HPLC fractions collected (i.e., peaks 16, 50

640

and 75) are 16, 65, and 75 min (2A-3) and ABTS scavenging activity of the collected fractions

641

(2A-4). Bars represent the standard deviation from triplicate determinations.

642 643

Fig. 2B. Size exclusion chromatography-high performance liquid chromatography (SEC-HPLC)

644

profiles of simulated gastrointestinal digestion (SGM) of non-pretreated microwave assisted

645

enzymatic hydrolysis (NPME; 2B-1) at sample concentration (100 µL injection of 5 mg/mL

646

solution). Retention time ranges for the HPLC fractions collected (i.e., peaks 7-11) are 36-48 min

647

for GI-NPME and ABTS scavenging activity of the collected fractions (2B-2). C18 HPLC

648

profiles of NPME at sample concentration (20 µL injection of 2 mg/mL solution). Retention time

649

ranges for the HPLC fractions collected (i.e., peaks 16, 50 and 75) are 16, 65, and 75 min (2B-3)

650

and ABTS scavenging activity of the collected fractions (2B-4). Bars represent the standard

651

deviation from triplicate determinations.

26

652

Fig. 3A. Base peak chromatogram of the selected representative MS/MS spectra of peptides for

653

GI-MPCE including NGRLGYSEGVM or GNRLGYSWDD (1,182.65 Da) (3A-1 and 3A-2).

654

The x-axis shows the m/z of the precursor and fragment ions while the y-axis shows the relative

655

intensity. The deduced sequence can be seen on the figure (3A-3). Only one example of

656

chromatogram is shown here.

657

Fig. 3B. Base peak chromatogram of the selected representative MS/MS spectra of peptides for

658

GI-NPME including IRGPEEHMHR or RVAPEEHMHR (1,261.77 Da) (3B-1 and 3B-2). The x-

659

axis shows the m/z of the precursor and fragment ions while the y-axis shows the relative

660

intensity. The deduced sequence can be seen on the figure (3B-3). Only one example of

661

chromatogram is shown here.

662 663 664 665 666 667 668 669 670

27

Increment of Activity (Fold)

MPCE FPE

250

A

NPME FME

200 150 100 50 0 Gastric (2-h)

Intesinal (3-h)

GI (4-h)

671

Increment of Activity (Fold)

MPCE FPE

B

NPME FME

70 60 50 40 30 20 10 0 Gastric (2-h)

672 673

Intesinal (3-h)

GI (4-h)

Fig. 1.

674 675 676

28

677

7

A-1

8

10 9

11

ABTS (µmolTE eq/g protein)

A-2 250 200 150 100 50 0 7

8

9

10

11

Fraction number

29

678 679 680 681 682 683 684 685 686

75

A-3

687

16

65

30

688 689 690 691 692 ABTS radical scavenging (µmolTE eq/g protein)

693 694 695 696 697 698

300 200 100 0 16

699 700

A-4

400

65 Fraction number

75

Fig. 2A.

31

B-2

ABTS (µmolTE eq/g protein)

250 200 150 100 50 0 7

8

9

10

Fraction number

11

B-4

ABTS radical scavenging (µmolTE eq/g protein)

800 700 600 500 400 300 200 100 0 16

65

75

Fraction number

B-1

7

32 8

10 9

11

702

Fig. 3A.

A-1

33

703 704 705 706 707 708

709

B-2

710 711 712 713 714

715 716 717 718 719 720 721 722 723 724

Fig. 3B.

725

34

726 727 728

Table 1. Degree of hydrolysis, molecular weight distribution and fraction content of microwave treated trout frame protein hydrolysates before and after simulated gastrointestinal digestion. Sample

MPCE*

Degree of hydrolysis (%)

