Fabrication and characterization of emulsions stabilized by tannic acid-wheat starch complexes

Journal Pre-proof Fabrication and characterization of emulsions stabilized by tannic acid-wheat starch complexes Xianling Wei, Jing Li, Mohamed Eid, Bin Li PII:

S0268-005X(19)32415-4

DOI:

https://doi.org/10.1016/j.foodhyd.2020.105728

Reference:

FOOHYD 105728

To appear in:

Food Hydrocolloids

Received Date: 15 October 2019 Revised Date:

11 January 2020

Accepted Date: 30 January 2020

Please cite this article as: Wei, X., Li, J., Eid, M., Li, B., Fabrication and characterization of emulsions stabilized by tannic acid-wheat starch complexes, Food Hydrocolloids (2020), doi: https:// doi.org/10.1016/j.foodhyd.2020.105728. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

Author Statement: Xianling Wei: Conceptualization, Methodology, Investigation, Data curation, Writing- Original draft preparation. Jing Li: Validation, Visualization, Project administration. Mohamed Eid: Formal analysis, Writing- Reviewing and Editing. Bin Li: Supervision, Funding acquisition.

Graphical abstract

1

Fabrication and characterization of emulsions stabilized by tannic acid-wheat

2

starch complexes

3

Xianling Wei a, b, Jing Li a, b, Mohamed Eid a, b, Bin Li a, b, *

4

a

5

430070, China

6

b

7

University), Ministry of Education, China

College of Food Science and Technology, Huazhong Agricultural University, Wuhan

Key Laboratory of Environment Correlative Dietology (Huazhong Agricultural

8 9 10

*

Corresponding author: Bin Li

E-mail address: [email protected]

11

1

12

Abstract

13

Soybean oil-based emulsions stabilized by tannic acid (TA) and wheat starch (WS)

14

complexes were prepared via a simple high-intensity ultrasound emulsification

15

technique. The effects of mass ratio of TA/WS (0.005 to 0.75) and WS concentration

16

(0.25%, 0.5% and 1.0%) on TA/WS complexes and emulsions were investigated. As

17

the mass ratio increased, TA and WS could gradually form soluble and insoluble

18

complexes, and their hydrophobicity also improved. Consequently, the tendency of

19

the particle size of emulsions to decrease first and then increase appeared with the

20

increasing mass ratio, mainly due to the changes in the contents, size and

21

hydrophobicity of the complex emulsifier. Additionally, the emulsifying capacity of

22

TA/WS complexes improved with increasing concentration of complexes. Moreover,

23

the creaming was inhibited at a high mass ratio or high complex concentration due to

24

the formation of a thick interfacial network around oil droplets or obtention of

25

smaller-sized droplets. Emulsion gel was formed due to TA cross-linked WS on the

26

interface within different oil droplets, and the strength of the gel increased with the

27

mass ratio due to increasing amounts of complexes being absorbed on the interface.

28

When the mass ratio and WS concentration were no less than 0.05 and 0.5%,

29

respectively, emulsions had better oxidative stability. The complexes also had high

30

resistance against droplet coalescence. This study demonstrated that TA/WS

31

complexes could be employed as an emulsifier to improve the oxidative stability of

32

O/W emulsions and that easily oxidized, oil-soluble nutrients or medicines could be

33

protected and delivered through these emulsion systems.

34

Keywords: wheat starch; tannic acid; emulsions; emulsifying capacity; oxidative

35

stability

2

36

1. Introduction

37

Lipids and fat-soluble nutrients are often formulated into emulsions to improve their

38

stability and bioavailability (Wang et al., 2018; Xu, Tang, Liu, & Liu, 2018). These

39

emulsions

40

octenyl-succinic-anhydride-modified starches, Tween 80, modified silica particles and

41

cellulose nanocrystals (Alison et al., 2016; Kalashnikova, Bizot, Cathala, & Capron,

42

2011; Li et al., 2019; Zhao et al., 2018). Although these emulsifiers have good

43

emulsification, the demand of consumers for more natural food products has led to an

44

increasing interest among food enterprises to replace nonnatural ingredients with

45

other, more natural ingredients (McClements, Bai, & Chung, 2017). However, among

46

emulsifiers, just a few natural emulsifiers have outstanding emulsifying capacity, such

47

as ovalbumin (Xu et al., 2018); most have bad emulsifying capacity, especially

48

polysaccharides (Huang, Kakuda, & Cui, 2001; Kasprzak, Macnaughtan, Harding,

49

Wilde, & Wolf, 2018). In detail, these defects (high hydrophobicity or hydrophilicity)

50

lead to them disperse in oil or water phases, not at the oil-water interface, resulting in

51

lower emulsifying capacity.

52

Various methods were used to improve the emulsification of biomacromolecules

53

(proteins

54

modification, compound emulsifier and noncovalent interactions (Jiang et al., 2018;

55

Li, Ye, Lei, Zhou, & Zhao, 2018; Lu, Wang, Li, & Huang, 2018; Zou, Guo, Yin, Wang,

56

& Yang, 2015). Chemical modification can use unavoidably harsh chemicals, and/or

57

may remain at low concentrations in the final product, which are increasingly

58

unacceptable to consumers. The physical modification and compound emulsifier

59

methods usually only improve the emulsification activity and rarely confer new

60

features, such as antioxidation or pH response. However, the noncovalent interactions,

61

including hydrophobic, electrostatic, and hydrogen bonding reactions, could not only

62

improve the emulsification activity but also be exploited to obtain complexes with

63

important properties (improved hydrophobicity, antioxidation, and rheology).

64

Moreover, such methods are green and safe. The emulsifying properties of citrus

65

nanofibers and the oxidative stability of emulsions stabilized by citrus nanofibers

are

and

usually

stabilized

polysaccharides),

by

including

3

nonnatural

chemical

emulsifiers,

modification,

such

as

physical

66

were improved when citrus nanofibers complexed with TA, forming complexes

67

through hydrogen bonds (Wang et al., 2018). The emulsifying properties,

68

hydrophobicity of zein and rheological properties of emulsions stabilized by zein

69

were all controllable through regulating the ratio of zein/TA and complex

70

concentrations (Zou et al., 2015; Zou, Baalen, Yang, & Scholten, 2018). Renewable

71

natural starches, which are widely distributed and inexpensive, have attracted great

72

attention thanks to their beneficial roles as energy foods for human health. For

73

emulsifiers, the abundant hydroxyl groups in starch can lead to high hydrophilicity

74

and poor emulsification, which greatly limit their applications in emulsion systems.

75

Thus, to produce stable emulsions, the hydrophilicity of starches must be decreased

76

by the incorporation of hydrophobic components, such as OSA-modified starch,

77

starch nanocrystals obtained by acid hydrolysis (Jo, Ban, Goh, & Choi, 2018; Li et al.,

78

2018; Yang et al., 2018). However, at present, improvement of the emulsifying

79

properties of starches involves fewer noncovalent modifications; rather, chemical and

80

physical modifications are involved.

81

Lipids and fat-soluble nutrients in emulsions are unstable and susceptible to oxidative

82

degradation, resulting in the development of off-flavors and a loss of nutritional

83

quality, hence affecting the sensory properties and shelf stability of products (Johnson,

84

Inchingolo, & Decker, 2018; McClements & Decker, 2017). Therefore, antioxidants

85

must be used in emulsions to inhibit lipid oxidation and extend their shelf life. In

86

addition, the effectiveness of antioxidants in emulsions depends on not only their

87

surface activity but also their capacity to accumulate at the oil-water interface, where

88

oxidative reactions are most common (Decker et al., 2017). It is well known that plant

89

polyphenols are good antioxidants, which can delay the oxidative electron transfer of

90

lipids. However, they usually have bad surface activity due to the abundance of

91

hydroxyls. It has been reported that plant polyphenols can bind to polysaccharides

92

through noncovalent interactions, resulting in improvements to their hydrophobicity

93

and oxidation stability of emulsions (Jin et al., 2017; Wang et al., 2018). Hence, the

94

antioxidant effects of polyphenols on lipid oxidation in emulsions could be improved

95

by forming complexes with polysaccharides, which could then absorb at the oil-water 4

96

interface (Patel, Seijen-ten-Hoorn, Hazekamp, Blijdenstein, & Velikov, 2013; Wang et

97

al., 2018). Polysaccharides and polyphenols can interact with each other through

98

covalent or noncovalent bonds (Renard, Watrelot, & Le Bourvellec, 2017).

