Characterization and interfacial rheological properties of nanoparticles prepared by heat treatment of ovalbumin-carboxymethylcellulose complexes

Accepted Manuscript Characterization and interfacial rheological properties of nanoparticles prepared by heat treatment of ovalbumin-carboxymethylcellulose complexes

Wenfei Xiong, Cong Ren, Jing Li, Bin Li PII:

S0268-005X(17)32159-8

DOI:

10.1016/j.foodhyd.2018.03.048

Reference:

FOOHYD 4359

To appear in:

Food Hydrocolloids

Received Date:

27 December 2017

Revised Date:

23 February 2018

Accepted Date:

27 March 2018

Please cite this article as: Wenfei Xiong, Cong Ren, Jing Li, Bin Li, Characterization and interfacial rheological properties of nanoparticles prepared by heat treatment of ovalbumincarboxymethylcellulose complexes, Food Hydrocolloids (2018), doi: 10.1016/j.foodhyd.2018.03.048

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ACCEPTED MANUSCRIPT Graphical abstract for:

Characterization and interfacial rheological properties of nanoparticles prepared

by

heat

treatment

of

ovalbumin-carboxymethylcellulose

complexes Wenfei Xionga,b, Cong Rena,b, Jing Li a,b, Bin Li a,b,c* a

College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, Hubei, China

b

Key Laboratory of Environment Correlative Dietology (Huazhong Agricultural University), Ministry of Education, China

c

Functional Food Enginnering & Technology Research Center of Hubei Province, Wuhan 430068, China

*Corresponding author: Tel: +86-27-63730040; Fax: +86-27-87282966 E-mail address: [email protected] (Bin Li)

ACCEPTED MANUSCRIPT 1

Characterization and interfacial rheological properties of nanoparticles

2

prepared

3

complexes

4 5 6 7

Wenfei Xionga,b, Cong Rena,b, Jing Li a,b, Bin Li a,b,c*

by

heat

treatment

of

ovalbumin-carboxymethylcellulose

a

College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, Hubei, China

b

Key Laboratory of Environment Correlative Dietology (Huazhong Agricultural University), Ministry of Education, China

c

Functional Food Enginnering & Technology Research Center of Hubei Province, Wuhan 430068, China

8

Abstract: The objective of this study was to investigate the physicochemical and interfacial

9

rheological properties of ovalbumin (OVA)-carboxymethylcellulose (CMC) nanoparticles. The

10

OVA/CMCs nanoparticles were prepared by heating (90 ℃, 30 min) the electrostatic self-assembly

11

complexes between OVA and CMC of different charge density (CMC 0.7 and CMC 1.2) at pH 4.4.

12

The results showed that the OVA/CMC 0.7 nanoparticles exhibited larger size and lower surface net

13

potential than OVA/CMC 1.2 nanoparticles. Atomic force microscopy (AFM) imaging and ultra-

14

small angle X-ray scattering (USAXS) results suggested that the shape of the particles was

15

approximately spherical, and the structure of OVA/CMC 1.2 nanoparticles was more compact than

16

that of OVA/CMC 0.7. The pyrene fluorescent probe indicated that the OVA/CMC 1.2

17

nanoparticles had a stronger hydrophobicity than OVA/CMC 0.7 nanoparticles in the range of pH

18

4-7. As the pH and the ionic strength increased, the average diameter of OVA/CMC nanoparticles

19

would increase (<400 nm), while the average size of the nanoparticles did not change significantly

20

after 30 days of storage at room temperature. The interfacial rheological experiments showed that

21

the permeation and rearrangement rates of OVA/CMC nanoparticles decreased significantly at oil-

22

in-water interface, and the surface pressure and interfacial dilatational modulus were lower than the

23

native OVA/CMC complexes. These findings suggest that OVA/CMC nanoparticles formed by heat

24

induction can be used to construct lipid-soluble nutrient delivery vehicles.

25

Keywords: Ovalbumin; Carboxymethylcellulose; Charge density; Nanoparticles stability;

26

Interface adsorption

27 28 29 30 31 1

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1. Introduction

33

The design and development of edible biopolymer nanoparticles has gained increasing

34

research interest in recent years, which are due to their potential application as encapsulation,

35

delivery systems, fat replacer, emulsifier and textural modifiers in food industry (Jones, &

36

McClements 2010a; McClements 2018; Zeeb, Mi-Yeon, Gibis, & Weiss. 2018). Based on safety

37

consideration, natural food grade biopolymers, such as proteins and polysaccharides, have been

38

widely used to prepare nanoparticles with different structures and to be used in the construction of

39

oral delivery systems for phytochemicals (Jones, Lesmes, Dubin, & McClements, 2010; Jones, &

40

McClements, 2010b; Xiao, Cao, & Huang, 2017). In addition, certain polysaccharides (e.g. dietary

41

fiber) also have potential health benefits such as reducing cholesterol, preventing cancer or

42

improving colon health (Dikeman & Fahey, 2006; Grabitske & Slavin, 2009). Thus, the

43

consumption of biopolymer particles rich in these polysaccharides may be beneficial in bringing

44

about more health effects, which further increasing interest in the development of various

45

biopolymer particles for commercial application (Jones, & McClements, 2011).

