Study on the emulsifying stability and interfacial adsorption of pea proteins

Accepted Manuscript Study on the emulsifying stability and interfacial adsorption of pea proteins

Maoshen Chen, Juhui Lu, Fei Liu, John Nsor-Atindana, Feifei Xu, H. Douglas Goff, Jianguo Ma, Fang Zhong PII:

S0268-005X(18)31067-1

DOI:

10.1016/j.foodhyd.2018.09.003

Reference:

FOOHYD 4640

To appear in:

Food Hydrocolloids

Received Date:

11 June 2018

Accepted Date:

03 September 2018

Please cite this article as: Maoshen Chen, Juhui Lu, Fei Liu, John Nsor-Atindana, Feifei Xu, H. Douglas Goff, Jianguo Ma, Fang Zhong, Study on the emulsifying stability and interfacial adsorption of pea proteins, Food Hydrocolloids (2018), doi: 10.1016/j.foodhyd.2018.09.003

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ACCEPTED MANUSCRIPT

Study on the emulsifying stability and interfacial adsorption of pea proteins

Maoshen Chena,b, Juhui Lua,b, Fei Liua,b, John Nsor-Atindanaa,b, Feifei Xua,b, H. Douglas Goffc, Jianguo Maa,b, Fang Zhonga,b*

a State

Key Laboratory of Food Science and Technology，Jiangnan University, Wuxi

214122, China. b

School of Food Science and Technology, Jiangnan University, Wuxi 214122, China.

c

Department of Food Science, University of Guelph, Guelph, ON N1G 2W1, Canada.

ACCEPTED MANUSCRIPT

1

Study on the emulsifying stability and interfacial adsorption of

2

pea proteins

3 4

Maoshen Chena,b, Juhui Lua,b, Fei Liua,b, John Nsor-Atindanaa,b, Feifei Xua,b, H.

5

Douglas Goffc, Jianguo Maa,b, Fang Zhonga,b*

6 7

a State

8

214122, China.

9

b

School of Food Science and Technology, Jiangnan University, Wuxi 214122, China.

10

c

Department of Food Science, University of Guelph, Guelph, ON N1G 2W1, Canada.

Key Laboratory of Food Science and Technology，Jiangnan University, Wuxi

11 12 13

*Corresponding author：Fang Zhong

14

Tel: +86-510-85197876

15

Email address: [email protected]

1

ACCEPTED MANUSCRIPT 16

Abstract:

17

In order to expand the natural food emulsifier applications of pea proteins, the

18

emulsifying stability and competitive relationship of interfacial adsorption were

19

investigated. The protein extracted at 90 oC had the highest nitrogen solubility index

20

and surface hydrophobicity. The molecular weight distributions analyzed by high-

21

performance size exclusion chromatography demonstrated that over 60% of the

22

protein had Mw > 500 kDa. The non-reducing SDS-PAGE results showed that the

23

percentage of aggregates were about 37%, and the proportion of proteins were

24

aggregates > vicilin > legumin > convicilin. Increasing the protein concentration from

25

1.0 to 30 mg/mL increased the emulsifying ability and stability of pea protein

26

stabilised emulsions significantly. At the same time, the concentration of interfacial

27

adsorbed proteins increased. However, the ratio of adsorbed protein to the protein in

28

the initial dispersion (AP%) was decreased significantly from 84% to 21%. When the

29

protein concentration was higher than 10 mg/mL, the interfacial adsorption of pea

30

proteins would reach the saturated adsorption point. The content of aggregates

31

adsorbed onto the interface at low concentration was higher than its proportion in the

32

initial pea protein. With the increase of protein concentration at the interface, the

33

proportion of adsorbed aggregates decreased, while the proportions of vicilin and

34

legumin increased. At saturated adsorption, the contents of proteins on the interface

35

were vicilin > legumin > aggregates > convicilin, and the proportion of aggregates

36

was lower than its proportion in the initial pea protein extraction.

37

Keywords: Pea proteins, Emulsion, Emulsifying property, Interfacial adsorption 2

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1. Introduction

39

Pea (Pisum sativum) is one of the most important legumin plants in the word, and is

40

rich in starch and protein. Pea seeds contain more than 50% starch and 20%-30%

41

protein (Aluko, Mofolasayo, & Watts, 2009; Gueguen, 1983; Koyoro & Powers,

42

1987). Because of its high starch content, pea is mainly used to produce pea starch

43

products, such as bean vermicelli. As the by-product of pea starch, pea protein is a

44

valuable protein source with a well-balanced amino acid profile and rich in lysine

45

(Schneider & Lacampagne, 2000). However, compared with soybean proteins, the

46

application of pea proteins is limited by its functional properties (Shand, Ya,

47

Pietrasik, & Wanasundara, 2007). Thus, pea proteins are a waste by product and

48

mainly used as the protein source for animal fodders.