45.02±0.15

Fraction number 1

Molecular weight (Da) 28,708±10.14

Content (%) 11.75±0.98

2

20,977±9.57

6.34±0.54

3

14,052±8.42

6.15±0.24

4

11,280±8.51

6.16±0.82

5

8,101±6.53

5.05±0.17

6

6,402±7.52

7.51±0.48

7

4,146±6.53

13.12±0.14

8

1,842±5.21

14.33±0.22

9

1,456±4.23

4.93±0.27

10

632±1.68

11.46±0.98

11

372±1.59

6.71±0.29

12

206±1.50

3.58±0.11

<1,800 Da

26.68±3.44

1

5,661±10.83

15.92±0.54

2

4,043.72±15.97

3.67±0.43

3

3,479.75±14.35

10.95±0.18

4

2,389.90±8.41

2.62±0.48

5

1,785.01±8.95

23.92±0.48

6

1,444.54±7.41

11.20±0.55

7

609.82±8.75

7.89±0.17

8

364.51±9.83

13.23±0.44

9

203.66±5.97

8.72±0.17

<1,800 Da

64.96±6.46

1

27,137±10.98

8.07±0.28

2

18,935.56±9.87

6.59±0.23

3

11,929.15±8.64

6.28±0.24

4

9,337.43±8.10

5.78±0..14

Sum of fraction 9-12

GI-MPCE

80.77±1.94

Sum of fraction 5-9

NPME

55.92±4.18

35

5

6,241.27±7.52

5.00±0.18

6

4,727.50±5.23

7.59±0.19

7

2,901.81±5.17

11.40±0.56

8

1,237.58±4.85

16.58±0.99

9

990.57±3.59

5.49±0.10

10

431.04±3.78

9.41±0.54

11

361.22±0.56

3.23±0.11

12

249.28±0.98

8.77±0.45

<1,800 Da

43.48±5.07

1

8,123±13.22

11.57±0.63

2

5,762.45±5.89

7.22±0.26

3

4,183.52±9.70

4.02±0.04

4

3,776.80±12.45

12.61±0.62

5

2,430.22±3.19

3.01±0.04

6

1,802.32±2.51

20.99±0.56

7

1,454.28±1.46

10.46±0.19

8

631.92±1.26

8.21±0.17

9

376.78±1.05

11.73±0.28

10

218.25±1.29

7.79±0.19

<1,800 Da

59.18±6.41

Sum of fraction 8-12

GI-NPME

98.98±0.78

Sum of fraction 6-10 729

Values in table are presented as the mean of two replicates ± SD.

730 731 732 733 734

*MPCE = hydrolysates produced from microwave pretreatment for 5 min at 90°C, followed by conventional enzymatic hydrolysis for 4 min; MPCE (before GI digestion) and GI-MPCE after 4 h of simulated gastrointestinal digestion; NPME = hydrolysates produced from no microwave pretreatment followed by microwave-assisted enzymatic hydrolysis at 55ºC for 2 min. NPME (before GI digestion) and GI-NPME after 4 h of simulated gastrointestinal digestion.

735 736

36

737 738 739

Table 2 Amino acid composition and summary of the selected amino acid groups of microwave treated trout frame protein hydrolysates before and after simulated gastrointestinal digestion. Amino acid

740 741 742 743

Relative abundance (% mole) a

Gly

MPCE 20.48

GI-MPCE 20.42

NPME 18.77

GI-NPME 21.21

Ala

11.95

11.54

12.93

11.80

Ser

1.47

1.96

1.06

1.93

Pro

6.63

6.93

6.31

7.26

Val

5.05

5.23

5.54

5.16

Thr

2.29

2.76

1.96

2.65

Ile

3.23

3.41

3.68

3.44

Leu

5.92

5.92

6.56

5.81

Asp

9.81

10.00

9.31

9.64

Lys

7.04

6.40

7.68

6.13

Glu

13.32

14.01

13.00

14.12

MetS

3.29

2.78

3.49

2.79

His

1.64

1.14

1.71

1.15

Phe

2.22

2.90

2.45

2.66

Arg

4.63

4.15

4.56

3.84

Tyr

0.56

0.00

0.54

0.00

Cya

0.46

0.46

0.44

0.43

EAAb

30.68

30.54

33.07

29.79

HHA

39.31

39.17

41.94

39.35

AAA

4.42

4.04

4.70

3.81

AXA

14.80

14.21

14.94

14.29

PCAA

13.31

11.69

13.95

11.12

NCAA

13.57

14.72

12.33

14.22

BCAA

14.20

14.56

15.78

14.41

a

The relative abundance of amino acids was calculated by dividing the quantity (moles) of each individual amino acid by the sum of the concentration of all amino acids. The value, converted to a percentage, is expressed as the relative abundance amino acid (% mole). Treatment captions (MPCE, GI-MPCE, NPME, GI-NPME) are described in Table 1. 37

744 745 746 747 748 749

b

EAA= essential amino acids: Arg, His, Ile, Leu,Lys, Met, Phe, Thr, Try, Val, HHA = hydrophobic amino acids: Ala, Val, Ile, Leu, Tyr, Phe, Trp, Pro, Met, Cys); AAA = aromatic amino acids: Phe, Trp, Tyr, His; AXA = antioxidant amino acids: Trp, Tyr, Met, Cys, His, Phe, and Pro; PCAA = positively charged amino acids: Arg, His, Lys; NCAA = negatively charged amino acids: Asp, Glu, Thr, Ser; BCAA= Branched chain amino acids: Leu, Ile, Val.

750 751 752 753

38

754 755

Highlights •

After simulated gastrointestinal digestion, hydrolysates produced from microwave

756

pretreatment followed by conventional hydrolysis (MPCE) showed higher antioxidant

757

activity than that of non-pretreated, microwave-assisted hydrolysis (NPME) samples.

758

•

After simulated gastrointestinal digestion, molecular weight of peptides was <1,800 Da

759

for both (MPCE) and (NPME), and these peptides provided an increase in ABTS•+radical

760

scavenging activity.

761 762

•

Peptides <1,300 Da with predominantly hydrophobic amino acids in sequence and with high ABTS•+ scavenging activity were found in both (MPCE) and (NPME).

763

39