Effects of pressure-assisted enzymatic hydrolysis on functional and bioactive properties of tilapia (Oreochromis niloticus) by-product protein hydrolysates

Journal Pre-proof Effects of pressure-assisted enzymatic hydrolysis on functional and bioactive properties of tilapia (Oreochromis niloticus) by-product protein hydrolysates Ashutosh Kumar Hemker, Loc Thai Nguyen, Mukund Karwe, Deepti Salvi PII:

S0023-6438(19)31345-3

DOI:

https://doi.org/10.1016/j.lwt.2019.109003

Reference:

YFSTL 109003

To appear in:

LWT - Food Science and Technology

Received Date: 14 June 2019 Revised Date:

18 December 2019

Accepted Date: 28 December 2019

Please cite this article as: Hemker, A.K., Nguyen, L.T., Karwe, M., Salvi, D., Effects of pressureassisted enzymatic hydrolysis on functional and bioactive properties of tilapia (Oreochromis niloticus) byproduct protein hydrolysates, LWT - Food Science and Technology (2020), doi: https://doi.org/10.1016/ j.lwt.2019.109003. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

Authors’ contribution • • •

•

Ashutosh Kumar Hemker: Prepared experimental plans, logistics; conducted the experiments and data analysis; drafted the manuscript. Dr. Loc Thai Nguyen: Initiated the research ideas; collaborated the research activities and preparation of the manuscript. Dr. Deepti Salvi: Jointly developed the research ideas, collaborated the experimental activities using high pressure processing unit, involved in the preparation of the manuscript. Prof. Mukund Karwe: Involved in development of the research ideas and experimental plans; supervised the research activities, and edited the manuscript.

1 2

Effects of pressure-assisted enzymatic hydrolysis on functional and bioactive properties

3

of tilapia (Oreochromis niloticus) by-product protein hydrolysates

4 5 6 Ashutosh Kumar Hemkera, Loc Thai Nguyena*, Mukund Karweb, Deepti Salvib,c*

7 8 9 a

10

Department of Food, Agriculture and Bioresources, Asian Institute of Technology, Pathum

11

Thani, Thailand b

12 13 14

c

Department of Food Science, Rutgers University, New Brunswick, New Jersey, USA

Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, North Carolina, USA

15 16 17 18 19 20 21 22 23 24

*Corresponding author: Loc T. Nguyen. Email: [email protected]; Tel: +66 25245448;

25

Fax: +66 25246200. Deepti Salvi. Email: [email protected]; Tel: 919-513-0176. 1

26

Abstract

27

Fish by-product protein can be converted into valuable food and nutraceutical

28

ingredients via proteolysis. The existing process suffers from many limitations such as

29

extended reaction time and nonselective hydrolysis. In this study, protein from tilapia fish by-

30

products was transformed into functional peptides using pressure-assisted enzymatic

31

hydrolysis. Proteins were extracted from the tilapia by-products by isoelectric solubilization

32

and precipitation method. The effects of pressure (38 - 462 MPa) and hydrolysis time (6-35

33

min) on the properties of the hydrolysates were investigated using a central composite design.

34

Pressure enhanced protein hydrolysis with a maximum trichloroacetic acid-solubility index

35

(TCA-SI) of 23 % obtained at 250 MPa for 35 min. Pressure and time were also vital in

36

improving soluble protein content (5.7 mg/mL), reducing power (44 µg AAE/g), and

37

solubility (71 %) of the hydrolyzed products. Improved antioxidant activity, indicated by a

38

significant decrease in IC50 values from 653 µg/mL to 304 µg/mL, was recorded. The

39

combined process facilitated the release of low-molecular-weight peptides and essential

40

amino acids. However, water and oil holding capacities were found to be decreased. Pressure-

41

assisted enzymatic hydrolysis could provide an effective approach for recovering bioactive

42

peptides from fish by-products for industrial applications.

43 44

Keywords: Fish by-products; protein hydrolysis; response surface methodology (RSM);

45

high-pressure processing; physicochemical properties.

46

2

47

1. Introduction

48

Tilapia (Oreochromis niloticus) is a major aquaculture species in many countries. The

49

annual tilapia production is about 4.5 million tons (Tveterås, 2014). Fishery industries

50

generally produce frozen fillets as the main commodity whereas by-products such as head,

51

skin, bones, and viscera, accounting for 50-70 % of the live fish weight, are considered as

52

waste (FAO, 2016). Around 30 % of these are utilized as fertilizer, silage production, and

53

animal feed (Hsu, 2010) while a minor portion is employed as intermediate ingredients in

54

food, nutraceutical, and pharmaceutical sectors (Klompong, Benjakul, Kantachote, &

55

Shahidi, 2007). Larger quantities are rejected and dumped as waste in the absence of effective

56

management systems. Fish by-products can be sources of valuable constituents such as

57

proteins, phospholipids, vitamins, polyunsaturated fatty acids, and bioactive compounds

58

(Shirahigue et al., 2016). Fish protein hydrolysates have been successfully incorporated into

59

foods, such as meat products, cookies and cereals (Chalamaiah, 2010). Kristinsson and Rasco

60

(2000) reported the applications of FPH as milk replacers in infant formulas. Dekkers,

61

Raghavan, Kristinson, & Marshall (2011) described enzymatic conversion of fish by-products

62

into fish silage and fish sauce. Effective use of these by-products, therefore, will help

63

mitigate environmental pollution and generate additional incomes for fish processors (Chi et

64

al., 2014; FAO, IFAD, & WFP, 2015).

65

Recently, biologically active peptides derived from fish protein have attracted

66

increasing attention. These peptides are inactive within the sequences of the parent proteins.

67

When released by enzymatic hydrolysis, they exert various bioactivities such as inhibition of

68

the

69

inflammatory, cytomodulatory, antimicrobial, immune-modulatory (Halim, Yusof, & Sarbon,

70

2016). In addition, the cleavage of protein molecules can modify their functional properties

71

such as emulsification, and gel formation abilities (Queirós, Saraiva, & da Silva, 2018). The

angiotensin-I-converting

enzyme

(ACE),

3

antioxidant,

anti-proliferative,

anti-

72

existing enzymatic hydrolysis of fish protein suffers from many limitations such as extended

73

reaction time and nonselective hydrolysis. Therefore, innovative hydrolysis technology to

74

convert fish by-products into functional peptides is highly desired. Global market share of

75

peptides is very significant, of about USD 14.4 billion per annum (Uhlig et. al., 2014).