46

The application of protein and polysaccharide mixtures to fabricate biopolymer nanoparticles

47

with specific structures is mainly based on the electrostatic interaction between them (Jones, &

48

McClements, 2010; Schmitt, & Turgeon, 2011). Therefore, it is necessary to precisely control the

49

factors that affect the electrostatic interaction of protein/polysaccharide, such as pH, ionic strength,

50

the ratio of protein/polysaccharide, biopolymer concentration, polysaccharide conformation and

51

charge density (Jones, & McClements 2011). For protein and anionic polysaccharide system,

52

protein/polysaccharide can form soluble complexes and coacervates by self-assembly as the pH of

53

solution changes from neutral to acidic. Generally, when the pH of the solution is within the range

54

of soluble complex formation, biopolymer particles of different scales can be formed by

55

electrostatic attraction interaction between protein and polysaccharide (Jones, Lesmes, Dubin, &

56

McClements, 2010; Jones, & McClements, 2010a). However, these particles are not stable and will

57

dissociate with the changing of pH value or the increasing of ionic strength. It has been shown that

58

by heating the formed protein/polysaccharide complexes above the denaturation temperature of the

59

protein, the particle stability can be greatly improved, and making them more suitable for use as

60

carriers for the nutrient delivery system (Jones, Lesmes, Dubin, & McClements, 2010; Jones, &

61

McClements 2011). On the other hand, soluble globular proteins are unfolded due to thermal

62

induction, exposing more hydrophobic groups, thereby enhancing their hydrophobic interactions 2

ACCEPTED MANUSCRIPT 63

with lipid-soluble active ingredients and increasing the loading capacity (Visentini, Sponton, Perez,

64

& Santiago, 2017a). For those reasons, this approach is widely used to construct biopolymer

65

nanoparticle delivery systems for the encapsulation and protection of lipid-soluble bioactive

66

compounds (Luo, Pan, & Zhong, 2015, Cho, Jung, Lee, Kwak, & Hwang, 2016; Zhou, Hu, Wang,

67

Xue, & Luo, 2016; Zhou, Wang, Hu, & Luo, 2016; Chang et al., 2017). Moreover, biopolymer

68

particles can also be used to construct Pickering emulsion delivery systems in addition to being

69

directly used for the encapsulation of lipid-soluble active ingredients, where the diffusion,

70

adsorption, rearrangement of the particles at the oil-in-water interface and the viscoelastic

71

properties of the adsorbed layer for its emulsifying ability and emulsion stability are very important

72

(Murray, 2002; Liu, & Tang, 2014). Indeed, the interfacial rheological behavior of biopolymer

73

particles is closely related to their structural and physicochemical properties, and exploring this

74

correlation is crucial for regulating the emulsifying properties of biopolymer particles (Dickinson,

75

2008). However, there have been very few reports on this topic so far.

76

Ovalbumin (OVA) is the most abundant protein in egg white protein (EWP) and plays a

77

leading role in the foaming and gelation of EWP (Weijers, Sagis, Veerman, Sperber, & Linden,

78

2002). OVA is a globular protein composed of 385 amino acids, and most of the hydrophobic

79

amino acids are buried within the structure of the protein, resulting in poor binding capacity

80

between native OVA and lipid-soluble bioactive ingredients (Visentini, Sponton, Perrez, &

81

Santiago, 2017b). By heat-induced unfolding of OVA structure to expose its internal hydrophobic

82

groups, its binding capacity with lipid-soluble ligands (such as linoleic acid and retinol) can be

83

greatly enhanced (Sponton, Perez, Carrara, & Santiago, 2015a; Sponton, Perez, Carrara, &

84

Santiago, 2015b; Sponton, Perez, Carrara, & Santiago, 2016; Visentini, Sponton, Perrez, &

85

Santiago, 2017a). However, the instability of the protein in the vicinity of the isoelectric point

86

greatly limits its application. Yu et al., (Yu, Hu, Pan, Yao, & Jiang, 2006) prepared nanogels with

87

better stability in the range of pH 2-10.5 by heating the OVA/chitosan electrostatic self-assembly

88

complexes. This result further demonstrates the effectiveness of the heat treatment in improving the

89

biopolymer particle stability.

90

In the present work, nanoparticles with good stability (pH, ionic strength, storage time) were

91

prepared by heating OVA/carboxymethylcellulose (CMC) electrostatic self-assembly soluble

92

complexes. Meanwhile, in order to compare the effect of polysaccharide charge density on the

93

formation and properties of nanoparticles, we studied two different CMCs with substitution degrees 3

ACCEPTED MANUSCRIPT 94

of 0.7 (CMC0.7) and 1.2 (CMC1.2), respectively. The focus of this study is to characterize the

95

morphology, structure and surface hydrophobicity of the nanoparticles by means of atomic force

96

microscopy (AFM), ultra-small angle X-ray scattering (USAXS) and pyrene fluorescence probe,

97

respectively. More importantly, to evaluate the potential of nanoparticles for application in

98

emulsion delivery systems. The adsorption behavior of the nanoparticles at the oil-in-water

99

interface and the viscoelastic properties of the interface adsorption layer were also investigated by

100

interfacial dilatational rheology. Guidance can be provided for the use of nanoparticles as particle or

101

emulsion delivery systems for the construction of lipid-soluble nutrients, especially the correlation

102

between the physicochemical properties of nanoparticles and their interfacial rheological behavior

103

can be established.

104

2. Materials and methods

105

2.1. Materials

106

Ovalbumin (OVA, A5503, > 98 % pure by agarose electrophoresis, and a molecular weight of

107

45 kDa) was purchased from Sigma Co. (St. Louis, MO) without further purification.

108

Carboxymethylcellulose (CMC, the degree of substitution (DS) is 0.7 and 1.2, respectively, and the

109

molecular weight is about 250 kDa.) was purchased from Aladdin Chemistry Co., Ltd. (Shanghai,

110

China). Other reagents were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai,

111

China). All reagents were analytical grade unless otherwise stated.