99

Noncovalent bonds, including hydrogen bonding, electrostatic interactions and

100

hydrophobic interactions, are more common in food systems (Chai, Wang, & Zhang,

101

2013; Li et al., 2019; Renard et al., 2017; Wang et al., 2018). TA is a naturally derived

102

polyphenolic

103

macromolecules at multibinding sites through multiple interactions, including

104

hydrogen bonding and hydrophobic interactions (Patel et al., 2013; Wei, Li, & Li,

105

2019). Patel et al. (2013) studied the colloidal complexes of TA and methylcellulose,

106

and the complexes exhibited excellent surface activity and were further used for

107

stabilization of emulsions and foams. In addition to the well-known antioxidant

108

properties of TA, a simple mixing approach based on the interaction between

109

macromolecules and plant polyphenols has been successfully applied to improve the

110

emulsification performance and oxidation stability of emulsions (Karefyllakis,

111

Altunkaya, Berton-Carabin, van der Goot, & Nikiforidis, 2017; Wang et al., 2018).

112

However, no attempt has been made to improve the emulsification performance and

113

oxidation stability of emulsions stabilized by starches through complexing with plant

114

polyphenols.

115

The emulsifying properties of emulsifiers are mainly affected by the particle size,

116

hydrophobicity and content in emulsions (Li et al., 2018; Saari, Rayner, & Wahlgren,

117

2019). There is usually a three-step interaction process, with increasing ratios of

118

polyphenol/macromolecules, which gradually form soluble complexes, insoluble

119

complexes, and large aggregates, and even precipitation (Charlton et al., 2002; Pascal

120

et al., 2007; Patel et al., 2013). In this progression, the size of the complexes always

121

increases with increasing ratio, which might influence the hydrophobicity and content

122

of the complexes, leading to a fundamental impact on their emulsification. In addition,

123

the network structure formed by polyphenol-crosslinked macromolecules can improve

124

the rheological and antioxidant properties of emulsions (Wang et al., 2018; Zou et al.,

125

2015). In our recent work, we affirmed that TA and WS used the same three-step

compound

and

is

capable

5

of

crosslinking

or

complexing

126

interaction process and analyzed the structures and particle sizes of TA/WS

127

complexes (Wei et al., 2019). However, the emulsifying properties of complexes and

128

the effects on structure formation, rheological properties and oxidation stability of

129

emulsions have been unreported.

130

In this work, TA was used as an efficient tool to control the self-assembly behavior of

131

WS to fabricate stable and edible TA/WS complexes and consequently prepare

132

soybean oil-based emulsions. We attempted to investigate the effects of the TA/WS

133

ratio and WS concentration on the complexes (size and hydrophobicity), their

134

emulsification performance and the protection of lipid droplets against oxidation. For

135

this purpose, the WS solution was prepared and modified with TA to form complexes,

136

which then acted as emulsifiers to fabricate emulsions. The complexes were

137

characterized by particle size distribution and contact angle. The emulsions were

138

characterized by optical microscopy, particle size distribution, confocal laser scanning

139

microscopy (CLSM), rheology, color, and physical stability measurements.

140

Furthermore, the oxidative status of the emulsions was also assessed by monitoring

141

the formation of primary and secondary oxidative products. Based on the noncovalent

142

interactions between WS and TA, the emulsifying, rheological and antioxidant

143

properties were all controllable, indicating that these substances could be developed

144

as novel functional food ingredients, which would extend their application in the food

145

industry.

146 147

2. Materials and methods

148

2.1 Materials

149

Wheat grains and soybean oil were purchased from a local supermarket (Wuhan,

150

China). TA was purchased from Aladdin Chemistry Co., Ltd. (Shanghai, China).

151

Fluorescent dyes (Nile Red and Nile Blue A) were purchased from Yuanye

152

Biotechnology Co., Ltd. (Shanghai, China). All other chemical reagents were of

153

analytical grade and used without further purification.

154

2.2 Preparation of WS and TA stock solutions

155

WS was isolated and purified from wheat grains according to the method of Wang et 6

156

al. (2015). The apparent amylose and protein contents of WS were 31.1% and 0.37%,

157

respectively. WS stock solution was prepared according to the published method with

158

minor modifications (Chai et al., 2013). Briefly, WS (8.0 g) was added into 90% (v/v)

159

dimethyl sulfoxide with a final WS concentration of 2.0% (w/v) and was then

160

incubated in a boiling water bath for approximately 60 min with continuous stirring.

161

Ethanol was then added to precipitate WS with a final ethanol concentration of 80%

162

(v/v). After centrifugation at 4000 g for 15 min, the WS precipitation was repeatedly

163

washed with ethanol and centrifuged three times to remove the residual dimethyl

164

sulfoxide. The precipitation was freeze-dried, ground and passed through a 100-mesh

165

sieve. The obtained sample (2.0 g) was dissolved in acetate buffer solutions (10 mM,

166

pH 4.0). It was then heated at 95°C for 60 min with continuous stirring and

167

centrifuged at 4000 g for 10 min at 25

168

The stock solution of TA (5.0%, w/v) was prepared using the same buffer at room

169

temperature.

170

2.3 Preparation of TA/WS complexes and emulsions

171

TA/WS complexes were prepared with magnetic stirring (1000 rpm) at room

172

temperature (25℃). In brief, TA solution (2 mL) at different concentrations was added

173

into WS solution (2.0%, 5 mL) with magnetic stirring. The final TA/WS mass ratios

174

were 0.005, 0.01, 0.025, 0.05, 0.125, 0.25, 0.50, and 0.75, respectively. TA/WS

175

complex dispersions were obtained after stirring for 10 min. Size measurements were

176

performed by Dynamic Light Scattering (DLS) using a Malvern Zetasizer Nano S

177

(Malvern, Worcestershire, Malvern-UK). To perform contact angle measurement,

178

TA/WS complex dispersions were frozen immediately in liquid nitrogen and then

179

freeze-dried using a lyophilizer (LGJ-30FD, Beijing, China).

180

The above-mentioned TA/WS complex dispersions were utilized to prepare soybean

181

oil-based emulsions. All emulsions were prepared using an oil/water ratio of 30:70

182

(v/v). In brief, 3 mL of soybean oil was added to 7 mL of TA/WS complex dispersions

183

in a glass vial (25 mL), and the mixtures were then sonicated using an ultrasound

184

processor (FB705, Fisher Scientific, USA) in an ice-water bath to produce emulsions.

185

The ultrasound probe (FB-4220) consisted of a cylindrical titanium alloy microprobe

to obtain the WS stock solution (2.0%, w/v).

7

186

with a flat tip and was 13 mm in diameter. The ultrasound frequency, amplitude and

187

pulse duration were set at 20 kHz, 95% and on-time 5 s + off-time 20 s, respectively.

188

Samples were sonicated for 1 min (effective processing time with omitting the pulsing

189

times) at 665 W. The final emulsions contained 30% oil phase, 1.0% WS and

190

0.005-0.75% TA (the mass ratios of TA to WS ranged from 0.005 to 0.75). To evaluate

191

the effect of WS concentration (0.25%, 0.5% and 1.0%) on the properties of

192

emulsions, the WS and TA solutions were all diluted 2 and 4 times to prepare

193

complexes and emulsions. Emulsions were sealed in glass bottles for 30 days at room

194

temperature for stability evaluation and creaming index measurements. These

195

emulsions were stabilized by WS (0.25%, 0.5% and 1.0%) were also prepared as the

196

control group.