76

Market demand is expected to increase due to the increasing number of health-conscious

77

consumers. Therefore, novel bioactive peptides with unique functionalities could have great

78

potential and value in the nutraceutical and food ingredient markets.

79

High-pressure processing (HPP) is considered as one of the most important

80

innovations in the last 50 years. The unique features of HPP offer food industry means to

81

produce novel foods, textures, and tastes. It was reported that HP treatment has unique effects

82

on proteolysis. Pressure-assisted protein unfolding reduces hydrolysis time (Graham, Penac,

83

Frias, Gomez, & Martinez-Villaluenga, 2015), enhances proteolysis via increased exposure of

84

susceptible peptide bonds to enzymatic cleavage (Girgih, Chao, He, Jung, & Aluko, 2015).

85

High pressure stabilizes and increases the activity of some enzymes during the hydrolysis of

86

proteins (Maresca and Ferrari, 2017). Pressure treatment also increases protein digestibility

87

(Quirós, Chichón, Recio, & López-Fandiño, 2007), facilitates the enzymatic release of

88

antioxidant peptides (Girgih et al., 2015), and enhances the formation of antioxidant peptides

89

in the hydrolysates (Zhang, Jiang, Miao, Mu, & Li, 2012). Moreover, some antimicrobial

90

peptides are only active under high pressure (Masschalck, Houdt, Haver, & Michiels, 2001).

91

These results suggest the feasibility of producing unique, bioactive peptides via pressure-

92

assisted enzymatic proteolysis (Chao, He, Jung, & Aluko, 2013; Girgih et al., 2015).

93

Nevertheless, the application of HPP for hydrolyzing the protein from fish by-products has

94

not been fully explored. The main objective of this study was to investigate the effects of

95

pressure-assisted enzymatic hydrolysis on physicochemical, functional, and bioactive

96

properties of fish protein hydrolysate (FPH) based on tilapia by-products. The roles of 4

97

process parameters (pressure and holding time) in modulating key properties of the

98

hydrolysates were subsequently evaluated.

99

2. Materials and Methods

100

2.1 Chemicals and sample preparation

101

Alcalase enzyme from Bacillus licheniformis was supplied by EMD Millipore Corp.

102

(Burlington, MA, USA). Bovine serum albumin (BSA) was purchased from Sigma Aldrich

103

(St. Louis, MO, USA). Coomassie brilliant blue dye, 2,2-diphenyl-1-picrylhydrazyl (DPPH),

104

phosphate buffer saline (PBS), sodium azide, potassium ferricyanide, sodium thiosulfate,

105

trichloroacetic acid (TCA), Tris hydrochloride and all other chemicals were of analytical

106

grade and provided by Brenntag Ingredients Public Company Limited (Bangkok, Thailand).

107

Fresh tilapia fish were procured from the local market. The fish were eviscerated and

108

the by-products (head, tail, and fins) were collected separately. The fish by-products were

109

then mixed with distilled water at 200 g/L and ground by a blender (BE-128, Otto Kingglass

110

Co., Bangkok, Thailand) to form a fine suspension. Protein was isolated by isoelectric

111

solubilization and precipitation method (Tahergorabi, Beamer, Mata, & Jaczynski, 2012).

112

The suspension was adjusted to pH 11.5 with the help of a pH meter (Jenway 3510, Cole-

113

Parmer Instrument Co., Staffordshire, UK) using 1 mol/L NaOH. The sample was mixed for

114

10 min by magnetic stirrer (MS 12-C, Bosstech Co., Bangkok, Thailand) and was then

115

centrifuged (Centrikon T-324, Kontron Instruments, Milano, Italy) at 10,000 x g for 10 min.

116

As the centrifugation was completed, the solubilized protein solution was collected while

117

lipid and insoluble layers were discarded. In the next stage, pH of the protein solution was

118

adjusted to 5.5 using 1 mol/L HCl, followed by mixing and centrifugation as previously

119

described. Fish protein isolate (FPI) thus obtained was freeze-dried (Scanva CoolSafe 55-4,

5

120

LaboGene ApS, Lillerod, Denmark), vacuum-packed (Turbovac 50005, Omcan Inc.,

121

Mississauga, ON, Canada) and kept at -20 ºC for further analysis.

122

2.2 Experiments

123

Pressure-assisted enzymatic hydrolysis was conducted using a high hydrostatic

124

pressure processing unit equipped with a 2-litre stainless steel vessel (Dx91, Engineered

125

Power Systems, MA, USA). The pressure chamber was thermostatically controlled by an

126

external heating tank. The temperature was measured by three probes installed inside the

127

pressure chamber. The system can deliver a maximum pressure of 690 MPa. For processing

128

the samples, isolated protein was re-suspended in distilled water at a concentration of 20

129

g/100 mL. Alcalase enzyme (3 mL/100 mL) was added to the mixture immediately before

130

HPP treatment. As suggested by the enzyme manufacturer, pH of the protein suspension was

131

adjusted to pH 8.0 using 1 mol/L NaOH and temperature of the hydrolysis process was fixed

132

at 55 ± 1°C. The samples were vacuum-packed in polyethylene bags and subsequently

133

processed at various combinations of holding time and pressure (Table 1). Treated samples

134

were heated at 95 °C for 10 min to inactivate the residual enzyme activity. The samples were

135

then cooled to room temperature and neutralized with 1 mol/L HCl. Obtained HPP treated

136

fish protein hydrolysate (HPP-FPH) were lyophilized in a freeze drier (Scanva CoolSafe 55-

137

4, LaboGene ApS, Lillerod, Denmark), and stored at 4 °C until further use. Samples of fish

138

protein hydrolyzed at the atmospheric pressure (AP-FPH) were used for comparison.

139

2.3 Characterization of the fish protein hydrolysate

140

The isolated protein and HPP hydrolysates were analyzed in triplicates for different

141

physicochemical, functional and bioactive properties.

142

2.3.1 Physicochemical properties

143

Soluble protein content

6

144

The soluble protein content was quantified by Bradford method (1976). One gram of

145

protein sample was blended at a concentration of 20 g/100 mL distilled water followed by

146

centrifugation at 3200 x g for 10 minutes. The supernatant (100 µL) was mixed with 5 mL

147

Coomassie brilliant blue dye solution (25 mL/100 mL) and incubated for 15 minutes. The

148

absorbance was recorded at 595 nm by a UV-Vis spectrophotometer (UV-1800, Shimadzu

149

Corporation, Kyoto, Japan). The protein content was determined by using the calibration

150

curve against a standard solution of Bovine serum albumin (BSA).

