Highly surface-active chaperonin nano-barrels for oil-in-water

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Highly surface-active chaperonin nano-barrels for oil-inwater Pickering emulsions and lipophilic compounds delivery Baomei Xu, Chengkun Liu, Haiyan Sun, Xiaoqiang Wang, and Fang Huang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b02379 • Publication Date (Web): 21 Aug 2019 Downloaded from pubs.acs.org on August 23, 2019

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Journal of Agricultural and Food Chemistry

Highly surface-active chaperonin nano-barrels for oil-in-water Pickering emulsions and lipophilic compounds delivery Baomei Xu, Chengkun Liu, Haiyan Sun, Xiaoqiang Wang* and Fang Huang*

State Key Laboratory of Heavy Oil Processing, College of Chemical Engineering, China University of Petroleum (East China), Qingdao 266580, P.R. China

*To

whom correspondence may be addressed: [email protected]; [email protected] Tel: +86-532-86981560, FAX: +86-532-86981560

1

ACS Paragon Plus Environment


1

Abstract

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Stabilization of Pickering emulsions via particles of biological origin exhibits a great

3

potential to be widely applied in food, cosmetic or biomedicine formulation, due to

4

their excellent biocompatibility, biodegradability as well as functional properties. This

5

paper describes the successful development of bio-derived GroEL protein nano-barrel

6

as a Pickering stabilizer, and its protection properties on β-carotene in dispersed oil

7

phase, as a model of labile bioactive compounds. It is shown that GroEL nano-barrel

8

is highly surface-active and allows the formation of Pickering emulsion by physical

9

adsorption at oil/water interface. The optimized formulation for generating stable

10

sub-micron oil droplet by ultrasonication includes a GroEL concentration of 0.05-0.45

11

wt.% with an oil/water volume ratio of 0.05-0.35. The as-prepared Pickering emulsion

12

shows pH responsive emulsification/demulsification transition, and excellent stability

13

at temperatures less than 65 °C and ionic strength (with NaCl addition) up to 500 mM.

14

Meanwhile, the emulsion tends to form gel-like network structure with the oil/water

15

ratio increasing. Finally, we demonstrate that possible factors of oxidant, reducing

16

agent, UV radiation and sucrose have sequentially decreasing to no effect on the

17

stability of β-carotene encapsulated in GroEL-stabilized Pickering emulsion, and that

18

higher GroEL concentration can significantly reduce β-carotene degradation rate, thus

19

ensuring more efficient long-term storage. We believe that the emulsion system

20

supported by GroEL nano-barrel could be developed to a vial tool for delivering

21

lipophilic bioactive compounds.

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Keywords: GroEL nano-barrel, Interfacial property, Pickering emulsion, Stability,

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Rheological behavior, β-carotene

24

25

26

27

28

29

30

31

32

33

34

35

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Introduction

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Pickering emulsions are mixtures of two immiscible liquids that are kinetically

39

stabilized by solid particles adsorbing at the liquid/liquid interface 1-2. Compared with

40

conventional surfactant-stabilized emulsions, Pickering emulsions are more stable

41

against coalescence and Ostwald Ripening 3, and can obtain many novel properties

42

from particulate stabilizers as well. Different types of inorganic particles have been

43

shown to act as effective Pickering stabilizers, such as calcium carbonate 4,

44

palygorskite 5, clay 6, carbon nanotubes

45

owing to the poor biocompatibility and biodegradability, their applications are largely

46

restricted. On the other hand, the stabilization of emulsions via particles of biological

47

origin is currently attracting vast research attention, because these materials are free of

48

end-of-life

49

unattainable by their purely synthetic counterparts

50

nanoparticles are particularly interesting, whose naturally amphiphilic nature and

51

nutritional value impart a dual benefit in the preparation of high-grade Pickering

52

emulsions, especially for food, cosmetic and medical industries 12-13.

53

While

environmental

most

commonly

impact,

7-8

while

available

and silica nanoparticles

offering

protein

9-10.

biocompatible 11.

However,

advantages

Among them, proteins

nanoparticles

display

superior

54

mono-dispersity in aqueous solutions, they are much vulnerable to the process

55

variables of industrial emulsions (e.g., high temperatures or broad pH shift)

56

Moreover, the surface of a protein usually presents a highly heterogeneous

57

distribution of charge and hydrophobicity, leading to unpredictable protein orientation

58

at interfaces and compromised surface activity

15.

14.

Surface modification or even

4


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intended denaturation is frequently performed to further improve protein’s packing

60

around dispersed phase droplets and thus its emulsive-ability 16-17, which, however, is

61

uncontrollable to a large extent and adds an extra layer of complexity to formulating

62

protein-supported emulsions. Consequently, it is highly desirable to develop

63

inherently robust protein nanoparticles with fine-tuned architecture and high surface

64

activity to efficiently stabilize high-grade Pickering emulsions.