49

Commercially, pea seeds are processed by physical cleaning, wet milling,

50

kneading, separation (to extract pea starch), alkali extraction, acid precipitation and

51

spray drying to obtain pea proteins. Other extraction methods, such as salt extraction-

52

dialysis and micellar precipitation to obtain less denaturation proteins with better

53

solubility, emulsifying property and foaming property have been reported (J-L

54

Mession, Assifaoui, Cayot, & Saurel, 2012; Stone, Karalash, Tyler, Warkentin, &

55

Nickerson, 2015). Limited by the cost of large scale production, the alkali extraction,

56

acid precipitation and spray drying are still the main methods used to extract and

57

produce pea proteins. However, high temperature of spray drying above the

58

denaturation temperature usually causes partial unfolding and subsequent aggregation

59

of protein (Wang, et al., 2012). 3

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The emulsifying property is one of the significant functional properties of pea

61

proteins. Knowledge of emulsifying properties will broaden the application of pea

62

proteins in food systems as a natural emulsifier. The emulsifying property of protein

63

is affected by many factors, including the protein structure, composition, as well as

64

environmental conditions, such as protein concentration, pH, ionic strength，the

65

volume fraction of oil phase and pretreatment temperature (Kulmyrzaev, Chanamai,

66

& McClements, 2000; Lakemond, de Jongh, Hessing, Gruppen, & Voragen, 2000).

67

Heat treatment can expose the hydrophobic groups inside the protein molecules and

68

improve the emulsifying property of soy protein isolate (Corredig, 2009). Meanwhile,

69

thermal treatment would increase the extent of protein aggregation of pea proteins

70

significantly (Peng, et al., 2016).

71

It has been reported that the major storage proteins in pea proteins are globulins,

72

including legumin and vicilin (Klassen & Nickerson, 2012; Tzitzikas, Vincken, de

73

Groot, Gruppen, & Visser, 2006). At present, in order to investigate the effects of

74

composition and structure on the emulsifying properties of pea proteins, many studies

75

have been concentrated on the emulsifying property of vicilin and legumin. The

76

results have found that vicilin and legumin had significant effects on the emulsifying

77

properties (Castellani, Belhomme, David-Briand, Guérin-Dubiard, & Anton, 2006;

78

Rangel, Domont, Pedrosa, & Ferreira, 2003). Vicilin was shown to be a better

79

emulsifier than legumin and is a smaller more flexible molecular size. Pea proteins

80

containing more vicilin demonstrated better emulsifying property (Dagorn‐Scaviner,

81

Gueguen, & Lefebvre, 1987; Kimura, et al., 2008). The effects of protein aggregates 4

ACCEPTED MANUSCRIPT 82

on the emulsifying ability and stability of the emulsions and the competitive

83

adsorption at interface of individual protein have been extensively studied in milk

84

protein and other proteins (Ye, 2008, 2011). Although aggregates make up the highest

85

proportion of pea proteins, their effects on the emulsifying ability and stability have

86

been ignored. Few studies have focused on the competitive relationship of interfacial

87

adsorption between the different protein subunits including their aggregates of pea

88

protein, which is important to understand the molecular mechanism of emulsifying

89

property.

90

In this work, the effects of pretreatment temperature on the nitrogen solubility

91

index and surface hydrophobicity of two pea proteins were investigated. Meanwhile,

92

the molecular weight distribution and composition of two pea proteins extracted at 90

93

oC

94

dodecyl sulfate-polyacrylamide gel electrophoresis, respectively. The effects of pea

95

protein concentrations on the emulsifying ability and stability were evaluated.

96

Percentage and composition of adsorbed proteins in emulsions were further analyzed

97

to understand the competitive relationship of interfacial adsorption between the

98

different protein subunits.

99

2. Materials and methods

100

were studied by high-performance size exclusion chromatography and sodium

2.1 Materials

101

Pea protein powder, PPI 1 (Nutralys, S85F) and PPI 2 (F85M) was obtained from

102

RoquetteFrères (S.A., Lestrem, France).Soybean oil was purchased from Yihai Kerry

103

(China). All other reagents and chemicals were all analytical grade. 5

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2.2. Chemical composition

105

Total moisture and ash content of pea protein powder were evaluated according to

106

AOAC (1990). Protein determination was performed by Kjeldahl analysis (N%*6.25)

107

according to AOAC Official Method. Carbohydrate determination was performed

108

according to phenol-sulfuric acid colorimetric method. All measurements were

109

performed at least three replicates. The contents of protein, ash, lipid and

110

carbohydrate of PPI 1 were 82%, 5.6%, 0.36% and 12%. The contents of protein, ash,

111

lipid and carbohydrate of PPI 2 were 84%, 5.0%, 0.34% and 11%.

112

2.3 Determination of protein solubility and extraction of soluble pea proteins

113

Protein solubility was determined by mixing 1.60 g of pea protein powder with 20

114

mL 10 mM pH 7.0 phosphate buffered solution (PBS) and stirring in the water bath

115

for 60 min at 30 – 90 oC. After extraction, the solution was separated by

116

centrifugation at 1600 g/min for 15 min. The protein content in the supernatant was

117

measured by Kjeldahl analysis (N%×6.25).