151

TCA-solubility index

152

TCA-SI was determined by trichloroacetic acid (TCA) precipitation method (Hoyle &

153

Merritt, 1994). Briefly, 5 grams of the protein hydrolysate was blended with 5 mL of 20

154

g/100 mL TCA. The mixture was incubated for 30 min and then centrifuged at 2700 x g for

155

10 min. Total and soluble protein content was determined by Bradford method (1976) using

156

UV-Vis spectrophotometer (UV-1800, Shimadzu Corporation, Kyoto, Japan) at the

157

wavelength of 595 nm. BSA was used as the standard. TCA-SI was calculated as below:

158 159

TCA - SI (%) =

Soluble protein content (mg/ml) × 100% Total protein content (mg/ml)

[1]

Amino acid profile

160

Free amino acids of FPI and FPH were determined by an ACQUITY TQD

161

LC/MS/MS system (Waters Corp., Milford, MA, USA) following the method of Chaimbault,

162

Petritis, Elfakir, & Dreux (1999). The stationary phase was a BEH C18 column (Agilent

163

Technology, Santa Clara, CA, USA). The mobile phase consisted of acetonitrile (A) and

164

heptaflourobutyric acid (B).

165

UV spectral analysis

166

UV spectra of the FPH were analyzed to elucidate their conformational changes

167

resulting from the treatments (Zhang et al., 2012). FPH suspensions at 2 g/L prepared with 7

168

0.05 mol/L Tris-HCl buffer (pH 8.0) were centrifuged at 3900 x g for 20 min. The

169

supernatant was scanned by a UV–Vis spectrophotometer (UV-1800, Shimadzu Corporation,

170

Kyoto, Japan) over the wavelength range of 250 nm - 360 nm at a rate of 10 nm/min.

171

2.3.2 Functional properties

172

Solubility

173

Solubility of the FPH was determined based on the methods of Liu et al. (2014). The

174

FPH (0.1 g) was dissolved in 100 mL water and pH of the solution was adjusted to 7.0. The

175

sample was mixed by magnetic stirring for 30 min at 30 ºC, followed by ultrasonication (CP

176

8892, Cole-Parmer Instrument Co., Vernon Hills, IL, USA) for 30 min. The mixture was then

177

centrifuged at 3900 x g for 20 min. The protein content in the supernatant was determined by

178

Bradford method (1976) using BSA as the standard. Solubility of the FPH was calculated by

179

the following equation:

180 181

Solubility (%) =

Protein content in supernatant × 100% Total protein content

[2]

Water holding capacity (WHC) and oil holding capacity (OHC)

182

WHC and OHC were determined by adopting the method of Diniz & Martin (1997).

183

For WHC, 400 mg of freeze-dried protein hydrolysates was added to 10 mL of distilled

184

water. The mixture was stirred for 5 min and placed in a water bath at 40 °C for 30 min. The

185

sample was centrifuged at 3900 x g for 30 min and the weight of absorbed water was

186

measured. OHC was determined by mixing 100 mg of sample to 10 mL soybean oil. The

187

weight of adsorbed oil was measured after centrifuging the mixture at 700 x g for 30 min.

188

WHC and OHC were expressed as g water or oil per g of sample, respectively.

189

Emulsifying properties

190

Emulsifying activity index (EAI) and emulsifying stability index (ESI) was

191

determined by the method of Sathe & Salunkhe (1981). Protein suspension at 2 g/L was 8

192

stirred for 10 min and their pH values were adjusted to 7. The solution (30 mL) was mixed

193

with 10 mL soybean oil and homogenized at a speed of 1200 rpm for 15 min (Servodyne

194

50,000-25, Cole-Parmer Instrument Co., Vernon Hills, IL, USA). Fifty microliters of the

195

prepared emulsion was pipetted out from the bottom of the tube and diluted with 5 mL of 1

196

g/L sodium dodecyl sulfate (SDS) solution. A 1 g/L SDS solution was used as the blank. The

197

absorbance was measured immediately (A0) and after 10 min (A10) at 500 nm using UV-Vis

198

spectrophotometer (UV-1800, Shimadzu Corporation, Kyoto, Japan).

199

EAI (m 2 /g) =

200

ESI (min) =

2 × 2.303 × 100 × A 0 0.25 × C × 10,000

[3]

A 0 ×10 A 0 − A10

[4]

201

where A0 is absorbance at 0 min, A10 is absorbance after 10 mins, and C is initial protein

202

concentration (g/mL).

203

2.3.3 Bioactive properties

204

DPPH radical scavenging activity

205

DPPH radical scavenging activity was determined by the method of Blois (1958). The

206

percentage of DPPH radical scavenging activity was calculated as follows:

207

DPPH radical scavenging activity (%) =

A control - A sample A control

× 100

[5]

208

where Acontrol is the absorbance of control sample (reagents and water). Asample is the

209

absorbance of the protein sample. The concentration of the sample required to decrease the

210

DPPH concentration by 50 % was determined by the software GraphPad Prism (v. Prism 7)

211

and denoted as IC50 (µg/mL).

212

Reducing power

213

Reducing power was determined by the method of Oyaizu (1986). Protein sample was

214

mixed with 0.2 mol/L phosphate buffer (pH 6.6) and 1 g/100 mL potassium ferricyanide in 9

215

equal proportions. The sample was incubated for 20 min at 50 °C followed by addition of 2.5

216

mL 10 g/ 100 mL TCA. Incubated and centrifuged at 3900 x g for 10 min. Upper layer (2.5

217

mL) was blended with 2.5 mL water and 0.5 mL of 1 g/L ferric chloride solution. The

218

absorbance was measured at 700 nm in an UV-Vis spectrophotometer (UV-1800, Shimadzu

219

Corporation, Kyoto, Japan). The reducing power was expressed as µg ascorbic acid

220

equivalent (AAE)/g of the hydrolysate.

221

Peroxide value (PV)

222

PV was determined using the method of AOCS Official Method Cd 8-53 (1997).

223

Peroxide value of the hydrolysate was quantified by its ability to oxidize potassium iodide

224

and was calculated by the following equation:

225

PV (meq/kg) =

[S - B] × N × 1000 Weight of sample

[6]

226

where S is the titration volume of sample, B is titration volume of the blank, and N is

227

normality of sodium thiosulfate.