197

2.4 Characterization of TA/WS complexes

198

2.4.1 Contact angle measurement

199

The three-phase contact angles (θow) of TA/WS complexes were measured using an

200

OCA 15EC (Dataphysics Instruments GmbH, Germany), as described in a previous

201

work (Li et al., 2019). In brief, the freeze-dried complex powders were prepared as

202

pellets 10 mm in diameter and 1 mm in thickness, and the pellets were placed in an

203

optical glass cuvette containing soybean oil. Next, a drop of Milli-Q water (2 µL) was

204

deposited on the surface of the pellets using a high-precision injector. After 5 min for

205

equilibration, the water-drop image was recorded using a high-speed video camera,

206

and the profile of the droplet was numerically solved and fitted to the Laplace-Young

207

equation. The contact angles were measured on each of five pellets per sample, and

208

three measurements were performed for each pellet. The contact angles of the WS and

209

TA powders were also measured as the blank.

210

2.5 Characterization of TA/WS complexes emulsions

211

2.5.1 Optical microscopy observations of emulsions

212

The fresh emulsions were observed with an optical microscope (Sunny CX40, China)

213

equipped with a video camera providing still images. The samples were diluted 5

214

times using acetate buffer, and one drop of the diluted sample was put on a glass slide

215

and covered with a cover slip for microscopic observation using a 40× magnification 8

216

lens.

217

2.5.2 Particle size distribution of emulsions

218

The particle size distribution profiles of various fresh emulsions and several selected

219

emulsions (storage up to 30 days for emulsion stability analysis) were determined

220

using a Mastersizer 2000 (Malvern Instruments Ltd., UK). The samples were diluted

221

with acetate buffer (pH=4) prior to analysis to minimize the effect of multiple light

222

scattering on data interpretation. The pump speed and obscuration rate were set at

223

2000 rpm and 10%, respectively. The refractive indices of the oil and water phase

224

were set at 1.46 and 1.33, respectively.

225

2.5.3 Creaming index (CI) and appearance of emulsions

226

Creaming stability was measured to evaluate the relative stability of emulsions as

227

described by Cai et al. (2018). Fresh emulsions (20 mL) were transferred into glass

228

tubes, tightly sealed with plastic caps and kept at 25°C. The CI was calculated by

229

measuring the total heights of the emulsion (HE, mm) and the serum layer (HS, mm).

230

The movement of any creaming boundary was tracked over time until it was

231

unchanged. The CI as a function of time (CI%(t)) was calculated according the

232

following equation (Eq. (1)):

233

CI%(t) = HS/HE×100 (1)

234

The appearance of emulsions (storage up to 30 days) was recorded with a camera

235

(Nikon D90, Japan).

236

2.5.4 CLSM of emulsions

237

CLSM was utilized to study the microstructures of emulsions. A confocal microscopy

238

(FV 3000, Olympus, Japan) with a 40× magnification lens was used. The sample

239

subjected to CLSM was stained in advance according to the method described by Li et

240

al. (2018) with a slight modification. Approximately 10 µL of Nile Red (1 mg/mL in

241

dimethyl sulfoxide) was used to stain the oil phase (argon laser with an excitation line

242

at 488 nm), and 10 µL of Nile Blue A (1 mg/mL in Milli-Q water) was used to stain

243

the starch (He-Ne laser with an excitation line at 633 nm). Immediately prior to use,

244

the Nile Red and Nile Blue A solutions were mixed and added into 0.2 mL of

245

emulsion (fluorescent dyes:emulsion=1:10, v/v), and they were thoroughly mixed. 9

246

The stained sample (5 µL) was then immediately placed on a glass slide and covered

247

with a cover slip for microscopic observation.

248

2.5.5 Rheological measurements of emulsions

249

The dynamic viscoelastic properties of emulsions were determined using a

250

strain-controlled rheometer (AR2000ex, TA, USA) fitted with parallel plate geometry

251

(60 mm in diameter) at a gap of 1 mm. Briefly, fresh emulsions were loaded and

252

maintained on the plate for 5 min to achieve thermal equilibrium. Strain sweep tests

253

were first conducted to determine the linear viscoelastic region (LVR) at a fixed

254

frequency of 1 Hz (strain = 0.01%-100%). Thereafter, a strain of 0.5% within the LVR

255

was selected to perform frequency sweep tests, with the frequency ranging from 0.1 to

256

10 rad/s. Flow measurements (shear rate = 0.1 to 300 s-1) were conducted to study the

257

shear thinning behavior. All these rheological tests were performed in triplicate at

258

25 .

259

2.5.6 Surface loads of TA and WS and mass ratio of TA/WS at the oil-water

260

interface

261

The surface loads, Γ (mg/m2), of TA and WS at the oil-water interface of the lipid

262

drops were determined according to the method described elsewhere with some

263

modifications (Zhu et al., 2018). Briefly, fresh emulsions (10 mL) containing 1.0%

264

WS and 0.005-0.75% TA were centrifuged at 10000 g for 30 min at 25 °C using a

265

centrifuge (H1850R, Cence Instruments Ltd., China). After centrifugation, oil droplets

266

were separated from the continuous phase of emulsions. The bottom aqueous phase,

267

which contained non-adsorbed TA and WS, was extracted with the aid of a syringe (5

268

mL). The aqueous phase was filtered through a 0.45-µm filter to remove any residual

269

lipid droplets. The concentrations of WS and TA in the initial aqueous phase before

270

emulsification and remaining after centrifugal separation were quantitatively

271

determined using the weight and UV-vis spectrophotometer method (UV-1800, Rely,

272

China; absorbance at 276 nm), respectively. The surface load was calculated using the

273

following equation (Eq. (2)):

274

Γ (mg/m2) =

1-Φ 6Φ

(Cinitial-Cserum) (2) 10

275

where Φ is the oil phase volume fraction, Cinitial is the initial TA or WS

276

concentration in the aqueous phase before emulsification (mg/L), Cserum is the TA or

277

WS concentration in the serum phase after centrifugation (mg/L), and d32 is the lipid

278

droplet size (mm).

279

The mass ratio of TA/WS at the oil-water interface was calculated using the following

280

equation (Eq. (3)): TA/WS =

281

(3)

282

where ΓTA and ΓWS are the surface loads of TA and WS, respectively.

283

2.5.7 Color measurement of emulsions

284

The fresh emulsions (20 mL) were measured for color in the L*, a* and b* system

285

using a chroma meter (CR-400, Konica Minolta, Japan). The chroma meter was

286

calibrated using a white standard porcelain plate (L*=93.63, a*=0.07, b*=2.98). The

287

emulsions were poured into a transparent disposable petri dish for measurement. In

288

this color system, L* represents the lightness (whiteness), and a* and b* are color

289

coordinates, where +a*, -a*, +b* and -b* are red, green, yellow and blue directions,

290

respectively.

291

2.5.8 Lipid oxidation in emulsions

292

The fresh emulsions (20 mL) were immediately placed in a sealed screw-cap glass

293

tube and stored in the dark for 30 days at 25 . An 1.5-mL aliquot of emulsion was

294

periodically taken out to determine both the primary oxidative product (lipid

295

hydroperoxides) and the secondary oxidative product (2-thiobarbituric acid reactive

296

substances, TBARS) to evaluate the lipid oxidation according to the published method

297

with some modifications (Zhu et al., 2018).

298

For the lipid hydroperoxides analysis, emulsion samples (0.2 mL) were mixed with

299

1.5 mL of extracting solvent (3:1, v/v, isooctane/2-propanol) and then vortexed three

300

times for 1 min. After centrifugation at 4000 g for 2 min, the organic solvent phase

301

was collected. This phase (200 µL) was then added to 2.8 mL of a methanol:1-butanol

302

mixture (2:1, v/v), followed by the addition of 50 µL of 3.94 M ammonium

303

thiocyanate and 50 µL of ferrous iron solution (prepared by mixing equal amounts of 11

304

0.132 M BaCl2 and 0.144 M FeSO4). After 20 min, the absorbance of the samples was

305

measured at 510 nm using a UV-visible spectrophotometer (UV-1800, Rely, China).

306

The concentrations of lipid hydroperoxides in the samples were quantified using the

307

standard curve made from cumene hydroperoxides.