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Chaperonin GroEL from Escherichia coli is a barrel-shaped assembly of two

66

heptameric rings of ~57 kDa subunits, with a height of ~14.6 nm and a diameter of

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~13.7 nm (Figure 1A-1B) 18. Evolutionarily optimized for coping with thermal stress

68

in the cell, GroEL nano-barrel is highly resistant against thermal or chemical

69

denaturation, which for example becomes denatured only at a temperature of up to

70

70 °C or in the presence of more than 3.2 M urea

71

nano-barrel represents a promising tool to push the limits of stability of protein-based

72

Pickering emulsions. Moreover, GroEL harbors a ring of hydrophobic binding surface

73

along the inner edge of its apical cavities (Figure 1C), poised to interact with foreign

74

hydrophobic surfaces or molecules. The strong hydrophobic contribution to the

75

binding to GroEL has been examined with a number of protein substrates in their

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disaggregation and assisted folding, or other hydrophobic guest molecules with no

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physiological relevance in the design of smart delivery nano-machines

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promiscuity of GroEL apical hydrophobic cavity makes the chaperonin an appealing

79

choice for oil dispersion, especially given the precise placement of binding sites on

80

the nano-barrel termini that is envisioned from a thermodynamic standpoint to favor a

19-20.

5


In this context, GroEL

21-23.

The


81

high surface coverage of oil droplets by “end-on” GroEL.

82

In the present study, GroEL nano-barrel was shown to be highly surface-active. As

83

a model oil phase we used rosemary oil and found that GroEL can act as efficient

84

Pickering emulsion stabilizers, packing around dispersed oil droplets. We next

85

optimized formulation composition and examined the effect of key factors on

86

emulsion stability. The emulsion rheology related closely to its performance and

87

application was also analyzed. Finally, we explored the protective features of

88

GroEL-based emulsion toward lipophilic bioactive molecules using β-carotene as a

89

model compound.

90

Materials and Methods

91

Materials. Bovine serum albumin (BSA) was purchased from Solarbio Life

92

Science (Beijing, China) (Catalog # A8010). Rosemary oil, Nile red and β-carotene

93

(≥97% purity) were purchased from Sigma-Aldrich (Catalog # W299200; 19123;

94

22040). Alexa Flour 488 C5-Malemide was purchased from Thermo Fisher Scientific

95

(Catalog # A10254). All the reagents were used without further purification. All

96

aqueous solutions were prepared with ultrapure water (resistivity > 18 MΩ·cm) from

97

a Millipore Milli-Q system.

98

GroEL Preparation and Characterization. Chaperonin GroEL used as a

99

Pickering emulsifier in this study was overexpressed in Escherichia coli strain BL21

100

(DE3) and purified to ~95% purity, as described previously 24-25. The purified GroEL

101

was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 6


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quantified using the Bradford Protein Quantification Kit. The hydrodynamic diameter

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distribution and zeta potential of GroEL were measured on a Malvern Zetasizer Nano

104

ZS spectrometer. The integrity and morphology of GroEL were examined by

105

transmission electron microscopy (TEM) with a JEOL JEM 1400Plus electron

106

microscope operated at 120 kV. GroEL was also labeled with Alexa Fluor 488

107

(AF488) for fluorescence imaging. After desalting to remove free dyes, the

108

AF488-labeled GroEL was characterized by UV-vis spectroscopy on a Shimadzu

109

UV-1700 UV-vis spectrophotometer.

110

Surface Tension Measurement. Surface-active substances are capable of changing

111

the surface tension of a liquid or the interfacial tension between two phases. To assess

112

the surface activity of GroEL dissolved in aqueous solution, surface tension

113

measurement was carried out with a series of GroEL concentrations on an EasyDyne

114

tensiometer (Kruss) at 25.0 ± 0.1 °C by using the Wilhelmy plate method. The values

115

of surface tension γ were obtained after a period of 10 min to ensure reaching

116

equilibrium. The surface activity of BSA was also evaluated with the same method

117

and compared with that of GroEL.

118

Preparation of GroEL-Stabilized Pickering Emulsion. GroEL in a Tris-HCl

119

buffer solution (50 mM Tris-HCl, pH 7.5 with 1 mM EDTA-2Na, 1 mM DTT and 3

120

mM NaN3) was dribbled slowly into a vigorously stirred mixture of water and

121

rosemary oil with a certain volume ratio. This usually produces only a crude oil

122

emulsion, in which the dispersed droplets have a broad diameter distribution. The 7



123

coarse emulsion was further homogenized by ultrasonication using an ultrasound

124

system (KQ-100KDE from Kun Shan Ultrasonic Instruments, China) for 2 minutes at

125

40% power. Preliminary TEM analyses indicated that the ultrasonication operation

126

did not change GroEL’s overall architecture and integrity. To determine the type of

127

the as-prepared emulsion, a droplet of rosemary oil or water was blended with a

128

droplet of GroEL-supported emulsion, followed by optical microscopic examination

129

of the mixture.

130

Fluorescence Imaging of Pickering Emulsion. The presence of GroEL adsorption

131

layer at the oil/water interface was examined by fluorescence imaging. Nile red was

132

used to stain the oil phase, GroEL being labeled with green-fluorescent dye AF488, as

133

previously reported

134

GroEL was placed on a microscope slide and then covered with a coverslip.

135

Fluorescence micrographs were next captured on a Leica microscope (DMI3000 B)

136

with a blue band excitation.

26.

The emulsion composed of the stained oil and the labeled

137

Optimization of GroEL-Stabilized Pickering Emulsion. The emulsion

138

composition was optimized to obtain emulsions with low oil droplet size distribution

139

but high stability. In this process, the concentration of GroEL was varied within the

140

range of 0.05 to 0.45 (wt.%) and oil/water (o/w) volume ratio from 0.05 to 0.50. After

141

a storage period of 15 days at room temperature, the droplet sizes in different

142

emulsion samples were examined and compared to evaluate the optimal formulation.