228

2.4 Statistical analysis

229

The effects of holding time (0, 6, 10, 20, 30, 35 min) and applied pressure (0.1, 38,

230

100, 250, 400, 462 MPa) on the enzymatic hydrolysis of tilapia by-product protein were

231

optimized by central composite design (CCD) with the help of the Design-Expert Software

232

(version 7.0.0, Stat-Ease Inc., MN, USA). The complete experimental design had 13 runs

233

including five replications of the central point (20 min, 250 MPa) (Table 1). Response

234

variables included physicochemical properties (TCA-SI, amino acid profile, UV spectra),

235

functional properties (protein solubility, water and oil holding capacity, emulsifying

236

properties), and bioactivities (antioxidant activity, reducing power, and peroxide value). The

237

influence of time (A) and pressure (B) on the response variables was analyzed using a

238

quadratic polynomial regression equation as below: 10

239

Y = β 0 + β1A + β 2 B + β12 AB + β11A 2 + β 22 B 2

[7]

240

Where, Y was the response variable, β0 was constant, βi, βii and βij were the linear, quadratic

241

and interactive coefficients, respectively. Additional cubic terms were used for OHC, ESI

242

and PV. The coefficients of the regression equation were estimated by Design Expert

243

Software (Table S3).

244

The results were expressed as the mean values with standard deviations. Comparisons

245

between groups were performed using Kruskal–Wallis test followed by Dunn's post hoc test

246

(SPSS statistics 22, IBM, Armonk, NY, USA). P < 0.05 was considered as significant.

247

3. Results and discussions

248

3.1. Physicochemical properties of the fish protein hydrolysate

249

3.1.1 Soluble protein content

250

The effects of pressure-assisted hydrolysis on soluble protein content of FPH are

251

presented in Fig. 4a. Both pressure and holding time had significant influence on soluble

252

protein content of HPP-FPH (p < 0.05). Soluble protein content of HPP-FPH generally

253

increased with increasing pressure and holding time. The highest concentration (5.7 mg/mL)

254

was obtained at 250 MPa and 35 min, which was significantly higher than that of the original

255

crude protein sample (1.3 mg/mL). Pressure treatment also yielded higher soluble protein as

256

compared to ambient hydrolysis for the same period (Table 1 and Fig. 1a). Soluble protein

257

residues produced are generally associated with the formation of hydrolysates with low

258

molecular mass (Dong et al., 2008). Improved soluble protein content of HPP-FPH samples

259

could be attributed to the activation of enzyme and unfolding of protein structure under

260

moderate pressures. When pressure exceeded 400 MPa, no further increase in soluble protein

261

content was observed. At this threshold, irreversible aggregation and precipitation of proteins 11

262

induced by pressure might have an adverse impact on enzyme activity (Chicón, Belloque,

263

Recio, & López-Fandiño, 2006). The active sites of enzymes are governed by their three-

264

dimensional structures. Therefore, any conformational changes can influence the activity and

265

substrate specificity of the enzymes (Klompong et al., 2007; Claeys, Indrawati, Van Loey, &

266

Hendrckx, 2003). In addition, the aggregation of the substrate proteins subjected to high-

267

pressure levels reduced the accessibility of the enzyme to peptide bonds, and eventually

268

slowed down the hydrolysis process (Maresca & Ferrari, 2017).

269

3.1.2 TCA-SI

270

TCA-SI of the original crude protein was 5 %. Hydrolysis at ambient pressure

271

increased TCA-SI of protein samples consistently with time. After 35 min, the maximum

272

TCA-SI obtained was 13 % (Fig. 1b). Pressure treatment had far more influence on TCA-SI

273

than hydrolysis at ambient pressure (Table 1 and Fig. 4b). Increase in holding time affected

274

TCA-SI of HPP-FPH significantly (p < 0.05). FPH processed at 250 MPa, 35 min exhibited

275

the highest TCA-SI (23 %) among all the samples. The underlying mechanisms could be

276

ascribed to the activation of enzyme and unfolding of protein substrate as previously

277

discussed. The results agree with the data reported by Zhang et al. (2012) for pressure-

278

assisted hydrolysis of chickpea protein.

279

3.1.3 Free amino acid composition

280

Free amino acid profiles of the control, AP-FPH and HPP-FPH are shown in Table 2.

281

Significant amounts of free amino acid were produced via hydrolysis at ambient and under

282

pressure condition. The application of pressure was previously demonstrated to enhance the

283

proteolysis, indicated by increased soluble protein content and TCA-SI. Therefore, as

284

expected, free amino acids generated in HPP-FPH were considerably higher than in AP-FPH.

285

Total free amino acid was found to increase by 30 % when pressure increased from 250 MPa 12

286

to 400 MPa. Dominant amino acids in HPP-FPH were leucine (0.147 mg/g), glutamic acid

287

(0.1429 mg/g) and phenylalanine (0.0549 mg/g) while cysteine and proline were present in

288

the lowest quantity. The distribution of free amino acid in the tilapia protein hydrolysates was

289

similar to that of hydrolyzed porcine myofibrillar proteins (Saiga, Tanabe, & Nishimura,

290

2003) and hydrolyzed whole herring by-products (Sathivel et al., 2003). Pressure treatment

291

has been reported to increase the neutral (valine, leucine, isoleucine and phenylalanine) and

292

basic (lysine) free amino acids in the hydrolysates of food proteins (Kim, Son, Maeng, Cho,

293

& Kim, 2016).

294

3.1.4 Analysis of UV spectra

295

UV spectra of the FPH are illustrated in Fig. 3 under (a) atmospheric pressure and (b)

296

high-pressure condition. Peak wavelengths of FPI were observed from 280 nm - 300 nm

297

which was typical of fish protein (Li et al., 2009) (Fig. 3a). These peaks were reportedly

298

associated with tyrosine and tryptophan present in the sample (Beaven and Holiday, 1952).

299

At ambient condition, the hydrolysis significantly increased the absorbance intensity of the

300

FPH as compared to FPI. However, the hydrolysis time did not affect the patterns of AP-FPH

301

samples. On the other hand, spectra of pressure-treated samples exhibited significant changes

302

in both peak wavelengths and absorbance intensity. A broad peak was observed between 290

303

nm - 320 nm (Fig. 3b). The absorbance intensity of HPP-FPH was strongly dependent on

304

pressure-time combinations. As pressure accelerated hydrolysis process, the higher release of

305

soluble proteins and free amino acids can contribute to higher absorbance intensity. In

306

addition, changes in UV spectra of HPP-FPH possibly reflect their conformational changes.