112

2.2. Preparation of OVA/CMC nanoparticles

113

In this work, the OVA/CMC complex is a mixture formed by electrostatic self-assembly

114

between OVA and CMC, and the OVA/CMC nanoparticle is biopolymer particles formed by the

115

heat treatment of the OVA/CMC complex. According to our previous study (Xiong et al., 2017), the

116

OVA powder and CMC powder was dissolved in deionized water to prepare the protein (10%, w/v)

117

and polysaccharide stock solution (2%, w/v), respectively. Meanwhile, sodium azide (0.02%, w/v)

118

was added in the OVA and CMC solution to inhibit bacteria growth. OVA/CMC mixtures with

119

weight ratio (OVA: CMC, r) of 4:1 was prepared by mixing corresponding stock solutions. The

120

total biopolymer concentration was fixed in 0.6% (w/v). Subsequently, the pH value of the

121

OVA/CMC mixtures were adjusted to 4.4 with 0.5 M HCl to form the OVA/CMC complexes, and

122

then the OVA/CMC nanoparticles was fabricated by heating complexes at 90 ℃ for 30 min. When

123

the heat treatment was completed, the samples were quickly cooled in ice water and then placed in

124

4 ℃ refrigerator for study. 4

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2.3. Determination of nanoparticle composition

126

Nanoparticle composition was carried out by measuring the OVA and CMC content of the

127

resulting supernatant after centrifugation. Centrifugation was carried out at 15,000 g for 60 min

128

(10 ℃) using an ultracentrifuge (Sorvall RC5B Plus, Thermo Scientific, Waltham, MA) (Jones,

129

Lesmes, Dubin, & McClements, 2010). The protein concentration was determined using the

130

bicinchoninic acid (Walker, 1994), and the polysaccharide content was determined using the

131

Phenol-Sulfuric Acid assay (Kochert, 1978).

132

2.4. Zeta potential and particle size determination

133

Zeta potential and particle size of the freshly prepared samples were directly determined by

134

microelectrophoresis instrument and dynamic light scattering (ZS Zetasizer Nano, Malvern

135

Instrument Ltd., UK). All measurements were carried out at 25 ℃ and repeated three times.

136

2.5. Morphological studies by atomic force microscopy (AFM)

137

The freshly prepared OVA/CMC nanoparticle dispersions were diluted 10 times using

138

deionized water and readjusted to pH 4.4. Three microliters of the diluted sample were dripped onto

139

freshly cleaved mica surface and dried at room temperature for 12 h. Morphological images were

140

collected at the tapping mode using a nano-probe cantilever tip (BrukerNanoprobe, Camarillo, CA)

141

at a frequency from 50 to 100 kHz on a Multimode VIII microscope (Bruker Corporation, Billerica,

142

MA) (Guan, Wu, & Zhong, 2016). Images were analyzed using the AMF instrument software

143

(Nanoscope Analysis version 1.50, Bruker Corporation, Billerica, MA).

144

2.6. Ultra-small angle X-ray scattering (USAXS) experiments

145

The USAXS experiments were performed on a beamline BL16B1 at Shanghai Synchrotron

146

Radiation Facility (SSRF). The wavelength of the X-rays was 1.24 Å, and the detector was a two-

147

dimensional (2048×2048 pixels) gas-filled detector placed at 5230 nm distance from the sample.

148

The final USAXS profiles were obtained through the average of 15 measurements followed by the

149

subtraction of solvent and beam line background. The obtained data were analyzed by FIT2D

150

software, and the relation between light scattering vector(q) and light scattering intensity (I(q)) was

151

calculated by the formula q = (4π⋅sinθ)/λ, where θ and λ is scattering angle and scattering

152

wavelength, respectively. The Kratky plot (q2I(q) vs q) was used to detect the folding conformation

153

of samples (Shi et al., 2017).

154

2.7. Pyrene fluorescence measurement

155

The pyrene fluorescence emission spectra was conducted using a fluorescence spectrometer 5

ACCEPTED MANUSCRIPT 156

(F-4600, Japan) at 25 ℃ (Yu, Hu, Pan, Yao, & Jiang, 2006). Briefly, recrystallized pyrene was

157

dissolved in acetone to prepare stock solution (2×10-5 g/mL). The nanoparticle solutions (0.6%,

158

w/v) were adjusted to various pH values (pH 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0) with 0.1 M HCl or 0.1

159

M NaOH. Subsequently, the pyrene solution was diluted 100 times by using nanoparticle solutions

160

with different pH values, and the resultant solutions were equilibrated at 4 °C for 60 h before

161

analysis. The fluorescence excitation wavelength was 335 nm, the emission wavelength range was

162

350-500 nm, and the excitation and emission slit width was 5 nm and 2.5 nm, respectively.

163

2.8. Stability of OVA/CMC nanoparticles

164

In order to determine the pH, salt stability and storage time of biopolymer particles formed by

165

heating OVA/CMC complexes. The biopolymer nanoparticle suspensions were prepared according

166

to the method of 2.2. Then, the pH (4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0) and/or ionic strength (20, 50,

167

100, 200, 400 mM) of the suspensions were adjusted to various values and examined the stability of

168

the nanoparticles to aggregation using dynamic light scattering measurements. Additionally, the

169

stability of OVA / CMC nanoparticle suspensions stored at room temperature for 30 days was also

170

evaluated by tracking the change in their average particle size.

171 172

2.9. Dynamic interfacial surface pressure (π) and interface viscoelasticity measurement 2.9.1. Measurement of interfacial surface pressure

173

The interfacial surface pressure (π) and adsorption kinetics of OVA/CMC nanoparticles at the

174

oil/water interface were performed by using automated drop tensiometer (Tracker-H, Teclis,

175

France) at 25 ℃. For this test, the bulk phase (OVA/CMC complexes or nanoparticles) and oil

176

phase (medium chain fatty acid, MCT) were placed in the cuvette and syringe, respectively. The

177

total biopolymer concentration was fixed at 0.6% (w/v), and the test model was raising hanging

178

drop. Before test, dispersions and oil were allowed to stay for at least 1 h to reach 25 ℃. The

179

temperature of the system was maintained constant by circulating water from a thermostat. The oil

180

droplet volume was 10 µL during the test. The oil/water interface pressure (π, mN m-1) was

181

calculated as follow formula:

182

π (mN m-1) = γ0-γ

183

Where γ0 (mN m-1) and γ (mN m-1) is the interfacial tension of pure oil-distill water (26 mN/m)

184

and sample solutions, respectively. The surface tension was determined by the drop shape analysis

185

(Benjamins, Cagna & Lucassen-Reynders, 1996). All the measurements were performed up to 3 h.