308

For TBARS analysis, emulsion samples (1.0 mL) were mixed with 2.0 mL of TBA

309

reagent containing 15% (w/v) trichloroacetic acid (TCA) and 0.375% (w/v)

310

thiobarbituric acid (TBA) in 0.25 M HCl, followed by boiling for 15 min. The cooled

311

solution was centrifuged at 4000 g for 10 min to obtain the supernatant. The

312

absorbance of the supernatant was measured at 532 nm, and the TBARS content was

313

evaluated according to a standard curve produced from 1,1,3,3-tetramethoxypropane.

314

2.6 Statistical analysis

315

One-way analysis of variance (ANOVA) with a 95% confidence interval was used to

316

assess the significance of the results. Statistical analysis was carried out using

317

SPSS18.0 and Origin 8.5.1 software.

318 319

3. Results and discussion

320

3.1 Sizes and three-phase contact angles of the TA/WS complexes

321

In our recent work, we affirmed that when TA was added dropwise to WS at pH=4,

322

they could gradually form soluble and insoluble complexes with increasing mass ratio

323

of TA/WS (Wei et al., 2019). To facilitate the discussion, we defined the transition

324

ratio from soluble complexes to insoluble complexes as Rt, which decreased with

325

increasing WS concentration.

326

Fig. 1 presents the particle size distribution of TA/WS complexes with different mass

327

ratios. Fig. 1A, B and C reflect the differences of particle size distribution among WS

328

concentrations (0.25%, 0.5% and 1.0%). The WS solution (control team not shown)

329

represented a bimodal size distribution trend the same as the ratio 0.005. The

330

small-sized peak in the size range of 15-70 nm was the soluble WS, while the

331

large-sized peak in the range of 150-1000 nm was the insoluble component (cellulose

332

and protein impurities remaining in the starch fractions). In terms of TA/WS

333

complexes, when the mass ratio of TA/WS was less than 0.05, the size distribution 12

334

had almost not changed, suggesting that TA was bonded to WS but did not change the

335

WS disperse state (mainly form TA/WS soluble complexes). Compared to the ratios

336

between 0.005 to 0.05, the particle sizes of 0.125 to 0.75 were larger and increased

337

with the ratio. Additionally, the content of larger particles increased, while the content

338

of smaller particles decreased. These findings indicate that because of the

339

cross-linking of soluble complexes, the content and the size of the insoluble

340

complexes increased with the TA/WS in the range from 0.125 to 0.75. It can be

341

inferred that the Rt ranged between 0.05 and 0.125 for these WS systems. The

342

tendency and mechanism were the same as those in published papers (Charlton et al.,

343

2002; Patel et al., 2013; Wei et al., 2019).

344

To evaluate the hydrophobicities of WS, TA and TA/WS complexes, the three-phase

345

contact angles (θow) were measured. The hydrophobicity (hydrophily) is indicated by

346

θow > 90° (< 90°). In general, a θow ∼ 90° is the best value for a stable oil-water

347

interface. As shown in Fig. 2, the θow values of blank WS and TA were 38° and 55°,

348

respectively, indicating their hydrophilic nature. However, compared to the blank WS,

349

the θow of TA/WS complexes increased progressively and approached 90° with mass

350

ratios of TA/WS ranging from 0.005 to 0.50, suggesting that hydrophobicity of WS

351

was markedly promoted after complexing with TA. When the ratio increased from

352

0.50 to 0.75, the θow decreased slightly from 86° to 81°. Similar results were also

353

observed in systems where polyphenol interacted with starch and zein (Feng et al.,

354

2018; Zou et al., 2015). The reason of the increase of hydrophobicity of WS could be

355

explained by the fact that when the mass ratio of TA/WS was less than 0.50, the

356

phenolic hydroxyl groups of TA combined with the hydroxyl groups of WS (hydrogen

357

bonding). This combination may lead to an increase in surface hydrophobicity

358

because of the decrease in free hydroxyl groups on the complexes’ surfaces. However,

359

when the TA/WS ratio increased to 0.75, the surface hydrophobicity decreased as a

360

result of the extra unbonded TA.

361

3.2 Influence of TA/WS mass ratio and WS concentration on emulsion

362

microstructures and size distributions

363

The microscopic images of emulsions stabilized by TA/WS complexes are shown in 13

364

Fig. 3. At a given Φ (0.3), the droplet size of emulsions was highly dependent on the

365

applied TA/WS mass ratio and WS concentration. As shown in Fig. 3, for a given WS

366

concentration, the droplet size of emulsions rapidly decreased with increasing TA/WS

367

ratio up to 0.05 and then increased with ratios between 0.125 and 0.75 for all

368

emulsions. This finding indicated that the emulsifying capacity of TA/WS complexes

369

first improved and then decreased. The emulsifying capacity could be regulated by the

370

changes in hydrophobicity, particle size and content of TA/WS complex emulsifier.

371

Thus, for given ratios from 0.005 to 0.05, the content of complexes increased, the

372

hydrophobicity of complexes improved and the size remained unchanged (Figs. 1 and

373

2), resulting in a decrease in emulsion droplet size. Similar results were also observed

374

in a system where the higher the emulsifier content, the smaller the emulsion size

375

(Saari et al., 2019). However, when the ratio increased from 0.125 to 0.75, large

376

insoluble complexes were formed by the cross-linking of several small soluble

377

complexes (Fig. 1). Therefore, even though the hydrophobicity improved (Fig. 2), the

378

size of complexes significantly increased (Fig. 1), inevitably resulting in the content

379

significantly decreasing, which finally led to a decrease in the interfacial areas

380

stabilized by complexes; therefore, the emulsion droplet size increased. Li et al. and

381

Lu et al. observed similar results that a large size and/or a low emulsifier content

382

would result in a large-sized emulsion droplet (Li et al., 2018; Lu et al., 2018).

383

Moreover, at any fixed TA/WS ratio, the emulsion droplet size gradually decreased

384

with increasing WS concentration from 0.25% to 1.0% (Fig. 3). When the ratio of

385

TA/WS was the same, the higher the WS concentration, the more TA/WS complexes,

386

resulting in a smaller emulsion droplet size (Liu, Zheng, Huang, Tang, & Ou, 2018;

387

Saari et al., 2019).

388

It is worth noting that when the ratio was less than 0.05, the emulsion droplets

389

dispersed well, suggesting that there was no interaction between the complexes on the

390

different emulsion droplets or that the interaction was too weak to work against the

391

emulsion dilution (emulsions were mainly stabilized by soluble TA/WS complexes).

392

However, when the ratio was greater than 0.125, the emulsion droplets occurred at

393

different degrees of aggregation/flocculation, especially at higher TA/WS ratios and 14

394

WS concentrations. This could be attributed to the cross-linking of TA and WS

395

between different oil droplets and the formation of bridged droplets in emulsions (i.e.,

396

bridging flocculation). This bridge interaction became stronger at higher TA/WS

397

ratios and higher WS concentrations, which could not be destroyed through dilution

398

(i.e., emulsions were stabilized by insoluble TA/WS complexes; irreversible

399

flocculation) (Liu et al., 2018).

400

For emulsion systems with different WS concentrations, the emulsion size analysis

401

(the smallest size was at the ratio of 0.05, and the size started to increase at the ratio of

402

0.125) again confirmed that the Rt was between 0.05 and 0.125 for all emulsion

403

systems (Fig. 3). To further study the effect of WS concentration on Rt, the emulsion

404

sizes (d3,2) at the ratios of 0.05 and 0.125 and the degrees of interfacial area reduction

405

of 10-mL emulsions with ratios increasing from 0.05 to 0.125 were measured and are

406

presented in Table 1. The droplet sizes increased from 9.99 µm to 10.59 µm, 4.82 µm

407

to 6.83 µm and 2.73 µm to 4.76 µm for 0.25%, 0.5% and 1.0% WS systems. We

408

assumed that the reduction of the degree of emulsifying capacity varied linearly

409

within ratios between 0.05 and 0.125. In addition, if the reduction degree was 0%, the

410

Rt was 0.125, and if the reduction degree was 100%, the Rt was 0.05. Compared with

411

the ratio of 0.05, if a larger degree of interfacial area reduction presented at a ratio of

412

0.125, the closer the Rt was to 0.05 (i.e., a larger cross-linking range). Therefore, we

413

could calculate the Rt according to the degrees of interfacial area reduction of 10-mL

414

emulsions. The Rt values were 0.121, 0.103 and 0.093 for 0.25%, 0.5% and 1.0% WS

415

systems, respectively, meaning that less TA was needed to link WS at higher WS

416

concentrations, similar to our previous work (Wei et al., 2019).