307

The spectra could be affected by the unfolding of the hydrolysates under pressure, which

308

exposed hydrophobic amino acid residues like tyrosine and tryptophan. The aggregation of

309

protein under high pressure could be conducive to the shift of the HPP-FPH peak 13

310

wavelengths. Increase in intensity and broadening of the peak absorbance has been observed

311

by Zhang et al. (2012) when chickpea protein isolates were processed up to 400 MPa.

312

However, the absorbance intensity decreased when pressure increased from 500 MPa to 600

313

MPa. The rise in pressure might result in a sequence of conformational changes due to altered

314

balance of stabilizing interactions (Zhang et al., 2012). Chao et al. (2013) also noted

315

decreased intrinsic fluorescence of protein solution at 400 MPa. This phenomenon was

316

attributed to protein aggregation or excessive protein-protein interactions, which shielded the

317

fluorescent amino acid residues.

318

3.2 Functional properties of fish protein hydrolysates

319

3.2.1 Solubility

320

In general, hydrolysis process significantly affected the solubility of FPH. At ambient

321

condition, the solubility of FPH after 35 min increased by 16 % as compared to crude protein

322

(Fig. 1c). Liu et al. (2014) attributed the enhanced solubility to increased TCA-SI. The impact

323

of pressure treatment on protein solubility was even more pronounced (Fig. 4c). The

324

solubility of HPP-FPH was influenced by applied pressure and holding time (Table S4).

325

Maximum solubility of 69 % was obtained at 100 MPa and 30 min (Table 1). Applied

326

pressure could have increased the susceptibility of protein to hydrolysis and release of small

327

peptides. In addition, protein unfolding exposes polar amino acids, which can form hydrogen

328

bonds with water. Consequently, the solubility of the resultant products can be improved

329

(Connolly, Piggott, & Fitz Gerald, 2014). The difference in solubility of obtained

330

hydrolysates could be due to the different lengths of peptide residues and their hydrophobic-

331

hydrophilic balance. Other studies also observed an increase in solubility of proteins

332

subjected to pressure treatment. However, pressures higher than 400 MPa were found to

333

reduce the solubility. This effect could be due to the formation of insoluble macro-aggregates 14

334

(Queirós et al., 2018). The impacts of pressure treatment on proteins depend on type, nature

335

and conformational stability of the protein molecules (Graham et al., 2015; Queirós et al.,

336

2018).

337

3.2.2 Water holding capacity

338

At atmospheric pressure, hydrolysis did not have any significant effect on the WHC

339

of FPH (Fig. 1d). The effects of pressure and time on WHC are presented in Fig. 4d.

340

Pressure-assisted hydrolysis reduced WHC (0.9 g/g) of FPH compared to FPI (1.3 g/g) (Table

341

1). Both pressure and holding time were found to influence WHC significantly (Table S4).

342

Increasing pressure and holding time tended to decrease WHC. The trend may be caused by

343

pressure-induced conformational changes of FPH. Li, Zhu, Zhou, & Peng (2011) suggested

344

that the decline of WHC at 600 MPa was related to extensive denaturation and increased

345

surface hydrophobicity of the protein.

346

3.2.3 Oil holding capacity

347

Hydrolysis at ambient pressure treatment was found to decrease OHC of FPH (Fig.

348

1e). During hydrolysis, protein molecules were broken into smaller fragments indicated by

349

increased TCA-SI. Therefore, the process adversely affected the integrity of protein

350

structures and their physical entrapment of the oil (Sathivel, Smiley, Prinyawiwatkul, &

351

Bechtel, 2005). For pressure treatment, OHC was affected by both process variables (Table

352

S4). OHC was dependent on the level of applied pressure and holding time (Fig. 4e). It was

353

also interesting to note that the OHC of pressure-treated FPH was not strongly related with

354

TCA-SI (Table 1). FPH subjected to pressure treatment may have experienced complex

355

changes in both molecular sizes and conformation. Thus, OHC resulted from the net effect of

356

these changes, which, in turn, were affected by a specific pressure-time combination.

15

357

3.2.4 Effects of pressure-assisted hydrolysis on surface activity of the peptides

358

The formation of emulsions depends on the surface hydrophobicity, solubility and

359

capability to decrease the interfacial tension of the proteins (Queirós et al., 2018). Pressure

360

treatment can modify vital properties of the protein molecules such as volume, surface

361

hydrophobicity/hydrophilicity, solubility, adsorption and interactions at the interfaces. The

362

process conditions, i.e., pressure level and holding time, strongly affect these properties and

363

eventually the surface activity of the peptides.

364

Emulsifying activity (EAI) and stability (ESI)

365

The EAI and ESI of the crude protein were 27 m²/g and 13 min, respectively.

366

Atmospheric hydrolysis tended to slightly reduce emulsifying properties of FPH (Fig. 1f &

367

g). The result concurs with past investigations for protein hydrolysates of Pacific whiting

368

(Pacheco-Aguilar, Mazorra-Manzano, & Ramirez-Suarez, 2008) and yellow stripe trevally

369

(Klompong et al., 2007). The production of peptides with low molecular weights during

370

hydrolysis weakened their interfacial activities (Klompong et al., 2007). The effects of

371

pressure treatment are shown in Fig. 4f & g. During pressure treatment, emulsifying activity

372

of the samples was predominantly affected by holding time whereas emulsifying stability

373

depended on both pressure and holding time (Table S4). The obtained FPH had EAI ranging

374

from 14-20 m²/g and ESI from 19-32 min (Table 1). EAI was maximum (20 m²/g) at 250

375

MPa and 20 min, then decreased with higher pressure level (Table 1). EAI and ESI of the

376

peptides

377

hydrophobicity/hydrophilicity, and ability to diffuse to the interface surface and form a film

378

to prevent the aggregation of droplets. The hydrolysis, in general, leads to loss of EAI and

379

ESI due to reduction in length of the peptides. Amphiphilicity is a crucial property for

380

interfacial and emulsifying activity of peptides. Under pressure treatment, the structure of

were

governed

by

various

factors

16

such

as

the

molecular

sizes,

381

FPH was significantly modified. Increased interactions of hydrophobic groups with the oil