118

2.4 Surface hydrophobicity

119

Protein dispersions were prepared with 10 mM pH 7.0 PBS solution to a serial

120

concentration of 0.02 mg/mL to 0.20 mg/mL. An aliquot (50 μL) of 1-anilino-8-

121

naphthalenesulfonate (ANS) solution (8 mM ANS in 10 mM pH 7.0 PBS solution)

122

was added to 4 mL of each dilution. The relative fluorescence intensity was measured

123

at 390 nm (excitation) and 470 nm (emission) using an F7000 fluorescence

124

spectrophotometer (Hitachi Co., Japan). The slit width of both was 5 nm. The index of

125

surface hydrophobicity was expressed as the initial slope of the plot of fluorescence

126

intensity versus protein concentration (Haskard & Li-Chan, 1998). 6

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2.5 Determination of molecular weight distribution

128

The molecular weight distribution of pea proteins was measured by high-

129

performance size exclusion chromatography (HPSEC) (Jung & Wicker, 2014). The

130

HPSEC system consisted of the Waters 1525 liquid chromatography and a multiangle

131

laser-light scattering detector (MALLS, Dawn HELEOS-II, Wyatt Technology). The

132

Ultrahydrogel™ 2000 column (7.8 mm × 300 mm) and Ultrahydrogel™ 500 column

133

(7.8 mm × 300 mm) were used. The supernatants of pea proteins were diluted to 1.0

134

mg/mL and passed through 0.45 μm filters. The elution was performed with 50 mM

135

pH 7.0 PBS solution containing 0.15 M NaCl and 0.02% (w/v) sodium azide at a flow

136

rate of 0.5 mL/min. The absorbance was monitored at 280 nm. Bovine serum albumin

137

(BSA) solution (5.0 mg/mL) with the Mw of 67 kDa was used for calibration. The

138

data was analyzed by the ASTRA software while the dn/dc value of 0.180 was used in

139

the calculations (Jung & Wicker, 2014). The percentage of each fraction was

140

calculated as % area relative to the 100% integrated area of the total spectrum.

141

2.6 Protein composition

142

Protein composition of pea proteins was determined by sodium dodecyl sulfate-

143

polyacrylamide gel electrophoresis (SDS-PAGE). The concentration of the stacking

144

and separating gels were 4.0% and 12.5%. Pea protein powder was dissolved in the

145

sample buffer in the presence (reducing conditions) or absence (non-reducing

146

conditions) of 5% (v/v) 2-mercaptoethanol (β-ME). After heating for 5 min in a

147

boiling water bath and centrifuging at 3500 g/min for 5 min, each sample (7.5 μL)

148

was loaded to a cell. The gel was stained with Coomassie Brilliant Blue (G-250) and 7

ACCEPTED MANUSCRIPT 149

scanned using a computing densitometer (Molecular Imager ChemiDocXRS+, Bio-

150

Rad, USA). The intensities of bands were integrated using Image Lab software (Bio-

151

Rad, USA).

152

2.7 Emulsion preparation

153

The pea protein solution was diluted from 1 to 30 mg/mL. The suspension was pre-

154

homogenized with soybean oil at oil fraction of 10% (w/w) using a high-speed

155

dispersing and emulsifying unit (model IKA-ULTRA-TURRAX® T25 basic, IKA®

156

Works, Inc., Wilmington, NC) at 10,000 rpm for 2 min, then immediately

157

homogenized through a homogenizer (Panda PLUS 2000, GEA NiroSoavi, Inc., Italy)

158

for 2 passes with overall pressure at 45 MPa. The sodium azide was added into

159

emulsions as an antimicrobial agent at the concentration of 0.02% (w/v).

160

2.8 Determination of particle size distribution and flocculation index

161

The particle size distribution of oil droplets in emulsions was determined by the

162

Laser Particle Size Analyzer (Mastersizer 2000, Malvern Instruments, Malvern, UK),

163

using deionized water or 1% (w/v) sodium dodecyl sulfate (SDS) solution as the

164

dispersant. The relative refractive index of emulsions was taken as 1.107, that is, the

165

ratio of the refractive index of soybean oil (1.472) to that of the continuous phase

166

(1.330). The droplet size measurement was reported as the volume-average diameter

167

(d43) and the surface-average diameter (d32). The flocculation index (FI) of emulsions

168

was calculated as follows (Castellani, et al., 2006):

169 170

FI = (d43 in water)/(d43 in 1% SDS)-1 2.9 Determination of emulsifying stability 8

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The heat stability of emulsions was determined by the average diameter of oil

172

droplets after heat treatment of emulsions at 90 oC for 30 min. Besides, fresh

173

emulsions (15 mL) were added into sample bottles and then stored vertically at 25 oC.

174

The average diameter of emulsions was measured after storage of 7 days.

175

2.10 Determination of percentage of adsorbed proteins and interfacial protein

176

concentration

177

Percentage of adsorbed proteins (AP%) and interfacial protein concentration (Γ) of

178

various fresh emulsions were determined using the method described by Liang et al.

179

(Liang & Tang, 2013) with slight modifications. Each fresh emulsion (1 mL) was

180

centrifuged at 7500 g/min for 30 min. After the centrifugation, two phases were

181

observed: cream layer (or concentrated oil droplets) at the top of the tube and aqueous

182

phase of the emulsion. The cream layer was carefully removed using a syringe, and

183

the supernatant was filtered through a 0.22 mm filter (Millipore Corp.). Protein

184

concentration of the filtrate was determined with the Lowry method using BSA as the

185

standard. The AP (%) and Γ (mg/m2) was calculated as:

186

AP= (C0 - Cs)/ C0×100%

187

Γ= (C0 - Cs) (1-Φ) (d32 in 1% SDS)/(6Φ)

188

Where C0 was the protein concentration in the initial pea protein dispersion, Cs was

189

the protein concentration of the unadsorbed layer, Φ is the volume fraction of the oil

190

phase.