143

Measurement of Droplet Size and Zeta Potential. Influence of emulsion 8


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composition, pH, temperature or ionic strength on the size distribution or/and zeta

145

potential of GroEL-stabilized Pickering emulsion was examined on a Malvern

146

Zetasizer Nano ZS spectrometer. For each independent measurement, the emulsion

147

was opportunely diluted to avoid multi-scattering phenomena. The droplet size was

148

measured based on dynamic light scattering (DLS) technique, while the zeta potential

149

was measured with a DTS1070 capillary cell, exposed to an electric field of 150 V at

150

a scattering angle of 173°. The change of zeta potential of GroEL with varied pH was

151

also characterized in the same way.

152

Estimation of Surface Protein Coverage of Pickering Droplet. In this

153

experiment, the o/w ratio was fixed at 0.05 while GroEL mass fraction was varied

154

from 0.05 to 0.45 (wt.%). Size of Pickering droplet was determined by DLS at each

155

GroEL mass fraction examined. The surface coverage was calculated as previously

156

described

157

was first estimated based on the volume and droplet size. The total area (Sp) that

158

GroEL nano-barrel can cover was evaluated based on the applied GroEL amount by

159

supposing that GroEL nano-barrels are closely packed with an “end-on” orientation to

160

form a monolayer at oil/water interface. Surface coverage was then calculated with S0

161

divided by Sp.

26.

Briefly, for a specific volume of emulsion the total interfacial area (S0)

162

Influence of pH, Storage Temperatureor Ionic Strength on GroEL-Stabilized

163

Pickering Emulsion. The tolerance of GroEL-stabilized Pickering emulsion against a

164

dramatic pH variation between 4.5 and 8.5 was investigated. The pH of the emulsion 9



165

prepared at neutral pH was adjusted by the addition of concentrated HCl or NaOH.

166

After each cycle of pH adjustment from 8.5 to 4.5 and then back to 8.5, the droplet

167

size distribution was measured by DLS. The emulsion microstructure at various pHs

168

was also examined by optical microscopy.

169

To investigate the influence of storage temperature on GroEL-stabilized Pickering

170

emulsion, the size distribution of dispersed droplets as well as the zeta potential was

171

measured over time at different incubation temperatures. The incubation temperatures

172

were set at 4 °C, 25 °C, 37 °C, 50 °C and 65 °C, respectively.

173

To investigate the effect of ionic strength on GroEL-stabilized Pickering emulsion,

174

emulsions with a NaCl concentration of 10 mM, 50 mM, 100 mM, 200 mM, 500 mM

175

or 800 mM were prepared separately, and stored at room temperature. The size

176

distribution of dispersed droplets and zeta potential in the as-prepared emulsions were

177

measured over time.

178

Rheological Analysis of GroEL-Stabilized Pickering Emulsion. Rheological

179

behavior of emulsions can offer insight not only to their workability but also to their

180

performance such as stability. The rheological properties of GroEL emulsions were

181

measured on a Thermo Scientific Haake MARS III modular rheometer at 25 °C. The

182

temperature was controlled by a Peltier temperature module. An oscillatory strain

183

sweep test was first performed to determine the linear viscoelastic region, in which all

184

the following rheological analyses were conducted. Dynamic frequency sweep

185

measurements were performed with a frequency range and strain of 0.01-10 Hz and

186

1%, respectively. 10


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Stability of β-Carotene in GroEL-Stabilized Pickering Emulsion. The

188

β-carotene was used as a model of labile bioactive compounds to test the protective

189

property of GroEL-stabilized emulsion. To prepare the GroEL emulsified β-carotene,

190

β-carotene was first dissolved in rosemary oil to a final concentration of 0.1 wt.%, and

191

then mixed with water with a o/w ratio of 0.10. Subsequent addition of GroEL and the

192

emulsification procedure were the same as aforementioned. The stability of β-carotene

193

in the as-prepared emulsion was evaluated in the presence of strong oxidant of

194

NaClO, reducing agent of sodium ascorbate, UV radiation or common stabilizer of

195

sucrose.

196

Effect of NaClO. Diluted NaClO solution was added to the β-carotene containing

197

emulsion to a final concentration of 0.02%. The samples were tightly sealed and kept

198

at 25 °C in the dark. The β-carotene retentions were determined over time.

199

Effect of sodium ascorbate. Sodium ascorbate was added to the β-carotene

200

containing emulsion to a final concentration of 5 μg/ml. The samples were tightly

201

sealed and kept at 25 °C in the dark. The β-carotene retentions were determined over

202

time.

203

Effect of UV radiation. The β-carotene retentions were determined following UV

204

irradiation with a portable UV analyzer (model WFH-2048 from Hangzhou Qiwei

205

Instruments, China) at room temperature for 0, 2, 4, 6, 8 or 10 h. The distance

206

between the sample and the UV lamp was kept at 5 cm. The temperature of emulsions

207

did not change significantly after UV radiation.

208

Effect of sucrose. The β-carotene containing emulsions with a sucrose concentration 11



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of 0, 2%, 4%, 6%, 8%, and 10% (wt.%) were prepared respectively, and subsequently

210

kept at 25 °C in the dark. The β-carotene retentions were determined after a storage

211

time of 2 hours.