382

droplets can help form smaller emulsified particles (Chao et al., 2018). In addition, the

383

exposure of hydrophobic group to a certain extent could help enhance the interactions

384

between peptide molecules (Li et al., 2011). If the interaction occurred at the interface, the

385

emulsifying property would be increased. However, if the aggregation occurred before the

386

interfacial adsorption, the emulsifying property would be lower (Queirós et al., 2018). The

387

threshold pressure, which induces protein aggregation, is dependent on the type of protein

388

substrate (Chao et al., 2018). Jung, Murphy, & Johnson (2005) demonstrated that limited

389

hydrolysis improved hydrophobicity and emulsification capacities of denatured protein but

390

decreased emulsification capacity of native-state proteins. However, further reduction of

391

peptide length and denaturation of peptides could impair their ability to stabilize the

392

emulsion. Pressure treatment from 200 MPa to 400 MPa was reported to increase EAI

393

(Queirós et al., 2018; Li et al., 2011) but higher pressure can decrease EAI and ESI (Wang et

394

al., 2008). Therefore, pressure could be used to manipulate the EAI and ESI of the FPH at

395

appropriate process conditions.

396

3.3 Bioactive properties of fish protein hydrolysates

397

3.3.1 DPPH scavenging activity

398

IC50 is the concentration of sample required to scavenge 50 % of the DPPH free

399

radicals (Veenuttranon & Nguyen, 2018). Therefore, the lower the IC50 value, the higher the

400

antioxidant activity of the compound. The effects of ambient and pressure-assisted hydrolysis

401

are presented in Table 1, Fig. 2a and Fig. 5a. Antioxidant activity of AP-FPH and HPP-FPH

402

was improved as compared to the control sample (IC50 = 653 µg/mL). The effect could stem

403

from the release of low molecular peptides during hydrolysis (Franck et al., 2019). In

404

addition, tyrosine contributes significantly to the scavenging of free radicals since their 17

405

phenolic lateral chains act as potent electron donors (Picot et al., 2010). The release of

406

tyrosine during hydrolysis process could be one of the factors that improved the antioxidant

407

activity of the FPH. Pressure treatment enhanced the antioxidant activity of the FPH as

408

compared to ambient hydrolysis. The effects were mainly dependent on pressure and time

409

(Table S4). Improvement in antioxidant activity of FPH could be explained by higher TCA-

410

SI of HPP-FPH as compared to AP-FPH. The content of free amino acid of HPP-FPH was

411

also higher than the control and AP-FPH. Antioxidant activity of HPP-FPH was highest at

412

250 MPa and increased with holding time. Zhang et al. (2012) reported increased antioxidant

413

activity of chickpea hydrolysates obtained by alcalase treatment at 100 MPa - 200 MPa for 10

414

min. Chao, He, Jung, Rotimi, & Aluko (2013) also reported a similar increase in antioxidant

415

activity of high pressure treated pea protein hydrolysates by 20-25 %. Pressure treatment

416

could be a viable method to improve the antioxidant activity of protein hydrolysates.

417

3.3.2 Reducing Power

418

Reducing power indicates hydrogen donating capacity of the hydrolysates. Hence,

419

reducing ability is directly correlated to antioxidant activity of the bioactive compounds

420

(Halim et al., 2016). Reducing power was not influenced by hydrolysis at atmospheric

421

condition but exhibited significant increase under pressure (Table 1, Fig. 2b and Fig. 5b). As

422

compared to the control sample (28 µg AAE/g), maximum reducing power of HPP-FPH (100

423

MPa, 30 min) obtained by the hydrolysis was 43.5 µg AAE/g (Table 1). Reducing power was

424

affected by pressure and holding time of the treatment (Table S4). Wang and Xiong (2005)

425

attributed the increase in reducing power of hydrolyzed proteins to increased availability of

426

hydrogen ions (protons and electrons) due to peptide cleavages. Donation of protons could

427

occur through side-chain groups or peptide structure. Pressure significantly facilitated the

428

hydrolysis and cleavages of protein molecules, hence increase the capacity of FPH to interact

429

with and donate electrons to ferric ion. 18

430

3.3.3 Peroxide value (PV)

431

PV reflects the oxidative status of FPH samples. Oxidation can occur due to

432

enzymatic activity in the samples as well as the presence of residual fat molecules. The

433

reaction can be triggered by availability of free oxygen. Hydrolysis at atmospheric pressure

434

did not have significant influence on peroxide values of FPH (Fig. 2c). On the contrary,

435

pressure-assisted hydrolysis significantly affected oxidative status of the FPH. PV of peptides

436

subjected to ambient hydrolysis ranged from 8-12 meq/kg whereas the samples processed

437

under pressure had PV varying from 4-10 meq/kg. Both pressure and holding time had

438

significant impact on PV (Table S4). The effects were dependent on the range of pressure and

439

hydrolysis time (Table 1, Fig. 5c). The lowest PV was obtained at 250 MPa and 35 min. The

440

effects of pressure on protein oxidation may depend on different factors such as pressure

441

level, process time, chemical composition, fat profile, handling and preprocesses, etc.

442

(Truong, Buckow, Stathopoulos, & Nguyen, 2015). Enhanced oxidative protection of FPH

443

could be due to antioxidant activity of low molecular peptides released.

444

4 Conclusions

445

This study provided information on the pressure-assisted enzymatic hydrolysis of

446

tilapia by-product protein as a new substrate. Pressure and holding time were found to have

447

significant impacts on the physicochemical, functional and bioactive properties of hydrolyzed

448

products. The HP process accelerated the hydrolysis and facilitated the release of free amino

449

acids. The treatment also considerably improved solubility, and emulsifying properties as

450

well as antioxidant activity of FPH. However, decrease in water holding capacity was

451

noticed. Further investigations are needed to elucidate the mechanisms and the role of process

452

parameters in conformational changes of FPH and their relationship to the final properties of

453

the products. The properties of HPP-FPH were strongly dependent on the range of process

19

454

parameters used. Therefore, optimization is required to obtain desirable characteristics of

455

HPP-FPH for a specific industrial application.

456

Acknowledgements

457

This project was supported by the GAIA expand award of Rutgers, The State

458

University of New Jersey, USA. The authors are also thankful to Washington State

459

University, USA for facilitating the high-pressure processing of protein samples. The author

460

would also like to acknowledge help from Sawali Naware in high-pressure processing of

461

protein samples.