191

2.11 Determination of interfacial protein composition

192

The composition of proteins adsorbed or unadsorbed at the interface of fresh 9

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emulsions was analyzed by SDS-PAGE (Peng, et al., 2016). Emulsions were

194

centrifuged at 7500 g/min for 30 min. The cream phase was carefully removed from

195

the top. The aqueous phase was withdrawn using a syringe and passed through a 0.45

196

μm filter. Samples of the cream and aqueous phase were dissolved in an equal volume

197

of the SDS-PAGE sample buffer in the presence (reducing conditions) of 5% (v/v) β-

198

ME. An aliquot (7.5 μL) of each sample was loaded into a cell and electrophoresed.

199

The intensities of bands were integrated using Image Lab software.

200

2.12 Statistical analysis

201

All experiments were conducted at least three times. Data reported are mean values

202

± standard deviations. The data were analyzed using SPSS following an analysis of

203

variance (ANOVA) one-way linear model. The means were compared by a least

204

significant difference test with a confidence interval of 95%.

205

3. Results and discussion

206

3.1 Solubility and surface hydrophobicity analysis

207

The hydrophobic regions of proteins rapidly adsorb to the surface of oil droplets

208

during the formation of O/W emulsions. Rearrangement of the protein conformation

209

leads for the formation of a membrane (the interfacial layer) on the surface of oil

210

droplets. It has been reported that the protein concentration and order of addition

211

significantly affects the flocculation stability of protein-stabilized emulsions (Kim,

212

Decker, & Mcclements, 2005). Protein solubility is important because higher protein

213

concentrations improve the creaming stability of emulsions by soy proteins (Shao &

214

Tang, 2014). The temperature solubilities of the two pea proteins are shown in Fig. 1 10

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(a). When the temperature was below 50 oC, the nitrogen solubility index of PPI 1 and

216

PPI 2 were both less than 50%, showing poor solubility. The solubility of pea protein

217

increased with increasing temperature. Pea proteins had the greatest solubility at 90

218

oC

with PPI 1 performing better than PPI 2.

219

Surface hydrophobicity improves the ability of the protein to adsorb to the interface

220

and is closely related to the emulsifying properties (Mahmoudi, Axelos, & Riaublanc,

221

2011). Because hydrophobic regions are usually concentrated in the interior of water

222

soluble proteins but can be exposed upon protein denaturation, denaturation

223

temperature is an important processing characteristic. It is well established that heat

224

treatment can expose hydrophobic groups buried in globular proteins as a result of

225

partial unfolding. The effects of heating temperature on the surface hydrophobicity of

226

pea proteins are shown in Fig. 1 (b). The pea protein extracted at 90 oC had the

227

highest surface hydrophobicity value. The denaturation temperatures of vicilin and

228

legumin were at 88.4 oC and 88.9 oC (Jean-Luc Mession, Chihi, Sok, & Saurel, 2015).

229

Peng et al (Peng, et al., 2016) has found that emulsions showed higher creaming

230

stability when the pea protein was heated at 95 oC, compared to unheated protein.

231

Thus, the pea protein extracted at 90 oC was used in this study to prepare the

232

emulsion.

233

3.2 The molecular weight distribution and components of pea protein

234

The molecular weight distribution of pea proteins extracted at 90

oC

was

235

determined by HPSEC-UV-RI (Barackman, Prado, Karunatilake, & Furuya, 2004).

236

The elution profiles of pea proteins are illustrated in Fig. 2. Accordingly, the elution 11

ACCEPTED MANUSCRIPT 237

profiles of pea proteins showed three major elution peaks at 23.6-33.8 min, 33.8-40.2

238

min and 40.2-49.2 min. According to the distribution, three fractions of molecular

239

weight were observed and divided as follows: the high molecular weight (HMw):

240

Mw > 2500 kDa, the medium molecular weight (MMw): 500 kDa < Mw < 2500 kDa,

241

and the low molecular weight (LMw): Mw < 500 kDa. The HMw, MMw and LMw

242

fractions of PPI 1 were 40%, 24% and 36% respectively. For PPI 2, the respective

243

fractions for HMw, MMw and LMw were 45.44%, 20.53% and 34.03%. The

244

percentages of soluble proteins of PPI 1 and PPI 2 in HMw and MMw fractions were

245

very high, which might be due to the soluble protein aggregates extracted after heat

246

treatment, or the soluble proteins further denatured and aggregated at extraction

247

temperature.

