Protein Oxidation in Plant Protein-Based Fibrous Products: Effects of

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Food and Beverage Chemistry/Biochemistry

Protein oxidation in plant protein-based fibrous products: effect of encapsulated iron and process conditions Patricia Duque Estrada, Claire C. Berton-Carabin, Miek Schlangen, Anniek Haagsma, Anna Paola T R Pierucci, and Atze Jan van der Goot J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02844 • Publication Date (Web): 26 Sep 2018 Downloaded from http://pubs.acs.org on October 2, 2018

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Journal of Agricultural and Food Chemistry

Protein oxidation in plant protein-based fibrous products: effect of encapsulated iron and process conditions

Patrícia Duque Estrada†, Claire C. Berton-Carabin† , Miek Schlangen†, Anniek Haagsma†, Anna Paola T. R. Pierucci§, Atze Jan van der Goot†* †

Food Process Engineering, Wageningen University & Research, PO Box 17, 6700 AA, Wageningen, The Netherlands

§

Laboratório de Desenvolvimento de Alimentos para Fins Especiais e Educacionais, Instituto de

Nutrição Josué de Castro, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho, 373, Centro de Ciências da Saúde, Bloco J sub solo, Sala 08, CEP: 21941 090, Cidade Universitária, Rio de Janeiro, RJ, Brazil.

*Corresponding author. Phone: +31 (0) 3174- 87012; E-mail: [email protected] (van der Goot, A. J.)

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Abstract

2

Plant protein-based fibrous structures have recently attracted attention because of their potential for

3

meat replacer formulation. It is, however, unclear how the process conditions and fortification with

4

micronutrients may affect the chemical stability of such products. Therefore, we aimed to

5

investigate the effect of process conditions and of the incorporation of iron (free and encapsulated)

6

on protein oxidation in a soy protein-based fibrous product. First, the physicochemical stability of

7

iron-loaded pea protein particles, used as encapsulation systems, was investigated when exposed to

8

100 or 140 ºC. Secondly, protein oxidation was measured in the iron-fortified soy protein-based

9

fibrous structures made at 100 or 140 °C. Exposure to high temperatures increased the carbonyl

10

content in pea protein particles. The incorporation of iron (free or encapsulated) did not affect

11

carbonyl content in the fibrous product, but the process conditions to make such products induced

12

the formation of carbonyls to a fairly high extent.

13

Keywords: protein oxidation, plant protein, fibrous structures, iron, encapsulation.

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1. Introduction

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Nowadays there is a global trend to reduce meat consumption and move towards a more plant

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protein-based diet for environmental, health and animal welfare reasons1. To help the population

30

towards such a transition, research has focused on developing products with similar fibrous

31

structures as in meat. At Wageningen University & Research, a range of shearing devices has been

32

developed to make fibrous structures using plant proteins, by applying a simple shear flow and high

33

temperatures (100-140 °C)2,3. Another technique widely applied to make meat analogues is

34

extrusion. Here, materials are processed using a similar temperature range, giving this study a

35

potentially broader relevance. So far, most of this research has been done using soy protein

36

ingredients4–6. Although the structure formation in such fibrous products has already been well

37

described, it is still unclear how the process conditions may affect the chemical stability, and thus

38

the sensory and nutritional quality of these products.

39

Oxidation is often associated with deterioration in food products7. Lipid oxidation in foods has been

40

largely studied over the past decades, whereas protein oxidation has been considered to a much

41

lower extent, and mainly in meat 8. Protein oxidation results in a number of chemical modifications

42

affecting both amino acid side chains and the peptide backbone. Such changes include thiol

43

oxidation, aromatic hydroxylation, and formation of carbonyl groups9,10. Moreover, oxidation can

44

induce additional cross-links, backbone fragmentation and conformational changes in the secondary

45

and tertiary structure of the protein7,11. The formation of cross-links results in aggregation and

46

reduces solubility

47

protein oxidation in plant protein-based products is a rather new research topic, which is now highly

48

relevant given the increasing interest in the development and application of such products.

49

In fact, it can be hypothesized that different factors may induce protein oxidation in plant protein-

50

based fibrous structures: first, the process conditions, which often involves a thermomechanical

51

process; and second, the incorporation of nutritionally relevant micronutrients, such as iron, which

52

have a pro-oxidant activity. For instance, ferrous sulphate has commonly been used for food

12–14

. Despite the relevancy of such chemical modifications for food quality,



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fortification due to its high bioavailability and low cost15, but its presence in food products can lead

54

to lipid and protein oxidation. Besides, ferrous sulphate has an unpleasant metallic taste. To

55

mitigate those undesirable effects, iron encapsulation could be a solution. Previous research has

56

shown that encapsulating ferrous sulphate in pea protein spray-dried particles is a promising

57

method, considering iron bio-accessibility16 and the potential to mask the metallic taste16,17.