417

To provide further support for the results obtained from optical microscopy, the

418

droplet size distributions of emulsions were measured and are shown in Fig. 4. As

419

shown, most emulsions showed single peak distributions, suggesting that the droplet

420

size of emulsions was uniform. In this study, at any fixed WS concentration, the peak

421

first moved to a smaller size with increasing TA/WS ratio up to 0.05 and then moved

422

to a larger size as the ratio increased from 0.125 to 0.75, which was the same as the

423

optical microscopy results (Fig. 3). At the same ratio (less than 0.05), the droplet size 15

424

decreased with increasing WS concentration, which was same as the optical

425

microscopy results (Fig. 3). In theory, the emulsion size should decrease with

426

increasing WS concentration. However, as shown in Fig. 4, the emulsion sizes in 1%

427

WS at 0.25, 0.5 and 0.75 were larger than those in 0.5% WS due to the strengthening

428

of the bridge interaction at higher TA/WS ratios and higher WS concentrations, which

429

could not be destroyed even when diluting at a high shearing rate of 2000 rpm. Thus,

430

the size distributions of emulsions stabilized by complexes at high ratios and high WS

431

concentrations were actually several bridge-emulsions with aggregation/flocculation.

432

Thus, the droplet size could be modulated through TA/WS ratios and WS

433

concentrations.

434

3.3 Influence of the mass ratio of TA/WS and WS concentration on the creaming

435

stability of emulsions

436

Creaming is one of destabilization processes of emulsion driven by gravity, which is

437

followed by emulsion separation with water and/or oil phases. Fig. 5 shows the

438

variation of CI as a function of storage time of emulsions with various ratios of

439

TA/WS and WS concentrations. Fig. 6 shows the visual observations of these

440

emulsions after storage for 30 days. As shown in Fig. 5, in the storage stability test,

441

the values of CI all first rapidly increased and then reached a subsequent plateau with

442

time, suggesting all emulsions were unstable, which could be attributed to the

443

emulsion aggregation/flocculation. In general, the creaming rate and CI can be

444

regulated by oil density, aqueous density, droplet size and interactions among droplets.

445

Given the oil and aqueous phases, a small droplet size and/or a strong interaction

446

would restrain creaming (McClements, 2015). The creaming rate and CI decreased

447

with increasing TA/WS ratio from 0.005 to 0.05, suggesting the emulsion stability

448

improved due to the gradually decreased size in this range (Figs. 3 and 4); the

449

increased viscosity of the emulsion might be another reason for the lower CI (Fig. 8B)

450

(Winuprasith & Suphantharika, 2015). However, in range of 0.05 and 0.75, for the

451

0.25% WS system, the CI first increased and then decreased (starting at the ratio of

452

0.25); for the 0.5% WS system, the CI first remained unchanged and then decreased

453

(starting at the ratio of 0.25); for the 1.0% WS system, the CI directly decreased 16

454

(starting at the ratio of 0.125). The decreased CI was in contrast to the increased

455

droplet size (Fig. 3), which could be explained by the fact that the strong interaction

456

among droplets prevented creaming of large droplets (the ratio > Rt) (Winuprasith &

457

Suphantharika, 2015). The declining CI indicated that the interaction became stronger

458

with increasing TA/WS ratio, which was in line with the optical microscopy, emulsion

459

photography and dynamic rheological data (Figs. 3, 6 and 8). In addition, at higher

460

ratios, reaching the creaming plateau required less time due to the formation of

461

stronger and more bridged droplets (Figs. 3 and 7).

462

At the fixed ratio of TA/WS, the CI decreased with increasing WS concentration,

463

indicating that the emulsion stability improved, which could be due to the smaller size

464

and/or the stronger and more bridged interaction in emulsions stabilized by higher WS

465

concentrations (Figs. 3 and 4). The TA/WS ratios of 0.25 in 0.25% WS, 0.25 in 0.5%

466

WS and 0.125 in 1.0% WS were the falling start points of CI, consistent with the

467

optical microscopy and emulsion photography results (Figs. 3 and 6). These findings

468

indicated that less TA was needed to form stronger emulsion gels in higher WS

469

concentration systems to inhibit creaming.

470

As shown in Fig. S1, WS was actually not a good emulsifier, with severe yellow

471

creaming and oiling off of emulsions stabilized by WS (Kasprzak et al., 2018).

472

However, the emulsification of WS improved significantly after adding TA, as

473

demonstrated by the white creaming and disappearing of oiling off of emulsions

474

stabilized by TA/WS complexes (Fig. 6). As shown in Fig. 6, all emulsions exhibited

475

a sharp boundary between the top cream layer and the bottom serum layer. At lower or

476

higher ratios, the emulsions creamed, leaving transparent sera, whereas at middle

477

ratios, the serum was cloudy. The turbidity of the serum was an indication of whether

478

an emulsion was flocculated and the degree of flocculation. In general, the higher

479

degree of transparency in the serum phase, the higher degree of droplet flocculation.

480

Our results showed that the emulsions were partly flocculated at lower ratios, fully

481

flocculated at higher ratios and slightly flocculated at middle ratios, which was

482

confirmed by optical microscopy and CLSM (Figs. 3 and 7). Even though the

483

emulsions stabilized by complexes had noticeable creaming and phase separation, 17

484

they simply exhibited aggregation/flocculation without coalescence, as demonstrated

485

in the 30-day storage experiment (the droplet size did not change; data not shown).

486

These findings indicated that the interfacial layer consisting of TA and WS could

487

effectively enhance the stability of adjacent droplets against coalescence by forming a

488

strong steric barrier. Similar results have reported that emulsions stabilized by starch

489

had outstanding stabilization against coalescence (Kasprzak et al., 2018). Moreover,

490

these emulsions with higher ratios and WS concentrations transformed from liquid to

491

gel states, contributing to their long-term stabilization and the weakening of emulsion

492

creaming.

493

3.4 CLSM of emulsions

494

The microstructures of emulsions stabilized by TA/WS complexes with mass ratios of

495

0.005, 0.05, 0.125 and 0.75 in the 1.0% WS system without dilution were

496

characterized by CLSM (Fig. 7). The oil droplets (labeled in green) covered by the

497

complexes (labeled in red) were rendered visible by the fluorescent dye, indicating

498

that O/W emulsions had been prepared.

499

For emulsions stabilized by low-ratio complexes, few complexes were absorbed

500

around the oil droplets (low fluorescence intensity, Fig. 7a and b). In contrast,

501

interfacial complex networks were formed around oil droplets at high-ratio emulsions

502

(high fluorescence intensity, Fig. 7c and d), which was in agreement with the higher

503

surface load at higher ratio (Fig. 9), indicating that the droplet surface coverage

504

increased with ratio.

505

Obviously, the degree of aggregation/flocculation increased with increasing ratio,

506

especially at 0.125 and 0.75, due to the formation of strong interfacial complex

507

networks presenting as emulsion gels (Figs. 6 and 8A). Wang et al. also observed that

508

TA/nanofiber complexes could form gel-like emulsions at high TA/nanofiber ratios

509

(Wang et al., 2018). In addition, their easy destruction through dilution and lower

510

values of A and z (Fig. 3 and Table 2) indicated that the structure of emulsion

511

aggregation/flocculation was weak at lower ratios (between 0.005 and 0.05). This

512

phenomenon could be explained by bridging or depletion flocculation of the lipid

513

droplets stabilized by the complexes. In bridging flocculation (high ratios), 18

514

soluble/insoluble complexes might simultaneously adsorb to the interfaces of two or

515

more oil droplets, thereby causing them to be held together in a cluster to form strong

516

interfacial complex networks. In depletion flocculation (low ratios), the non-adsorbed

517

complexes were excluded in a narrow region surrounding each oil droplet, thereby

518

generating an osmotic attraction that forced the droplets together.