186

2.9.2. Measurement of interfacial viscoelasticity

187

(1)

The dynamic interface viscoelasticity of OVA/CMC nanoparticles at the oil−water interface 6

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was investigated using sinusoidal interfacial compression and expansion procedure at 25 ℃.

189

Specially, OVA/CMC complexes or nanoparticles and oil were placed in the cuvette and syringe,

190

respectively. The total biopolymer concentration was fixed at 0.6% (w/w). The sinusoidal

191

interfacial compression and expansion were performed by the changing of drop volume at 10% of

192

deformation amplitude within the linear regime, and the oscillation frequency was 0.1 Hz. The

193

numbers of active and blank were 5 cycles during the experiments. The other parameter settings and

194

experimental procedures were the same as 2.7.1.

195

2.10. Statistical analysis

196

All measurements were carried out in triplicate unless otherwise stated. One-way analysis of

197

variance (ANOVA) with a 95% confidence interval was used to assess the significance of the results

198

obtained. Statistical analysis was performed using SPSS software version 19.0.

199

3. Results and discussion

200

3.1 Fabrication of OVA/CMC nanoparticles

201

To

prepare

nanoparticles

from

electrostatic

self-assembled

complexes

of

202

protein/polysaccharide, it was necessary to determine the pH interval of the soluble complexes of

203

protein/polysaccharide (Schmitt, Sanchez, Desobry-Banon, & Hardy, 1998; Jones, Decker, &

204

McClements, 2010; Jones, & McClements, 2010b). According to our previous work, when the mass

205

ratio of OVA to CMC was 4:1, both CMC 0.7 and CMC 1.2 interacted with OVA through

206

electrostatic attraction to form soluble complexes at pH 4.4 (Xiong et al., 2017). Therefore, this

207

condition was used to prepare OVA/CMC nanoparticles in this study.

208

Obviously, heating caused the unfolding and aggregating of protein, resulting in an increase in

209

the size of OVA/CMC complexes, whereas there was no significant change in the polydispersity

210

index (PDI) of the OVA/CMC complexes after heat treatment (Table 1). It is worth noting that the

211

particles size of OVA/CMC 0.7 both before and after heat treatment were greater than that of

212

OVA/CMC 1.2 particles, respectively (Table 1). This phenomenon can be explained by the

213

difference in charge density between two types of polysaccharides. Based on our past work, the

214

higher charge density of CMC 1.2 produced stronger binding capacity with OVA than CMC 0.7,

215

and formed more compact nanoparticles (Xiong et al., 2017). The same phenomenon was also

216

observed in the study of the preparation of nanoparticles using β-lactoglobulin/pectin (Jones,

217

Lesmes, Dubin, & McClements, 2010). In addition, an increase in aggregated size of OVA/CMC

218

complexes upon heating was also observed with an AFM imaging, and the particle size of 7

ACCEPTED MANUSCRIPT 219

OVA/CMC 0.7 was larger (Fig.1). After heating, it was found from these images that the size of the

220

spherical particles was approximately 150-300 nm, which was also approximately in agreement

221

with the result of the dynamic light scattering technique.

222

On the other hand, heating resulted in a significant increase in the zeta potential of OVA/CMC

223

nanoparticles (Table 1), indicating an obvious change in the structure of OVA/CMC complexes

224

after heat treatment. At the same time, this enhancement of electrostatic repulsion could also

225

improve the stability of the formed biopolymer particles after heat treatment. Furthermore，to

226

further investigate the mechanism of OVA/CMC nanoparticles formed by heating (90 ℃, 30 min)

227

at pH 4.4, the compositions of the biopolymer complexes and particles were measured by analyzing

228

polysaccharide and protein concentrations after centrifugation. After high-speed centrifugation,

229

both the OVA/CMC complexes and the nanoparticles were separated to the bottom of centrifuge

230

tube and the results of protein and polysaccharide content in the supernatant were presented in

231

Table 1. The supernatant after centrifugation contained a large number of OVA (over 30%) and

232

CMC (over 80%) before the heat treatment. Meanwhile, the mass ratio of OVA to CMC 0.7 and

233

CMC 1.2 in the pellet after centrifugation without heat treatment was 13:1 ((0.48-0.22)/(0.12-0.1))

234

and 16.5:1 ((0.48-0.15)/(0.12-0.1)), respectively. These results indicate that an appropriate fraction

235

of OVA and CMC did not precipitate after centrifugation, suggesting that they may remain in the

236

supernatant as individual molecules or as soluble biopolymer complexes. However, the OVA

237

content in the supernatant of the heat-treated samples was significantly reduced after centrifugation.

238

The mass ratio of protein to polysaccharide in OVA/CMC 0.7 and OVA/CMC 1.2 pellet after

239

centrifugation with heat treatment was 9.25: 1 and 13.3: 1, respectively. This difference should be

240

attributed to higher charge density of CMC 1.2 than CMC 0.7. In addition, by comparing the mass

241

ratio of OVA to CMC in the pellets before and after heating, we could find that some aggregates of

242

the electrostatic complexes formed by the protein/polysaccharide after heat treatment separated into

243

pellets at the bottom of the centrifuge tube. However, the content of CMC in the supernatant after

244

heat treatment did not decrease significantly, suggesting that OVA was the main component of

245

aggregates.