212

The degradation of β-carotene in the Pickering emulsion over storage time was also

213

studied. Emulsions with different concentrations of GroEL (0.05-0.45 (wt.%)) were

214

prepared and kept at 25 °C in the dark. The β-carotene retentions were determined

215

following a storage time of up to 35 days.

216

The β-carotene retention was defined as Cx/C0, in which C0 represents β-carotene

217

initial concentration, Cx being β-carotene concentration remaining after each specific

218

emulsion treatment. The amounts of β-carotene were quantified according to the

219

procedure previously described 27. Briefly, emulsions containing β-carotene were first

220

destabilized with absolute ethanol, followed by saponification with KOH and then

221

extraction with n-hexane. The amount of β-carotene in n-hexane was determined on a

222

Shimadzu UV-1700 UV-vis spectrophotometer using an extinction coefficient of

223

1,023 M-1·cm-1 at 450 nm.

224

Results and Discussion

225

GroEL Nano-Barrel as a Pickering Emulsion Stabilizer. The bacterial

226

chaperonin GroEL, a naturally abundant protein cage, can easily be prepared through

227

induced stable expression and one-step chromatographic purification, with a typical

228

yield of some 100 mg purified protein per liter culture

229

scalable for bio-applications. In addition to the common features required for 12


28,

making it conveniently

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high-grade Pickering emulsions such as non-toxic biodegradability, GroEL

231

nano-barrel is also highly mono-disperse in aqueous solution with a polydispersity

232

index determined to be 0.225 by DLS (Figure 1S), and possesses a key structural

233

element that is envisioned to be adapted readily for oil phase stabilization. That is,

234

GroEL bears a hydrophobic inner rim at each mouth of its cavity (Figure 1C), which

235

is flexible in structure and responsible for interactions with hydrophobic molecules or

236

their aggregates

237

may also render GroEL enhanced amphiphilicity or significantly influence its

238

interfacial behavior. Bearing this in mind, we investigated the interfacial property of

239

GroEL through surface tension measurements at the water-air interface, and compared

240

with the commonly available BSA. As seen from Figure 1D, while the surface tension

241

of both systems decreased clearly with the increase of protein concentration, GroEL

242

led to a much greater surface tension decrease than BSA of the same concentration,

243

with a much lower plateau value as well, which was reached after 40 mg/mL of

244

GroEL. Thus GroEL has a higher surface activity than BSA, which is translatable to a

245

better emulsifying performance, from a conventional viewpoint that surface tension or

246

the action of surface-active materials tightly governs the formation of emulsions 29.

21-23,

despite of the high water solubility of GroEL as a whole. This

13



247

Figure 1. The ability of GroEL nano-barrels to act as Pickering emulsion stabilizers. (A) and (B)

248

are top-view and side-view TEM micrographs of GroEL tetradecameric cage, adapted with

249

permission from our previous publication

250

side-view is visible as a rectangular particle with four stripes (marked by numbers), which

251

correspond to thicker protein regions as shown in the cross section. (C) Cross section of GroEL

252

highlighting the hydrophobic lining of its apical domain with yellow. The diagram was generated

253

with Pymol (PDB code 1SS8). (D) Surface tension measurement of GroEL aqueous solutions with

254

varying concentrations at 25 °C, compared with BSA. (E) Fluorescence microscopy image of the

255

emulsion of rosemary oil and water prepared using GroEL nano-barrel as an emulsifier. GroEL

256

was labeled with AF488, the oil phase stained with Nile red. (F) Change of oil-surface protein

257

coverage with GroEL concentration at a constant o/w ratio of 0.05 (v/v) at neutral pH. Each data

258

point shows the mean of triplicate measurements. Error bars represent the standard deviation.

30.

Copyright (2017) American Chemical Society. The

14


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We next prepared the emulsions of rosemary oil and water using GroEL as an

260

emulsifier, and further examined the interfacial behavior of GroEL through

261

fluorescence imaging at the oil/water interface. To facilitate imaging, Nile red was

262

used to stain the oil phase, GroEL being labeled with the green-fluorescent dye

263

AF488. The labeling was confirmed by UV-vis spectroscopy (Figure S2). Figure 1E

264

shows the fluorescence image of the as-prepared emulsion, revealing a clear layer of

265

GroEL nano-barrels (green) coating the rosemary oil droplet (red). The presence of a

266

physical barrier formed through particle interfacial adsorption is the distinctive feature

267

of Pickering emulsions. The interfacial adsorption of GroEL was also investigated by

268

measuring surface protein coverage of oil droplet. We found that the surface coverage

269

increased with GroEL concentration till reaching a maximum value of ~35% in the

270

concentration range tested (Figure 1F), which is comparable to those determined

271

previously with a similar method

272

defect and inter-particle electrostatic repulsion, have been shown to dramatically

273

lower interfacial adsorption and surface coverage

274

indicated that full surface coverage with active particles is not necessarily required for

275

emulsion stabilization once the adsorbed particle layer forms a rigid network

276

GroEL has an isoelectric point of 4.7 34, thus being largely negatively charged in the

277

aqueous phase (pH=7.5) of the emulsion. The strong inter-GroEL electrostatic

278

repulsion may partially explain the lower surface protein coverage of oil droplet. On

279

the other hand, this would greatly contribute to emulsion stability against droplet

280

coalescence through introducing inter-droplet electrostatic repulsion. The remarkable

26.

Several factors, like curvature effect, packing

26, 31-32.