58

Moreover, the incorporation of such ferrous sulphate pea protein spray-dried particles (here referred

59

to as iron-loaded pea protein particles) in cooked black beans17 and in a banana candy 16showed a

60

good consumer acceptability. It should be noted, though, that the iron-loaded pea protein particles

61

were incorporated after the food was processed, which means that the iron-loaded pea protein

62

particles were not exposed to further food process conditions. Therefore, it would be relevant to

63

assess if additional processing can lead to undesirable chemical reactions, such as protein oxidation,

64

when such particles are exposed to process conditions such as high temperatures, which can be

65

necessary to make a food product.

66

In this work, we investigated protein oxidation in a plant protein-based fibrous product. We aimed

67

to assess the effect of process conditions used to make fibrous product, and quantified the effect of

68

incorporation of iron-loaded pea protein particles on protein oxidation.

69 70

2.

Material and methods

71

2.1.

Materials

72

Pea protein concentrate (PPC, 81% protein, N x 6.25) was obtained from NUTRALYS (S85F)

73

(Roquette, The Netherlands). Soy protein concentrate (SPC, 63.20% protein, N x 5.71) (Alpha 6

74

ZP) was obtained from Solae (Europe S.A). Food grade ferrous sulphate heptahydrate, 2-propanol,

75

sodium chloride (NaCl, S9625), petroleum ether, diaminoethane tetraacetic acid (EDTA),

76

tris(hydroxymethyl) aminomethane (Tris), potassium chloride (KCl), 2,4-dinitrophenylhydrazine

77

(DNPH), trichloroacetic acid (TCA), sodium dodecyl sulphate (SDS) and guanidine hydrochloride

78

(CH5N3HCl) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Hydrochloric acid (HCl) 4 4 ACS Paragon Plus Environment

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N was purchased from AVS TITRINORM (VWR Chemicals, USA) and solvents such as ethanol

80

(ACS 99%) and ethyl acetate (ACS 99%) were purchased from Emsure (Merck Millipore, The

81

Netherlands). Bicinchoninic acid (BCA) Protein Assay kit was obtained from Thermo Scientific

82

(Piercetm, The Netherlands). Demineralized water was used to make the feed solution and to prepare

83

the soy protein matrix. Ultrapure water obtained from a Millipore Milli-Q system (Darmstadt,

84

Germany) was used for all the other experiments.

85 86

2.2.

Methods

87

2.2.1.

88

Iron-loaded pea protein particles were produced according to Bittencourt et al.16 with a few

89

modifications. A feed solution (6.34% solids content) with PPC as the wall material and ferrous

90

sulphate heptahydrate as a core material was prepared. First, 10 w/v% PPC was slowly added to

91

distilled water and the pH was adjusted to 7.0 with 1 M HCl. Then, the feed solution was heated to

92

80 °C for 30 min in a water bath. The feed solution was then homogenized with an IKA® T18 Ultra-

93

Turrax (Thermo Fisher Scientific, Inc., Waltham, MA, US) at 8000 rpm for 5 min and left overnight

94

in a shaking water bath at 40 °C and 100 rpm. The next day, the feed solution was diluted to 9

95

w/v% and homogenized at 9000 rpm for 1.5 min. The pH was adjusted to 6.5 with 1 M HCl. A 1 M

96

ferrous sulphate solution was prepared with demineralized water and was slowly stirred in the feed

97

solution to obtain a final concentration of

98

homogenized at 9000 rpm for 2 min. A final dilution was made to 7 w/v% protein and stirred. The

99

feed solution was then spray dried using a Mini Spray Dryer Büchi B-290 (BÜCHI Labortechnik

100

AG, Flawil, Switzerland) with an inlet temperature of 180 °C and an outlet temperature of around

101

100 °C. The setting was: aspirator at 90%, pump at 20% and feed rate of 6 mL/min. The samples

102

were stored in plastic vials, in the dark, and frozen prior to further analysis. Spray dried particles

103

without iron were prepared following the same procedures as described above, except that the

104

ferrous sulphate solution was replaced by demineralized water. The yield defined as the ratio solid

Production of iron-loaded pea protein particles

22.5 mM. Subsequently, the feed solution was



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in particles and solids in feed solution was 54.5 ± 2.4% for samples prepared with iron and 62.3 ±

106

0.9% without iron.