462

Declarations of interest

463

None

464

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28

Table 1. Physicochemical and functional properties of fish protein hydrolysates obtained from pressure-assisted enzymatic hydrolysis Protein concentration (mg/ml) Std. Order

Time (min)

TCA-SI (%)

Solubility (%)

WHC (g/g)

OHC (g/g)

EAI (m²/g)

ESI (min)

Pressure (MPa) Mean value

±SD

Mean value

±SD

Mean value

±SD

Mean value

±SD

Mean value

±SD

Mean value

±SD

Mean value

±SD

1

10

100

1.6

±0.1

11

±2

58

±2

0.9

±0.1

2.6

±0.1

17

±1

30

±1

2

30

100

2.9

±0.2

20

±1

69

±4

0.8

±0.1

3.0

±0.2

19

±1

26

±2

3

10

400

`3.0

±0.1

16

±1

54

±2

0.7

±0.1

2.8

±0.1

17

±3

27

±2

4

30

400

5.0

±0.2

18

±1

68

±1

0.7

±0.1

2.1

±0.3

17

±1

21

±2

5

6

250

2.0

±0.1

14

±1

44

±3

0.9

±0.1

2.1

±0.3

18

±2

32

±5

6

35

250

5.7

±0.2

23

±2

67

±2

0.7

±0.2

3.1

±0.5

16

±2

20

±2

7

20

38

2.9

±0.1

18

±1

54

±4

0.9

±0.2

2.3

±0.2

14

±2

19

±1

8

20

462

4.9

±0.2

22

±1

65

±1

0.8

±0.1

3.0

±0.2

14

±1

26

±2

9

20

250

5.0

±0.1

19

±1

59

±1

0.8

±0.2

2.2

±0.2

20

±1

24

±1

10

20

250

5.0

±0.1

16

±1

59

±1

0.8

±0.1

2.3

±0.2

20

±1

29

±1

11

20

250

4.8

±0.1

18

±1

60

±1

0.8

±0.1

2.2

±0.3

20

±1

26

±1

12

20

250

5.0

±0.2

21

±1

62

±1

0.8

±0.1

2.3

±0.1

19

±1

26

±1

13

20

250

5.0

±0.1

20

±1

62

±1

0.8

±0.1

2.1

±0.2

22

±1

23

±1

Mean value ± SD

5.0

±0.1

19

±1

61

±2

0.8

±0

2.2

±0.1

20

±1

26

±2

Pooled standard deviation

-

0.1

-

1

-

2

-

0.2

-

0.2

-

1.5

-

2

*

*

Mean value ± standard deviation of the values at the central points. Pooled standard deviation indicates the analytical precision. TCA-SI: trichloroacetic acid-solubility index;

WHC: Water Holding Capacity; OHC: Oil Holding Capacity; EAI: Emulsifying Activity Index; ESI: Emulsifying Stability Index.

Table 2. Free amino acid composition of fish protein hydrolysates obtained from different processing conditions Free amino acid (mg/g protein) Amino acid

FPI

AP-FPH

HPP-FPH

HPP-FPH

0 min

30 min

35 min, 250 MPa

30 min, 400 MPa

Alanine

0.0183 (± 0.0002)a

0.0279 (± 0.0003)d

0.0202 (± 0.0002)b

0.0250 (± 0.0002)c

Arginine

0.0047 (± 0.0001)a

0.0352 (± 0.0002)b

0.0381 (± 0.0003)c

0.0486 (± 0.0003)d

Aspartic acid

0.0030 (± 0.0001)a

0.0091 (± 0.0002)b

0.0141 (± 0.0002)c

0.0197 (± 0.0003)d

Glutamic acid

0.0221 (± 0.0003)a

0.0752 (± 0.0002)b

0.1179 (± 0.0009)c

0.1429 (± 0.0005)d

Glycine

0.0343 (± 0.0004)d

0.0303 (± 0.0002)c

0.0277 (± 0.0003)a

0.024 (± 0.006)b

Histidine

0.0064 (± 0.0001)a

0.0086 (± 0.0001)b

0.0112 (± 0.0001)c

0.0146 (± 0.0003)d

Isoleucine

0.0022 (± 0.0001)a

0.0053 (± 0.0001)b

0.0177 (± 0.0004)c

0.0283 (± 0.0004)d

Leucine

0.0092 (± 0.0002)a

0.0894 (± 0.0008)b

0.1181 (± 0.0003)c

0.147 (± 0.001)d

Lysine

0.0133 (± 0.0003)a

0.0342 (± 0.0001)c

0.0321 (± 0.0003)b

0.0386 (± 0.0004)d

Methionine

0.0001 (± 0.0001)a

0.0083 (± 0.0001)b

0.0217 (± 0.0004)c

0.0304 (± 0.0003)d

Phenylalanine

0.0076 (± 0.0001)a

0.0316 (± 0.0002)b

0.0416 (± 0.0007)c

0.0549 (± 0.0007)d

Proline

0.0060 (± 0.0003)c

0.0054 (± 0.0001)a

0.0057 (± 0.0001)b

0.0061 (± 0.0001)c

Serine

0.0006 (± 0.0001)a

0.0280 (± 0.0001)d

0.0227 (± 0.0002)b

0.0277 (± 0.0001)c

Threonine

0.0058 (± 0.0001)a

0.0272 (± 0.0006)b

0.0326 (± 0.0006)c

0.0463 (± 0.0007)d

Tyrosine

0.0052 (± 0.0001)a

0.0180 (± 0.0002)b

0.0343 (± 0.0004)c

0.0407 (± 0.0004)d

Valine

0.0050 (± 0.0001)a

0.0121 (± 0.0001)b

0.0322 (± 0.0008)c

0.0513 (± 0.0009)d

0.27 (± 0.01)a

0.93 (± 0.01)b

1.35 (± 0.02)c

1.76 (± 0.01)d

Total (mg amino acid /g protein)

FPI: Fish Protein Isolate; AP-FPH: Atmospheric Pressure-Fish Protein Hydrolysate; HPP-FPH: High Hydrostatic Pressure-Fish Protein Hydrolysate. Data show mean values (±SD) for three replicates. Different letters in rows indicate significant difference at P < 0.05.