15


Moreover, it has been

26, 33.


281

coalescence stability, especially at very low interfacial particle coverage, is a general

282

feature for a variety of Pickering emulsions stabilized by different types of particles

283

35.

284

Taken together, the above results establish that GroEL nano-barrel is highly

285

surface-active and can act as physical stabilizers in Pickering emulsions through

286

adsorbing at oil/water interface. So far the knowledge about the molecular

287

mechanisms of protein-mediated emulsification is still quite scarce. That protein

288

surface hydrophobicity is closely involved in this process is the only general

289

consensus that has been reached up to now 35. We believe that the same holds true for

290

the emulsification with GroEL as well. The protein barrel carries a well-defined

291

hydrophobic rim at each end, ready to interact with the oil phase. This facilitates the

292

regular packing of GroEL at oil/water interface and the lowering of surface tension,

293

and probably leads to the observed high surface activity and emulsifying activity.

294

However, the contributions of other small hydrophobic patches on GroEL surface and

295

its conformational flexibility at oil/water interface to its emulsifying performance as

296

well as the possible cooperation of GroEL’s two hydrophobic ends are important

297

aspects that deserve further investigation.

298

Based on the fluorescence imaging, the rosemary oil appeared to be the dispersed

299

phase surrounded by water. We further checked the type of GroEL-stabilized

300

Pickering emulsion, water-in-oil or oil-in-water, through dilution test based on the

301

principle that an emulsion can be diluted with its continuous phase. When rosemary

302

oil was added to the GroEL emulsion, the emulsion was not diluted and the separation 16


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303

was apparent; in contrast, when water was added, the emulsion was diluted indicating

304

that water was the continuous phase (Figure S3). Hence dilution test confirmed that

305

the GroEL-stabilized Pickering emulsion belongs to an oil-in-water emulsion.

306

Optimization of Formulation and Operational Conditions of GroEL-Stabilized

307

Pickering Emulsions. Mixing of rosemary oil, water and GroEL with vigorous

308

stirring usually produces a coarse Pickering emulsion, with dispersed droplet size

309

ranging from several hundred nanometers to several microns (Figure 1E and Figure

310

S3). This emulsion can be homogenized by ultrasonication, leading to the further

311

break-up of dispersed droplets and formation of sub-micron emulsions. Formulation

312

composition was next optimized to acquire stable Pickering emulsion with sub-micron

313

droplets via altering the o/w volume ratio as well as GroEL concentration. Pickering

314

droplet size in a series of samples was measured and compared after a shelf life of 15

315

days (Figure S4). Overall, the formulation composition showed a clear influence on

316

the droplet size distribution. In the tested GroEL concentration range, the droplet size

317

increased with o/w ratio. At lower GroEL concentrations (0.05-0.25 wt.%), this trend

318

was more obvious until reaching the highest value of ~950 nm, while at higher GroEL

319

concentrations (0.25-0.45 wt.%), the highest value achieved was only ~500 nm. From

320

another angle, the droplet size decreased with GroEL concentration especially at

321

higher o/w ratios, underlining the important role of GroEL in dispersing or stabilizing

322

oil droplets. When the o/w ratio falls in the range of 0.05-0.35, Pickering emulsions

323

with relatively small and stable oil droplets (200-450 nm) are formed over the entire

324

GroEL concentration range tested (0.05-0.45 wt.%). The decrease of emulsion droplet 17



325

size helps reduce the chance of gravitational separation, thus leading to the

326

enhancement of emulsion kinetic stability based on the Stokes’ law27. Thus the above

327

formulas will produce more stable GroEL emulsions than other o/w ratios (e.g.,

328

0.35-0.5).

329

important aspect to be considered for emulsions stability and its practical applications.

330

Here the influence of operational conditions (pH, storage temperature or ionic

331

strength) on GroEL emulsion stability has also been investigated. Figure 2A-2C

332

shows the influence of pH on the emulsion stability, which was found closely related

333

to GroEL surface charge that varies with pH. As seen from Figure 2A and inserts, at a

334

pH value close to GroEL isoelectric point of 4.7

335

potential measurement to bear little net surface charge and the emulsion became

336

demulsified and separated into two obvious phases, emulsion and serum layers;

337

subsequent pH adjustment to 8.5 led to the restoration of a stable emulsion, in which

338

GroEL carried substantial net negative charge. The microstructures corresponding to

339

these two states were checked by optical imaging (Figure 2B and 2C), revealing an

340

obvious change from flocculation to highly monodisperse sub-micron droplets. The

341

flocculation occurs when the attractive interactions between individual droplets (e.g.,

342

van der Waals forces) dominate the long-range repulsive interactions (including

343

electrostatic and steric forces), but not the short-range repulsive interactions 35-36. Our

344

results can be interpreted in support of an important role of GroEL-introduced

345

electrostatic repulsive force between oil droplets in the coalescence stability of the

346

emulsion. Hence from a practical application standpoint, the pH of GroEL-stabilized

In addition to formulation composition, operational conditions are another

34,

GroEL was detected by zeta

18


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347

Pickering emulsions should be kept at a value far from 4.7 as long as the structure of

348

GroEL nano-barrel is retained.