107 108

2.2.2. Exposure of iron-loaded pea protein particles to high temperatures

109

Iron-loaded pea protein particles were exposed to different temperatures in a thermostat oven

110

HeraThermtm (Thermo Fisher Scientific, Inc., Waltham, MA, US). Two grams of sample was

111

evenly spread as a thin layer in an aluminum tin, after which the tin was put in the oven at 100 ˚C or

112

140 ˚C for 30 min. These temperatures were chosen according to the temperatures applied to make

113

plant protein-based fibrous product (section 2.2.4). Pea protein particles samples without exposure

114

to temperature were considered as a control. Afterward, the samples were analyzed for

115

physicochemical characterization.

116 117

2.2.3. Physical characterization of iron-loaded pea protein particles

118

The morphology of iron-loaded pea protein particles was analyzed by scanning electron microscopy

119

(SEM). Samples were deposited on carbon conductive tape on aluminum SEM stubs and analyzed

120

using a Phenom G2 Pure ‘Semseo’ microscope (Thermo Fisher Scientific, Inc., Waltham, MA, US).

121

SEM pictures were analyzed with the ImageJ software (1.40g, National Institute of Health,

122

Bethesda, MD, US) to calculate the content of broken particles.

123

Particle size distribution (PSD) was determined by laser diffraction particle size analyzer

124

(Mastersizer 2000, Malvern Instruments Ltd., UK) using 2-propanol as the continuous phase,

125

particle refractive index of 1.45, particle absorption index of 0.001 and dispersant refractive index

126

of 1.39. Samples were gently agitated to ensure homogeneity before the measurement.

127

Particles were analyzed for morphology and PSD just after spray drying, and after being exposed to

128

temperature.

129

Iron retention in the particles was determined after spray drying, and after being exposed to

130

temperature using an Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES, iCAP 6 ACS Paragon Plus Environment

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6500 duo, Thermo Fisher Scientific, Inc., Waltham, MA, US) method. First, 1 w/v% samples were

132

suspended in 10 mL aqua regia (7.5 mL HCL 37% and 2.5 mL HNO3 65%) and the organic phase

133

was digested in a microwave digestion system (Milestone Srl, Sorisole, Italy). After microwave, the

134

samples were diluted 200x. A calibration curve was prepared with an iron stock solution in aqua

135

regia and within a range that went up to 150 mg/L. The samples were measured in the axial

136

direction and the wavelength of 240.88 nm was used for iron quantification. Iron retention was

137

calculated by the following equation:

138 139

% =

100

eq. 1

140 141

2.2.4. Preparation of a plant protein-based fibrous product

142

The fibrous product was prepared using the high temperature shear cell, HTSC (Wageningen, NL)

143

with SPC as the protein source. Samples were prepared with iron-loaded pea protein particles,

144

ferrous sulphate (consider as free iron) solubilized in the water used to make the fibrous product,

145

and no iron.

146

The fibrous product was composed of 45 wt% SPC, 54 wt% demi water, 1% NaCl and 3.5 mg of

147

elemental iron per 100 g of product (except for the control with no iron). The iron content was

148

based on 25% of the Nutrient Reference Value for female adults between 19-50 years, which is a

149

high risk group for iron deficiency18. Per run, 90 g of total weight were used. The model system was

150

prepared according to Grabowska et al.5 at 100 °C or 140 °C, 30 rpm for 15 min, followed by a

151

cooling step at 25 °C for 5 min. After preparation, samples were prepared by cutting small pieces of

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8 mm out of the product. The pieces were mixed to obtain a homogeneous distribution. Then, 9 g of

153

samples were placed in a sous-vide bag and sealed using a V.300 Premiun Line vacuum machine

154

(Lava, Bad Saulgau, Germany). Bags were placed in the oven at 35 °C for accelerated storage

155

stability analysis and analyzed after 1 and 7 days.

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2.2.5. Quantification of protein-bound carbonyls: DNPH method

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We first measured the carbonyl content in pea protein particles prepared with iron and without iron

159

after exposure to 100 and 140°C. To investigate the effect of processing and iron incorporation, we

160

quantified the carbonyl content in fibrous products prepared with iron-loaded pea protein particles,

161

ferrous sulphate solution and without iron. As a reference, the carbonyl content was measured in a 6

162

wt% protein suspension of PPC and SPC.

163

The carbonyl content was measured using the method described by Soglia et al.12, with

164

modifications to account for the low protein solubility of the samples (pea protein particles and

165

fibrous products). Therefore, the samples were sequentially dispersed in buffers, which resulted in

166

three different protein fractions: in fraction 1 the most soluble proteins were dissolved, in fraction 2

167

the salt-soluble proteins and in fraction 3 some less soluble proteins (Figure 1)12.