248

The components of pea protein extracted at 90

oC

were analyzed by

249

electrophoresis. As shown in Fig. 3, pea protein extracted at 90 oC consisted of

250

multiple components. The major storage proteins in pea proteins are globulins,

251

including legumin (11S) and vicilin (7S) (Klassen, et al., 2012; Tzitzikas, et al.,

252

2006), and the ratio of 7S to 11S is varied between 0.5 to 1.7, as the mean value is

253

around 1.1 (Schroeder, 1982). The 11S legumin is about 320-380 kDa, and has a

254

hexametric quaternary structure (six subunits), with one subunit consisting of an

255

acidic polypeptide (leg A, 38-40 kDa) and a basic polypeptide (leg B, 19-22 kDa) that

256

are linked together by a disulfide bridge (O'Kane, Happe, Vereijken, Gruppen, & van

257

Boekel, 2004b). The 7S vicilin is about 150-180 kDa, and has a trimer quaternary

258

structure (three subunits), consisting of polypeptides about 47-50 kDa, 30-34 kDa and 12

ACCEPTED MANUSCRIPT 259

< 19 kDa, and lack of lysine (Gatehouse, Lycett, Croy, & Boulter, 1982). The

260

convicilin is about 71-75 kDa, also been regarded as a subunit of the 7S vicilin

261

(Barac, et al., 2010; Croy, Gatehouse, Tyler, & Boulter, 1980; O'Kane, Happe,

262

Vereijken, Gruppen, & van Boekel, 2004a). Since the Mw of the largest pea proteins

263

are < 500 kDa, larger eluting materials are presumed to be soluble protein aggregates.

264

The composition of pea proteins was determined by non-reducing electrophoresis,

265

and the composition of protein subunits was determined by reducing electrophoresis.

266

The protein compositions of pea proteins determined by SDS-PAGE are listed in Tab.

267

1. The non-reducing SDS-PAGE results showed that the proportions of proteins were

268

aggregates > vicilin > legumin > convicilin. The percentages of aggregates in two pea

269

proteins were all about 37%. The reducing SDS-PAGE results showed that the

270

proportions of proteins were vicilin > legumin > convicilin > aggregates. The

271

aggregates were dissociated partly into subunits by SDS and β-ME. It is well known

272

that pea proteins have acidic and basic (AB) subunits under non-reducing conditions.

273

AB subunits and convicilin in pea proteins were involved in the formation of

274

polymers linked by disulfide bonds after heat treatment (Peng, et al., 2016). Wang et

275

al. (2012) proposed that the presence of intermolecular disulfide bonds in heated soy

276

proteins were caused by oxidation and/or SH-SS interchange reactions. The

277

percentages of aggregates determined by reducing electrophoresis were decreased to

278

less than 10%, these results implied that the intermolecular interaction generating the

279

protein aggregates were not just the disulfide bond and the hydrophobic interaction.

280

3.3 Emulsifying ability of pea protein and stability of pea protein stabilised 13

ACCEPTED MANUSCRIPT 281

emulsion

282

3.3.1 Particle size distribution

283

The particle size distribution of oil droplets in the flocculation state was determined

284

by using deionized water as the dispersant, while particle size of single oil droplet was

285

determined by using 1% SDS as the dispersant, which could inhibit the flocculation

286

between the oil droplets (Liang, et al., 2013). The effects of protein concentration on

287

the particle size distribution of emulsions are presented in Fig. 4. As shown in Fig. 4,

288

when the protein concentrations were smaller than 2.5 mg/mL, the particle size

289

distribution was very wide and the particle sizes were very high. When the protein

290

concentrations were at 5 mg/mL, the distribution of emulsion using water as

291

dispersant appeared to have three peaks: a small peak at 0.04-0.3 μm, a middle peak at

292

0.3-3 μm and a large peak at 3-15 μm. When using 1% SDS as the dispersant, there

293

was only one peak when the protein concentrations were smaller than 2.5 mg/mL, and

294

the distribution appeared to be two peaks at 0.04-0.3 μm and 0.3-3 μm at 5 mg/mL.

295

These results indicated that oil droplets were flocculated when protein concentrations

296

were ≤ 5mg/mL. With the increase of protein concentration, obvious decrease in

297

particle size was observed. When the protein concentration was further increased to

298

10, 20 and 30 mg/mL, the particle size in water dispersant was less than 10 μm, and

299

the distribution yielded 2 peaks at 0.04-0.3 μm and 0.3-3 μm. It has also been reported

300

that the size distribution of emulsions prepared with heat treatment pea protein

301

showed a bimodal or trimodal distribution in water (Peng, et al., 2016).

302

3.3.2 Average diameter and flocculation index of emulsions 14

ACCEPTED MANUSCRIPT 303

The flocculation condition could be described by the average diameter and

304

flocculation index (FI) of emulsions. The effects of protein concentration on the

305

average diameter and FI of emulsions are listed in Tab. 2. When the protein

306

concentration was increased from 1.0 to 30 mg/mL, the diameter of emulsions made

307

by PPI 1 decreased from 15.56 μm to 0.60 μm using water as the dispersant.

308

Emulsions stabilized by pea protein at higher concentrations showed smaller d43

309

values. Similar observations have been reported for pea proteins (Peng, et al., 2016;

310

Roesch & Corredig, 2002). Furthermore, the diameter of emulsions stabilized by PPI

311

1 decreased from 3.22 μm to 0.59 μm using 1% SDS as the dispersant. This is

312

presumably due to the stabilization of a larger interfacial area by a higher protein

313

concentration in the continuous phase, which is consistent with the smaller oil droplet

314

size (Liu & Tang, 2013). The FI values decreased from 3.83 to 0.02. as protein

315

concentrations increased from 1.0 to 30 mg/mL. The decrease in FI could be

316

interpreted as a result of inhibited flocculation (Liu & Tang, 2013). When the protein

317

concentration was ≤ 5 mg/mL, the d43 (H2O) was significantly higher than d43 (SDS).