349

Next, the tolerance of GroEL-stabilized emulsion against a dramatic pH variation

350

was investigated by moving pH back and forth between 4.5 and 8.5. The Pickering

351

droplet size was recorded following each pH adjustment from 8.5 to 4.5 and then back

352

to 8.5 (Figure 2D). After only several adjustments, the change of droplet size was not

353

evident indicating that the oil phase is re-dispersible after an abrupt pH change or

354

after demulsification. However, the droplet size increased substantially after 10

355

adjustments and could not change into the original state. Thus, only to a limited

356

extent, the Pickering emulsion exhibits reversible emulsification/demulsification

357

transition with pH change. Similar pH-responsive behavior has been reported for

358

several Pickering emulsion systems

359

developed can stand more cycles of pH change suggestive of being more stable. One

360

possible explanation for the irreversibility after 10 consecutive cycles of pH change is

361

that the structure or packing of barrel-shaped GroEL at the oil/water interface may get

362

disrupted after this intense treatment, resulting in the desorption of the protein and

363

hence irreversible droplet fusion.

26, 37-38,

but the GroEL-based system we

19



364

Figure 2. Effect of pH on GroEL-supported Pickering emulsion stability. (A) Zeta potentials of

365

GroEL nano-barrel at different pH values at 25 °C. (B) and (C) are microscopy images of the

366

emulsions at pH 4.5 and pH 8.5, corresponding to the inserted digital images in (A). (D) Oil

367

droplet size distribution after each cycle of pH switch from 8.5 to 4.5 and then back to 8.5. Each

368

data point shows the mean of triplicate measurements. Error bars represent the standard deviation.

369

To examine the effect of storage temperature or ionic strength on the emulsion

370

stability, we incubated the emulsions at different temperatures or ionic strengths and

371

recorded the change of droplet size or zeta potential over time. As seen from Figure

372

3A, the droplet size showed no appreciable change during the storage period of 15

373

days at 4 °C, 25 °C, 37 °C or 50 °C. When the temperature was increased to 65°C,

374

however, the droplet size increased from 250 nm to 650 nm after 15 days storage.

375

This suggests a reduced stability of the emulsion under prolonged high temperature,

20


Page 20 of 37

Page 21 of 37


376

whereas the final droplet size still falls in the sub-micron range. On the whole, the

377

zeta potential increased with time at all storage temperatures (Figure S5A). Compared

378

to other temperatures, the greater absolute value of final zeta potential at 4 °C or

379

25 °C indicative of more negative surface charge may be advantageous for a

380

prolonged storage of more than 15 days.

381

Figure 3B shows the influence of ionic strength on the Pickering droplet size

382

examined through the addition of different concentrations of NaCl. It is observed that

383

the droplet sizes did not change significantly over time at a NaCl concentration of no

384

more than 500 mM. On the other hand, when NaCl concentration was increased to

385

800 mM, the droplet size increased from 390 nm to 590 nm after 15 days storage.

386

This might be largely attributed to the well-known protein salting-out in concentrated

387

salt solutions. In other words, the concentrated aqueous NaCl possibly caused a

388

decrease of GroEL solubility and its aggregation to some extent, thus leading to the

389

reduced emulsion stability as indicated by the increase of dispersed droplet size. We

390

also observed that the corresponding zeta potential increased obviously over time, and

391

increased with NaCl concentration as well (Figure S5B). The responsive behavior of

392

the zeta potential to ionic strength is probably due to salt screening, which results in

393

the compression of the electrostatic double layer of charged species on oil droplet

394

surface like GroEL nano-barrels, and hence the observed reduction of zeta potential

395

(absolute value)

396

based on our results. However, the zeta potential change at relatively lower ionic

397

strengths (e.g., ＜800 mM) did not produce a profound effect on the droplet size of

39.

This effect was enhanced with time or ionic strength increasing

21



398

the emulsions. Combined, at temperature ≤ 50 °C and ionic strength (NaCl

399

concentration) up to 500 mM, GroEL-stabilized Pickering emulsions show excellent

400

stability during the storage period of 15 days.

401

Figure 3. Change of the droplet size in GroEL-stabilized Pickering emulsions with time in a

402

temperature series (A) or an ionic strength series (B). Each data point shows the mean of triplicate

403

measurements. Error bars represent the standard deviation.

404

Rheological Analysis. Rheological properties tightly control emulsion performance

405

like creaming and sedimentation

406

emulsions has been assessed by dynamic frequency sweep measurement. We first

407

prepared emulsions with different o/w ratios of 0.05 and 0.50 (v/v), which were both

408

stabilized by 0.15 wt.% GroEL but produced dramatically different droplet sizes, 250

409

nm vs. 850 nm (Figure S4). An oscillatory strain sweep test was performed to

410

determine the linear viscoelastic region, in which all the subsequent rheological

411

analyses were conducted. Figure 4A demonstrates shear stress-shear rate curves for

412

the two emulsions stabilized by GroEL nano-barrels, which have been fitted with

413

Herschel-Bulkley model. It is fundamentally a power law model of a non-Newtonian

414

fluid 41, for which the equation is as below:

40.