168

The following procedure was applied to the pea protein particles samples. First, 2 g of sample was

169

dispersed in 20 mL buffer 1 (100 mM Tris and 5 mM EDTA pH 7.5) and left stirring overnight at

170

300 rpm at 4 °C. The samples were then centrifuged at 18,000×g at 2 °C for 20 min (Sorval Lynx

171

4000, Thermo Fisher Scientific, Inc., Waltham, MA, US). The supernatant was collected as protein

172

fraction 1. Next, buffer 2 (100 mM Tris, 50 mM NaCl and 5 mM EDTA pH 7.5) was added to the

173

pellet (1:3, w/v) and vortexed for 30 s at 2500 rpm. The homogenate was centrifuged at 18,000×g at

174

2 °C for 20 min. The supernatant was collected as protein fraction 2. Further, 0.15 M KCL solution

175

was added to the pellet (1:3, w/v) and homogenized as described before, forming a suspension

176

which constituted protein fraction 3.

177

For the fibrous product, 9 g (9.5 w/v% protein) sample were homogenized with buffer 1 (1:3, w/v)

178

using an Ultra Turrax at 13600 rpm for 1 min in an ice bath. Samples meant to determine the effect

179

of seven days of storage were taken one day before measurement and soaked in buffer 1 at 4 °C

180

overnight. The dispersion was centrifuged at 18,000×g at 2 °C for 20 min. The supernatant

181

collected was protein fraction 1. Next, buffer 2 (1:3, w/v) was added to the pellet and homogenized

182

for at 13,600 rpm 30 s in an ice bath. Afterward, the sample was centrifuged as described before. 8 ACS Paragon Plus Environment

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The supernatant was collected as protein fraction 2. Further, 0.15 M KCL solution was added to the

184

pellet (1:3, w/v) and homogenized as described before, forming a suspension which constituted

185

protein fraction 3.

186

We attempted to accordingly analyze those three protein fractions from pea protein particles and

187

fibrous products suspensions. However, the soluble protein concentration was less than 1 g/L in

188

protein fraction 3 for pea protein particles, and in protein fraction 2 for fibrous products. For this

189

reason, for the quantification of protein-bound carbonyls, only protein fraction 1 and 2 were

190

considered for pea protein particles, and only protein fractions 1 and 3 for fibrous products. The

191

soluble protein concentration was determined in each fraction by the BCA method. Protein fractions

192

were filtered with a 0.22 µm syringe filter (Millex PES, Merck Millipore, Burlington, MA, US).

193

Then 1 mL of working reagent (50:1 reagent A/B) was added to 50 µm of samples and were

194

incubated in a Thermomix at 37 °C and 300 rpm for 30 min. After incubation, samples were cooled

195

down and kept at 4 °C for 5 min and at room temperature for 10 min. Calibration curves (0 to 1 g/L)

196

with bovine serum albumin were prepared in the buffers used to separate the protein fractions and

197

were prepared under the same conditions as the samples. Then, the absorbance was measured at 562

198

nm with a UV-visible spectrophotometer (HACH Lange DR 3900, Tiel, the Netherlands) and

199

polystyrene cuvettes. To calculate the percentage of soluble protein concentration in each fraction,

200

we considered that all proteins in the fibrous product and pea particles were solubilized (Figure 1).

201

Carbonyl content was determined with DNPH method adapted from Soglia et al.12, Vuorela et al.19

202

and Levine et al.20. First, 0.8 mL of protein fraction 1 or 1 mL of protein fraction 2 from pea protein

203

particles samples were precipitated with 40% TCA (1:1, v/v). For fibrous product, 1 mL of protein

204

fractions 1 and 3 were precipitated with 20% TCA (1:1, v/v). Then, samples were centrifuged at

205

15,000×g for 5 min. The supernatant was removed with a Pasteur pipette and 400 µL of 5 w/v%

206

SDS was added to the pellet. The samples were heated in a thermomixer (Eppendorf ThermoMixer

207

C & Grant QBT4, Hamburg, Germany) at 99 °C for 10 min, then put in an ultrasonic bath

208

(Elmasonic P & Branson 5210, Elma, Singen, Germany) at 40 °C for 30 min using 80 kHz, and 9 ACS Paragon Plus Environment


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90% sweep. Afterward, 0.8 mL of 0.3 w/v% DNPH in 3 M HCl was added to the samples and 0.8

210

mL of 3 M HCl to the blanks. Then, the samples were incubated at room temperature in the dark for

211

60 min and vortexed at 2500 rpm for 5 s every 10 min. After incubation, 400 µL of 40 w/v% TCA

212

was added to precipitate the proteins, and the samples were centrifuged at 1,5000×g for 5 min. The

213

supernatant was removed and the samples were washed three times with 1 mL ethanol/ethyl acetate

214

(1:1) and centrifuged at 1,5000×g for 5 min every time. After the washing steps, the pellet was

215

dissolved in 1.5 mL of 6 M guanidine hydrochloride and vortexed at 2500 rpm for 5 s. Samples

216

were incubated in a thermomixer at 37 °C overnight.