246

3.2. Structure characterization of OVA/CMC nanoparticles

247

In this section, ultra-small-angle X-ray scattering (USAXS) was used to investigate the

248

microstructure of OVA/CMC nanoparticles. Fig. 2A showed the USAXS intensities I(q) results for

249

biopolymer particles as a function of scattering vector (q) both before and after heat treatment. 8

ACCEPTED MANUSCRIPT 250

Obviously, all the USAXS curves decreased sharply at small q range (0.003 < q < 0.02 nm-1), while

251

decreased slightly at large q range (0.02 nm-1< q < 0.2 nm-1). On the other hand, it was observed

252

that the scattering intensity of OVA/CMC nanoparticles prepared by heat treatment was

253

significantly enhanced in the small q range (0.003 < q < 0.02 nm-1), indicating that the structure of

254

OVA/CMC nanoparticles changed greatly after heat treatment. Additionally, OVA/CMC 1.2

255

nanoparticle exhibited higher scattering intensity than OVA/CMC 0.7 nanoparticles in the same

256

scattering vector range. This observation may be ascribed to the different charge densities of two

257

CMCs. CMC 1.2 carries more negative charges on its chains, which will lead to a relatively larger

258

number of protein molecules bound on polysaccharide chains (Xiong et al., 2017). Consequently,

259

protein molecules will screen the charges of CMC1.2 to a larger extent. In OVA/CMC 1.2

260

nanoparticles, CMC chains carrying protein molecules will then aggregate more tightly to form a

261

network than OVA/CMC0.7 nanoparticles. On the other hand, the power law relationship exponent

262

between the scattering intensity (I(q)) and scattering vectors (q) at small q range can be used as the

263

fractal dimension (df ) of particle aggregation (Shi et al., 2017), and the results were presented in

264

Fig. 2A. After heat treatment, the df values of OVA/CMC 0.7 and OVA/CMC 1.2 nanoparticles

265

increased from 2.43 and 2.14 to 3.29 and 2.90, respectively. This result indicated that the thermally

266

induced OVA/CMC nanoparticles have a gel-like structure with ideal df of 3.0, and further

267

demonstrated that the nanoparticles have a larger size and density after heat treatment than the

268

native complexes (Shi et al., 2017). Additionally, the fractal dimensions of OVA/CMC 0.7

269

nanoparticles before and after heating were larger than that of OVA/CMC 1.2 nanoparticles, which

270

should be attributed to the larger number and size of complexes or aggregates in OVA/CMC0.7

271

system. Because CMC 1.2 with higher charge density can bind more OVA molecules and form

272

more compact, smaller particles, and this phenomenon can be confirmed by the average diameter of

273

the particles (Table 1).

274

Furthermore, the scattering intensity patterns can be better distinguished using the Kratky plots

275

(I(q)q2 vs q), which is used to detect the folding conformation of proteins and densely packed

276

complexes (Shi et al., 2017). Fig. 2B exhibited the Kratky plot which was employed extensively to

277

characterize the biopolymer particle structure and highlight the scattering characteristics in small q

278

range (0.003 < q < 0.02 nm-1). Kratky plots showed a peak at finite q (0.003 < q < 0.004 nm-1),

279

implying that the presence of a folded domain (Shi et al., 2017). On the other hand, by comparing

280

the change of Kratky curves before and after heating in the small q range (0.003 < q < 0.02 nm-1), it 9

ACCEPTED MANUSCRIPT 281

was further proved that heating rendered the structure of OVA/CMC nanoparticles more compact.

282

Indeed, the upturn in large q range (0.02 nm-1< q < 0.2 nm-1) possibly corresponds to an increase of

283

loops in proteins, which may be attributed to the change of protein secondary structure (Doniach,

284

2001).

285

3.3. Pyrene probe fluorescence spectra

286

Pyrene, a strongly hydrophobic fluorescent probe, has 5 emission peaks after excitation at 335

287

nm, the ratio of the fluorescence intensity I1 at peak 1 (373 nm) to the fluorescence intensity I3 at

288

peak 3 (385 nm) (I1/I3) strongly depends on the polarity of the surrounding microenvironment,

289

lower I1/I3 value reflects higher hydrophobicity of the microenvironment (Aguiar, Carpena, Molina-

290

Bolívar, & Ruiz, 2003). The change of I1/I3 values of OVA/CMC nanoparticles as a function of pH

291

was presented in Fig.3. The I1/I3 values of OVA/CMC 0.7 and OVA/CMC 1.2 nanoparticles were

292

about 1.4 and 1.2 in the pH range of 4-7, respectively. This result indicated that the OVA/CMC

293

nanoparticles was relatively hydrophobic, which may be due to the fact that heat treatment induced

294

the hydrophobic residues inside the protein to be exposed on the surface. Ovalbumin-chitosan

295

nanogels formed by the heating method also showed similar properties (Yu, Hu, Pan, Yao, & Jiang,

296

2006). In addition, it is noteworthy that the I1/I3 values of OVA/CMC 1.2 nanoparticles were

297

significantly lower than that of OVA/CMC 0.7 nanoparticles throughout the experimental pH range,

298

which meant that the surface of OVA/CMC 1.2 nanoparticles had a stronger hydrophobic

299

performance. This phenomenon may be attributed to the fact that CMC 1.2 has a higher charge

300

density, which can combine more ovalbumin molecules. On the other hand, the heat-induced

301

OVA/CMC 1.2 nanoparticles are more compact and smaller in size, exhibiting larger specific

302

surface area than OVA/CMC 0.7 nanoparticles, thereby resulting in more pyrene-bound

303

hydrophobic region.