519

At low ratios, the fluorescence of complexes was uniform, without bright spots (Fig.

520

7a and b). However, large amounts of complex aggregates presented between different

521

oil droplets at high ratios (Fig. 7c and d), again confirming that the Rt was between

522

0.05 and 0.125. Dickinson previously reported that the increasing hydrophobicity of

523

modified particles resulted in them aggregating with each other in the aqueous phase,

524

after which the aggregates adsorbed on the oil droplet surface rather than individual

525

small particles absorbing and forming larger thicknesses (Dickinson, 2013). In

526

addition, when insoluble complexes began to form, TA also bridged complexes

527

between different droplets and formed bridged droplets in emulsions, which partially

528

contributed to the stiffness of emulsions (G’ > G’’) (Fig. 8A).

529

3.5 Rheological properties of emulsions

530

To further investigate the stability mechanisms of emulsions with different ratios,

531

viscoelasticity assessments of emulsions in the 1.0% WS system were performed and

532

are shown in Fig. 8A. All emulsions presented a predominantly elastic-dominated

533

behavior (G’ > G’’). The viscoelasticity of emulsions at low ratios was mainly due to

534

the interaction of soluble complexes between the oil droplet surface and the aqueous

535

phase, whereas the viscoelasticity of emulsions at high ratios was due to the insoluble

536

complexes between different oil droplet surfaces. Moreover, in comparison to those

537

emulsions at low ratios, the G’ value increased markedly at high ratios, indicating that

538

the network of emulsions showed an excellent elasticity, resulting from the formation

539

of emulsion gels (Figs. 6C and 7). Wang et al. also reported that the elasticity of

540

emulsions stabilized by citrus nanofiber improved after complexing with TA (Wang et

541

al., 2018). According to the Bohlin model, these emulsion systems could be explained

542

by the power law equation: G* = A

, where G* was the dynamic complex 19

543

modulus, ω was the frequency, z was the coordination number to evaluate the number

544

of rheological units connected with others in the emulsion network, and A was the

545

proportional coefficient related to the strength of the interaction between these units

546

(Anvari & Chung, 2016). The values of A and z are presented in Table 2. The increase

547

of the z value at low ratios was due to the decrease in emulsion size and the increase

548

in surface area, which resulted in the increase of interaction sites of complexes

549

between oil droplet surface and the aqueous phase. However, the values of A did not

550

increase significantly with increasing ratios from 0.005 to 0.05, reflecting that there

551

was little internal structure change in this range. At high ratios, the z value gradually

552

increased with increasing ratio, suggesting more complex structures and greater

553

numbers of interaction sites between oil droplets and complexes. Moreover,

554

significant increases in A values occurred between 0.125 and 0.75, indicating that the

555

interactions between sites enhanced significantly. These findings could be attributed

556

to the cross-linking that was triggered and that formed a continuous network among

557

and around the well-dispersed oil droplets at high ratios, leading to the formation of

558

strong emulsion gels. Anvari et al. also reported that TA could cross-link fish

559

gelatin/gum arabic coacervate to improve the gels’ viscoelasticity (Anvari & Chung,

560

2016). The increases in G’ and G’’ with the TA/WS ratio reflected the enhanced

561

viscoelastic properties, which showed improved rigidity and stronger emulsion

562

structure, resulting in lower values of CI (Figs. 5C and 6C).

563

The emulsion viscosity profiles are shown in Fig. 8B. The viscosities decreased with

564

increasing shear rate for all emulsions, suggesting pseudoplastic behavior, which is a

565

common behavior presented in emulsions and can be attributed to the deformation and

566

disruption of emulsion aggregation/flocculation (Liu et al., 2018). Compared to low

567

ratios, the viscosity of emulsions stabilized by complexes at high ratios decreased

568

more rapidly with increasing shear rate, indicating that the structure of emulsions was

569

stronger and easier to destroy at higher ratios because of the formation of emulsion

570

gels. The increasing viscosity at 100 s-1 with increasing ratio is summarized in Table 2.

571

The increase in viscosity could be related to the size, polydispersity of the oil droplets

572

and the state of emulsions. At ratios of 0.005 to 0.05, a large amount of smaller-sized 20

573

oil droplets could provide a larger surface area, which would strengthen the

574

interaction of complexes between oil droplets and the continuous phase. Thus, a

575

smaller oil droplet size would support the higher viscosity at low ratios. However,

576

there were increasing numbers of bridged droplets against the free flow of emulsions

577

at high ratios. Thus, even though the size of oil droplets increased, the stronger

578

network structure could provide higher viscosity at high ratios (0.125 to 0.75), as

579

shown by optical microscopy and CLSM (Figs. 3 and 7). In addition, a high emulsion

580

viscosity could restrain droplet movement, reduce collision probability and improve

581

emulsion stability.

582

3.6 TA and WS surface loads and the mass ratio of TA/WS at the oil-water

583

interface

584

Surface load is a predictor of the strength of the oil-water interfacial membrane in

585

emulsions (Liu et al., 2019). The surface loads of TA and WS and their ratio at the

586

oil-water interface as a function of the TA/WS ratio in the emulsions are shown in Fig.

587

9. At low ratios (0.005 to 0.05), the WS surface load was larger than that of TA,

588

indicating that WS played a dominant role at the oil-water interface. In addition, the

589

changeless WS surface load and slight increase in the TA surface load suggested that

590

the interfacial thickness increased insignificantly, as indicated by the lack of change in

591

fluorescence intensity in CLSM (Fig. 7a and b). However, TA and WS surface loads

592

increased abruptly as the ratio increased from 0.05 to 0.125, indicating that the

593

interfacial thickness increased significantly due to the formation of emulsion gels

594

stabilized by insoluble complexes (Figs. 7b and c and 8A). The gradual and gigantic

595

increases in TA and WS surface loads from 0.125 to 0.75 suggested that the interfacial

596

thickness increased with increasing ratio, resulting in a stronger interfacial network, in

597

line with the rheological results (Fig. 8). Wang et al. also discovered that the surface

598

loads increased with increasing TA/nanofiber ratio due to the TA cross-linkage of

599

citrus nanofibers (Wang et al., 2018). As shown in Fig. 9C, the TA/WS ratio at the

600

oil-water interface was larger than that in the aqueous phase at the ratios of 0.005 to

601

0.25 because most TA was used to bind WS to form high hydrophobic complexes and

602

was then adsorbed to the oil-water interface (Fig. 2). However, the ratios at the 21

603

interface were smaller than those in the aqueous phase at the ratios of 0.50 and 0.75,

604

possibly because the TA was in excess. The gradual increase of TA/WS ratio at the

605

interfacial membrane indicated that increasing amounts of TA persistently absorbed to

606

the WS as the ratio increased, resulting in the formation of soluble and insoluble

607

complexes, strengthening the interfacial structure.

608

3.7 Color of emulsions

609

The color of the emulsions stabilized by TA/WS complexes was worth examining due

610

to the relatively high color intensity of TA, which often determined if the product was

611

fit for its intended use. The colors (L*, a* and b*) of emulsions stabilized by TA/WS

612

complexes with different ratios and WS concentrations are shown in Fig. 10. At any

613

fixed WS concentration, the lightness (L* value) of emulsions rapidly increased with

614

increasing TA/WS ratio up to 0.05 and then decreased at the ratio 0.125 for all

615

emulsions, whereas the value of b* was opposite that of L* value as the ratio

616

increased. In addition, the a* value experienced only a slight change in all cases.

617

These findings indicated that the emulsions became brighter and that their color

618

tended to be lighter (0.005 to 0.05) and then became darker and more intense in color

619

(0.125 to 0.75), which was confirmed by visual examination of the emulsions (Fig. 6).