217

Then, the samples were centrifuged at 5000×g for 10 min and the absorbance of the supernatant was

218

measured at 370 nm, using a UV-visible spectrophotometer and polystyrene cuvettes. Samples with

219

absorbance outside the range of 0.12-0.55 were diluted in 6 M guanidine hydrochloride. The soluble

220

protein concentration in 6 M guanidine hydrochloride was determined by BCA method, as

221

described previously. Carbonyl content was calculated following the equation:

222

((

"#$%&'

)

*+,-./012 3 *+,41.56 7 = 89

eq. 3

223 224

Where ABS is the absorbance of the samples and of the blank at 370 nm, and : is the molar

225

absorptivity coefficient of carbonyls, set as 22000 M-1cm-1.

226 227

2.3. Statistical analyses

228

Pea protein particles samples were prepared in duplicates. Samples exposed to temperatures were

229

prepared in duplicate and used immediately. The sample was measured twice with an average of 3

230

readings from the Mastersizer to determined particle size distribution. Fibrous products were

231

prepared in duplicates and the DNPH method was done in triplicate per sample and per blank. The

232

BCA assay was done in triplicates.

233

Statistical analysis was done using the Statistical Package for the Social Sciences (SPSS software v.

234

23 (IBM Inc., Armonk, NY, US). Analysis of variance (ANOVA) with a post hoc Turkey test was 10 ACS Paragon Plus Environment

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performed to compare means of particle diameter size from pea protein particles samples treated at

236

different temperatures and to compare means of carbonyl content (pea protein particles and fibrous

237

product) between samples. The one-sample T-test was performed to compare the means of samples

238

prepared with iron and no iron exposed to the same temperature (p > 0.05).

239 240

3. Results and discussion

241 242

In this section, the properties of pea protein particles obtained after spray drying are first

243

characterized, followed by a description of the chemical stability of the particles. Then, the effect of

244

particle addition to fibrous product and the effect of thermal treatment on protein oxidation is

245

presented and discussed.

246 247

3.1. Characterization of iron-loaded pea protein particles

248

The iron retentions of the iron-loaded pea protein particles after spray drying and exposed to 100 °C

249

and 140 °C were: 59.86 ± 0.91%, 61.45 ± 0.5 and 61.71 ± 0.58%, respectively. Our results of the

250

iron retention were lower than the value of 67% described by Ferreira (2014)21, but higher than the

251

value of 43.9% described by Bittencourt et al.16. The differences are probably due to adaptations in

252

spray drying setting.

253

The morphology of iron-loaded pea protein particles was monitored just after particle preparation

254

and after exposure to 100 °C and 140 °C for 30 min (Figure 2). Iron-loaded pea protein particles

255

presented more invaginations, were less spherical and had a smoother surface compared to pea

256

protein particles without iron. Pea protein particles exposed to 100 °C and 140 °C looked more

257

broken than the control samples. The roughness and invaginations found in our pea protein particles

258

have been described by other authors as typical for particles made with PPC as encapsulation

259

material22,23. The authors argued that a high protein content can produce spray dried particles with

260

less invagination, suggesting that the presence of carbohydrates in protein concentrates can result in 11 ACS Paragon Plus Environment


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more invaginations. In the present work, we can see that the presence of iron also affected the

262

particle morphology.

263

Directly after spray drying, the PSD of pea proteins particles with and without iron (Figure 3A)

264

showed 3 peaks: around 1 µm, 0.18 µm, and 10 µm. This result shows that pea protein particles

265

were polydisperse, independently of the presence of iron. After exposure to high temperature, larger

266

particle sizes were measured (Figure 3B,C). Especially in case of iron-loaded pea protein particles,

267

aggregation was observed after heating at 140 °C. Several physical and chemical mechanisms,

268

including protein oxidation induced by iron and heat, could cause protein aggregation seen in

269

Figure 3C. The average particles sizes, d3,2, of iron-loaded pea protein particles exposed to 140 °C

270

(1.69 ± 0.17 µm) or 100 °C (0.78 ± 0.32 µm) were larger than those of the sample not exposed to

271

heat (0.37 ± 0.03 µm). The span values also showed an increase in polydispersibility: 8.33 ±. 014

272

(no heat) to 35.84 ± 14.38 (iron-loaded pea protein particles at 140 °C).