The rheology of GroEL-stabilized Pickering

22


Page 22 of 37

Page 23 of 37


415

σ = 𝜎0 + 𝑘𝛾𝑛

416

in which σ represents the shear stress (Pa), 𝜎0 the yield stress (Pa), 𝛾 the shear rate

417

(s-1), 𝑘 the consistency index and 𝑛-the flow index, which is a measure of the extent

418

of shear thinning (𝑛 < 1) or shear thickening (𝑛 > 1) of the fluid. If 𝑛 = 1 and 𝜎0 =

419

0, this model reduces to the Newtonian fluid

420

parameters for the two different emulsions (Table S1). In general, yield stress 𝜎0 and

421

consistency index 𝑘 increase with an increase of w/o ratio from 0.05 to 0.50. The 𝜎0

422

increase indicates that more effort is needed to make the emulsion flow. Flow index 𝑛

423

was estimated to be 0.94 or 0.67 for emulsions with an o/w ratio of 0.05 or 0.50. The

424

emulsion having a low percentage of oil approaches a Newtonian fluid, in which the

425

shear stress is directly proportional to the shear rate, as the value of 𝑛 is very close to

426

1 and 𝜎0 very close to 0. In contrast, the emulsion with a high o/w ratio displays

427

shear-thinning effect since 𝑛 is well below 1. This effect is characterized by

428

decreasing viscosity with increasing shear rates, caused by the fluids internal

429

structure. Shear thinning observed in the GroEL-stabilized Pickering emulsion with

430

high o/w ratio suggests that there were weak attractive forces among dispersed

431

droplets or even the formation of gel-like structure

432

this behavior are glues, shampoos, polymer solutions and so on.

42.

Curve-fitting yields rheology

26, 43.

Typical materials showing

433

Oscillation rheology-testing is also performed to further examine the strength of

434

GroEL-based emulsions of different o/w ratios. Figure 4B presents their frequency

435

sweeps from 0.01-10 Hz at 1% strain in the linear viscoelastic region. At an o/w ratio

436

of 0.05, the equilibrated storage modulus (G′) and loss modulus (G″) of the Pickering 23



437

emulsion were extremely low and crossed within the frequency range tested,

438

indicating little or even no network formation in the emulsion. When the o/w ratio

439

was increased to 0.50, the G′ and G″ values of the emulsion rose to ca. 200 Pa and 20

440

Pa, respectively. That is, G′ exceeded G″ by 1 order of magnitude. Meanwhile, both

441

values were independent of the frequency and kept relatively constant. These

442

observations suggest that the emulsion with a o/w ratio of 0.50 has formed gel-like

443

network

444

stabilized by other proteins 26, 35.

445

Figure 4. Rheological properties of GroEL-stabilized Pickering emulsions with two different o/w

446

ratios of 0.05 and 0.5. (A) Shear stress plotted as a function of shear rate. (B) Dynamic frequency

447

sweeps. GroEL concentration was kept at 0.15 wt.%.

44.

The gel-forming propensity has been reported previously for emulsions

448

Gelation or even just thickening is considered to be a beneficial property for

449

emulsions since this inhibits the coalescence of dispersed droplets and other cases

450

resulting in the destabilization of emulsions. When emulsions form a gel-like structure

451

as an extreme case, their stability is expected to be great for a prolonged storage time.

452

Thus the tunable rheological behaviors of GroEL emulsions depending on the o/w

453

ratio are of importance in terms of specific end-use applications. 24


Page 24 of 37

Page 25 of 37


454

GroEL-Stabilized Pickering Emulsions for Lipophilic Compounds Delivery.

455

Inclusion of liposoluble bioactive compounds in oil-in-water emulsions is a current

456

trend for food, cosmetic and medical industries, which, however, represents a

457

challenge because of the labile nature of these compounds and the instability of

458

emulsion-based delivery systems

459

was encapsulated in GroEL-stabilized emulsion; this system was then evaluated for its

460

potential to decrease β-carotene degradation against different possible environmental

461

stresses, which is important for designing efficient emulsified delivery vehicles.

45-46.

As a model lipophilic compound, β-carotene

462

Oxidation is the major cause of carotenoids degradation. Figure 5A shows the

463

influence of strong oxidant of NaClO on β-carotene stability dispersed in

464

GroEL-supported emulsion. ClO¯ is present in tap water and thus has a high chance of

465

mixing with emulsions. As seen, β-carotene exhibited a rapid response to the addition

466

of NaClO, with a retention of ~72% after 2 hours. Similar quick degradation trend has

467

been observed in the presence of other oxidizing agents

468

extended system of 11 conjugated pi bonds, while the Cl in NaClO is electrophilic and

469

will react with nucleophiles like the pi bonds in β-carotene, thus making such

470

structures very vulnerable. The physical barrier created by GroEL at the rosemary

471

oil/water interface may not be able to completely restrain the reactions between

472

NaClO and β-carotene encapsulated in the dispersed droplets, as observed with

473

surfactant-stabilized conventional emulsions 47. Therefore, emulsion delivery systems

474

need keep emulsified β-carotene from coming into contact with a strong oxidant as far

475

as possible. On the other hand, the dependence of the emulsion dispersed β-carotene 25


47.

β-carotene contains an


476

on reducing agent of sodium ascorbate was also examined. Sodium ascorbate

477

functions as an antioxidant in food and cosmetics 48. Treatment with sodium ascorbate

478

showed much less influence on the stability of β-carotene, when compared to the

479

effect of NaClO, as more than 90% β-carotene was retained after 10 hours (Figure

480

5B). While sodium ascorbate with a very strong reducing power helps to prevent

481

oxidative degradation (e.g., from O2 in the surroundings or ClO¯ in tap water), it

482

might also cause a slow reduction of the double bonds in β-carotene, thus leading to

483

the observed slight decrease in the pigment retention over time. However, the

484

contribution of oxidative degradation due to incomplete inhibition by sodium

485

ascorbate to this result cannot be ruled out.