304

3.3. pH, ionic strength and storage time stability of OVA/CMC nanoparticles

305

The effects of pH and salt on the stability of biopolymer particles formed by heating

306

OVA/CMC soluble complexes were determined in this section. Initially, the suspensions of

307

biopolymer particles were formed by heating OVA (0.48%, w/v) and CMC (0.12% CMC 0.7 or

308

CMC 1.2, w/v) soluble complexes at pH 4.4 (90 ℃, 30 min), then were cooled to room temperature

309

with ice-water bath. Subsequently, the pH and/or ionic strength OVA/CMC nanoparticles

310

suspensions were adjusted, and the stability of the biopolymer particles against aggregation was

311

examined by monitoring the changes of particle size and zeta potential. The effect of pH on the 10

ACCEPTED MANUSCRIPT 312

stability of OVA/CMC nanoparticles was presented in Fig. 4. Obviously, when the pH was adjusted

313

from 4 to 7, the average particle sizes of OVA/CMC 0.7 and OVA/CMC 1.2 nanoparticles

314

progressively increased from 285 to 395 nm and 185 to 275 nm, respectively (Fig. 4A), while the

315

PDI did not change significantly (Fig. 4B). This result suggested that a small number of dissociation

316

of the heat-induced aggregates may occur at pH 5 or above, which should be attributed to the

317

electrostatic repulsion between negatively charged OVA and CMC in the pH range from 5 to 7 (Fig.

318

4C) (Jones, Lesmes, Dubin, & McClements, 2010). On the other hand, as pH decreased from 4 to 3,

319

the mean diameter of the heat-induced particles increased significantly (data not shown), and visible

320

precipitates could be observed, which was due to the extensive aggregation of the biopolymer

321

complexes occurred. The origin of this effect can be attributed to the reduction in net charge on

322

biopolymer particles through complexation between newly formed positively charged OVA

323

segments and free CMC (Jones, Lesmes, Dubin, & McClements, 2010).

324

The salt stability of the OVA/CMC nanoparticles was presented in Fig. 5. The average

325

diameter of OVA/CMC nanoparticles gradually increased with the rise of sodium chloride

326

concentration (Fig. 5A), while PDI showed an opposite trend (Fig. 5B). This finding indicated an

327

appreciable increase in aggregation with the addition of NaCl. Indeed, the presence of salt ions

328

greatly reduced the range and magnitude of electrostatic interactions in the protein-polysaccharide

329

system, which would result in the dissociation of some particles and weaken the electrostatic

330

complexation between the biopolymers (Jones, Lesmes, Dubin, & McClements, 2010). This point

331

could be confirmed by the reduction of the zeta potential of the OVA/CMC suspensions as the

332

increase of salt ion concentration (Fig. 5C). Moreover, OVA/CMC nanoparticles had excellent

333

storage stability after storage at 25 ℃ for 30 days, the mean particle size and PDI did not change

334

significantly (P>0.05). The excellent stability of OVA/CMC nanoparticles means that they have

335

potential value as carriers for nutrient delivery.

336

3.4. Interfacial adsorption and dilatational rheological properties

337

3.4.1 Adsorption kinetics and structural rearrangements at the oil-in-water interface

338

To evaluate the emulsification properties and mechanism of biopolymer particles, the

339

absorption behavior of OVA/CMC complexes and nanoparticles at the oil-in-water interfaces were

340

examined in this section. Generally, dynamic adsorption behavior of colloid particles in the oil-in-

341

water interface in turn mainly involve diffusion, actual adsorption (penetration and unfolding) and

342

conformational reorganization (Dickinson 2008). In all cases, the interfacial surface pressure was 11

ACCEPTED MANUSCRIPT 343

increased with adsorption time (Fig. 7A), which could be related to the particle adsorption at the

344

interface (Perez, Carrara, Sánchez, Santiago, & Patino, 2009; Wan, Wang, Wang, Yuan, & Yang,

345

2014). The change in interfacial surface pressure (π) with adsorption time (t) can be correlated by a

346

modified form of Ward and Tordai equation (Ward, & Tordai, 1946):

347

π=2C0KBT(Dt/3.14)1/2

348

Where C0 is the concentration in the continuous phase, KB is the Boltzmann constant, T is the

349

absolute temperature, and D is the diffusion coefficient. If the adsorption process is controlled by

350

the protein diffusion, a plot of π versus t1/2 will then be linear, and the slope of this plot will be the

351

diffusion rate (Kdiff), as shown in Fig. 7A. As calculated using Equation (2), the diffusion rate of

352

OVA/CMC 0.7 and OVA/CMC 1.2 nanoparticles formed by heating was 0.2833 and 0.2852

353

mN/m/s1/2, respectively, it was basically equal to the samples before heat treatment (OVA/CMC 0.7

354

and OVA/CMC 1.2 was 0.2786 and 0.2789 mN/m/s1/2, respectively). However, the initial surface

355

pressure of OVA/CMC 0.7 (π0=2.48 mN/m) and OVA/CMC 1.2 (π0=1.18 mN/m) nanoparticles and

356

the final surface pressure (π10800) after 3 h adsorption was less than those of the samples before

357

heating. Those results can be contributed to the change of structure of OVA/CMC nanoparticles

358

after heating. On one hand, heat treatment induced OVA unfolding and aggregation, leading to a

359

significant increase in the size and surface net potential of OVA/CMC nanoparticles (Table 1).

360

These changes increased the electrostatic repulsion and steric hindrance between the nanoparticles,

361

resulting in the nanoparticles exhibiting lower π0 values than the samples before heating (Delahaije,

362

Gruppen, Giuseppin, & Wierenga, 2014). On the other hand, the enhancement of surface

363

hydrophobicity after heating was beneficial to improve the adsorption rate of nanoparticles from

364

bulk to oil-water interface (Wierenga, Meinders, Egmond, Voragen, & de Jongh, 2003; Delahaije,

365

Wierenga, van Nieuwenhuijzen, Giuseppin, & Gruppen, 2013; Delahaije, Gruppen, Giuseppin, &

366

Wierenga, 2014). Moreover, since CMC 1.2 had a higher charge density than CMC 0.7, the

367

electrostatic repulsion between the OVA/CMC 1.2 nanoparticles was larger than that of OVA/CMC

368

0.7, thereby reducing the adsorption amount of OVA/CMC 1.2 nanoparticles at the interface.