273 274

3.2 Chemical characterization of iron-loaded pea protein particles: protein oxidation

275

Carbonyl content was determined as a measure of protein oxidation to assess the chemical stability

276

of iron-loaded pea protein particles being exposed to 140 °C and 100 °C for 30 min (Figure 4 A, B).

277

Here we present the results of carbonyl content in protein fractions 1 and 2 only, due to the fact that

278

the soluble protein concentration was too low in fraction 3.

279

In both protein fractions (Figure 4 A, B), the carbonyl content was higher after spray drying than in

280

the starting PPC suspension: for the latter, values of about 12.7 mmol carbonyl per kg soluble

281

protein (protein fraction 1) and 9.7 mmol carbonyl per kg soluble protein (protein fraction 2) were

282

obtained, represented as dotted lines in graphs Figure 4 A,B. After expose of the pea protein

283

particles to high temperature (100 or 140 °C for 30 min), there was an increase in carbonyl content

284

for both protein fractions, without significant difference between both heat treatments (Figure 4A).

285

The presence of iron in the pea protein particles clearly decreased protein solubility, which in fact

286

could be due to protein aggregation as a result of protein oxidation. However, we could not see any 12 ACS Paragon Plus Environment

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increase in carbonyl content compared to pea protein particles without iron. Considering that only a

288

small fraction of the protein in the iron-loaded pea protein particles was soluble in guanidine

289

hydrochloride, most probably there were more carbonyls formed than in particles without iron. For

290

protein fraction 2, iron-loaded pea protein particles exposed to 140 °C had significantly higher

291

carbonyl content compared to samples exposed to 100 °C and to the control (Figure 4B). However,

292

there was no effect of temperature on carbonyl content for the pea protein particles without iron.

293

Protein oxidation values for iron-loaded pea protein particles were only significantly different from

294

those of the pea protein particles without iron in protein fraction 2 at 140 °C.

295

As described above, spray drying temperature (180 °C) alone induced protein oxidation in the PPC

296

used as a wall material to encapsulate iron. Costa (2014)24 showed that spray drying reduced free

297

sulfhydryl content in pea protein isolate, which could be an indication of protein oxidation.

298

Comparable to our findings, Tang et al.25 described a similar increase in carbonyl content when

299

heating soy proteins at 100 °C for 30-90 min compared to no heat treatment. However, the values

300

found were much lower than ours: ~3 mmol carbonyls per kg soluble protein for proteins not heated

301

and ~ 6 mmol carbonyls per kg soluble protein for soy proteins heated for 90 min. In our study,

302

PPC itself was already oxidized and then exposed first to spray drying temperature, and later to

303

further heat treatment, which also increased protein oxidation.

304

These results showed that spray drying leads to less chemically stable iron-loaded pea protein

305

particles and that the presence of iron reduces pea protein soluble concentration. Some

306

improvement might be necessary to increase the particles’ oxidative stability; the chemical status of

307

the starting protein material also seems important since the PPC powder was already oxidized,

308

which indicates that protein oxidation started either during protein fractionation process or during

309

storage of the powders.

310 311

3.3. Chemical stability of fibrous products with iron incorporation: protein oxidation



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Plant protein-based fibrous products prepared with a HTSC at different temperatures were used as

313

a model product to study fortification with iron either as free iron or encapsulated (iron-loaded pea

314

protein particles) as described in the previous sections. Sequential suspensions of the fibrous

315

products were prepared in different buffers, and 3 protein fractions were obtained. Protein fraction 1

316

and fraction 3 had more soluble proteins than fraction 2 and for this reason, carbonyl content was

317

only measured in fractions 1 and 3.

318

Figure 5 shows the carbonyl content in protein fractions 1 and 3 of fibrous products prepared at 100

319

or 140 °C. The presence and form of iron (free versus encapsulated) did not seem to substantially

320

affect the carbonyl formation in the fibrous product: the only significant difference was found in

321

protein fraction 1 between samples prepared at 140 °C. Carbonyl content increased significantly

322

over storage time (up to 168 h) in protein fraction 1 of samples prepared without iron (only at 100

323

°C) and encapsulated iron (at both temperatures). The effect of temperature was only pronounced in

324

protein fraction 1 for samples prepared with encapsulated iron after 24 h of storage.