486

Figure 5. Chemical stability of β-carotene in GroEL-stabilized Pickering emulsion. The amount of

487

β-carotene was monitored in the presence of strong oxidant NaClO (A), reducing agent sodium

488

ascorbate (B), UV radiation (C) or sucrose (D). Data reported as mean of triplicate. The error bar 26


Page 26 of 37

Page 27 of 37

489 490


shows standard deviation.

β-carotene is sensitive to light and becomes unstable under illumination

49.

The

491

effect of light on β-carotene stability in GroEL-stabilized Pickering emulsion was next

492

probed by UV radiation (Figure 5C). Obvious decrease in β-carotene retention was

493

observed with time, presumably due to the formation of β-carotene radical cations 50.

494

After a radiation period of 12 hours, ca. 25% β-carotene was lost. Thus it is crucial to

495

keep the emulsion system from illumination to reduce the effect of light. Sucrose is

496

routinely used as additives to sweeten foods and also used as a protein stabilizer 51. It

497

was observed that the addition of sucrose maintained the stability of β-carotene

498

(Figure 5D). Meanwhile, the increase of sucrose concentration from 2 wt.% to 10

499

wt.% also showed no obvious effect in this aspect. Nevertheless, sucrose has been

500

found to have a pronounced influence on the thermal stability of oil-in-water

501

emulsions stabilized by protein particles 52.

502

The β-carotene retention over a long storage time was also monitored in the

503

presence of different GroEL concentrations (Figure 6). The results highlighted

504

decreasing β-carotene retention (or continuous β-carotene loss) during 35 days of

505

storage at room temperature, which was describable by zero-order kinetics reaction

506

model. The slopes of the linear plots in Figure 6 are listed in Table S2, which can be

507

used to estimate β-carotene loss rate at different GroEL concentrations 27. It is evident

508

that the rate of β-carotene loss was stringently dependent on GroEL concentration.

509

When GroEL concentration was insufficient to ensure stability of the system (0.05 -

510

0.30 wt.%), the rate of loss was higher (from 1.55% per day to 1.02% per day), while 27



511

at elevated GroEL concentrations (0.35 - 0.45 wt.%), this parameter decreased to a

512

steady value (0.92% per day). Hence, for an efficient long-term storage of β-carotene,

513

GroEL should be used at a sufficient concentration to form stable protective barriers

514

around dispersed oil droplets containing the bioactive compound. This is closely

515

related to the shelf life of potential end products of the GroEL-emulsified β-carotene

516

delivery system. One possible explanation for the observed β-carotene loss along the

517

long-term storage is the presence of atmospheric O2 in GroEL emulsions, which

518

causes the autoxidation of β-carotene with relative ease 47.

519

Figure 6. Kinetics of degradation of 0.1% (w/w) β-carotene included in oil-in-water Pickering

520

emulsions (o/w = 0.10) prepared with various concentrations of GroEL nano-barrels. Data

521

reported as mean of triplicate. The error bar shows standard deviation.

522

In conclusion, stable Pickering emulsion based on bio-derived GroEL protein

523

nano-barrel was successfully developed. A key element of the present design is the

524

inherently high thermo- and chemo-stability of GroEL as well as the promiscuity of

525

its apical hydrophobic cavity that can accept diverse hydrophobic molecules or 28


Page 28 of 37

Page 29 of 37


526

surfaces. We demonstrated that GroEL is highly surface-active, and allows the

527

formation of Pickering emulsion by providing a protective barrier around dispersed oil

528

droplets. GroEL-stabilized Pickering emulsions with sub-micron droplet size

529

(200-450 nm) could be easily prepared when the o/w volume ratio is set to 0.05-0.35

530

with a GroEL concentration of 0.05-0.45 wt.%. The as-prepared emulsion shows

531

reversible emulsification/demulsification transition with pH moving back and forth

532

between 4.5 and 8.5, and excellent stability at storage temperatures less than 65 °C

533

and ionic strength (NaCl concentration) up to 500mM. With the o/w ratio increasing,

534

GroEL emulsions tend to form a gel-like mcirostructure. In addition, we also

535

evaluated the protection properties of GroEL-stabilized emulsion on β-carotene

536

encapsulated in its oil phase. While strong oxidant NaClO and UV radiation caused

537

clear degradation of the occluded β-carotene, reducing agent sodium ascorbate and

538

sucrose showed much less or no influence on the stability of β-carotene. Finally, it

539

was shown that higher GroEL concentration (e.g., 0.35-0.45 wt.%) is necessary for an

540

efficient long-term storage of β-carotene. Taken together, the stabilization of emulsion

541

via GroEL nano-barrel has been proven a valid alternative to synthetically generated

542

emulsion stabilizers, which also offers a promising strategy for the delivery of

543

lipophilic bioactive compounds in the fields of food, cosmetics and biomedicine.

544

Acknowledgements

29



Page 30 of 37

545

This work was supported by the National Natural Science Foundation of China

546

(21503278), China Postdoctoral Science Foundation (2014M560588, 2015T80756),

547

and the Fundamental Research Funds for the Central Universities.

548

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Graphical Abstract 368x131mm (150 x 150 DPI)


Highly surface-active chaperonin nano-barrels for oil-in-water

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