318

When the protein concentration was increased from 10.0 mg/mL to 30.0 mg/mL, the

319

particle size of oil droplets was not decreased. These phenomena implied that protein

320

membranes outside of the oil droplets were tighter and inhibited the flocculation of

321

the oil droplets effectively. Meanwhile, PPI 1 had a little better emulsifying ability

322

than PPI 2, according to the average diameter of emulsions.

323

3.3.3 Heat stability and storage stability of emulsions

324

During storage, the two phases of emulsions tend to separate to reduce the free 15

ACCEPTED MANUSCRIPT 325

energy, resulting into the flocculation, coalescence and creaming. Meanwhile, heat

326

treatment is a significant step during the process of food emulsions. In order to

327

investigate the emulsifying stability of pea proteins, the emulsions were heated at 90

328

oC

329

measured. The effects of heat treatment or storage on the average diameter of

330

emulsions with different concentrations proteins are listed in Tab. 3. Compared with

331

Tab. 2, the average diameter of emulsions was increased obviously after the heat

332

treatment and storage when the protein concentrations were ≤ 5 mg/mL. However,

333

when the protein concentration was ≥ 10 mg/mL, pea proteins had a good emulsifying

334

stability. It can be inferred that when the protein concentration was too low, it might

335

difficult to cover the oil droplets and effectively inhibit the flocculation, coalescence

336

and creaming of emulsions. PPI 1 performed slightly better than PPI 2 as far as their

337

emulsifying ability was concerned.

338

3.4 Interfacial adsorption properties of pea proteins

339

3.4.1 Percentage of adsorbed proteins in emulsions

for 30 min or stored at 25 oC for 7 d, and the average diameters of emulsions were

340

The percentages of adsorbed protein (AP%) are showed in Tab. 4. With the

341

increasing of protein concentration, the concentration of interfacial adsorbed proteins

342

(C0-Cs) was increased from 0.84 mg/mL to 6.46 mg/mL for PPI 1, and increased from

343

0.78 mg/mL to 6.94 mg/mL to PPI 2. This result demonstrated that more pea proteins

344

were adsorbed to the interface of the emulsion. However, the AP% was decreased

345

with the increase of protein concentration from 84.33% to 21.53% for PPI 1 and from

346

78.33% to 23.13%. These results illustrated that the pea proteins taking part in 16

ACCEPTED MANUSCRIPT 347

emulsifying process were decreased with the increase of protein concentration.

348

Similarly, emulsions prepared with soy proteins and pea proteins both exhibited a

349

decrease in AP% values with increasing protein concentrations (Li, Kong, Zhang, &

350

Hua, 2011; Peng, et al., 2016).

351

The interfacial protein concentrations (Г) of emulsions are illustrated in Tab. 4.

352

When the protein concentration increased from 1.0 mg/mL to 10.0 mg/mL, the value

353

of Г increased from 0.94 mg/m2 to 2.31 mg/m2 for PPI 1, indicating that pea proteins

354

could stabilize larger interfacial area. When protein concentration was higher, more

355

proteins could be adsorbed at the O/W interface (Shao & Tang, 2014). When the

356

protein concentration further increased to 10.0 mg/mL and 30.0 mg/mL, the value of

357

Г had no significant change. These results demonstrated that the adsorption of soluble

358

pea proteins has reached the saturated point and the quantity of pea proteins adsorbed

359

to the oil droplets has reached the highest value. At the saturated adsorption of 10.0

360

mg/mL, 20.0 mg/mL and 30.0 mg/mL, the value of AP% was 61.84%, 31.35% and

361

21.53%, which indicated that 38.16%, 68.65% and 78.47% of pea proteins in the

362

system did not take part in emulsifying process.

363

3.4.2 Composition of adsorbed proteins in emulsions

364

In order to further study the interfacial adsorption property of pea proteins in O/W

365

emulsions and the competitive adsorption relationship between different subunits of

366

pea proteins, the non-reducing SDS-PAGE shown in Fig. 5 was used to analyze the

367

subunits of adsorbed proteins in emulsions at different protein concentrations.

368

The compositions of adsorbed protein in emulsions are listed in Tab. 5. With the 17

ACCEPTED MANUSCRIPT 369

increasing of protein concentration, less aggregates but more vicilin and legumin were

370

adsorbed onto the interface. As shown in the Tab. 1, the percentages of protein

371

aggregates in PPI 1 and PPI 2 were 37.21% and 37.65%, respectively. However, when

372

the protein concentration was at 1.0 mg/mL, the percentages of protein aggregates

373

adsorbed onto the interface in emulsion for PPI 1 and PPI 2 were more than its

374

proportion in the pea protein extraction shown in Tab. 1, indicating that protein

375

aggregates could be effectively adsorbed to the interface. Meanwhile, the percentages

376

of vicilin and legumin adsorbed onto the interface were less than its proportion in the

377

pea protein extraction. These phenomena indicated that when the protein

378

concentration was too low, it was difficult to cover the oil droplets, and most of the

379

proteins were adsorbed to the interface when emulsifying, and the adsorption of

380

protein aggregates with high molecular size might cause the steric hindrance and

381

inhibit the adsorption of vicilin and legumin.