325

Protein fraction 3 showed higher carbonyl content compared to protein fraction 1 of samples

326

prepared at both temperatures. Our results show that the process used to make the fibrous products

327

increased carbonyl content in this fraction, for both temperatures tested. Interestingly, protein

328

oxidation did not increase with the incorporation of free or encapsulated iron into the product. This

329

result was not expected since studies have shown that even low iron concentrations can increase

330

protein oxidation in meat, especially after heat treatments26. As an example, Promeyrat et al.

331

(2013)27 showed that heat treatment (45-90 °C) affected carbonyl content in myofibrillar proteins

332

suspension only weakly. However, in the presence of heat and a mix of pro-oxidants (iron,

333

hydrogen peroxide, and ascorbate) there was a synergistic effect on oxidation. In this study, iron

334

was applied without such synergistic pro-oxidant components, which suggested that iron alone is a

335

not a powerful pro-oxidant in these fibrous plant protein-based products. Besides, carbonyls are

336

often measured in only one protein fraction, using a salt-rich buffer (NaCl or KCl), to allow for the

337

extraction of myofibrillar proteins in meat products. 14 ACS Paragon Plus Environment

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338

The chemical status of the starting plant protein ingredient and the effect of processing on protein

339

oxidation in fibrous products was determined via the carbonyl content in the commercial SPC

340

powder (6 wt% protein suspension). We observed that all fibrous products had a higher carbonyl

341

content than the SPC suspension: the latter showed values about 15 mmol carbonyl per kg soluble

342

protein for protein fraction 1, and 20 mmol carbonyl per kg soluble protein for protein fraction 3.

343

For protein fraction 1 at day 0, all samples had similar carbonyl content compared to SPC

344

suspension. Conversely, protein fraction 3 had a substantially higher carbonyl content compared to

345

SPC suspension. This shows that protein fraction 3 was the most oxidized protein fraction and that

346

both temperatures applied in the HTSC increased the carbonyl content. We conclude that the

347

commercial SPC was readily oxidized and that when processes conditions are applied, such as

348

temperatures above 100 °C, the formation of carbonyls further increased. Chen et al.28 found quite

349

low carbonyl content of 5.78 mmol carbonyl per kg soluble protein, in non-commercial soy protein

350

isolate (SPI). Most non-commercial SPI and SPC are prepared by freeze-drying, while commercial

351

processes for SPC and SPI use spray drying29. As we showed before (section 3.2), spray drying

352

temperature induces protein oxidation. So far, there is no literature associating spray drying with

353

protein oxidation, but this process has been associated with other protein modifications, resulting in

354

protein denaturation, reduced protein solubility and altered surface hydrophobicity29.

355

The results on carbonyl content in SPC and PPC commercial powders, pea protein particles and in a

356

fibrous product shows the need to find: firstly, how protein oxidation starts during production of

357

the ingredients and how to control it; secondly, efficient antioxidant strategies for the production of

358

spray dried particles; and thirdly, a mild process to generate fibrous products. Protein oxidation is

359

an important parameter to control the quality and safety of food products. In the current study, the

360

addition of iron did not result in clear additional oxidation.

361 362

3.4 On the interpretation of the DNPH method results



Page 16 of 33

363

The protein oxidation values reported above should be interpreted carefully, because of the low

364

protein solubility of most samples. The average of soluble protein concentration was 4.2 ± 0.01 g/L

365

in buffer 1 (4.4%), and 2.9 ± 0.04 g/l in buffer 3 (3.1%) (Figure 1) for the fibrous products tested.

366

However, the soluble protein concentration in 6 M guanidine hydrochloride for protein fraction 1

367

was reduced to half (Figure 6). Protein loss during the washing steps of the DNPH method is

368

reported as expected, which increases the standard deviation and lowers the reproducibility30. In

369

samples with very low solubility, such as our fibrous product, we only measured carbonyls in a

370

small fraction of the total proteins and the extent of protein oxidation in the insoluble protein

371

fraction (i.e., the majority of proteins) remained unknown. Fibrous products were already quite

372

insoluble. That fact alone could be a sign of oxidation because protein oxidation induces protein

373

aggregation and can decrease solubility as a result of that.

374

The modifications included in the DNPH method have contributed to increase carbonyl detection,

375

especially in protein fraction 3. The improvement in DNPH was described by Soglia et al.12 as

376

beneficial to increase protein solubility and the exposure of carbonyls buried within the protein

377

structure, which resulted in a threefold higher carbonyl content compared to the traditional DNPH

378

method. Ooizumi and Xiong (2004)31 described the formation of oxidized aggregates as a reason of

379

decreased myosin solubility from chicken muscle. Even though the modifications in the DNPH

380

increased the detection of carbonyls, this method has some points of attention. The DNPH method

381

measures total carbonyl content and it is not possible to determine which amino acid was oxidized.