369

Therefore, lower π0 and π10800 values were shown in the OVA/CMC 1.2 system both before and

370

after heat treatment, despite the higher surface hydrophobicity of OVA/CMC 1.2 nanoparticles after

371

heat treatment.

372 373

(2)

Furthermore, the rates of penetration and rearrangement of adsorbed layer at the interface can be analyzed by the first-order equation: 12

ACCEPTED MANUSCRIPT 374

ln[(πf-πt)/ (πf-π0)] =-kit

(3)

375

Where πf, π0, and πt are the interfacial pressures at the final adsorption time, at the initial time, and

376

at any time of each stage, respectively, and ki is the first-order rate constant. The application of Eq.

377

(3) to the adsorption of the biopolymer particles at the interface was presented in Fig. 7B. Clearly,

378

there were two linear regions in these plots. Generally, the first slope is usually regarded as a first-

379

order rate constant of penetration (KP), while the second slope takes to a first-order rate constant of

380

molecular reorganization (KR) (Perez, Carrara, Sánchez, Santiago, & Patino, 2009). Interestingly,

381

the differences in CMC charge density did not have any effect on the rate of OVA molecule

382

penetration (KP) and rearrangement (KR) at the interface. The KP and KR were -3.0 and -51 (×10-4, s-

383

1)

384

treatment, respectively. This change should be attributed to the increased hydrophobicity and

385

flexibility of the protein after heating (Wang et al., 2012).

386

3.4.2. Dilatational rheological properties at the oil-in-water interface

before heat treatment, respectively, and the KP and KR were -2.0 and -37 (×10-4, s-1) after heat

387

In general, the viscoelastic properties of the adsorbed layer at oil-in-water interfaces can be the

388

prediction of emulsions stability (Murray, 2002). Therefore, the surface dilatational modulus (E)

389

can reflect the mechanical strength of the protein interfacial absorbed layer, which derives from the

390

change in interfacial tension (γ) (dilatational stress) resulting from a small change in surface area

391

(dilatational strain) (Lucassen& van den Tempel, 1972). The surface dilatational modulus (E,

392

E=Ed+iEv) includes real (storage component, Ed=|E|cosδ) and imaginary parts (loss component,

393

Ev=|E|sinδ), in which the phase angle (δ) between stress and strain is a representation of the relative

394

viscoelasticity of interfacial absorbed layer (Rodríguez Patino, Rodríguez Niño & Carrera Sánchez,

395

1999).

396

The evaluation of surface dilatational modulus (E) with interfacial surface pressure (π) in the

397

surface layer for the adsorption of was presented in Fig. 8A. The E values were increased

398

immediately due to the adsorption of protein at the interface in all cases, and the slopes of the E

399

versus π curves were higher than 1.0, suggesting a non-ideal behavior for stronger molecular

400

interactions between the film-forming components of protein and polysaccharide, not just between

401

the amount of protein molecules or OVA/CMC particles adsorbed at the oil-in-water interface

402

(Camino, & Pilosof, 2011; Wang, et al., 2012). However, the E-π curve slopes of the heat-treated

403

samples increased, especially OVA/CMC 1.2 nanoparticle system, which meant that a greater

404

amount of proteins were needed at the interface to establish intermolecular interactions. In addition, 13

ACCEPTED MANUSCRIPT 405

the dilatational modulus (E) of the heat-induced nanoparticle-adsorbed layer was significantly

406

smaller than that of the native OVA/CMC complex system, which indicated that the interaction

407

between the nanoparticles on the interface was weak. This result should be attributed to the increase

408

in the size and surface net potential of biopolymer particles after heat treatment, resulting in

409

enhanced electrostatic repulsion and steric hindrance between the particles (Delahaije, Gruppen,

410

Giuseppin, & Wierenga, 2014). At the same time, it is noteworthy that the native OVA/CMC 1.2

411

system exhibited a larger interfacial modulus than the OVA/CMC 0.7 system, probably due to the

412

smaller size of the OVA/CMC 1.2 particles and resulting in closer alignment at the interface.

413

Furthermore, the dynamic dilatational elastic modulus (Ed) of interfacial layers as a function of

414

absorption time was presented in Fig. 8B. Clearly, the Ed values were gradually increased due to the

415

adsorption of proteins on the interface and the establishment of intermolecular interactions. After

416

adsorption for 180 min, the dilatational elastic modulus (Ed) of all samples was significantly greater

417

than the viscosity modulus (Ev) (data not shown), indicating that the interface absorbed layer mainly

418

exhibited the elastic behavior. Nevertheless, samples before and after heat treatment showed

419

distinctly different plot profiles. The non-heat-treated OVA/CMC complex system quickly

420

exhibited higher Ed values, and remained essentially unchanged with the adsorption. However, the

421

Ed values of the OVA/CMC nanoparticle system formed by the heating method showed a slow

422

increasing trend, and the final Ed values were significantly lower than that of the native OVA/CMC

423

complex system. This phenomenon should be closely related to the adsorption, diffusion and

424

structure changes of the biopolymer particles before and after heat treatment. Although the surface

425

hydrophobicity of the heated particles was significantly increased, while the increase of its size and

426

surface net potential greatly hindered its diffusion, adsorption and rearrangement to the interface.

427

More importantly, the above changes in particle size and surface potential would greatly reduce the

428

amount of interfacial adsorption and the interaction between interfacial layer molecules, thereby

429

exhibiting lower interfacial modulus values (Delahaije, Gruppen, Giuseppin, & Wierenga, 2014).