382

When the protein concentrations were at 20 mg/mL and 30 mg/mL, the percentages

383

of protein aggregates adsorbed onto the interface in emulsion were less than its

384

proportion in the pea protein extraction shown in Tab. 1. Meanwhile, the percentages

385

of vicilin and legumin adsorbed onto the interface were more than its proportion in the

386

pea protein extraction. When the protein concentration was sufficient, the vicilin and

387

legumin with smaller molecular size might be adsorbed to the interface at a faster

388

speed, thus covered the oil droplets better, while the aggregates with higher molecular

389

size might have a lower speed and at the inferior position. When the adsorption

390

reached the saturation state, the vicilin and legumin maintained the superior position 18

ACCEPTED MANUSCRIPT 391

on the interface, and the competitive adsorption relationship of protein subunits

392

tended to be stable. When the adsorption reached the saturation state, the quantity of

393

proteins on the interface were vicilin > legumin > aggregates > convicilin, and the

394

proportion of aggregates was lower than its proportion in the pea protein extraction.

395

Meanwhile, the proportion of aggregates of PPI 2 on the interface was a little higher

396

than PPI 1.

397

4. Conclusions

398

The present work indicated that the nitrogen solubility index and surface

399

hydrophobicity of pea protein increased with the increase of temperature. The

400

molecular weight distributions analyzed by high-performance size exclusion

401

chromatography were divided into three fractions: HMw > 2500 kDa, 500 kDa <

402

MMw < 2500 kDa and LMw < 500 kDa, and the fraction with Mw > 500 kDa was

403

over 60%. The non-reducing SDS-PAGE results showed that the percentage of

404

aggregates were about 37%, and the proportions of proteins were aggregates >

405

vicilin > legumin > convicilin. The reducing SDS-PAGE results showed that the

406

proportions of proteins were vicilin > legumin > convicilin > aggregates. With the

407

protein concentrations increase from 1.0 to 30 mg/mL, the emulsifying ability and

408

stability of pea protein increased significantly. Meanwhile, the concentration of

409

interfacial adsorbed proteins (C0-Cs) was increased, demonstrating that more pea

410

proteins were adsorbed to the interface of the emulsion. However, the percentage of

411

interfacial adsorbed proteins was decreased significantly.

412

When the protein concentration was higher than 10 mg/mL, the interfacial 19

ACCEPTED MANUSCRIPT 413

adsorption of pea proteins would reach the saturated adsorption point. At low protein

414

concentration, the contents of proteins adsorbed onto the interface were aggregates >

415

vicilin > legumin > convicilin, and the content of aggregates was higher than its

416

proportion in the initial pea protein. With the increase of protein concentration,

417

aggregates would lose the superiority in interfacial adsorption, while the proportion of

418

vicilin and legumin increased. At saturated adsorption, the contents of proteins

419

adsorbed onto the interface were vicilin > legumin > aggregates > convicilin, and the

420

proportion of aggregates was lower than its proportion in the initial pea protein

421

extraction.

422

Acknowledgements:

423

This research was supported by National Natural Science Foundation of China

424

(31601437,

21676122),

the

National

425

(2016YFD0400802). This work was supported by national first-class discipline

426

program of Food Science and Technology (JUFSTR20180204). The research is also

427

supported by 111 Project B07029, and program of "Collaborative Innovation Center

428

of Food Safety and Quality Control in Jiangsu Province".

20

Key

R&D

Program

of

China

ACCEPTED MANUSCRIPT 429

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23

ACCEPTED MANUSCRIPT Tables Tab. 1. The composition of pea proteins

Electrophoresis

Aggregates

Convicilin

Vicilin

Legumin

%

%

%

%

PPI 1

37.21

11.57

31.57

19.65

PPI 2

37.65

9.07

29.18

24.09

PPI 1

7.31

18.28

43.01

31.41

PPI 2

9.89

14.47

38.51

37.13

Proteins

Non-reducing

Reducing

PPI: Pea protein powder.

24

ACCEPTED MANUSCRIPT Tab. 2. Effects of protein concentration on the average diameter and flocculation index of emulsions Protein concentration

Average diameter of emulsions (μm)

Proteins

FI （mg/mL）

d43(H2O)

d43(SDS)

1.0

15.56±0.47f

3.22±0.04g

3.83±0.08h

2.5

4.56±0.24d

1.33±0.02f

2.43±0.13f

5.0

1.25±0.05b

0.72±0.01d

0.74±0.05d

10.0

0.57±0.01a

0.50±0.01ab

0.14±0.01b

20.0

0.52±0.01a

0.51±0.01ab

0.02±0.01a

30.0

0.60±0.01a

0.59±0.02c

0.02±0.01a

1.0

13.04±0.35e

3.57±0.05h

2.65±0.05g

2.5

3.25±0.48c

1.09±0.14e

1.98±0.06e

5.0

1.13±0.05b

0.72±0.02d

0.57±0.03c

10.0

0.55±0.01a

0.49±0.02ab

0.12±0.02ab

20.0

0.48±0.01a

0.45±0.02a

0.07±0.03ab

30.0

0.57±0.01a

0.55±0.02bc

0.04±0.02a

PPI 1

PPI 2

PPI: Pea protein powder. FI: Flocculation index In the same column of each index, the different superscript letters represent significant difference (p < 0.05).