620

It is well known that the lightness and color of emulsions are mainly determined by

621

the scattering and absorption of incident white light by emulsion, which are mainly

622

influenced by emulsion droplet size, emulsion concentration and the presence of

623

colorants in the emulsion. The droplet size decreased first and then increased as the

624

TA/WS ratio increased (Fig. 3). The light scattering efficiency of the droplet increased

625

with decreasing droplet size; thus, the light could not penetrate further into emulsions

626

and was absorbed to a smaller extent, which resulted in the highest L* and lowest b*

627

at the smallest droplet size (TA/WS=0.05). Moreover, the increased color intensity of

628

emulsions at high ratios of TA/WS might also be caused by higher contents of TA in

629

the emulsions since TA itself was yellow-brown in color. In the presence of TA, some

630

of the light was absorbed by the chromophores of TA and therefore did not contribute

631

to the light scattering, resulting in a lower lightness and a higher color intensity. A

632

variety of studies have shown that the lightness decreased and the color intensity 22

633

increased as the concentration of chromophoric material increased in the emulsion

634

(Winuprasith & Suphantharika, 2015). However, this effect was not observed at lower

635

ratios in this study, which might be covered by the decreased droplet size. In addition,

636

at fixed TA/WS ratios, higher L* and lower b* were observed at higher WS

637

concentrations, mainly due to the smaller droplet size (Fig. 3). However, at a higher

638

ratio (TA/WS=0.75), an abnormal phenomenon was observed. A higher b* was

639

obtained at higher WS concentrations, which was due to the presence of more TA at

640

higher WS concentrations, resulting in a higher color intensity.

641

3.8 Primary and secondary oxidative products of emulsions

642

Soybean oil with a high concentration of unsaturated fatty acids (approximately 80%)

643

is susceptible to oxidation (Kupongsak & Sathitvorapojjana, 2017; Park, Mun, & Kim,

644

2019). Thus, the oxidative stability of soybean oil-based emulsions was evaluated

645

through monitoring the formation of oxidative products during storage, as shown in

646

Fig. 11. In emulsion systems, the oil-water interface plays an important role in

647

determining the rate and extent of lipid oxidation because it is the region where

648

hydrophobic, hydrophilic and amphiphilic molecules involved in the reaction come

649

into close proximity. Thus, the influence of the TA/WS ratio and the WS

650

concentration on the interfacial composition of the oil droplets in the emulsions would

651

be expected to affect lipid oxidation. In this study, lipid hydroperoxides and TBARS

652

contents increased over time in all emulsions, suggesting that lipid oxidation was

653

occurring. For the 1.0% WS concentration emulsion system (Fig. 11A and a), the rates

654

of lipid hydroperoxides and TBARS formation were the highest in the WS control

655

emulsion. This result suggested that WS was the least effective in retarding lipid

656

oxidation. However, when the TA/WS ratio increased from 0.005 to 0.05, the rate of

657

lipid oxidation gradually decreased, as evidenced by decreased rates of lipid

658

hydroperoxides and TBARS formation. This result was in agreement with a previous

659

study that examined the influence of TA/nanofiber complexes on the oxidative

660

stability of O/W emulsion (Wang et al., 2018). This effect was mainly because the

661

antioxidative TA was incorporated into the droplet’s shell of emulsions through a

662

simple noncovalent interaction with WS (Fig. 9), and then the antioxidant interfacial 23

663

shells around the oil droplets formed, resulting in emulsions with improved oxidative

664

stability. However, there was an increase in lipid oxidation when the ratio increased

665

from 0.005 to 0.01, which could be attributed to the significantly decreased particle

666

size of the droplets (Fig. 3), which increased the oxidative rate of lipids by increasing

667

the oil-water interfacial area (Zhu et al., 2018). Obviously, the rate of lipid oxidation

668

was almost changeless (there was a slight decrease) when the ratio increased from

669

0.05 to 0.75, indicating that the content of TA in the emulsion system was enough to

670

inhibit lipid oxidation in this range (Wang et al., 2018). Another reason for the best

671

oxidative stability of high TA/WS ratio emulsion systems was that a thicker interfacial

672

network and larger droplet size would provide a promising potential against lipid

673

oxidation via the production of a physical barrier and lower interfacial area (Fig. 7)

674

(Hu, McClements, & Decker, 2003; Kargar, Fayazmanesh, Alavi, Spyropoulos, &

675

Norton, 2012). Compared to the WS control emulsion, the concentrations of lipid

676

hydroperoxides and TBARS decreased by approximately more than 70% and 82%,

677

respectively, when the ratio was greater than 0.05 after 30 days of storage. Therefore,

678

fabrication of an antioxidant interface via TA/WS complexes is a promising approach

679

to improve the oxidant stability of soybean oil-based emulsions (Fan, Liu, Gao, Zhang,

680

& Yi, 2018).

681

At the fixed ratio of 0.05, the effect of WS concentration on the lipid oxidation was

682

also investigated and is shown in Fig. 11B and b. Hydroperoxides and TBARS

683

increased with time in the 0.25% WS system, whereas they remained nearly

684

changeless (there were slight increases) in the 0.5% and 1.0% WS systems, indicating

685

that lipid oxidation was more effectively retarded at higher WS concentrations.

686

Both adsorbed and non-adsorbed TA can inhibit lipid oxidation in emulsions (Aewsiri,

687

Benjakul, Visessanguan, Wierenga, & Gruppen, 2010). As shown in Fig. 11C and c,

688

compared to the WS control emulsion, the rate of lipid oxidation was lower in the TA

689

control emulsion, suggesting that TA in the aqueous phase could also restrain lipid

690

oxidation to some extent. However, the emulsion stabilized by TA/WS complexes

691

exhibited the lowest lipid oxidation degree, which confirmed that the absorbed TA

692

was more effective in inhibiting lipid oxidation than the non-adsorbed ones (all 24

693

emulsions contained the same total TA level).

694

3.9 Physical stability of emulsions

695

The influence of storage time (fresh and 30 days) on the particle size distribution of

696

emulsions stabilized by TA/WS complexes with the fixed ratio of 0.05 at various WS

697

concentrations was investigated and is shown in Fig. 12. The results showed that all

698

fabricated emulsions were extremely stable against droplet coalescence, given that no

699

increase in size occurred after 30 days of storage. These results suggested that the

700

complexes on the oil-water interface and the gel-like networks of the complexes in

701

these emulsion systems played crucial roles in the stability of these emulsions against

702

droplet coalescence (Kasprzak et al., 2018). However, severe phase separation at

703

lower ratios was observed due to the aggregation/flocculation of oil droplets (Fig. 6),

704

which could be due to the low zeta-potential of complexes at pH=4 (< 1 mV), the low

705

viscosity of emulsions (Fig. 8B) and the large size of oil droplets (d3,2 > 2 µm).

706

Therefore, the next step would be to introduce charged macromolecules to increase

707

the electrostatic repulsion between droplets, high-viscosity polysaccharides to reduce

708

droplet movement and collision probability, or small-molecular-weight emulsifiers

709

(phospholipids, etc.) to reduce the size of emulsion droplets, which could improve the

710

anti-flocculation phenomenon of emulsions (Jiang et al., 2018; Lu, Zheng, & Miao,

711

2018; Yang et al., 2018).