430

4. Conclusions

431

In summary, a convenient and green heating method (90 ℃, 30 min) was used to process the

432

soluble OVA/CMC complexes formed by electrostatic attraction at pH 4.4, and negatively charged

433

biopolymer nanoparticles (200–260 nm) in an aqueous suspension were obtained. Furthermore, the

434

charge density of CMC played an important role in the size, structure and physicochemical

435

properties of the formed particles by heat treatment of the electrostatic associative complexes. 14

ACCEPTED MANUSCRIPT 436

Compared with OVA/CMC 0.7 nanoparticles, OVA/CMC 1.2 (with higher charge density)

437

nanoparticles had smaller size, tighter structure, stronger surface absolute potential and

438

hydrophobicity. Meanwhile, thermally-induced OVA/CMC nanoparticles had excellent pH (4-7),

439

ionic strength (0-400 Mm) and storage stability (30 days, 25℃), the particle size of OVA/CMC 0.7

440

and OVA/CMC 1.2 nanoparticles were maintained below 400 and 270 nm, respectively. In

441

addition, the increase of nanoparticle size and surface net potential after heat treatment has a strong

442

influence on the diffusion, adsorption and rearrangement rates at the oil-water interface, resulting in

443

lower surface pressure and viscoelastic properties of the interface adsorption layer.

444

Acknowledgments

445

The authors acknowledge the financial support from the National Key R&D Program of China

446

(Program No. 2017YFD0400200), the Natural Science Foundation of China (NSFC, Grant No.

447

31772015) and Wuhan Yellow Crane Special Talents Program.

448

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18

ACCEPTED MANUSCRIPT

1 2

Fig. 1. AFM micrographs (2D, 3D) of OVA/CMC 0.7 (A, A’ Unheated; B, B’ Heated) and OVA/CMC 1.2 (C, C’

3

Unheated; D, D’ Heated) nanoparticles formed at pH 4.4 without salt ionic (images are 5×5 µm). The mass ratio

4

of OVA:CMC=4:1.

5 6 7 8 9 10 11 12 1

ACCEPTED MANUSCRIPT

13 14

Fig. 2. USAXS scattering intensity profile (A) and Kratky plot (B) of OVA/CMC nanoparticles.

15 16 17 18 19 20

21 22

Fig. 3. I1/I3 ratio of pyrene fluorescence of OVA/CMC nanoparticles solution as a function of pH. The ratio of

23

OVA to CMC is 4:1 (w/w).

2

ACCEPTED MANUSCRIPT

24 25

Fig. 4. Effect of pH on the average particle size (A), polydispersity index (PDI)(B) and Zeta-potential (C) of

26

OVA/CMC nanoparticles.

27 28

Fig. 5. Effect of ionic strength on the average particle size (A), polydispersity index (PDI) (B) and Zeta-potential

29

(C) of OVA/CMC nanoparticles.

30 31

Fig. 6. Average particle size and polydispersity index (PDI) of nanoparticles, measured after storage at 25 ℃ for

32

1 (A, C is OVA/CMC 0.7 and OVA/CMC 1.2, respectively) and 30 days (B, D is OVA/CMC 0.7 and OVA/CMC

33

1.2, respectively).

3

ACCEPTED MANUSCRIPT

34 35

Fig. 7. (A)Square root of time (t1/2) dependence of surface pressure (π) for native and heat-treated OVA/CMC

36

nanoparticles adsorbed layers at the oil-in-water interface. Kdiff represent diffusion rate. (B) Typical profile of the

37

molecular penetration and configurational rearrangement steps at the oil-water interface for native and heat-treated

38

OVA/CMC nanoparticles. Kp and Kr represent first-order rate constants of penetration and rearrangement,

39

respectively. Total polymer concentration in the bulk phase is 0.6% (OVA:CMC=4:1, w/w).

40 41

Fig. 8. Surface dilatational modulus (E) as a function of surface pressure (π) for native and heat-treated

42

OVA/CMC nanoparticles at the oil-in-water interface (A). Time-dependent dilatational elasticity (Ed) for native

43

and heat-treated OVA/CMC nanoparticles adsorbed layers at the oil-in-water interface (B). Total polymer

44

concentrations in the bulk phase is 0.6% (OVA:CMC=4:1, w/w). Frequency is 0.1 Hz. Amplitude of

45

compression/expansion cycle is 10%.

46

4

ACCEPTED MANUSCRIPT Highlights  The biopolymer nanoparticles were fabricated by heating OVA/CMC complexes.  The charge density of CMC had an important effect on the properties of the nanoparticles.  OVA/CMC 1.2 nanoparticles have a smaller size and a tighter spherical structure than OVA/CMC 0.7.  The surface hydrophobicity of OVA/CMC1.2 nanoparticles was higher than OVA/CMC0.7.  The viscoelasticity modulus of OVA/CMC nanoparticle was lower than native OVA/CMC complexes.

1

ACCEPTED MANUSCRIPT 1

Table 1 Characteristics of particles in dispersions with mass ratio of OVA:CMC=4:1 at pH 4.4 before and after

2

heating at 90 ℃ for 30 min1. polydispersity Samples

Size(nm)

OVA

CMC

(%, w/v)

(%, w/v)

Zeta potential (mV) index (PDI)

OVA/CMC 0.7-Unheated

201.9±19.9a

0.33±0.03a

-23.7±1.23a

0.22±0.03a

0.10±0.01a

OVA/CMC 0.7-Heated

260.3±11.9b

0.34±0.08a

-31.0±0.96b

0.11±0.02b

0.08±0.02a

OVA/CMC 1.2-Unheated

170.4±11.5c

0.39±0.03a

-26.8±1.40c

0.15±0.02c

0.10±0.01a

OVA/CMC 1.2-Heated

203.5±18.6a

0.38±0.05a

-34.6±1.63d

0.08±0.01d

0.09±0.01a

3

1

4

indicate significant difference (P <0.05).

Data are mean ± standard deviation (SD) from triplicates. Different superscript letters (a, b, c, d) in the samples

1