382

According to Estévez32 most of the carbonyls are derived from the oxidation of proline, threonine,

383

lysine, and arginine, which for instance are higher in soybeans (mg/100 g food) than in beef (except

384

proline)33.

385 386

Protein oxidation in fibrous products and in plant protein concentrate-based materials has been

387

shown to be a relevant measurement to investigate the chemical stability of these protein-rich

388

products when submitted to process conditions used to make meat analogue products. In the current 16 ACS Paragon Plus Environment

Page 17 of 33


389

study we found that spray drying increased protein oxidation in pea protein particles, with or

390

without iron. The presence of iron decreased pea protein solubility, which may be associated to

391

protein oxidation. Moreover, the starting PPC powder obtained from the manufacturer presented a

392

certain level of protein oxidation, which could be due to the protein fractionation process and the

393

storage conditions. In fibrous products, the presence of iron-loaded pea protein particles and free

394

iron did not increase protein oxidation in both process temperatures, 100°C or 140°C. However,

395

these high temperatures applied to make fibrous products increased substantially protein oxidation

396

compared to the SPC powder, which was already oxidized but in a lower extend.

397 398 399

Abbreviations used ABS

absorbance

BCA

bicinchoninic acid

DNPH

2,4-dinitrophenylhydrazine

:

molar absorptivity

EDTA

diaminoethane tetraacetic acid

HCL

hydrochloric acid

HTSC

high temperature shear cell

ICP-OES

inductively coupled plasma-optical emission spectroscopy

KCL

potassium chloride

NACL

sodium chloride

PPC

pea protein concentrate

PSD

particle size distribution

SDS

sodium dodecyl sulphate

SEM

scanning electron microscopy

SPC

soy protein concentrate 17 ACS Paragon Plus Environment


TCA

trichloroacetic acid

Tris

tris(hydroxymethyl) aminomethane

Page 18 of 33

400 401 402

Funding sources

403

P.D.E. is supported by a PhD scholarship grant from the Conselho Nacional de Desenvolvimento

404

Científico e Tecnológico (CNPq/Brazil), process number 233663/2014-2.

405 406

Notes

407

The authors declare no competing financial interest.

408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 18 ACS Paragon Plus Environment

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424 425 426 427 428

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Figure Captions: Figure 1. Schematic representation of the separation of protein fractions from fibrous product and the average of soluble protein concentration (g/L) per supernatant. Figure 2. Scanning electron microscopy of pea protein particles with and without iron after spray drying (A and B), after exposure to 100 ºC (C and D) and to 140 ºC (E and F) for 30 min. Magnification x 2500. Figure 3. Particle size distribution of pea protein particles prepared with and without iron, just after spray drying (A) and after exposure to 100 °C (B) and 140 °C (C) for 30 min. For clarity only one representative curve is shown per sample, but similar trends were observed for the replicates (n = 2 independent replicates, 2 measurements per sample). Figure 4. Carbonyl content (mmol/kg soluble protein) and soluble protein concentration (g/L) in 6 M guanidine hydrochloride for iron-loaded pea protein particles (Fe, dashed bars), and control without iron (No Fe, white bars). A: carbonyl content in protein fraction 1, B: carbonyl content in protein fraction 2, C: soluble protein concentration in protein fraction 1 and D: soluble protein concentration in protein fraction 2. The dotted line represents the carbonyl content for the starting PPC suspension. Results are expressed as mean and error bars as standard deviation. A significant difference (p < 0.05) at different temperatures for each sample is expressed as *. Different letters mean significant difference (p < 0.05) between samples with no iron and iron. Figure 5. Carbonyl content (mmol/kg soluble protein) in protein fractions 1 and 3 of fibrous products prepared at 100 ºC and 140ºC at 30 rpm for 15 min. Samples were prepared with no iron (No Fe, white bars), free iron (Fe, dashed bars) and iron-loaded pea protein particles (Fe-pea protein particles, black bars). A: protein fraction 1 at 100 ºC; B: protein fraction 1 at 140 ºC; C: protein 23 ACS Paragon Plus Environment


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fraction 3 at 100 ºC; D: protein fraction 3 at 140 ºC. The dotted line represents the carbonyl content for the starting SPC suspension. Results are expressed as mean and error bars as standard deviation (n = 2 independent replicates, 3 measurements per sample). Different letters indicate significant differences between samples within the same storage time. The data series that do not include letters did not show any significant difference (p

Protein Oxidation in Plant Protein-Based Fibrous Products: Effects of

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