Table 3. Bioactive properties of fish protein hydrolysates produced by pressure-assisted enzymatic hydrolysis Antioxidant activity IC50 (µg/ml)

Std. Order

Peroxide value (meq/kg)

Pressure (MPa) Mean value

±SD

Mean value

±SD

Mean value

±SD

1

10

100

472

±15

28.6

±2.8

8

±2

2

30

100

352

±10

43.5

±4.9

10

±2

3

10

400

304

±40

32.7

±5.1

7

±2

4

30

400

312

±20

39.1

±1.8

8

±3

5

6

250

331

±20

30.4

±3.6

10

±1

6

35

250

312

±20

37.5

±4.5

4

±1

7

20

38

386

±30

34.9

±1.1

9

±2

8

20

462

386

±30

27.8

±3.6

6

±1

9

20

250

362

±40

35.3

±2.2

8

±2

10

20

250

304

±40

35.1

±1.8

8

±2

11

20

250

362

±30

36.6

±1.5

8

±2

12

20

250

333

±40

30.4

±2.2

8

±1

13

20

250

324

±40

35.3

±2.6

8

±1

Mean value ± SD

337

±25

34.5

±2.4

8

±0

Pooled standard deviation

-

28

-

3.1

-

1

*

*

Time (min)

Reducing power (µg AAE/g dry extract)

Mean value ± standard deviation of the values at the central points. Pooled standard deviation indicates the

analytical precision.

1

Figure Captions

2

Figure 1. Effects of time of hydrolysis at atmospheric pressure on physicochemical and functional

3

properties of fish protein hydrolysates. a) Soluble protein content, b) Trichloroacetic acid-solubility

4

index (TCA-SI), c) Solubility, d) Water holding capacity (WHC), e) Oil holding capacity (OHC), f)

5

Emulsifying activity index (EAI), g) Emulsifying stability index (ESI).

6 7

Figure 2. Effects of time of hydrolysis at atmospheric pressure on bioactive properties of fish

8

protein hydrolysates. a) Antioxidant activity, b) Reducing power, c) Peroxide value.

9 10

Figure 3. a) UV spectra of fish protein hydrolysates produced by enzymatic hydrolysis under

11

atmospheric pressure at: 0 min (♦), 6 min (■), 10 min (▲), 20 min (˟), 30 min (ӿ), 35 min (●).

12

b) UV spectra of fish protein hydrolysates produced by enzymatic hydrolysis under high pressure. 6

13

min,250 Mpa (●); 10 min,100 Mpa (□); 10 min,400 Mpa (∆); 20 min,38 Mpa (◊); 20 min,250 Mpa

14

(○); 20 min,462 Mpa(♦); 30 min,100 Mpa (■); 30 min,400 Mpa (▲); 35 min,250 Mpa (˟)

15 16

Figure 4. Effects of pressure and holding time on physicochemical and functional properties of fish

17

protein hydrolysates.

18 19

Figure 5. Effects of pressure and holding time on bioactive properties of fish protein hydrolysates.

15

(a)

(b) 12 TCA-SI (%)

Soluble protein content (mg/ml)

2.2 1.9 1.6 1.3

9 6 3

1

0 0

5

10

15 20 Time (min)

25

30

35

0

15 20 Time (min)

25

30

35

5

10

15 20 Time (min)

25

30

35

(d)

50 45 40 35 30

1.6 1.4 1.2 1.0

0

5

10

15 20 Time (min)

25

30

35

0

30

4.0 EAI (pH 7) (m²/g)

(e) OHC (g/g)

10

1.8

(c) WHC (g/g)

Solubility (pH 7) (%)

55

5

3.5 3.0 2.5

(f)

27 24 21 18 15

2.0 0

5

10

15 20 Time (min)

25

30

0

35

5

10

20 21

22 23 24 25 26

ESI (pH 7) (min)

15

(g)

14 13 12 11 10 0

5

10

15 20 Time (min)

27 28

Figure 1 2

25

30

35

15 20 Time (min)

25

30

35

700

45

(a)

Reducing power (µg AAE/gm)

Antioxidant activity (µg /ml)

29

600 500 400 300 0

5

10

15 20 Time (min)

25

30

(b) 40 35 30 25

35

0

5

10

25

30

35

30

32 33 34 35 36 37

Peroxide value (meq/kg)

31 14

(c)

11 8 5 0

5

10

15 20 Time (min)

38 39

Figure 2

40 41 42 43 44 45 46

3

15 20 Time (min)

25

30

35

1.0

Absorbance

(a)

0.5

0.0 250

260

270

280

260

270

280

290

300 310 320 Wavelength (nm)

330

340

350

360

47 2.5

(b)

Absorbance

2.0

1.5

1.0

0.5 250

290

300 310 320 Wavelength (nm)

48 49

Figure 3

4

330

340

350

360

50

(b) TCA solubility index

(a) Soluble protein content

51 52 53 54 55 (c) Solubility

(d) Water holding capacity

56 57 58 59 60 (e) Oil holding capacity

61

(f) Emulsifying activity index

62 63 64 65 66 67

(g) Emulsifying stability index

68 69 70 71 72 73 74

Figure 4 5

75 76

(a) Antioxidant activity (IC 50)

(b) Reducing power

77 78 79 80 81 82 (c) Peroxide value

83 84 85 86 87 88 89

Figure 5

6

1

Highlights

2

+ Pressure accelerated the protein hydrolysis and facilitated the release of free amino acids.

3

+ The solubility and antioxidant activity of fish protein hydrolysates were enhanced.

4

+ Water and oil holding capacities of fish protein hydrolysates were decreased.

5

+ Emulsifying properties varied with applied pressure and holding time.

6

AUTHOR DECLARATION •

•

•

•

•

We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us. We confirm that we have given due consideration to the protection of intellectual property associated with this work and that there are no impediments to publication, including the timing of publication, with respect to intellectual property. In so doing we confirm that we have followed the regulations of our institutions concerning intellectual property. We further confirm that any aspect of the work covered in this manuscript that has involved either experimental animals or human patients has been conducted with the ethical approval of all relevant bodies and that such approvals are acknowledged within the manuscript. We understand that the Corresponding Author is the sole contact for the Editorial process (including Editorial Manager and direct communications with the office). He/she is responsible for communicating with the other authors about progress, submissions of revisions and final approval of proofs. We confirm that we have provided a current, correct email address which is accessible by the Corresponding Authors and which have been configured to accept email from ([email protected]; [email protected]).

Signed on behalf of all authors as follows:

Loc Thai Nguyen Ashutosh Kumar Hemker Mukund Karwe Deepti Salvi