712

3.10 Potential mechanism of the effect of the TA/WS mass ratio on TA/WS

713

complexes and emulsions

714

The mechanism of the effect of the TA/WS mass ratio on the complexes and

715

emulsions is shown in Fig. 13. When TA was added to WS, the components gradually

716

formed soluble and insoluble complexes. Soluble complexes with the same size

717

distribution were formed when the TA/WS ratio was less than 0.05. However,

718

larger-sized insoluble complexes were formed when the TA/WS ratio increased from

719

0.125 to 0.75. The surface hydrophobicity of complexes increased with increasing

720

ratio, resulting in increasing amounts of complexes absorbed on the oil-water

721

interface. However, in the range of 0.005 and 0.05, the decreasing size of emulsions

722

with increasing ratio was due to the increased content of individual complexes 25

723

emulsifier. In the range of 0.125 and 0.75, the increased emulsion droplet size was due

724

to the decreased content and increased size of individual complexes emulsifier

725

resulting from the TA cross-linking WS. After 30 days of storage, the low-ratio

726

emulsions

727

flocculation/aggregation, whereas the high-ratio emulsions exhibited smaller CI due

728

to the bridging flocculation/aggregation induced by complexes on the oil-water

729

interface.

exhibited

significant

creaming

due

to

the

depletion

of

730 731

4. Conclusion

732

In summary, this study suggested that the TA/WS mass ratio and WS concentration

733

had substantial effects on emulsions stabilized by TA/WS complexes. In this study,

734

when TA was added into WS, they could gradually form soluble and insoluble

735

complexes for all WS systems. The Rt decreased with increasing WS concentration.

736

The size of the emulsions first decreased and then increased with increasing ratio,

737

mainly due to the changes in the content, size and hydrophobicity of the complexes.

738

Additionally, the emulsifying capacity of the complexes improved with increasing WS

739

concentration.

740

flocculation/aggregation, but creaming was inhibited at high ratios and WS

741

concentrations, where a thick interfacial network (emulsion gel) formed instead. The

742

emulsion gel formed due to the TA cross-linked WS on the interfaces of different oil

743

droplets (Ratio > Rt), and the gel strength increased with the ratio due to increasing

744

numbers of complexes absorbed on the interface. At the ratio of 0.05, the emulsion

745

had the highest lightness. When the ratio and WS concentration were no less than 0.05

746

and 0.5%, respectively, emulsions had better oxidative stability. The emulsions also

747

had high resistance against droplet coalescence. TA was an effective antioxidant;

748

therefore, TA emulsions could be used to protect and deliver easily oxidizing

749

oil-soluble nutrients or medicines.

All

emulsions

exhibited

obvious

creaming

due

to

750 751

Acknowledgements

752

This work was financially supported by the National Key R&D Program of China 26

753

(Program No. 2017YFD0400205) & Hubei Provincial Natural Science Foundation for

754

Innovative Group (2019CFA011).

755

27

756

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906

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907

33

908

Figure caption:

909

Fig. 1. Particle size distributions of TA/WS complexes with various mass ratios of TA

910

to WS (0.005 to 0.75) and WS concentrations (A: 0.25%; B: 0.5%; C: 1.0%).

911

Fig. 2. Typical three-phase contact angles (θow) of WS, TA and TA/WS complexes

912

with different TA/WS mass ratios (0.005 to 0.75) in a 1.0% WS system.

913

Fig. 3. Optical microscopy images of fresh emulsions stabilized by TA/WS complexes

914

with various mass ratios of TA to WS (subscripts 1 to 8 are for 0.005 to 0.75) and WS

915

concentrations (A: 0.25%; B: 0.5%; C: 1.0%).

916

Fig. 4. Particle size distributions of emulsions stabilized by TA/WS complexes with

917

various mass ratios of TA to WS (0.005 to 0.75) and WS concentrations (A: 0.25%; B:

918

0.5%; C: 1.0%).

919

Fig. 5. Variation of the creaming index of emulsions as a function of storage time.

920

These emulsions were stabilized by TA/WS complexes with various mass ratios of TA

921

to WS (0.005 to 0.75) and WS concentrations (A: 0.25%; B: 0.5%; C: 1.0%).

922

Fig. 6. Appearances of emulsions stabilized by TA/WS complexes with various mass

923

ratios of TA to WS (0.005 to 0.75) and WS concentrations (A: 0.25%; B: 0.5%; C:

924

1.0%) after 30 days of storage.

925

Fig. 7. CLSM images of emulsions stabilized by TA/WS complexes with different

926

TA/WS mass ratios (A, a: 0.005; B, b: 0.05; C, c: 0.125; D, d: 0.75) in a 1.0% WS

927

system. The complexes were stained red, and the oil phase was stained green.

928

Fig. 8. The storage (G’, closed symbols) and loss (G’’, open symbols) modulus versus

929

angular frequency (A), and the viscosity versus shear rate (B) of the emulsions

930

stabilized by TA/WS complexes with various mass ratios of TA to WS (0.005 to 0.75)

931

in a 1.0% WS system.

932

Fig. 9. TA (A), WS (B) surface loads and TA/WS at the oil-water interface (C) as a

933

function of TA/WS mass ratio in emulsions containing 1.0% WS. Different letters (a,

934

b, c....) indicate significant differences at p < 0.05.

935

Fig. 10. The color values (L*, a* and b*) of emulsions as a function of TA/WS mass

936

ratio in the emulsions containing different WS concentrations (0.25%, 0.5% and

937

1.0%). 34

938

Fig. 11. Effects of TA/WS mass ratio (A, a), WS concentration (B, b) and the position

939

of TA (C, c) on the formation of lipid hydroperoxides (A, B and C) and TBARS (a, b

940

and c) in emulsions during 30 days of storage at room temperature. The WS (A, a, C

941

and c) meant the emulsion was stabilized by WS. The TA and TA/WS complexes (C

942

and c) meant these emulsions were stabilized by TA, TA/WS complexes, respectively.

943

Fig. 12. Stability of emulsions assessed through the evolution of the droplet size

944

distributions over time. These emulsions were prepared with a fixed TA/WS mass

945

ratio (0.05) at different WS concentrations (0.25%, 0.5% and 1.0%).

946

Fig. 13. Schematic diagram illustrating emulsions stabilized by TA/WS complexes

947

with different TA/WS mass ratios (0.005, 0.05, 0.125 and 0.75) in a 1.0% WS system.

948

35

949

Table 1 The particle size (d3,2) of emulsion droplets at the TA/WS mass ratios of 0.05

950

and 0.125, and the extent of total surface area reduction of 10-mL emulsions as the

951

ratio increased from 0.05 to 0.125 at different WS concentrations. The d3,2 was

952

obtained by microscope statistical analysis. d3,2 (µm)

The extent of surface area

WS concentration (%) TA/WS = 0.05

TA/WS = 0.125

reduction (%)

0.25

9.99

10.59

5.67

0.5

4.82

6.83

29.35

1.0

2.73

4.76

42.56

953

36

954

Table 2 Bohlin’s parameters and the viscosity at 100 s-1 of the emulsions stabilized by

955

TA/WS complexes with various mass ratios of TA to WS (0.005 to 0.75) in a 1.0%

956

WS system. TA/WS

A (Pa s1/z)

z

R2

Viscosity (mPa.s)

0.005

0.09±0.09a

1.25±0.68a

0.93

7.23±0.19a

0.01

0.30±0.08a

2.34±0.05b

0.90

9.97±0.36b

0.025

1.18±0.52a

3.05±0.56bc

0.91

11.95±0.19c

0.05

0.72±0.27a

3.39±0.37c

0.92

14.42±0.77d

0.125

12.53±1.86b

5.76±0.40d

0.91

30.17±0.04e

0.25

22.52±6.40c

6.36±0.72de

0.95

35.34±0.42f

0.50

48.96±5.08d

6.70±0.54e

0.97

62.09±2.08g

0.75

66.14±5.45e

7.07±0.20e

0.95

111.75±0.35h

957

Results are mean ± SD. Different letters in the same column indicate a statistically significant

958

difference (p < 0.05).

37

Fig. 1.

Fig. 2.

Fig. 3.

Fig. 4.

Fig. 5.

Fig. 6.

Fig. 7.

Fig. 8.

Fig. 9.

Fig. 10.

Fig. 11.

Fig. 12.

Fig. 13.

Highlights > The hydrophobicity and emulsifying capacity of WS was improved by complexing with TA. > Emulsifying capacity of TA/WS complexes was regulated by TA/WS mass ratio. > The creaming was inhibited at high TA/WS mass ratio or high WS concentration. > The emulsions at high TA/WS mass ratio had better oxidative stability.

Conflict of interest The authors declared no conflict of interest.