25

ACCEPTED MANUSCRIPT Tab. 3. Effects of heat treatment or storage on the average diameter of emulsions Protein concentration

Heat treatment

Storage

（mg/mL）

(μm)

(μm)

1.0

15.65±0.25f

16.54±0.75e

2.5

5.72±0.23d

5.05±0.13b

5.0

1.37±0.04b

4.76±1.11b

10.0

0.57±0.01a

0.68±0.01a

20.0

0.56±0.02a

0.73±0.09a

30.0

0.60±0.02a

0.96±0.03a

1.0

13.38±0.69e

14.73±0.23d

2.5

4.26±0.55c

7.23±0.79c

5.0

1.51±0.04b

1.16±0.03a

10.0

0.72±0.02a

0.76±0.02a

20.0

0.54±0.02a

0.77±0.01a

30.0

0.58±0.02a

0.83±0.03a

Proteins

PPI 1

PPI 2

PPI: Pea protein powder; Heat treatment: 90 oC for 30 min; Storage: 25 oC for 7 d In the same column of each index, the different superscript letters represent significant difference (p < 0.05).

26

ACCEPTED MANUSCRIPT Tab. 4. Percentages of adsorbed protein and interfacial protein concentration of emulsions PPI 1

Protein concentration

PPI 2

C0-Cs

Г

C0-Cs

AP%

AP%

Г（mg/m2）

（mg/m2）

（mg/mL）

84.33±2.01e

0.94±0.01a

0.78±0.04a

78.33±3.92f

1.09±0.05a

1.78±0.04b

71.08±1.63d

1.03±0.02b

1.76±0.01b

70.52±0.38e

1.21±0.01b

5.0

3.52±0.02c

70.33±0.38d

1.74±0.03c

3.38±0.05c

67.74±0.97d

1.70±0.03c

10.0

6.18±0.07d

61.84±0.74c

2.31±0.02d

6.52±0.10d

65.23±0.99c

2.36±0.03d

20.0

6.27±0.20d

31.35±1.02b

2.35±0.10d

6.63±0.08e

33.10±0.40b

2.44±0.03d

30.0

6.46±0.65d

21.53±2.17a

2.38±0.21d

6.94±0.24f

23.13±0.81a

2.45±0.08d

（mg/mL）

（mg/mL）

1.0

0.84±0.02a

2.5

PPI: Pea protein powder; AP%: Percentage of adsorbed proteins; Γ: Interfacial protein concentration; C0: the protein concentration in the initial pea protein dispersion; Cs: the protein concentration of the unadsorbed layer; In the same column of each index, the different superscript letters represent significant difference (p < 0.05).

27

ACCEPTED MANUSCRIPT Tab. 5. Composition of the adsorbed proteins in emulsions Protein concentration Proteins

Aggregates%

Convicilin%

Vicilin%

Legumin%

1.0

56.17

8.06

22.82

12.94

2.5

52.27

7.38

23.45

16.90

5.0

36.12

9.16

30.29

24.43

10.0

30.43

10.92

39.03

19.62

20.0

14.03

9.50

45.04

31.43

30.0

9.99

10.39

49.31

30.31

1.0

41.56

9.20

23.74

25.49

2.5

55.21

7.15

16.78

20.87

5.0

49.73

9.25

22.69

18.33

10.0

41.90

7.92

25.14

25.03

20.0

13.96

7.31

46.20

32.53

30.0

15.12

8.91

43.34

32.64

（mg/mL）

PPI 1

PPI 2

28

ACCEPTED MANUSCRIPT Figure Captions Fig. 1. Effects of heat temperature on nitrogen solubility index (a) and surface hydrophobicity (b) of pea protein. PPI: Pea protein powder; Bars refer to mean values; Error bars refer to standard deviation; Different letters indicate significant difference (p < 0.05) Fig. 2. The molecular weight distribution of pea protein extracted at 90 oC. PPI: Pea protein powder. Fig. 3. Reducing (+βME) and non-reducing (-βME) SDS-PAGE profiles of pea protein extracted at 90 oC. PPI: Pea protein powder. Fig. 4. Effects of protein concentration on the particle size distribution of emulsions when dispersant is water (a, b) or 1% SDS (a’, b’). PPI: Pea protein powder. Fig. 5. Non-reducing electrophoresis of adsorbed proteins in emulsions with pea proteins extracted at 90 oC. (Lane 1: marker, Lane 2-7: pea protein with concentration at 1.0, 2.5, 5.0, 10.0, 20.0, 30.0 mg/mL)

29

ACCEPTED MANUSCRIPT Figures Fig. 1.

30

ACCEPTED MANUSCRIPT Fig. 2.

31

ACCEPTED MANUSCRIPT Fig.3.

32

ACCEPTED MANUSCRIPT Fig. 4.

33

ACCEPTED MANUSCRIPT Fig. 5.

34

ACCEPTED MANUSCRIPT Highlights: Heat treatment improved solubility and surface hydrophobicity. Initial protein composition by SDS-PAGE were aggregates > vicilin > legumin > convicilin. Increasing concentration increased emulsifying ability and stability. Interfacial protein concentration at saturation was vicilin > legumin > aggregates > convicilin,